Management of Acute Gouty Arthritis With A Polyherbal Unani Formulation

Management of Abstract
Acute Gouty out is one of the most common types of inflammatory joint

Arthritis with a
Polyherbal
Unani
G

diseases; affects an estimated 1-1.5% of the world population. Modern drugs
used for subsiding acute attacks or lowering serum uric acid are associated
with potent adverse effects. Moreover, these commonly used therapeutic agents
often, and for various reasons, do not achieve the desired lowering of serum
urate levels to below 6.0 mg/dl. On the basis of conventional Unani Usool-e-
Formulation

Ilaj of Niqris (gout), five herbal drugs from the list of classical Unani anti-

arthritic drugs have been selected and formulated in capsule form and a single

Rais ur Rahman,

1
blind placebo controlled clinical trial was carried out to evaluate the efficacy

2
Dania Siddiqui,

and safety of this capsule in the management of gouty arthritis. Six week

2
Naseem Akhtar,

treatment with test drug produced remarkable effects on various efficacy

2
D.S. Dua

and parameters. No any adverse effect was observed during the course of treatment.

*Yasmeen Shamsi
3

Keywords: Gout, Niqris, Wajaul Mafasil, Suranjan, Cocchicum luteum

Department of AYUSH
1

Ministry of Health & F.W.,

Government of India, Introduction

GPO Complex, INA,

New Delhi - 110023 Gouty arthritis is among the earliest diseases that have been recognized as

a clinical entity. First identified by the Egyptians in 2640 BC, podagra (acute

Department of Moalijat
2

Ayurvedic and Unani Tibbia College, gout occurring in the first metatarsophalangeal joint) was later recognized by

Karol Bagh, New Delhi-11005 Buqrat in the fifth century BC, who referred to it as the unwalkable disease.

Buqrat also noted the link between the disease and an intemperate lifestyle,

Department of Mahiyatul Amraz

3

Faculty of Medicine (U),

referring to podagra asarthritis of the rich, as opposed to rheumatism, an

Jamia Hamdard, New Delhi-110062 arthritis of the poor(Nuki et al., 2006). Six centuries later to Buqrat, Jalinoos

was the first to describe tophi (James et al., 2000).

Gout is one of the most common types of inflammatory joint diseases; affects

an estimated 1-1.5% of the world population (Praveen et al., 1994). The

prevalence of gout is rising as a result of a changing pattern of lifestyle

(Arromdee et al., 2002). In most cases, no identifiable underlying cause of

gout is present, but evident factors are usually present that could contribute

to increase in urate (uric acid) levels, such as renal function disorders, obesity,

and the use of thiazide diuretics (Roubenoll, 1990). Although, hyperuricemia is

a risk factor for the development of gout, the exact relationship between

hyperuricemia and acute gout is unclear. Acute gouty arthritis can occur in the

presence of normal serum uric acid concentrations. Conversely, many persons

with hyperuricemia never experience an attack of gouty arthritis (McCarty,

1994).

3
*Author for correspondence

Hippocratic Journal of Unani Medicine 1

July - September 2014, Vol. 9 No. 3, Pages 1-13
Several approaches to the treatment of gout are available depending on the

patients presentation of the disease, patients specific risk factors (high serum

urates, previous attacks and radiographic signs), the clinical phase of the

disease (acute, recurrent, tophaceous) and general risk factors, such as obesity

and alcohol consumption.

Acute gout is usually treated by reducing inflammation of the affected joint with

non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids etc.

Although these agents are generally effective, they also present significant

risks in patients who have pre-existing renal, cardiovascular, metabolic and

gastrointestinal diseases (Nuki, 1999; Emmerson, 1996).

Antihyperuricemic drugs such as allopurinol, benzbromarone, sulfinpyrazone

and probenecid can have potent side effects (Singer et al., 1986, Arellano et

al., 1993). Benzbromarone was withdrawn from the market in Europe in 2003,

but was registered again in some countries in 2004 (Sutaria et al., 2006). Its

use is now restricted for patients with gout who are allergic to allopurinol or

those in whom allopurinol is contraindicated (Jansen et al., 2004). Furthermore,

these commonly used therapeutic agents often, and for various reasons, do

not achieve the desired lowering of serum urate levels to below 6.0 mg/dl.

The side effects/drawbacks of all above mentioned drugs call for the development

of novel drugs with similar or better efficacy and lesser toxicity than presently

available drugs.

On the basis of conventional Unani Usool-e-Ilaj of Niqris (gout), five herbal

drugs from the list of classical Unani anti-arthritic drugs have been selected

and formulated in capsule form and a clinical trial was carried out to evaluate

the efficacy and safety of this capsule in the management of gout.

Materials and Methods

Study Drug

Study drug was a combination of five herbs namely, Suranjan (Colchicum

luteum), Elva (Aloe barbadensis), Qurtum (Carthamus tinctorius), Halaila-e-

Zard (Terminalia chebula), Zanjbeel (Zingiber officinale). All these five drugs in

equal proportion were finely powdered and encapsulated in hard gelatin capsule

in the quantity of 1gm.

Placebo

Placebo was supplied to the patients in the form of similar capsules of 1gm

each containing wheat flour.


Study Design

This was a randomized, single blind, placebo controlled study, conducted in

the Department of Moalejat, A & U Tibbia College & Hospital, Karol Bagh, New

Delhi, From September 2009 to December 2011.

Participants

(i) Inclusion Criteria

Both male and female patients aged between 18 -65 years fulfilling the criteria

of American College of Rheumatology (ACR) for diagnosing acute gouty arthritis

including clinical features, laboratory and radiographic findings were included

in the study, who had serum uric acid level more than the upper limit of normal

range ( > 7 mg/dl)

(ii) Exclusion Criteria

Patients were excluded if they had renal or hepatic insufficiency or cardiovascular

disorders. Patients taking thiazide group of diuretics/ aspirin/NSAIDs and

Pregnant and lactating women were too excluded from the study.

Ethical Consideration

All patients were included in the study after obtaining written informed consent

and study was conducted according to Good Clinical Practice guidelines.

Dosage and Administration

Following 5-7 days washout period of anti-inflammatory/ analgesic drugs (e.g.,

NSAIDs, Corticosteroids) or antihyperuricemic drugs (eg allopurinol) or any

other medication used for the treatment of arthritis (e.g. Ayurvedic, Homeopathic

or Unani drugs), patients were randomly assigned to receive either drug or

placebo capsule in the dose of 2 capsule thrice daily with plain water up to a

period of six weeks. Randomization was done by lottery method.

Follow up and Drug Compliance

Clinical as well as laboratory evaluation was performed and recorded at the

baseline, week 1, week 4 and week 6. Compliance with treatment drug/placebo

was evaluated at each follow up visit by capsule count.

Criteria for the Assessment of Efficacy

To assess the response of treatment on patients of gouty arthritis in both

groups, the following parameters were used.


Subjective Parameters

Pain (Wong-Bakers Faces rating scale; with 0=doesnt hurt, 2=hurts a

little bit, 4=hurts a little more, 6=hurts even more, 8=hurts a lot, 10=as

much as the patient can imagine), (Cheng et al., 2004; Taylor et al.,

2007).

Tenderness (0-4 point scale; with 0=no tenderness, 1=patient says it is
painful, 2=patient says it is painful, winces, and pulls back, 4=patient

does not allow palpation), (Cheng et al., 2004; Taylor et al., 2007).

Joint swelling (0-4 point scale; with 0=no swelling,1=barely perceptible,

2=mild, 3=moderate, 4=severe, bulging beyond the joint margins) (Cheng

et al., 2004; Taylor et al., 2007).

Movement/Mobility (0-4 point scale; with 0=full voluntary

movement,1=partial voluntary movement, 2=full movement when the

joint is moved by the examiner, 3=partial movement when the joint is

moved by the examiner, 4=no movement at all)

Serum uric acid, C-Reactive proteins, Erythrocyte Sedimentation Rate

and total leukocyte count

Assessment of Safety

To establish the safety of test drug , the following investigations were carried

out at baseline, after one week and just after the termination of treatment.

Liver function test S.Bilirubin, S.G.O.T, S.G.P.T. & S.Alkaline

Phosphatase

Kidney function test Blood Urea, S. Creatinine.

Haemogramme Hb%, TLC, DLC, E.S.R.

Statistical Analysis

The differences in pretreatment and post treatment obtained in both groups

(test group and control group) were compared by applying Mann-Whitney U

Test.

Results

Total 46 cases, 21 in test group and 25 in control group were randomly

enrolled in the study. Two subjects from test group and 4 patients from control

(placebo) group dropped out of the study due to unknown reason. Twenty

patients in test group and 20 in control group completed the treatment up to

the end of study (6 weeks).The demographic data and other characteristics at

baseline are presented in Table 1.


Table 1: Demographic Data Baseline Characteristics of Study Patients

Variable Test Group (N=20) Control Group (N=20)

Age (Years) Mean SD 42.70 11.15 43.80 9.28

Gender Male- N (%) 14 (70%) 15(75%)

Female-N (%) 06 (30%) 05 (25%)

Family H/o Gout-N (%) 2 (10%) 3 (15%)

Pain Score (Mean SD) 5.95 1.54 5.95 1.50

Tenderness (Mean SD) 2 1.12 1.60 1.14

Swelling (Mean SD) 2.20 0.95 1.55 0.94

Movement (Mean SD) 1.70 0.86 1.40 0.59

S. Uric Acid 8.04 1.98 7.81 0.91

C.R.P. 3.92 2.35 4.37 2.18

T.L.C. 9300 1554.6 8785 1786.3

E.S.R. 26.65 10.49 28.70 11.3

SD = standard deviation; ESR = erythrocyte sedimentation rate;

CRP = C-reactive proteins; TLC= total leukocyte count

The effects of 6 weeks treatment with test drug and control on various clinical

and laboratory parameters are described below:

Clincal Findings

Joint Pain

In test group, the mean score of pain ( SD) at baseline was 5.95 1.54,

which was reduced to 4.50 1.91 on the 7th day, 3.30 1.72 on 28th day and

1.85 1.98 on the termination of treatment (42nd day). While in control group,

the mean pain score was 5.95 1.50 at baseline, which gradually increased

to 6.05 1.47 on 7th day, 6.0 1.07 on 28th day, and 6.35 1.23 at the end

of treatment. On applying Mann- Whitney test, extremely significant difference

between the two groups was detected on 28th day (p<0.0001) and 42nd day

(p<0.0001), (Table-2).

Joint Tenderness

In test group the baseline tenderness score (mean SD) of 2.0 1.12

decreased to 1.75 0.97 (7th day), 1.05 0.94 (28th day) and 0.60 0.59

(42nd day). Whereas, there was very gradual and insignificant decrease in

mean tenderness score in control group, from baseline (1.60 1,14) to 1.50

1 on 7th day, 1.55 0.99 on 28th day, 1.50 0.21 on termination of


Table 2: Effect of Test Drug and Control on Pain

Pain Control Group (N=20) Test Group (N=20)

0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 5.95 6.05 6.0 6.35 5.95 4.50 3.30 1.85

S.D. () 1.50 1.47 1.08 1.23 1.54 1.90 1.72 1.99

S.E.M. () 0.34 0.33 0.24 0.28 0.34 0.43 0.38 0.44

% change - 1.68 0.84 6.72 - 24.36 46.9 68.91

P value at 7th day (T/C) <0.01 (LS) (Mann-Whitney Test)

P value at P value 28th day (T/C) <0.0001 (ES) (Mann-Whitney Test)

P value 42nd day (T/C) <0.0001(ES) (Mann-Whitney Test)

SD = Standard Deviation,; S.E.M. Standard Error of Mean; LS = Less Significant,

HS = Highly Significant

treatment. The difference in tenderness in between the groups as analysed by

Mann-Whitney Test was not significant at 28th day (p>0.05) and moderately

significant at 42nd day (p<0.01), (Table-3).

Joint Swelling

In Placebo group, the mean score of swelling increased from 1.55 0.94

(baseline) to 1.70 0.98 on 7th day, 1.70 0.98 on 28th day and1.75 1.02

on 42nd day. On the other hand the mean score of swelling at baseline in test

group was 2.20 0.95, 1.80 0.83 on 7th day, 1.15 0.67 on 28th day, and

0.45 0.51 on 42nd day. The difference in between the groups as analysed

statistically was found to be non-significant (p>0.05) on 7th day & 28th day, but

extremely significant on 42nd day (p<0.001), (Table-4).

Table 3: Effect of Test Drug and Control on Tenderness

Tenderness Control Group (N=20) Test Group (N=20)

0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 1.60 1.50 1.55 1.50 2 1.75 1.05 0.60

S.D. () 1.14 1.0 0.99 0.94 1.12 0.97 0.94 0.59

S.E.M. () 0.25 0.22 0.22 0.21 0.25 0.22 0.21 0.13

% change - 6.25 3.12 6.25 - 12.5 47.5 70

P value at 7th day (T/C) 0.48 (NS) (Mann-Whitney Test)


P value at 42nd day (T/C) 0.003 (MS) (Mann-Whitney Test)

SD = Standard Deviation; S.E.M. Standard Error of Mean; LS = Less Significant,

NS Not Significant; MS = Moderately Significant


Table 4: Effect of Test Drug and Control on Swelling

Joint Control Group (N=20) Test Group (N=20)

Swelling 0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 1.55 1.55 1.70 1.75 2.20 1.80 1.15 0.45

S.D. () 0.94 0.94 0.98 1.02 0.95 0.83 0.67 0.51

S.E.M. () 0.21 0.21 0.22 0.23 0.21 0.18 0.15 0.11

% change - 0 9.68 12.9 - 18.18 47.73 79.54


P value at 28th day (T/C) 0.05(NS) (Mann-Whitney Test)

P value at 42nd day (T/C) 0.0002 (ES) (Mann-Whitney Test)

SD = Standard Deviation; S.E.M. = Standard Error of Mean; NS = Not Significant;

ES = Extremely Significant

Restriction of Movements

In Control group, the mean score ( SD) of restriction in movements in control

group was 1.40 0.59 at baseline, 1.45 0.60 at 7th day, 1.45 0.60 at 28th

day, 1.65 0.74 at 42nd day. On the other hand, in test group the restriction

of movement was 1.70 0.86 (baseline), 1.35 0.59(7th day), 0.85 0.67

(28th day), and 0.55 0.51 (42nd day).According to Mann-Whitney test, the

difference in between the groups was moderately significant (p<0.01) on 28th

day and extremely significant (p<0.001) on 42nd day (Table-5).

Table 5: Effect of Test Drug and Control on Restriction of Movements

Restriction Control Group (N=20) Test Group (N=20)

of

Movement 0 day 7th day 28th day 42nd day 0day 7th day 28th day 42nd day

Mean 1.40 1.45 1.45 1.65 1.70 1.35 0.85 0.55

S.D. () 0.59 0.60 0.60 0.74 0.86 0.59 0.67 0.51

S.E.M 0.13 0.13 0.13 0.17 0.19 0.13 0.15 0.11

% change 3.57 3.57 17.86 20.59 50 67.65


P value at 28th day (T/C) 0.008(NS) (Mann-Whitney Test)

P value at 42nd day (T/C) 0.0001 (ES) (Mann-Whitney Test)

SD = Standard Deviation; S.E.M. = Standard Error of Mean;, NS = Not Significant;

ES = Extremely Significant


Laboratory Findings

Serum Uric Acid

In control group, the mean values of serum uric acid were 7.81 0.91 at

baseline, 7.62 1.13 mg/dl, 7.62 1.14 mg/dl, 7.71 1.82 mg/dl, on day 7th,

28th and 42nd respectively. On the contrary, in test group the change in the

serum levels of uric acid decreased from baseline of 8.04 1.98 mg/dl to 6.21

1.62 mg/dl on 7th day, 6.27 1.52 mg/dl on day 28 and 5.55 1.57 mg/dl

on day 42. The difference in mean values between the two groups on all the

three follow ups was extremely significant (p<0.001) as analysed by using

Mann Whitney U test (Table-6).

C - Reactive Protein

The C-Reactive protein values observed in test group were 3.92 2.35 mg/

dl at baseline 3.91 2.17 mg/dl on 7th day, 2.99 1.86 mg/dl on 28th day and

2.30 1.42 mg/dl on 42nd Whereas, in control group the mean values of CRP

were recorded as 4.37 2.18 mg/dl at baseline, 4.30 2.20 mg/dl on 7th day,

3.95 2.14 mg/dl on 28th day and 3.70 2.03 mg/dl on 42nd day. The inter-

group difference was not significant (p>0.05) on day 7 and day 28, but was

less significant (p<0.05) on day 42 (Table-7).

Total Leucocyte Count (T.L.C.)

In Control group, the mean TLC count in control group was 8080 1395/mm3

at baseline, 9105 1325 /mm3 on day 7th, 9180 1405 /mm3 on day 28th, and

8950 1325/ mm3 on day 42nd. While in test group it was recorded as 9300

1554 mm3/ 8080 1395/ mm3 on day 7th, 8380 1505/ mm3 on day 28th,

and 7860 1622/ mm3 on day 42nd. The difference in between the group was

Table 6: Effect of Test Drug and Control on Serum Uric Acid

S. Uric Control Group (N= 20) Test Group (N=20)

Acid (mg/dl) 0 day 7th day 28th day 42nd day 7th day 28th day 42nd day

0 day

Mean 7.81 7.62 7.62 7.71 8.04 6.21 6.27 5.55

S.D.() 0.91 1.13 1.14 1.18 1.98 1.62 1.52 1.57

S.E.M() 0.20 0.25 0.25 0.26 0.44 0.36 0.34 0.35

% change 2.43 2.43 1.28 22.76 22.01 30.97

P value at 7th day (T/C) 0.0008 (ES) (Mann-Whitney Test)

P value at 28th day (T/C) 0.0007 (ES) (Mann-Whitney Test)

P value at 42nd day (T/C) <0.0001 (ES) (Mann-Whitney Test)

SD = Standard Deviation; S.E.M. = Standard Error of Mean; ES = Extremely Significant


Table 7: Effect of Test Drug and Control on C-Reactive Proteins

C-reactive Control Group (N= 20) Test Group (N=20)

protein

(mg/dl) 0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 4.37 4.3 3.95 3.70 3.92 3.91 2.99 2.30

S.D.() 2.18 2.20 2.14 2.03 2.35 2.17 1.86 1.42
S.E.M() 0.48 0.49 0.48 0.45 0.52 0.49 0.42 0.32

% change 1.6 9.61 15.33 0.25 23.72 41.32



P value at 42nd day (T/C) 0.03 (LS (Mann-Whitney Test)

SD= Standard Deviation; S.E.M..=Standard Error of Mean;, NS= Not Significant; LS=

Less Significant

less significant (p<0.05) at day 7th, not significant (p<0.05) at day 28th,and

again less significant(p<0.05) at day 42nd (Table-8).

Erythrocyte Sedimentation Rate (E.S.R.)

In control group, E.S.R noted was 28.70 11.33/1 hr at baseline, 29.45

11.07/1 hr at 7th day, 29.75 10.42/1 hr at day 28th and 30.10 11.19/1 hr

at 42nd day. On the other hand the E.S.R observed in test group was 25

11.53/1 hr at 7th day, 25.30 10.27/1 hr at 28th day, 22.30 12.02/1 hr on

42nd day. The difference in ESR values between the two groups were a not

significant (p>0.05) on 7th and 28th day, whereas mildly significant difference

was observed (p<0.05) on 42nd day, (Table 9).

Table 8: Effect of Test Drug and Control on Total Leukocyte Count

T.L.C. Control Group (N=20) Test Group (N=20)

(cu.mm) 0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 8785 8080 8380 7860 9300 9105 9180 8950

S.D. () 1786.3 1395.7 1505.3 1622 1554.6 1325.3 1405.5 1325.7

S.E.M. () 399.43 312.09 336.59 362.69 347.62 296.34 314.27 296.43

% change - 8.02 4.61 10.53 - 2.09 1.29 3.76

P value at 7th day (T/C) 0.01 (LS) (Mann-Whitney Test)


P value at 42nd day (T/C) 0.01 (LS) (Mann-Whitney Test)


LS = Less Significant


Table 9: Effect of Test Drug and Control on ESR

E.S.R. Control Group (N=20) Test Group (N=20)

(mm/1sthr) 0 day 7th day 28th day 42nd day 0 day 7th day 28th day 42nd day

Mean 28.70 29.45 29.75 30.10 26.65 25.00 25.30 22.30

S.D. () 11.33 11.08 10.42 11.19 10.49 11.53 10.27 12.02

S.E.M. () 2.533 2.48 2.33 2.5 2.35 2.58 2.29 2.69

% change - 2.61 3.65 4.88 - 6.19 5.06 16.32



P value at 42nd day (T/C) 0.02 (LS) (Mann-Whitney Test


LS = Less Significant

Safety

During the course of the study, no adverse events were reported by the

patients or clinically detected by the investigator. No any significant change

from base line was observed in haemoglobin, SGOT, SGPT, S.Bilirubin, B.Urea

and S. Creatinine values in both the groups. The test formulation as well as

placebo was found well tolerated as indicated by 85% drug compliance

Discussion

Gout is one of the most common types of inflammatory joint diseases, modern

drugs used for subsiding acute attacks or lowering serum uric acid are

associated with potent adverse effects. Furthermore, these commonly used

therapeutic agents often, and for various reasons, do not achieve the desired

lowering of serum urate levels to below 6.0 mg/dl. In the present study, the

unani formulation consisting of Suranjan (Colchicum luteum), Elva (Aloe

barbadensis), Qurtum (Carthamus tinctorius), Halaila-e-Zard (Terminalia

chebula), Zanjbeel (Zingiber officinale) not only relieved various signs and

symptoms of gouty arthritis but also exerted remarkable effects on lowering

serum uric acid level and various other inflammatory markers. The relief in joint

pain, tenderness can be attributed to the analgesic activity of

Suranjan (Colchicum luteum) (Hakeem, 1991; Rainsford, 1999), Elva (Aloe

barbadensis) (Sharma et al., 2002; Sheshadri, 1976) and Zanjbeel (Zingiber

officinale) (Khare, 2004; Sharma et al, 2002). Anti-inflammatory activity of

Qurtum (Carthamus tinctorius) ( Linda, 2001), Halaila-e-Zard (Terminalia

chebula) (Sharma et al., 2002), Zanjbeel (Zingiber officinale) (Linda, 2001;

Sharma et al., 2002; Wagner et al.,1985 ) can also be considered in decreasing


pain, swelling and tenderness. Acute attack of gouty arthritis is initiated by the

precipitation of urate crystals in the synovial fluid resulting in inflammatory

response and the acute inflammatory cells (nutrophils) phagocytose urate

crystals and release a glycoprotein which further aggravates inflammation.

Colchicine, an active constituent of Suranjan (Colchicum luteum), has been

proved inhibitory to the glycoprotein released by the nutrophils in acute gouty

inflammation. Colchicine by binding with fibrillar protein tubulin has been found
to inhibit nutrophil migration in the inflammed joint. All this explains relief in

pain, swelling and tenderness because of Suranjan (Colchicum luteum) (Jean

Brunneton, 1995; Robert et al., 1983). The diuretic activity of Qurtum (Carthamus

tinctorius) (Kritikar & Basu, 1984) and laxative action of Halaila-e-Zard (Terminalia

chebula) (Barthakur et al., 1991) and Elva (Aloe barbadensis) (Blumenthal et

al., 1998) might also be helpful in reducing monosodium urate precipitation by

increased excretion of uric acid through urine and faeces respectively.

In the light of above discussion and on the basis of observations and results

obtained in this study, large scale, standard control, double blind randomized

clinical is warranted to further support the efficacy and tolerability of test

formulation in the treatment of gouty arthritis.

Conclusion

Unani formulation produced remarkable effects on various efficacy parameters

in cases of acute gouty arthritis. The drug was found safe and well tolerated,

as no adverse events were reported by the patients or clinically detected by

the investigator during the course of six week therapy.

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Hippocratic Journal of Unani Medicine

14
Clinical Abstract

Evaluation of nfertility is a common condition with important psychological,

Effect of
Sufoofe Muallif
in the
I

economical, demographical and medical implications. WHO estimates that 60-
80 million couples worldwide are currently suffering from infertility. Quilatte
Haiwane Manwiya (oligospermia) is one of the most common causes of male
infertility. In conventional system, various drugs are being used to increase
sperm count, but these drugs are expensive and their long term use produces
Management of

several adverse effects too. Therefore, keeping in the mind about the need of

Quilatte the hour an open, uncontrolled clinical trial was carried out on 30 infertile

patients of Quilatte Haiwane Manwiya. Each patient was given trial formulation

Haiwane

Sufoofe Muallif - 5 grams once daily for 60 days. The assessment of efficacy

Manwiya of treatment was carried out on the basis of objective parameters (Semen

analysis for sperm count, motility, morphology and hormone profile for serum
(Oligospermia)

testosterone, LH, FSH). The results were analyzed statistically by using Wilcoxon

matched-paired signed ranks test, one tail paired-T test and Kruskal- Wallis

1*Shaikh Imtiyaz,

test. After the trial, significant improvement was found in various objective

2Mohd. Anwar

and parameters; sperm count (10.53 1.30 to 18.06 2.57, P<0.001), sperm

3Mohd. Nayab motility (21.70 3.80 to 46.56 5.06, P<0.001) and sperm morphology (76.2

6.40 to 91.9 4.22, P<0.01). The changes in hormone profile (serum

1&3Department of Ilaj bit Tadbeer,

Testosterone, LH, FSH) were not significant (P>0.05). Safety parameters were

National Institute of Unani Medicine,

Kottigepalaya, Magadi Main Road, remained within normal limits after the trial. This study suggests that Sufoof

Bangalore-560091 Muallif has exhibited a good response in the improvement of semen markers;

sperm count, sperm motility and sperm morphology. Furthermore, no adverse

2Department of Ilaj bit Tadbeer,

Ajmal Khan Tibbia College, effect was observed and safety parameters remained within normal limits. Thus

Aligarh Muslim University, it might be concluded that the Test drug is safe and effective for the treatment

Aligarh-202002
of Quilatte Haiwane Manwiya (oligospermia).

Keywords: Quilatte Haiwane Manwiya, Oligospermia, Unani medicine, Sufoofe

Muallif, Quillate mani

Introduction

Infertility is defined as the failure of a couple to achieve conception after one

year of regular unprotected intercourse (Bradon et al., 2002; Jonathan, 2002).

Infertility is a distressing problem for about 10-15% of worlds population, with

the incidence increasing over the years (Leon Speroff et al., 1999). However,

it affects both men and women; male factor contributes for about 30-40%

cases of infertility (Sengupta et al., 1998). A recent study has indicated that

there is a decrease in sperm density over a period of past fifty years (Dhaliwal

et al., 2001). Despite of remarkable advancement in pharmacotherapy, infertility

1


continues to raise significant economic and personal burden to the society.

Male infertility, with its clinical and psychosocial implications, poses a significant

challenge to the physicians and to the society as a whole.

Quilatte Haiwane Manwiya (oligospermia) is one of the most common causes

of male infertility. It is a condition in which the sperm count is decreased to less

than 20 million/ml of semen (Walsh, 2002). The important causes include

varicocoele, cryptorchidism, klinefelters syndrome, damaged testes due to

trauma or infections (tuberculosis, syphilis, mumps), neoplasm of testes, kidney

and liver diseases, smoking, alcoholism, wearing tight under garments, working

at high temperature places like welding, dyeing, blast furnace, cement and

steel factories etc (Smith, 1997).

In conventional system, various drugs like testosterone, clomiphene citrate,

tamoxifene and HCG are being used to increase sperm count, but these drugs

are expensive and their long term use produces several adverse effects like

gynaecomastia, hepatic carcinoma, deep vein thrombosis, lowering of HDL and

rise in LDL etc (Tripathi, 2006). However, in Unani system of medicine there

are ample of single as well as compound drugs which are used for male sexual

weakness (Zoafe bah) and infertility since the time immemorial.

In Unani system of medicine, most of Unani scholars particularly Ibn Sina (980-

1037 AD), Zakaria Razi (865-925 AD), Ismail Jurjani (1110 AD) and Rabban

Tabri (810-895 AD) have elaborately discussed sexual diseases in their

respective treatises. They have mentioned the causes, symptoms, complications,

treatment and management of various sexual diseases under the caption of

Zoafe bah. In fact, Zoafe bah is a broad term which encompasses different

disease entities like Istirkhae Qazeeb (Erectile dysfunction) Surate Inzaal

(Premature ejaculation) and Qillate Mani (oligospermia) etc. The concept of

Qillate Mani (Qillat- less, Mani-semen) can be correlated with the concept of

oligospermia (Oligo-less, spermia-spermatozoa). The literal meaning of

oligospermia is Qillate haiwane manwiya. The important causes of Qillate mani

are kasrate istifragh, excessive use of mudirrat, sue mizaj of alaate mani,

excessive use of drugs like afyoon (opium) and bhang and excessive riding etc

(Ahmad, 1954; Kabiruddin, YNM; Majusi, 2010; Razi, 2007).

The exact aetiopathogenesis of Qillate haiwane manwiya is not described in

classical Unani literature but on the basis of its cause it can be concluded that

Sue mizaj of alaate mani alter the production of mani due to excessive Baroodat,

Yaboosat, Hararat or Ratubat in alaate mani. For the spermatogenesis, there

is a need of balance in temperament of particular organs. In case of Sue mizaj

of the alate mani the process of spermatogenesis slow down production of

Mani and thus causing oligospermia. (Kabiruddin, YNM)


Keeping all these facts in mind, a protocol of an observational clinical trial has

been planned to conduct a preliminary clinical study on Quilatte Haiwane

Manwiya (oligospermia). A commonly used Unani pharmacopoeial compound

drug, Sufoofe Muallif, was selected for the study. Its ingredients of are

Talmakhana, Salab misri, Singhara, Gonde kekar, Mazu sabz, Mastagi Rumi,

Nishasta gandum and Shakar safed (Lateef, 1986). These drugs possess

properties like Muwallide Mani, Mughallize Mani, Muqawwie bah, Mumsik and
Musammine badan which form a rational basis for proposed hypothesis that

the ingredients of selected compound formulation owing to their above cited

properties may be beneficial in patients of oligospermia.

