ANTIHELMINTHIC AND HEPATOPROTECTIVE

PROPERTIES OF four different PLANT EXTRACT

By Subhrajyoti Banerjee
INSTITUTE OF GENTIC ENGINEERING
BSc(H)BIOTECHNOLOGY Roll:21008215019
6th SEMESTER DISSERATION Reg- 152102410019

Supervised by- Mudhimita J. Mukhopadhya

 AIM OF THE PROJECT
I. To STUDY
ANTIHELMINTHIC AND
HEPATOPROTECTIVE
PROPERTIES OF
DIFFERENT PLANT LEAF
EXTRACTS
II. 1. Carica papaya,
III. 2. Piper betle,
IV. 3. Andrographis paniculate,
V. 4. Murraya koenigii
 Introduction

 Anthelmintic are drugs that are used to treat infections with parasitic worms. This includes
both flat worms, e.g., flukes and tapeworms and round worms.
 The World Health Organization estimates that a staggering 2 billion people harbor parasitic
worm infections.
 Albendazole, mebendazole, triclabendazole and related synthetic anthelmintic drugs were
used in helminthiasis but are contraindicated for certain groups of patients which increased
the need for development of novel anthelmintic drugs.
 Utilization of traditional medicine including herbal extracts should be considered in this
scenario to kill these parasitic worms and it will help to find out newer molecular entities.
What are helminths ?

 Helminths are commonly known as parasitic worms, are large multicellular

organisms, which can generally be seen with the naked eye when they
are in their mature state but in their immature state they generally
overlooked and appears to be invisible to our naked eyes for their
microscopic size. They are often referred to as intestinal worms even
though not all helminths reside in the intestines. For example, schistosomes
are not intestinal worms, but rather reside in blood vessels.
Lifecycle of the helminths:

nematode cycle cestode cycle trematode cycle

egg - larvae (L1-L4) - egg - metacestode - adult egg-miracidium-sporocyst-redia-
adult cercaria-(metacercaria)-adult

Only adult stages were taken for

experimental purpose
About the plants

 1. Murraya koenigii :- The curry tree (Murraya koenigii) is a tropical to

sub-tropical tree in the family Rutaceae, (citrus, and satinwood), which is
native to India and Sri Lanka.
About the plants

 2. Andrographis paniculate :- It is an annual herbaceous plant in the

family Acanthaceae, native to India and Sri Lanka. It has been traditionally
used to treat infections and some diseases. Mostly the leaves and roots
were used for medicinal purposes.
About the plants

 3. Carica papaya :- the plant Carica papaya,

one of the 22 accepted species in the genus Carica
of the family Caricaceae.
 The papaya is a small, sparsely branched plant,
usually with a single stem growing from 5 to 10 m (16
to 33 ft) tall, with spirally arranged leaves confined to
the top of the trunk.
 The lower trunk is conspicuously scarred where
leaves and fruit were borne. The leaves are large, 50–
70 cm (20–28 in) in diameter, deeply palmately
lobed, with seven lobes.
About the plants

 4. Piper betle :- It is the leaf of a vine belonging to

the Piperaceae family, which includes pepper and
kava.
 Betel leaf is mostly consumed in Asia, and
elsewhere in the world by some Asian emigrants, as
betel quid or in pan, with Areca nut and/or tobacco.

 In India and Sri Lanka a sheaf of betel leaves is

traditionally offered as a mark of respect and
auspicious beginnings.
Experimental worms:

 1. Indian earthworm Pheretima

posthuma were used to study
Anthelmintic activity.
 The earthworms were bought from
a local zoological specimen
dealer and washed with normal
saline to remove all fecal matter.
 The earthworms in 10-15 cm in
length were used for experimental
protocol due to their anatomical
and physiological resemblance
with the intestinal roundworms
parasites of human beings.
Experimental worms:

 2. Aquarium worms Tubifex

tubifex (Annelida) were collected
from the local market.
 The average sizes of the worms
were 1-1.5 cm.
 The anthelmintic assay was carried
with minor modifications.
 The assay was also performed on
the aquarium worm, Tubifex tubifex,
because they belong to same
group of Annelida.
 Experimental animal:

 Laboratory rats share origins with their cousins in

domestication, the fancy rats.
 In 18th century Europe, wild Brown rats ran rampant and
this infestation fueled the industry of rat-catching. Rat-
catchers would not only make money by trapping the
rodents, but also by selling them for food, or more
commonly, for rat-baiting. The rat found early use in
laboratory research in five areas: W. S. Small suggested
that the rate of learning could be measured by rats in a
maze; a suggestion employed by John B. Watson for his
Ph.D. dissertation in 1903.
 The first rat colony in America used for nutrition research
was started in January 1908 by Elmer McCollum, and
then nutritive requirements of rats were used by Thomas
Burr Osborne and Lafayette Mendel to determine the
details of protein nutrition.
 STUDY OF
ANTIHELMINTIHIC ASPECT
OF THE PLANT LEAVES
MATERIALS AND METHODS: -

