Journal of Research in Biology An International Scientific Research Journal

Original Research

Effects of extruded diets with different energy levels on fat Body composition and profile of fatty
acids of Rainbow trout (Oncorhynchus mykiss) fed extruded diets with different energy levels
Journal of Research in Biology

Authors: ABSTRACT:
Aba Mustapha 1,
Belghyti Driss1, We have evaluated the inuence of energy level on the ratio of digestible protein
Benabid Mohammed2 and digestible energy on the body composition and fatty acid profile composition of fillet
in rainbow trout. Two extruted diets with different energy levels were used. The
comparison of the two foods with different formulations is perfomed in isoenergetic
Institution: conditions. Following this study, two diets were formulated: the extruded diet A with
1. Biology and Health 41,4% crude protein, 27,4% lipids and 20,4% carbohydrate while the extruded food B with
Laboratory, Environmental 39.7% CP, 24,4% fat and 15,7 carbohydrates with digestible energy of 21.32 Mj kg -1 and
and Parasitology Team/UFR 19.32 Mj kg-1 respectively. The initial average weight of the trouts was 100 g from the
Doctoral "Parasitology same batch of eggs which were divided randomly into six fiberglass conical tanks at open
compared: Medical and circuit.
Veterinary Applications", The diet was assigned to six tanks of 50 fish each with three replicates for each
Sciences Faculty, diet and the experiment was conducted for 127 days. The ratio DP/DE of body composition
Ibn Tofail University, influenced by diet and profile of fatty acid and their distribution in the fillet of fish was
Knitra B.P. 133, 14000, calculated. The test focused on the ventral fillet of fish. Our results demonstrated that the
Morocco. total fatty acids expressed in g/100g wet matter (WM) reveal significant variations
(p<0.05). The fatty acid composition of the fish muscle varied with the high level fat for the
2. National Center of low ratio DP/DE, and the best ratio n3/n6 (p<0.05) is found out. The lipid content increased
Hydrobiology and with increasing dietary lipid levels and depended on the protein content in feeds and the
Pisciculture (NCHP) ratio DP / DE. Fatty acid (FA) composition of the muscle reflected the fat levels in the diet.
Azrou Morocco.

Keywords:
Corresponding author: Fat, fatty acid, protein digestible, energy digestible, fillet, n-3/n-6 Ratio,
Aba Mustapha. rainbow trout.

Email: Article Citation:

aba_mustapha@yahoo.fr
Web Address:
http://jresearchbiology.com/
documents/RA0275.pdf.
Aba Mustapha, Belghyti Driss, Benabid Mohammed.
Effects of extruded diets with different energy levels on fat Body composition and
profile of fatty acids of Rainbow trout (Oncorhynchus mykiss) fed extruded diets with
different energy levels.
Journal of Research in Biology (2012) 2(7): 617-625
Dates:
Received: 11 Aug 2012 Accepted: 22 Sep 2012 Published: 11 Oct 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

617-625 | JRB | 2012 | Vol 2 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
The success of the Fish culture is based on the
provision of feed supplied in rations containing optimal
levels of energy and nutrients for growth and body
composition (Hardy and Barrows, 2002). The
optimization of the ratio protein / energy (P: E) has been
therefore having an important role in protein and energy
utilization (Kaushik, and Medale, 1994).
So, one of the factors affecting optimization of
the efficiency of food is the balance between digestible
protein (availability of amino acids) and digestible
energy non-protein food. This balance is represented by
the ratio of digestible protein (DP) and digestible energy
(DE) of the food (DP / DE). To get a better optimization
of the ratio DP / DE, the rate of this ratio can be reduced
if an additional power source (fat or digestible
carbohydrates) is provided to save the protein.
(Aba et al., 2012). Many studies have shown that
increasing dietary DE by increased non-protein energy
food resulted in better retention of protein and a decrease
in the excretion of ammonia nitrogen, (Dias et al., 1999,
Watanabe et al., 2001; Bureau et al., 2002, Aba et al.,
2011).
Fish and fish products are considered as valuable
foodstuffs because of their nutritional properties that are
beneficial for human health. The nutritional benefits of
fish stems, from its exceptionally advantageous fatty acid
profile. In recent years increasing attention has been
focused on significance the of n-3 polyunsaturated fatty
acids (PUFAs) in nutritional food consumed by humans
(Hossain, 2011). As the worlds fish stock is limited,
farmed fish is being proposed as an alternative
(Medale, 2010).
There are variations in lipid content with varying
quatity of fat deposits in the muscle in diferent species.
Thus, in salmonids, the ventral part of the net is usually
fat than the dorsal, and the anterior oilier than the
posterior (Katikou et al., 2001; Testi et al., 2006).
618
Aba et al., 2012
Fish is the main food source of omega 3 and
omega 6PUFAs (n3, n6), in the human diet
(Simopoulos, 2008). So, numerous studies have revealed
that n-3 fatty acids are essential for the best development
of the body and are beneficial for human health
(Dewailly et al., 2001; Demers, 2001). Polyunsaturated
fatty acids, omega 3 are effective against cardiovascular
problems. They have been shown to have beneficial
effect in a range of cardiovascular risk factors, which
result in primary cardiovascular prevention, reduction in
total and cardiovascular mortality (Calder and Yaqoob,
2010), and various inammatory (Wall, 2010) conditions
such as arthritis, asthma, psoriasis and bowel diseases
(Dewailly et al., 2007; Gogus and Smith, 2010), and are
essential for brain development (Blanchet et al., 2005).
The n3 play a role in improving learning ability
(Yonekubo, 1992) and visual function (Birch
et al., 2000). Long chain n3 PUFAs cannot be
synthesized by humans and must be obtained through
diet (Alasalvar et al.,2002)
The n3/n6 ratio has been suggested to be a useful
indicator for comparing relative nutritional values of fish
oils (Piggot and Tucker,1990).Todays western diet is
generally deficient in n-3 fatty acids and excessive in n-6
resulting in a low n-3/n-6 ratio. It was proposed in
general that human diet should have the n-3/n-6 ratio
close to 1 whereas in the western diet it exceeds 1:15
(Simopoulos, 2008).
Rainbow trouts are members of Salmonidae
family. It is also widely used as farmed fish in many
countries because of its rapid growth and value as a food.
Freshwater fishes have a greater capacity to desaturate
and elongate short chain fatty acids than marine fishes
(Moreira et al., 2001).
Rainbow trout (Oncorhynchus mykiss) is one of
the most popular fish species in nature, but in many
countries it is also recognized and accepted as cultivated/
farmed fish species, due to its fast growth and excellent
nutritional quality (Tikeogly, 2000).
Journal of Research in Biology (2012) 2(7): 617-625
Aba et al., 2012

The quality assessment of rainbow trout must be anesthetized after 24 h of fasting in order to measure the
considered from two aspects. One important attribute is size and the weight of each fish. The quantities of food
its nutritional value as a source of n-3 PUFA but sensory distributed were weighed to estimate the consumption by
attributes like colour, need to be taken into account as fish between two weighings.
well. (Aba et al., 2012) The rate of feeding
There isnt much researches in Morocco that The experimental test was aimed at comparing
gives information on the whole nutritional value of the effect of two non-isoenergetic foods of different
rainbow trout. In view of these facts, the objective of the formulations on the growth performance of fish and their
present study is to determine the, fat, fatty acid, flesh quality in isoenergetic condition. The amount of
compositions of the rainbow trout. And the aim of the food distributed is consistent with the feeding tables of
present study is to investigate the impact of the chemical the tow extruded foods (A,B) that have different
composition of feed (feeds with different levels of digestible energies 21.32 Mj kg-1,19.32 Mj kg-1,
energy and lipid) on the fat content of body composition respectively. These rates of rationing depend on the
and dietary quality of fillets of rainbow trout. temperature of the water close to the site. We have set
the rates according to the temperature of the site which is
MATERIALS AND METHODS about 14C so that the quantitative ratio for the same
Experimental design food energy intake is: amount of food extruded (ExA)
The experiment was conducted between 1.10 = amount of extruded (ExB) food.
June 1, 2010 and October 5, 2010 at the National Gross energy was calculated using the
Center of Hydrobiology and Fish Culture (NCHP) in following values: crude protein = 23.73 Mj Kg-1
Azrou (Morocco). crude lipids = 39.5 Mj kg -1 and
This test was conducted in fiberglass conical carbohydrate = 17.2 Mj kg-1proposed by Brett and
tanks of 0,8 m3 volume at open circuit with an initial Groves (1979). The calculation of digestible energy is
load of 5 kg fed with spring water at a constant obtained by the coefficient of digestibility of protein, fat
temperature of around 14C 0.2 and a flow rate of and carbohydrates (gelatinized or raw) (Guillaume and
3
1,6 m /h, with a time renewal of water two times per Medale, 2001).
hour with oxygen levels above 80% saturation. The Proximate composition of experimentals diets are
average content of dissolved oxygen in the outlet of the shown in Table 1.
ponds was 7.1 ppm, and pH around 7. Extruded Extruded
Parameters
diet A diet B
Biological materials Dry matter 94.4 % 96.1 %
300 juveniles trout females triploid of average Protins 41.1% 39.7 %
weight of 100 g 3g from the same batch of eggs were Lipids 27.4% 24.4 %
divided randomly into six fiberglass conical tanks. carbohydrates 20.4% 15.7 %
The test was conducted in triplicate culture, the Moisture 5.6 % 3.9 %
Gross Energy
fish were fed manually and the daily ration was split into 23.73 21.70
(GE, Mj Kg-1)
two meals distributed at 09 am and 03 pm, seven days a Digestible energy
21.32 19.32
(DE, MJ Kg-1)
week for 127 days, according to the feeding table
DP / DE (g MJ- 1)
17.35 18.48
provided by the supplier of food (LeGouessant). Every (DP:Digestible Protein)
two weeks eight fish of each batch have been Ratio P /L 41/27 40/24

Journal of Research in Biology (2012) 2(7): 617-625 619


Chemical analysis of fat
Four fish in each group were randomly selected,
filleted and each fillet was divided into four discrete
sections; antero ventral part, antero dorsal part, postero
dorsal part, postero ventral part.
These fillets were divided into four parts
(Fig 1): Each part of fillets from all fish was pooled. The
pooled fillet parts were ground and homogenized in a
blender and then the proximate composition of the fillets
was analyzed. Crude lipid analysis was conducted by
ether extraction, and total lipid was extracted after
homogenization in chloroform/methanol (2/1 v/v)
containing 0.01% butilated hydroxytoluene as
antioxidant basically according to Folch et al., (1957).
Chemical analysis of fatty acids
Total lipid was extracted from fish fillets and
feed samples, by homogenization in chloroform/
methanol (2/1, v/v) containing 0.01% butylated
hydroxytoluene (BHT) as antioxidant, according to the
method of Folch et al., (1957).Fatty acid methyl esters
were prepared from total lipid by acid-catalyzed
transesterification using 2 mL of 1% H2SO4 in methanol,
plus 1 mL of toluene, as described by Christie (24), and
fatty acids were analyzed by gas liquid chromatography
(Auto System XL Perkin Elmer) using a 30 0.25 mm
capillary column (FID detector CP-2330 Supelco,
Deutschland). The conditions of the method were: carrier
gas, helium; flame ionization detection temperature,
220C; split rate: 1/50, oven temperature programmed to
rise from 120C/2 min to 220C/15 min at a rate of
5C/1 min; injector temperature, 240C. The
identification of the individual methyl esters Were
achieved by comparison of their retention times with
commercial standards (Sigma-Aldrich Chemie GmbH,
Deutschland).
Levels of polyunsaturated fatty acids (PUFAs),
the total content and the ratio of n3 and n6 fatty acids
were determined and expressed in grams per 100 g fillet
of wet matter. All analytical determinations were
620
Aba et al., 2012
Table 2 : content of fat and fatty acids n3 and n6 for
two diets (g/ 100g of fillet WM)
Parameters Extruded diet A Extruded diet B
Fat 8.50b 0 .02 7.03a 0.03
Omega 3 2.8b 0.06 1.90a 0,05
Omega 6 2.6b 0.04 2.10a 0.02
Ratio n 3 / n 6 1.08b 0.03 0.90a 0.02
done in triplicate.
Statistical studies
Results are expressed as mean ( SD). Our
results are compared statistically (Development Core
Team R, 2011). All parameters of growth and yield were
subjected to Analysis of Variance test (ANOVA).
Tukeys multiple procedure was used to compare the
differences among mean values. Differences were
regarded as significant, when P<0.05.
RESULTS
Values are means of four replications. Data are
expressed as mean SD. Values in a row with different
superscripts are significantly different from each other
(P<0.05).
The present study shows, food with different fat
and this energy concentrations, has a significant impact
on body composition of rainbow trout which is expressed
as the percentage of the total of 100g of fillet. The values
of fat are significantly the highest in the group of fish
receiving feed with the lowest DP/DE (P < 0.05).
The content of polyunsaturated fatty acids n3 and
n6 are limited to the anterior ventral part of fillets of
rainbow trout expressed in g/100 g fillet of wet matter.
Fatty acid compositions of fish were well reflected by the
content composition of dietary lipids. The content of the
PUFAs from n-3 fatty acids in the fillets of fish from fed
by the extruded diet A was higher than that fed with the
diet B (P<0.05, Table 2),and the content of the n-6 fatty
acids is lower for the diet B than the diet A (P<0.05).
The ratio n-3 / n-6 is affected by the content of fat in this
part (P<0.05). The values of the n3/n6 index were the
most advantageous in the fillets of fish fed with the
Journal of Research in Biology (2012) 2(7): 617-625
Aba et al., 2012
extruded diet A.
DISCUSSION
The content of fat in foods reflects significant
the lipid content in the ventral part of the fillets of fish,
particularly salmonids that are recognized by excessive
deposition at this part (Corraze and Kaushik, 1999;
Mdale 2010)
Lipid content in fish fillets of rainbow trout
increased along with increases in the lipid content and
energy of feeds. The distribution of lipids in the fillet of
fish is uneven and depends on the species, and location
(Katikou et al., 2001) reported differences in the lipid
content between dorsal and ventral as well as anterior
and posterior sections of Salmo salar , the anterior parts
contain more fat than the posterior parts and the same for
the ventral and dorsal parts . Thakur et al., (2003)
determined varying amounts of lipid in the anterior
dorsal, dorsal, and tail sections of muscle in cultivated
Seriola quinqueradiata .
The studies in Brown trout of Toussaint et al.,
(2005) have shown that differences in lipid contents exist
in the fillet of this fish .Feeding a high-energy diet
resulted in significantly higher lipid deposition,
especially in the anterior ventral region of the muscle
(Toussaint et al., 2005). It is evident from our results,
that different levels of fat in parts of the fillet result of
the increased ability of the fish to capture more fatty
acids from the diet A, diet with high lipid content and
low a ratio of DP/DE. The activity of the Lipoprotein
lipase (LPL) at preferential sites of storage was higher
when the dietary fat intake was high. (Arantzamendi,
2002; Kolditz et al., 2008) .
In recent years fish nutrition has foccused on one
main objective, the content of polyunsaturated fatty acids
n-3 and n-6 acid (Wall et al., 2010). In general, lipid
content in the different fillet portions was correlated to
PUFA content (Medale, 2010). The fatty acid
composition of fillets is reported in table 2, as
Journal of Research in Biology (2012) 2(7): 617-625
percentages of total fatty acids (g/100 g fillet).
Polyunsaturated n-3 (PUFA n-3) fatty acids and the
n-3 / n-6 ratio were significantly higher in the trout fed
with diet A.
Freshwater fish normally contain more omega 6
PUFAs, whereas marine fish are rich in omega 3 fatty
acids (Tocher,2003).
The lipids of the muscle from rainbow trout
examined in the present study consisted mainly of
polyunsaturated fatty acids (PUFA). The n-3 FAs and
n-6 FAs are two bio-chemical families within the
PUFAs, and they also have different biological effects
(James and Cleland, 1996).
The omega 3/omega 6 ratio has been an indicator
to be useful for comparing the relative nutritional value
of different fish species. The n-3/n-6 ratio depends on the
type of diet consumed (Steffens and Wirth 2005). The
Omega-3/Omega-6 ratio has been suggested to be a
useful indicator for comparing the relative nutritional
value of fishes. (Sargent et al., 2002).
The ratio of n-3 n-6 PUFAs is an important
index that enables a comparison of the relative nutritive
value of lipids. According to Valfre et al. (2003), in
freshwater sh species, the ratio is varied from 1 to 4. In
general n-3 n-6 PUFAs ratios are higher in wild sh
species than in the farmed ones (Grigorakis et al., 2002).
The ability to elongate and desaturate fatty acids is not
the same in all the fish species, and freshwater fish such
as rainbow trout is capable of this bioconversion.
(Tocher, 2003). The current recommendation regarding
the consumption of PUFA in human nutrition is 1:4
(the ratio of n-3 to n-6 PUFA) (Ralph, 2000).
Flesh quality can be influenced by the
bi och em i ca l com posi t i on of fi sh fi l l et s
(Hernndez et al., 2002). A n 3 /n 6 ratio of 1:1 is
considered to be optimal for nutritional purposes but
ratio within 1:5-1:0.68 would constitute a healthy human
diet (Simopoulos and Cleland, 2003). The ratio of n3 /n6
in total lipids of freshwater fishes changes mostly
621

between 0.55 and 5.60, whereas for marine fishes is
between 4.7-14.4 (Henderson and Tocher, 1987; Hearn
et al., 1987). Hence, our findings are in accordance with
those earlier studies. Our studies indicate that the values
of the n3/n6 ratio were the most advantageous in the
fillets of fish fed with the extruded diet A, therefore the
ratio of the extruded diet A is favourable for health
compared to the extruted diet B.
Our studies indicate that the values of ratio n3/n6
were more favourable in the fish fillets fed with the
extruded diet A. Our results for the ratio n3 / n6 showed
a rate of 1.08 and 0.90 respectively for the extruded
foods A and B. The values are higher than the values
found by Stancheva and Merdzhanova (2011) with a
ratio of 0.84, despite the fact that the ratio of
omega-3 / omega-6 is in freshwater fish ranging from
0.55 to 5.60. So, from this study it is evident that
extruded diet A is rich in n 3 and n6 when compared to
the extruded diet B. The diet A has a large deposit at the
fish fillets, which generated high levels of omega 3 and a
better ratio n3/n6 (Kolditz,2008; Medale, 2010).
CONCLUSION
Fish is a source of protein and fat and its
consumption is recommended for its beneficial effects on
health. In fact, fish is the main source of polyunsaturated
fatty acids long chain (PUFA) n-3 (or omega 3) in
human food, but its contents of these products depend
mainly on the feed, the content of fat in food, the ratio of
DP/DE , by its energy content and its composition will
reflects the levels of lipids and fatty acids in the flesh of
fish.
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 Journal of Research in Biology
Occupational Diseases In Textile Dyers A Brief Review
Journal of Research in Biology
Authors:
Clara G Sargunar1 and
Ravishankar S2.
Institution:
1.Assistant Professor, PG
and Research Dept. of
Zoology, Government Arts
College, Coimbatore, Tamil
Nadu, India.
2. Associate Professor, PG
and Research Dept. of
Zoology, Kongunadu Arts
and Science College,
Coimbatore, Tamil Nadu,
India.
Corresponding author:
Clara G Sargunar.
Email:
clarags@gmail.com
Phone No:
09363114874.
Web Address:
http://jresearchbiology.com/
documents/RA0282.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
REVIEW
ABSTRACT:
The textile industry not only accounts for a large percentage of India's industrial
production and export earnings, but also generates employment in both organized and
unorganized sectors. The industrial operations, specifically dyeing, encompasses many
occupational diseases, which have either long term or short term health effects, depending
on the type of exposure and its duration. The exposures to such chemicals may occur,
through any route of entry, like inhalation or absorption. This study attempts a brief review
of the occupational diseases caused by textile dyes and suggests mitigation measures.
Occupational skin diseases like Allergic Contact Dermatitis (ACD) and Irritant
Contact Dermatitis (ICD), involving exposed sites, cause significant morbidity in textile
industry workers. Occupational eczema and urticaria from reactive dyes, has also been
reported. Dyes used by textile industries are known to be carcinogenic, teratogenic and
mutagenic, with genotoxic risk to textile dyers. The International Agency for Research on
Cancer (IARC) has classified various dyes as being associated with cancer in humans.
The spraying of Acramin F system, led to Organizing Pneumonia (OP). Reactive dyes
(Lanasol Yellow 4G) and carmine dye have been implicated as etiologic agents of
occupational asthma and allergic rhinitis. Occupational exposure to vat dyes may result in
sub-clinical adverse effects on the liver.
Strategies like, implementation of safety measures according to the type of work,
periodic screening coupled with worker-oriented educational approaches, further
epidemiological study, and modern Occupational Health Safety (OHS) legislation will help
deal with this problem.
Keywords:
Occupational disease, textile dyes, carcinogen, mutagen.
Abbreviations:
A CD All ergi c Co nt act De rmat iti s , I CD I rrit an t Cont ac t De rmatiti s ,
IARC International Agency for Research on Cancer, OP Organizing Pneumonia, SCEs Sister
Chromatid Exchanges, CA Chromosomal Aberration, PRR Pooled Relative Risk, ALP Alkaline
Phosphatase, ALT Alanine Transaminase, AST Aspartate Transaminase, SHE Sentinel Health
Event, OHS Occupational Health Safety
Article Citation:
Clara G Sargunar and Ravishankar S.
Occupational Diseases In Textile Dyers - A Brief Review.
Journal of Research in Biology (2012) 2(7): 626-633
Dates:
Received: 10 Sep 2012 Accepted: 01 Oct 2012 Published: 12 Oct 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
626-633 | JRB | 2012 | Vol 2 | No 7
www.jresearchbiology.com