Methodology

The present observational, open uncontrolled clinical study was conducted at

National Institute of Unani Medicine hospital, Bangalore, India. Before initiation

of the project, a comprehensive protocol was framed and put forth for ethical

clearance from the Institutional Ethical Committee. This study was conducted

from February, 2012 to March, 2013. The protocol of the trial was framed for

the study and accordingly findings are recorded on CRF. The inclusion criteria

consisted of being aged from 21 to 50 years infertile males, having sperm

count less than 30 million/ml (Mushtaq, 2007) and willing to follow up and for

semen examination. Those individuals who were suffering with chronic renal

diseases, chronic cardiac ailments, chronic liver diseases, thyroid dysfunctions,

any organic disease of testes and related organs of spermatogenesis, etc or

receiving any medication were not included in the study. Patients fulfilling the

inclusion criteria were given the information sheet having details regarding the

nature of the study, the drug to be used, method of treatment etc. Patients

were given enough time to go through the contents of informed consent sheet.

They were given the opportunity to ask any question and if they agreed to

participate in the study, they were asked to sign the informed consent form.

Eligible patients were selected from OPD of NIUM Hospital, Bangalore. Complete

history and examination including general physical and systemic examinations

were carried out with special attention to endocrine and genital examination,

and recorded on a prescribed proforma which was designed in accordance

with the objectives of the study. Several investigations were carried out with the

aim to exclude the patients with pathological conditions such as Semen Analysis,

Hormone Analysis (Serum Testosterone, FSH, LH), Hb%, TLC, DLC, ESR,

Blood Sugar-F/PP, KFT (Blood urea, serum creatinine) , LFT (SGOT, SGPT,

Alkaline Phosphatase), Urine (Routine and Microscopic). The sample size of

the study was limited to 30 patients and the treatment period was determined

as 60 days. Sufoof Muallif was given to patients in a dose of 5 gram/day for

60 days. Follow up of the patient was done after every 15 days period upto


60 days. At each visit patient was inquired about any side effect of the drug

and safety parameters were evaluated before and after treatment. Assessment

of the efficacy of test formulation was carried out on the basis of objective

parameters. Semen analysis (Semen volume, sperm count, sperm motility and

morphology) and hormone analysis (Serum Testosterone, LH, FSH) of each

patient was performed before and after treatment. The objective parameters

were analysed by using Wilcoxon matched-paired signed ranks test, one tail
paired-T test and Kruskal Wallis test.

Trial Formulation (Sufoofe Muallif)

Talmakhana (Asteracatha longifolia) 100 gms

Salab misri (Orchis latifolia) 80 gms

Singhara (Trapa bispinosa) 150 gms

Gonde kekar (Acacia arabica) 150 gms

Mazu sabz (Quercus infectoria) 75 gms

Mastagi rumi (Pistacia lentiscus) 75 gms

Nishasta gundum (Starch) 100 gms

Shakar safed (Sugar) 730 gms

Method of preparation and mode of administration of test drug

Proper identification of the ingredients of the Unani formulation was done by

chief pharmacist, National Institute of Unani Medicine, Bangalore, to ensure

their originality and authenticity. The single drugs then were cleaned by weeding

out unwanted material and impurities and pounded to fine powder.

Results and Observations

Overall 30 patients completed the trial according to the study protocol. Sufoof

Muallif was generally well tolerated and no remarkable adverse events were

reported in the test group. The mean score for sperm count pretreatment (0

day) was 10.531.30 while the same post-treatment (60th day) was 18.06

2.57 (p<0.01). The mean and SEM scores for sperm motility on 0 day were

21.703.80 and for 60th day were 46.565.06 (p<0.01). The mean and SEM

scores for sperm morphology on 0 day were 76.2 6.40 and for 60th day were

91.9 4.22 (p<0.01). The mean and SEM scores for serum testosterone on

0 day were 4.66 0.32 and for 60th day were 4.67 0.32 (p>0.05). The mean

and SEM scores for serum LH on 0 day were 5.8 0.44 and for 60th day were

6.3 0.47 (p>0.05). The mean and SEM scores for serum FSH on 0 day were

6.7 0.89 and for 60th day were 7.08 0.89 (p>0.05).The baseline and after

treatment values of objective parameters are depicted in Table 1.


Table 1

Mean SEM

Sperm Count Sperm Sperm Serum Serum Serum

(million/ml) Motility Morphology Testosterone LH FSH

BT AT BT AT BT AT BT AT BT AT BT AT

Test 10.53 18.06 21.70 46.56 76.2 91.9 4.66 4.67 5.8 6.3 6.7 7.08
Group

1.30 2.57 3.80 5.06 6.40 4.22 0.32 0.32 0.44 0.47 0.89 0.89

P-Value p<0.001 p<0.001 p<0.01 p>0.05 p>0.05 p>0.05

Discussion

The improvement in the sperm count would be because of Muqqawie bah,

Muwallide mani, Mughallize mani and Musammine badan activities of most of

the ingredients of Test drug i.e. Talmakhana (Asteracatha longifolia), Salab

misri (Orchis latifolia), Singhara (Trapa bispinosa) and Nishasta (Starch). These

results are in conformity with the properties of the drugs as indicated by Unani

scholars such as Ibn Rushd, Ibn Sina, Hakeem Abdul Hakeem, Najmul Ghani

and Kabeeruddin etc. (Ibn Rushd, 1987; Ghani,YNM; Kabeeruddin, 2010;

Haleem, 2009; Kabeeruddin, 2007). Modern scientific clinical and experimental

studies have also proved aphrodisiac and spermatogenic properties of some

ingredients of Test drug like Asteracantha longifolia, Trapa bispinosa and Orchis

latifolia (Chauhan et al., 2009; Agarwal et al., 2003; Mayank et al., 2008).

Chemical analysis of various test drugs show that apart from different chemical

constituents they possess various nutritional elements such as carbohydrate,

Figure 1


protein, minerals like Ca, Mg, Fe, Zn and Cu etc, and vitamins such as thiamine,

riboflavin, pantothenic acid and pyridoxine etc (Patra et al., 2009; Singh et al.,

2010). These chemical constituents and vitamins are essential for the process

of spermatogenesis (Zakai et al., 2011). Therefore, the effect of Test formulation

might be due to presence of these elements. The studies of Tikkiwal et al. and

Wong et al., 2002 reveal that Zinc and folic acids are responsible for

improvement in sperm count and motility.
The improvement in the sperm motility might be due to Muqqawie bah, Muwallide

mani, Mughallize mani properties of the various constituents of test formulation

which have been documented in Unani literature (Ibn Rushd, 1987; Ghani,YNM;

Kabeeruddin, 2007, 2010; Haleem, 2009). Different researches have

documented the antioxidant (Trommer et al., 2005; Kaur et al., 2008; Chryssavgi

et al., 2008) and immunomodulatory (Patel, 2010) activities of Mazu, Mastagi,

Singhara and Samaghe Arabi which are ingredients of Sufoofe Muallif. By

virtue of these antioxidant and immunomodulatory properties test drug is

effective in improving sperm motility. As it has been proved by the studies of

Carmely et al. (2009) and Bansal et al. (2009) etc. that antioxidant and

immunomodulating agents play a major role in improving the process of

spermatogenesis, increasing sperm motility and viablility.

Before and after treatment values of sperm morphology were subjected to

statistical analysis by using Wilcoxon matched-paired signed ranks test and it

was found that the difference between the Mean SEM scores of sperm

motility pre and post treatment was statistically significant (p<0.01).

Hormone analysis of each patient was performed for serum testosterone, LH

and FSH. The values of LH and FSH were analyzed statistically by using one

tail paired-T test and it was found that the difference between the Mean SEM

scores of serum testosterone was not significant (p> 0.05).

Zakai et al. (2011) mentioned that for the management of oligospermia, the

rational approach is to focus on enhancing those factors which promote sperm

formation which is closely linked to nutritional status. Therefore, it is critical that

men with low sperm counts have optimal nutritional intake. In addition to

consuming a healthful balanced diet, there are several nutritional factors that

deserve special place viz vitamin C and other antioxidants, fats and oils, zinc,

folate, vitamin B, arginine, and carnitine spermatogenesis. As various drugs of

our Test formulation specially Talmakhana and Singhara have Musammine

badan property, and also the chemical analysis proved the presence of various

important nutritional factors like carbohydrate, proteins, minerals and vitamins

such as thiamine, riboflavin, pantothenic acid and pyridoxine etc (Patra et al.,

2009; Singh et al., 2010). The chemical constituents present in the ingredients


of Test formulation may further facilitate the process of spermatogenesis and

thereby effective in improving sperm count and motility.

In Unani system of medicine the principle of treatment is based on the concept

of organ protection, strengthening and maintenance of the Quwa (faculties) at

its equilibrium (etedal). The faculties at their equilibrium are balanced inherently

to maintain the normal function of that organ or system. It has been mentioned

that each organ has been gifted with special Quwat for its optimal functioning.

Unsiyaen (Testes) are the azae raesa (vital organs) for Quwwate tanasuliya

(Reproductive power). In case of derangement of function (zoaf) of any organ,

the drugs enhancing its power (muqawwi advia) are advocated. This is the

reason why in Unani system of medicine, for every organ and system there is

a group of tonic drugs (muqawwi advia) proposed that safe guard its larger

interest and bring it near to its equilibrium, if some derangement in its structure

or function takes place. Therefore, most of the sexual diseases are being

treated on the basis of concept of Taqwiyate aaza. The ingredients of Test

formulation are bestowed with the properties like Muqawwie bah, Muwalllide

mani and Muqawwie aam (general tonic) etc, by virtue of these actions these

drugs potentiated the functions of testes. Thus it may be presumed that the

observed significant differences in the values of sperm count and motility

would be due to Muqqawie bah, Muwallide mani and Mughallize mani activities

of the ingredients of Sufoofe Muallif. Thus we can say that scientific studies

and reported effects of individual ingredients of Test drug are in confirmatory

to a great extent with that of our hypothesis as well as the inferences we drew

out of the present study.

In order to determine toxicity of test drug, safety parameter i.e. complete

haemogram, LFT and KFT were carried out before and after the treatment in

each patient. It was found that all the safety parameters were within the normal

levels after the completion of trial. This suggests that Test formulation can be

used safely at mentioned therapeutic dose.

This discussion is helpful to draw the conclusion that the Test formulation is

safe and effective for the management of oligospermia and can be used for

long period without any adverse effect. However, the long term studies with a

larger sample size are required to elucidate other pharmacological actions of

Test formulation.

Study Limitations and strengths:

The main limitation of this trial was the duration of the trial and by longer follow

up; we might reach to more favourable changes in objective parameters. The

strength of this trial is its novelty in the infertile group of males.


Conclusion

On the basis of above results and observations it may be concluded that the

Test drug is safe and effective and can be used potentially in the management

of oligospermia. Further long term and large scaled phase III and IV trials are

advocated to explore other important chemicals and pharmacological actions

of the Test formulation.
Acknowledgment

The authors are thankful to Director, National Institute of Unani Medicine,

Bangalore, for providing research facilities to carry out this work.

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Effect of Habbe Abstract

Aftimoon in the randomized single blind standard controlled study was

Patients of
Dyslipidemia
with Tasallube
A

conducted to evaluate the efficacy of Habbe Aftimoon in the patients of
dyslipidemia with Atherosclerosis. Thirty diagnosed patients were selected and
randomly allocated to Control and Test groups (comprising 20 patients in Test
group and 10 in Control group). Habbe Aftimoon in a dose of 4 Habb twice a
day in Test group whereas Lipotab 2 tablets once a day was given in Control
Sharaeen

group for 60 days. All the patients were advised low fatty diet and moderate

(Atherosclerosis) exercise. Before and after the treatment, both groups were assessed on

subjective and objective parameters. The outcome of treatment were analyzed

statistically by using Paired t test, Wilcoxon test, Friedman test with post test,

1Mohd.Aslam,

2*Mohd. Anwar, one way ANOVA with post test and Kruskal Wallis test with Dunns multiple

3Hamid Ali compare test.

and

2M. Shoaib The Test drugs exhibited statistically significant result in subjective parameters

(Palpitation and Body weight) in intra group and inter group comparison. In

1Department
of Moalejat, objective parameters, reduction in S. Cholesterol and increase in L ank ASI

Allama Iqbal Unani Medical

and ABI were observed, and this difference was found statistically significant

College & Hospital,

Muzaffarnagar-251002 (U.P.) in intra group comparison. The control drug exhibited significant improvement

in palpitation and reduces body weight in intra and inter group comparison. In

2Department of Ilaj bit Tadbeer,

objective parameters, S. Cholesterol and S. Triglyceride were significantly

A.K. Tibbiya College,

Aligarh Muslim University, decreased in intra group comparison, where as the changes in other subjective

Aligarh-202002 and objective Parameters were remain insignificant in both groups.

3Department
The study revealed that Test drug is effective in some objective and subjective

of Ilaj bit Tadbeer,


parameters in patients of dyslipidemia with Atherosclerosis particularly in reducing

Kottigepalaya, Magadi Main Road,

body weight and S. Cholesterol level. No adverse effect and toxicity was seen

Bangalore-560091

during and after the study. Thus, it can be concluded that Test drug is effective

and safe in the management of Atherosclerosis in dyslipidemic patients up to

some extent. So, it may be recommended for delaying complications of

Atherosclerosis.

Keywords: Atherosclerosis, Dyslipidemia, Habbe Aftimoon, Unani Medicine

Introduction

Tasallube Sharaeen (Atherosclerosis) is one of the commonest conditions

which underlying pathologic process causes several cardiovascular and

cerebrovascular complications. It is well known fact that Hyperlipidaemia and

Obesity are two important risk factors associated with Atherosclerosis (Longo

et al., 2012).

2


Since very beginning the concept of Tasallube Nabz also exist in Unani system

of Medicine, as most of the ancient Unani scholars like Majoosi, Ibn Sina, Ibn

Rushd, Ibn Zuhr and Samar Qandi have elucidated the cause of narrowing and

stiffness of vessels in their treaties (Ibn Sina, 1993; Khawaja Rizwan, 2010;

Majoosi, 1889; Ibne Zuhr, 1986) Now Tasallube Sharaeen is used as standard

term for arteriosclerosis and atherosclerosis is main type of arteriosclerosis

(Anonymous, 2012).
Atherosclerosis is predicted to become the leading cause of death in India by

2020 (Satishchandra et al., 2011) Tobacco smoking, obesity, hypertension,

diabetes mellitus, elevated plasma homocysteine and LDL are the principal

factors responsible for deposition of lipid in large and medium sized arteries

(Longe, 2002). Slow and progressive lipid deposition narrows down the arterial

lumen by forming atherosclerotic plaque, which initially causes ischemia of the

irrigated organs, but in advance stages results in peripheral vascular diseases,

myocardial and cerebral infarction and stroke etc. (Nicholas et al., 2006).

Now a day many pharmacological and non-pharmacological modes of treatment

are available for prevention of atherosclerosis. Among non-pharmacological,

life style modifications like decreasing daily calorie intake, and increase in

physical activity is indeed helpful for most of the patients but in several

circumstances pharmacological management of atherosclerosis is inevitable.

For this purpose several cholesterol lowering agents such as Levostatin,

Atorvastatin, Simvastatin, Clofibrate, Bezafibrate and Niacin etc are widely

prescribed in conventional Medicine. But long term use of these drugs produces

several adverse effects, such as Hepatotoxicity, Myopathy, Dyspepsia, Renal

failure and Cholelithiasis. (Kumar, 2005; Goodman & Gillman, 2011)

High prevalence of the disease, multi factorial causes and life threatening

complications and most important inability of contemporary system of Medicine

to deliver safe and effective drug for the management of atherosclerosis

effectively, warrant search of alternative treatment to alleviate such a complex

disease of serious complications.

Unani system of Medicine offers different approach of treatment i.e. Ilaj bil

Ghiza (Diet therapy), Ilaj bit Tadbeer (Regimental therapy) and Ilaj bid Dawa

(Pharmacotherapy) (Kabeeruddin, 1954) which are the mainstay of treatment

of hyperlipidaemia and atherosclerosis. These three could be used alone or

in combination. Fundamentally, combination of Ilaj bil Ghiza, and Ilaj bit Tadbeer

are very useful for the prevention of atherosclerosis. The principle of treatment

should be to reduce caloric intake, to burn extra calories deposited in body,

to eliminate Mawade fasida and correction of Sue mizaj barid, use of Qalilul

taghaziya kasirul kammiyat Ghiza along with Riyazate kasira and Hammam


(Razi, 1997). Moreover, Unani physician also recommends Ilaj bid Dawa for the

management of Samne Mufrit. A large number of, drugs available in Unani

Medicine which possess action like Muhazzil, Munzij, Mushil, Mufatteh, Jaali

and Muhallil properties could also be used for prevention of Tasallube Sharaeen.

Unfortunately, there is no convincing treatment available for the management

of atherosclerosis in contemporary system of Medicine. Therefore, search of

safe and effective drug for its management is quite necessary. In Unani system

of medicine, Habbe Aftimoon is recommended for the treatment of amraze

balghamia and saudawia. (Akbar Arzani, 2009) The ingredients of Habbe

Aftimoon are Aftimioon (Cuscuta reflexa L.), Gule Surkh (Rosa damascene L.),

Mastagi (Pistacia lentiscus L.), Post Halela Zard (Terminalia chebula L.), Bisfaij

(Polipodium vulgare L.), Ustokhudoos (Levendula stoechas L.) and Namak

Hindi (sodium chloride) (Kabeeruddeen, 2006).

Tasallube Sharaeen is also categorized as amraze saudawia. Thus this drug

may be proven useful in this condition also, but its efficacy has not been

evaluated on scientific parameters particularly for the management of

Atherosclerosis. Therefore, a single blind randomized standard control clinical

trial was designed to evaluate the efficacy of Habbe Aftimoon in the management

of Atherosclerosis in dyslipidemic patients.

Methodology

A single blind standard controlled clinical trial was conducted from March 2012

to January 2013 in National Institute of Unani Medicine Hospital, Bangalore.

The study protocol was designed according to the need of the trial, and

approval was obtained by the Institutional Biomedical Ethics Committee of

NIUM, Bangalore. After providing detailed oral information about the study,

written consent was obtained from the participants. The patients belonging age

of 2065 years, having dyslipidemia with Tasallube Sharaeen (Atherosclerosis)

were selected for the study. Diagnosis of Atherosclerosis was confirmed by

computerised device (Atherowin & Canwin) on the basis of assessment of right

brachial arterial stiffness index, left brachial arterial stiffness index, right ankle

arterial stiffness index, left ankle arterial stiffness index, right brachial pulse

wave velocity, left brachial pulse wave velocity, carotid femoral pulse wave

velocity and ankle brachial index.

Individuals below 20 years and above 65 years of age , and those having

history of AIDS, Tuberculosis, Hypothyroidism, uncontrolled Diabetes Mellitus,

established I.H.D., advanced Kidney, Liver and Heart diseases and Pregnant

and lactating women were not included to the study. According to subjective

and objective criterion a total of 50 patients were registered for the study from


the OPD and IPD of NIUM Hospital. During screening 11 patients did not fulfil

inclusion criteria and excluded from the study, remaining 39 patients were

randomly allocated into Test (Group A) and standard Control (Group B) groups

respectively by using simple randomization sampling method. In the Test group

4 tablets of Habbe Aftimoon twice a day (Each tablet contains 750 mg of Test

drug) was given orally for 60 days whereas Lipotab 2 tab was administered

once a day for the same duration. All the patients were advised low fatty diet
with low caloric value (1600-200 kcal per day) and aerobic exercise for 30-45

minute per day (Agarwal, 2014). All patients were asked to come fortnightly for

the assessment of progression or regression of symptoms. During the

whole duration of protocol concomitant treatment was not allowed in both

groups.

The assessment of efficacy of Test and Control drugs were carried out on the

basis of subjective and objective parameters. Subjective parameters include

symptoms like Palpitation, Xanthelesma, and Nabz sulb which were assessed

at fortnightly, while other objective parameters i.e. body weight, lipid profile,

arterial stiffness, pulse wave velocity and ankle brachial index were measured

before starting treatment and after the completion of treatment.

In order to asses safety of the Test and Control drug , complete Haemogram

(TLC, DLC, Hb%, ESR), Liver Function Test (S. Bilirubin, SGOT, SGPT, Alkaline

Phosphates) and Kidney Function Test (Blood Urea & S. Creatinine) were also

carried out before and after treatment .

During study seven patients from Test group and two patients from Control

group were lost to follow-up, leaving behind 20 patients in Test and 10 patients

in Control group. Therefore, statistical data were calculated on 30 patients

only who were completed entire course of treatment. Data was statistically

analyzed by pairedt test, Wilcoxon matched pair test and Friedman test for

intra group comparison and one way ANOVA and Kruskal-Wallis test with Dunns

multiple pair comparison for inter group comparison

Results

Out of 30, 14 (46.66 %) are male and 16 (53.33 %) are female. 05 (25 %),

10 (50%), and 05 (25%) subjects in test group belongs to 20-35 year, 36-50

year, and 51-65 year age groups respectively, similarly 01 (10%), 04 (40%),

and 05 (50%) subjects in standard control group belongs to 20-35 year, 36-

50 year, and 51-65 year age groups respectively (Table 1).

The effect of Test and control drug on various subjective and parameters are

depicted in Table 2 and 3A, 3B.


Table 1: Demographic Data

Factor No. of patients Total Percentage

No. of (%)

Test group Control group Patients

Age 20-35 5 1 6 20

36-50 10 4 14 46.7
51-65 5 5 10 33.3

Sex Male 9 5 14 46.67

Female 11 5 16 53.33

Diet Mixed diet 19 9 28 93.33

Vegetarian 1 1 2 6.67

Socio- Upper (I) 0 0 0 0

economic Upper Middle (II) 7 3 10 33.33

status Lower Middle (III) 4 2 6 20

Upper Lower (IV) 8 5 13 43.34

Lower (V) 1 0 1 3.33

Table 2: Effect of Drugs on Subjective Parameter

Parameter Group Assessment day P value

0 day 15th 30th 45th 60th (a) P<0.01 with respect

day day day day to zero days test group.

Palpitation Control 3(1,4) 3(1,4) 2.5(1,3) 1*,#,(1,2) (b) P<0.01 with respect
2(1,3)

to 15 day test group.

Test 3(2,4) 3(1,4) 3(1,3) 1a,b,c 1a,b,c

(1,3) (1,3) (c) P<0.01 with respect

to 30 days test group.

Xanthelesma Control 0(0,2) 0(0,2) 0(0,2) 0(0,2) 0(0,2)

* P< 0.01 with respect

Test 0(0,2) 0(0,2) 0(0,2) 0(0,2) 0(0,2)

to zero day control

Nabz sulb Control 3(1,3) 2.5(1,3) 2.5(1,3) 2.5(1,3) 2.5(1,3) group.

n=10
# P<0.01 with respect to

Test 2(1,3) 2(1,3) 2(1,3) 2(1,3) 2(1,3) 15 days control.

After two months administration of Habbe Afteemoon and Lipotab, significant

difference was observed only in body weight, total cholesterol, L ank ASI, and

ABI in Test group, while in standard control group significant difference was

observed only in body weight, total cholesterol and serum triglycerides. Whereas,

no significance difference was observed in other objective parameters neither

in test nor in standard control group.


Table 3A: Effect of drugs on objective parameter (20 in Test and 10 in Control group)

Parameters Group Assessment day P value

Before After

Treatment Treatment

Body Control(10) 83.84.15 81.9 4.2* * P<0.01 with respect to before

weight Test(20) 79.02.4 76.82.6+ treatment in control group

+ P<0.01 with respect to before

treatment in test group.

S. Control 2058.93 1809.35a a P<0.05 with respect to before

Cholesterol Test 2158.73 1955.93 b,c treatment in control group.

b P<0.01 with respect to before

treatment in test group.

c p< 0.05 with respect to after

treatment in control group.

Triglyceride Control 27425.7 21428.9d d P<0.01 with respect to before

Test 21811.3 20412.6 treatment in control group.

Low Density Control 92.711.9 8910.3 P>0.05 Inter group comparison,

Lipoprotein Test 96.19.33 103.16.37 with respect to before & after

(LDL) treatment in test & control group.

High Density Control 37.92.56 46.43.82 P>0.05 Inter group comparison,

Lipoprotein Test 51.13.5 44.42.32 with respect to before & after

(HDL) treatment in test & control group.

Furthermore, safety markers i.e. Haemogram (TLC, DLC, Hb%, ESR), Liver

Function Test (S. Bilirubin, SGOT, SGPT, Alkaline Phosphates) and Kidney

Function Test (Blood Urea & S. Creatinin) remained normal before and after

treatment. (Table 4)

Discussion

Tasallube Sharain (Atherosclerosis) is one of the commonest causes of the

premature vascular diseases, causing Ischemic Heart Disease, Cerebrovascular

accidents, Stroke and Hypertension. Obesity and Hyperlipidaemia are two risks

factor which are associated with atherosclerosis. In modern system of medicine

several Hypolipidaemic agents are being used for the prevention of

atherosclerosis. However, these drugs are neither drug of choice of

Atherosclerosis nor producing convincing therapeutic effects. Furthermore, the

side effects of these Hypolipidaemic agents are also causes of concern.

In view of the above facts, the development of Hypolipidaemic agents from

herbal sources is quite necessary. There is no dearth of such drugs in Unani

System of Medicine. Habbe Aftimoon is one of the compound formulations

which is effective in the treatment of Amraze Saudawia. Apart from this the


Table 3B: Effect of drugs on objective parameter (20 in Test and 10 in Control group)

Parameters Group Assessment day P value

Before After

Treatment Treatment

Right Brachial Control 304.77 28.92.87 P>0.05 Inter group comparison,

Arterial with respect to before & after

Stiffness Index Test 24.41.66 23.32.31 treatment in test & control group.

(R Bra ASI)

Left Brachial Control 273.06 28.143.39 P=0.924 Inter group comparison,



(L Bra ASI)

Right ankle Control 42.083.29 42.082.82 P=0.66 Inter group comparison,



(R Ank ASI)

Left ankle Control 33.94.19 39.43.3 e!P<0.05 with respect to before

Arterial treatment in test group. Inter group

Stiffness Index Test 31.52.04 35.9e2.54 comparison, p=0.09

(L Ank ASI)

Right Brachial Control 2344359 2004216 P=0.75 Inter group comparison,

Pulse Wave with respect to before & after

Velocity Test 1592267 1966180 treatment in test & control group.

(R ba PWV)

Left Brachial Control 1116367 1426188 P>0.05 Inter group comparison,

Pulse Wave with respect to before & after

Velocity Test 1058295 1645238 treatment in test & control group.

(L ba PWV)

Carotid Control 1209213 1229142 P=0.62 Inter group comparison,

Femoral Pulse with respect to before & after

Wave Velocity Test 87018 1270119 treatment in test & control group.

(C F PWV)

Ankle Control 1.11.031 1.10 .031 f!P<0.05 With respect to before

Brachial treatment in test group.

Index (ABI) Test 1.01.029 1.10 f.021

ingredients of Habbe Aftimoon possess some important pharmacological

properties such as Hypolipidaemic, Antioxidants, Mohazzil actions. These

pharmacological actions are effective in the delaying process of Atherosclerosis.

Therefore, A single blind standard control study was designed to evaluate the

efficacy of Habbe Aftimoon in the patients of Tasallube Sharain (Atherosclerosis).


Table 4: Effect of Test and Control drugs on Safety parameters

Parameters Test Control

No=20 No=10

B.T A.T B.T A.T

Hb% gm% 12.41.31 12.46.33 12.34.41 11.92.44

TLC cells/cu 8185330 8062386 8290438 7170480

DLC P 57.61.6 57.31.4 581.5 59.42.45

L 37.91.4 37.61.2 35.71.48 35.12.32

Cells/cu DLC E 3.2.18 3.50.23 4.29 3.50.22

M 2.2.20 2.3.21 2.30.21 2.30.21

Cells/cu B 00.00 00.00 00.00 00.00

ESR (mm/1hrs) 27.44.2 27.54.6 21.54.18 23.65.20

FBS (mg/dl) 992.98 1033.96 1017.8 1037.62

PPBS (mg/dl) 16712.3 157.79.36 15116.7 14616.2

B. Urea (mg/dl) 31.31.6 29.11.5 29.32.42 35.52.75

S. Creatinin (mg/dl) 0.89.02 0.840.03 0.850.02 0.940.05

S. Bilirubin (mg/dl) 0.640.05 0.650.05 0.520.04 0.520.05

SGPT(IU/L) 29.52.5 31.63.82 19.43.59 20.51.68

SGOT (IU/L) 25.11.8 23.71.6 20.51.7 18.72.34

Alkaline Phosphates 1316.5 1214.9 1395.5 1326.9

(IU/L)

After 60 days treatment, significant improvement has been noticed in subjective

and objective parameters such as S. Cholesterol, Left ankle Arterial Stiffness

Index (L Ank ASI), Arterial Brachial Index (ABI), and body weight significantly in

test group and total cholesterol, triglycerides, and body weight in standard

control group respectively in atherosclerotic patients.

From above, it is evident that test drug is effective in intra group comparison.

Such effect may be due to ingredients of test drug Habbe Aftimoon which

contain antioxidant, Anti-atherosclerotic and Antioxidant properties. (Cheng et

al., 2003; Naik et al., 2006; Lee et al.,1639; Naik et al., 2004; Chang et al.,

2010; Selvaraj et al., 2007) and hypolipidaemic properties of Halela zard

(Chang et al., 2010) Mastagi (Stella et al., 2009; Xiuzhen Han et al., 2007;

Dedoussis et al., 2004, Duke 2008) and Ustukhodoos (Nikolaevskii et al.,

1990; Yumi et al., 2008; Parejo et al., 2002; Ferreira et al., 2006; Gulcin et al.,

2004).