 Collection of the plant leaves: -

Fresh leave of the mentioned plant as Carica papaya, Piper betle, Andrographis paniculate,
Murraya koenigii were collected after authentication, fresh leaves of both plants were
collected in bulk, washed under running tap water, dried under shade for a period of 10 days
and then pulverized in electrical grinder to obtain coarse powder.
The dried powder was stored in airtight bottles.
 Extraction methodology: -

 Ethanolic extract of leaves: coarse leaf powder were taken of

about 5g.
 suspended in 50ml of 50% ethanol (meaning the solvent was of
25ml distilled H2O and 25ml 99.9% ethanol).
 This suspension was then kept for 24 hours and as to reduce the
evaporation the suspension was kept in in an airtight container of
about 200ml capacity at least. As the atmospheric temperature
was high as 35oC so in order to decrease the rate of evaporation
the container containing the suspension was kept in refrigerator.
 After 24 hours the suspension was filtered and the extract was
collected in another airtight container.
 The suspension was kept in refrigerator even after the filtration in
order to decrease their evaporation.
 The same procedure was repeated for all the mentioned leaves
was performed obeying the same precaution.
 Extraction methodology: -

 Aqueous extract of leaves: the desire amount of coarse

plant powder was taken like 5g of the coarse leaf powder
and suspended in 50ml of double distilled water and kept
for about 24 hours.
 kept in refrigerator so that it does not get degraded due to
higher room temperature.
 After 24 hours this was taken for filtration to separate the
extract from the suspension.
 After filtration the extract was kept in an airtight container of
about 200ml in refrigerator to prevent degradation or
denaturation.
 The same was repeated for all the plant leaves and was
stored separately.
 Anthelminthic Screening:

 two types of worms were taken and the experiment was

divided into two divisions one with Tubifex tubifex and one with
Pheretima posthuma.
 Plant extracts were taken in petri dishes and worms were
submerged in them.
 Their paralyzing time and death time were recorded.
 Also, the worms were subjected to a recovery stage where
they were submerged in distilled water for recovery.
 All the worms whether they be Tubifex sp. or Pheretima
posthuma were first washed in distilled water before the
experiment.
 The observations were made for time taken to paralyze(it is said
to be when the worms did not recover when subjected to
distilled water for recovery)and death (it is when the worms lost
their mobility and followed by their body deformity and
discoloration).
Qualitative analysis of condensed
tannin:

 The plant extracts

were subjected to
FeCl3 by add few
drops of the plant
extract into ferric
chloride and it was
observed that the
colour changed to
greenish giving a
conclusion of
positive
 Histopathological investigation

Histocytological screening:
• The livers were fixed in 10% formalin
for light microscopic examination.
Paraffin-embedded sections were
cut at 5-7 μm thickness and
subjected to:

• Hematoxylin and eosin stain

The observations were done by experts
and the conclusions were drawn by
them as well.
The pictures of the screened livers and
their best slides are provided below:
Histopathological investigation(conclusion)

 (a)Control group showing normal hepatic architecture.

 (b) Sodium arsenite induced necrotic liver cells and focal hemorrhages in the
periportal area considered as negative control.
 (c)Papaya leaf extract plus Sodium arsenite showing mild inflammatory cell
infiltration.
 (d)Pan leaf extracts plus Sodium arsenite showing moderate inflammatory cell
infiltration.
 (e)Kalmegh leaf extracts plus Sodium arsenite showing moderate inflammatory
cell infiltration.
 (f)and (g) Papaya extracts plus Sodium arsenite arrangement of cells in the liver
lobule with only mild inflammatory cell infiltration.
 (h)50% ethanol plus Sodium arsenite showing severe inflammatory cell infiltration.
 (i)Curry leaf extracts plus Sodium arsenite showing severe to moderate
inflammatory cell infiltration.
 Observation table:

Anthelminthic activity of Carica papaya against earthworm and Tubifex sp. –

Treatment Time taken by earthworm for Time taken by Tubifex sp. for
Paralysis(min) Death(min) Paralysis (min) Death (min)
Distilled water -- -- -- --
Aqueous extract
40% extract 46 72 35 48
60% extract 32 52 23 45
80% extract 21 26 15 25
Ethanolic extract
40% extract 32 50 27 36
60% extract 21 41 15 25
80% extract 12 21 12 18
Observation table:

Anthelminthic activity of Andrographis paniculate against earthworm and Tubifex sp. –