INTRODUCTION
Textiles account for a large percentage of India's
industrial production and export earnings. The textile
industry covers a wide range of economic activities,
including employment generation in both organized and
unorganized sectors. The industry comprises diverse
operations such as fiber synthesis, weaving,
manufacturing, dyeing and finishing.
There are numerous health and safety issues
associated with the textile industry. They include:
chemical exposure from the processing and dyeing of
materials, exposures to various solvents, exposure to
cotton and other organic dusts, musculoskeletal stresses,
and noise exposure. The exposures to chemicals may
occur through inhalation or absorption. Thus, textile
industries encompass many occupational hazards, which
have either long term or short term health effects
(occupational diseases), depending on the type of
exposure and its duration.
An occupational disease is any chronic ailment
that occurs as a result of work or occupational activity.
They are the illness caused by the substances or
conditions that the worker was brought into contact with
at the workplace or while he was working at his
respective work environment.
Occupational skin diseases of textile dyers
Occupational skin disease can cause significant
morbidity in textile industry workers. Both Allergic
Contact Dermatitis (ACD) and Irritant Contact
Dermatitis (ICD) have been reported among textile
workers. Soni and Sherertz (1996) characterized and
determined the relative frequency of work-related ACD
and ICD in textile workers. 29% were diagnosed as
having a predominantly work-related ACD. Allergens
included textile dyes. 38% were diagnosed as having
work-related ICD. The hands were the most common site
of involvement.
Singhi et al., (2005) studied the prevalence of
contact dermatitis among workers engaged in the 'tie and
627
Sargunar and Ravishankar, 2012
dye' industries in and around Jodhpur (Western
Rajasthan, India). 7.69% had dermatitis involving the
exposed sites, the hands and forearms. The nature of the
job had a significant bearing on the prevalence of skin
lesions. The dermatitis showed clearing on temporary
discontinuation of work and relapse on resuming the
work. Singh (1970) found benzanthrone to be the main
allergen causing dermatitis. Mathur (1981) described
49 cases of contact dermatitis among 250 workers in a
cottage dyeing industry in Jaipur (Rajasthan).
A study of the clinical and aetiological features
of contact dermatitis in Israel, to assess the sensitization
to textile dyes, revealed that 12.9% had allergic reactions
to a dye and/or resin allergen (Lazarov, 2003). Although
chronic dermatitis was the typical clinical presentation,
purpuric, hyperpigmented and papulopustular lesions,
erythema multiforme-like, nummular-like lesions,
lichenification and erythrodermia were observed.
The trunk and extremities, less frequently, the hands,
face, genital area and the soles were affected.
Estlander (1988) reported five cases of
occupational eczema, urticaria and respiratory disease
from reactive dyes, among workers in dye houses or
textile plants, who were exposed to reactive dyes for
eight months to four years before symptoms developed.
The efficacy of using barrier creams in lowering
the incidence of skin lesions was assessed among
workers of dyeing and printing factories in Como,
N. Italy (Duca et al., 1994). The use of a hydrocarbon
cream was found to be more effective than a silicone
cream.
Genotoxic and Mutagenic Effects of Textile Dyes
Mathur et al., (2012) have reviewed literature on
the mutagenicity of azo and non-azo textile dyes, and
discussed the genetic hazards associated with the
production and use of these dyes throughout the world.
Donbak et al., (2006) evaluated the genotoxic
risk of workers from textile dyeing plants
in Kahramanmaras, Turkey. Sister Chromatid
Journal of Research in Biology (2012) 2(7): 626-633
Sargunar and Ravishankar, 2012
Exchanges (SCEs) and Chromosomal Aberrations (CAs)
were investigated in peripheral blood lymphocyte
cultures. The frequency of CA was significantly higher.
There was a significant correlation between years of
exposure and CA frequency.
Mathur et al., (2005) tested seven dyes used in
textile printing and dyeing in Pali district, Rajasthan, for
their mutagenicity, by Ames assay, using strain TA 100
of Salmonella typhimurium. Three of these were
processing dyes or cremazoles (Orange 3R, Brown GR
and Blue S1) while remaining four were direct dyes
(Violet, Congo red, Royal blue and Bordeaux). Only one
dye, Violet showed absence of mutagenic activity. The
remaining six dyes were positively mutagenic, and
caused genetic damage through base pair substitution
mutations.
Carcinogenic effects of textile dyes
Many of the dyes used by textile industries are
known carcinogens (IARC, 1982; Jenkins, 1978) and
teratogens (Beck, 1983). The International Agency for
Research on Cancer (IARC) has classified various dyes
like benzidine as being associated with cancer in humans
(IARC, 1982). Two benzidine dyes, Direct Blue 6 and
Direct Black 38, have been reported to be such potent
carcinogens that, hepatocellular carcinomas and
neoplastic liver nodules occurred in rats after only
13 weeks of exposure (Robens et al., 1980). A number of
dyes have been tested for mutagenicity using Salmonella
assay. Several of them have been found to be
carcinogenic (Garner and Nutman,1977; Venturini and
Tamaro, 1979). Triple primary cancers involving kidney,
urinary bladder and liver in a dye worker have been
reported (Morikawa et al., 1997).
Workplace exposures account for 5 to 25% of all
bladder cancer cases. Olfert et al., (2006) reviewed the
literature between 1938 and 2004, and found that
occupational exposures to bladder carcinogens,
particularly beta-naphthylamine occur in a number of
industries, including dyestuff manufacture and use.
Journal of Research in Biology (2012) 2(7): 626-633
Mastrangelo et al., (2002) analyzed
epidemiologic studies for textile industry workers, to
evaluate whether the cancer risk varied within the textile
industry in relation to the job held. The increased bladder
cancer Pooled Relative Risk (PRR) in dyers was
attributed to textile dye exposure.
The association of aniline dyes with bladder
cancer was first described in 1895. Dye intermediates,
such as beta-naphthylamine (BNA) and benzidine have
been implicated in the development of bladder cancer;
ortho-toluidine and ortho-dianisidine are also suspected
agents.
Morrison et al., (1985) evaluated the relationship
between occupational history and the development of
cancer of the lower urinary tract ("bladder cancer").
In Boston, Massachusetts, USA, elevated risk of bladder
cancer associated with employment related to dyes was
observed.
Gonzales et al., (1988) investigated the possible
causes of an unusually high mortality rate from bladder
cancer in Mataro, Spain, with focus on occupational
exposures. An increased risk for past employment in the
textile industry was found. Further analyses indicated
that the risk was elevated for subjects who worked in
dyeing or printing, and who were most probably exposed
to azo-dyes.
Urothelial tumors were detected in 10.3% male
dye workers in Wakayama City (1951-1990), formerly
engaged in manufacturing of benzidine and/or
beta-naphthylamine. The mean period from exposure to
such carcinogenic chemicals to the onset of the disease
was estimated to be 25 years (Shinka et al., 1991).
Further, the biological behavior of occupational
urothelial tumors may be different from that of urothelial
tumors in the general population.
Frumin et al., (1990) reported six cases of
bladder cancer from different fabric dyeing plants in
New Jersey and North Carolina. The average latency
from onset of exposure to diagnosis was 23.3 years.
628

The results of You et al., (1990) suggested that,
in Shanghai, the main cause of bladder cancer was
occupational exposure, especially to benzidine. The risk
of bladder cancer, however, existed only in the
presynthesis stage of dye manufacture. Bladder cancer
was also reported in dye-manufacturing workers in South
Korea (Kim et al., 2007).
However, in a recent study, Serra et al., (2008)
investigated the risk of bladder cancer in Spanish textile
workers (1998-2001), but found no overall increased risk
for textile workers.
Occupational respiratory ailments in dyers
A study by Alanko et al., (1977) reports four
cases of immediate-type occupational allergy to reactive
dyes. All the patients had symptoms of asthma and
allergic rhinitis. The identification of specific IgE
showed that the mechanism of the hypersensitivity was
immunological, reactive dyes probably acting as haptens.
Romano et al., (1991) reported a case of
occupational asthma, in a wool and cotton dyer handling
reactive dyes. A bromoacrilamidic dye (Lanasol Yellow
4G) was identified as being responsible for the
sensitization. A very short (4-minute) exposure produced
a severe immediate obstructive ventilatory defect
followed by arterial hypotension and urticaria.
Carmine dye has been implicated as an etiologic
agent of occupational asthma. Lizaso et al., (2000)
identified three allergens of around 17, 28, and 50 kD
implicated in occupational asthma of three carmine
workers.
The outbreak of severe respiratory illness during
1992 among aerographic textile printing workers
in Valencia, Spain, was linked to the
inhalation of a reformulated aerosolized product,
Acramin-FWN. Clinical, laboratory, and pathological
data confirmed Organizing Pneumonia (OP). The
common clinical findings were cough, epistaxis,
dyspnoea, oppressive chest pain, and crackles. The
organizing pneumonia tended to evolve into progressive
629
Sargunar and Ravishankar, 2012
interstitial fibrosis. Once respiratory failure developed,
the prognosis was poor (Romero et al., 1998). Another
report by Moya et al., (1994) states that 27% of Spanish
textile dye sprayers developed bronchiolitis obliterans
organizing pneumonia.
Hoet et al., (1999) studied the pulmonary disease
in textile printing sprayers in Spain and Algeria
(Ardystil syndrome), caused by spraying of Acramin F
system. The study showed that, the three polycationic
paint components, Acramin FWR (a polyurea), Acramin
FWN (a polyamide-amine), and Acrafix FHN
(a polyamine) exhibited considerable cytotoxicity.
Effect of textile dyes on liver function and general
health
Soyinka et al., (2007) investigated the possible
effects of occupational exposure (textile dyeing and
finishing), to vat dyes on liver function in Abeokuta,
South Western Nigeria. The activity of
Alkaline Phosphatase (ALP) and the concentrations of
total protein and albumin were lower, while
Alanine Transaminase (ALT) and Aspartate
Transaminase (AST) were significantly higher, in the
exposed group. The results indicated that occupational
exposure to vat dyes resulted in sub-clinical adverse
effects on the liver, involving inhibition of its synthetic
function.
Mortality from diabetes and ischaemic heart
disease was found to be increased across a wide range of
textile occupations among people born in the Indian
subcontinent, with increased risk specific to men
(Zanardi et al., 2011).
The occupational health problems of desert
textile workers and their association with nutritional and
environmental factors were explored in Jodhpur and Pali,
Rajasthan by Singh et al., (2005). 25.5% of dyers
suffered the most from aches, probably due to a higher
percentage of severe anaemia, besides physical labour.
Mitigation measures and Conclusion
Occupational environment is the sum of external
Journal of Research in Biology (2012) 2(7): 626-633
Sargunar and Ravishankar, 2012
condition and influences which prevail at the place of
work and which have a bearing on the health of the
working population (Jaiswal, 2007). An occupational
Sentinel Health Event (SHE) is a disease, disability, or
untimely death which is occupationally related and
whose occurrence may: 1) provide the impetus for
epidemiologic or industrial hygiene studies; or 2) serve
as a warning signal that materials substitution,
engineering control, personal protection, or medical care
may be required (Rutstein et al., 1983).
Ramaswamy (1987) draws attention to the issues
which need to be considered when dealing with health
hazards due to toxic exposure in the work environment:
(i) Growth pattern of industries inherent with such
hazards. (ii) Statutory and other safeguards available for
controlling such hazards. (iii) Documented statistics on
the magnitude of such health hazards in the typical
industries/processes. (iv) Reasons for the shortfall in
efforts to control such health hazards in the past.
(v) Future strategy to control the hazards, and monitor
the levels from time to time.
The need of the hour is compulsory
implementation of worker safety measures according to
the type of work in the textile industry, Occupational
exposure to hazardous chemicals can be prevented or
minimized by using protective equipment such as gloves,
goggles, masks, etc.
Researchers should focus on collecting
epidemiological data and evidences as to the nature of
conditions caused, chemical/allergen responsible, the
type of workforce at risk, period of exposure, latency
period, if any, rate of incidence, etc. The mutagenicity
testing of textile dyestuffs is crucial for accurately
predicting health risks for consumers and workers
exposed to dyes (Mathur et al., 2012).
Based on the epidemiological data collected,
periodic screening programs should be conducted for
both the organized and unorganized workforce, in
addition to extension of health and nutrition education
Journal of Research in Biology (2012) 2(7): 626-633
and welfare programs. Soni and Sherertz (1996)
emphasize the importance of patch testing with standard
screening allergens and textile dye and finish allergens,
in the diagnostic evaluation of patients with dermatitis
who work in the textile industry. As bacterial
mutagenicity assays can be carried out in 48 hrs, they
have been suggested as rapid pre-screens for
di st i n gui shin g bet ween ca r ci n ogen i c an d
non-carcinogenic chemicals (Mathur et al., 2005). Ames
test (Ames et al., 1975) can easily and quickly assess
mutagenic potential of these dyes. Screening of high risk
populations with urinary cytology tests was found to be
effective for early diagnosis and treatment of urothelial
tumors, and it improved patient prognosis (Shinka et al.,
1991). Screening programs will be more successful if
coupled with worker-oriented educational approaches, as
compliance with health screening programs will be
greater in the case of better-informed employees
(Frumin et al., 1990).
Agnihotram (2005) suggests strategies such as
modern Occupational Health Safety (OHS) legislation,
enforcement machinery at sub-district level, training to
health professionals, and international collaboration, to
deal with the situation.
The interaction between man and his working
environment may lead to betterment of health, when
work is fully adapted to human needs and factors, or to
ill health, if work stresses are beyond human tolerance.
The administrative machinery, industrial community and
society at large are thus faced with the daunting task of
ensuring the safety of the workforce which toils for its
economic prosperity.
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633 Journal of Research in Biology (2012) 2(7): 626-633

 Journal of Research in Biology
Formulation and evaluation of alginate based mesalazine matrix tablets for
intestinal delivery
Journal of Research in Biology
Authors:
Lone KD1 Dhole JA2 and
Dhole NA3.
Institution:
1. JSPMs Rajarshi Shahu
College of Pharmacy and
Research, Tathawade, Pune.
2. Department of Botany,
Yeshwant Mahavidyalaya,
Nanded.
3. School of Life Sciences,
S.R.T.M. University,
Nanded-4316063.
Corresponding author:
Lone KD.
Email:
krishnalone@gmail.com
Web Address:
http://jresearchbiology.com/
documents/RA0280.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
The aim of this study is to develop alginate based mesalazine matrix tablets
for intestinal delivery. Sodium alginate is a biocompatible natural polymer, with pH
sensitive gel forming ability. Matrix tablets of Mesalazine were prepared using Sodium
alginate with three different concentrations by wet granulation method. The granules
were evaluated for angle of repose, bulk density, tapped density, compressibility index
and Hausners ratio. The tablets were subjected to weight variation, hardness, friability
and drug content test. The in vitro release characteristics of mesalazine from alginate
tablets were compared with those of the commercial product Asacol.
The cumulative amount of released drug of S3 formulation was found to be
almost the same as the of commercial product in acidic and basic media. The release
profiles were affected by variable concentrations of Sodium alginate and hence, the
release of Mesalazine was prevented in upper GIT with increase in the proportion of
Sodium alginate. Mesalazine-alginate matrix tablet formulations can deliver drug to
the small and large intestine. Thus, it may be a promising system for the treatment of
Ulcerative colitis.
Keywords:
Mesalazine, Sodium alginate, Intestinal drug delivery, ulcerative colitis.
Article Citation:
Lone KD, Dhole JA and Dhole NA.
Formulation and evaluation of alginate based mesalazine matrix tablets for intestinal
delivery.
Journal of Research in Biology (2012) 2(7): 634-640
Dates:
Received: 01 Sep 2012 Accepted: 02 Oct 2012 Published: 13 Oct 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
634-640 | JRB | 2012 | Vol 2 | No 7
www.jresearchbiology.com

INTRODUCTION:
Oral administration is the most convenient and
proffered means of drug delivery into systemic
circulation. (Hwang et al., 1998). The
pH environment of GI tract varies from acidic to slight
alkaline. pH-sensitive hydrogels may be an alternative
for site specific drug delivery (Wilding, 2000). A variety
of synthetic and natural polymers with acidic or basic
pendent groups have been used to fabricate pH sensitive
hydrogels. Among them, alginate is one of the most
commonly used natural polymer. (Lin et al., 2005).
Alginates are naturally occurring substances mostly used
in pharmaceutical dosage formulations, particularly as
vehicle for controlled drug delivery. The formulation of a
matrix, upon hydration, causes a gelatinous layer which
can act as a drug diffusion barrier (Bajpai, 2004).
Mesalazine has been used for several years in the
treatment of inflammatory bowel disease. When pure
mesalazine, administered directly in proximal part of
small bowel or orally as conventional tablet, it is rapidly
and almost completely absorbed, with little drug
reaching the distal small intestine and
(Prakash, 1999). Therefore premature absorption of
mesalazine can be prevented by the preparation of
enteric coated tablets. Orally administered
delayed-release mesalazine, act locally from within the
lumen of the inflamed bowel and is partly absorbed in
systemic circulation. To prevent proximal
intestinal absorption, and to allow mesalazine to reach
the inflamed small bowel/colon, a variety
mesalazine delivery systems have been developed
(Altamash et al., 2005). The aim of the present study was
to develop a site specific matrix tablet of mesalazine with
sodium alginate and to investigate the in vitro release
characteristics of the tablets and compare them with
those of the commercial product Asacol.
635
Lone et al., 2012
MATERIALS AND METHODS
Materials
Mesalazine was obtained from Sarex Overseas,
natural Mumbai, India, Sodium alginate from Alembic
Pharmaceuticals Ltd., Baroda, Povidone (PVP-K30) and
microcrystalline cellulose from Signet chemical
corporation, Mumbai for free of cost. Talc and
Magnesium stearate were procured from Emcure House
M.I.D.C. Pune. All the other chemicals and reagents used
were of analytical grade. The commercially available
mesalazine product, Asacol was procured from A. Birla
hospital (Pune).
Measurement of viscosity
Viscosity measurement of 1% W/V aqueous
dispersion of sodium alginate was carried out using
Brookfield viscometer (Brookfield viscometer LVTD
USA) at 25C.
Preparation of Mesalazine matrix tablets
For preparing matrix tablets, the content of
Mesalazine was maintained at 250 mg, in each type of
formulation. The accurately weighed quantities of
colon selected polymers and the drug were mixed in various
proportions and the mixtures were assigned different
formulation codes presented in table-1 (Aultons, 1998).
The active ingredients Mesalazine, the polymers,
sodium alginate were passed through screen (60 #). The
physical mixtures of the drug, polymers and excipients
small were prepared by blending the accurately weighed
quantities of each of them with Mesalazine in geometric
of proportions in glass mortar for 15 minutes. Ethnolic
solutions of PVP K-30 were used as binders which were
added gradually to powder blends with trituration until a
coherent moist mass was formed. This mass was passed
through screen (22#) to get moderately coarse granules.
The wet granules were dried at 50C for 1 hour. The
dried granules were again passed through screen (44#) to
obtain fine granules. The resulting granules were
lubricated with magnesium stearate and then evaluated
for the following flow properties bulk density, tapped
Journal of Research in Biology (2012) 2(7): 634-640
Lone et al., 2012