This improvement may be due to Muhallil (Resolvent), Mulattif (Demulcent)

and Munzij balghame wa sauda properties of Ustukhudoos (Kulkarni et al.,


2004; Ghani, 2010; Nabi, 2007) Mufatteh, Muhallil and Mushile Balgham wa

sauda properties of Aftimoon, Bisfaij, GuleSurkh, Mastagi, and Halela zard.

(Ghani, 2010; Nabi, 2007; Ibn Sina, 1993) These findings are in accordance

with the description given by Razi, Ibne Sina, Ibne Baitar, N.Ghani, Mohd. Azam

khan etc. Further, some recent studies revealed that Mastagi and Ustukhodoos

possess anti atherosclerotic action. (Duke, 2008; Catherine et al., 2001)

Individual drugs that constitute the ingredients of Test drugs have been reported

to possess some interesting pharmacological effects that directly or indirectly

support our contentions regarding the efficacy of the Test drugs. Afteemoon

Bisfaij, Halela zard, Ustokhudoos, Mastagi possesses Mufatteh sudad, Muhallil,

Mulattif and Mushile sauda wa balgham properties. (Ghani, 2010) Ustokhodoos

(Catherine et al., 2001) and Halela zard (Selvaraj et al., 2007; Duke, 2002)

possesses Anti-arteriosclerotic and hypolipidaemic properties, Halela zard

(Prajapati et al., 2005, Cheng et al., 2003) Gule Surkh, (Prajapati et al., 2005;

Said, 1997; Boskabady, et al., 2001) and Mastagi (Benhammou et al., 2008;

Tassou et al., 1995) possesses antioxidant and cardiotonic properties. These

effects are in the same line, as we have mentioned above that the drugs are

producing effects because of hypolipidaemic, antioxidant and cardio tonic

properties. Thus, on the basis of the scientific studies and the reported effects

of the individual ingredients of Test drugs are in conformity to a greater extent

with that of our hypothesis as well as the inferences we drew out of the present

study.

In the light of above discussion, it can be concluded that the Test drugs

produced significant hypocholesterolemic and anti obesity effect without

demonstrating any sign of toxicity or adverse effect.

Although, the Test drug did not produces any significant effect in most of the

objective parameters except body weight, S. Cholesterol, L Ank ASI, and ABI.

However, the Test drug exhibited improvement in some objective parameter

such as on Serum Cholesterol, body weight. Obesity and hypercholesterolemia

are considered an important risk factor for development of atherosclerosis.

The Test drug (Habbe Aftimoon) is quite effective in reducing body weight and

serum Cholesterol level, thereby it may play pivotal role in delaying of

atherosclerosis process. Therefore, it can be concluded that Test drug can be

used for the prevention of atherosclerosis and delaying the progression of the

disease. As a matter of fact, there is no curative treatment available in any

system of medicine. The conventional system of medicine is using hypolipidaemic

and thrombolytic agents for the prevention of atherosclerosis. (Goodman and

Gillman, 2011) Hence, the Test drug Habbe Aftimoon can be safely used for

the same purpose.


Conclusion

On the basis of above result and discussion, it can be concluded that the

compound formulation Habbe Aftimoon is effective in reducing lipid profile in

the patients of atherosclerosis associated with dyslipidemia. Hence, this drug

could be effectively used for prevention of atherosclerosis and to reduce

progression of its manifestation. Since the diverse mechanism is involved in
the development of Atherosclerosis and disease is complex in nature. Therefore,

some elaborate studies are required to ascertain other pharmacological action

of Test drug relatively for longer duration.

Acknowledgement

The authors are very much thankful to Director of National Institute of Unani

Medicine, Bangalore, for providing necessary facilities and conducive

environment in the Institute to carry out this research work

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38
Efficacy of Abstract

Roghan-e- yperlipidaemia is an important factor for the

Kalonji (Nigella
sativa oil) in the
Treatment of
H

development of atherosclerotic lesion which is responsible for various
complications such as ischemic heart diseases, cerebrovascular diseases and
hypertension etc. These complications are major cause of mortality and
morbidity. The association of Hyperlipidaemia with development of atherosclerotic
lesion has prompted the researchers of various field of medicine to develop
Primary

safe and efficacious drug for the management of Hyperlipidaemia. Although, a

Hyperlipidaemia number of plant origin hypolipidaemic drugs have been screened for

hypolipidaemic activity but none of them offers convincing treatment. In Unani

1M. Nazim, system Medicine a large number of drugs are reported to possess possible

1*B.D.
Khan, treatment for some metabolic diseases particularly obesity (Saman-e-Mufrat)

1Misbahuddin Siddiqui

and its related complications. Most of these drugs have not been subjected for

and

evaluation on scientific parameters. Keeping these facts in mind, a single blind

2M. Shoaib

standard controlled non randomized trial was planned to evaluate the efficacy

1Department of Moalijat of a Roghane Kalonji (Nigella sativa oil) in the management of primary

2Department of Ilaj-bit-Tadbeer hyperlipidaemia.

A.K. Tibbiya College,

Aligarh Muslim University, The present study was conducted on 60 diagnosed patients of primary

Aligarh-202002

hyperlipidaemia at Ajmal Khan Tibbiya College Hospital, Aligarh Muslim University

Aligarh. The patients were allocated in two groups. Group A (Test group)

comprising of 40 patients and Group B (Placebo group) comprising of 20

patients. In group A the Roghane Kalonji in the dose of 6 ml twice a day was

given whereas in group B Atrovastatin in the dose of 10 mg once a day was

administered for 3 months. The subjective and the objective parameters (lipid

profile) were assessed on 0, 15th, 30th, 60th, 90th days. The test drug was

found to be statistically significant in lowering serum cholesterol and serum

triglyceride level in the patients of Hyperlipidaemia (p<0.05).

Keywords: Hyperlipidaemia, Roghan-e-Kalonji, Lipid profile.

Introduction

Hyperlipidaemia is a major public health problem worldwide. It accounts about

3.2 million visits to a doctor per year. It is characterized by an excess of fatty

substances such as cholesterol, triglycerides and lipoproteins in the blood. It

is an important risk factor for development of atherosclerosis and ischaemic

heart diseases. More than half of the coronary heart diseases are attributed

to the abnormalities in lipids and lipoproteins metabolism. It also reflects the

adverse impact of the sedentary life style and dietary factors like dietary fat

1


intake greater than 40% of the total calories, saturated fat intake greater than

10% of total calories. (Akbar et al., 1930; Biff et al., 2003; Fauci et al.,2008;

Hongdao, 2006; Joshep, 2003; Jurjani, 1878; Khan et al., 2002, Masson et al.,

2003; Rath et al., 1991). In classical Unani text Shaham is broadly classified

into two types Samin and Riwaj. It is essential for the nourishment of the body

and essential for normal health. According to Unani concept, when the amount

of lipid increase in the blood it leads to increased viscosity and stickiness

(ghilzat and Lazojat) of blood that reduces lumen of vessels which results in

Tasallube Sharaeen. (Maseehi, 1963; Ibne Sena, 1929; Razi, 1999).

As such, there is no direct reference to this disease per se, but hyperlipidemia

is usually associated with obesity. The ancient Unani physicians like Buqrat

(460 BC) and Ibn-Sina (980-1037 AD) have described Saman-e-Mufarat in

their Lexicon. They have mentioned the etiological factors, symptoms

complications like paralysis, constriction of vessels, coma and sudden death

owing to obesity in their clinical observations. Due to the lack of diagnostic

facilities and means of evaluation of lipids in blood, ancient Unani physicians

may have considered obesity and hyperlipidaemia as one disease thus

describing obesity as a whole not specifying excess of fat in blood (Al-Qamri

Mansoori, 1255H; Aqsarai, 1907; Arzani, 1954; Baitar, 1999; Hussain, 1980;

Ibnul-Qaf, 1986; Jalinoos, 1903; Majoosi, 1889; Mansoor Ibne Mohammad,

1989; Maseehi, 1963; Razi, 1999; Rushd, 1987; Ibne Sena, 1929).

The association of hyperlipidaemia with development of atherosclerotic lesion

has prompted the researchers of various field of medicine to develop safe and

efficacious drug for the management of Hyperlipidaemia. Although, a number

of plant origin hypolipidaemic drugs have been screened for hypolipidaemic

activity but, none of them offers convincing treatment. Even though, in mainstream

Medicine Statin (HMG-co Reductase inhibitor) is being used but long term

administration is associated with several side effects. (Lazar et al., 2011; Siig

et al., 2004; Ziajka, 1998) Therefore, search of safe and effective drug is

imperative. In Unani system medicine a large number of drugs are reported to

be used for the treatment of obesity (Saman-e- Mufrat) and its related

complications. Such as Zanjbeel, (Rafiquddin, 1985), Muqil (Anonymous, 2001;

Tripathi, 1984), Kundur (Anonymous, 2004) etc., among them Kalonji (Nigella

sativa L.) (Rafiquddin, 1985) is one the important drugs, which is extensively

used for the remedy of many diseases. The Holy Prophet said that black seed

(kalonji) is remedy for all diseases except death. (Ibne-al-Qayyum, 1985) Recent

studies revealed that seed oil contains major active constituent Thymoquinone

which posses anti inflammatory, antioxidant, anti hypolipidaemic and cardio

protective properties. Nigella sativa oil contains omega-3 fatty acids and other

polyunsaturated and monounsaturated fatty acids in large amount (Ali, 2003).


Consumption of some Omega-3 fatty acids such as Eicosapentaenoic (EPA)

and Decosahexaenoic (DHA) acids present in fish oil has shown a preventive

action against cardiovascular diseases (Anonymous, 2010).

Some practicing Unani physicians are using Kalonji oil in the treatment of

cardiovascular diseases and dyslipidaemic with better results, but clinical efficacy

of Kalonji oil has not been carried out, so far, particularly in relation to

hypolipidaemic property. Keeping these facts in view, a single blind standard

controlled non randomized clinical trial was planned to evaluate efficacy of a

Roghane Kalonji in the management of primary hyperlipidaemia.


For the assessment of efficacy of the Roghan-e-kalonji in the management of

primary hyperlipidaemia, patients were selected from Ajmal Khan Tibbiya College

Hospital OPD, Aligarh, during the period 2010-2012. In the present study, the

patients who attended OPD with the symptoms of palpitation, chest pain, joints

pain, obesity, dyspnoea on exertion, xantholesma and were enrolled for the

screening of hyperlipidaemia. The patients belonging to the age group of 20-

60 years of either sex, ready to participate in the study and whose serum

cholesterol, serum triglyceride level was found abnormal, were included in the

clinical study. The patients of secondary hyperlipidaemia such as hypothyroidism,

diabetes mellitus, nephrotic syndrome and obstructive liver diseases were

excluded from the study on the basis of relevant symptoms and investigations.

Similarly, the patients using oral contraceptive pills and having history of chronic

alcoholism were also excluded from the study. Diagnosis of hyperlipidaemia

was confirmed on the basis of history, clinical examination and analysis of Lipid

profile markers i.e. S. Cholesterol, Triglyceride, VLDL and HDL.

Study Procedure

After complete physical general, systemic examination and biochemical

investigations, patients who fulfilled all the inclusion criteria and signed written

consent, were included in the clinical trial. Total 60 patients were selected for

the study. The patients were allocated into two groups, i.e. Test group (40

patients- Group A) and control group (20 patients- Group B). In the group A

the Test drug was administered in the dose of 6 ml three times a day for a

period of 90 days, while in the group B the Atrovastatin was given in the dose

of 10 mg once a day for same duration. Assessment was done on 0, 15th, 30th,

60th, 90th day of treatment for subjective and objective parameters. In all

patients lipid profile (Serum Cholesterol, Serum Triglyceride, and HDL

cholesterol) was carried out before and after treatment. The data was statistically

analyzed by using Students t test.


In order to assess toxicity of the drugs, safety parameters like liver function

Test (Serum Bilirubin, AST, ALT and Alkaline Phosphate), Kidney Function Test

(Blood Urea, Serum Creatinine) and complete Haemogram were also carried

out.

Results

In this study out of sixty patients of hyperlipidaemia 19 patients were 25-35

years of age, 19 patients were 36-45 years of age, 12 patients were 46-55

years of age, 07 patients were 56-65 years of age group and 03 patients were

>65years of age. The highest prevalence was found in 4th decade. The

percentage of female patients is 76.66% was slightly higher than the male

patients i.e. 23.33%. The demographic data and other observation are depicted

in Table 1.

The effects of test drug on objective parameters i.e. Lipid profile (Serum

Cholesterol, Serum Triglyceride, HDL, LDL, and VLDL) and body weight are as

follows.

Table 1: Demographic Data of patients in Test and Control group n=60(40+20)

N Fp% N Fp%

Age group Mizaj

25-35 19 31.66% Balghami 46 76.66%

36-45 19 31.66% Damvi 08 13.33%

46-55 12 20.0% Safravi 06 10.0%

56-65 07 11.66% Saudavi 0 0%

>66 03 5.0%

Gender Marital Status

Male 14 23.33% Married 53 88.88%

Female 46 76.66% Unmarried 07 11.66%

Religion Dietary Habit

Muslim 50 83.33% Vegetarian 09 15.0%

Hindu 10 16.66% Mixed Diet 51 85.0%

Occupation History of

Students 03 5.0% Xanthomata 07 11.66%

Service 09 15.0% Xanthelesma 13 21.66%

Labour 02 3.33% Arcus cornea 07 11.66%

Business 17 28.33%

House wife 29 48.33%

History of IHD Family History of HLD

Present 35 58.33% Present 28 46.66%

Absent 25 41.66% Absent 32 53.33%


Effect on Serum Cholesterol

In Test group mean serum cholesterol level was 183.32 mg/dl 31.40 before

treatment and at the end of study it was167.60 mg/dl 26.52, showing mean

reduction 15.72 mg/dl 4.88 and which was found to be significant (P<0.001)

(Table 2).
Effect on Serum Triglyceride

In Test group mean serum triglyceride level was 299.07 mg/dl 97.64 before

treatment and at the end of study it was 235.8282.92 mg/dl, showing mean

reduction 63.25 mg/dl 14.72 and which was found to be significant (P<0.001)

(Table 2).

Effect on HDL

In Test group mean HDL level was 33.67 mg/dl 6.96 before treatment and at

the end of study it was 38.08 mg/dl 6.45, showing mean elevation 4.41 mg/

dl 0.51 and which was found to be significant (P>0.05) (Table 2).

Effect on LDL

In Test group mean LDL level was 89.83 mg/dl 33.83 before treatment and

at the end of study it was 82.35 mg/dl 29.17, showing mean reduction 7.48

mg/dl 4.66 and where was found to be significant (P<0.05) (Table 1).

Effect on VLDL

In Test group mean VLDL level was 59.81 mg/dl 19.52 before treatment and

at the end of study it was 47.16 mg/dl 16.58, showing mean reduction 12.65

mg/dl 2.94 and which was found to be significant (P<0.001) (Table 2).

Table 2: Effect of Test drug on Objective parameter in Test and control group

S. Parameter Group-A n=40 Group-B n=20

No.

Before After P-Value Before After P-Value

Treatment 90 days Treatment 90 days

(Base line) (Base line)

1 S. Cholesterol 183.3231.40 167.60 26.52 <0.001 185.20 29.27 141.6017.53 <0.001

2 S. Triglyceride 299.0797.64 235.8282.92 <0.001 290.40 58.41 195.25 39.12 <0.001

3 HDL 33.676.96 38.086.45 >0.05 39.426.93 40.156.34 >0.05

4 LDL 89.8333.83 82.35 29.17 <0.05 87.6922.87 62.40 17.63 <0.001

5 VLDL 59.8119.52 47.1616.58 <0.001 58.0811.68 39.057.82 <0.001

6 Body weight 67.274.22 64.453.73 <0.001 67.004.40 65.204.67 <0.001

7 SBP 136.2210.18 131.308.10 <0.001 142.606.96 133.46.55 <0.001

8 DBP 86.756.65 84.255.21 <0.001 86.306.33 83.404.55 <0.05


Effect on Body weight

In Test group mean WHR level was 67.274.22 kg before treatment and at the

end of study it was 64.453.73 kg, showing mean reduction 2.820.49 kg and

which was found to be significant (P<0.001) (Table 2).

Effect on Safety Parameters
The safety parameters of the test drug like, AST, ALT, Blood Urea, Serum

Creatinine, Hb% and ESR were remained within the normal limits before & after

treatment, in both groups (Table 3).

Discussion

Hyperlipidaemia is a major risk factor for the development of atherosclerotic

heart disease. Reducing plasma cholesterol level particularly LDL cholesterol

reduces the risk of coronary heart disease and other associated complications.

Indeed, dietary modification plays an important role for the prevention of

atherosclerotic diseases but in certain circumstances use of hypolipidaemic

drugs are imperative. Although, a large number of drugs are being used in

contemporary systems of Medicine but search of new plant origin hypolipidaemic

drug is still thrust area of research. The seed of Nigella sativa L. (black seed)

and its oil have been used since long time for the treatment of many diseases

including hyperlipidaemia in traditional system of medicine. The Holy Prophet

Muhammad (PBUH) said, The black seed (kalonji) is the remedy for every

disease except death (Ibne-al-Qayyum 751H). Recent experimental studies

revealed that this drug possess lipid lowering effects in dyslipidaemic patients.

Further studies exhibited that Nigella sativa oil (Roghan-e-Kalonji) contains

Table 3: Effect of Test drug on Safety parameter in Test and control group

S. Parameter Group-A n=40 Group-B n=20

No.

Before After Before After

Treatment 90 days Treatment 90 days

(Base line) (Base line)

1 S. Bilirubin 0.980.2083 0.940.1585 1.0530.23 1.140.17

2 AST 23.134.66 23.744.23 23.493.72 25.354.60

3 ALT 21.773.87 22.443.29 20.564.27 23.026.59

4 S. Alkaline 120.8015.84 122.0712.85 122.010.57 131.013.88

Phosphates

5 Blood Urea 29.227.51 26.315.91 26.865.45 27.844.75

6 S. Creatinine 0.950.144 0.900.108 0.930.13 0.940.11


chemical constituents such as polyunsaturated fatty acids, Omega-3 Fatty

acids, Eicosapentaenoic acid (EPA), Decosahexaenoic acid (DHA) and Alpha

Linolenic acid (ALA) (Anonymous, 2010).

Several preclinical studies revealed that Omega-3 fatty acids decrease the

triglyceride levels either by decreasing hepatic synthesis or secretion of VLDL

particles by inhibiting various enzyme transcription factors or EPA and DHA

increase the activity of lipoprotein lipase, leading to an increase in chylomicron

clearance. (Harris et al., 2008)

Furthermore, researches on Nigella sativa seed oil (Roghan-e-kalonji) revealed

that the seed oil contains major active constituent Thymoquinone. Much of the

biological activity of the seed has been possess due to Thymoquinone, which

shows antioxidant, anti-inflammatory, anti-hyperlipidaemia and cardioprotective

properties. (Ali, 2003; Badary et al., 2000).

The present study demonstrates that the Test drug (Nigella sativa oil) significantly

reduces. Serum Cholesterol, Serum Triglyceride, LDL Cholesterol. These findings

of the study are in accordance to the previous experimental studies as reported

by the Dakha Khani et al., 2000 who revealed that the administration of Nigella

sativa oil for four weeks duration showed significant decrease in serum

Cholesterol, Triglycerides and increases of HDL Cholesterol. Further in other

clinical study administration of 2.5ml of Nigella sativa oil in morning and evening

produce significant hypolipidaemic effects (Dakha Khani et al., 2000).

This drug also exhibited significant effect in reducing Blood pressure and other

subjective parameters. The safety parameters like, AST, ALT, Blood Urea, Serum

Creatinine, Hb% and ESR were remained within the normal limits before & after

treatment, in both groups. This indicates that oral administration of the test

formulation is safe for therapeutic use.

The above mentioned effect of Test drug are mainly due to chemical constituent

present in the seeds of Kalonji particularly Thymoquinone and omega -3 Fatty

acid and unsaturated Fatty acids. Further antioxidant, anti-inflammatory,

hypolipidaemic and cardioprotective properties facilitate the effect of Test drug

in the patients of hyperlipidaemia (Ali, 2003).

Conclusion

In the present study the Test drug was found to be significant in lowering

serum cholesterol and serum triglyceride level in the patients of hyperlipidaemia

without producing any adverse effect. Therefore, it can be concluded that the

Test drug possesses significant hypolipidaemic effect and thus it can be used

as a safe and cost effective therapy in the management of hyperlipidaemia.


However, further study is required to explore hidden potential of Test drug on

larger population.

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Clinical Abstract

Evaluation of a yperlipidemia is a major public health problem

Unani
Formulation for
the Treatment
H

throughout the world. Consequent atherogenic disorders occupied the first
place in five major killer diseases due to high mortality and high morbidity in
the world.
Adouble blind, placebo controlled, randomized clinical trial was conducted to
of Fart-e- test the safety and efficacy of a unani formulation in subjects with hyperlipidaemia.

Unani formulation significantly improved lipid profile as compared to placebo.

Tadassum-Fid-

During 60 days therapy, no noticable adverse/side effectes were detected in

Dam (Hyprer- both treatment groups.

lipidaemia): A

Keywords: Hyperlipidaemia; Badranjboy; Bombyx mori; Abresham;

randomized,

Cardioprotective

Double Blind,

Introduction

Placebo

Hyperlipidemia is considered as lifestyle disorder in present age and it is a

Controlled

major public health problem throughout the world. It is characterized by

Clinical Study

increased lipids in blood due to either increase in rate of synthesis or decrease

in rate of breakdown of lipoproteins (Clayton et al., 1999). Hyperlipidemia is a

1Rais-ur-Rahman, common condition which may either results from primary abnormality in lipid

2Afshan

Qaiser metabolism or is a secondary manifestation of some other conditions (Christofer

and

et al., 2004). Worldwide, the prevalence of hyperlipidaemia is about 39,000

3*Yasmeen Shamsi

per 100,000 patients. In developed countries, the prevalence of hyperlipidaemia

1Department of AYUSH, is about 57,000 per 100,000 patients. In developing countries, the prevalence

Ministry of Health and F.W., of hyperlipidaemia is about 26,000 per 100,000 patients (Michael Gibson,

Government of India,

2013). Epidemiological evidence suggests 1% increase in Coronary Heart

GPO Complex, INA,

Disease (CHD) risk for each 1% increase in Low Density Lipoprotein (LDL), 2-

New Delhi-110023

3% reduction in CHD risk for each 1% increase in High Density Lipoprotein

2Department
of Moalijat,
(HDL) (Annonymous, 2001). According to the available data, the atherogenic

A&U Tibbia College, Karol Bagh,

disorders occupied the first place in the five major killer diseases due to high

New Delhi-110005

mortality and high morbidity in the world. The diseases are usually considered

3Department of Mahiyatul Amraz,

as disease of atherogenic pathology and affect the various parts of the body

Faculty of Medicine (U),

viz. ischemic heart disease, cerebrovascular accidents and peripheral vascular

Jamia Hamdard,

disorders etc. Various epidemiological studies suggest that the development of

New Delh-110062

atherosclerosis and ischemic heart disease is strongly associated with

hyperlipidemia.

To overcome the problem of hyperlipidaemia, day by day several synthetic

drugs of better efficacy are being introduced in the modern system of medicine.

3


In this series nicotinic acid was the first drug to be introduced by the Altschul

et al in 1955 (Rudolf et al., 1960). Use of hypolipidaemic agents and low fatty

diet is the corner stone of the management of hypercholesetrolaemia. Several

hypolipidaemic drugs have been already introduced in main stream medicine

such as Hydroxy methyl glutaryl CO-A (HMG CO-A) reductase inhibitors

(levostatin, atorvastatin), bile acid sequestrents (colestipol) and fibric acid

derivetives (gemfibrozil, and fenofibrates) etc. But the long term use of these
drugs causes various side effects like myalgia, arthragia, dyspepsia and

cholelithiasis loss of libido, impotence etc (Tripathi, 1994). Such side effects

limit the use and the efficacy of these drugs. Hence, there is a need to develop

a drug from herbal source which should be safe, cost effective, easily available

and efficacious. Keeping in view all the above mentioned drawbacks of modern

medicine in the management of hyperlipidaemia, need was felt to conduct a

clinical trial with dried aqueous extract of Abresham (Bombyx mori), Badranjboya

(Nepeta hindostana) and Arjun Bark (Terminalia arjuna) in the ratio of 1:2:1.

Preclinical study of this unani formulation has already been conducted on

isoproterenol treated rats, which showed remarkable hypolipidemic and

cardioprotective effects of test drug comparable to control (Tajuddin et al.,

2006 & 2007).


This study was double blind, randomized, placebo controlled clincal trial ,

carried out in the department of Moalejat , A &U Tibbia College, Karol Bagh,

New Delhi, from September 2012 to March 2013. The aim of the study was to

evaluate the efficacy and safety of unani formulation in the treatment of Fart-

e-Tadassum Fiddum (Hyperlipidaemia) on scientific parameters.

Study Drug

The study drug was a combination of three Unani drugs supplied by Dehlvi

Naturals, India, in the form of capsule. Placebo capsules were also supplied by

Dehlvi Naturals, India, the composition of test drug is given in Table-1.

Table 1: Composition of Unani Formulation

Each 500 mg capsule contains dried aqueous extract of:

Unani Name Scientific Name Part Used Quantity

Abresham Bombyx mori Raw Silk Cocoon 125 mg

Badranjboya Whole Plant 250 mg

Nepeta hindostana

Arjun Terminalia arjuna Bark 125 mg


Partcipants

Eligible subjects as per the inclusion/exclusion criteria were enrolled in the

study after obtaining written informed consent according to Helsinki declaration.

Inclusion Criteria

Subjects (men and women) aged 18-65 years were eligible for the study if they
had a history of dyslipidemia regardless of strict diet control and had fasting

LDL-Cholesterol= 130-159 mg/dl, Total Cholesterol= 200-239 mg/dl,

Triglycerides= 150-190 mg/dl and HDL-Cholesterol: <40 mg/dl (Anonymous,

2001).

Exclusion Criteria

The exclusion Criteria were pregnancy, lactation, intake of oral contraceptives

or any other medication that might affect serum lipids (thyroid or steroid

hormones, beta blockers, diuretics etc), H/O cardiovascular disease, impaired

hepatic, renal function, malignancy, secondary hyperlipidaemia and body mass

index >30 kg/m2.

Treatment

At baseline, lipid profile determinations and laboratory safety tests were

performed and the eligible cases as per the inclusion/exclusion criteria were

randomly assigned to receive either drug or placebo capsule in the dose of 1

capsule twice daily for a period of 60 days. All the patients were instructed to

maintain low cholesterol diet. Clinical examination and laboratory tests were

done at each visit. Adverse events were recorded and compliance with study

medications was assessed at each follow up visit.

Primary Outcome Measures

The primary efficacy end point was percentage reduction from baseline in

LDL-Cholesterol, Triglycerides and Total-Cholesterol.

Secondary Outcome Measures

Percentage of change from baseline in HDL, LDL/HDL ratio and TC/HDL ratio.

were the secondary outcome measures.

Safety Assaessment

For the assesment of safety, Liver function test, Kidney function test, Haemogram

and ECG were carried out at baseline, on first follow up visit i.e., on on 15th

day of treatment and at the end of therapy i.e., on 60th day. Data from the


physical/clinical examination, laboratory tests and interview for adverse events

as recorded in CRF were included in the analyses of safety and tolerability.


The changes between pre-treatment and post treatment values of primary and

secondary outcome obtained in test group were compared with those obtained

in placebo group by using unpaired t test. Statistical calculations were performed

with GraphPad InStat statistical softwere version 3.10 . Statistical analysis was

done only for those patients who completed the course of treatment for 60

days.

Results

Total 70 patients were registered out of them 10 cases (4 receiving test drug

and 6 receiving placebo) were dropped out from the study, only 30 patients in

test group and 30 in the placebo group completed the treatment..

Pretreatment and post treatment (after 60 days) maean values of lipid profile

components are their percent changes areshown in Table-2 & Figure-1.

Sixty days treatment with test drug was significantly effective than placebo on

the primary efficacy measures in reducing LDL-C by 15.98% as compared with

4.21% in the placebo group (p < 0.001). Test drug also significantly reduced

total cholesterol (TC) and triglycerides by 10.30% (compared with 0.94% in

placebo group) and 12.05% (compared with 1.77% in placebo group)

respectively (p value was <.0001 in both cases). The mean reduction in total

lipids was 10.24%, in drug group, compared with placebo group (0.64%).

The test drug was also found significantly effective than placebo on the

secondary efficacy measures in reducing LDL/HDL ratio by 26.87% compared

with 2.47% in the placebo group and TC/HDL ratio by 24.22% (compared with

6.27% in placebo group). A significant rise in HDL was observed in test group

(10.57%) compared to placebo group (3.00%), (p<0.001) respectively (p value

was <.0001 in both cases).

Safety and Tolerability

Unani formulation (also placebo) treatment for 60 days did not impair physical

safety indicators such as body weight, pulse rate or blood pressure.

Laboratory safety indicators e.g., kidney function test (blood urea, serum

creatinine), Liver function test (ALT, AST, serum bilirubin, serum alkaline

phosphalase) and haemogram remained within the normal limits in all study

patients.