Treatment Time taken by earthworm for Time taken by Tubifex sp. for
Paralysis(min) Death(min) Paralysis (min) Death (min)
Distilled water -- -- -- --
Aqueous extract
40% extract 52 70 48 72
60% extract 44 63 37 64
80% extract 38 51 27 37
Ethanolic extract
40% extract 43 58 32 45
60% extract 36 42 26 38
80% extract 28 39 19 28
Observation table:

Anthelminthic activity of Murraya koenigii against earthworm and Tubifex sp. –

Treatment Time taken by earthworm for Time taken by Tubifex sp. for
Paralysis(min) Death(min) Paralysis (min) Death (min)
Distilled water -- -- -- --
Aqueous extract
40% extract 55 69 77 105
60% extract 39 54 57 82
80% extract 20 45 35 55
Ethanolic extract
40% extract 42 60 38 48
60% extract 37 55 26 40
80% extract 25 43 18 31
 Observation table:

Anthelminthic activity of Piper betle against earthworm and Tubifex sp. –

Treatment Time taken by earthworm for Time taken by Tubifex sp. for
Paralysis(min) Death(min) Paralysis (min) Death (min)
Distilled water -- -- -- --
Aqueous extract
40% extract 47 55 43 55
60% extract 35 48 32 48
80% extract 28 39 25 44
Ethanolic extract
40% extract 35 41 29 42
60% extract 28 37 19 28
80% extract 21 31 17 20
Graphical comparison(EARTHWORM)

Comparison of ethanolic extract for Paralysis

time
50
45
40
35
30
25
20
15
10
5
0
Papaya Pan Kalmegh Curry
40% 60% 80%
Graphical comparison(EARTHWORM)

Comparison of ethanolic extract for Death

time
70

60

50

40

30

20

10

0
Papaya Pan Kalmegh Curry
40% 60% 80%
Graphical comparison(Tubifex sp.)

Comparison of ethanolic extract for Paralysis

time
40

35

30

25

20

15

10

0
Papaya Pan Kalmegh Curry
40% 60% 80%
Graphical comparison(Tubifex sp.)

Comparison of ethanolic extract for Death

time
60

50

40

30

20

10

0
Papaya Pan Kalmegh Curry
40% 60% 80%
 STUDY OF
HEPATOPROTECTIVE
ASPECT OF THE PLANT
LEAVES
MATERIALS AND METHODS: -

 Collection of the plant leaves: -

Fresh leave of the mentioned plant as Carica papaya, Piper betle, Andrographis paniculate,
Murraya koenigii were collected after authentication, fresh leaves of both plants were
collected in bulk, washed under running tap water, dried under shade for a period of 10 days
and then pulverized in electrical grinder to obtain coarse powder. The dried powder was
stored in airtight bottles.
 Extraction methodology: -

 Ethanolic extract of leaves: the coarse leaf powder were

taken of about 10g
 were suspended in 50ml of 50% ethanol (meaning the solvent
was of 25ml distilled H2O and 25ml 99.9% ethanol). This
suspension was then kept for 24 hours and as to reduce the
evaporation the suspension was kept in in an airtight container
of about 200ml capacity at least. As the atmospheric
temperature was high as 35oC so in order to decrease the rate
of evaporation the container containing the suspension was
kept in refrigerator.
 After 24 hours the suspension was filtered and the extract was
collected in another airtight container. The suspension was
kept in refrigerator even after the filtration in order to decrease
their evaporation.
 The same procedure was repeated for all the mentioned
leaves was performed obeying the same precaution.
Hepatoprotective screening:
 A total of 7 albino rats were taken of around 50-60g 1.
of their body weight and divided into the seven 2.
categories. 3.
 Each category was made on the basis of the plant
extract that they were presumed for administration. 4.
5.
 The rats were not differently caged as there were 6.
only one for each extract, one for negative, one for
7.
positive control, one for extract vector control.
 The rats were differentiated with distinct marks on
them using picric acid, which are as beside:
Head mark for Papaya leaf extract
Lower back mark for Pan leaf extract
Full back mark for Kalmegh leaf
extract
No mark for Curry leaf extract
Abdomen mark for Positive control
Jaw mark for Negative control
Big no mark for Vector control
Hepatoprotective screening:

 The dosage for above mentioned

administration was performed
accordingly as 1mg/g/day for 15
consecutive days. On the 14th day they
were intoxicated with Sodium
arsenite(NaAsO2) so as to monitor how it
damages the hepatocytes and their body
is able to resist against it.
 After 24hrs these rats were sacrificed and
their liver was extracted for
histocytological screening and blood
collected for liver function test.
 Liver function test:

 ASAT (GOT):-
Results(in IU/L): - for
 For working reagent: One part of the reagent 2 was 1. Headmark for Papaya leaf extract=152.06
mixed with four part of the reagent 1 was taken and 2. Lower back mark for
mixed well. Pan leaf extract =169.86
 For assay: we add 1ml of the working reagent with
3. Full back mark for
100μl of the serum/plasma and the od was measured Kalmegh leaf extract =178.2
after incubation period of every 60 seconds for three 4. No mark for Curry leaf extract =182.7
consecutive times under 340nm against distilled water. 5. Abdomen mark for Positive control =292.5
6. Jaw mark for Negative control =118.4
 Calculation: (tf at 37 C is 1)
o
7. Big no mark for Vector control =197.08
 At 340nm in IU/L = (change in absorbance per min)x1746xtf
 This was repeated for all samples.
 Liver function test:

 ASLT (GPT):-
Results: - for
 For working reagent: One part of the reagent 2 was 1. Head mark for Papaya leaf extract =91.3
mixed with four part of the reagent 1 was taken and 2. Lower back mark for
mixed well. Pan leaf extract =102.26
 For assay: we add 1ml of the working reagent with
3. Full back mark for
100μl of the serum/plasma and the od was measured Kalmegh leaf extract =118.43
after incubation period of every 60 seconds for three 4. No mark for Curry leaf extract =119.79
consecutive times under 340nm against distilled water.5. Abdomen mark for Positive control =261.4
6. Jaw mark for Negative control =74.5
 Calculation: (tf at 37 C is 1)
o
7. Big no mark for Vector control =146.31
 At 340nm in IU/l = (change in absorbance per
min)x1746xtf
 This was repeated for all samples.
 Liver function test:

Bilirubin (total and direct): -

For direct Bilirubin: The mixtures were mixed well and
the OD was recorded for d1 and d2 after 1min under
spectrophotometer at 540nm.
For total Bilirubin: The mixtures were mixed well and
the OD was recorded for t1 and t2 after 5 mins under
spectrophotometer at 540nm
Calculations : -
Total Bilirubin (mg/ml) = absorbance of (t1-t2)x26.31
Direct Bilirubin (mg/ml) =absorbance of (d1-
d2)x26.31
 Liver function test: bilirubin
Reagent Total Total Direct Direct
1(ml) 2(ml) 1(ml) 2(ml)
(sample (sample Results: - for
blank) blank) 1. Head mark for Papaya leaf extract = 0.98
Diazo A 0.5 0.5 0.5 0.5 2. Lower back mark for
Diazo B 0.05 - 0.05 -
Pan leaf extract =1.02
Mixed thoroughly and then proceeded
3. Full back mark for
Activator 0.5 0.5 - -
Kalmegh leaf extract =1.09
Distilled 1.0 1.0 1.5 1.5
water
4. No mark for Curry leaf extract =1.14
Serum 0.1 0.1 0.1 0.1 5. Abdomen mark for Positive control =2.2
/plasma 6. Jaw mark for Negative control =0.84
7. Big no mark for Vector control =1.76
Rat
liver
Graphical comparison

COMPARISON AGAINST EACH OTHER

SGOT SGPT BILIRUBIN

292.5
261.4

197.08
178.2 182.7
169.86
152.06 146.31
118.43 119.79 118.4
102.26
91.3
74.5

0.98 1.02 1.09 1.14 2.2 0.84 1.76

Papaya Pan Kalmegh Curry Positie Negative Vector

control control control
Discussion

 Different plant extracts were assessed for anthelminthic activity using

Tubifex sp. and Pheretima posthuman.
 According to the present study the extracts of both ethanolic and
aqueous of Papaya showed maximum anthelminthic activity followed by
Pan, Kalmegh and Curry.
 According to review the anthelminthic property is mainly due to the
presence of tannin.
 The present experiment showed the presence of condensed tannins in all
the plant extracts.
 The present experiment also showed the hepatoprotective activity of the
plant extract. The experiment on rats showed protection by Papaya , Pan
followed by Kalmegh and Curry
Conclusion

 In the present study Papaya (Carica papaya) and Pan

(Piper betle) showed a potent anthelminthic property in
comparison to other plant as mentioned above.
 The hepatoprotective study showed maximum
protection by Papaya (Carica papaya) and Pan (Piper
betle).
 These studies showed a pioneer view for using these
plants as promising hepatoprotective and anthelminthic
drug and related researches in future.
Reference

 1. http://shodhganga.inflibnet.ac.in/
 2. https://en.wikipedia.org/wiki/Helminths
 3. https://www.ncbi.nlm.nih.gov/pmc/articles/
 4. https://scialert.net/fulltext/?doi=rjphyto.2014.57.63