Table-1. Formulations of the matrix Hardness:

tablets of Mesalazine. Hardness of three tablets of each formulation
Ingredients Formulation codes
Sr. No. type was determined using Monsanto hardness tester
(%W/W) S1 S2 S3
1 Mesalazine 50 50 50 following the procedure described in standard text book
2 Sodium alginate 15 30 45 (Liberman, 2001).
3 PVP K-30 3 3 3
4 Magnesium state 1 1 1 Friability:
5 Aerosil 1 1 1 The friability of 10 tablets of each formulation
6 Talc 30 15 0
type was noted using Roche friabilator following the
Total weight of one tablet is 500mg
procedure described in standard text book
density, compressibility index (C.I.) and Angle of (Liberman, 2001,). And the weight loss (% w/w) was
repose (). calculated using the following formula,
The granules of each formulation type were % Friability = (Loss in weight/ Initial weight) x 100
compressed into matrix tablets using S.S. punches Drug contents
(diameter 13 mm flat surface) on rotary tablet press. The The contents of Mesalazine were estimated using
compression force was maintained in such a way that the five tablets of individual formulations. The tablets were
hardness of resulting tablets ranged between weighed individually, and were crushed in mortar. From
2
7-8 Kg/m . The batch size prepared for each formulation this, the powder equivalent to 250 mg of Mesalazine was
was of 25 tablets. taken in volumetric flasks and dissolved in sufficient
Evaluation of matrix tablets of Mesalazine quantity of phosphate buffer (pH 7.2) and the final
Weight variation volume was made up to 100 ml. Appropriate dilutions of
This test was performed as per the procedure the resulting solutions were carried out and the contents
described in Indian Pharmacopoeia (1996) using of Mesalazine were estimated using UV absorbance of
10 tablets of each formulation type. The tablets were these solutions at 331 nm using previously prepared
weighed individually and their mean weight was calibration curve of Mesalazine in phosphate buffer
calculated. The deviation of individual weight from the pH 7.2 (Acarturk and Demiroz, 2007).
mean was expressed as standard deviation. The In vitro release of Mesalazine from matrix tablets
compliance of tablets with recommended allowances for The test was conducted using three matrix tablets
variations in weight was judged on the basis of official of each type of formulation using USP (23) dissolution
specifications. apparatus (Apparatus I). (Watts and Illum, 1997) The

Table-2: The experimental conditions used for in vitro release of Mesalazine from matrix tablets.
Volume withdrawn and
Type and volume of Speed of Duration max used for
Phases Recording absorbance
frequency of withdrawn
Dissolution Medium Rotation (rpm) (min)
aliquots
Phase I
0.1N HCl 500ml
Acid stage 100 rpm 120 303.0 10ml at intervals of 30min
pH- 3
Phase II
Buffer Phosphate buffer 900ml
100 rpm 60 330.0 10ml at intervals of 30min
stage-1 pH- 6

Phase III
Buffer Phosphate buffer 900ml
50 rpm 90 331.0 10ml at intervals of 30min
stage-2 pH-7.2

Journal of Research in Biology (2012) 2(7): 634-640 636

 Lone et al., 2012

Table-3: Flow properties of granules of Mesalazine with individual pH sensitive

HPMC polymers and their combination with sodium alginate.
Code Loose bulk density (g/ml) Tapped bulk density (g/ml) Carrs Compressibility Index. (%) Angle of repose()
S1 0.1550.0190 0.1830.004 15.300.005 25.810.021
S2 0.1470.0190 0.1840.011 19.780.002 24.250.022
S3 0.1640.0195 0.1850.005 11.350.013 25.970.016
All values are expressed as mean SD, n=3
tablets of each type of matrix formulations were kept in angle of repose 24.25-25.97. All the flow characteristics
baskets which were placed successively in below are satisfactory for subsequent processing of granules for
mentioned dissolution media. The dissolution apparatus preparation of matrix tablets of Mesalazine.
was run maintaining test conditions are stated below in Evaluation of Mesalazine Tablets
table 2. (Liew et al., 2006). The release profiles of matrix The matrix tablets of Mesalazine prepared with
tablets were compared with those of the commercial Sodium alginate were characterized for various tablet
product Asacol (Acarturk and Demiroz, 2007). characteristics as per the monograph mentioned in
Stability Studies table 4.
Stability studies were carried out to assess the The pharmacopoeial specifications for deviation
stability of all formulated sustain release Mesalazine in weight from average weight for tablets weighing more
tablets (Melia and Davies1995). The prepared tablets than 250 mg are 5%. The percentage deviation in the
were kept at 452C, 755% RH for 45 days. At 15 weight of prepared tablets (weighing 500 mg) was within
days intervals the tablets were evaluated for all physical the specified limits for all the formulation types and
parameters. The percentage of Mesalazine content and hence, they complied with the test for weight variation.
Invitro drug release studies were also determined. Diameter of the matrix tablets was in the range of
12.87-12.89mm. Thickness of the matrix tablets was in
RESULTS AND DISCUSSION the range of 3.32-3.38 mm. Hardness of the matrix
Evaluation of Mesalazine Granules: tablets was in the range of 8-8.5 Kg/cm2, Friability of the
The values for loose bulk density, tapped bulk matrix tablets of Mesalazine was within 0.33-0.48%.
density, compressibility index and angle of repose of The matrix tablets of different formulations
granules of Mesalazine prepared with Sodium alginate, possessed consistent dimensions and hardness values and
revealed different behaviour of each formulation blend all of them complied with the specified limits for
expressed in table 3. However, all these values are still friability (<1%). The higher values of hardness may be
suggestive of good flowability of blends. justified since the drug is targeted for colon and the
Values of loose bulk density and tapped bulk inclusion of pH sensitive polymer would ensure its
densities for Mesalazine granules ranged between release in the destined organ. The pharmacopoeial
0.147-0.164, and 0.183 to 0.185 respectively. The Carrs specifications for permissible allowances for deviation in
index values ranged between 11.35-19.78, the values of
Table-4: Characterization of matrix tablets of Mesalazine.
Code Avg. Wight Diameter Thickness Hardness Friability Drug
(mg) (mm) (mm) (Kg/cm) (%) Content (%)
S1 4991.63 12.88 3.38 8.0 0.44 97.350.122
S2 5010.95 12.87 3.34 8.0 0.48 98.880.183
S3 5011.25 12.89 3.32 8.5 0.33 98.560.155
All values are expressed as mean SD, n=3
637 Journal of Research in Biology (2012) 2(7): 634-640
Lone et al., 2012

Table-5: Dissolution data of matrix tablets of Mesalazine with variable concentration of sodium alginate
Dissolution Phase
Cumulative (Avg.) % drug release
and duration
Time (min)
S1 S2 S3
15% 30% 45%
0 0 0 0
Acid Stage pH= 3
30 9.500.48 7.720.98 3.211.20
( 120 min)
60 11.470.84 9.461.12 5.611.10
90 13.970.37 11.200.44 6.460.49
120 15.641.94 13.312.12 9.781.62
Buffer Stage-1 pH-6 150 18.901.19 15.181.41 11.781.13
( 60 min) 180 22.870.94 18.541.74 13.521.41
210 29.261.69 26.421.38 21.282.23
240 43.622.45 37.921.90 32.491.91
Buffer Stage 2 pH= 7.2 270 55.281.56 48.771.43 44.691.26
(150 min) 300 69.121.13 60.171.43 57.121.20
330 83.240.82 71.342.78 69.212.30
360 97.411.21 86.551.32 83.141.78
*The dissolution studies were extended by 60 minutes in buffer stage 2 (pH 7.2) for estimating the time
taken for complete release of drug contents.
% of drug contents for tablets of Mesalazine is not less In vitro release of Mesalazine from matrix tablets
than 98% and not more than 101% of the labelled prepared with variable concentration of Sodium
amount. (USPNF 2004). The percent of drug contents for alginate.
Mesalazine formulations with sodium alginate ranged The formulations S3 qualified the first stage of
between 97.35-98.88 Hence, the tablets are complied release while all formulations qualified the second stage
with the official specifications. of drug release. The release profiles were affected by
In vitro release of Mesalazine from matrix tablets variable concentrations of matrix forming polymer and
The USP specifications for % of cumulative hence, the release of Mesalazine retarded with increase
release of drug from colon targeted dosage forms are; in proportion of sodium alginate (table 5, fig. 1). Sodium
Acid stage: Not more than 12% of LA. alginate has pH sensitive gel forming ability because of
Buffer stage 1: Not more than 30% of LA (LA is that it could effectively prevent the escape of drug at
labeled amount) both the acid stage and buffer stage 1.
Cumulative (Avg.)%drug release

Cumulative (Avg.)%drug release

Fig 1: In vitro release of Mesalazine from matrix Fig 2: Comparative evaluation of Mesalazine
tablets prepared with Sodium alginate. matrix tablets with marketed formulation Asacol*

Journal of Research in Biology (2012) 2(7): 634-640 638

 Lone et al., 2012

Table-6: Comparative evaluation of Mesalazine matrix tablets with marketed formulation Asacol*
Dissolution Phase
Cumulative (Avg.) % drug release
and duration
Time (min) S1 S2 S3
Asacol*
15% 30% 45%
0 0 0 0 0
Acid Stage pH=3
30 0 9.500.48 7.720.98 3.211.20
( 120 min)
60 0 11.470.84 9.461.12 5.611.10
90 0 13.970.37 11.200.44 6.460.49
120 0 15.641.94 13.312.12 9.781.62

Buffer Stage-1 pH=6 150 0 18.901.19 15.181.41 11.781.13

( 60 min) 180 0 22.870.94 18.541.74 13.521.41
210 0.73 29.261.69 26.421.38 21.282.23
240 1.147 43.622.45 37.921.90 32.491.91

Buffer Stage 2 pH=7.2 270 42.18 55.281.56 48.771.43 44.691.26

(150 min) 300 79.82 69.121.13 60.171.43 57.121.20
330 92.12 83.240.82 71.342.78 69.212.30
360 98.54 97.411.21 86.551.32 83.141.78
*The dissolution studies were extended by 60 minutes in buffer stage 2 (pH7.2) for estimating time
taken for complete release of drug contents.

The comparison of in vitro release profile of the CONCLUSION:

formulation with those of the commercial product Asacol
showed that the matrix tablets released 22.87% of drug
during the first two hours, in acidic and one hour in
buffer stage 1 at pH-6. No mesalazine release was found
from Asacol* (Fig 2). The increasing amount of alginate
results in the increase in viscosity of gel layer,
which retards the drug diffusion from the tablet.
(Liew et al., 2006)
Stability Studies:
Mesalazine matrix tablets of all the formulations
o o
were stored at 45 2 C, 755% RH upto 45 days. Tablet
evaluation tests were carried out at every 15 days
intervals. All the formulations were physically stable.
There were no deviations found in the tests and all were
within the limits. There were no significant change in the
drug content and Invitro drug release profiles. It showed
that all the formulations are chemically stable.
The results of experimental studies of
Mesalazine matrix tablets proved that the granules of
Mesalazine showed good flow properties and that the,
tablet evaluation tests are within the acceptable limits.
The dissolution profile indicates that formulation
S3 prevents escape of Mesalazine in acidic pH, of not
more than 12% of the labeled amount. All the other
formulations minimize escape of Mesalazine in buffer
stage 1 (pH 6) i.e. not more than 30% of the labeled
amount. Sodium alginate offered better protection from
escape of Mesalazine in precolonic pH stages. Hence
matrix tablets of Mesalazine with optimum concentration
of Sodium alginate were successfully developed. It was
concluded that Mesalazine-alginate matrix tablet
formulation can deliver the drug to small and large
intestine.

639 Journal of Research in Biology (2012) 2(7): 634-640

Lone et al., 2012

ACKNOWLEDGEMENT: Melia CD, Davies MC, Mitchell JR. 1995. Structure

The authors are thankful to Sarex Overseas
Mumbai, India for providing sample of Mesalazine and
Alembic Pharmaceuticals Ltd. Baroda for sample of
Sodium alginate free of cost. Authors wish to thank
JSPMs Rajarshi Shahu college of Pharmacy and
Research, Pune for providing necessary facilities to carry
out the work.
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Acarturk F and Demiroz FT. 2007. Evaluation of
alginate based mesalazine tablets for intestinal drug
delivery. Eur. J. Pharm. Sci., 67: 491-497.
Altamash I, Qureshi, Ruddell D. Cohen. 2005.
Mesalamine Delivery system: Do they really make much
difference? Adv. Drug Del. Rev., 57:281-302.
Aultons ME. 1998. Pharmaceutics: The science of
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dosage form design.2 ed. London, England: Churchill
Livingstone, 208.
and behavior in hydrophilic matrix sustained release
dosage forms: 3. The influence of pH on the sustained-
release performance and internal gel structure of sodium
alginate matrices. Journal of Controlled Release.
33:143-152.
Pharmacopoeia of India. 1996. New Delhi: Ministry of
Health and Family Welfare, Government of India,
Controller of Publication;
Prakash A. 1999. oral delayed released Mesalazine,
Drugs 57:383-408
Watts PJ and Illum L. 1997. Colonic Drug delivery,
Drug Dev. Ind. Pharm., 23:893-913.
Wilding I. 2000. Sight specific drug delivery in
gastrointestinal tract, cric. Rev. Ther. Drug Carr.
Systems 17:557-620.

Bajpai SK. 2004. Investigation of swelling/degradation

behavior of alginate beads cross linked with Ca2+ and
Ba2+ ions, Reac. Func. Poly., 59:129-140.

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drug delivery systems, Cric. Rev. Ther. Drug Carr.
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Liberman HA, Lachman L and Kanig JL. 2001. The

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Journal of Research in Biology (2012) 2(7): 634-640 640

 Journal of Research in Biology
Effect of foliar fertilizer and some growth regulators on vegetative and
anatomical characters of dill (Anethum graveolens L.)
Journal of Research in Biology
Authors:
Majeed Kadhim Abbas
and Intedhar Abbas
Marhoon.
Institution:
College of Agriculture
Al-Qadisiya University
Diwaniya Iraq
Corresponding author:
Majeed Kadhim Abbas.
Email:
Majeed_edu@yahoo.com
Phone No:
964 780 2344057.
Web Address:
http://jresearchbiology.com/
documents/RA0244.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
This experiment was conducted to study the effect of foliar fertilizer (Oligo green
HF), gibberellin and naphthalene acetic acid and their interaction on some vegetative and
anatomical characters of (Anethum graveolens L.) Dill local names. The foliar fertilizer,
(contains Fe, Zn, Mn, Cu and B), was used at rates of 0, 50, 100, and 150mg/L while the
GA3 was used at the concentrations of 250, and 500mg/L and NAA at concentrations of
600 and 1000mg/L. The results showed that foliar fertilizer and both growth regulators
increased plant height, stem diameter, and shoot dry weight in proportion to increase in
the concentrations used. Number of branches were not affected by the application of
growth regulators but they increased significantly by the foliar fertilizer treatments. Also,
the lower concentrations of the two growth regulators showed no effect on number of
leaves while all rates of the foliar fertilizer increased the number significantly. All rates of
foliar fertilizer caused significant increase in cortex thickness, number and thickness of
vascular bundles, and vascular units diameter. Also, all growth regulators concentrations
increased cortex thickness significantly but they have no effect on vascular bundles
number.
Vascular bundles thickness and vascular units diameter decreased significantly
under the effect of both growth regulators. Pith thickness decreased significantly as the
foliar fertilizer rates increased but it increased significantly due to the use of the growth
regulators. Interaction between foliar fertilizer and gibberellin or naphthalene acetic acid
had significant effect on most characters studied. It can be concluded that all characters
studied are positively affected by factors under consideration.
Keywords:
Cortex thickness; Growth regulators; Micronutrients; Pith thickness; Vascular
bundles.
Article Citation:
Majeed Kadhim Abbas and Intedhar Abbas Marhoon.
Effect of foliar fertilizer and some growth regulators on vegetative and anatomical
characters of dill (Anethum graveolens L.)
Journal of Research in Biology (2012) 2(7): 641-651
Dates:
Received: 21 May 2012 Accepted: 01 Jun 2012 Published: 17 Oct 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
641-651 | JRB | 2012 | Vol 2 | No 7
www.jresearchbiology.com

INTRODUCTION
Dill (Anethum graveolens L.) is an annual,
aromatic herb plant belongs to umbelliferae family. It is
cultivated in central and southern regions of Iraq as a
winter crop. Dill plant is native to India and it has spread
to Mediterranean and Europe later and then to America
and Japan (Bailer et al., 2001). The plant has a potential
importance as a medicinal herb that contains volatile oils
such as B-camphene, -pinene, anethole, lonone,
umbelliferone, and carvone (Dhalwal et al., 2008 and
Sharma, 2004). Foliar fertilizer is a mean of increasing
agricultural production due to the rapid absorption of
nutrients by the plant. Micronutrients are needed in small
quantities for normal plant growth and development
(Abunyewa and Mercer-Quarshie, 2004; and Ali et al.,
2001) and to proceed biological processes such as
photosynthesis, respiration, synthesis of chlorophyll and
stimulation of many enzymes (Whitehead, 2000;
Sabir et al., 2002 and Refaat and Balbaa, 2001).
Plant growth regulators are used widely to improve plant
performance. Gibberellic acid is one of those growth
regulators that has positive effect on plant growth
through the effect on cell division and elongation
(Batlang et al., 2006; and Yamaguchi and Kamiya 2000).
Naphthalene acetic acid is also used to improve plant
growth (Ayan et al., 2004 and Resmi and
Gopalakrishnan, 2004). Anatomical characteristics of
stems such as cortex thickness and vascular bundles
diameter have been used as criteria for species
characterization (Khandelwal et al., 2002 and Kaplan
et al., 2007 and Sidhu and Saini, 2011). Several branches per plant, leaves number, and shoot dry weight
investigators study the effect of micronutrients and some
growth regulators on the anatomaical characters of
several plants and they found a pronounce effects on
thickness of epidermis; cortex and number and diameter
of vascular bundles (Agamy, 2004; and Jaleel et al.,
2009). The aim of the present study was to investigate
the effect of foliar application of some micronutrients,
Gibberellin, and Naphthalene acetic acid on some
642
Abbas and Marhoon,2012
vegetative and anatomical characters of Dill plant
(Anethum graveolens L.).
MATERIALS AND METHODS
This experiment was carried out during the
growing season of 2009-2010 in the fields of college of
agriculture/ Al-Qadisiya University to study the effect of
foliar fertilizer and two growth regulators, Gibberellin
and Naphthalene acetic acid, on some growth and
anatomical characters of Dill (Anethum graveolens L.)
cv. Local. The soil used for cultivation characterized as
silty clay with, PH 7.8, EC 2.7, and 1.08% organic
matter. The foliar fertilizer (Oligo green HF, Green HAS
Company) contains; Fe 5%, Zn 2%, Mn 2%, Cu 1%, and
B 0.5%. The foliar fertilizer was used at rates of 0, 50,
100, and 150mg/L. Gibberellin (Flagro Comp.) was used
at concentrations of 250 and 500mg/L while Naphthalene
acetic acid (Green River Comp) was used at
concentrations; 600, and 1000mg/L in addition to control
(spray with distilled water only). Seeds of dill were
planted at 15/10/2009 on 3m long rows with 75cm
between them. The total area of the experimental unit
was 9m2. Seeds were planted at a rate of 5-6 seeds per
cavity and at a distance of 25cm in between. After two
weeks, seedlings were thinned to only two. At
16/11/2009, the two growth regulators and the foliar
fertilizer were sprayed at the early morning. Spray was
repeated one month after the first spray. All agricultural
practices were done as usual. Vegetative characters of
plants such as; plant height, stem diameter, number of
were taken on three plants of each replicates at
13/1/2010. Three plants were chosen randomly at the
beginning of blooming at 28/1/2010 for anatomical
study.
Preparation of samples
Cross sections of the middle part of the stem
were taken. The fresh samples were preserved in glass
tubes containing ethyl alcohol at a concentration
Journal of Research in Biology (2012) 2(7): 641-651
Abbas and Marhoon,2012
of 70% to keep them fresh until use. Hand sectioning
was used to prepare thin sections. Sections were
transferred carefully by a brush into clean slides
containing drops of Safranin dye and left for a period of
5-7 minutes. Then, stained sections were transferred to
other slides containing drops of glycerin for immersion.
Samples were examined under light microscopy and
measurements were taken in micrometer using Ocular
Micrometer. Thickness of cortex, vascular bundles, pith
and vascular units diameter, and number of vascular
bundles were recorded. Sections were filmed using
lucida camera, wild type, erected on microscope under
the force of (X40). Experiment was designed as a
factorial experiment with two factors (4X5) in a
completely randomized design with three replicates.
Means were compared using LSD at 5%.
RESULTS
Results of Table 1 clearly show that all
treatments have significant effect on plant height. All
rates of foliar fertilizer used increased plant height
significantly. The tallest plants were achieved at the rate
of 150mg/L (82.27cm) while the shortest (70.76cm) were
at the control. For growth regulators, an obvious increase
in plant height was observed as correspondence to the
increase in concentrations of the two growth regulators
used. Gibberellin is more effective on plant height than
naphthalene acetic acid. The highest concentration of
gibberellin gives the tallest plants (84.83cm) compare to
79.68cm for the highest concentration of naphthalene
and 62.92cm for the control. Interaction between foliar
fertilizer and the two growth regulators shows significant
effect. Combination of 150mg/L of the foliar fertilizer
with 500mg/L of gibberellin gives the tallest plants
(93.33cm). In general, combination of 500mg/L with all
rates of foliar fertilizer gives tallest plants compare to
other combinations.
With regard to stem diameter, it was clear that
foliar fertilizer and the two growth regulators used
Journal of Research in Biology (2012) 2(7): 641-651
caused significant increase in diameter (Table 2).
Foliar fertilizer at 150mg/L records the highest stem
diameter (8.74mm) compare to 5.40mm for the control.
Also, the highest value of the diameter is achieved at
500mg/L of GA3 (7.75mm) and at 1000mg/L of NAA
(7.81mm). For the interaction, it is shown that the
highest two diameters (9.57 and 9.67mm) were reached
at the combinations of foliar fertilizer at 150mg/L with
500mg/L of GA3 or 1000mg/L of NAA, respectively.
The least stem diameter was at the control (4.33mm). All
foliar fertilizer rates increased significantly the number
of branches (Table 3). The percent increase over the
control was 26.25, 56.56, and 95.94% at the fertilizer
rates of 50, 100, and 150mg/L, respectively. However,
the use of the two growth regulators has no effect on
number of branches. For the interaction, the highest
number of branches was obtained at the combination of
150mg/L of the foliar fertilizer along with all
concentrations of both growth regulators.
Number of leaves increases in proportional
manner with the increase in the rate of fertilizer used
(Table 4). Plants sprayed with 150mg/L of the fertilizer
recorded the highest number of leaves per plant (33.67)
which was significantly differ from other treatments. For
the effect of the two growth regulators, it was noted that
the lowest concentration of both did not cause significant
increase in leaves number, while the highest
concentration of both caused significant increase.
However, the higher concentrations did not differ
significantly from each other in their effect. The highest
number of leaves per plant (32.1) was obtained at the
treatment of GA3 at 500mg/L followed by 1000mg/L
treatment (31.83). For the interaction, it was found that
the use of the highest rate of the fertilizer with either
GA3 at 500mg/L or NAA at 1000mg/L gave the highest
number of leaves per plant (41.00 and 39.70,
respectively).
Results in Table 5 show that as the foliar
fertilizer rate increases, the dry weight increases also.
643
 Abbas and Marhoon,2012