Table 1: Effects of Test Drug (Unani Formulation) and Control (Placebo) on Lipid Profile

Lipid Profile MeanSEM MeanSEM % t p

0 Day 60th Day Change value value

LDLCholesterol (mg/dl)

Test Drug 145.231.61 122.022.2 15.98% 2.69 0.001

Control 140.30 2.3 134.384.04 4.21%

Total Cholesterol (mg/dl)

Test Drug 217.801.63 195.371.77 10.30% 7.28 0.0001

Control 216.971.82 214.931.64 0.94%

Triglycerides (mg/dl)

Test Drug 171.011.50 150.401.63 12.05% 5.138 0.0001

Control 163.971.75 161.071.84 1.77%

Total Lipids

Test Drug 607.34.07 545.133.82 10.24% 7.77 0.0001

Control 598.233.77 594.405.06 0.64%

HDLCholesterol (mg/dl)

Test Drug 38.230.84 42.270.87 10.57% 2.206 0.001

Control 40.871.54 42.101.39 3.00%

LDL/HDL ratio (mg/dl)

Test Drug 3.870.14 2.830.10 26.87% 2.056 0.04

Control 3.240.13 3.160.13 2.47%

TC/HDL ratio

Test Drug 5.781.94 4.382.03 24.22% 3.832 0.02

Control 5.11.18 4.781.17 6.27%

N=30 in each group; LDL=Low Density Lipoprotiens; HDL= High Density Lipoprotiens;

TC=Total Cholesterol, SEM=Standard error of Mean

Figure 1: Mean Percent Change in Various Components of Lipid Profile After Treatment


Discussion

In the present clinical trial the effects of test drug have been tested on all the

components of lipid profile in a double blind, randomized fashion and the

safety of the drug has also been established. Unani formulation (test drug)

significantly reduced LDL,TC, triglycerides, LDL/HDL and TC/HDL comparable

with control(placebo). Unani formulation also significantly improved HDL level
than that observed in control group. Individual ingredients of unani formulation

have been reported to possess important pharmacological actions that directly

or indirectly support our contention regarding efficacy of test drug on human

being. Abresham, Arjun chhaal and Badranjboya possess diverse

pharmacological activities like cardio tonic, anti inflammatory, anti-oxidant,

anxiolytic, fibrinolytic, and anti-platelet activities as evident by previous

pharmacological studies (Collabawalla, 1951; Ghani, ynm; Mahdi et al., 2011;

Singh et al., 2001; Maulik et al., 1997).

The effect of unani test formulation on various components of lipid profile

could be due to hypolipidaemic, cardiotonic and cardiac stimulant activities of

Arjun (Terminalia arjuna) and cardiotonic, anti hypercholesterolemic and

antiatherogenic effects of Abresham (Bombyx mori) & Badranjboya (Nepeta

hindostan) (Monahan, 2007; Halleys Khan et al., 2011; Ghani, ynm).

All these support the cardiovascular protective effects of test drug. Both

Abresham (Bombyx mori) and Arjuna (Terminalia arjuna) have been reported

to have potent antioxidant activity; these findings also support cardioprotective

effects of test drug. Badranjboya (Nepeta hindostana) is known to prevent

myocardial infarction which supports its lipid lowering action.

The results of present study suggest that the unani test formulation is safe and

efficacious in treating hyperlipidaemia.This formulation can be valuable in

prevention of atherosclerosis and cardiovascular disease by anti-platelet,

fibrinolytic, anti-oxidant and cholesterol lowering activities of its ingredients.

Conclusion

The results of this study can be concluded as:

The unani formulation significantly reduced LDL Cholesterol, Total

Cholesterol, Triglycerides, LDL/HDL ratio, and TC/HDL ratio as compared

with placebo.

Improvement in HDL Cholesterol obtained with unani fornulation

treatment was significantly greater than that of placebo.

The unani test formulation was well tolerated and no adverse/side

effect were observed during the entire period of protocol therapy.


References

Anonymous, 2001. Executive summary of the Third Report of the National

Cholesterol Education Program (NCEP) Expert panel on detection, evaluation

and treatment of high blood cholesterol in adults (Adult treatment panel III),

JAMA may 16; 285 (19): 2486 -97.

Christofer R, Edwin A, Nicholas R. Nikkia, 2004. Davidsons principles and
practice of medicine, 19th edition. Churchill living stone, pp.308-311.

Clayton, L., Thomas, Tabers Cyclopedic, 1999. Medical Dictionary, 16th Ed.

F.A. Davis Company, USA, pp.863-1039.

Collabawalla, H., 1951. An evaluation of the cardiotonic and other properties

of Terminalia arjuna. Indian Heart Journal 3:205-230.

Ghani Najmul, ynm. Khzainul Advia: Sheikh Mohd. Basher and Sons Pub. Urdu

Bazar Lahore, pp.209, 217.

Halleys Khan, Z.M., Hossain Md. Faruquee , Md. Munan Shaik, 2011.

Phytochemistry and Pharmacological Potential of Terminalia arjuna L.

Medicinal Plant Research, 3(10:70-77.

Maulik, G., Maulik, N., Bhandari, V., 1997. Evaluation of antioxidant effectiveness

of a few herbal plants. Free Radical Research 27: 221-228.

Michael Gibson, C., 2013. Hyperlipidemia epidemiology and demographics.

Wikidoc Editor-In-Chief: C. Michael Gibson, Hardik Patel ib.wikidoc.org/

Hyperlipidemia epidemiology and demographics.

Mir Mahdi Ali, Arumugam, A., Sarasa, Bharati, 2011. Effect of crude extract of

Bombyx mori cocoons in Hyperlipidemia and atherosclerosis. Journal of

Ayurveda and Integrative Medicine 2(2):72-78.

Monahan, 2007. Effect of Hyperlipidemia on autonomic and cardiovascular

control in human. Journal of Applied Physiology 103(1): 162-169.

Rudolf Altschul, Abram Hoffer, 1960. The effect of nicotinic acid on

hypercholesterolaemia. Canad. M.A.J. 82:783-785.

Singh, K.P. and Jayasomu, R.S., 2001. Bombyx mori; A review of its potential

as medicinal insect. Pharmaceutical Biology 39:1-5.

Tajuddin, Nasiruddin, M. and Ahmad, N., 2007. Cardioprotective effect of Unani

formulation in rats. Indian Journal of Traditional Knowledge 6(4):663-667.

Tajuuddin, Nasiruddin M., Ahmad, N., 2006. Effect of a unani formulation on

lipid profile in rat. Indian J. Pharmacol 38(1):56-57.

Tripathi, K.D., 1994. Essentials of Medical Pharmacology, 3rd edition. Jaypee

Brothers Medical Publishers, pp.59345.



56
Pharmacognostic Abstract

Evaluation of n view of probability of adulteration especially in unorganized

Dammul
Akhawain with
Reference to
I

drugs, this study was designed for standardization of Dammul Akhawain
(Dragons blood) to generate data for future reference. The study was carried
out on a samples of Dragons blood obtained from the plant Pterocarpus
marsupium Roxb, considered as the standard sample. The study comprised of
morphology, physicochemical study, physical constants, preliminary
Standardization

phytochemical study and spectrophotometery. Since in literature parameters

for this have not been mentioned sufficiently, therefore, the findings of this

Ehteshamuddin,

study may be considered as standard for quality assessment of available

*Abdul Wadud,

sample of Dragons blood.

Ghulamuddin Sofi,

Mohammad Yusuf Ansari

and Keywords: Pharmacognosy, Unorganized drug, Standardization, Phytochemistry.

Shamim Irshad

Introduction


Kottigepalya, Magadi Main Road,

Adulteration, substitution, misidentification, quality inconsistency, and controversy

Bangalore-560091

pertaining to herbal drugs are challenging the wide acceptability of traditional

systems of medicine. Therefore, it is imperative to determine authenticity of

crude drugs used for the preparation of medicaments, because it is associated

with the safety of consumers. However, it becomes challenging when two or

more plants are claimed to be the source of one drug especially in case of

unorganized drugs, such as gum, resin, oleo-resin and oleo-gum-resin, as in

such circumstances herbarium and drug museum, which are the main sources

of information, become of little use (Bonakdar, 2002). Eventually, important

drugs compromise their efficacy in spite of possessing significant effects.

Among the various steps to be taken for solving these problems, quality

assessment of samples of all herbal drugs appears to be of utmost importance.

Conventional methods of standardization substantiated with analytical techniques

are considered most reliable tools for quality assessment of herbal drugs

(Shinde, and Dhalwal, 2007). Most of the regulatory guidelines also suggest

macroscopic, microscopic, physicochemical and phytochemical standardization

of medicinal plants materials. HPLC, HPTLC, and spectrophotometery etc. may

further reinforce the above methods (Xing et al., 2010).

Dragons blood (DB), as known in trade, is a bright red resin obtained from a

number of different taxa such as Croton, Dracaena, Daemonorops, Calamus

and Pterocarpus (Shinde, Dhalwal, 2007). Hence, a number of plants such as

Croton draconoides Mll Arg, Pterocarpus marsupium Roxb, Calamus rotang

Linn, Croton draco Schltdl & Cham, Croton lechleri Mll Arg, Croton urucurana



Baill, Croton xalapensis Kunth, Daemonorops draco Blume, Daemonorops

didymophylla Becc, Daemonorops micranthus Becc, Daemonorops motleyi Becc,

Daemonorops rubra (Reinw ex Blume) Mart, Daemonorops propinquus Becc,

Dracaena cinnabari Balf.f, Dracaena cochinensis Hort ex Baker, and Pterocarpus

officinalis Jacq etc. have been accounted as the source of DB (Xing et al.,

2010). Such a big list of sources for one drug creates enormous degree of

uncertainty. Even if two or more sources, as happens in case of some drugs,

are considered the problem persists as it is not clear which source the available

sample belongs to.

DB is a red resin and the name refers to reddish resinous product applied to

many red resins described in literature (www.en.wikipedia.org, 2010). However,

red gum resin of some of the above mentioned sources have the official status

in their respective countries which they are indigenous to, such as red gum

resin of Pterocarpus marsupium Roxb., family Fabaceae is known as Indian

Dragons blood (www.thefullwiki.org, 2010), red gum resin of Dracaena cinnabari

is considered as Socotra Dragons blood. Similarly, Dracaena cochinensis is

said to be the source of Chinese Dragons blood (www.aseanbiodiversity.info,

2010).

DB is an important drug used in Unani Medicine as Qabiz (astringent), Habis

(styptic), Muqawie meda (stomachic) , Mohallil-e-Auram (resolvent), Dafe-Zaheer

(anti dysentery) (Ibn Sina, 2007). Its liniment is useful in anal fissure, prolapsed

rectum and inflammation of rectum. It is styptic and astringent for stomach

whether used alone or with other astringent drugs, when used as enema it

causes constipation (Khan,1314 AH).

In this study, red gum- resin of Pterocarpus marsupium Roxb. (DB) has been

considered as the standard sample viewing that the same is commonly available

in Indian crude drug markets. Therefore, it was obtained from the plant

Pterocarpus marsupium Roxb, and was evaluated pharmacognosticaly to

establish standard parameters for further studies.


Materials

The sample of DB was collected from Pterocarpus marsupium Roxb. grown in

Erepalya, Bidadi, Hobli, District Ramnagar, Karnataka, India. It was authenticated

by Dr. Shiddamallayya N and Dr. V. Rama Rao, vide Drug Authentication/

SMPU/NADRI/BNG/2010-11/961. A voucher specimen has been deposited in

the Drug Museum of National Institute of Unani Medicine (NIUM), Bangalore,

vide Voucher specimen No.01/IA/Res./2011.


Methods

Organoleptic evaluation

The organoleptic characters like color, odor, taste, luster, texture, fracture,

consistency and cut surface of were examined by naked eye (Anonymous,

1968).

Physicochemical evaluation

For estimation of extractive values and ash values, methods described in

British Pharmacopoeia (Anonymous, 1968) were applied. Moisture content was

determined by TGA method (Anonymous, 1992).

Determination of pH value

The pH value of 1% and 10% aqueous solution was estimated by the method

described by (Khandelwal, 2008; Brewster and Mcewen, 1971).

Determination of melting point

Melting point was estimated by melting point apparatus model C-LMP-1,

Campbell electronics.

Solubility test

Solubility was tested by the method described in British Pharmacopoeia

(Anonymous, 1968).

Qualitative Phytochemical evaluation

For preliminary phytochemical studies, powder of the DB was extracted in

different solvents viz. petroleum ether, di-ethyl ether, chloroform, ethanol,

acetone, benzene and distilled water. The extracts were subjected to various

qualitative phytochemical tests for estimation of alkaloids, glycosides,

carbohydrates, phenol compounds, tannins, phytosterols, proteins and amino

acids etc.

Alkaloid was tested by Dragendroffs test, Mayers test, Hagers test and

Wagners test (Anonymous, 1992). Protein and amino acids were tested by

Ninhydrin test, Biurettes reaction, Millions reaction and Xanthoproteinic reaction

(Khandelwal, 2008; Brewster, and Mcewen, 1971). Glycosides were tested by

Molishs test (Paech and Tracey, 1955). Cardiac glycosides were tested by

Keller-killiani test. Bufadenoloids were tested by Liebermanns test. Flavonoids

were tested by Ammonia test (Anonymous, 1992). Saponin was tested by


Honey comb frothing test (Arthur and Chan, 1962). Tannins were tested by

Ferric chloride test (Brewster and Mcewen, 1971). Phenols were tested by

Ferric chloride test and Lead acetate test (Khandelwal, 2008). Phytosterols /

Terpenes were tested by Hosses reaction, Liebermann Burchards reaction,

and Moleschotts reaction (Khandelwal, 2008).

Test for Inorganic constituents
Ash of DB was prepared. To the ash 50% v/v hydrochloric acid and 50% v/v

Nitric acid were added, and kept for an hour and then filtered. Various tests

were performed with the filtrate for qualitative estimation of inorganic constituents

such as sulphate, phosphate, iron, chloride, carbonate and nitrate (Brewster

and Mcewen, 1971).

Spectrophotometery

The alcoholic extract was subjected to Spectrophotometery by using UV-VIS

Spectrophotometer. The test was performed at room temperature with the

following settings: Number: 18-1885-01-0259; Spectral Bandwidth: 2.00 nm;

scan Range: 190.00 to 360.00 nm; Measure Mode: Abs; Interval: 1.00 nm,

Speed: Medium.

Results

The results of macroscopic evaluation are shown in table 1 and figure 1&2.

The extractive values taken in ethanol, chloroform, diethyl ether, pet. ether,

benzene, acetone, and distilled water; mean percentage of total ash, acid

insoluble ash, water insoluble ash and water soluble ash; moisture content as

obtained by TGA method; mean percentages of pH value in 1% and in 10%

aqueous solution, and the melting point are given in table 2. The Preliminary

Table 1: Morphology of Dragons blood

S.No. Characteristics Result

1. Color Yellowish red

2. Luster Lustrous

3. Fracture Transverse

4. Texture Brittle

5. Consistency Liquid

6. Odor Odorless

7. Taste Astringent

8. Cut surface Smooth


Figure 1: Samples of Dragons blood Figure 2: Powder of dried sample of

Dragons blood

Table 2: Physical constants of Dragons blood

S.No. Parameters Values (Mean SEM)

1. Extractive Solvents Pet. ether 0.00

Values Di-ethyl ether 0.00

Chloroform 0.00

Benzene 0.00

Acetone 28.26 0.86

Ethanol 63.33 1.46

Distilled water 68.442.25

2. Ash Values Total Ash 4.030.19

Acid insoluble Ash 2.590.14

Water insoluble Ash 12.940.16

Water soluble Ash 0.450.08

3. Moisture content 0.00%

4. pH 1% solution 4.66 0.17

10% solution 4.78 0.05

Phytochemical screening of the different solvent extracts was done systemically

for phytochemical constituents. Glycoside, amino acid and phytosterol were

positive. Iron, nitrate and phosphate were also detected. Spectrum scanning

gave three peaks and three valleys (Figure 3).

Discussion

Due to crude nature of herbal drugs, traders often take advantage of it and

indulge in malpractices; however, it may happen due to ignorance as well.

Usually it is noticed that commercial samples of some drugs do not match with

their description in literature (Kartik et al., 2010).


Table 3: Solubility of Dragons blood in different organic solvents at different temperature

S.No. Solvents Result

TemperatureC

20 40 60 80

1. Ethanol Soluble

2. Pet. Ether Insoluble

3. Di-ethyl ether Insoluble

4. Chloroform Insoluble

5. Benzene Insoluble

6. Acetone Insoluble

7. Distilled water Insoluble

Figure 3: Spectrum Scan curves of Dragons blood

So literature survey can give preliminary ideas about probable sources of

drugs to draw some conclusion. Most of the literature consulted revealed that

gum-resin of Pterocarpus marsupium (www.en.wikipedia.org), Daemonorops

draco (www.botanical.com), Dracaena cinnabari (Al-Awthan et al., 2010), and

Dracaena cochinensis (www.aseanbiodiversity.info, 2010; www.aseanbiodiversity.

info, 2010) may be considered as the sources of DB for different countries. It


was also concluded that gum-resin of Pterocarpus marsupium Roxb is the

Indian Dragons blood.

Morphological studies of crude drugs give some idea at very first sight. Color,

odor, taste, luster, fracture, and consistency etc. (Evans, 2008; Pearson and

Prendergast, 2007) are some prominent characters, therefore, these characters

were observed.

It is important to note that physical constants of a drug are good criteria for

identification. These constants are extractive values, ash values, and moisture

content. These parameters are widely accepted for checking purity of drugs

(Anonymous, 1992). These parameters too were applied in this study.

The constituent of a particular drug sample can be estimated in terms of %

with respect to air dried weight by extracting in various solvents known as

extractive value which was applied to the sample. In literature gum resin of

Pterocarpus marsupium has been reported to be 80 - 90 % soluble in alcohol.

Our finding demonstrated no yield in diethyl ether, pet. ether and benzene.

However the yield % was 28.26 0.86, 63.33 1.46, 68.442.25 in ethanol,

acetone and distilled water, respectively.

Ash value is taken in terms of total ash, acid insoluble, water insoluble and

water soluble ash. In some cases there may be considerable difference in total

ash value in the same drug which may either be due to variation in the amount

of oxalate or some adulteration with metallic and the like materials or earthy

material. In such cases acid in soluble ash is taken into consideration. This

parameter was also applied in our study.

The water content (moisture) of crude drugs is another important parameter

for checking purity of drugs. In this study, Thermo gravimetric Analysis (TGA)

method was applied for estimation of moisture content. This method is suitable

for all types of substances as it provides quantitative measurement of mass

change in materials. It records changes in mass from dehydration, decomposition

and oxidation of a sample with time and temperature (El-Sayd and Makawy,

2010). We found 0.00% moisture in the sample.

DB is a gum-resin and for these types of drugs, melting point, pH and solubility

may be considered important parameters. Gum-resin is insoluble in water and

petroleum ether but more or less soluble in alcohol, chloroform, and ether.

Crude drugs containing mixed chemicals are described with certain range of

melting points. pH of solution of a substance at 1 % w/v and 10% w/v of water

soluble portion can give accurate estimation of purity of a drug. These

parameters were also. No data regarding melting point and pH of gum resin

of Pterocarpus marsupium are available in literature; therefore, we considered

our findings as stand. In literature melting point of gum resin of Calamus draco


is shown to be 76C. (www.henriettesherbal.com, 2010). The melting point of

standard sample did not coincide with the reported finding. Similarly, pH and

solubility were estimated at 20, 40, 60, and 80C. Except solubility, no data on

gum- resin of Pterocarpus marsupium, which is 80-90 % soluble in cold water

and almost soluble in alcohol, are available (Kokate, 2007), we considered our

results as standard.

The analysis of physiologically active compounds is important parameter for

checking the authenticity of a drug. These compounds are alkaloids, glycosides,

flavonoid, phytosterol, essential oil, resin, tannin, etc. These were also estimated.

Analysis of inorganic constituents may also be considered parameter for

checking the authenticity of drug. In literature Pterocarpus marsupium is shown

to contain flavonoids, tannins, and phytosterol. Our findings are in confirmation

with the report.

Spectrophotometery may be a sophisticated tool for standardization of crude

drugs. Spectrum scanning curves were obtained to get preliminary information.

It demonstrated peaks and valleys of different absorbance and wave length,

indicating presence of different constituents.

Conclusion

Detailed data on DB regarding physical, chemical and other properties are not

available to compare our findings, therefore, our findings may be considered

as standard for Indian Dragons blood for future reference.

Acknowledgement

The authors are thankful to the Director, National Institute of Unani Medicine,

Bangalore, for providing facilities for experimentation.

References

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Dracaena cinnabari Resin and Effects of the Aqueous Extract on Isolated

Smooth Muscle Preparations, Perfused Heart, Blood Pressure and Diuresis

in the Rat. Jordan J Pharmaceuti Sci. 3 (1):8 -17.

Anonymous, 1968. British Pharmacopoeia, General Medical Council.

Pharmaceutical Press, London, pp.l1276-77; 1285- 88.

Anonymous, 1992. Quality Control Methods for Medicinal Plant Materials, rev.

1, Organisation Mondiale De La Sante. World Health Organisation, p.159.

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Alkaloids, Essential oil, and Saponin. Trop-sci. 4: 147.


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the Dietary Supplement Health and Education Act. Family Medicine 2002,

July - Aug.; 34(7):522-527. [cited 2010Aug.5] http :// www. ncbi. nlm. nih.

gov/ pubmed /12144007.

Brewster, R.C. and Mcewen, W.E., 1971. Organic Chemistry, 3rd ed. Prentice-

Hall of India private Ltd, New Delhi, p. 406.

El-Sayd, N.I., and Makawy, M.M., 2010. Comparison of Methods for Determination

of Moisture in Food, Research J Agriculture and Biol Sci. 6(6): 906-11.

Evans, W.C., 2008. Trease and Evans Pharmacognosy, Elsevier, A Division of

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Ibn Sina, 2007. Al-Qanun Fit Tib. Part 1st, Vol.2nd. (Urdu translation by Kantoori,

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Kartik, C.P., Surendra, K.P., Ranjit, K.H., Kumar, K.J., 2010. Traditional

Approaches towards Standardization of herbal Medicines-A review. J

Pharmaceut Sci and Technol 2 (11): 372-379.

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pp. 18-19.

Khandelwal, K.R., 2008. Practical Pharmacognosy-Techniques & Experiments,

Nirali Prakashan, Pune, pp. 146-59,193.

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Pearson, J. and Prendergast, H.D.V., 2007.Collection corner Daemonorops,

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Hepatoprotective Abstract

Activity of iver diseases have become one of the major causes of

Extracts and
Chemically
Defined
L

morbidity and mortality in man and animals all over the globe and hepatotoxicity
due to drugs appears to be the most common contributing factor. About 20,000
deaths occur every year due to liver disorders. Hepatocellular carcinoma is
one of the ten most common tumors in the world with over 2, 50,000 new cases
each year. Plants have potent biochemical components of phytomedicine. Plant
Molecules from

based natural phyto-constituents can be derived from any morphological part

Herbal Drugs : of the plant and may contain active components. The beneficial medicinal

effects of plant materials typically result from the combinations of secondary

Review#

products present in the plant. The medicinal actions of plants are unique to a

particular plant species or groups and are consistent with this concept as the

1*Manoj Kumar Pandey,

combination of secondary products in a particular plant is taxonomically distinct

2Nitin Rai

and as such different types of drugs such as acetaminophen; chloroquinine and

2Rajeev Kr. Sharma isoniazid are inducing hepatoxicity in the world. Herbal plants have been used

traditionally by herbalists worldwide for the prevention and treatment of liver

1Pharmacopoeia Commission for

disease so. Herbal drugs were prescribed even when their biologically active

Indian Medicine, Raj Nagar,

Ghaziabad-201002 compounds were unknown because of their effectiveness, few side effects and

relatively low cost.

2Pharmacopoeial
Laboratory for

Indian Medicine, Kamla Nehru Nagar, In this review, an attempt has been made to compile the reported

Ghaziabad-201002

hepatoprotective activity of plants from India and abroad and may be useful to

develop evidence based medicine to cure different kind of liver diseases in

man and animal.

Keywords: Hepatic disorder, Hepatoprotective herbs, Ayurvedic formulations

Introduction

Liver has a pivotal role in regulation of physiological processes. It is involved

in several vital functions such as metabolism, secretion and storage.

Furthermore, detoxification of a variety of drugs and xenobiotics occurs in liver.

The bile secreted by the liver has, among other things, an important role in

digestion. Liver diseases are among the most serious ailment. They may be

classified as acute or chronic hepatitis (inflammatory liver diseases), hepatosis

(non inflammatory diseases) and cirrhosis (degenerative disorder resulting in

fibrosis of the liver). Liver diseases are mainly caused by toxic chemicals

(certain antibiotics, chemotherapeutics, peroxides oil, aflatoxin, carbon-

tetrachloride, chlorinated hydrocarbons, etc.), excess consumption of alcohol,

infections and autoimmune/disorder.

#Invited Paper

1


The liver functions as a great metabolic factory and is particularly concerned

with metabolizing drugs, especially those given orally. It plays a key role in the

metabolism of lipids, proteins and carbohydrates, as well as in

immunomodulation. The sheer complexity and varied nature of its interactions

continually expose it to a variety of toxins, therapeutic agents etc., making it

susceptible to literally hundreds of diseases. Some of these diseases are rare;

others are common, such as hepatitis, cirrhosis, pediatric liver disorders, alcohol
related disorders, liver cancer, and weakened liver function on older people.

Cirrhosis is the third leading cause of death in adults aged between 25 and

59, and seventh leading cause of death overall. It has been estimated that

approximately 14 - 16 million people are infected with this virus in South East

Asia region and about 6% of the total population in the region are carriers of

this virus.

The situation in India is more serious so far as viral hepatitis is concerned. It

is reported that one among every 25 Indians is a carrier of hepatitis B virus

and fifth major cause of mortality of people in the age groups of 15-45 years.

1% of total adult death attributed in India is due to infection caused by hepatitis

virus B. Besides, incidence 60% chronic liver diseases and 80% of primary

liver cancer are due to residual effects of hepatitis B infection. With of lack of

safe and effective treatment for liver diseases, researches have been turned

towards alternative therapies with ethnic drugs of herbal origin used traditionally,

especially in lights of new findings.

Treatment of liver diseases is still largely influenced by holistic approach in

different system of medicines. The modern allopathic has very limited effective

remedies. However much of remedies claims to be available in folk lore

traditional system of medicine based on plants. More than 600 numbers of

plants based commercial products are available in different parts of world

market for the treatment of variety of liver diseases. In India alone there are

more than 60 poly herbal preparations available in market.

In vivo and in vitro investigations established conclusively that many such

plants species does posses prophylactic and therapeutic activity. Recent

development of both in vivo and in vitro investigation procedure laid the

foundation for scientific exploration of such plants species as well as helped

in validating the folk lore claims. This is evident from the voluminous scientific

publications on such investigations on traditional herbal remedies particularly

in last two decades. Isolation of novel active phytoconstituents from many such

plant species possessing significant potency of antihepatotoxic, will lead for

further development of ideal remedies for various liver diseases.

Hence, there is an ever-increasing need for safe hepatoprotective agents.

Herbal-based therapeutics for liver disorders have been in use in India for a


long time and popularized world over by leading pharmaceuticals. Despite the

significant popularity of several herbal medicines in general, and for liver

diseases in particular, they are still unacceptable as treatment modalities for

liver diseases. The limiting factors that contribute to this eventuality are lack

of standardization of the herbal drugs, identification of active ingredient(s)/

principles(s), randomized controlled clinical trials (RCTs) and toxicological

evaluation.
A large number of plants and formulations have been claimed to have

hepatoprotective activity. Nearly 160 phytoconstituents from 101 plants have

been claimed to possess liver protecting activity. In India, more than 87 plants

are used in 33 patented and proprietary multi ingredient plant formulations.

About 600 commercial herbal formulations with claimed hepatoprotective activity

are being sold all over the world. However, only a small proportion of

hepatoprotective plants as well as formulations used in traditional medicine are

pharmacologically evaluated for their safety and efficacy

Numerous plants and polyherbal formulations are used for the treatment of

liver diseases. However, in most of the severe cases, the treatments are not

satisfactory. Although experimental evaluations were carried out on a good

number of these plants and formulations, the studies were mostly incomplete

and insufficient. The therapeutic values were tested against a few chemicals-

induced subclinical levels of liver damages in rodents. Even common dietary

antioxidants can provide such protection from liver damage caused by oxidative

mechanisms of toxic chemicals. However, experiments have clearly shown that

plants such as Picrorrhiza kurroa, Andrographis paniculata, Eclipta alba, Silibum

marianum, Phyllanthus maderaspatensis and Trichopus zeylanicus are

sufficiently active against, at least, certain hepatotoxins.

Single plant may not have all the desired activities. A combination of different

herbal extracts/fractions is likely to provide desired activities to cure severe

liver diseases. Development of such medicines with standards of safety and

efficacy can revitalise treatment of liver disorders and hepatoprotective activity.

The traditional medicinal plants species have been subjected in various

experimental models of investigation and attempt has made to calibrate their

therapeutical activity. The herbs used in hepatic disease have been extensively

exploited all over the world and large numbers of the plant species has been

documented as hepatoprotective antihepatotoxic, cholegoge and choleric. A

diverse nature of chemical compounds has been identified from such plants

species through bio-assay guided investigation. However, in some plant species

the total extract or fraction of extract has reported to possess better and

potent biological activity compared to isolated pure compound(s) from the


extracts. Plant derived extracts and chemically defined molecules are

enumerated in Table 1 and 2.

Table 1: Plant Extracts with Hepatoprotective Activity

Family and Origin Plant Extracts Type of Hepato- References

Botanical name Parts Studied assay toxicity

used Inducing
Agents

Acanthaceae India Leaves Alcohol CCl4 Babu et al.

In vivo

Acanthus ilicifolius L. (2001)

Andrographis lineata India Leaves Aqueous, In vivo CCl4 Sangameswaran

Nees methanol et al. (2008)

India Leaves Alcohol CCl4 Rana and

Andrographis In vivo

paniculata (Burm.f.) Avadhoot (1991)

Nees

Yemen Whole Ethanol CCl4 Fleurentin et al.

Anisotes trisulcus In vivo

(Forssk.) plant (1986)

Asteracantha Sri Whole Aqueous In vivo CCl4 and Hewawasam

longifolia L. Lanka plant PCM et al. (2003)

Hygrophila auriculata India Seeds Methanol In vivo PCM and Singh and Handa

(K.Schum.) Heine Thioacetamide (1995)

Hypoestes triflora Rwanda Leaves Aqueous In vivo CCl4 Van Puyvelde

(Forssk.) Roem. and et al. (1989)

Schult

Rhinacanthus nasuta India Root Methanol In vivo CCl4 Suja et al. (2003)

(L.) Kurz.

Adoxaceae Viburnum Southern Leaves Aqueous- In vivo CCl4 Mohammed et al.

tinus L. Europe methanol (2005)

Aizoaceae Trianthema India Leaves Ethanol In vivo PCM and Kumar et al.

portulacastrum L. Thioacetamide (2004)

Apiaceae Apium India Seeds Methanol In vivo PCM and Singh and Handa

graveolens L. Thioacetamide (1995)

Carum copticum L. Pakistan Seeds Aqueous- In vivo CCl4 and PCM Gilani et al.

methanol (2005a)

Apocynaceae China, Leaf Aqueous In vivo CCl4 and GAIN Xiong et al.

Apocynum venetum L. Japan (2000)

Araliaceae Taiwan Whole Methanol CCl4 and Lin and Huang

In vivo

Acanthopanax plant Acetami- (2002)

senticosus (Rupr. and nophen

Maxim.) Harms

Asclepiadaceae India Stem Ethyl In vivo CCl4 Sethuraman et al.

bark acetate (2003)

Sarcostemma

brevistigma Wight

Asteraceae Argentina Aerial Aqueous In vivo Bromobenzene Kadarian et al.

parts (2002)

Achyrocline

satureioides (Lam.)

DC.




used Inducing

Agents

Pakistan Aerial Aqueous- CCl4 and Gilani and

Artemisia absinthium In vivo

L. parts methanol Aceta- Janbaz (1995a)

minophen

Artemisia maritima L Pakistan Aerial Aqueous- In vivo CCl4 and Janbaz and Gilani
parts methanol Aceta- (1995)

minophen

Artemisia vulgaris L. Pakistan Aerial Aqueous- In vivo GAIN and LPS Gilani et al.

parts methanol (2005b)

Bidens chilensis DC Taiwan Whole Methanol CCl4 and PCM Chih et al.

In vivo

plant (1996)

Bidens pilosa L. Taiwan Whole Methanol In vivo CCl4 and PCM Chih et al. (1996)

plant

Cichorium intybus L. India Seeds Alcohol In vivo CCl4 Ahmed et al.

(2003)

Crassocephalum Japan Whole Aqueous In vivo GAIN, LPS Aniya et al.

crepidioides Benth plant and in and CCl4 (2005)

vitro

Elephantopus mollis Taiwan Whole Aqueous In vivo Acetaminophen Lin et al.

Kunth. plant and GAIN (1995b)

Elephantopus Taiwan Whole Aqueous In vivo Acetaminophen Lin et al. (1995b)

scaber L. plant and GAIN

India Leaf Methanol CCl4 Umadevi et al.

Flaveria trinervia In vivo

(Spreng.) C.Mohr (2004)

Gundelia Iran Stalk Hydro- In vivo CCl4 Jamshidzadeh

tourenfortii L. alcoholic and in et al. (2005)

vitro

Pseudoelephantopus Taiwan Whole Aqueous In vivo Acetaminophen Lin et al. (1995b)

spicatus (Juss. Ex plant and GAIN

Aublet) Gleason

Taiwan Whole Methanol CCl4, Aceta- Lin et al. (1994)

Wedelia chinensis In vivo

(Osbeck) Merr. plant minophen and

GAIN

India Leaf Ethanol CCl4 Murugaian et al.

Wedelia In vivo

calendulacea L. (2008)

Balanophoraceae Ghana Roots, Aqueous In vivo GAIN and Gyamfi et al.

Thonningia leaves and in CCl4 (1999)

sanguinea Vahl. vitro

Bixaceae Mali Rhizome Ethanol In vivo CCl4 Diallo et al.

Cochlospermum and hydro- (1992)

tinctorium Perri ethanol

ex Rich. extract

Bixa orellana L. Bangla- Seed Methanol In vivo CCl4 Ahsan et al.

desh (2009)

Brassicaceae India Whole plant Aqueous In vivo CCl4 Mantena

Coronopus didymus L. et al. (2005)




used Inducing

Agents

Burseraceae Saudi Aerial Ethanol CCl4 Al-Howiriny et al.

In vivo

Commiphora Arabia parts (2004)

opobalsamum (L.)

Engl.
Caesalpiniaceae India Bark Methanol In vivo CCl4 and PCM Gupta et al.

Bauhinia racemosa (2004)

Lam

Capparidaceae India Leaves Ethanol In vivo CCl4 Gupta et al.

Cleome viscosa L (2009)

Casuarinaceae Bangla- Leaves, Methanol In vivo CCl4 Ahsan et al.

Casuarina desh bark (2009)

equisetifolia Forst

Celasteraceae Salacia Sri Lanka, Root, Aqueous, CCl4 Yoshikawa et al.

In vivo

reticulata Wight India stem methanol (2002)

Chenopodiaceae Beta India Root Ethanol In vivo CCl4 Agarwal et al.

vulgaris L. (2006)

Combretaceae Japan Leaves Methanol In vivo GAIN Banskota et al.

Combretum Kurz. and in (2003)

vitro

Terminalia arjuna L. India Bark Aqueous In vivo CCl4 Manna et al.

(2006)

Terminalia belerica India Fruits Ethanol In vivo CCl4 Jadon et al.

Roxb (2007)

Terminalia catappa L. Okinawa Leaves Aqueous In vivo GAIN and LPS Kinoshita et al.

Island and in (2007)

vitro

India Fruits Ethanol Anti TB drugs Tasduq et al.

Terminalia chebula In vivo

Reiz. and in (2006)

vitro

Compositae Ambrosia Egypt Whole Aqueous- In vivo Acetaminophen Ahmed and

maritima L. plant methanol Kharter (2001)

Crepis rueppellii Yemen Whole Ethanol In vivo CCl4 Fleurentin et al.

(Sch.) Bip. plant (1986)

Eclipta alba Hassk. India Whole Alcohol In vivo CCl4 Singh et al.

plant (1993)

India Whole Aqueous CCl4 Hewawasam et al.

Epaltes divaricata In vivo

(L.) Cav. plant (2004)

Convolvulaceae Korea Seeds Aqueous In vivo DMN Kim et al. (2007a)

Cuscutae semen Lam.

Erycibe expansa Thailand Stem Methanol In vitro GAIN Matsuda et al.

Wall. and G.Don (2004)

Crassulaceae India Leaves Juice of In vivo CCl4 Yadav and Dixit

Kalanchoe pinnata leaves, and (2003)

Pers. ethanol

in vitro

extract of

marc




used Inducing

Agents

Cucurbitaceae Luffa India Fruits Pet.ether, CCl4 Ahmed et al.

In vivo

echinata Roxb. acetone, (2002)

methanol

Cyperaceae Cyperos Indonesia, Tubers Aqueous- In vivo CCl4 Gilani and

scariosus R.Br. Pakistan methanol Jambaz (1995b)

Ebenaceae Diospyros India Bark Methanol In vivo CCl4 Mondal et al.

malabarica (Desr.) (2005)

Kostel

Euphorbiaceae Nigeria Leaves Ethanol In vivo Acetaminophen Olaleye et al.

Alchornea cordifolia (2006)

Schum and Thonn.

Croton oblongifolius India Aerial Pet.ether, In vivo CCl4 Ahmed et al.

Roxb. parts acetone, (2002)

methanol

Emblica officinalis India Fruits Hydro- In vitro Anti TB drugs Tasduq et al.

Gaertner alcoholic (2005)

India Whole n-hexane CCl4 and Asha et al. (2007)

Phyllanthus In vivo

maderaspatensis L. plant Thioacetamide

Phyllanthus niruri L. Brazil Leaves Aqueous In vivo PCM Sabir and Rocha

(2008)

Phyllanthus India Aerial parts Ethanol In vivo CCl4 Das et al.

reticulatus Poir. (2008)

Fabaceae Acacia India Bark Ethyl acetate In vivo CCl4 Ray et al.

catechu (L.f.) Willd. (2006)

Bauhinia variegata L. India Stem Alcohol In vivo CCl4 Bodakhe and

bark Ram (2007)

Cajanus cajan L. India Leaves Methanol In vivo Alcohol Kundu et al.

(2008)

Cassia fistula L. India Leaves n-heptane In vivo CCl4 Bhakta et al.

(1999)

Cassia occidentalis L. India Leaves Aqueous- PCM and Jafri et al. (1999)

In vivo

ethanol ethyl alcohol

Glycine max (L.) Merr Taiwan Seed Water In vivo Acetaminophen Wu et al. (2001)

Taiwan Seed Water Acetaminophen Wu et al. (2001)

Phaseolus aureus In vivo

Roxb.

Phaseolus calcaratus Taiwan Seed Water In vivo Acetaminophen Wu et al. (2001)

Roxb

Phaseolus radiatus L. Taiwan Seed Water In vivo Acetaminophen Wu et al. (2001)

Pterocarpus India Stem Methanol In vivo CCl4 Mankani et al.

marsupium Roxb. bark (2005)

India Leaves Ethanol H2O2 CCl4 Meera (2009)

Trigonella foenum-

graecum L.




used Inducing

Agents

Fumariaceae Fumaria India Whole Methanol, In vivo PCM, Rao and Mishra

indica (Hausskn.) plant Pet.Ether, Rifampicin, (1997)

Pugsley aqueous CCl4

Fumaria parviflora Pakistan Shoots Aqueous- In vivo PCM Gilani et al.

Lam. methanol (1996)

Gentianaceae India Whole Alcohol In vivo CCl4 Senthilkumar

Enicostemma littorale plant et al. (2005)

Blume.

Swertia japonica Japan Whole Butanol In vivo GAIN Hase et al.

(Roem. and Schult.) plant (1997b)

Makino.

Lamiaceae Ocimum India Leaves Ethanol H2O2 CCl4 Meera et al.

basilicum L. (2009)

Moraceae Ficus India Leaves Methanol CCl4 Krishna et al.

In vivo

carica L. (2007)

Ficus hispida L India Leaves Methanol In vivo PCM Mandal et al.

(2000)

Moringaceae Moringa Malaysia Leaves Hydro- Acetaminophen Fakurazi et al.

In vivo

oleifera L. alcoholic (2008)

Myrtaceae Careya India Stem Methanol In vivo CCl4 Sambath et al.

arborea Roxb. bark (2005)

Nyctaginaceae India Roots Aqueous In vivo Thioacetamide Rawat et al.

Boerhaavia diffusa L. (1997)

Nymphaceae India Flowers Alcohol In vivo CCl4 Bhandarkar and

Khan (2004)

Nymphaea stellata

Willd.

Oleaceae Phillyrea Jordan Leaves Aqueous In vivo CCl4 Janakat and

latifolia L. Al-Merie (2002)

Ophioglossaceae India Rhizomes Methanol In vivo CCl4 Suja et al. (2004)

Helminthostachys

zeylanica (L.) Hook

Orchidaceae Taiwan Whole Aqueous In vivo CCl4 Wu et al. (2007)

Anoectochilus plant and

formosanus Hayata

in vitro

Polygalaceae India Leaves Chloroform In vivo GAIN Dhanabal et al.

Polygala arvensis (2006)

Willd.

Rhamnaceae Taiwan Bark Methanol, In vivo CCl4 Lin et al. (1995a)

Ventilago leiocarpa ethanol,

Benth. butanol

and

aqueous

Ziziphus mauritiana Nigeria Leaves Ethanol In vivo CCl4 Dahiru et al.

Lam. (2005)




used Inducing

Agents

Rubiaceae Hedyotis India Whole Methanol PCM Sadasivan et al.

In vivo

corymbosa (L.)Lam. plant (2006)

Mitracarpus scaber Mali Whole Methanol In vivo CCl4 Germano et al.

Zucc. plant and (1999)
in vitro

Morinda citrifolia L. America Whole Methanol In vivo CCl4 Wang et al.

plant (2008)

Rutaceae Aegle India Leaves Fine In vivo Alcohol Singanan et al.

marmelos (L.) Corr. powder (2007)

Serr. inphysio-

logical

saline

Glycosmis Bangla- Leaves, Methanol In vivo CCl4 Ahsan et al.

pentaphylla Corr. desh bark (2009)

Scrophulariaceae India Whole Alcohol In vivo Morphine Sumathy et al.

Bacopa monniera (L.) plant (2001)

Pennell

Picrorrhiza kurroa Himalayas Rhizome, Ethanol In vivo GAIN Anandan and

(Roule.) Sans roots Devaki (1999)

Smilacaceae Smilax Saudi Roots Ethanol In vivo CCl4 Rafatullah et al.

regelii Killip and Arabia (1991)

Morton

Solanaceae Jordan Leaves, Aqueous CCl4 Janakat and

In vivo

Nicotiana glauca flowers Al-Merie (2002)

Graham.

Solanum nigrum L India Fruits Ethanol CCl4 Raju et al. (2003)

In vivo

Jerusalem Leaves Methanol CCl4 Vijayan et al.

Solanum In vivo

pseudocapsicum and in (2003)

Hassl. vitro

Solanum trilobatum L. India Whole Methanol In vivo CCl4 Shahjahan et al.

plant (2004)

Umbelliferae Taiwan Leaves Aqueous In vitro Acetamino- Liu et al. (2006)

Bupleurum kaoi Liu phen and CCl4

(Chao et Chuang)

Daucus carota L. Europe, Roots Aqueous In vivo CCl4 Bishayee et al.

Asia, (1995)

Africa

Foeniculum vulgare Turkey Seeds Essential In vivo CCl4 Ozbek et al.

Miller oil (2003)

Valerianaceae India Rhizomes Ethanol In vivo CCl4 Ali et al. (2000)

Nardostachys

jatamansi D.C.

Vitaceae Rhoicissus South Roots Aqueous In vivo CCl4 Opoku et al.

tridentata(L.f.) Wild Africa (2007)

and R.B. Drumm


Table 2: Chemically defined Molecules with Hepatoprotective activity

Chemical substance Plant Plant part Class References

3,4-di-O- Lactuca indica L. Aerial parts Quinic acid Kim et al.

caffeoylquinic acid (2007b)

3,5-di-O-caffeoyl- Lactuca indica L. Aerial parts Quinic acid Kim et al.

muco-quinic acid (2007b)

5-O-(E)-p- Lactuca indica L. Aerial parts Quinic acid Kim et al.

coumaroylquinic acid (2007b)

-Amyrin Protium heptaphyllum Trunk wood Triterpene Oliveira et al.

(Aubl.) March resin (2005)

-Amyrin Trunk wood Triterpene Oliveira et al.

Protium heptaphyllum

(Aubl.) March resin (2005)

Anastatin A Anastatica Whole plant Flavonoid Yoshikawa et al.

hierochuntica L. (2003)

Anastatin B Anastatica Whole plant Flavonoid Yoshikawa et al.

hierochuntica L. (2003)

18 -glycyrrhetinic Glycyrrhiza uralensis Rhizomes Glycyrrhetinic Shim et al.

acid Fisch. acid (2000)

Tetrahydroswer- Swertia japonica Makino Aerial parts Xanthione Hase et al.

tianolin (1997b)

Gentiopicroside Swertia japonica Makino Aerial parts Iridoid Hase et al.

(1997b)

Sweroside Swertia japonica Makino Aerial parts Iridoid Hase et al.

(1997b)

Andrographolide Andrographis paniculata Aerial parts Diterpene Chander et al.

(Burm.f) Nees (1995)

Erycibenin A Erycibe expansa Stem Pterocarpane Matsuda et al.

(2004)

5,7,4-trihydroxy-3- Erycibe expansa Stem Isoflavone Matsuda et al.

Methoxyisoflavone (2004)

Genistein Erycibe expansa Wall. Stem Isoflavone Matsuda et al.

Ex G.Don. (2004)

Orobol Erycibe expansa Wall. Stem Isoflavone Matsuda et al.

Ex G. Don. (2004)

Mangiferin Salacia reticulata Abst. Roots Phenolic Yoshikawa et al.

compound (2002)

(-)-4-O-methy- Salacia reticulata Abst. Roots Phenolic Yoshikawa et al.

lepigallocatechin compound (2002)

Thymoquinone Nigella sativa L. Aerial parts Quinone Daba and Abdel-

Rahman (1998)

Lithospermate B Salvia miltorhiza Bunge Roots Caffeic acid Hase et al.

(1997a)



Taxiresinol Enciostemma littorale Aerial parts Tetrahydro- Nguyen et al.

furan (2004)

(7R)-7- Enciostemma littorale Aerial parts Tetrahydro- Nguyen et al.

hydroxylariciresinol furan (2004)

Onitin Equisetum arvense L. Aerial parts Phenolic Oh et al. (2004)

compound
Luteolin Equisetum arvense L. Aerial parts Flavonoid Oh et al. (2004)

Quercetin-3-O--D- Aerial parts Flavonol Sung et al.

Saururos chinensis

glucuronopyranoside (Lour.) Baill. glycoside (1997)

Quercetin-3-O--D- Saururus chinensis Aerial parts Flavonol Sung et al.

glucuronopyranosyl (Lour.) Baill. glycoside (1997)

methyl ester

Scropolioside-A Scrophularia koelzii Aerial parts Iridoid Garg et al.

Pennell glycoside (1994)

3-(S)-3-_- Whole plant Aliphatic Du et al. (2000)

Goodyera

Dglucopyrano- schlechtendaliana glycoside

syloxybutanolide Reichb.G. matsumurana

Schltr.G. discolor

Kergawl

3-(S)-3-_-D- Goodyera schlechten- Whole plant Aliphatic Du et al. (2000)

glucopyranosyloxy- daliana Reichb.G. glycoside

4-hydroxybutanoic matsumurana Schltr.G.

acid discolor Kergawl.

Agathisflavone Canarium manii King Aerial parts Biflavonoid Anand et al.

(1992)

(S)-bakuchiol Psoralea corylifolia Aerial parts Monoter- Hyun et al.

Babchi penephenol (2001)

Monomethyl fumarate Fumaria indica Pugsley Whole plant Fumaric acid Rao and Mishra

(1998)

Wighteone Cudrania Roots Flavonoid Lin et al. (1996)

cochinchinensis (Lour.)

Kudo et Masam.

Naringenin Cudrania Roots Flavonoid Lin et al. (1996)

cochinchinensis (Lour.)

Kudo et Masam.

Torilin Cnidium monnieri (L.) Aerial parts Sesquiterpene Oh et al. (2002)

Cusson

Torilolone Cnidium monnieri (L.) Aerial parts Sesquiterpene Oh et al. (2002)

Cusson.

Allicin Allium sativum L. Cloves Allyl Vimal and Devaki

thiosulfinates (2004)

Kaempferol Rhodiola sachalinensis Roots Phenolic Song et al.

A.Bor. compound (2003)



Salidroside Rhodiola sachalinensis Roots Phenolic Song et al. (2003)

A.Bor. compound

1-O-galloyl-6-O- Combretum Seeds Gallic acid Adnyana et al.

(4-hydroxy-3,5- quadrangulare Kurz (2001)

dimethoxy)benzoyl-

-d-glucose

Picroliv Picrorhiza kurroa Aerial parts Iridoid Visen et al.

Royle ex Benth. glycoside (1991)

Indigtone Indigofera tinctoria L. Aerial parts Aliphatic Singh et al.

nitrocompound (2001)

Acanthoic acid Acanthopanax Root bark Diterpene Park et al. (2004)

koreanum Nakai

Myristin Aerial parts Cetyl ester Morita et al.

Myristica fragrans

Houtt. (2003)

Rutin Artemisia scoparia Aerial parts Flavonoid Janbaz et al.

Waldst. and Kit. (2002)

Troxerutin Artemisia scoparia Aerial parts Flavonoid Zhang et al.

Waldst. and Kit. (2009)

Neoandrographolide Andrographis paniculata Aerial parts Diterpene Chander et al.

(Burm.f.) Wall. Ex Nees (1995)

5-0-methyl-(E)- Acer mono Maxim. Leaves Stilbene Yang et al.

resveratrol.3-0--D glycoside (2005)

glucopyranoside

5-0-methyl-(E)- Acer mono Maxim. Leaves Stilbene Yang et al.

resveratrol.3-0- glycoside (2005)

_-Dapiofuranosyl-

1->6)--D

glucopyranoside

Corilagin Terminalia catappa L. Leaves Tannin Kinoshita et al.

(2007)

y-Amyrone Sedum sarmentosum Aerial parts Triterpene Amin et al.

Bunge (1998)

3-epi-y-amyrin Aerial parts Triterpene Amin et al.

Sedum sarmentosum

Bunge (1998)

y-Amyrin Sedum sarmentosum Aerial parts Triterpene Amin et al.

Bunge (1998)

18-hydroperoxy- Sedum sarmentosum Aerial parts Triterpene Amin et al.

olean.12-en-3-one Bunge (1998)

Rubiadin Rubia cordifolia L. Roots Anthraquinone Rao et al. (2006)

3,4,5-trihydroxy- Terminalia belerica Fruit Gallic acid Jadon et al.

benzoic acid Roxb. (2007)

Kinsenoside Anoectochilus Whole plant Furanone Wu et al. (2007)

formosanus Hay.


Discussion and Conclusion

The liver is the most important organ in the body. It has a pivotal role in

regulation of physiological processes. It is involved in several vital functions

such as metabolism, secretion and storage. Liver diseases are among the

most serious ailments. They may be classified as acute or chronic hepatitis

(inflammatory liver diseases), hepatosis (non inflammatory diseases) and
cirrhosis (degenerative disorder resulting in fibrosis of the liver).

Modern society has innate knowledge about the herbal treatment of liver disease

from many cultures. Research into plants traditionally used in the treatment of

liver disease has significantly advanced in the past 15 years, and much of

what has been discovered supports traditional knowledge.

Considering the enormous biodiversity resources over the worlds traditional

system and the high incidence of liver complications, the present review

extensively focuses on collection of data for different plants,

which are available in India and all over the world. These medicinal plants claimed

as liver protective agents are classified according to their biological source,

phytoconstituents; part used and plants in formulations. People from India are

still dependent on conventional therapies to treat liver complications. Because

of their easy availability and low cost. Since large mass of populations used

preferable herbal preparation, therefore there is need to be evaluate for their

proportion, their dose and rational behind combination in different polyhedral

preparation.

These herbal drugs have shown the ability to maintain the normal functional

statues of the liver with or without fewer side effects. These are the reason

thats why herbal hepatoprotectives are mostly preferred by medical

practitioners.

It has been seen that herbal hepatoprotective drugs have less side effect or

interaction as compared to synthetic medicine but in other hand scientific

evidence from tests done to evaluate the safety and effectiveness of traditional

hepatoprotective medicine products and practices is limited and further study

of products and practices is needed.

Pharmacokinetic and toxicity studies have not disclosed any issues that could

limit the therapeutic use of these drugs. Also the study is required to identify

glycosides, flavonoids, triterpenes and phenolic compounds as classes of

compounds with hepatoprotective activity.

Further studies including clinical trials need to be carried out to ascertain the

safety of these compounds as a good alternative to conventional drugs in the

treatment of liver diseases


Since the traditional system of medicine recommends various hetptoprotective

agents and preparations to treat hepatic disorders. The management of lives

disease is still challenges to modern medicine. The modern allopathic drugs

have very little to offer for alleviation of hepatic ailments and some these drugs

adversely affect the liver function. A phytotherapeutic approach to modern

drug development provides many invaluable drugs from traditional medicinal

plants. Search for pure phytochemical as drug is time consuming and extensive.
Numerous plants and polyherbal formulations are being used for the treatment

of liver diseases.

Today, unfortunately the herbal resources have declined rapidly because more

than 80% of our total medicinal plants used by Indian pharmaceutical industry

are collected from their wild sources and they are not being grown or

domesticated so far. To meet their burgeoning demand is the need of the day.

Moreover, our natural resource base of medicinal plants is being depleting day

by day. Hence there is an urgent need to encourage field scale cultivation of

prioritized medicinal plants through government initiatives before its too late.

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Prevalence of Abstract

Anti- his study was carried out to detect the elevated serum Anti-

streptolysin
O antibodies at
Bhadrak
T

streptolysin O (ASO) in patients which were coming in OPD of Regional Research
Institute of Unani Medicine with various clinical conditions during the period of
January 2011 to December 2012. The serum samples were tested for ASO
antibodies by latex agglutination test. Total 205 patients including 30.24 %
male and 69.7 % female were tested for ASO serum levels. 60 were found
Region, India

positive and 145 were negative. In 60 positive cases 16 were male and 44

were female. The prevalence of ASO antibody in total cases was 29.26 %. The

*Kishore Kumar,

prevalence in male was 25.8 % and in female was 30.76 %. The presence of

Subhan A. Khan,

elevated streptococcal antibody titers in such a population probably reflects a

Mahe Alam,

medium background prevalence of streptococcal infections.

Hakimuddin Khan,

Chander Pal,

Qamar Uddin, Keywords: Anti-streptolysin O, Agglutination, Streptococcal infections, Serum

Mukesh Kumar

and

Introduction

L. Samiulla

Streptococcus pyogenes (Group A Streptococcus/GAS) is one of the most

Regional Research Institute of

common and ubiquitous human pathogens. It causes a wide array of infections,

Unani Medicine (CCRUM),

Mathasahi, Bhadrak-756100, the most frequent of which are acute pharyngitis (strep throat) and impetigo

Odisha
(pyoderma). It is also associated with two main non-suppurative sequelae:

acute rheumatic fever (ARF) and acute glomerulonephritis (AGN) (Bisno, 1991).

Rheumatic fever causes inflammation of tissues and organs and can result in

serious damage to the heart valves, joints, central nervous system and skin.

Susceptibility to rheumatic fever in certain individuals has been ascribed to a

number of factors. These include genetic determinants, for example, HLAs and

the presence of certain markers such as the B-cell alloantigens. One of the

factors originally considered in rheumatic fever susceptibility is an innate state

of immune hyperresponsiveness, particularly to streptococcal antigens (Meiselas

et al., 1961; Stollerman, 1972; Rejholec, 1957).

The detection of streptococcal infection is done by positive culture for group

A Streptococcus from the throat or through anti-streptococcal antibody tests

(Homer and Shulman, 1991). The throat cultures are spontaneously negative

due to previous antibiotic treatment, or positive due to a carriage state. Hence

streptococcal antibody tests have become a method commonly used to provide

evidence of preceding streptococcal infection (Fink, 1991).

During infection, the host may produce antibodies to one or more extracellular

products of group A streptococci, and these antibodies are useful markers of

recent streptococcal infection. Tests that measure antibodies to the extra-



cellular products are commercially available and more commonly used. The

anti-streptolysin O (ASO) assay was the first such test to be developed and

is still widely used. ASO is not only useful in the diagnosis of streptococcal

infections or complications, but also in the follow-up process and in evaluating

the effectiveness of treatments. It measures the ability of serum to neutralize

streptolysin O.

Material and Methods

Serum Collection

Serum samples were collected from 205 patients between the periods of January

2011 to December 2012 at Regional Research Institute of Unani Medicine in

Bhadrak, India. Blood samples from patients were obtained using a standard

2-mL syringe. Samples were allowed to coagulate for 30 minutes at room

temperature in a serum-collecting tube. The sample was centrifuged at 3000

rpm for 5 minutes. The serum was then separated by using micropipette.

Procedure

All the serum samples were tested by ASO kit (Span Diagnostics P. Ltd. India).

The instructions, reagents and accessories to follow were supplied with the kit.

Test serum and reagents were kept at room temperature before testing. 40l

patients serum within the circled area was placed on the clean and dry special

glass slide provided in the kit. One drop of well mixed ASO latex reagents was

added to serum. The reagent and serum using the applicator stick were mixed.

The slide was rotated and observed for agglutination macroscopically within

two minutes.

Results

Total 205 patients were included in this study. From them, 30.24 % male and

69.7 % female tested for ASO serum levels (Figure 1), 60 were positive and

145 were negative.

In 60 positive cases 16 were male and 44 were female. In 145 negative cases

46 were male and 99 were female (Figure 2). Highest positive case (27 patients)

was found in the age group of 21- 40 while 23 patients were positive in age

group of 41-60 (Figure 3). The prevalence of total case was 29.26 %. The

prevalence of total female was 30.76 % and the prevalence of total male was

25.8 % (Table 1).

The number of positive cases in different age group is given in table 2. The

highest prevalence of male among positive cases found in the age group 41-


Figure 1: Blood sample collection in General OPD attendance at RRIUM, Bhadrak

Figure 2: Sex wise distribution of ASO Positive and ASO Negative cases at RRIUM,

Bhadrak

Table 1: Sex wise prevalence of all streptococcal infection among General OPD at

RRIUM, Bhadrak

Sex ASO Positive ASO Negative Total (%) Prevalence Rate

(%)

Male 16 46 62 (30.24) 25.84

Female 44 99 143 (69.75) 30.76

Total 60 145 205 (100) 29.6

60 (6/16, 37.5%) and the highest prevalence of female among positive cases

found in the age group 21 40 (22/44, 50 %) (Figure 4, Figure 5). The

prevalence rate of male and female in age group 21-40 was 31.25 % and 50

% respectively. This indicates that the prevalence rate is higher in females

then male in this age group.


Figure 3: Age wise distribution of total Anti- streptolysin O (ASO) positive cases

Figure 4: The comparative chart of ASO positive cases between Male and Female

in different age group

The prevalence rate of male and female in age group 41-60 was 37.5 % and

38.6 % respectively which was almost similar. The overall data clearly indicates

that the prevalence of ASO positive case is slightly higher in females than

males.