Table 1. Effect of foliar fertilizer (FF) and growth regulators (GR)

concentrations and their interaction on plant height (cm) of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 55.67 74.00 79.67 70.00 74.00 70.76
50 59.67 77.33 80.00 73.42 78.70 73.82
100 64.67 79.60 86.33 74.61 82.30 77.50
150 71.67 83.66 93.33 79.00 83.70 82.27
Mean 62.92 78.65 84.83 74.26 79.68
LSD p<0.05 foliar fertilizer growth regulators interaction
2.17 2.42 4.72

Table 2. Effect of foliar fertilizer (FF) and growth regulators (GR)

concentrations and their interaction on stem diameter (mm) of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 4.33 5.00 6.10 5.33 6.23 5.40
50 5.00 6.02 7.33 6.67 7.33 6.47
100 7.00 7.53 8.01 7.67 8.00 7.64
150 8.00 8.13 9.57 8.33 9.67 8.74
Mean 6.08 6.67 7.75 7.00 7.81
LSD p<0.05 foliar fertilizer growth regulators interaction
0.63 0.78 1.43

It is noted also that all concentrations of growth
regulators increased the percent dry weight. GA3 at
500mg/L gave the highest percent (18.06%). However,
the two growth regulators did not differ from each other
in their effect when they used at lower concentration.
The combination of foliar fertilizer at 150mg/L and
gibberellin at 500mg/L was superior in its effect.
This combination gave the highest percent (21.17%)
followed by the combination between the foliar fertilizer
at 150mg/L and NAA at 1000mg/L (20.12%).
Also, it was clear that all combinations that contain the
higher concentration of foliar fertilizer and higher
concentration of GA3 or NAA were the most effective
which indicates the positive response of plant to such
higher concentrations.
Stem cross-section reveals several distinct zones
(Figure 1) starting from the cuticle layer, the outer layer,
that surrounds a single row of cubic shape of epidermal
cells. The epidermis is followed by layers of cortex
which consist of paranchyma tissue. Cortex can be

Table 3. Effect of foliar fertilizer (FF) and growth regulators (GR)

concentrations and their interaction on number of branches of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
(0) GA3 GA3 NAA NAA mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 5.15 5.17 5.19 5.18 5.20 5.18
50 6.58 6.64 6.67 6.38 6.44 6.54
100 8.15 8.18 8.20 7.95 8.08 8.11
150 10.22 10.21 10.23 9.97 10.12 10.15
Mean 7.52 7.55 7.57 7.37 7.46
LSD p<0.05 foliar fertilizer growth regulators interaction
0.46 N.S 0.71

644 Journal of Research in Biology (2012) 2(7): 641-651

Abbas and Marhoon,2012

as a very narrow region, and the xylem. Xylem tissue

Figure 1. Transverse cross-section of Dill stem (X40).
distinguished into two distinct zones; the outer zone is
located directly under the epidermis and it is
characterized by having chlorenchyma and an ample
amount of chloroplasts. The inner zone is composed of
several rows of an ordinary parenchyma cells with thin
cell walls. Also, the cortex contains an oil ducts which
are located directly underneath the collenchymaus tissue.
Following the cortex there is the vascular
cylinder which consists of vascular bundles. The
vascular bundles are collateral. They are composed from
soft area represents the phloem, then vascular cambium
consists of vessels and tracheids. Vessels are arranged as
rows that consist of several circles or ovule vascular
units. The units become smaller in size whenever they
headed towards the pith. The pith contains large storage
parenchymaus cells. Parenchyma cells that are nearby
the wood become smaller in compare to that near the
cavity. It is also noted in the stem cross-sections under
view that the cavity occupies the center of the stem.
Results of Table 6 show clearly that all foliar fertilizer
rates increased significantly the cortex thickness. The
highest thickness (385.84m) was achieved at 150mg/L
of the fertilizer while the lowest thickness was at the
control treatment (277.48m). Also, both growth
regulators had significant effect on cortex thickness with
the highest concentration of both gave the highest
thickness. For the interaction, the combination of the
highest rate of foliar fertilizer along with either
gibberellin or naphthalene acetic acid at their
higher concentrations gave the highest cortex

Table 4. Effect of foliar fertilizer (FF) and growth regulators(GR) concentrations and their
interaction on leaves number of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 19.33 20.33 23.33 19.30 23.30 21.12
50 21.33 24.33 28.33 21.00 28.30 24.66
100 23.00 27.00 36.00 23.70 36.00 29.14
150 28.33 30.00 41.00 29.30 39.70 33.67
Mean 23.00 25.42 32.17 23.33 31.83
LSD p<0.05 foliar fertilizer growth regulators interaction
2.21 2.95 3.62

Table 5. Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on percent dry weight of shoot of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 13.12 13.82 14.82 13.12 13.86 13.75
50 12.95 14.82 16.31 13.83 14.98 14.58
100 15.40 17.13 19.95 17.12 17.98 17.52
150 18.31 19.33 21.17 18.67 20.12 19.52
Mean 14.95 16.28 18.06 15.69 16.74
LSD p<0.05 foliar fertilizer growth regulators interaction
0.42 0.48 0.94

Journal of Research in Biology (2012) 2(7): 641-651 645

 Abbas and Marhoon,2012

Table 6. Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on cortex thickness (m) of stem of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 224.11 261.31 316.05 263.95 322.00 277.48
50 249.27 312.53 337.72 325.07 339.93 312.90
100 298.81 353.56 388.19 371.18 400.02 362.35
150 322.33 381.33 415.12 390.31 420.10 385.84
Mean 273.63 327.18 364.27 337.63 370.51
LSD p<0.05 foliar fertilizer growth regulators interaction
10.03 10.81 13.88

thickness (415.12 and 420.10m respectively). the diameter of the vascular bundles units (Table 9).
Vascular bundles number and thickness were However, all concentrations of both growth regulators
increased significantly in proportional to the increase in decreased the diameter, whereas the 500mg/L of GA3
rates of foliar fertilizer (Table 7 and 8). The 150mg/L recorded the lowest diameter (46.69 m). The
rate gave the highest number and thickness of the combination of foliar fertilizer of150mg/L plus 0mg/L of
vascular bundles (18.80 and 684.01m, respectively). In the growth regulators recorded the larger diameter of the
contrast, the two growth regulators had no effect on vascular bundles units, while the combination of 0mg/L
number of vascular bundles in the vascular cylinder, of the fertilizer plus 500mg/L of GA3 recorded the
while they decrease the thickness at all concentrations lowest diameter.
especially the higher ones. Combination of foliar Results in Table 10 show that the increase in
fertilizer at 150mg/L with 0mg/L of the growth rates of foliar fertilizer was accompanied with a decrease
regulators gave the highest number of vascular bundles in pith thickness. The 150mg/L recorded the least
(18.85). The lowest number was obtained at 0mg/L of thickness (636.57mm) compare to the highest thickness
the fertilizer plus GA3 at 500mg/L (12.49). Treatments (886.07m) for control. All growth regulators treatments
of foliar fertilizer at 150mg/L regardless of the growth increase pith thickness and the increase in concentration
regulators used gave the thicker vascular bundles while of each caused an increase in thickness. The interaction
the lowest thickness was obtained using the combination between the two factors had significant effects.
of 0mg/L of the foliar fertilizer with gibberellin or Combination of foliar fertilizer at 0mg/L along with
naphthalene at their higher concentration. It was evident NAA at its higher concentration gave the highest pith
that all rates of the foliar fertilizer increased significantly thickness, and combination of 0mg/L of the fertilizer
Table 7. Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on number of vascular bundles Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 12.55 12.53 12.49 12.59 12.57 12.55
50 14.13 14.10 14.09 14.21 14.19 14.14
100 17.17 17.15 17.12 17.23 17.21 17.18
150 18.85 18.84 18.82 18.76 18.74 18.80
Mean 15.68 15.66 15.63 15.70 15.68
LSD p<0.055% foliar fertilizer growth regulators interaction
0.53 N.S 0.83
646 Journal of Research in Biology (2012) 2(7): 641-651
Abbas and Marhoon,2012

Table 8 Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on vascular bundle thickness (m) of stem of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 438.01 408.16 365.55 420.05 382.13 402.78
50 520.39 488.70 458.13 490.11 478.00 487.07
100 642.22 601.41 574.00 610.33 570.19 599.63
150 725.13 685.30 661.15 688.57 659.92 684.01
Mean 581.44 545.89 514.71 552.27 522.56
LSD p<0.05 foliar fertilizer growth regulators interaction
12.27 15.63 19.18

along with 500mg/L comes after. The combination of sub-apical meristem (Ezz El-Din and Khalil, 2004).
150mg/L of the fertilizer along with 0mg/L of the growth Similar results reported by (Gul et al., 2006; and Shah,
regulators gave the least pith thickness (584.98m) 2007). With regard to stem diameter, it has been stated
which was significantly less than other combinations. that the regulatory effect of growth regulators on cell
division and elongation at the stem radial direction
DISCUSSION resulted in an increase in stem diameter (Hooykass et al.,
The positive effect of foliar fertilizer in present 1999). This was noticed in our results (Table 2), and
study on plant height and growth in general may be due agreed with earlier findings by (Ntui et al., 2007) on
to the role of micronutrients in the fertilizer. It is known pumpkin and (Akter et al., 2007) on mustard. However,
that iron has a direct role in increasing chlorophyll other investigators did not notice any significant effect of
content and synthesis of cytochrome and ferridoxin some growth regulators on stem diameter on other plants
(Focus, 2003). Zinc has positive effect in the synthesis (Al-Shamery, 2008; and Leite et al., 2003).
of tryptophan, a precursor of IAA, that necessary for cell Some micronutrients such as manganese has a
elongation (Hopkins and Hner, 2004). Our results come hormonal regulation role in plant. Manganese may
in agreement with the results of (Swaefy, 2002) on activate IAA-oxidase and this would regulate the
Trachspermum ammi and (El-Sherbeny et al., 2007) on auxin content in plant tissues and in turn breaks
Ruta plant. Also, the obvious increase in plant height due the apical dominance and increases branching
to the use of the two growth regulators may be a (Hooykass et al., 1999). In addition, microelements have
attributed to the stimulatory effect of the growth indirect role in increasing plant content of carbohydrates
regulators on cell division and elongation at the and this may have stimulated the lateral buds to grow
Table 9. Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on diameter of vascular units (mm) of stem of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
GA3 GA3 NAA NAA
(0) mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 51.83 46.67 38.93 48.65 40.21 45.26
50 54.33 50.82 45.30 50.93 48.37 49.95
100 58.81 52.31 48.32 54.35 50.86 52.93
150 62.66 56.71 54.21 58.03 56.11 57.54
Mean 56.91 51.63 46.69 52.99 48.89
LSD p<0.05 foliar fertilizer growth regulators interaction
1.13 1.84 2.16

Journal of Research in Biology (2012) 2(7): 641-651 647

 Abbas and Marhoon,2012

Table 10 Effect of foliar fertilizer (FF) and growth regulators (GR) concentrations and their
interaction on pith thickness (mm) of stem of Dill (Anethum graveolens L.)
GR Conc.(mg/L)
(0) GA3 GA3 NAA NAA mean
FF Conc.(mg/L) (250) (500) (600) (1000)
0 781.27 894.18 917.21 899.87 937.21 886.07
50 723.33 787.46 819.71 795.88 823.13 789.90
100 653.85 711.89 792.32. 730.81 797.79 737.33
150 584.98 618.73 667.80 631.44 679.90 636.57
Mean 685.86 753.22 799.26 764.50 809.51
LSD p<0.05 foliar fertilizer growth regulators interaction
11.28 13.67 26.15
and differentiate (Hartmann et al., 2002). This positive comes in accordance with the results of of (Ezz El-Din
effect was obvious in the current results at which all and Khalil, 2004) on plantago and (El-Sherbeny et al.,
foliar fertilizer rates had positive effect on number of 2007) on Ruta plant. The present results also show that
branches (Table 3). Other investigators (Ezz El-Din and spray with GA3 or NAA cause an increase in the dry
Khalil, 2004) stated same results on other plants. matter of shoot and this may be correlated with the
However, the two growth regulators show no effect on increase in vegetative growth as mentioned earlier by
number of branches. This comes in agreement with the (Raifa et al., 2005).
results of (Khandelwal et al., 2002) on henna and Microelements may play a role, as cofactors, in
(Balraj et al., 2002) on chilli. On the other hand, activation of several enzymes such as those involved in
(Shah et al., 2007) found an increase in number of photosynthesis. This would reflect in increasing the
branches of black cumim due to the use of GA. Dufour accumulation of carbohydrate in cells (Mostafa, 1996).
and Guerin, 2005, mentioned that the positive Also, it is known that growth regulators activates some
effect of micronutrients on leaves number of enzymes in addition to de novo synthesis of proteins
Antharium andreanum may be due to their stimulatory which all contribute to stimulate growth (Collett et al.,
effects on carbohydrates synthesis and hormonal 2000). The increase in cortical thickness as a result of the
regulation and, as a result, growth in general. Our results treatments shown in this experiment (table 6) may be due
agreed with that statement in which number of leaves to the effect on the size of the parenchyma cells because
increases in proportional manner with the increase in the of the stimulation in the whole growth. These results
rate of fertilizer used (Table 4). Also, it was noted that come in agreement with the results of
the lowest concentration of both growth regulators did (Sharma et al., 1992) on potatoes stem that sprayed with
not cause significant increase in leaves number which gibberellin and auxin at different concentrations.
may indicate a suboptimal concentration. These Vascular bundles number and thickness are increased
results are in agreement with the earlier results of significantly as the rates of foliar fertilizer increased
(Raifa et al., 2005). The increase in dry weight that was (Table 7 and 8). The increase in size of vascular bundles
seen in this study (table 5) may be a result of the due to the fertilizer may be as a result of the
stimulation of metabolism and then an increase in the enhancement of the activity of cambium to form and
accumulation of dry matter. In addition, the increase in differentiate new vascular bundles. The result is in
vegetative growth due to the fertilizer would increase in agreement with the result of (Mohammed, 2005) who
photosynthesis rate and in turn the accumulation of was found an increase in stem diameter of
carbohydrates and, as a result the dry matter. This result treated Dill plant with some microelements, and also
648 Journal of Research in Biology (2012) 2(7): 641-651
Abbas and Marhoon, 2012

with the results of (Agamy, 2004) on sweet
fennel. In contrast, the two growth regulators had no
effect on number of vascular bundles in the vascular
cylinder, while they decrease the thickness at all
concentrations especially the higher ones. This may be
due to the negative effect on the diameter of vascular
units without the effect on the activity of cambium.
(Leite et al., 2003) mentioned that the decrease in stem
diameter of soybean treated with gibberellin was due to
the decrease in size of vascular cylinder layer. Also, our
result comes in agreement of the results of
(Starman et al., 1990) when they study the effect of
gibberellin on leaves of sunflower. A negative effect of
foliar fertilizer in decreasing pith thickness was noted
(Table 10), this may be due to increase in size of
vascular cylinder that occupies larger area of stem
diameter in the expense of pith region. Also, the results
show that the increase in rates of foliar fertilizer was
accompanied with a decrease in pith thickness. However,
all growth regulators treatments increase pith thickness
and this may explain the positive effect of those
promoters in increasing cell division and elongation.
Agamy RA. 2004. Effect of mineral and/ or biofertilizers
on morphological and anatomical characters,
chemical constituents and yield of sweet fennel
(Foeniculum vulgare Mill. cv. Dulce) plants grown in
calcareous soil. Egypt J Appl Sci., 19(3):55-75.
Akter A, Ali E, Islam MM, Karim R, Razzaque AH.
2007. Effect of GA3 on growth and yield of mustard. Int
J Sustain Crop Pord., 2(2):16-20.
Ali Z, Ahmed G, and Rahmam N. 2001. Effect of
zinc and manganese on the yield and quality of tomato.
Pakistan J Biol Sci., 4(2):156-157.
Al-Shamery WM. 2008. Effect of gibberellin and cultar
in the growth of two faba bean (Vicia faba L.) cultivars
grown on different salinity levels. MSc thesis. Al-
Qadisiya University. Iraq.
Ayan AK, Kurtar ES, Cirak C, Kevseroglu K.
2004. Bulb yield and some plant characters of summer
snowflake (Leucojum aestivum L.) under shading as
affected byGA3 and NAA at different concentrations. J
Agron.,3(4):29-300.

Bailer J, Aichinger T, Hackl G, Hueber KD, Dachler

CONCLUSION
M. 2001. Essential oil content and composition in
It was obvious that foliar fertilizer and both
commercially available dill cultivars in comparison to
growth regulators have positive effect on most vegetative
caraway. Industrial Crop and Products. 14:229-239.
characters studied especially at higher concentrations.
Foliar fertilizer caused significant increase in cortex Balraj R, Kurdikeri MB, Revanappa KA. 2002.
thickness number and thickness of vascular bundles, and Effect of growth regulators on growth and yield of chilli
vascular units diameter. Pith thickness decreased due to (Capsicum annuum L.) at different pickings. Ind J Hort.,
the use of foliar fertilizer but it increased due to the use 59(1):84-88.
of growth regulators. Interaction between the two factors
Batlang V, Emongor VE, Pule-Meulenburg F. 2006.
under study had significant effect on most vegetative and
Effect of benzyladenine and gibberellic acid
anatomical characters.
on yield and yield components of cucumber
(Cucumis sativus L. cv. 'tempo'). J Agron.,
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

A study on the effect of intensive health education on the biochemical profile of the
Diabetics living in the selected rural areas of Tirunelveli District in Tamil Nadu.
Journal of Research in Biology

Authors: ABSTRACT:
Pauline Suganthy
Vijayabarathy and Background: Non-communicable disease diabetes mellitus is on the rise globally and developing
Pushparani D. countries are also witnessing the burden. Once diabetes is diagnosed, adequate treatment
requires a significant amount of resources for patients of low economic standing. Being a
chronic disease, there is great scope for cost effective strategies like motivation and health
awareness in controlling this lifelong disease. The objective of this study was to determine the
effect of intensive health education on the control of this chronic life long disease.
Methods: Nine villages were screened for diabetes among subjects aged above 20 years.
Among the identified cases, data on blood pressure, anthropometric and biochemical profile
were assessed. Their knowledge on the disease was also assessed. Based on their level of
Institution:
knowledge an education programme was planned. Fifty volunteers (25 males and 25 females)
Associate Professor in
were chosen from the cases and were given an intensive personalized health education with
Applied Nutrition and the aim of motivating them to have effective control over the disease. The blood parameters
Public Health, Sadakathullah such as HbA1c, Fasting blood glucose, HDL, LDL, urea, creatinine, microalbuminuria, along with
Appa College, Body Mass Index (BMI) and Blood Pressure (BP) were assessed both before and after three
Tirunelveli - 627011. months of intervention through health education. The results were statistically analyzed for
interpretation using t-test.
Results: Through the study 68 new cases and 163 old cases of diabetes were identified. Among
the new cases 60.3% were pre-diabetics, 62.6% did not have control over the disease, 48.9%
had varied grades of hypertension and 21.6% were pre-hypertensive. More than 90% of the
cases had no idea on the complications of uncontrolled diabetes mellitus (DM) and
hypertension (HT). The effect of intervention showed significant difference in BP, HbA1c, LDL,
Corresponding author: urea, creatinine and microalbuminuria but HDL, Waist Hip Ratio (WHR) and BMI did not have
Pauline Suganthy any significant difference.
VIjayabarathy. Conclusion: Personalized diet counseling and health awareness on DM and HT had been found
to be an effective means for achieving good control over this chronic lifelong disease and in
preventing the dreaded complications of the disease among the rural population with low
socioeconomic background.