Discussion

The serological test for ASO is commonly used to aid in the diagnosis of post-

streptococcal non-suppurative sequelae such as ARF and glomerulonephritis

(Kimoto et al., 2005; Batsford et al., 2002).


Figure 5: The prevalence rate of ASO among positive cases in different age group

Table 2: The prevalence rate of ASO among positive cases in different age group

Age group Total No. of No. of Positive No. of Positive

Positive cases Male (%) Female (%)

0-20 6 4 (25) 2 (4.5)

21-40 27 5 (31.25) 22 (50)

41-60 23 6 (37.5) 17 (38.6)

61-80 4 1 (6.25) 3 (6.8)

Total 60 16 (100) 44 (100)

Conventional laboratory practice is to measure levels of antibodies to various

combinations of the extra-cellular Group A Streptococcus antigens. However,

Blyth and Robertson (2006) showed that the addition of anti-streptokinase

antibodies measurement did not increase the sensitivity and specificity of

serological testing for the diagnosis of acute post-streptococcal disease (Blyth

and Robertson, 2006). ASO test method is based on an immunologic reaction

between streptococcal exotoxins bound to biologically inert latex particles and

streptococcal antibodies in the test sample. Visible agglutination occurs when

increased antibody level, are present in the test specimen. A positive ASO titer

indicates nonspecific immune stimulation due to past streptococcal exposure

resulting in polyclonal gammopathy (Sainani and Sainani, 2006).

Fujikawa and Okuni (1979) observed that ASO elevation occurs only in 60%

of rheumatic fever. But after one more test is added either


antideoxyribonuclease-B titre or streptokinase test, they were able to diagnose

rheumatic fever with 95% accuracy (Fujikawa and Okuni, 1979). In this study,

60 samples were positive out of 205 samples. Our study showed 29.26 %

prevalence of ASO positive cases whereas the same study which was conducted

in Nepal showed 45.45 % prevalence of ASO positive cases (Khan et al.,

2012). Similar study was also performed where 20.89 % prevalence was

observed (Kandel et al., 2007). Further in an another study conducted at CMS

Teaching Hospital, Bharatpur, Nepal, 4230 serum samples of rheumatic fever,

glomerulonephritis, rheumatic heart disease and rheumatic arthritis were tested

for ASO antibodies by latex agglutination test during the period of January

2003 to December 2009. Among them 1944 samples were positive and 2286

samples were found negative with overall prevalence of 45.95 %. (Dewasy et

al., 2010)

This clearly indicates that the ASO levels vary with age group of the study

population and geographical distribution. This study, together with data derived

from the present study indicates that the levels of the streptococcal antibodies

in healthy populations can vary substantially, depending on the frequency of

streptococcal infections in those populations. The presence of elevated

streptococcal antibody titers in such a population probably reflects a medium

background prevalence of streptococcal infections.

Acknowledgement

The authors are thankful to the Director General, Central Council for Research

in Unani Medicine, New Delhi for his cooperation and taking keen interest in

this work. We also thank to all staff of Biochemistry laboratory of RRIUM,

Bhadrak, for helping in biochemical investigations.

References

Batsford, S., Brundiers, M., Horbach, E. and Monting, J., 2002. Streptococcal

Cysteine Proteinase as a Seromarker of group A Streptococcal

(Streptococcus pyogenes) Infections. Scand J. Infect. Dis. 34: 40712

Bisno, A. L., 1991. Group A Streptococcal Infections and Acute Rheumatic

Fever. N. Engl. J. Med. 325: 78393

Blyth, C. C. and Robertson, P.W., 2006. Streptococcal anti-bodies in the

Diagnosis of Acute and post-streptococcal disease: streptokinase versus

streptolysin O and deoxyribonuclease B. Pathology. 38: 1526.

Dewasy, B.L., Singh, Y .I., Jha, B.K. and Kapil, J., 2010. A rapid screening test

by the latex antistreptolysin O test for streptococcal infections in CMS

Teaching Hospital, Bharatpur, Nepal. Journal of college of Medical Sciences-

Nepal 6: 24-28


Fink, C. W., 1991. The Role of the Streptococcus in Post-streptococcal Reactive

Childhood Polyarteritis Nodosa. J. Rheumatol. Suppl. 29: 1420.

Fujikawa, S. and Okuni, M., 1979. The determination of ADNase-B titres Jpn.

Circ. J. 43: 417418.

Homer, C. and Shulman, S. T., 1991. Clinical aspects of acute rheumatic fever

J. Rheumatol. Suppl. 29: 213.
Kandel, N. P., Koirala, B., Shrestha, S., Kumar, R. and Basnyat, S. R., 2007.

Laboratory Tests for Infective Endocarditis among Patients Visiting Shahid

Gangalal National Heart Centre, Bansbari, Kathmandu, Nepal. Journal of

Nepal Health Research Council 5: 1-4.

Khan, S., Singh, P. and Siddiqui, A. H., 2012. Prevalence of anti-streptolysin

O antibodies at Banke region Nepal. Bali Medical Journal 1(3): 98-100.

Kimoto, H., Fujii, Y., Hirano, S., Yokota, Y. and Taketo, A., 2005. Expression of

Recombinant Streptolysin O and Speci Production. J. Mol. Microbiol.

Biotechnol. 10: 6468.

Meiselas, L.E., Zinglae, S.B. and Lee, S.L. 1961. Antibody production in rheumatic

diseases, the effect of brucella antigen. J. Clin. Investig. 40: 1872-1881.

Rejholec, V., 1957. Incidence of rheumatic fever in relation to immunologic

reactivity. Ann Rheum. Dis. 16: 23-30.

Sainani, G. S. and Sainani, A. R., 2006. Rheumatic Fever - How Relevant in

India Today? Journal of the Association of Physicians of India 54: 42-47.

Stollerman, G.H., 1972. Hypersensitivity and antibody responses in streptococcal

disease. In: L.M. Wannamaker and J.M. Matesen (Eds.) Streptococci and

streptococcal diseases: recognition, understanding and management.

Academic Press, New York, pp. 501-513.



98
Review on Abstract

Pharmacopoeial he dried mature roots of Withania somnifera (L.) Dunal are

Standards of
Asgand
(Withania
T

specified source of drug named Asgand or Ashwagandha. The drug is highly
regarded for varied therapeutic values in Unani, Ayurvedic, Siddha and
Homoeopathic system of medicine. The drug has been compared to Panax
ginseng for its endurance - enhancing properties. The drug enjoys the official
status in Unani Pharmacopoeia of India, Ayurvedic Pharmacopoeia of India,
somnifera (L.)

Siddha Pharmacopoeia of India, Homoeopathic Pharmacopoeia of India and

Dunal) Indian Pharmacopoeia. Pharmacopoeial monographs are regulatory standards

and mandatory for compliance to ensure the quality, safety and efficacy of

1*Nitin Rai drug. This communication reviews the pharmacopoeial monographs and

and analytical parameters specified in pharmacopoeial and non pharmacopoeial

2Rajeev Kr. Sharma publications with a view on harmonization of quality specifications on W.

somnifera of drug in pharmacopoeias.

1Pharmacopoeial Laboratory

for Indian Medicine,

Kamla Nehru Nagar, Keywords: Withania somnifera (L.) Dunal, Pharmacopoeias, Pharmacopoeial

Ghaziabad-201002

harmonization.

2Pharmacopoeia Commission


Introduction

Raj Nagar, Ghaziabad-201002

W. somnifera (L.) Dunal. (Family-Solanaceae) is specified botanical source of

Asgand or Ashwagandha in various pharmacopoeias. The drug has been

used in Unani system of medicine since a long time. It is an official drug and

one of the ingredient of a number of important Unani classical and patent

formulations. The Ashwgandha is also used in ayurvedic, siddha, homoeopathic

and modern system of medicine. W. somnifera (L.) Dunal. is attributed medicinal

as aborfacient, alexipharmica, alternative, aphrodisiac, astringent, deobstruent,

diuretic, hypnotic and sedative, narcotic, pungent, restorative and tonic. It is

medicinally used in carbuncles, cough, debility of old age, dropsy, emaciation

of children, general weakness, promotes urination, functional obstruction of

body, rheumatism, scrofula, senile decay, ulcers and vulnerary treatment. The

drug finds mention in classical literature of unani, ayurvedic, siddha and

homoeopathic system of medicine. The tender shoots of the plant are also

used as a vegetable and seeds are used as masticatory. The green berries

are bruised and rubbed on ringworm in human beings and on animal sores

and girth-golls in horses. They are also employed to curdle milk. The drug is

substituted with W. coagulans Dunal. (Kirtikar and Basu, 1933; Chopra et al.,

1956; Nadkarni, 1954; Anonymous, 1976).

1


Systematics of source plant : W. somnifera (L.) Dunal belongs to family

Solanace. Solanaceae Juss. Lindl. Veg. Kingd. 618, Endl. Gen. 662. Miers,

Illustr. S. Amer. Pl. 1849-1857, Gen. Pl. II: 882.

This family comprises about 20 genera and 2,000 species found in tropical

and temperate regions of the world with the prime centers in Central and South

America. In India, this family is spread over 15 genera and 88 species, mostly

in the Himalayas and the mountains of Southern India.

Genus: Withania Pauquy. Diss. de Bellad. Paris, 1824, ex Endl. Gen. 666;

Gen. Pl. II: 893, FBI 4:239.The genus consists of ten species which are

distributed in South America, South Africa, Canaries and Mediterranean to

India. In India, 2 species are distributed in drier regions. The generic name of

the plant is supposed to be in honour of H. Withan, a British geologist and

writer on Paleontobotany, in the nineteenth century.

W. somnifera (L.) Dunal. In DC. Prodr. 13: 453. 1852; FBI 4:239; FUGP 2:128,

Physalis somnifera Linn. Sp. Pl. 182. 1753, P. flexuosa Linn. Sp. Pl. 182. 1753.

Synonyms: Physalis flexuosa Linn and P. somnifera Linn. An erect much

branched, evergreen, perennial, undershrub with glutinous cells sap. Roots

fairly long, stem very short, stellate hairy often with hoary mealy exteremites.

Leaves simple, short petioled, alternate, or in sub-opposite pairs at a node,

exstipulate, elliptic-oblong or ovate rounded with an acute decurrent base.

Flowers greenish or yellowish, small, sessile or short pedicellate, axillary fascicles

or in crowded fascicles of two to five. Calyx gamosepelous, five or six lobed,

campanulate, stellate-hairy, lobes ovate-triangular, acute, corolla small,

gamopetalous, campanulate, three to six lobed to more than half way down,

ovate, stellate, hairy outside. Stamens five, inserted on the tube of the corolla

near its base, anthers broadly elliptic oblong and dehiscing longitudinally, pistil

bicarpellary, many ovuled, syncarpous, ovary ovate-globose, style glabrous,

linear filiform and stigma bifid. Fruit berry, globose, enclosed within the enlarged

calyx, stellate hairy, seed very many and discoid. Flowering and Fruiting:

January to September.

Distribution : The drug plant is distributed throughout the drier subtropical

regions of India. In global distribution it is reported from Arabia, Mediterranean

regions, Pakistan to Persia, the Canaries and to tropical and South Africa

(Chopra et al., 1958; Sharma and Kachroo, 1983).

Observations

The Pharmacopoeial monographs on herbal drugs published in Unani, Ayurveda,

Siddha, Homeopathic and Indian Pharmacopoeias are dealt in detail for content

review (Rai et al., 2012, 2013 and Tiwari et al., 2013).It was observed that


drugs Asgand or Ashwagandha (botanically specified as W. somnifera (L.)

Dunal. ) is subjected for regulatory standards in Unani Pharmacopoeia of

India, Ayurvedic Pharmacopoeia of India, Siddha Pharmacopoeia of India,

Homeopathic Pharmacopoeia of India, Indian Pharmacopoeia, British

Pharmacopoeia and United State Pharmacopoeia (Table -1). Non regulatory

quality standard are also published on the drugs in Indian Herbal Pharmacopoeia

and Quality Standards of Indian Medicinal Plants. (Anonymous, 1940; 1955;

1966; 1971; 1976; 1986; 1996;1998; 2000a & b; 2002; 2007; 2008 a & b;

2010; 2011; 2013 a & b and 2014.) The monographs published in these

pharmacopoeial and non-pharmacopoeial publications are reviewed as per the

pharmacopoeial parameters to assess the variability in quality standards of

drug.Table-2 exhibit the format of pharmacopoeial monographs in various edition

of pharmacopoeias.Table-3 & 4 provide the account of pharmacopoeial

standards on drug published in pharmacopoeias. Non- regulatory quality

standard published in Indian Herbal Pharmacopoeia and Quality Standards of

Indian Medicinal Plants are enumerated in Table-5.

Conclusion

India is the only country which recognizes the five pharmacopoeias of different

systems of medicines under regulatory frame work (Anonymous, 1940). Indian

Table 1: Pharmacopoeial status of Withania somnifera (L.) Dunal.

Botanical Name Morphological Pharmacopoeial/ Regulatory/

(as specified in Part specified Monograph Title Pharmacopoeial

Pharmacopoeial as drug References

Monograph)

Withania somnifera Dried mature Ashwagandha IP 2007 IP 2010 &

(L.) Dunal root IP 2014

Ashwagandha IP 2010 & IP 2014

dry extract

Amukkara SPI-I

Withania radix, IP 55& IP 66

Aswagandha

Asvagandha API- I & VIII

Asgand UPI-I

Withania somnifera HPI-I & VIII

Withania Somnifera British Pharmaco-

Root poeia (BP) 2013

Asvagandha Root The United States

Pharmacopoeia

(USP 36/NF 31) 2013

Abbreviations: API-Ayurvedic Pharmacopoeia of India, UPI-Unani Pharmacopoeia of India, SPI-

Siddha Pharmacopoeia of India, HPI-Homeopathic Pharmacopoeia of India, IP-Indian

Pharmacopoeia, BP -British Pharmacopoeia, USP-United States Pharmacopoeia India.


Table 2: Monograph pattern in various edition of Indian Pharmacopoeia.

Sl. Quality Unani Ayurvedic Siddha Homoeopathy Indian

No. Specification Pharmaco- Pharmaco- Pharmaco- Pharmacopoeia Pharmacopeia

poeia of poeia of poeia of of India (IP 2014)

India, India, India, (HPI)

PartI Part-I Part-I Volume-I

(UPI) (API) (SPI) & VIII

Volume-I Volume-I Volume-I
& VIII

1. Pharmacopoeial Title

2. Definition-Botanical Botanical

Name (family), Name, Family,

Part used as Part used,

distribution Distribution

are under

independent

headings

3. Synonyms

4. Regional Language Common Names

Name

5. Description Description-

Macroscopic Macroscopic

Microscopic Microscopic

Powder Powder-

independent

headings

6. Identity, Purity &

Strength

Foreign Matter

Total Ash

Acid insoluble ash

Alcohol/ethanol

soluble extractive

Water soluble

Extractive

Loss on drying

Heavy metals

Microbial

contamination

Pesticide residues

Aflatoxins

7. Assay

8. Thin Layer In certain

Chromatography monographs

9. Constituents



No. Specification Pharmaco- Pharmaco- Pharmaco- Pharmacopoeia Pharmacopeia

poeia of poeia of poeia of of India (IP 2014)

India, India, India, (HPI)

PartI Part-I Part-I Volume-I

(UPI) (API) (SPI) & VIII

Volume-I Volume-I Volume-I

& VIII

10. Properties and Category

Action (as per

system of

medicine)

11. Important

Formulations

12. Therapeutic Uses

13. Dose

14. Identification Macroscopic

Microscopic

& TLC

15. History and authority

16. Preparation

17. Heavy metals

18. Loss on drying

19. Microbial

contamination

20. Storage

Table 3: Comparative account of pharmacopoeial standards on Withania somnifera (L.)

Dunal published various edition of Indian Pharmacopoeia.


No. Specification Pharmacopoeia Pharmacopoeia Pharmacopoeia Pharmacopoeia Pharmacopeia

of India (UPI), of India (API), of India (SPI), of India (HPI) (IP 2014)

Part-I, Part-I, Part-I, Volume-I & VIII

Volume-I Volume-I & VIII Volume-I

1. Official Title Asgand Asvagandha Amukkara Withania Ashwagandha

somnifera Indian Ginseng;

(With. Som.)

Withania

somnifera

2. Botanical Withania Withania Withania Withania Withania

Species somnifera Dunal. somnifera (L.) somnifera (L.) somnifera somnifera (L.)

Dunal. Dunal. Dunal. Dunal.

3. Synonyms Physalis Physalis

somnifera L., somnifera L.,

P. flexuosa L., P. flexuosa L.

P. arborescens

DC.







4. Morphological Dried mature Dried mature Dried roots Root Root

part/Official roots roots

part

5. Description I. Macroscopic I. Macroscopic I. Macroscopic I. Macroscopic I. Macroscopic

II. Microscopic II. Microscopic II. Microscopic II. Microscopic II. Microscopic

III. Powder III. Powder

6. Identity, Purity

& Strength

Foreign Matter 2.0 %, Not 2.0 %, Not 2.0 %, Not 2.0 %, Not

more than more than more than more than

Total Ash 7.0 %, Not 7.0 %, Not 7.0 %, Not 7.0 %, Not


Acid insoluble 1.0%, Not 1.0%, Not 1.0%, Not 1.2%, Not

ash more than more than more than more than

Alcohol/ 15.0%, Not 15.0%, Not 15.0%, Not 10.0%, Not

ethanol soluble less than* less than* less than less than

extractive (Vol. VIII)

Water soluble 7.0%, Not 27.0%, Not 15.0%, Not

Extractive less than less than less than

(Vol. VIII)

Loss on drying 12.0 %, Not 12.0%, Not

more than more than

Heavy metals Pharmacopoeial Pharmacopoeial

limits (Vol. VIII) limits

Microbial Pharmacopoeial Pharmacopoeial

contamination limits (Vol. VIII) limits

Pesticide Pharmacopoeial

residues limits (Vol. VIII)

Aflatoxins Pharmacopoeial

limits (Vol. VIII)

7. Assay Total alkaloids- Total alkaloids- Assay by HPLC, Total withanoloid-

0.2 % Not 0.2 % Not Quantification 0.2 % Not

less than less than not given less than

8. Chromato- TLC profile TLC/HPTLC TLC profile TLC profile

graphy profile (Vol. VIII)

(TLC/HPTLC/

HPLC)

*Alcohol (25 percent) soluble extractive

Pharmacopoeia (IP) is the first official pharmacopoeia having its first edition in

the year 1955 followed by the publication of other pharmacopoeias viz. Ayurvedic

Pharmacopoeia of India (1986), Unani Pharmacopoeia of India (1998), Siddha

Pharmacopoeia of India (2008b) and Homoeopathic pharmacopoeia of India

(1971). All these Pharmacopoeias provide regulatory standards under Drugs

& Cosmetics Act 1940 & Rules thereunder for quality control of drugs of



Dunal published other Pharmacopoeias

Sl. Quality American Herbal British The United States

No. Specification Pharmacopoeia Pharmacopoeia Pharmacopoeia

(AHP) 2000 (BP) 2013 (USP 36/NF 31) 2013

1. Official Title Asvagandha Root Withania Somnifera Asvagandha Root

Root
2. Botanical Species Withania somnifera Withania somnifera Withania somnifera

(L.) Dunal. (L.) Dunal. (L.) Dunal.

3. Morphological part/ Dried root Dried mature root Dried mature root

Official part

4. Description I. Macroscopic I. Macroscopic I. Macroscopic

II. Microscopic II. Microscopic II. Microscopic

III. Powder III. Powder

5. Identity, Purity & Strength

Foreign Matter 2.0%, Not more than 2.0%, Not more than

Total Ash 7.0%, Not more than 7.0%, Not more than 7.0%, Not more than

Acid insoluble ash 1.0%, Not more than 1.0%, Not more than 1.0%, Not more than

Alcohol/ethanol 10.0%, Not less than

soluble extractive

Water soluble

Extractive

Loss on drying 10.0%, Not more than 12.0%, Not more than 12.0%, Not more than

Heavy metals - - Pharmacopoeial limits

Microbial Pharmacopoeial limits - Pharmacopoeial limits

contamination

Pesticide residues - - Pharmacopoeial limits

Aflatoxins - - Pharmacopoeial limits

6. Assay Total alkaloids-0.2% Withaferin A-0.1% Withanolides- 0.3%

Not less than Not less than and Not less than

Withanolide 0.1%

Not less than

7. Chromatography HPLLC profile TLC profile TLC profile

(TLC/HPTLC/HPLC)

Ayurvedic, Siddha, Unani, Homoeopathic and modern systems of medicine.

Pharmacopoeial standards on W. somnifera (L.) Dunal. in respect of total ash,

acid insoluble ash, alcohol/ethanol soluble extractives and water soluble

extractive varies in different pharmacopoeial monographs (Table-6). To facilitate

uniformity in regulatory quality specifications, harmonization of pharmacopoeial

standards is very much needed when acceptability of herbal drugs in uniform

manner is accelerating (Rai and Sharma, 2014).



Dunal published on non Pharmacopoeial.

Sl. Quality Indian Herbal Quality Standards of

No. Specification Pharmacopoeia Indian Medicinal Plants

(IHP) 2002 (QCIMP) 2011

1. Official Title Withania Somnifera Withania somnifera (L.)

Dunal.(Asvagandha)
2. Botanical Species Withania somnifera (L.) Withania somnifera (L.)

Dunal. Dunal.

3. Synonyms Physalis somnifera L., Physalis somnifera L.

P. flexuosa L.,

P. arborescens DC.

4. Morphological part/ Dried roots Dried roots

Official part

5. Description I. Macroscopic I. Macroscopic

II. Microscopic II. Microscopic

III. Powder

6. Identity, Purity & Strength

Foreign Matter 2.0%, Not more than 1.0%, Not more than

Total Ash 7.0%, Not more than 8.0%, Not more than

Acid insoluble ash 1.2%, Not more than 2.0%, Not more than

Alcohol/ethanol soluble 20.0%, Not less than 18.0%, Not less than

extractive 20.0%, Not less than*

Water soluble Extractive 22.0%, Not less than

7. Assay Quantification not given Withaferine A - 0.036

to 0.094

8. Chromatography HPLC profile TLC/HPLC profile

(TLC/HPTLC/HPLC)

*Alcohol (25 percent) soluble matter.

Pharmacopoeial standards on W. somnifera (L.) Dunal. incorporated in various

pharmacopoeias are required to be harmonized with the monographs appeared

in other contentmpoarory pharmacopoeias. The quality of herbal drugs is

always prime issue and dealt with regulatory provisions of pharmacopoeial

monographs. The harmonized pharmacopoeial monographs will be yardstick to

ensure the quality, safety and efficacy of herbal drugs without any ambiguity.

References

Anonymous, 1940. Drugs & Cosmetics Act and Rules, Govt. of India, Ministry

of Health & Family Welfare, New Delhi.


Table 6: Variation in standards in respect of Pharmacopoeial standards.






1. Foreign Matter 2.0%, Not 2.0%, Not 2.0%, Not 2.0%, Not


2. Total Ash 7.0%, Not 7.0%, Not 7.0%, Not 7.0%, Not


3. Acid insoluble 1.0%, Not 1.0%, Not 1.0%, Not 1.2%, Not

ash more than more than more than more than

4. Alcohol/ 15.0%, Not 15.0%, Not 15.0%, Not 10.0%, Not

ethanol soluble less than less than less than less than

extractive (Vol. VIII)

5. Water soluble 7.0%, Not 27.0%, Not 15.0%, Not

Extractive less than less than less than

(Vol. VIII)

6. Microbial Pharmacopoeial Pharmacopoeial

contamination limits (Vol. VIII) limits

7. Pesticide Pharmacopoeial

residues limits (Vol. VIII)

8. Aflatoxins Pharmacopoeial

limits

(Vol. VIII)

9. Assay Total alkaloids- Total alkaloids- Quantification Total

0.2% Not 0.2% Not not given withanoloid-

less than less than 0.2% Not

less than

Anonymous, 1955. Pharmacopoeia of India (The Indian Pharmacopoeia) first

ed., Govt. of India, Ministry of Health and Family Welfare, New Delhi.

Anonymous, 1966. Pharmacopoeia of India (The Indian Pharmacopoeia) second

ed., Govt. of India, Ministry of Health and Family Welfare, New Delhi.

Anonymous, 1971. Homoeopathic Pharmacopoeia of India Vol. I. Government

of India, Ministry of Health & Family Welfare, New Delhi.

Anonymous, 1976.The Wealth of India (Raw Materials), Vol. X (Sp-W). C.S.I.R.,

New Delhi.

Anonymous, 1986. The Ayurvedic Pharmacopoeia of India, Part- I, VolumeI,

First edition, Govt. of India, Ministry of Health & Family Welfare, New Delhi,

pp.15-16.

Anonymous, 1996. Pharmacopoeia of India (The Indian Pharmacopoeia) Vol-

I&II. Fourth ed. Manager of Publications, Govt. of India, New Delhi.

Anonymous, 1998. The Unani Pharmacopoeia of India, Part-I, Vol.-I, Govt. of

India, Ministry of Health & Family Welfare, New Delhi pp. 7-8.


Anonymous, 2000a. Homoeopathic Pharmacopoeia of India Vol. VIII. Government

of India, Ministry of Health & Family Welfare, New Delhi, pp.136-37.

Anonymous, 2000b.American Herbal Pharmacopoeia and Therapeutic

Comendium-Aswgandha root, Published by ABC, USA, pp. 1-24.

Anonymous, 2002. Indian Herbal Pharmacopoeia. Revised New Edition.

Published by IDMA-Mumbai, pp. 2479-2481.


I, II& III. Fifth ed. The Indian Pharmacopoeia Commission, Govt. of India,

Ministry of Health and Family Welfare, New Delhi.

Anonymous, 2008a. The Ayurvedic Pharmacopoeia of India, Part- I, Volume

VIII, First edition, Govt. of India, Ministry of Health & Family Welfare, New

Delhi.

Anonymous, 2008b. The Siddha Pharmacopoeia of India, Part-I, Vol.-I, Govt.

of India, Ministry of Health & Family Welfare, New Delhi, pp. 1-3.


I, II& III. sixth ed. The Indian Pharmacopoeia Commission, Govt. of India,

Ministry of Health and Family Welfare, New Delhi.

Anonymous, 2011.Quality Standard of Indian Medicinal Plants, Vol. IX. Indian

Council of Medicinal Research. New Delhi, pp. 356-367.

Anonymous, 2013a. (The United States Pharmacopoeia/National Formulary)USP

36/NF 31, 2013 Vol. 1, Published by the United States Pharmacopoeial

Contention, Rockville, USA.

Anonymous, 2013b. British Pharmacopoeia, CD-ROM, pp.1-5.


III. seventh ed. The Indian Pharmacopoeia Commission, Govt. of India,

Ministry of Health and Family Welfare, New Delhi, pp. 3183-3185.

Chopra; R.N., Nayar, S.L. and Chopra, I.C., 1956. Glossary of Indian Medicinal

Plants, C.S.I.R., New Delhi.

Chopra, R.N; Chopra, I.C; Handa, K.L. and Kapoor, L.D. 1958. Chopras

Indigenous Drugs of India. U.N. Dhur & Sons Pvt. Ltd., Calcutta.

Kirtikar, K.R. and Basu, B.D., 1933. Indian Medicinal Plants, Vol. 1-4. L.M.

Basu, Allahabad.

Nadkarni, A.K., 1954. K.M. Nadkarnis Indian Materia Medica. ,Vol.I ,Popular

Book Depot, Bombay.

Rai, Nitin, Lalit Tiwari & Rajeev Kr. Sharma, 2012. Quality standards on medicinal

plants with special reference to regulatory aspects. In: Modern Technologies

for Sustainable Agriculture (eds. Birendra Prasad and Sunil Kumar). Chapter

9, pp. 147-175.


Rai Nitin, Lalit Tiwari and Rajeev Kr. Sharma, 2013. Regulatory status of herbal

drugs and related substances in Indian Pharmacopoeia (IP). Hippocratic J.

Unani Medicine 8(2): 149-170.

Rai Nitin and Rajeev Kr. Sharma,2014. Harmonization of Indian Pharmacopoeial

Standards. Hippocratic J. Unani Medicine 9(2): 75-108.

Sharma, B.M. and P. Kachroo,1988. Flora of Jammu and plants of
neighbourhood Vol.I. Bishan Pal Singh Mahendra Pal Sing, Dehradun.

Tiwari, Lalit; Nitin Rai and Rajeev Kr. Sharma, 2013. Regulatory Standards on

Homoeopathic Drugs: Indian Perspective. Int. J. Adv. Pharma. Sci. & Tech

1(1): 1-20.



110
Acute & Sub- Abstract

Acute Toxicity he present study was carried out during 2011-2012 to

Study of Qurse-
Nazla in
Experimental
T

investigate the safety of Qurse-nazla by conducting acute and sub-acute toxicity
in Swiss albino mice & rats respectively. Acute toxicity was determined by
administering aqueous extract of Qurse-nazla orally to two groups of mice of
six each at a dose 1gm/kg and 2gm/kg body weight. The animals were observed
for gross behaviour and mortality for 24 hours after drug administration. The
Animals

formulation was well tolerated by the animals and no abnormality was observed

in the general behaviour (salivation, lacrymation, lethargy, sleep and coma) of

*Mohd. Nadeem,

the animals and no overnight mortality was recorded. Similarly, sub-acute toxicity

Mohd. Urooj,

was determined in albino rats by orally administration of aqueous extract to

Habibur Rehman

three groups of seven animals each at a dose ranges from 1gm/kg and 3gm/

and

Shariq Ali Khan kg body weight for 28 days. The results of haematology and biochemistry

profile done on 29th day were found to be normal and no changes were

Pharmacology Research Unit

observed in organ to body wt ratio of liver, heart, kidney and spleen.