Email:
paulinepeterma@yahoo.com
Web Address:
http://jresearchbiology.com/
documents/RA0289.pdf.
Keywords:
Diabetes mellitus, pre-diabetes, hypertension.
Article Citation:
Pauline Suganthy Vijayabarathy and Pushparani D.
A study on the effect of intensive health education on the biochemical profile of the
Diabetics living in the selected rural areas of Tirunelveli District in Tamil Nadu.
Journal of Research in Biology (2012) 2(7): 652-659
Dates:
Received: 10 Oct 2012 Accepted: 17 Oct 2012 Published: 27 Oct 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

652-659 | JRB | 2012 | Vol 2 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Diabetes is increasing faster in the developing
countries. India ranks first in diabetes followed by China
and U.S. Diabetes Mellitus is a silent disease and it
continues to be one of the most costly and burdensome
chronic disease. Each year another 6 million people
develop diabetes and India leads the world with large
number of diabetic subjects and termed as DIABETES
CAPITAL OF THE WORLD(Mohan et al., 2007).
A national survey of diabetes conducted in six major
cities in India in the year 2000 has shown that the
prevalence of diabetes in urban Indian adults was 12.1%
(Ramachandran et al., 2001). The onset of diabetes
among Indians is about a decade earlier than their
western counterparts and this has been noted in Asian
Indians in several studies (Ramaiya et al., 1990).
Diabetes Mellitus is a metabolic disorder and
lifelong disease marked by high levels of glucose in
blood. It is due to the consequences of abnormalities of
beta cells of pancreas that convert blood glucose into
energy (Kathleen Mahan and Sylvia Escott-Stump.
2004). Diabetes mellitus basically produces changes in
the blood vessels and hence can affect almost every
part of the body. Long standing diabetes mellitus
is associated with an increased prevalence of
micro-vascular and macrovascular diseases. The onset of
Type-2 diabetes is usually insidious and the patient may
remain asymptomatic until late stages of the disease
(Gen et al., 2009). The insidious onset of the disease and
long duration of asymptomatic disease before the
symptoms develop makes the prevalence of
complications quite high even at the time of diagnosis of
Type-2 diabetes. Persistent elevations in blood sugar
(and, therefore, HbA1c) increase the risk for the
long-term vascular complications of diabetes such as
coronary disease, heart attack, stroke, heart failure,
kidney failure, blindness, erectile dysfunction,
neuropathy (loss of sensation, especially in the feet),
gangrene, and gastroparesis (slowed emptying of the
653
Pauline and Pushparani, 2012
stomach). Poor blood glucose control also increases the
risk of short-term complications of surgery such as poor
wound healing.
Once diabetes is diagnosed, adequate treatment
requires a significant amount of resources for patients i.e.
access to glucocheck instrument, medications, regular
access to health care and referral to specialists for
management of complications. For a person of low
economic standing in India, diabetes care can account for
25% or more of their family income for each person with
diabetes (Gen et al., 2009). The management of
long-term complications of diabetes is costly and also
leads to enormous productivity loss with significant
social burden to the patient and family. Lifestyle
changes/interventions and drugs are the current strategies
that exist to prevent or reduce the onset of diabetes.
A country like India with its large burden of
diabetes and vulnerability to chronic complications, must
evolve strategies for primary prevention of diabetes and
its complications. Early screening and therapeutic
interventions are the primary steps towards achieving
this goal. Being a chronic disease, quality diabetes care
involves more than just the primary provider. There is
great scope for motivation and health awareness in
controlling this lifelong disease. No longer it is
satisfactory to provide patients who have diabetes with
brief instructions and a few pamphlets and expect them
to manage their disease adequately. Instead, education of
these patients should be an active and concerted effort
involving the physician, nutritionist, diabetes educator,
and other health professionals. Moreover, diabetes
education needs to be a lifetime exercise.
Hence a study was under taken with the objective
of identifying the diabetic cases in selected rural areas of
Tirunelveli district, to know the level of awareness about
the disease and its control and to assess the effect of
health education on their health status.
Journal of Research in Biology (2012) 2(7): 652-659
Pauline and Pushparani, 2012

Table 1 Characteristics of Cases under study

Percentage Percentage
S.no Particulars Male Female % Total
% %
Old cases 60 (63.8%) 103 (75.2%)
1 Diabetes 10.3 9.3 231 9.7
New case 34 (36.2%) 34 (24.8%)
2 Non-diabetic 821 89.7 1337 90.7 2158 90.3
3 Total screened 915 100 1474 100 2389 100

MATERIALS AND METHODS anti-hypertensive medications (WHO, 2010). Positive

Locale of the study
To carry out this study, nine village panchayats
namely Seevalaperi, Naduvakurichi, Maruthur,
Udaiyarkulam, Kansapuram, Keelapaatam, Melapattam,
Notchikulam and Thirumalaikozhundhupuram were
selected along with nine hamlets, from Palayamkottai
union in Tirunelveli district based on the findings in a
voluntary medical camp conducted by the National
Social Service Scheme (NSS) of Sadakathulah Appa
College unit No:151.
Selection of the Respondents
Door-to-door survey of the houses in these
villages was carried out based on the willingness and
availability of the subjects for the study. After obtaining
oral consent, all available individuals of age 20 years and
above were screened by testing the urine glucose
(Benedicts method) and measuring the blood pressure
with sphygmomanometer. The blood pressure of
participants was measured in the supine position after
five minutes of rest. Hypertension was defined as either
systolic blood pressure above 139 mmHg and/or diastolic
blood pressure above 89 mmHg and/or treatment with
cases in the urine glucose test were confirmed with
gluco check strips. With this pre-test, the positive cases
were selected for the assessment of fasting blood
glucose level. The fasting glucose test is greatly
preferred because of the ease of administration,
convenience, acceptability to patients, and low cost.
Fasting is defined as no caloric intake for at least eight
hours (ADA, 2000). Five milliliter of blood was drawn
from each selected case under fasting condition. The
level of blood glucose was ascertained using glucose kit
and semi auto analyzer and were classified as per the
American diabetes association guideline for diabetes,
normoglycemia is Fasting Plasma Glucose (FPG)
<100 mg/dl (<5.6 mmol/L), Impaired Fasting Glucose
(IFG) is FPG 100-125mg/dl (5.6-7 mmol/L) and for
diabetes it is>125mg/dl (>7 mmol/L) (Mahan and
Escott- Stump, 2008 and Diabetes care, 2007).
On the test day the diabetic cases were
administered a set of 10 questions to assess their
knowledge on both diabetes mellitus and hypertension
because hypertension coexisted with diabetes in most of
the cases. A set of possible right answers were recorded

Table 2 Classification of Fasting Blood Glucose

New DM cases Old DM cases
Fasting Blood
Male Female Total Male Female Total
glucose
No % No % No % No % No % No %
Normal
- - - - - - 8 13.3 9 8.7 17 10.4
<100mg/dl
Pre-diabetics 23 67.6 18 52.9 41 60.3 17 28.3 27 26.2 44 27.0
100125mg/dl
Diabetics 11 32.4 16 47.1 27 39.7 35 58.3 67 65.1 102 62.6
>125mg/dl
Total 34 100 34 100 68 100 60 100 103 100 163 100
Pearsons Chi-Square: 29.530; df=2; p= 0.000 which is <.05(0 cells (0%) have expected count less than 5.

Journal of Research in Biology (2012) 2(7): 652-659 654

 Pauline and Pushparani, 2012

Table 3 Sex wise classification of hypertension among diabetes mellitus

DBP* in mm/Hg Male Female Total
No % No % No %
Normal < 81 29 30.8 39 28.5 68 29.4
Pre Hyper tension 81-89 29 30.8 21 15.3 50 21.6
Mild Hyper tension 90-99 27 28.7 47 34.3 74 32.0
Moderate Hyper tension100-109 3 3.2 19 13.9 22 9.5
Severe Hyper tension >=110 6 6.4 11 8.0 17 7.4
Total 94 100 137 100 231 100
Chi square=14.499; p<.05 *DBP-Diastolic Blood Pressure

in a scoring sheet for all the questions and the field
investigator was trained to score three options such as
correct answer, wrong answer and No idea based on the
answers given by the subjects.
Among the subjects 25 male and 25 female cases
who had no control over blood glucose were chosen for
the study. After getting oral permission anthropometric
measurements, BP and initial fasting blood parameters
such as HbA1c, HDL LDL, urea, creatinine and urine
microalbumin were recorded using standard kits and
semi autoanalyzer (CPC-Stat fax 3000).
The subjects were given intensive health
education on the importance and the methods of
management of diabetes and hypertension with special
emphasis on diet, regular medication and exercise, with
the help of power point slides using a laptop for
individualized counseling and LCD projectors for a
group. They were also given live cooking demonstrations
on the preparation of high fiber diet, low sodium diet and
low carbohydrate and low cholesterol diet. Each subject
was given a handout printed in the vernacular language
containing the instructions for effective control of
diabetes and hypertension. After three months of
personal follow up, counseling and motivation, the same
biochemical and anthropometric parameters were tested.
The results were analyzed using SPSS soft ware for
significance using pared sample t test.
RESULTS AND DISCUSSIONS
The results of the study are as follows. The
number of the participants in the diabetic and non
diabetic group are shown in Table 1.
Total sample of the study population who
volunteered for the study was 2389. Out of which 90.3%

Table 4 deals with the results of the pre test administered on the knowledge of diabetes mellitus and
hypertension. Level of awareness of diabetics
S.No Questions Correct % Wrong % No idea %
1 What is DM? 84 36.4 64 27.7 83 35.9
2 What foods Should be avoided /
17 7.4 183 79.2 31 13.4
included for DM?
3 What are the methods of controlling
12 5.2 4 1.7 215 93.1
DM?
4 How often should the blood be tested
9 3.9 23 10.0 199 86.1
to keep DM under control?
5 What are the complications of DM? 11 4.8 10 4.3 210 90.9
6 What is Hypertension? 32 13.9 36 15.6 163 70.6
7 What foods Should be avoided /
13 5.6 94 40.7 124 53.7
included for HT?
8 What are the methods of controlling HT? 6 2.6 16 6.9 209 90.5
9 How often should BP be checked to
13 5.6 34 14.7 184 79.7
control HT?
10 What are the complications of HT? 4 1.7 5 2.2 222 96.1
655 Journal of Research in Biology (2012) 2(7): 652-659
Pauline and Pushparani, 2012

were non diabetics and the rest 9.7% were diabetics.

Among the diabetics 63.8% of the males and
Significance 75.2% of the females were old cases and 36.2% of the
0.251**
1.000**

0.268**
0.001*
0.001*
0.000*
0.000*

0.000*
0.000*
0.000*
0.002*
males and 24.8% of the females were newly identified as
diabetics. There are many studies to support the
increasing trend of diabetes in rural areas also. A study
T value

12.452

10.233
-1.120
conducted by Deo et al., (2006) reported a high
1.162
0.000
3.457
3.512

7.879

9.780
7.286
3.264
prevalence (9.3%) in rural Maharashtra. In another study,
the prevalence of known diabetes was 6.1% in
49
49
49
49
49
49
49
49
49
49
49
df

individuals aged above 40 years which was unexpectedly

high at that time for a rural area with low
Table 5 Biochemical profile of DM cases Before and after intervention
Std.error

socio-economic status and decreased health awareness

0.0878
0.0033

0.0558

0.0143
0.1158
Mean

0.625
0.342
2.388

0.339
0.278
0.282

(Rao et al., 1989).

Table-2 deals with the level of fasting glucose
among the new and the old cases of diabetics.
0.440 0.3949
29.74 16.888

0.104 0.1009

Of the newly identified cases, 60.3% were

0.102 0.620

0.378 0.819
-0.38 2.398
2.16 4.419
1.20 2.416

2.84 1.963
2.76 1.996
0.0 0.0237
Mean SD
Difference

pre-diabetics and the rest were diabetics. If not

intervened at this point pre-diabetes will be heading
towards irreversible stage of diabetes. The occurrence of
pre-diabetes in the rural area is much higher than the
findings of Viswanathan et al., (1996) which state that
128.88 11.239

258.12 47.374

134.14 15.957
23.510 3.905

38.28 14.068

15.918 5.003
5.898 1.2343

1.376 0.4293
0.844 0.060

83.70 5.874

40.38 5.458

the prevalence of pre-diabetes in the rural population is

Mean SD

at 7-8%, which indicates the presence of a genetic basis

After

for Type-2 diabetes in ethnic Indian population

(Viswanathan et al., 1996) Among the diabetics 10.4%
had very good control and 62.6% did not have control
over their blood glucose level, indicating the need for
*p value < 0.05 significant at Five percent level
131.04 13.544

136.98 16.481
23.612 4.288

41.04 14.263

16.296 5.470
1.480 0.4794
287.86 52.04
84.90 7.203
0.844 0.054

6.338 1.332

health education on diabetic control. It was found that

40.0 5.704
Mean SD

there was significant association between the cases and

Before

**not significant at Five percent level

the fasting blood glucose level with Pearsons

chi-square value 29.530 and p-value <0.05.
From table-3 it is evident that 39% of the total
Diastolic pressure mm/hg

cases were identified as pre-hypertensive cases.

Systolic Pressure mm/hg

About 7.4% of the diabetic cases were found to have

severe hypertension. Only 29.4% of the diabetics had
Creatinine mg/dl

normal blood pressure. The Pearson chi-square value is

UMA mg/dl
Particulars

HDL mg/dl
LDL mg/dl
FBG mg/dl

Urea mg/dl

14.499 with a significance of 0.013 (P value <0.05).

HBA1C
WHR
BMI

Hence it is concluded that there is association between

hypertension and sex among DM cases.

Journal of Research in Biology (2012) 2(7): 652-659 656


More specifically, it has been reported that individuals
with blood pressure values of 130-139/85-89 mmHg
were significantly in higher risk of developing
cardiovascular diseases compared to subjects with lower
blood pressure values (Vasan et al., 2001).
It is clear from the table-4 that more than ninety
percent of the cases did not have any idea on methods of
controlling HT and DM and the complications of
uncontrolled HT and DM. Nearly 79.2% did not have
clear knowledge on diet restrictions for DM and 40.7%
did not have knowledge on food restriction for HT.
Based on the findings of the awareness study, health
education was given with audio visual aids and personal
diet counsel with special emphasis on positive
motivation in adhering to diet to prevent or delay the
complications
The biochemical profile of diabetics study group,
both before and after health education intervention is
given in Table-5. It indicates that there was significant
difference in the biochemical profile with regard to
systolic, diastolic pressure, fasting blood glucose,
HbA1c, LDL, serum urea and creatinine and urine
microalbumin among diabetic patients before and after
intervention. The current goal of LDL<100 mg/dl or the
optional goal of <70 mg/dl is recommended for high-risk
patients and to address other components of diabetic
dyslipidemia. Intensive treatment of LDL cholesterol in
patients with diabetes can substantially affect long-term
health outcome. (Richard and Nesto, 2008). A low level
of high-density lipoprotein cholesterol (HDL-C) is a key
feature of the metabolic syndrome and Type-2 diabetes.
HDL particles exert an anti-atherogenic effect and hence
low HDL-C levels are associated with increased
cardiovascular disease risk. (Rosenson, 2000). Serum
creatinine and urea concentrations change inversely with
changes in Glomerular Filtration Rate (GFR) and are
therefore useful in gauging the degree of
renal dysfunction. Microalbuminuria is the earliest
manifestation of diabetic nephropathy which leads to
657
Pauline and Pushparani, 2012
irreversible kidney damage. Control over these
parameters reflect good control of diabetes and
prevention of complications such as diabetic
nephropathy. The three months of intervention did not
have any significant difference on BMI, Waist Hip Ratio
and HDL, probably because these anthropometric indices
may require longer time for any significant changes.
Emphasis on proper diet control, regular medication and
exercise to avoid or postpone complications through
intensive heath education has had a positive impact in the
control and maintenance of diabetic cases. HbA1c is
primarily a treatment-tracking test reflecting average
blood glucose levels over the preceding 90 days
(approximately). The mean HbA1c level after the
intervention was 5.898 (SD1.2343). The current
recommended goal for HbA1c in patients with diabetes
is <7.0%, as defined as "good glycemic control".
glycated hemoglobin (hemoglobin A1c, HbA1c, A1C,
or Hb1c; sometimes also HbA1c) is a form
of hemoglobin that is measured primarily to identify the
average plasma glucose concentration over prolonged
periods of time. The 2010 American diabetes
association standards of medical care in diabetes added
the A1c 48 mmol/mol (6.5%) as another criterion for
the diagnosis of diabetes (Standards of medical care in
diabetes, 2010 )
People with diabetes that have HbA1c levels
within this goal have a significantly lower incidence of
complications from diabetes, including retinopathy and
diabetic nephropathy. This study has shown that an
intensive personalized health education can help in
controlling and preventing the complications of diabetes
mellitus. Similar findings have been recorded in a study
conducted by (Mohan et al., 2005) which has shown that
increasing awareness and empowerment of community
can possibly help in the prevention of diabetes and other
non communicable disorders.
Journal of Research in Biology (2012) 2(7): 652-659
Pauline and Pushparani, 2012
CONCLUSION
A focused individualized awareness creation along
with motivation brought in significant changes in the
biochemical status of type-2 diabetics. Patients with
type-2 diabetes who had a baseline hemoglobin A1c
(HbA1c) of greater than 8% may achieve better glycemic
control when given individual education rather than
usual care. Hence the study concludes that the quality
diabetic care involves more than just the primary medical
care. Education can empower patients to take on greater
responsibility for the management of their disease. This
forms the basis for integration of medical and social
sciences into professional education so that partnerships
with patients can be realized. Within this frame work,
responsibility for management of the disease resides with
patients and the primary role of the health care
professional becomes one of supporter and educator.
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Deo SS, Zantye A, Mokal R, Mithbawkar S, Rane S,
Thakur K. 2006. To identify the risk factors for high
prevalence of diabetes and impaired glucose tolerance in
Indian rural population. Int J Diab Dev Ctries 26:19-23.
American Diabetes Association. 2007. Diagnosis and
classification of Diabetes Mellitus (Position Statement),
Diabetes care. 30:S48.
"Executive summary: Standards of medical care in
diabetes-2010". Diabetes Care 33 (Suppl 1): S4-10.
2010.
Kathleen Mahan L and Sylvia Escott-Stump. 2004.
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Scourge, MJAFI, 65(1):50-54.
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Remam. 2007. Gene-environment interactions and the
diabetes epidemic in India.
Ramachandran A, Snehalatha C, Kapur A, Vijay V,
Mohan V, Das AK, Rao PV, Yajnik CS, Prasanna
Kumar KM, Nair JD. 2001. High prevalence of
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44(9):1094-101.
Ramaiya KL, Kodali VR, Alberti KG. 1990.
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Mather HM. 1989. The Eluru survey: prevalence of
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Lowering in Type 2 Diabetes: What Is the Optimum
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Rosenson RS. 2005. HDL-C and the diabetic patient:
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659 Journal of Research in Biology (2012) 2(7): 652-659

 Journal of Research in Biology An International Scientific Research Journal

Original Research

Establishing continuity in distribution of

Diploknema butyracea (Roxb.) H. J. Lam in Indian subcontinent
Journal of Research in Biology

Authors: ABSTRACT:
Koushik Majumdar1,
BK Datta1 and
Uma Shankar2. The known distribution of Indian Butter Tree (Diploknema butyracea (Roxb.)
H. J. Lam) is discontinuous, i.e., in the sub-Himalayan tracts in the north and Andaman
Institution: and Nicobar Islands in the south. Here, we present the first record of its distribution
1. Department of Botany,
from Tripura upholding the continuity between the north and the south.
Tripura University,
Suryamaninagar 799 130,
Tripura, India.

2. Department of Botany, Keywords:

North-Eastern Hill Indian Butter Tree, distribution, restoration of Jhum fallows,
University, Shillong Northeast India.
793 022, India.

Corresponding author: Article Citation:

Koushik Majumdar.
Email:
majumdark80@gmail.com
Web Address:
http://jresearchbiology.com/
documents/RA0268.pdf.
Koushik Majumdar, BK Datta and Uma Shankar.
Establishing continuity in distribution of Diploknema butyracea (Roxb.) H. J. Lam in
Indian subcontinent.
Journal of Research in Biology (2012) 2(7): 660-666
Dates:
Received: 28 Jul 2012 Accepted: 20 Aug 2012 Published: 27 Oct 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

660-666 | JRB | 2012 | Vol 2 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Majumdar et al., 2012

INTRODUCTION Recently, Diploknema butyracea has been reported from

D. butyracea is an economically important, but Andaman Islands (Kureel et al., 2008; Rajkumar and
lesser-known, underutilized tree. The tree is best known Parthasarathy, 2008). Interestingly, this species has not
for the yield of edible oil from kernels, phulwara butter, been reported from Manipur (Singh et al., 2000)
which is used in chocolate, soap and candle manufacture, Meghalaya (Balakrishnan, 1981; Joseph, 1982;
as fertilizer and fish intoxicant (Mitra and Awasthi, Haridasan and Rao, 1985), Mizoram (Singh et al., 2002),
1962; Mukerji, 1951; Awasthi and Mitra, 1961). Hence, Tripura (Deb, 1981) and Bangladesh (Ahmed et al.,
it is known as Indian butter tree. The bark which is rich 2009) which lie between the Himalayan foothills and
in tannin is used in dyeing. The leaf is good for fodder, Andaman Islands. Here, we report the first distributional
branches for fuel wood and trunk for timber. The nectar record of Diploknema butyracea in the State of Tripura,
from the flowers is harvested through honeybees or India (Fig 1).
directly to produce a jaggery which is highly prized in
Uttarakhand (Bahar, 2011). The medicinal properties of MATERIALS AND METHODS
the tree are also well recognized and various tissues are Three adult trees of Diploknema butyracea were
used for the treatment of rheumatic pain, ulcers, itching, found and Gps -mapped at Betlingshib (23 48' 39.60" N
haemorrhage, inflammation of tonsils, leprosy and and 92 15.4' 6.40" E) in Jampui Hills of North Tripura
diabetes (Awasthi and Mitra, 1968; Mishra et al., 1991; district. This area is bordering the State of Mizoram
Khetwal and Verma, 1986). In Nepal, Chepang tribes at 940 m altitude and experiences tropical climate.
manage D. butyracea for their livelihood. They harvest The annual rainfall is high (>2,109) mm and mean daily
the fruits, extract kernel butter (locally called cheuri temperature ranges between 10C and 25C. The habitat
ghee) and market the products (Shakya, 2000).
The butter is used for burning in lamps, in sweetmeats,
and for soap manufacture. The cake obtained after
extraction of the fat contains saponins and is toxic
(Mitra and Awasthi, 1962; Mukerji, 1951; Awasthi and
Mitra, 1961).
Globally, the genus Diploknema Pierre of
Sapotaceae is represented by eleven taxa, with eight
accepted species and three synonyms. Only three species
are found in India, viz., Diploknema butyracea (Roxb.)
H.J. Lam, Diploknema butyracea var. andamanensis P.
Royen, and Diploknema butyraceoides (M.B. Scott) H.J.
Lam. Of these, Diploknema butyracea, named as East
India Butter Tree by William Roxburgh in 1805, is
native to the sub-Himalayan tracts of Bhutan, China,
India and Nepal (Brandis, 1906). In India, it occurs in
subtropical forests of Himalaya spanning Garhwal and Figure 1 Distribution of Diploknema butyracea in
Himalayan subtropical region (line) and Andaman
Kumaon Hills, Sikkim, Darjeeling, Arunachal Pradesh Islands (oval) and location of this report from
and Assam (Awasthi et al., 1975; Negi et al., 1988). Tripura (circle).

661 Journal of Research in Biology (2012) 2(7): 660-666

Majumdar et al., 2012

Table 1 An inventory of the specimens of Diploknema butyracea collected by

different workers from northeastern India
Sl No. BSI sheet number* Collection Date* Collection number* Collectors name* Locality*
1 17507 ??.03.1875 16 Not known Cachar, Assam
2 Not known 05.12.1913 3137 Not known Nizamghat, Assam
3 14833 10.11.1957 10332 R. S. Rao Badasu to Sone
godam, Lohit
4 14834 15.04.1958 15630 G. Panigrahi Chingri, Kameng
5 51280 12.03.1965 41294 N. P. Balakrishnan Shali, Eastern
Bhutan
6 53414 06.09.1965 43488 A. S. Rao Kheri, Tawang
7 51278 19.10.1965 44067 N. P. Balakrishnan Gumkhara, Eastern
Bhutan
8 This study 02.04.2009 0700 K. Majumdar Jampui Hills,
Tripura
*Source: Botanical Survey of India, Shillong.

is characterized by semi-evergreen forest that is facing RESULTS AND DISCUSSION

destruction for shifting cultivation by local tribes In northeastern India, Diploknema butyracea
(Lusai, Reang and Mizo), horticultural expansion for (Fig 2) was first collected from Cachar Valley, Assam in
orange and betel nut, and construction of new 1875 and then from Nizamghat in 1913 (Table 1).
roads. Only a few remnant patches of pristine Subsequent collections were between 1957 and 1965
vegetation surrounded by secondary successional from Arunachal Pradesh and eastern Bhutan.
vegetation survive today. The associated species An inventory at the herbarium of the BSI, Shillong
include Baccaurea ramiflora, Bombax ceiba, revealed lack of any collection after 1965. The specimen
Castanopsis armata, C. indica, Diospyros sps, collected from Jampui Hills, 45 years after the last
Engelhardia spicat a, Garci nia acuminata, taxonomic collection, indicates the first record of
Lithocarpus spicata, Saraca asoka. The identity of occurrence in Tripura. This record of occurrence in
collected specimens was determined by a critical isotype Tripura has significance in biogeography of
examination at the Botanical Survey of India, Shillong Diploknema butyracea since its known distribution
and study of taxonomic descriptions in floras. exhibited discontinuity between sub-Himalayan tracts
The voucher specimens were deposited in the herbarium and Andaman Islands (Fig 1).
of Tripura University.
Table 2 A chronological inventory of taxonomic identities assigned to Diploknema butyracea
Taxonomic identities Source Year
Bassia butyracea Roxb. Asiat. Res. 8: 499-502 1805
Wall. Cat. 4164 1828
Fl. Ind. 2: 527 1832
Mixandra butyracea (Roxb.) Pierre ex L. Planch. tude Sapot. 26 1888
Illipe butyracea (Roxb.) Engl. Bot. Jahrb. Syst. 12: 509 1890
Vidoricum butyraceum (Roxb.) Kuntze Revis. Gen. Pl. 2: 407 1891
Mixandra butyracea (Roxb.) Pierre ex Dubard Rev. Gn. Bot. 20: 196 1908
Madhuca butyracea (Roxb.) J.F. Macbr. Contr. Gray Herb. n.s., 53: 18 1918
Diploknema butyracea (Roxb.) H.J. Lam Bull. Jard. Bot. Buitenzorg III, 7: 186 1925

Aesandra butyracea (Roxb.) Baehni Boissiera 11: 29 1965

Journal of Research in Biology (2012) 2(7): 660-666 662

 Majumdar et al., 2012

Figure 2 A photographic illustration of Diploknema butyracea: (a) tree; (b) bark; (c) leaf adaxial side,
(d) leaf abaxial side; (e) arrangement of fruits; (f) flower; (g) a single fruit; (h) transverse section of the
fruit and (i) seed.
As per the Himalayan Glaciation theory, some of important Indian trees; where some forest trees showing
the Himalayan flora pushed southwards resulting into the discontinuous distribution in Burma, Assam, Andaman,
discontinuity in distribution of some tree species Eastern Ghats, Sri Lanka and Western Ghats.
(Medlicott and Blanford, 1879). However, Puri et al., Furthermore, since our study site is in close proximity
(1983) suggested ten types of distribution pattern of to the State of Mizoram (India) and Bangladesh,

663 Journal of Research in Biology (2012) 2(7): 660-666

Majumdar et al., 2012

occurrence in these territories is plausible. ACKNOWLEDGEMENTS

Diploknema butyracea has been assigned different Financial support for this work was received
names in the taxonomic literature and a chronology of through grant no BT/PR7928/NDB/52/9/2006 from the
these names indicates that the species was prominently Department of Biotechnology, Government of
known as Aesandra butyracea before and after settling India, New Delhi. The authors are grateful to
down to Diploknema butyracea (Table 2). Dr. K. N. Ganeshaiah, UAS, Bangalore for
The regeneration of D. butyracea is also poor encouragement. Thanks are due to the Wildlife Warden,
because: 1) the seeds are recalcitrant and lose viability Tripura for permission to undertake survey in forest
quickly after dispersal (Tewari and Dhar, 1996) and areas; the Botanical Survey of India (BSI), Eastern
2) nutritious oil-rich seeds attract sizeable predators such Circle, Shillong for access to herbarium; and to Prof. S.
as rove beetles, termites and ants. Attempts have been Sinha, Head, Department of Botany, Tripura University
made to regenerate the species through clonal for facilities.
propagation (Tewari, 1997) as well as seed germination
(Sundriyal and Sundriyal, 2003). REFERENCES
Ahmed ZU, Hassan MA, Begum ZNT, Khondker M,
CONCLUSION Kabir SMH, Ahmad M and Ahmad ATA. (eds).
Success in regeneration of this species has 2009. Encyclopedia of Flora and Fauna of
opened vistas for inducting D. butyracea to grow during Ba n g l a d e s h : An g i os p e r m s : D i c o t y l e do n s :
fallow phase in shifting cultivation systems, locally Ranunculaceae-Zygophyllaceae. Vol. 10, Asiatic Society
known as jhum in northeastern India. Of late, of Bangladesh, Dhaka. 1-580.
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linked with protection of NTFP species in the fallow
Chemurgy of Sapotaceous Plants: Madhuca species of
lands (Uma Shankar, 2003; 2005). Hence, multipurpose
India. Economic Botany 29:380-389.
tree species are increasingly attracting attention of the
farmers to help them with supplement income. Awasthi YC and Mitra CR. 1961. Flavonoids of
Although D. butyracea is not a redlist species, its Madhuca butyracea Nut-Shell. Journal of Organic
abundance in wild is poor. It has been observed that Chemistry, 27:1636.
economically important trees face the risk of destruction
Awasthi YC and Mitra CR. 1968. Madhuca latifolia;
by the people for livelihood benefits and hence may
triterpenoid constituents of the trunk bark.
become locally extinct (Sundriyal and Sundriyal, 2003).
Phytochemistry, 7(8):1433-1434.
This threat is greater if the species is infrequent in the
wild and seeds form the economic part of the plant. Bahar N. 2011. Cheura [Diploknema butyracea (Roxb.)
Domestication of such species on fallow lands, marginal H. J. Lam.]: an important tree for poverty alleviation.
lands, agroforestry systems and homegardens may be Indian Forester, 137:1344-1345.
helpful for conservation.
Balakrishnan NP. 1981. Flora of Jowai. Vol. I,
Botanical Survey of India, Howrah,

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Brandis D. 1906. Indian Trees: An Account of Trees,
Shrubs, Woody Climbers, Bamboos and Palms
Indigenous or Commonly Cultivated in the British Indian
Empire. Fifth impression (1971). Bishen Singh
Mahendra Pal Singh: Dehradun, India.
Deb DB. 1981. The Flora of Tripura State, Vol. I, Today
and Tomorrows Printers and Publishers, New Delhi.
Haridasan K and Rao RR. 1985. Forest flora of
Meghalaya. Vol. I, Bishen Singh Mahandra Pal Singh
Publishers, Dehra Dun.
Joseph J. 1982. Flora of Nongpoh and vicinity, East
Khasi Hills District Meghalaya. Forest Department,
Government of Meghalaya.
Khetwal KS and Verma DL. 1986. Flavonoids from the
flowers of Diploknema butyracea. Fitoterapia 57:128.
Kureel RS, Gupta AK and Pandey A. 2008.
Cheura - a wonder tree borne oilseed. National Oilseeds
and Vegetable Oils Development Board, Ministry of
Agriculture, Government of India, 8.
Medlicott HB and Blanford WT. 1879. Manual of the
Geology of India, Part 2. Calcutta, Geological Survey of
India.
Mishra G, Banerji R and Nigam SK. 1991.
Butyrpenoidal sapogenin from Madhuca butyracea.
Phytochemistry, 30:2087-2088.
Mitra CR and Awasthi YC. 1962. Constituents of
Madhuca latifolia and M. butyracea nuts. Journal of
Scientific and Industrial Research 21(D):102-103.
Mukerji B. 1951. Indian Pharmaceutical Codex,
C.S.I.R., New Delhi. I, 144.
Negi KS, Tewari JK, Gaur RD and Pant KC. 1988.
Indian butter tree - Asiandra butyracea (Roxb.) Baehni;
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Puri GS, Meher-Homji VM, Gupta RK and Puri S.
1983. Phytogeographical ecology. 115-210. In: Forest
ecology. Second edition. Oxford and IBH Publishers,
New Delhi, India.
Rajkumar M and Parthasarathy N. 2008.
Tree diversity and structure of Andaman giant evergreen
forests, India. Taiwania , 53:356-368.
Shakya MR. 2000. Chepangs and Chiuri - the use of
non-timber forest products in Nepal. ITFC.
http://practicalaction.org/?id=food_chain.
Singh NP, Chauhan AS and Mondal MS. (eds.) 2000.
Flora of Manipur. Vol. I Ranunculaceae-Asteraceae,
Botanical Survey of India, Calcutta.
Singh NP, Singh KP and Singh DK. (eds.) 2000. Flora
of Mizoram. Vol. I Ranunculaceae-Asteraceae, Botanical
Survey of India, Calcutta.
Sundriyal M and Sundriyal RC. 2003. Underutilized
edible plants of the Sikkim Himalaya: need for
domestication. Current Science, 85:731-736.
Tewari A and Dhar U. 1996. An investigation on seed
germination on Indian butter tree-Asiandra butyracea
(Roxb.) Baehni. Seed Science and Technology,
24:211-218.
Tewari A. 1997. Clonal propagation of Indian Butter
tree (Diploknema butyracea) through tissue culture.
Ph. D. Thesis, Kumaon University, Nainital.
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resources for subsistence and trade: utilization,
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Ursolic acid from leaf extracts of Barleria lupulina acting as

anti clastogenic and anti tumor agent.
Journal of Research in Biology

Authors: ABSTRACT:
Pranab Kumar Das1 and
Pradip Kumar Sur2

Institution:
1.UGC Research Scholar,
Cytogenetics Laboratory,
Dept. of Zoology,
Kanchrapara College,
Kanchrapara-743145,
Dist-(N)24 Pgns ,
West Bengal, India.
In our previous study, we have reported the radio-protective, anti clastogenic
and anti tumor activities of the leaf extract of Barleria lupulina on mice and fish. In the
present work, biological active component from the leaf extract has been isolated, by
column chromatography. Phytochemical analysis, TLC and NMR studies confirm the
presence of ursolic acid as the main anti cancer component, besides other such
components, as sitosterol, sitosterol-3-O-glucoside are also present.

2. Associate Professor,
Cytogenetics Laboratory,
Dept. of Zoology,
Kanchrapara College,
P.O. Kanchrapara-743145, Keywords:
Dist-(N)24 Pgns , Barleria lupulina; Acanthaceae family; anti-cancer, anti-tumor activities;
West Bengal, India. ursolic acid; sito sterol; sitosterol-3-O-glucoside.

Corresponding author: Article Citation:

Pranab Kumar Das.
Email:
drpksur@rediffmail.com
prnv_20042003@rediffmail.com
Web Address:
http://jresearchbiology.com/
documents/RA0281.pdf.
Pranab Kumar Das and Pradip Kumar Sur.
Ursolic acid from leaf extracts of Barleria lupulina acting as anti clastogenic and
anti tumor agent.
Journal of Research in Biology (2012) 2(7): 667-675
Dates:
Received: 06 Sep 2012 Accepted: 24 Sep 2012 Published: 29 Oct 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

667-675 | JRB | 2012 | Vol 2 | No 7

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
Tumor formation and cancer is a life threatening
disease and a problem to mankind all over the world.
Tumors in malignant form lead to cancer. Search for
anti cancer and radio protective agents from plant
extracts is aimed by researchers all over the globe. Since
the discovery of Patt et al., (1949) that cysteine protects
against X-irradiation in rats and mice, several workers
are engaged in active research for the search of
radio-protective component, even from plant extracts.
Jagetia et al., (2003) proved that naringin, a citrus
flavonone protects mice from -irradiated chromosomal
damage. Kanchanapoom et al., (2001) reported that
Barleria lupulina plant is externally used as an
anti-inflammatory agent. Its anti diabetic potential has
been stated by Suba et al., (2004). Kanchanapoom et al.,
(2001) isolated iridoid glucosides from the aerial parts of
Barleria lupulina.
In our previous experiments (Sur and Das, 2012),
we reported for the first time the anti cancer,
anti clastogenic, radio-protective properties of the leaf
extract of Barleria lupulina Lindl; on laboratory models
as mice and anti tumor property on fish. Investigation of
active component in the leaf extract is carried out in this
study. Ursolic acid has been found to be the main
component in the leaf extract, which pertains to its anti
cancer property.
MATERIALS AND METHODS
Plant Material
Barleria lupulina of Acanthaceae family
(common name hophead Philippine violet),
identified from the Central National Herbarium,
Botanical Survey of India, Howrah-711103 (Ministry of
Environment and Forests, Govt. of India). Leaves of this
plant (Fig 1) were cleaned, washed and dried in shade.
After sufficient drying, they were subjected
Soxhlet extraction in ethanol and its leaf extract (LE)
was obtained as described in our previous study
668
Das and Sur, 2012
(Sur and Das, 2012).
Instrumentation
NMR (Bruker Corp.) was operated at 400 MHz.
Data were recorded by dissolving 15 mg of sample
in 1 ml deuterated chloroform (CDCl3). Column
chromatography was operated using a glass column of
1 meter length, and 10 cm diameter packed with
silica gel C (mesh size 100-200, EMerck). Column was
packed following wet packing method, with
100% petroleum ether. Solvent systems used for elution
were petroleum ether (non-polar solvent), chloroform,
ethyl acetate (medium polar solvents), methanol and
ethanol (highly polar solvents) according to increasing
order of their polarity. Elution was started with
100% petroleum ether (isocratic non-polar solvent), then
the concentration of petroleum ether was gradually
decreased and that of chloroform was gradually
increased (gradient eluent). Then 100% chloroform was
used (isocratic eluent). After this, medium polar solvent
as ethyl acetate was used along with chloroform, for
elution (gradient eluent). This was followed by
100% ethyl acetate (isocratic eluent, medium polar
solvent). Methanol (a more polar solvent) was used next.
The concentration of methanol was gradually increased
and ethyl acetate was decreased. Gradually
100% methanol was used. And finally ethanol
(most polar solvent) was used to washing off the column.
was
to
Fig (i): Leaves of Barleria lupulina
Journal of Research in Biology (2012) 2(7): 667-675
Das and Sur, 2012

Table 1 Standards and mobile phase used in TLC to identify the active component by trial and error method
Sl No Phytochemical Standards Used Mobile phase used Ratio of mobile
Results phase