Regional Research Institute of

Unani Medicine (CCRUM),

Keywords: Acute toxicity, Sub-acute toxicity, Qurse-Nazla, Ocimum sanctum

Post Box 70, Aligarh-202002, U.P.

Linn., Cinnamomum zeylanicum Blume., Zingiber officinale Rose., Tinospora

cordifolia Wild.

Introduction

The World Health Organization is fully aware of the importance of herbal

medicines to the health of many people throughout the world, as stated in a

number of resolutions adopted by the World Health Assembly and the Regional

Committee for the Western Pacific. Thus herbal medicines have been recognized

as a valuable and readily available resource for primary health care, and WHO

has endorsed their safe and effective use. A comprehensive programme for

the identification, cultivation, preparation, evaluation, utilization and conservation

of herbal medicines has been developed. Meanwhile, it has been realized that

medicinal plants are a valuable resource for new pharmaceutical products and

thus a potential source of new drugs as well as for economic development

(Anonymous, 1993).

Determination of efficacy and safety of herbal remedies is necessary because

many people using these agents as self medication. Since there is limited data

available about the safety of the commonly used herbal remedies, therefore,

effort to elucidate health benefits and risk of herbal medicine should be

intensified. Toxicity testing in animal is typically the initial steps to determine the

effect profile of test substance and potential hazards which occurs due to short

term exposure of test substance. A toxicity study provides information on the



hazardous properties and allows the substance to be ranked and classified

according to the Globally Harmonized System (GHS) for the classification of

chemicals which cause acute toxicity (OECD, 2000).

Formulation

Qurse-nazla is a poly herbal formulation containing main constituents in equal

parts:

S.No. Common Name Botanical Name

1. Barg-e-Tulsi Ocimum sanctum Linn.

2. Darchini Cinnamomum zeylanicum Blume.

3. Zanjabeel Zingiber officinale Rose.

4. Satte Gilo Tinospora cordifolia Wild.

5. Satte Ajwain Ptychotica jowar DC

Methodology

This study was carried out in Pharmacology Research Unit, of Regional

Research Institute of Unani Medicine, Aligarh and conducted in accordance

with the protocol approved by Institutional Ethics Committee (RRIUM), Aligarh.

Procurement of Drug

The Qurse nazla formulation was procured in the form of Tablet from CRIUM

(Central research Institute of Unani Medicine) A.G colony Road Near ESI

Hospital, Eragadda Road, Hyderabad.

Animals

The study was carried out in Swiss albino mice (20-25 g) and rats (100-150g)

of either sex, for acute & sub-acute toxicity determination respectively. The

animals were procured from Mr. Rahat Hussain Enterprises Biological Suppliers

Babri Mandi Aligarh. They were acclimatized to the conditions for one week

before experimental study. The animals were maintained in a standard

environmental condition at a room temperature of (252 degree Celsius) with

12 Hrs light/Dark cycles, humidity (50-55%), and had free access to food

pellets. The study was conducted after approval of protocol from Institutional

ethics committee of RRIUM, Aligarh.

Preparation of Drug Extract

The tablets of the drugs were crushed into fine powder and a weighed quantity

was steeped in acidulated distilled water. The water soaked mass of the drug


was warmed over water bath and kept for 24 hours at room temperature.

During this period it was occasionally stirred. After 24 hours it was filtered

through a filter paper and filtrate was dried over water bath. The aqueous

extracts of the drug thus obtained was used in different doses selected according

to OECD guidelines for safety evaluations.
Acute Toxicity Study
The current study was carried out in accordance to Organization of Economic

Co-operation and Development (OECD) guideline for testing of chemicals.

Swiss Albino mice of either sex weighing 20-25 g were randomly selected and

divide into two groups of six mice each. Mice were kept fasted overnight

(12hrs) with free access to water prior to administration of dose ranging 1gm/

kg body weight and 2g/kg body weight as per limit test of OECD guideline. The

aqueous extract of the drugs was administered orally. The animals were kept

in polypropylene cages after drug administration and were observed for Gross

behaviour (salivation, lacrymation, lethargy, sleep and coma) & mortality at 1

hour, 2 hour, 3 hour, 4 hour, 5 hour, 6 hour, 12 hour, 24 hour and thereafter

once every day up to 14 days after drug administration.

Sub-Acute Toxicity Study

Swiss Albino rats of either sex weighing between 100-150 g were randomly

selected and divided into three groups of seven animals each. Rats were kept

fasted overnight (12hrs) with free access to water prior to administration of

dose ranging 1gm/kg and 3gm/kg body weight for 28 days as per limit test of

OECD guideline. Group I was kept as normal control which received distilled

water for 28 days, while in the IInd and IIIrd groups aqueous extract of the drug

was administered orally at a dose of 1gm/kg and 3gm/kg body weight for 28

days. The animals were observed for Gross behavior (salivation, lacrymation,

lethargy, sleep and coma) & mortality at 1 hour, 2 hour, 3 hour, 4 hour, 5 hour,

6 hour, 12 hour, 24 hour and thereafter once every day up to 28 days after

drug administration. On 29th day, blood was collected of all the three groups

of rats through retro-orbital plexus for estimation of SGOT,SGPT and Serum

alkaline Phosphatase, Serum urea and Serum Creatinine, Serum cholesterol,

Serum triglyceride and Serum HDL, Percentage hemoglobin, ESR, Total

leukocyte count and Differential leukocyte count. After collection of blood the

animals in all the three groups were sacrificed and liver, heart, kidney and

spleen were excised out for determination of organ to body weight ratio. SGOT,

SGPT were estimated by Reitmans and Frankel, method (Reitman et al.,

1957). Alkaline Phosphatase was estimated by Bessey and Brock, 1946 method

(Bessay et al., 1946). Serum urea was estimated by GLDH, Ureas method


given by (Tiffany et al., 1972), while Serum Creatinine was estimated by Jaffes

method given by (Bowers et al., 1980). Serum HDL was estimated by

Phosphotungstic Acid method given by (Burstein et al., 1970), while Serum

cholesterol and Triglyceride were estimated by CHOD-PAP method given by

(Roeschlau et al., 1974) and GPO-Trindev method given by (Mcgowan et al.,

1983). ESR and DLC were estimated by Westergreen and Leishman stain

methods given in Medical Laboratory Technology (Mukherjee, 1990). TLC was

estimated by Hemoaltometry method (Plum, 1936). Percent hemoglobin was

estimated by Sahlis Acid Haematin method given by (Newcomer, 1919).


Statistical analysis was performed by using unpaired t test calculating p value

at 5% level. All values are expressed as MeanSEM (standard error of mean).

p value less than 0.05 found to be considered statistically significant.

Results and Discussion

Acute Toxicity Study

The effect of oral administration of single dose of aqueous extract of Qurse

nazla in Swiss albino mice shows that the formulation was well tolerated by the

animals and no abnormality was observed in the general behaviour of the

animals and no overnight mortality was recorded. Herbs and supplements can

be toxic when used for inappropriate indication, or prepared inappropriately, or

used in large excessive dosages or for a prolonged duration of time. Since it

is polyherbal formulation other ingredients present in formulation helps in

reducing the toxic effect of active component.

Sub-Acute Toxicity Study

The values of all the parameters including liver functions, renal functions

hematology and organ body weight ratio found to be normal as compared to

control group. The effects of the studied drug on organ body weight ratio in

control and treated animals are presented in Table 4. There were no significant

changes observed in organ body weight ratio of the control and the animals

treated with various doses. Table 1 and 2 is a summary of the results of the

effects of the drug on the biochemical parameters. There were no significant

changes in AST and ALT levels in all the treated animals compared with the

control. Similarly lipid profile of treated animal was found to be normal as

compared to control group. Table 3 reflects the values for hematological

parameters of treated group as compared to control showed no significant

changes in Hb, TLC, ESR, and % lymphocyte except % polymorph count in


Table 1: Effect of Aqueous Extract of Qurse- Nazla on Liver Function & Renal Function

Test in Albino Rats

Groups Liver Function Test Renal Function Test

SGOT SGPT SALP Serum Urea Serum

Unit/L Unit/L KAU Mg/dl Creatinine

Mg/dl

Group I
Normal Control 178.0715.39 47.453.83 164.7420.08 33.70 4.03 1.29 0.07

(n = 7)

Group II

Drug treated 177.0914.52 45.402.43 166.8519.30 33.56 2.41 1.42 0.06

1g /kg(n = 7) p = 0.9638 p = 0.694 p = 0.9409 p= 0.9767 p = 0.1839

Group III

Drug treated 176.0213.50 47.864.85 169.4217.42 34.83 4.90 1.380.05

3g /kg(n = 7) p = 0.9219 p = 0.9482 p = 0.8632 p = 0.8616 p = 0.3161

Values are expressed as MeanSEM

n = number of rats in a group.

P* < 0.05, P** < 0.01, P*** < 0.001

Table 2: Effect of Aqueous Extract of Qurse- Nazla on Lipid Profiles in Albino Rats

Groups Serum Cholesterol Serum HDL Serum Triglyceride

Mg/dl Mg/dl Mg/dl

Group I

Normal Control 47.12 2.95 10.821 .30 43.60 3.34

(n = 7)

Group II

Drug treated 48.764.01 11.320.68 41.204.06

1g /kg p = 0.7475 p = 1.00 p = 0.6562

(n = 7)

Group III

Drug treated 49.313.96 12.171.55 43.565.87

3g /kg p = 0.6653 p = 1.00 p = 0.9554

(n = 7)



P* < 0.05, P** < 0.01, P*** < 0.001

group III animals where p value = 0.0319. This is not quite significant to effect

safety of the drugs. It can be concluded on the basis of above observation that

drug is quite safe.


Table 3: Effect of the Aqueous Extract of Qurse- Nazla on Haematological Parameters

in Albino Rats

Groups Haemoglobin TLC / ESR DLC

gm % Cumm mm/hr

% Polymorph % Lymphocyte

count count

Group I
Normal Control 14.080.20 4264.28147.88 1.710.28 30.853.23 65.142.46

(n = 7)

Group II

Drug treated 13.880.19 4350.00186.56 1.570.29 36.572.91 58.003.70

1g kg p = 0.4824 p = 0.7520 p = 0.7344 p = 0.2129 p = 0.1340

(n = 7)

Group III

Drug treated 14.650.33 4157.144157.14 1.420.29 40.852.56 57.713.88

3g /kg p = 0.1654 p = 0.9719 p = 0.4857 p* = 0.0319 p = 0.1318

(n = 7)



P* < 0.05, P** < 0.01, P*** < 0.001

Table 4: Effect of the Aqueous Extract of Qurse-Nazla on Organ to the Body Weight

Ratio on Albino Rats

Groups Organ weight in g/100 g body weight

Liver Heart Kidney Spleen

Group I

Normal Control 4.50 0.10 0.50 0.01 0.99 0.03 0.51 0.07

(n = 7)

Group I

Drug treated 4.880.22 0.51 0.01 1.15 0.08 0.57 0.03

1 g /kg p = 0.1418 p = 0.4930 p = 0.0857 p = 0.4461

(n = 7)

Group I

Drug treated 4.80 0.23 0.50 0.01 1.11 0.05 0.52 0.01

3g /kg p = 0.2547 p = 1.000 p = 0.0620 p = 0.889

(n = 7)



P* < 0.05, P** < 0.01, P*** < 0.001


Conclusion

The formulation was well tolerated by the animals and no abnormality was

observed in the general behavior of the animals and no overnight mortality

was recorded. There were no finding of any organ toxicity and hematological

changes as laboratory findings were normal. It can be concluded on the basis

of above observation that drug is quite safe in animals.
Acknowledgement

The authors would like to express their gratitude to Prof. Shakir Jamil, Director

General, Central Council for Research in Unani Medicine, New Delhi, for

providing research facilities to carry out this work.

References

Anonymous, 1993. Research Guidelines for Evaluating the Safety and Efficacy

of Herbal Medicine, Manila.WHO, pp. 35-40.

Bessay, O.A., Lowry, O.H., Brock, M.J., 1946. A method for the rapid

determination of alkaline phosphates with five cubic millimeters of serum.

J Biol.Chem. 164:321-329.

Bowers, L.D., Wong, E.T., 1980. Kinetic serum creatinine assay I. The role of

various factors in determining specificity. Clin.Chem. 26(5):551-554.

Burstain, M., Scholnic, H.R., Morphin, R., 1970. Rapid method for the isolation

of lipoprotein from human serum by precipitation with polyanions. J. Lipid.

Res 11:583-595.

Macgowan, M.W., Artiss, J.D., Strangberg, D.R., Zak, B., 1983. A peroxidase-

coupled method for the colorimetric determination of serum triglycerides.

Clin Chem. 29(3): 538-542.

Mukherjee, K.L., 1990. Medical Laboratory Technology, 3rd Edition, Tata

McGraw-Hill Publishing Company Limited, New Delhi, pp. 228-30.

Newcomer, H.S., 1919. Absorption Spectra of Acid Hematin, Oxyhemoglobin

and carbon monoxide hemoglobin: A new hemoglobinometer. J .Biol.chem.

37:465-496.

OECD, 2000. Guidance Document on the Recognition, Assessment and Use

of Clinical Signs as Humane Endpoints for Experimental Animals Used in

Safety Evaluation. Environmental Health and Safety Monograph Series on

Testing and Assesment No 19.

Plum, P., 1936. Accuracy of hematological counting method. Acta. Med.

Scandinav 90:342-364.


Reitman, S., and Frankel, S., 1957. A colorimetric method for determination of

serum glutamic oxaloacetic and glutamic pyruvic transaminases.

Am.J.Clin.Pathol. 28:56-63.

Roeschlau, P., Bernt, E., Gruber, W., 1974. Enzymatic determination of total

cholesterol in serum. Z Klin. Chem, Klin Biochem 12(5): 226.

Tiffany, T.O., Jansen, J., Burtis, C.A., Overton, J.B., Scott, C.D., 1972. Enzymatic

kinetic rate and end- point analyses of substrate, by use of a GeMSAEC

fast analyzer. Clin. Chem 18:829-840.


Standardization Abstract

and Quality udri Surkh botanically equated to seeds of Cheiranthus cheiri

Control
Methods of
Unani Single
T

Linn. belongs to Cruciferae (Brassicaceae) family. In Unani System of Medicine
Tudri Surkh is used as stomachic, diuretic, expectorant, demulcent,
emmenagogue and also in the ailments of asthma, cough and fever. Seed oil
is applied locally for bruises, nervous and rheumatic pains. It is one of the
ingredients in the Unani formulations namely Majoon-e-Alkula, Majoon-e-
Drug Tudri

Regmahi, Khamira Gaozaban Ambari, Khamira Gaozaban Ambari Jadwar Ood

Surkh Saleeb Wala, Khamira Gaozaban Ambari Jawaharwala, Khamira Gaozaban

Sada. In view of its medicinal importance, the present study was conducted to

(Cheiranthus

standardize the drug using pharmacognostic method, physico-chemical

cheiri Linn.)

parameters, TLC studies and WHO methods. Physico-chemical data observed

were moisture content (9.56%), total ash (6.60%), acid in-soluble ash (1.25%)

1*D. Ramasamy, and solubility in alcohol (13.69 %) and water (20.01%). TLC studies of

1Rampratap Meena, chloroform and alcohol extracts showed various spots at 254nm, 366nm and

1S.Mageswari,

in visible light. The Quality control parameters such as microbial content (TBC,

1P. Meera Devi Sri,

TFC, Enterobacteriaceae, Salmonellae Spp. and Staphylococcus aureus) and

2Shamsul
Arfin,

1Syed Jameeluddin Ahmed the heavy metals (Pb, Cd, As and Hg) were found within the permissible limits.

and Aflatoxins (B1, B2, G1 and G2) and pesticide residues were not detected in the

2Syed Shakir Jamil drug Tudri Surkh.

1Regional
Research Institute

of Unani Medicine, Keywords: Tudri Surkh, Pharmacognostic, Physico-chemical, TLC, Quality

1, West Madha Church Street, control parameters

Royapuram, Chennai-600013

Introduction

2CentralCouncil for Research

in Unani Medicine,

61-65 Institutional Area, Plants plays a vital role in maintaining human health and improving the quality

Janakpuri, New Delhi-110058 of human life from thousands of years and serves to human the valuable

components of medicines, seasonings, beverages, cosmetics and dyes. Herbal

medicine contains natural substances that can promote health and reduce

illness. Nowadays researchers are focusing on plant research and it has

increased all over the world. Enormous evidence has been collected to show

immense potential of medicinal plants used in various traditional systems (Pratap

et al., 2013).

The seed of Cheiranthus cheiri Linn. is known as Tudri Surkh in Unani System

of Medicine. In Unani medicine the seeds are used as stomachic, diuretic,

expectorant, demulcent, emmenagogue and also used in the ailments of asthma,

cough and fever. Seed oil is applied locally for bruises, nervous and rheumatic

pains besides a tonic to improve the male reproductive system. Flowers are

1


used in paralysis and impotence (Khare, 2007; Kritikar and Basu, 1998; Chopra

et al., 2006). Cheiranthus cheiri is a shrub like herb, indigenous in the North

Temperate zone, Central and Northern Europe; it is cultivated in Indian gardens

as an ornamental plant (wall flower).


Collection of drug

Seeds were collected from raw drug dealers, Chennai and identified by the

botanist and compared with the herbarium specimen of RRIUM, Chennai

(Specimen No. 00165).

Pharmacognostical studies

Botanical identification of the fruit was carried out using available literature

(Kritikar and Basu, 1998; Khare, 2007; Hooker, 1999). The pharmacognostical

studies such as macroscopical, microscopical and powder microscopy were

carried out using standard method (Johansen, 1940). Free hand sections of

the fruit were taken, microscopical drawings made using Camera Lucida and

observations recorded.

Physico-chemical parameters

Physico-chemical parameters like foreign matter, total ash, acid in-soluble ash,

loss on drying at 105C, solubility in alcohol and water were carried out as per

standard method (Anonymous, 1998).

TLC analysis

Preparation of extract

The powder of the drug (2g) was extracted using 30ml of chloroform and

alcohol extracts were concentrated upto 10ml in a standard flask. These extracts

were used for the TLC studies.

The TLC profile of chloroform and ethanol extracts were performed using pre-

coated silica gel 60 F254 TLC plate (E. Merck) as adsorbent. TLC studies of

both extracts were carried out using solvent systems like toluene: ethyl acetate:

Acetic acid (8: 2: 0.2) and toluene: ethyl acetate (1: 1) respectively. After

drying, the plates were examined under UV 254nm and 366nm and observed

the spots. Further the plates were dipped in vanillin-sulphuric acid reagent

followed by heating at 105C till appeared the bright spots appeared (Wagner

et al., 1984; Sethi, 1996).


Quality control parameters

The WHO parameters like microbial load, heavy metals, aflatoxin and pesticide

residues were carried out using standard methods of WHO & AOAC guidelines

(Anonymous, 1997, 1998, 2000).

Pharmacognostic studies

Macroscopic: Seeds are reddish brown, bright, 2.5 to 3.5mm long, 1.5 to 2 mm

wide, mucilaginous with warty surface; cotyledons incumbent, non-endospermic

with large embryo, musky odour and mucilaginous taste (Fig. 1 & 2).

Microscopic: T.S. of seed shows, epidermis consisting of single layer of

rectangular, flattened, thin walled cells containing colourless concentrically

striated mucilage; sclerenchyma cells palisade like consisting of single layer of

non-lignified cells with their radial and inner tangential walls thickened looks

like beaker shaped cells; pigmented cells consisting of single layer of elongated

parenchyma cells filled with yellowish brown contents; single layer of thick

walled cells followed by a layer of crushed parenchyma cells; cotyledons and

embryo consisting of oval to polygonal, thin-walled, parenchyma cells containing

aleurone grains and oil (Fig. 3,4 & 5).

Fig. 1: Seeds Fig. 2: Seed - surface view Fig. 3: T S of Seed

A diagrammatic sketch

Fig. 4: T. S. of seed Fig. 5: T S of cotyledons

Abbreviation: EP - Epidermis; SCL - Sclerenchyma; CT - Cotyledons;

MUEP - Mucilaginous epidermis; PIGC - Pigmented cells; TWC - Thick walled cells


Powder Microscopy: Reddish brown, sclerenchyma cells in surface view,

epidermal cell is surface view with mucilage, thick walled cells in surface view;

elongated pigmented cells in surface view, cotyledonary parenchyma cells in

surface view

(Fig. 6).

Chemical analysis
Analytical data shows 9.56 % of moisture content. Ash content of the drug

was 6.59 % and 1.25 % of acid in-soluble ash shows the siliceous matter in

the plant. Alcohol soluble extractives represent the extraction of polar constituents

like phenols, tannins, glycosides, alkaloids and flavonoids. The water soluble

extractive denotes the presence of inorganic contents. The results of physico-

chemical parameters are shown in Table (1).

Fig. 6: Powder microscopy

Table 1: Physico-chemical parameters of Tudri Surkh

S.No. Parameters Results (n=3)S.D.

1. Foreign matter (%) Nil

2. Loss on drying at 105C (%) 9.56

3. Ash (%) 6.59

4. Acid insoluble ash (%) 1.25

5. Alcohol soluble extractives (% 13.69

6. Water soluble extractives (%) 15.81


Thin Layer Chromatography

The Rf values of the TLC analysis of chloroform and alcohol extracts are

shown in Table - II and III. The plates were visualized using vannilin-sulphuric

acid reagent and heated at 105 till appear the colored spots. The TLC of the

chloroform extract at UV- 254 nm showed 5 spots, UV-366 nm showed 3 spots

and 5 spots showed after derivatization with vanillin - sulphuric acid (Fig. 7).
Alcohol extract showed at UV-254 nm 4 spots, UV-366 nm showed 3 spots and

after derivatization with vanillin sulphuric acid showed 6 spots (Fig. 8).

Quality control parameters

The microbial load and heavy metals were found within the permissible limit

(Table 4 and 5).

Fig. 7: Chloroform extract Fig. 8: Alcohol extract

Solvent system: Toluene : Ethyl acetate Solvent system: Toluene : Ethyl acetate

: Acetic acid (8 : 2 : 0.2) (1 : 1)

Detector: V. S. Reagent Detector: V. S. Reagent

Table 2: TLC data of the chloroform extract of Tudri Surkh

Solvent system Rf Values

UV 254 nm UV 366 nm V. S. Reagent

Toluene : Ethyl 0.78 Pink 0.63 Blue 0.56 Dark grey

acetate : Acetic acid

0.63 Light pink 0.26 Pale blue 0.45 Violet

(8 : 2 : 0.2)

0.49 Light pink 0.16 Blue 0.39 Grey

0.23 Pink 0.26 Violet

0.16 Pink 0.12 Grey


Table 3: TLC data of the alcohol extract of Tudri Surkh

Solvent system Rf Values

UV 254nm UV 366nm V. S. Reagent

0.91 Light pink 0.72 Blue 0.91 Grey

0.72 Pink 0.39 Pale blue 0.72 Violet

Toluene: Ethyl acetate

0.39 Pink 0.14 Blue 0.56 Light grey

(1 : 1)

0.14 Pink 0.39 Light grey

0.34 Violet

0.14 Violet

Table 4: Microbial load

S.No. Parameter Analyzed Results WHO Limits

2 105 CFU / gm

1 Total Bacterial Count 2 x 10 CFU / gm

2 Total Fungal Count Ansent 103 CFU / gm

3 Enterobacteriaceae Absent 103 CFU / gm

4 Salmonella Spp. Absent Nil

5 Staphylococcus aureus Absent Nil

Table 5: Heavy metals

S.No. Parameter Analyzed Results WHO & FDA Limits

1 Arsenic Nil 10 ppm

2 Cadmium Nil 0.3 ppm

3 Lead 0.0031 10 ppm

4 Mercury Nil 1.0 ppm

The aflatoxin such as B1, B2, G1 & G2 and analysed pesticide residues such as

organo chlorine group, organo phosphorus group, acephate, chlordane,

dimethoate, endosulphan, ethion, endosufon sulphate, fenthion, lindane,

methoxychlor, phorate sulfoxide and phorate sulfone were not detected from

the drug.

Conclusion

The evaluated standards such as macroscopic, microscopic, physico-chemical,

TLC analysis and quality control parameters were derived and described are


of diagnostic impor-tance in authentication and quality control of the seeds of

Cheiranthus cheiri.

Acknowledgement

The authors are deeply indebted to the Director General, CCRUM, New Delhi,

for providing necessary research facilities and encouragement for this study.
References

Anonymous, 1997. Official Analytical Methods of the American Spice Trade

Association (ASTA). Inc. 4th edn., New Jersey, pp. 149-152.

Anonymous, 1998. Quality Control Methods for Medicinal Plant Materials. World

Health Organisation, Geneva, pp.10-31, 61-63.

Anonymous, 2005. Official Methods of Analysis of AOAC International. In: Horwitz

W, Latimer, G.W. (eds). 18th Edn. AOAC International: Maryland, chapter 10

pp.18-23 and chapter 3, pp. 10-11.

Anonymous, 2006. National Formulary of Unani Medicine, Part I. Ministry of

Health & Family Welfare, Department of AYUSH, Govt. of India, New Delhi.

Pratap, Bhanu, G.S. Chakraborthy, Nandini, Mogha, 2013. Complete aspects

of Alstonia scholaris. International Journal of Pharm.Tech. Research 5(1):

17-26.

Hooker, J.D., 1999. The Flora of British India, Vol. I. Bishen Singh Mahendra

pal Singh, Dehra Dun, p. 132.

Johansen, D.A., 1940. Plant Microtechnique Mc. Graw Hill Book Company Inc.,

New York and London, pp. 181 - 186.

Khare, C.P. (Ed.), 2007. Indian Medicinal Plants, An Illustrated Dictionary,

Springer International Edition, pp. 140141.

Kritikar, K.R. and Basu, B.D., 1998. Indian Medicinal Plants, Vol. 1. Bishen

Singh Mahendra Pal Singh, Dehra Dun, IInd Edition, pp.143145.

Chopra, R.N., Nayar, S.L. and Chopra, I.C., 2006. Glossary of Indian Medicinal

Plants, National Institute of Science Communication and Information

Resources. Council of Scientific & Industrial Research, New Delhi, India,

pp. 6061.

Sethi, P.D., 1996. High Performance Thin Layer Chromatography. CBS Publisher

and Distributors, New Delhi.

Wagner, H., Bladt, S.A., 1996. Thin Layer Chromatography Atlas. In: Plant

Drug Analysis. 2nd edn. Springer-Verlag Berlin Heidelberg, Germany.



126
Development of Abstract

Quality he Unani system of medicine prescribes large number of

Standards on
Jawarish-e-
Kafoor Qawi A
T

classical herbal formulations to cure the different types of diseases. Jawarish-
e-Kafoor Qawi a Unani herbal formulation is prepared in combination of
ingredients like Kafoor, Zafran, Jauzbuwa, Filfil Siyah, Zanjabeel, Bisbasa,
Darchini, Narmushk, Qirfa, Filfilmoya, Faranjmushk and Qand Safaid. The Unani
Physicians prescribes the drug Jawarish-e-Kafoor Qawi to cure the ailments of
Classical Unani

Zof-e-Meda (Weakness of the stomach) and Nafkh-e-Shikam (Flatulence in the

Formulation# stomach). At present no pharmacopoeial standards on drug is available and

it is basic requirement for the research on quality control of this drug. There

1*Rampratap Meena, is lack of standardization and proper documentation of Unani drugs. Based on

2S. Mageswari, the available sources an attempt is made to evaluate the drug on

2D. Ramasamy,

pharmacopoeial parameters to develop standards for the drug Jawarish-e-

2P. Meera Devi Sri,

Kafoor Qawi. To evaluate the pharmacopoeial parameters of the drug, various

3Shamsul Arfin,

3Aminuddin parameters like powder microscopy, moisture content, ash values, bulk density,

and pH values, extractive values, TLC/HPTLC finger printing and other quality

4Nitin Rai control parameters viz. heavy metals, microbial content, aflatoxins and pesticide

residues are performed. The evaluated data will help to lay down

1Drug Standardisation Research

pharmacopoeial standards for the drug Jawarish-e-Kafoor Qawi.

Institute,

PLIM Campus, Sanjay Nagar,

Ghaziabad-201002

Keywords: Jawarish-e-Kafoor Qawi, Powder microscopy, Physico-chemical,

2Regional
Research Institute TLC/HPTLC, WHO parameters

of Unani Medicine,

1, West Madha Church Street,

Introduction

Royapuram, Chennai-600013

Jawarish-e-Kafoor Qawi (Anonymous, 2006) is one of the ancient commonly

3CentralCouncil for Research

in Unani Medicine,
used classical Unani formulations. This poly herbal formulation consists of 12

61-65 Institutional Area, ingredients (Table 1). This drug is prescribed for the treatment of Zof-e-Meda

Janakpuri, New Delhi-110058

(weakness of the stomach) and Nafkh-e-Shikam (flatulence in the stomach)

disorders. The development of traditional medicines particularly Unani medicines

4Pharmacopoeial Laboratory

with the perspective of safety, efficacy and quality will not only to preserve the


Kamla Nehru Nagar, traditional heritage but also to rationalize the uses of Unani medicines in the

Ghaziabad-201002
health care.

Standardisation of Unani herbal formulations is necessary step to assess the

quality of drugs. Due to lack of Standard Operating Procedures (SOPs) and

quality control methods, there are batch to batch variations among the similar

formulations. Pharmacopoeial study of a drug is an essential requirement to

establish the presence of each ingredient in the formulations (Bandaranayake,

# Accepted for presentation in National Seminar on Opportunities in Medicinal Plant Research,

Jadavpur University, Kolkata-32; November 29-30, 2014

1*Author for correspondence


2006; Myers and Cheras, 2004). The present study is an attempt to evaluate

the pharmacopoeial studies of the drug by applying modern parameters such

as microscopical, physico-chemical, thin layer chromatography and WHO

parameters such as microbial load, aflatoxin, heavy metals and pesticide residue.

Material and Methods

To evaluate the pharmacopoeial studies of Jawarish-e-Kafoor Qawi a systematic

scheme of standardization was developed.

(i) Ingredients authentications

Genuine raw drugs namely Kafoor, Zafran, Jauzbuwa, Filfil Siyah, Zanjabeel,

Bisbasa, Darchini, Narmushk, Qirfa, Filfilmoya, Faranjmushk and Qand Safaid

of the formulation were procured from raw drugs dealers of Chennai and Delhi

(Fig. 1). The raw drugs were authenticated as per pharmacopoeial and other

official standards (Anonymous, 2004, 2007, 2008 & 2009).

(ii) Drug formulation

The ployherbal semisolid drug was prepared in different batches at Laboratory

scale as per the ingredients composition and guidelines of NFUM, Part IV

(Anonymous, 2006) (Table 1).

(iii) Powder microscopy

The drug sample (5g) was weighed and mixed with 50ml of water in a beaker

with gentle warming, till the sample completely dispersed in water. The mixture

was centrifuged and decanted the supernatant. The sediment was washed

several times with distilled water, centrifuged again and decanted the

supernatant. A few mg of the sediment was taken and mounted in glycerine.

A few mg was taken in watch glass and added few drops of phloroglucinol and

concentrated hydrochloric acid, mounted in glycerine. The microscopic salient

features of the drug were observed in different mounts (Wallis, 1997; Johansen,

1940).

(iv) Physico-chemical analysis

The physico-chemical methods viz., moisture content, ash values, solubility in

different solvents, pH values, bulk density and sugar content etc., are useful

tools in standardisation of a herbal product for maintaining batch to batch

consistency. The drug samples were subjected for the standardisation of

physico-chemical parameters and analysed as per the standards method

(Anonymous, 1987).


Kafoor Zafran Jauzbuwa

Cinnamomum camphora (L.) Crocus sativus Linn. Myristica fragrans Houtt.

Nees & Eberm Dried stigmas and top of styles Endosperm

Natural Camphor

Filfil Siyah Zanjabeel Bisbasa

Piper nigrum Linn. Zingiber officinale Rosc. Myristica fragrans Houtt.

Fruit Rhizome Arillus

Darchini Narmushk Qirfa

Cinnamomum zeylanicum Blume. Mesua ferrea Linn. Cinnamomum cassia Blume.

Inner stem bark Stamens Stem bark

Filfilmoya Faranjmushk Sugar

Piper longum L. Ocimum sanctum L.

Fruit Seed

Fig. 1: Ingredients of Jawarish-e-Kafoor Qawi

(v) TLC/HPTLC finger printing

The formulations of the three batch samples were extracted with chloroform

and alcohol. The extracts were concentrated and made up to 10 ml in a


volumetric flask separately. These solutions were used for the TLC/HPTLC

finger print analysis.

The TLC/HPTLC finger print analysis of chloroform and alcohol extracts of the

formulations were performed using aluminium plate precoated with silica gel 60

F254 (E.merck) employing CAMAG Automatic TLC sample - IV applicator. The

chromatogram were developed using the developing systems toluene: ethyl

acetate (9: 1) and toluene: ethyl acetate (6: 4) for chloroform and alcohol

extracts respectively. The plates were dried at room temperature to record the

image of the plates at UV-254 nm, UV-366 nm using TLC visualizer and the

plates were scanned at 254 nm to record the finger print spectrum using TLC

Scanner - IV. Finally the plate were dipped in vanillin-sulphuric acid and heated

at 105 till coloured spots appeared (Wagner, and Bladt, 1984; Sethi, 1996).

(vi) Other quality control parameter

The usage of herbal products along with higher safety margins, WHO has

taken necessary step to ensure quality control parameters with the modern

techniques and application of suitable standards. The microbial load and heavy

metal parameters were carried out as per the WHO guidelines (Anonymous,

1998). Aflatoxin and pesticide residues were carried out by standard methods

(Anonymous, 2000).

Obseravtions

Jawarish-e-Kafoor Qawi is a dark brown semi-solid product with sweetish bitter

taste.

(i) Pharmacognostical observation (Powder microscopy):

The diagnostic characteristics of cellular elements in respect of each ingredients

is in Table 1 and Fig. 2.

(ii) Chemical analysis

The physico-chemical data such as moisture content was obtained in the drug

19.57%. The alcohol soluble extractive (44.56%) might be due to the extraction

of polar chemicals constituents and the water soluble extractives (65.54%)

indicate the presence of inorganic constituents. The obtained data are shown

in Table 2.

(iii) Thin Layer Chromatography analysis

The chloroform and alcohol extract of all the three batch samples showed

identical spots in UV 254nm and 366nm ranges and the Rf values of both


Table 1: List of the raw drugs and cellular elements of Jawarish-e-Kafoor Qawi

Formulation

S. Unani name Unani Name Part used Quantity Salient features of the drug

No.

1. Kafoor Cinnamomum Natural 25 g.

API-VI camphora (L.) camphor

Nees & Eberm.

2. Zafran Crocus Stamens & 25 g. Pollen grains size upto 120, spherical

UPI-VI sativus Linn. Stigmas in outline with clear exine and intine

3. Jauzbuwa Myristica Endosperm 25 g. Endosperm cells filled with numerous

UPI-I fragrans starch grains, crystalline fat and large

Houtt. aleurone grains (crystalloid proteins

upto 40), perisperm cells filled with

reddish brown contents

4. Filfil Siyah Piper nigrum Fruit 25 g. Stone cells polygonal interspersed

UPI-IV L. among parenchyma cells with circular

lumen, perisperm cells isolated or in

groups with angular walls filled with

aleurone grains and minute calcium

oxalate crystals

5. Zanjabeel Rhizome 25 g. Isolated starch grains, simple oval to

Zingiber

UPI-I officinale round shaped measuring upto 70,

Rosc hilum eccentric, lamellae distinct;

non-lignified septate fibres upto 50,

reticulate vessels and fragments of

reticulate vessels upto 70;

parenchyma cells filled with abundant

starch grains

6. Bisbasa Arillus 25 g. Thick walled elongated parenchyma

Myristica

UPI-VI fragrans cells in surface view upto 50 wide

Houtt.

7. Darchini Cinnamomum Inner stem 25 g. Fibres thick walled lignified with

UPI-I bark striated walls and narrow lumen of

zeylanicum

Blume. length upto 1000 and breadth upto

30, stone cells with horse shoe

shaped thickenings upto 70

8. Narmushk Mesua Stamens 25 g. Tricolporate golden yellow pollen

UPI-IV ferrea Linn grains upto 50

9. Qirfa UPI-III Cinnamomum Stem bark 25 g. Fibres thick walled lignified with

cassia Blume. striated walls and narrow lumen of

length upto 1000 and breadth upto

40 and very large stone cells upto

200, stone cells with horse shoe

shaped thickenings upto 70

10. Filfilmoya Piper longum Fruit 25 g. Parenchyma cells with elongated

API-IV L. spindle shaped stone cells; perisperm

cells isolated or in groups with angular

walls filled with aleurone grains and

minute calcium oxalate crystals

11. Faranjmushk Ocimum Seed 25 g. Fragments of irregular shaped thick

API-IV sanctum L. walled epidermal cells

12. Qand Safaid Sugar 800 g.


Jauzbuwa Bisbasa

Elongated thick walled

Zafran Endosperm cells filled parenchyma cells

with starch grains Perisperm cells

Pollen grains
Filfil Siyah & Filfilmoya Filfilmoya Filfil Siyah

Perisperm cells with angular Parenchyma cells with Parenchyma cells

walls filled with starch grains spindle shaped stone cells with stone cells

Narmushk Zanjabeel

Pollen grains

Reticulate vessels Septate fibres Starch grains

Qirfa / Darchini

Fibres Stone cells Parenchyma cells

Faranjmushk filled with starch grains

Thick walled irregular

epidermal cells

Fig. 2: Powder microscopy of Jawarish-e-Kafoor Qawi

the extracts are shown in Table 3 and 4. The plates were dipped in vanillin-

sulphuric acid and heated at 105C till appeared coloured spots.

(iv) Quality control parameters

The evaluated quality control parameters such as microbial load and heavy

metals were found within the permissible limit in the drug shown in Table 5


Table 2: Physico-chemical parameters

Parameters Batch Number (n=3)

I II III

Extractives

Alcohol soluble matter 44.71% 44.17% 44.80%

Water soluble matter 65.31% 65.79% 65.52%
Ash

Total ash 0.89% 0.73% 0.62%

Acid insoluble ash 0.18% 0.28% 0.35%

pH values

1% Aqueous solution 5.36 5.48 5.39

10% Aqueous solution 4.19 4.35 4.41

Sugar estimation

Reducing sugar 33.61% 33.26% 33.72%

Non-reducing sugar 9.47% 9.09% 9.50%

Moisture 19.24% 19.83% 19.46%

Bulk Density 1.3203 1.3114 1.3152

Table-3: Rf values of the chloroform extract

Rf Values

Solvent UV-254 nm UV-366 nm After derivatisation with

System vanillin sulphuric acid

reagent

0.95 Green 0.92 Fluorescent blue 0.93 Grey

0.80 Green 0.81 Blue 0.83 Violet

Toluene: Ethyl acetate (9:1)
0.72 Green 0.78 Red 0.73 Violet

0.67 Green 0.69 Red 0.66 Grey

0.60 Green 0.64 Fluorescent blue 0.59 Grey

0.48 Green 0.60 Red 0.57 Grey

0.35 Green 0.56 Red 0.48 Pink

0.27 Green 0.50 Blue 0.37 Grey


0.16 Green 0.23 Green 0.27 Pink

0.14 Blue 0.15 Grey


Table 4: Rf values of the alcohol extract

Rf Values

Solvent UV-254 nm UV-366 nm After derivatisation with

System vanillin sulphuric acid

reagent

Toluene: Ethyl acetate (6:4)

0.88 Green 0.86 Pink 0.87 Brown

0.79 Green 0.78 Blue 0.78 Grey

0.72 Green 0.59 Green 0.69 Light grey

0.48 Green 0.51 Green 0.65 Violet

0.39 Green 0.47 Blue 0.58 Light grey

0.23 Light blue 0.49 Light grey

0.10 Pink 0.10 Grey

and 6. The other parameters like aflatoxins B1, B2, G1 and G2 and pesticide

residues - organo chlorine group, organo phosphorus group, acephate,

chlordane, dimethoate, endosulphan, endosulfan, endosulfon, ethion,

endosufon sulphate, fenthion, heptachlor, lindane, methoxychlor, phorate

sulfoxide and phorate sulfone were not detected from the drug samples shown

in Table 7 and 8.

Table 5: Analysis of Microbial load

S.No. Parameter Analyzed Results WHO Limits

1 Total Bacterial Count 300 CFU / gm 105 CFU / gm

2 Total Fungal Count < 10 CFU/ gm 103 CFU / gm

3 Enterobacteriaceae Absent / gm 103 CFU / gm

4 Salmonella Absent / gm Nil

5 Staphylococcus aureus Absent / gm Nil

Table 6: Estimation of Heavy Metals

S.No. Parameter Analyzed Results WHO & FDA Limits

1 Arsenic Not detected 10 ppm

2 Cadmium Not detected 0.3 ppm

3 Lead 0.0018 ppm 10 ppm

4 Mercury Not detected 1.0 ppm


Table 7: Estimation of Aflatoxins

S.No. Aflatoxins Results WHO Limits

1 B1 ND 0.05ppb

2 B2 ND 0.05ppb

3 G1 ND 0.05ppb

4 G2 ND 0.05ppb

ND = Not Detected

Table 8: Analysis of Pesticide Residues

S.No. Pesticide Residues Results Limits

1 Organo Chlorine group ND (DL 0.005mg/Kg)

2 Organo Phosphorus group ND (DL 0.005mg/Kg)

3 Acephate ND (DL 0.005mg/Kg)

4 Chlordane ND (DL 0.005mg/Kg)

5 Dimethoate ND (DL 0.005mg/Kg)

6 Endosulphan ND (DL 0.005mg/Kg)

7 Endosulfan ND (DL 0.005mg/Kg)

8 Endosulfon ND (DL 0.005mg/Kg)

9 Ethion ND (DL 0.005mg/Kg)

10 Endosufon sulphate ND (DL 0.005mg/Kg)

11 Fenthion ND (DL 0.005mg/Kg)

12 Heptachlor ND (DL 0.005mg/Kg)

13 Lindane ND (DL 0.005mg/Kg)

14 Methoxychlor ND (DL 0.005mg/Kg)

15 Phorate sulfoxide ND (DL 0.005mg/Kg)

16 Phorate sulfone ND (DL 0.005mg/Kg)

ND Not detected

TLC/ HPTLC finger print studies

(i) TLC/ HPTLC finger print studies of chloroform extract

The TLC studies of chloroform extract are tabulated in Table 3. All the three

batch samples shows identical spots in UV-254 nm, UV-366 nm and visible light

(after derivatised with vanillin sulphuric acid reagent). In UV 254, 366 nm

and visible light it shows 10, 11 and 11 spots respectively with different Rf

values (Fig. 3). The finger print of the chloroform extract shows 13 peaks of


which peaks at Rf 0.17, 0.30, 0.38, 0.57, 0.62, 0.75, 0.82 and 0.91 were the

major peak whereas peaks at R f 0.04, 0.07, 0.22, 0.44 and 0.67 were

moderately smaller peaks (Fig.4). The HPTLC densitometry chromatogram of

chloroform extract of three batch samples were recorded at 254 nm (Fig. 5).
UV- 254 nm UV- 366 nm Visible Light

(After derivatisation with

vanillinsulphuric acid reagent)

Fig. 3: TLC photos of chloroform extracts of three batch samples at different wavelength

of light

Fig. 4: HPTLC finger print profile for chloroform extract at 254 nm


Fig. 5: HPTLC densitometry chromatogram of chloroform extracts of three batch

samples at 254 nm

(ii) TLC/ HPTLC finger print studies of alcohol extract

The TLC studies of alcohol extract are tabulated in Table 4. All the three batch

samples shows identical spot in UV-254 nm, UV-366 nm and visible light (after

derivatised with vanillin sulphuric acid reagent). In UV 254, 366 nm and

visible light it shows 6, 8 and 8 spots respectively with different Rf values (Fig.

6). The finger print of the alcohol extract shows 13 peaks of which peaks at

Rf 0.54, 0.74, 0.81 and 0.90 were the major peak whereas peaks at Rf 0.08,

0.12, 0.25, 0.32, 0.37, 0.44, 0.60, 0.67 and 0.99 were moderately smaller

peaks (Fig. 7). The HPTLC densitometry chromatogram of alcohol extract of

three batch samples were recorded at 254 nm (Fig. 8).


The evaluated data in respect of powder microscopy, physico-chemical, TLC/

HPTLC fingerprint and other quality parameters provides analytical parameters

on classical Unani formulation Jawarish-e-Kafoor Qawi can be used for in

house quality control of drug as well for development of pharmacopoeial

standards on drug.


UV- 254 nm UV- 366 nm Visible Light

(After derivatisation with

vanillin sulphuric acid reagent)

Fig. 6: TLC photos of alcohol extracts of three batch samples at different wavelength of

light

Fig. 7: HPTLC finger print profile for alcohol extract at 254 nm


Fig. 8: HPTLC densitometry chromatogram of alcohol extracts of three batch samples

at 254 nm

Acknowledgement

The authors are extremely thankful to Director General, CCRUM, New Delhi

and the Director, Pharmacopoeial Laboratory of Indian Medicine (PCIM/PLIM),

Ghaziabad, for their valuable guidance, encouragement and providing necessary

research facilities to carry out the present studies.

References

Anonymous, 1987. Physico-chemical standards of Unani Formulations Part

II. CCRUM, Min. of Health & Family Welfare, New Delhi, pp. 300 - 317.

Anonymous, 1998. Quality Control Methods for Medicinal Plant Materials. World

Health Organization, Geneva, pp. 25 - 28.

Anonymous, 2000. Association of Official Analytical Chemists (AOAC), 17th

Edition.

Anonymous, 2004. The Ayurvedic Pharmacopoeia of India, Part-I, Vol.-IV (English

Edition). Govt. of India, Min. of Health & Family Welfare, New Delhi, pp.

105-106; 146-147.


Anonymous, 2006. National Formulary of Unani Medicine, Part IV (English

Edition). Govt. of India, Min. of Health & Family Welfare, New Delhi, p. 59.

Anonymous, 2007. The Unani Pharmacopoeia of India, Part-I, Vol.-I (English

Edition). Govt. of India, Min. of Health & Family Welfare, New Delhi, pp. 26-

27; 38-39; 88-89.

Anonymous, 2007. The Unani Pharmacopoeia of India, Part-I, Vol.-III (English

83.

Anonymous, 2007. The Unani Pharmacopoeia of India, Part-I, Vol.-IV (English


39; 98-99.

Anonymous, 2008. The Ayurvedic Pharmacopoeia of India, Part-I, Vol.-VI (English

Edition). Govt. of India, Min. of Health & Family Welfare, New Delhi, pp.

210-211.

Anonymous, 2009. The Unani Pharmacopoeia of India, Part-I, Vol.- VI (English


24; 101-102.

Bandaranayake WM, 2006. Quality control, screening, toxicity and regulation

of herbal drugs. Modern Phytomedicines, pp. 25 - 57.

Johansen, D.A., 1940. Plant Microtechnique. Mc. Graw Hill Book Company Inc.,

New York and London, pp. 181-186.

Myers, S.P., Cheras, P.A., 2004. The other side of the coin: safety of

complementary and alternative medicine. Medical J. Australia 181: 222 -

225.

Sethi, P.D., 1996. High Performance Thin Layer Chromatography, Vol. X. (1st

Edition). CBS Publishers and Distributors, New Delhi, pp. 1-56.

Wagner, H., Bladt, S., and Zgainski, E.M., 1984. Plant Drug Analysis: A Thin

Layer Chromatography Atlas (2nd Edition). Springer-Verlag, Germany.

Wallis, R.E., 1997. Text Book of Pharmacognosy, 5th Edition. CBS Publishers

& Distributors, Delhi, pp. 494 496.


Short Research Communication:

Overview of Catharanthus roseus (L.)

G. Don Alkaloids

Catharanthus roseus (L.) G.Don; syn. Vinca rosea L.
Family : Apocynaceae

English name : Periwinkle

Hindi name :Sadabahar, Baramasi

Introduction

A tropical perennial herb, hardy and resistant to attack by fungi or insects. It

grows wild but now cultivated scientifically for ornamental and medicinal use.

Flowers white or deep rose coloured; follicles long. Earlier civilisations knew

about its use in medicine.

The plant originated from Malagasy (Madagascar), then spread to Mozambique,

followed by several South East Asian countries viz., South Vietnam, Philippines

and Australia, including India and Ceylon (Tiwari et al., 2011).

Alkalodial Composition

Catharanthus roseus (L.) G.Don contains two important classes of compounds,

viz; alkaloids and tannins. Although it is a poisonous plant, but under close

medical supervision its alkaloids have been employed successfully for the

treatment of cancers viz; Hodgkins carcinoma and leukemia.

There are over 130 organic constituents present in Catharanthus roseus (L.)

G.Don plant. These have indole or di-hydro-indole moieties present in their

molecular structure, which exhibited oncological activity. The chief alkaloids

which we shall focus upon here are known as Vinblastine and Vincristine. They

occur together naturally and the yield of the former alkaloid is pre-dominating.

The 17-de-acetyl vinblastine has been synthetically converted to the

corresponding lactone, which was found to be associated with chemotherapeutic

activity.

Applications

The alkaloidal components of Catharanthus roseus (L.) G.Don have been

claimed to be good for brain health. These alkaloids help in improving the

supply of nutrient (glucose) and oxygen to the brain, which it can effectively

utilize. Additionally these components are highly effective in the abnormal


coagulation of blood and in raising the level of serotonin. This molecule is well

known blood neurotransmitter in the Central Nervous System (CNS); its vital

function is to regulate the heart function, memory including sleep and appetite.

Lack of serotonin may lead to several mental disorders like schizophrenia. The

other application of Catharanthus roseus (L.) G.Don alkaloids are;

Hypotensive

Sedative

Tranquiliser

Anticancer

Relief from pain in muscles and wasp stings

Provides relief to depression of the CNS

Stop bleeding from nose/gums

Relief to mouth ulcers and sore throat

Decrease glucose concentration in blood (hypoglycaemia) (specially

from plant leaves)

Controls diarrhea

Taken internally it produces memory enhancing action

Reduces the inflammation of urinary bladder, stomach and intestines

Wound healing properties

According to literature reports there are over 100 alkaloids isolated from the

various parts of the plant (viz; leaves, stem, root, root bark etc.).Amongst them

two medicinally important alkaloids may be mentioned here viz; vinblastine and

vincristine. The yield of vincristine is extremely small (0.00025%) in comparison

to vinblastine (root : 0.78-1.22; root bark : 4.5-9.00)% respectively (Tiwari et

al., 2001). Amongst the other bio-active alkaloids may be mentioned reserpine

,ajmalicine (raubasine), serpentine and vincamine. The laboratory synthesized

derivative of vincamine (alternatively known as vincopectine) is a well known

triple action medicine, viz; vaso-dilator, blood thinner and memory enhancer.

Praubasine has been reported to be useful in checking fragility of capillaries

(tiny thin walled blood vessels).

One outstanding property of Catharanthus roseus (L.) G.Don extract is that it

exhibits anticancer activity against numerous cell type in general and against

multi drug resistant cancer tumors in particular (Favretto et al., 2001). This

observation points to the fact that some components of the Catharanthus

roseus (L.) G.Don exerts a synergic effect which counters the multi drug

resistance offered by the tumors cells.


In this article we shall highlight the pharmaceutical uses of the two clinically

important alkaloids viz; vinblastine and vincristine used for the treatments of

cancers.

Vinblastine

This alkaloid exhibits quite a different and wide spectrum of activity, and is
used for the following types of cancers;

Hodkgins lymphoma, cancer of breast, head, lungs, neck, ovaries and testicles.

The vinblastine is biosynthesized in the plant by joining together two alkaloidal

residues(viz; Cathaanthine and vindoline).

According to the regimen of chemothraphy directed from the Hodgkins

lymphoma, vinblastine is incorporated into belomycin and metotrenote. This

allows the use of lower dose of belomycin thereby reducing the overall toxicity

and providing larger resting periods between chemotherapy cycles (Gobbi et

al., 2003).

Caution should however, be exercised in the use of vinblastine, since it is

associated with wide adverse side effects, namely, hair loss, stomach and

intestinal disorders, elevation of blood pressure, depression, involuntary and

painful contraction of muscles, giddiness, headache etc.

Vincristine

Vincristine is the second vinca alkaloid, which is used for the treatment of

cancer. This cytotoxic drug acts by inhibiting the ability of the cell to divide,

which in turn stops the proliferation of the disease .The alkaloid has quite a

different and wide spectrum of activity. It is employed for the acute lymphobic

cancers in children, malignant tumor (Favretto et al., 2001) and breast cancer.

The use of the extract of Catharanthus roseus (L.) G.Don on a rabbit led Beer

& Noble (Western Ontario, Canada) to a chance discovery. They noticed that

there was reduction of white blood cells (WBCs)and which decreased further

with the administration of higher concentration of the drug. Besides, the blood

platelets count, including the immunity of the animal fell considerably. There

was practically no resistance in the body of the rabbit and the animal died due

to infection of Pseudomonas sp. This led the investigators to conclude that

Catharanthus roseus (L.) G.Don. alkaloid could be used to cure leukaemia

a medical condition wherein there is a large increase of WBCs than the normal.

This conclusion was found to be correct and was based on the positive results

obtained with the leukemic patients. The property of Croesuss alkaloid to

suppress immunity could be exploited in organ transplant operations to overcome


the problem of rejection by the body .At this stage a need was felt to devise

a method of obtaining vincristine alkaloid in sizeable amounts. Recourse was

taken to extractive procedure from vinca rosea, involving the use of tons

quantity of dried leaves. This, however, is a cumbersome and time consuming

process. Thanks to the efforts of synthetic organic chemists, who were able to

prepare the valuable vincristine alkaloid on a pilot plant scale, in order to cope

up with the demand of its use as a drug in cancer therapy as well as an

immuno-suppressant drug.

These two alkaloids (vinblastine and vincristine) are now available in the US

markets in the form of their respective sulphates.

Isolation of the alkaloids

The modern technique has been employed for the isolation of the above

mentioned alkaloid in a pure form;

Methods involving the;

Selective or differential extraction.

Method based on pH gradient based separation.

Flavonidal/terpenoidal composition

The flowers of Catharanthus roseus (L.) G.Don contained flavonids. The major

anthocyanidins present consist of Petunidin, malvidin, including kaempferol

and Quercetin (both are strong antioxidants). The whole plant is reported to

contain tri-terpenoid,viz ; ursolic acid, accompanied with loganin, deoxyloganin

severoside etc.

Acknowledgement

The authors thank Prof. G.S. Sandhu, Director, HIET, Ghaziabad, for the kind

interest shown by him during the present study.

References

Favretto, D., Piovan, A., Filippini, R., Caniato, R., 2001. Monitoring the production

yields of vincristine and vinblastine in Catharanthus roseus from somatic

embryogenesis. Semi-quantitative determination by flow- injection

electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom.

15(5): 364-9.


Gobbi, P.G., Brogila, C., Merli, F., DellOlio, M., Stelitanto, C., Lannito, E.,

Federico, M., Berte, R., Luisi, D., Molica, S., Cavalli, C., Dezza, L., Ascari,

E., 2003. Vinblastine, Belomycin and Methotrexate Chemotherapy plus

irradiation for Patients with Early Stage, Favorable Hodgkin Lymphoma.

Cancer 98 (11): 23932401.

Tiwari, D.N., Kumar, K. and Tripathi, A., 2001. SADABAHAR. Ocean book Pvt.

Ltd., Allahabad, p. 80.

July 2, 2014 Durga Nath Dhar

44 Duplex, Block-A

Rajat Vihar, Sector 62

NOIDA-201301

and

Shalin Kumar

Hi-Tech Institute of

Engg.& Technology

NH-24, Adhyatmik Nagar

Ghaziabad 201015



146
HIPPOCRATIC JOURNAL OF UNANI MEDICINE

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Management of Acute Gouty Arthritis With A Polyherbal Unani Formulation

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Management of Abstract

treatment with test drug produced remarkable effects on various efficacy

Keywords: Gout, Niqris, Wajaul Mafasil, Suranjan, Cocchicum luteum

Ministry of Health & F.W.,

Government of India, Introduction

GPO Complex, INA,

Department of Mahiyatul Amraz

Faculty of Medicine (U),

was the first to describe tophi (James et al., 2000).

an estimated 1-1.5% of the world population (Praveen et al., 1994). The

prevalence of gout is rising as a result of a changing pattern of lifestyle

(Arromdee et al., 2002). In most cases, no identifiable underlying cause of

and the use of thiazide diuretics (Roubenoll, 1990). Although, hyperuricemia is

presence of normal serum uric acid concentrations. Conversely, many persons

with hyperuricemia never experience an attack of gouty arthritis (McCarty,

Hippocratic Journal of Unani Medicine 1

non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids etc.

risks in patients who have pre-existing renal, cardiovascular, metabolic and

gastrointestinal diseases (Nuki, 1999; Emmerson, 1996).

Antihyperuricemic drugs such as allopurinol, benzbromarone, sulfinpyrazone

those in whom allopurinol is contraindicated (Jansen et al., 2004). Furthermore,

On the basis of conventional Unani Usool-e-Ilaj of Niqris (gout), five herbal

the efficacy and safety of this capsule in the management of gout.

Materials and Methods

Study drug was a combination of five herbs namely, Suranjan (Colchicum

luteum), Elva (Aloe barbadensis), Qurtum (Carthamus tinctorius), Halaila-e-

in the quantity of 1gm.

each containing wheat flour.

Hippocratic Journal of Unani Medicine 2

(i) Inclusion Criteria

of American College of Rheumatology (ACR) for diagnosing acute gouty arthritis

including clinical features, laboratory and radiographic findings were included

range ( > 7 mg/dl)

(ii) Exclusion Criteria

Patients were excluded if they had renal or hepatic insufficiency or cardiovascular

disorders. Patients taking thiazide group of diuretics/ aspirin/NSAIDs and

and study was conducted according to Good Clinical Practice guidelines.

Dosage and Administration

Following 5-7 days washout period of anti-inflammatory/ analgesic drugs (e.g.,

NSAIDs, Corticosteroids) or antihyperuricemic drugs (eg allopurinol) or any

or Unani drugs), patients were randomly assigned to receive either drug or

period of six weeks. Randomization was done by lottery method.

Follow up and Drug Compliance

Clinical as well as laboratory evaluation was performed and recorded at the

baseline, week 1, week 4 and week 6. Compliance with treatment drug/placebo

was evaluated at each follow up visit by capsule count.

Criteria for the Assessment of Efficacy

To assess the response of treatment on patients of gouty arthritis in both

groups, the following parameters were used.

Hippocratic Journal of Unani Medicine 3

Joint swelling (0-4 point scale; with 0=no swelling,1=barely perceptible,

2=mild, 3=moderate, 4=severe, bulging beyond the joint margins) (Cheng

et al., 2004; Taylor et al., 2007).

Movement/Mobility (0-4 point scale; with 0=full voluntary

movement,1=partial voluntary movement, 2=full movement when the

joint is moved by the examiner, 3=partial movement when the joint is

moved by the examiner, 4=no movement at all)

Serum uric acid, C-Reactive proteins, Erythrocyte Sedimentation Rate

and total leukocyte count

Liver function test S.Bilirubin, S.G.O.T, S.G.P.T. & S.Alkaline

Kidney function test Blood Urea, S. Creatinine.

Haemogramme Hb%, TLC, DLC, E.S.R.

The differences in pretreatment and post treatment obtained in both groups