1 Terpenoid Ursolic acid Methanol : chloroform 7:43

2 Steroid Sitosterol Benzene : petroleum ether: 4:2:3:1
chloroform: ethyl acetate

3 Glycoside Sitosterol-3-O-glucoside Benzene : chloroform: ethyl acetate: 18:2:1:4

methanol

Extraction and Isolation by column chromatography
After Soxhlation 1.75 gm of LE was dissolved in
adequate amount of 99.9% ethanol. Then it was mixed
with silica gel C, and dried to remove the solvent. This
was further used for column packing. Column was
packed with silica gel C mixed in 100% petroleum ether
by following wet packing method. Elution was done by
using varying concentrations of non-polar, medium polar
and polar solvents such as petroleum ether, chloroform,
ethyl acetate, methanol and ethanol according to their
increasing order of polarity.
After elution, each fraction was heated on water
bath to evaporate the solvents and concentrate
their constituents. Qualitative analysis of the
phyto-constituents present in the fractions was performed
by standard phytochemical tests described in Trease and
Evans (1997).
Preparative TLC (for identification of active
component)
Preparative TLC was performed by using
standard 60F254 TLC plates (pore size; 60 ,
uv fluorescence at 254 nm, Merck, Germany). The silica
gel which acted as the stationary phase and mobile phase
was chosen by trial and error method. Very small amount
of the fractions from column chromatography (test) were
put on the plates with the help of a capillary tube along
with the standard phyto-constituents; and TLC was run
using the mobile phase. Components of the fractions
were identified by comparing with Rf value of the
standards. Different standards and mobile phases were
used for the comparison study. Standards and mobile
phase that showed fruitful results are summed up in
(Table 1).
1 H NMR study of the fractions
1 H NMR of the fractions was performed by
dissolving 15 mg of each fraction in 1 ml (CDCl3). The
NMR machine (Bruker Corp.) was operated at 400 MHz.
RESULTS
Column Chromatography and Phytoconstituents
Present (Qualitative)
A total of 52 fractions were obtained from
column chromatography. The use of non-polar, medium
polar and polar solvents for elution is mentioned in
materials and methods. Compounds in pure form as
terpenoids were isolated in fractions 2-5 (eluted with
petroleum ether and chloroform), and in fractions 12 and
13 (eluted with ethyl acetate and chloroform); steroids
were isolated in fractions 6-11 (eluted with petroleum
ether and chloroform) and also in fractions 24-26
(eluted with ethyl acetate and chloroform). Further
eluting with ethyl acetate and methanol in varying
concentrations, the mixture yielded flavonoid in fractions
31-35, glycoside in fractions 28-30, 40-44 and finally
steroid again in fractions 45-52. Each fraction was tested
for their nature by performing qualitative phyto chemical
tests, simultaneously. The fractions 14-23, 27 and
36-39 were mixtures of compounds.
Preparative TLC (for Identification of active
component)
In fraction No. 13, terpenoid was identified as
ursolic acid (Rf value =1), in fraction No. 24-26, steroid

Journal of Research in Biology (2012) 2(7): 667-675 669

 Das and Sur, 2012

Table 2 Preparative TLC: Identification of terpenoid, steroid and glycoside by comparison with commercially
available standards
Sl No Fraction No Phytochemical Standard used Mobile phase used Rf value of Rf value of
Result (test) standard test
1 13 Terpenoid Ursolic acid Methanol: chloroform 1 1
(7:43)
2 24-26 Steroid sitosterol Benzene : petroleum 0.59 0.59
ether: chloroform:
ethyl acetate (4:2:3:1)
3 28-30 Glycoside Sitosterol-3-O-glucoside Benzene : 0.35 0.35
chloroform: ethyl
acetate: methanol
(18:2:1:4)

as sitosterol (Rf value = 0.59) and in Fraction
No.28-30, glycoside as Sitosterol-3-O-glucoside
(Rf value = 0.35) (Table 2 and Fig 2).
1 H NMR study of the fractions
The 1H NMR results of ursolic acid,
sitosterol and sitosterol-3-O-glucoside are shown in
Fig -3, Fig - 4 and Fig -5 respectively.
Five main signal peaks are observed in case of
ursolic acid. The highest signal peak (doublet) is
observed at 1.293 and 1.254 intensities, and chemical
shift between 1.5 to 1.1 ppm. In case of sitosterol,
five doublets, one doublet-of-doublets and one triplet is
observed, representing that the protons are arranged in
three different coupling patterns. Some peaks in this
NMR are at 5.823, 4.941, 4.307 etc signal intensities.

M-14,C-16 B-G Pet-2,C-3,Etone-1 B-18,C-2,Etone-1,Mech-4

Fig 2 Identification of terpenoid, steroid and glycoside by comparison with standards by TLC
(the corresponding Rf values are shown)

670 Journal of Research in Biology (2012) 2(7): 667-675

Das and Sur, 2012

Fig 3 1H NMR study of ursolic acid

Fig 4 1H NMR study of sitosterol

Thirdly, the NMR data of Sitosterol-3-O-glucoside and Sitosterol-3-O-glucoside (Mizushina et al., 2006) are
reveals three peaks, among which, the highest signal shown in Fig -6, Fig -7 and Fig-8 respectively.
intensity is 1.254 between 71.30 ppm chemical shifts.
Chemical structure of the active components DISCUSSION
The chemical structures of ursolic acid (Furtado Ursolic acid is a pentacyclic triterpenoid and is a
et al., 2008), sitosterol (http://webprod.hc-sc.gc.ca) major component of traditional medicinal herbs

Journal of Research in Biology (2012) 2(7): 667-675 671


Fig 5 1H NMR study of Sitosterol-3-O-glucoside
(Lai et al., 2007). The five signal peaks obtained in our
NMR result represents its pentacyclic nature. The highest
peak which is a doublet represents the single proton of
the OH group which is near the = O group at the right
hand side of Fig (vi). The other weak signal peaks are for
the protons at the side chains. This triterpenoid
has shown anti-ulcer (Ovesn et al., 2004),
anti-inflammatory, analgesic (Vasconcelos et al., 2006)
anti oxidant (Jun et al., 2010) activities etc. It has potent
anti tumor activity also (Furtado et al., 2008).
Ursolic acid acts on different stages of tumor
Fig 6 Structure of ursolic acid
672
Das and Sur, 2012
development as tumor initiation and promotion and also
plays an important role in tumor cell differentiation
apoptosis (Furtado et al., 2008). Pathak et al., (2007) had
shown that this terpenoid inhibits STAT 3 activation
pathway, leading to the suppression of Human Multiple
Myeloma Cells. Subbaramaiah et al., (2000) proved that
ursolic acid suppresses the activation of COX-2 gene
expression by inhibiting the PKC signal transduction
pathway.
sitosterol is widely available in a variety of
plant and plant parts. The five doublets, one doublet of
Fig 7 Structure of sitosterol
Journal of Research in Biology (2012) 2(7): 667-675
Das and Sur, 2012
Fig 8 Structure of Sitosterol-3-O-glucoside
doublets and one triplet is observed in our NMR study of
in this steroid represent the hexacyclic and pentacyclic
groups; the connecting chain and the methyl groups
respectively. This sterol is reported in the treatment of
benign prostatic hyperplasia (Berges et al., 1995).
Carine et al., (2006) proved that beta-sitosterol in
combination with polyphenols from cocoa inhibits
proliferation of prostatic cancer cell growth.
Sitosterol-3-O-glucoside falls under
class D glycoside. The three peaks
Sitosterol-3-O-glucoside our NMR study represent
the gluco-pyranose sugar, cyclo-hexagon
cyclo-pentagon groups and the free side chains
(Fig (viii). Gohar et al., (2009) had reported that
methanolic extracts of seeds of Ceratonia siliqua L. has
a rich source of natural anti oxidants, which contains -
sitosterol-3-O--D-glucoside along with other flavonol
glycosides.
In the present work, we are the first to report
the rich source of ursolic acid, sitosterol and
sitosterol-3-O-glucoside in leaves and other parts of the
plant, Barleria lupulina. It is discussed above that
ursolic acid and sito sterol show potent anti cancer
activity and sitosterol-3-O-glucoside has anti oxidant
activity. Leaf extract of Barleria lupulina containing
these components, has been found by us to have anti
clastogenic and anti tumor activity (in mice and fish)
Journal of Research in Biology (2012) 2(7): 667-675
which had been discussed in our previous work
(Sur and Das, 2012). Therefore in conjugation with
ursolic acid, these components together act as a potent
anti cancer agent in mice and fish models. We also report
the anti cancer activity in ethanolic extracts of leaves of
Barleria lupulina, due to the presence of ursolic acid; for
the first time.
Anticlastogenic, radio-protective, anti-tumor and
anti-cancer activities from this plant extract has been
applied to Govt. of India for patenting, by us
(Sur and Das, 2012).
ACKNOWLEDGEMENTS
The authors are highly thankful to the University
Grant Commission, BahadurShah Zafar Marg, New
Delhi-110002, for providing financial support in the
form of UGC Major Research Project in Zoology, 2011
entitled Assessment of protective role of some
ayurvedic formulations on artificial mutagenesis in
some mammalian models mice Mus musculus and
the shrew Suncus murinus) (F.No: 39-579/2010 (SR)).
of The authors also highly acknowledge Prof. Pandey
(The Principal) and Ms. Moulisha Biswas, Bengal
and Institute of Pharmaceutical Sciences, Kalyani, Nadia,
West Bengal, India, for helping in the work on column
chromatography, TLC and Phytochemical analysis.
Moreover Dr. Sukhen Das of Dept. of Physics, Jadavpur
University is acknowledged for his great cooperation in
Ph.D. registration of the first author.
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1995. -Sitosterol Study Group. Randomised,
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-sitosterol in patients with benign prostatic hyperplasia.
The Lancet. 345(8964):1529-1532.
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2006. In-vitro effects of polyphenols from cocoa and 2004. Pentacyclic triterpenoic acids: new
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Qin Y, Chan-min L, Qun S, Yong-jian W. 2010. Dannenberg AJ. 2000. Ursolic acid inhibits
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Lai MY, Leung HWC, Yang WH, Chen WH, Lee HZ.
2(5):439-447.
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gene involvement in ursolic acid-induced human lung Sur PK and Das PK. 2012. A Process of Extracting
non-small carcinoma cell apoptosis. Anticancer Res., Barleria lupulina Lindl. and the product
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id=3618&lang=eng

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 Journal of Research in Biology
Isolation and characterization of feather degrading
Journal of Research in Biology
Authors:
Arun Kumar JM1,
Lakshmi A2, Sangeetha
Rani V2, Sailaja B2.
Institution:
1. Asst Prof, Dept of
Biotechnology, Ballari
Institute of Technology and
Managment, Bellary.
2. B.E. Scholar, Dept of
Biotechnology, Ballari
Institute of Technology and
Managment, Bellary.
Corresponding author:
Arun Kumar JM.
Web Address:
http://jresearchbiology.com/
documents/RA0239.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
bacteria from poultry waste
ABSTRACT:
The aim of this study was to characterize keratinolytic bacteria isolated from
feather waste. Feather waste is generated in large amounts as a by-product of
commercial poultry processing. This residue is almost pure keratin, which is not easily
degradable by common proteolytic enzymes. The crude protein from feather has of
high nutrient value and could be used as animal feed for livestock and fish feed in
aquaculture. Feather constitutes over 90% protein, the main component being
beta-keratin, a fibrous and insoluble structural protein extensively cross linked by
disulfide bonds. This renders them resistant to digestion by animals, insects and
proteases leading to serious disposal problems. It is degraded only by keratinase
enzyme. These enzymes were produced by some species of Bacillus. In the present
study, B. licheniformis was used for degrading keratin substrate such as feathers.
Based on morphology and biochemical analysis, the isolates were identified as
Bacillus spp. Fermentation using feather as a substrate was carried out on minimal salt
media for seven days which resulted in almost complete degradation of feather. The
optimum conditions for keratinase production were temperature 37C, pH 7.0 and
initial substrate concentration 1%. Maximum enzyme activity was found to be 100 U/L
with the protein concentration of 4 g/ml.
Keywords:
Feather, keratin, feather degrading bacterium, poultry waste, keratinase,
keratinolytic activity.
Article Citation:
Arun Kumar JM, Lakshmi A, Sangeetha Rani V, Sailaja B.
Isolation and characterization of feather degrading bacteria from poultry waste.
Journal of Research in Biology (2012) 2(7): 676-682
Dates:
Received: 07 May 2012 Accepted: 26 May 2012 Published: 01 Nov 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
676-682 | JRB | 2012 | Vol 2 | No 7
www.jresearchbiology.com

INTRODUCTION
Feathers, which are almost pure keratin proteins,
are produced in large amounts as a waste by-product at
poultry-processing plant. A total of 5-7% weight of
mature chicken comprises of feathers (Avinash et al.,
2011). Feather waste constitutes beta-keratin,
insolvable protein (Swetlana and Jain, 2011). In addition
to this, feather waste is produced at the rate of 22 million
kg per year (US alone) (Savitha et al., 2007). A group of
proteolytic enzymes which are able to hydrolyze
insoluble keratins more efficiently than other proteases
are called keratinizes produced by some microorganisms.
These bacterial strains produce enzymes which
selectively degrade the beta-keratin found in feathers.
These enzymes make it possible for the bacteria to obtain
carbon, sulphur and energy for their growth and
maintenance from the degradation of beta keratin
(Muthusamy et al., 2011). Keratins are the most
abundant proteins in epithelial cells of vertebrates and
represent the major constituents of skin and its
appendages such as nail, hair, feather, and wool. Keratins
are grouped into hard keratins (feather, hair, hoof and
nail) and soft keratins (skin and callus) according to
sulphur content (Scott and Untereiner 2004). The protein
chains are packed tightly either in -helix (-keratins) or
in -sheet (-keratins) structures, which fold into final
3-dimensional form (Veslava et al., 2009)
Keratinases which are produced by these
keratinolytic organisms could be used to degrade feather
waste and further the digested products could be a
excellent material for producing animal feed, fertilizers
or natural gas (Tamilmani et al., 2008). Use of
keratinolytic microorganisms for feather degradation is
an economical, environmental friendly alternative.
Keratinolytic proteases offer considerable opportunities
for a low energy consuming technology
bioconversion of poultry feathers from a potent pollutant
to a nutritionally upgraded protein feed for live stock
(Jahan et al., 2010; Vigneshwaran et al., 2010). Most
677
Kumar et al., 2012
feather waste is land filled or burned which involves
expense and can cause contamination of air, soil and
water. Bacterial strains are known which are capable of
degrading feathers (Savitha et al., 2007). Traditional
ways to degrade feathers such as alkali hydrolysis
a and steam pressure cooking may not only destroy the
amino acids (methionine, lysine and histidine) but also
consume large amounts of energy. Utilizing poultry
feathers as a fermentation substrate in conjunction with
keratin-degrading microorganism or enzymatic
biodegradation may be a better alternative to improve
nutritional value of poultry feathers and reduce
environmental waste (Veslava et al., 2009). These
feathers constitute a sizable waste disposal problem.
Several different approaches have been used for
disposing of feather waste, including land filling,
burning, natural gas production and treatment for animal
feed. Most feather waste is land filled or burned which
involves expense and can cause contamination of air, soil
and water (Rai Sapna et al., 2011; Savitha et al., 2007).
Feathers hydrolysed by mechanical or chemical
treatment can be converted to feedstuffs, fertilizers, glues
and foils or used for the production of amino acids
and peptides (Jahan et al., 2010; Andrea et al., 1996).
An alternative to decrease this pollution is the utilisation
of feather constitutes that can be used as animal feed,
preventing accumulation in the environment and the
development of some types of pathogens (Veslava et al.,
2009).
MATERIALS AND METHODS
Enrichment
1 g of poultry waste was serially diluted in order
to reduce the initial number of micro organisms. This
dilution was then inoculated on minimal feather agar
for media. Feathers were washed, dried and hammer milled
prior to being added to the medium. The medium was
sterilized by autoclaving. All incubations were done at
37C.
Journal of Research in Biology (2012) 2(7): 676-682
Kumar et al., 2012

Screening (Sigma Co. St. Louis. Mo.) as a substrate by azocasein

Skim milk agar (Himedia) was prepared and the solution in 0.05 M Tris -HCI buffer at pH 8.5, which was
above dilutions were streaked on milk agar plates for incubated with 400 l crude enzyme solution for 30 min
testing the caseinolytic activity of the organism. Bacteria at 37C in a water bath with shaking. The reaction was
were inoculated onto plates and incubated at 37C for terminated by addition of 1.4 ml of 10% trichloroacetic
24 h. Strains that produced clear zones in this medium acid (TCA). After 15 min at 4C, the reaction mixture
were selected. was centrifuged at 10,000xg for 10 min. One ml
Subculturing supernatant was mixed with 1 ml of 0.5 N NaOH and the
The organism screened with Keratin agar plates absorbance was read at 440 nm. The control was treated
was subcultured by continuously growing the bacterium in the same way, except TCA was added before the
in minimal broth medium (3 days at 37C, 120 rpm) and addition of crude enzyme. One unit of caseinolytic
subsequently streaking on minimal agar medium activity was determined as the amount of enzyme that
(1.5% agar, 2 days 37C). produces an increase in absorbance of 0.01 per min
Identification of Isolated feather degrading bacteria under the assay conditions. The soluble protein
Gram Stain, Spore staining, Motility test and concentration in the culture supernatant was estimated
Catalase Test. according to the Bradford method (Bradford, 1976).
Characterization of the isolate using Biochemical Enzyme characterization
assays Ta kin g T em perat ur e, pH, Substrat e
IMViC Test, Hydrogen Sulfide Test, Urease concentration, Activator and Inhibitor as parameters,
Test, Lipid hydrolysis, Carbohydrate Fermentation, characterization of enzyme was done.
Starch hydrolysis and Gelatin liquefaction. Feather degradation
Production of keratinolytic enzyme 100 ml of Nutrient broth is prepared; 1 gm of
The bacterial isolate was cultivated in 100 ml feather is added to the media and sterilized. A loop full
minimal feather media. The samples were withdrawn and of inoculum is inoculated into the media and incubated
centrifuged at 6000 rpm for 10min. The supernatants for seven days at 37C. Residual feathers were harvested
were preserved at 4C and assayed for protein. from the fermentation media by filtering it over
Determination of keratinase activity whatman filter paper No: 3. The harvested feathers were
Azocasein hydrolysis was used as an alternative kept in hot air oven at 50C until weight stabilized to
to the azokeratin hydrolysis. Keratinolytic protease constant value. The difference between the weight of
a ct i vi t y wa s det erm in ed wi th a z oca sein residual feather obtained from the control and that of
Table 1: Biochemical Characterization. inoculated media has been used as measure of feather
Sl. No Bio-Chemical Test Bacillus sp degradation. Degradation was expressed in percentage.
1. Indole production Test Positive
2. Methyl Red Test Positive
3. Voges proskaeur Test Negative RESULTS
4. Citrate Utilization Positive Positive A screening program was employed to obtain
Positive/
5. Carbohydrate fermentation Presence of bacterial isolates capable of producing feather degrading
Test Air Bubbles extracellular keratinase enzyme using feather (keratin) as
6. Catalase Positive
sole carbon substrate. The potential isolate was then
7. Starch hydrolysis Positive
8. Urease Positive characterized and identified to its genus level.

Journal of Research in Biology (2012) 2(7): 676-682 678

 Kumar et al., 2012

Identification of bacteria was based on Table 2 : Rate of feather degradation

morphological, cultural and biochemical tests comparing Time in Concentration of Rate of
weeks feather in gms degradation in %
the data with standard species (Hoq et al., 2010).
1 1 25
Morphological and physiological characteristics of the 2 1 79
bacteria were compared with the Bergey's Manual of 3 1 85
Systemic Bacteriology. The isolate was Gram positive, enzyme decreases (Fig 3 and 4).
rod shaped and spore-former and were able to utilize Effect of Substrate on Keratinase enzyme
both glucose and sucrose but not lactose. They were also The activity of the enzyme at different
catalase positive, oxidase negative. MR test is positive concentration was carried out and the graph is plotted
and VP test negative for these organisms. The organisms (Fig 5).
were unable to utilize citrate and all were able to reduce Rate of degradation
nitrate to nitrite. The isolate showed typical Bacillus sp was able to grow and produce
characteristics of Bacillus licheniformis. keratinase in nutrient medium in which feather meal
Characterization of Enzyme served as an additional carbon and nitrogen source and
Characterization was done by determining the acted as enzyme inducer, resulted in 85% of feather
effect of pH, Temperature, Activator, Inhibitor and degradation in seven days at 37C. Kerotinolytic activity
Substrate. was measured in the absorbance at 440 nm by the
Effect of Temperature and pH on Keratinase enzyme standard enzyme assay method.
The activity of the enzyme at various
temperatures and pH was studied and graphs are plotted. DISCUSSION
The optimum temperature and pH is 37C and 7 A bacterium isolated from poultry waste has
respectively (Fig 1 and 2). been shown to degrade feather keratin. The identification
Effect of Activator and Inhibitor on Keratinase of the keratinolytic bacteria was based on cell
enzyme morphology, colony morphology, and several other
The enzyme samples were checked for the effect methods. These results suggested that the strain belong to
of activator and Inhibitor. Zinc Chloride is used as genus Bacillus (Saritha Agrahari and Neeraj Wadhwa
activator and EDTA is used as an inhibitor. It was 2010). The most studied keratinolytic bacteria are
observed that as the concentration of the activator Bacillus spp., which have been described to possess
increased, activity of the enzyme increased where as the feather-degrading activity (Kim et al., 2001; Lin et al.,
concentration of the inhibitor increases the activity of the 1999). Through the strategy of isolation of keratinolytic
Activity (U/L)
Activity (U/L)

Temperature (C) pH

Fig 1: Effect of Temperature Fig 2: Effect of pH

679 Journal of Research in Biology (2012) 2(7): 676-682
Kumar et al., 2012
Activity (U/L)
Concentration of Activator (ml)
Fig 3: Effect of Activator
microorganisms utilized in this work, bacteria presenting
high keratinolytic activity were selected. Considering
that feather protein has been showed to be an excellent
source of metabolizable protein (Klemersrud et al.,
1998), and that microbial keratinases enhance the
digestibility of feather keratin (Lee et al., 1991;
Odetallah et al., 2003), these keratinolytic strains could
be used to produce animal feed protein. The enzyme was
stable at the pH range of 6-8 (Cheng et al., 2007). The
activity decreased at pH 3.0 and 8.0. The isolated
bacterium showed Optimum keratinolytic activity at
37C and pH 7.0. The enzyme also showed to be stable
at 60C and pH 9.0 (Kurt Cotanch and Grant 2007). In
the present study, it has been recorded that the
Bacillus licheniformis degraded the feather at a rate of
85% at 37C in seven days where as Williams et al.,
(1990) reported that Bacillus licheniformis PWD-1
degraded chicken feather completely at 50C in 10 days.
(Bockle et al.,1995) demonstrated that Streptomyces
pactum DSM40530 partially degraded native chicken
feather at 50C, the maximum feather degrading activity
Activity (U/L)
Concentration of Inhibitor (ml)
Fig 4: Effect of Inhibitor
Journal of Research in Biology (2012) 2(7): 676-682
Activity (U/L)
Concentration of Substrate (l)
Fig 5: Effect of Substrate
was at 50C.
On increasing the feather concentration the
extent of feather degradation decreases because of a
decrease in keratinase activity. It indicates that at higher
substrate concentration repression of keratinase
production can take place. This observation is similar to
previous studies which concluded that a low
concentration of substrate is optimum for yielding
maximum enzyme activity (Avinash et al., 2011).
CONCLUSION
In the present study, we isolated the Bacillus sp.
capable of producing keratinase from habitats where
keratin containing substrate is decomposed under natural
conditions. The isolate exhibited highest keratinase
activity is the most potential isolate which degraded
feather at a rate of 85% after seven days at 37C. The
keratinolytic microorganisms isolated in this study
therefore could play an important role in the production
of animal feed protein in addition to the biodegradation
of poultry wastes. The degradation of feathers with
keratinolyticmicro organisms is the best eco friendly
approach in the poultry feather waste management. This
novel keratinolytic isolate could be a potential candidate
for the degradation of feather keratin and also in
de-haring process at leather industry.
680
 Kumar et al., 2012

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a Novel Thermophilic Anaerobic Species of the
with Blood, Projact thesis.
Order Thermotogales, Applied and Environmental
Microbiology, 62:2875-2882. Lee CG, Ferket PR, Shih JCH. 1991. Improvement of
feather digestibility by bacterial keratinase as a feed
Avinash Srivastava, Anshul Sharma and
additive. FASEB J., 59:1312.
VuppuSuneetha. 2011. Feather Waste biodegradation as
a source of Amino acids, European Journal of Lin X, Inglis G, Yanke L and Cheng KJ. 1999.
Experimental Biology, 1 (2):56-63. Selection and characterization of feather-degrading
bacteria from canola meal compost, J. Ind. Microbiol.
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Characterization of a keratinolytic serine trotease from
strectomycespactm DSM40530. APPL environ Muthusamy Govarthanan Selvankumar T and
microbial., 61:3705-3710. Arunprakash S. 2011. Production Of Keratinolytic
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Cheng-gang CA1, Bing-gan LOU and Xiao-dong
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ZHENG. 2007. Keratinase production and keratin
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Journal of Zhejiang University SCIENCE B, 9(1):60-67. Odetallah NH, Wang JJ, Garlich JD, Shih JCH. 2003.
Keratinase in starter diets improves growth of broiler
Williams CM. 1990. Isolation, Identification, and
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Characterization of a Feather-Degrading Bacterium,
Appl. Environ. Microbiol., 56(6):1509. Tamilmani P, Umamaheswari A, Vinayagam A and
Prakash B. 2008. Production of an Extra Cellular
Vigneshwaran C, Shanmuga S and Sathish Kumar T.
Feather Degrading Enzyme by Bacillus licheniformis
2010. Screening and Characterization of Keratinase from
Isolated from Poultry Farm Soil in Namakkal District
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Scott JA and Untereiner WA. 2004. Determination of
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Bacillus species from poultry waste, Process Biochem.,
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Degradation of Chicken Feather a Poultry Waste Product
Klemersrud MJ, Klopfenstein TJ, Lewis AJ. 1998. by Keratinolytic Bacteria Isolated from Dumping Site at
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Journal of Research in Biology (2012) 2(7): 676-682 682

 Journal of Research in Biology
Insect diversity and succession pattern on different carrion types
Journal of Research in Biology
Authors:
Rosina AK Kyerematen1,
Bernard A Boateng2,
Emmanuel Twumasi1.
Institution:
1. Department of Animal
Biology and Conservation
Science, University of
Ghana, Legon, Accra.
2. Department of Crop
Science, College of
Agriculture and Consumer
Sciences, University of
Ghana, Legon, Accra.
Corresponding author:
Bernard A Boateng.
Web Address:
http://jresearchbiology.com/
documents/RA0153.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
Insect fauna attracted on four different types of carrion; beef, chicken, pork,
and fish were conducted in a cassava growing field on the campus of the University of
Ghana for a period of twenty-eight days between February and March, 2005. Four
stages of decomposition were recognized; fresh, bloated, decay and dry. A total of
19 insect species belonging to five orders and thirteen families were recorded.
Coleoptera represented 23% of the total number of species. Diptera and
Hymenoptera constituted 35% and 41% respectively while Collembola and
Heteroptera a mere 1% of the insect orders that dominated the carrion fauna.
Caliphoridae were the first to arrive on all four types of carrion. Five species of
Histeridae, three of Formicidae and one each from Dermestidae, Scarabaeidae,
Cleridae, Mycetophagidae, Scolytidae were recorded on beef, chicken, pork and fish.
Representatives of four Dipteran families; Calliphoridae, Muscidae, Sarcophagidae and
Tachinidae were also recorded. One species each of the families Pyrrhocoridae and
Isotomidae were unique to pork and chicken respectively.
Keywords:
Insect diversity, succession pattern, carrion, decomposition, forensic
entomology, Ghana.
Article Citation:
Rosina AK Kyerematen, Bernard A Boateng, Emmanuel Twumasi.
Insect diversity and succession pattern on different carrion types.
Journal of Research in Biology (2012) 2(7): 683-690
Dates:
Received: 25 Nov 2011 Accepted: 07 Dec 2011 Published: 02 Nov 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
683-690 | JRB | 2012 | Vol 2 | No 7
www.jresearchbiology.com

INTRODUCTION
Decomposing carrion supports a wide diversity
of several organisms, many of which are insects.
Although a decomposing carcass is a temporary, rapidly
changing resource (Grassberger and Frank, 2004), it can
support a large, dynamic insect community. Apart from
ecological interest, carrion decomposition and succession
studies have proven important in forensic entomology.
When the sequence of insects colonising carrion is
known, an analysis of the arthropod fauna on a carcass
can be used to determine time since death in legal
investigations (Anderson and van Laerhoven, 1996). In
addition, if an insect can be found exclusively in a rural
or urban habitat, analysis of the carrion associated fauna
may help to determine whether the remains have been
moved from an urban to a rural environment or vice
versa (Erzinclioglu, 1983; Catts and Haskell, 1990;
Grassberger and Frank, 2004)
It is known that insects are usually the first
organisms to arrive on a body after death, and they
colonise in a predictable sequence. The body progresses
through a recognised sequence of decomposition stages,
from fresh to skeletal, over time (Peters, 2003). Each of
these stages of decomposition is attractive to a different
group of sarcosaprophagous arthropods, primarily insects
(Anderson and van Laerhoven, 1996). The occurrence of
the insects forms a succession of colonising species
which are eliminated as carrion decay progresses.
Succession is the idea that as each organism or group of
organisms feeds on the body, the corpse changes thereby
making it more attractive to another group of organisms
(Goff and Flynn, 1991). The first generation of initial
colonizers can provide a biological clock that more
precisely measures the time of death for up to two or
more weeks; medical examiners estimates are limited to
about a day or two (Greenberg and Kunich, 2002; Peters,
2003).
The nature and time of the insects succession
depends on several factors including the size of the
684
Kyerematen et al., 2012
carrion, seasonal and climatic conditions, and the
surrounding non-biological environment, such as soil
type (Snodgrass, 1967; Erzinclioglu, 1983; Carvalho and
Linhares, 2001). The organisms involved in the
succession vary according to whether they are upon or
within the carrion, in the substrate immediately below
the carrion or in the soil at an intermediate distance
below or away from the carrion and finally the kind of
carrion (Gullan and Cranston, 2010). This stems from the
fact that carrion of different animals have different fat
and muscle composition that affect the pattern and length
of decomposition. Furthermore, each succession will
comprise of different species in different geographic
areas, even in places with similar weather factors. This is
because few species are very widespread in distribution
and each biogeographic area will have its own specialist
carrion fauna. Blow flies are the most common dipterans
seen in abundance around carcasses, which serve as
oviposition sites and larval food sources. Adult flies are
attracted to the carrion until it is nearly dry, but other
species visit the carcasses only during specific stages of
decomposition; thus a succession of species can be
observed (Goddard and Lago, 1985).
According to Gullan and Cranston (2010) during
decomposition, the first wave of insects involves certain
blowflies (Diptera: Calliphoridae) and house flies
(Diptera: Muscidae) that arrive within hours or few
days at the most. The second wave is of flesh flies
(Diptera: Sarcophagidae) with additional house flies and
blowflies that follow shortly thereafter, as the carrion
develops an odour. All these flies either lay eggs or
oviposit on the carrion. At this stage blowfly activity
ceases as their larvae leave the carrion and pupate in the
ground. When the fat of the carrion turns rancid, a third
wave of species enter this modified substrate, notably
more dipterans, such as Phoridae, Drosophilidae and
Syrphidae (especially Eristalis sp., the rat-tailed
maggots) in the liquid parts. As the carrion becomes
butyric, a fourth wave of Diptera, Piophilidae and related
Journal of Research in Biology (2012) 2(7): 683-690
Kyerematen et al., 2012

Table 1 Insect orders, families and species from Carrion

Order Family Species Chicken Beef Pork Fish
Coleoptera Cleridae Necrobia rufipes 29 44 0 5
Coleoptera Dermestidae Dermestes frischii 46 37 20 83
Coleoptera Histeridae Carcinops pumilio 52 0 0 26
Coleoptera Histeridae Gnathoncus sp. 0 0 0 21
Coleoptera Histeridae Hister sp. 0 0 16 18
Coleoptera Histeridae Platysoma carolinus 45 38 0 0
Coleoptera Histeridae Teratosoma sp. 0 0 0 32
Coleoptera Mycetophgidae Typhaea stercorea 0 0 0 6
Coleoptera Scarabaeidae Geotrupes sp. 0 0 0 14
Coleoptera Scolytidae Xyleborus sp. 35 0 0 0
Collembola Isotomidae Isotoma sp. 18 0 0 0
Diptera Caliphoridae Lucilia rufifacies 55 86 48 88
Diptera Muscidae Musca domestica 81 99 73 84
Diptera Saracophagidae Sarcophaga sp. 56 62 35 52
Diptera Tachinidae Tachinid sp. 18 37 0 11
Heteroptera Pyrrhocoridae Dysdercus superstitious 0 0 9 0
Hymenoptera Formicidae Oecophylla longinoda 183 159 0 498
Hymenoptera Formicidae Monomorium sp. 86 0 0 0
Hymenoptera Formicidae Solenopsis xyloni 0 0 0 75
Total 704 542 211 1013

flies also eat the body. A fifth wave occurs as the
ammonic smelling carrion dries out, adult and larvae of
Dermestidae and Cleridae (Coleoptera) become
abundant, feeding on the keratin. In the final stage of dry
decay, some Tineid larvae (clothes moths) feed on the
remnant hair. The rather predictable sequence of
colonisation and extinction of carrion insects allows
forensic entomologist to estimate the age of corpse,
which can have medico-legal application in homicide
investigation. Differences in decomposition of carrion in
relation to biogeography and ecology of necrophagous
insect communities have been the subject of several field
studies. However, most of these studies have used single
animal species (Bornemissza, 1957; Arnaldos et al.,
2001; Grassberger and Frank, 2004). Forensic
entomology research is nascent in Ghana and as such no
work on decomposition and insect attractions have been
done. The data obtained from this study will therefore
provide basic information regarding carrion
decomposition fauna of this area. The objective of this
work therefore was to evaluate the attractiveness of four
carrion types to insects and the species diversity and
succession pattern of the insects.
MATERIALS AND METHOD
Study site
The study was conducted on a crop farm growing
cassava on the campus of the University of Ghana,
Legon (0539 N, 00011W). Legon is located in the
coastal savanna vegetation belt of Ghana. The annual
rainfall ranges from 740-890 mm. Relative humidity is
however high (60%-75%) throughout the year and thus
compensates for the scanty annual rainfall. There are two
rainfall maxima but the dry seasons are more marked
(Hall and Swaine, 1981).
Carrion placement
A kilogram each of four carrion types was used;
namely, pork, beef, chicken and fish. The fresh meat and
fish were obtained from a local abattoir and the market
respectively. These carrion were placed separately in
rectangular wire cages (size: 23 x 23 x 90 cm; mesh
sizes: 1 cm x 1 cm) to prevent dogs, rodents and other
vertebrates from eating them. The four cages were placed
on the ground in a crop farm on the campus of the
University of Ghana, Legon. The cages were so placed to
facilitate the entry of both crawling and flying insects to
access the carrion through the wire mesh. The cages

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 Kyerematen et al., 2012

Table 2 Total number of orders, families, species and RESULTS

insects collected during the study. Five insect orders; Coleoptera, Diptera,
No. of No. of Total no. Hymenoptera, Collembola and Heteroptera were
Carrion Order
families species of insects
recorded on the four carrion types. The most diverse
Fish Coleoptera 5 8 205 order was Coleoptera as several insects recorded
Diptera 4 4 235
belonged to six families of this order (Table 1) with five
Hymenoptera 1 2 573
Chicken Coleoptera 4 5 207 of the insect species collected belonging to the family
Diptera 4 4 210 Histeridae. Oecophylla longinoda (Hymenoptera:
Hymenoptera 1 2 269 Formicidae) was the most abundant insect on all carrion
Collembola 1 1 18
types except pork. In all, the fish carrion attracted the
Beef Coleoptera 3 3 119
highest number of insects (1013), followed by chicken
Diptera 4 4 264
Hymenoptera 1 1 159 (704), with pork attracting the least number of insects
Pork Coleoptera 2 2 36 (211) (Table 1).
Diptera 3 3 156 Insect diversity was highest on fish with fourteen
Heteroptera 1 1 9
species from ten families followed by chicken with
were separated 10 m apart to avoid the same insects twelve species from ten families and then beef with eight
moving from one cage to other. species from eight families (Table 2). Pork attracted the
Care was taken to protect the data collectors from lowest diversity of five species from five families. The
any pathogens, pollutants or contaminants by wearing most abundant insects were the Hymenoptera with over
protective clothing. Sign posts warning passersby about 1000 individuals followed by Diptera with more than
the potential hazards of the experiment were erected at 800 individuals (Table 2). Rare insects collected were
three locations, about five metres from the experimental from the orders Collembola and Heteroptera on chicken
set-up. and pork carrion respectively.
Adult and developing immature insects were The fish carrion consistently attracted the highest
carefully collected with a sweep net, forceps gloved insect numbers throughout the four-week period
hands and camel hair brush twice daily at 6:00 AM and (Table 3). Chicken also attracted higher numbers of
5:00 PM for 28 days. Adult beetles and flies were killed insect from first to third week than were recorded on
in an ethyl acetate kill jar. Pupae collected from soil were beef, but lower numbers were attracted to chicken than to
placed into empty vials, while larvae were put in 70% the beef in the fourth week. There were no insect records
alcohol. These were returned to the laboratory for further for pork in the third and fourth week because it was
processing. Soil samples of about 300 g were also taken away by an unidentified animal.
collected from beneath each of the carrion and taken to Fig-1 shows the number of insects collected daily
the laboratory for any possible insect emergence. during the time of decomposition for each carrion type.
Temperature and relative humidity were recorded with Varied amounts of insects were collected on the carrion
Tinytag datalogger (Gemini Dataloggers, UK). Detection types throughout the 28 days the study lasted. The initial
of gasses given off during decay was done by testing for numbers of colonizing insects decreased on the second
SO2, NH3, CO2 and H2S. day but the most drastic reduction occurred on chicken,
decreasing by about 79%. The numbers increased on the
third and fourth days for all carrion reaching 86 and 104
686 Journal of Research in Biology (2012) 2(7): 683-690
Kyerematen et al., 2012

Fig 1 Chronology of total insects attracted to the (Smith, 1986). Neither gross morphological changes nor
carrion types odour of the decay was detected in the fish, chicken, beef
and pork at this stage.
The bloated stage, which lasted for about a week,
commenced with the onset of noticeable swellings on the
Number of insects

carrion and ended when they deflated. Putrefaction, the

principal component of decomposition process, began at
this stage. The first visible signs of the bloating were
observed in the fish; chicken and beef showed slight
Day inflation due to buildup of gasses from the metabolic
Table 3 Total number of insects attracted weekly by activities of anaerobic bacteria. The pork did not bloat
various carrion types due probably to the part that was used for the
Carrion Week 1 Week 2 Week 3 Week 4
Fish 337 223 243 210 experiment.
Chicken 230 187 183 99 The decay stage began with the detection
Beef 187 100 141 114
Pork 97 104 - - of gases especially carbon dioxide, ammonia and
hydrogen sulphide. The carrion deflated and cracks were
for beef and fish respectively. The numbers again observed in the fish, chicken and beef carrion in one or
fluctuated until day 22 before decreasing gradually to the more places by feeding dipterous larvae. The stage
end of the study. ended when most of the remnants were relatively dry
(Reed, 1958). The dry stage was the final stage of
DISCUSSION decomposition. The carrion at this stage consisted of
Decomposition only dry skin, cartilage, and bones. Odour was typically
Throughout the period of study all the four of dried animal skin.
carrion types were observed to follow the normal pattern Insect Succession
of decomposition as seen in most carrion decomposition The resource-driven selection of the decomposer
studies. These were divided into four stages as fresh, community was repeatedly observed as the insect
bloated, decay and dry (Reed, 1958; Tantawi et al., succession associated with carrion decomposition on the
1996). The decomposition stages are a convenient means soil surface (Carter et al., 2007) As reported in most
to summarize physicochemical changes; they are arthropod succession studies on carrion (Reed, 1958;
subjective and do not typically represent discrete series Payne, 1965; Coe, 1978; Abell et al., 1982; Anderson
(Schoenly and Reid, 1987). The carrion stayed fresh for and van Laerhoven, 1996; Tantawi et al., 1996); the
about a day or two and several insects, mainly three insect orders, Diptera, Coleoptera, and
calliphoridae, were attracted to all of them (Galal et al., Hymenoptera, dominated the carrion communities in the
2009). Usually, fresh stage begins at the moment of present study as well. The house flies and the blow flies
death and continues until bloating is first evident. During were the first to arrive on all four carrion types just
this stage the process of autolysis; the breakdown of within hours of the set up (Gullan and Cranston, 2010).
complex proteins and carbohydrate molecules to simpler They dominated the carrion for the first three days. The
chemical compounds (Gill-King, 1997) primarily due to second wave was of Sarcophagids and the muscids and
the action of digestive enzymes or ferments occurs calliphorids that followed shortly thereafter as the carrion

Journal of Research in Biology (2012) 2(7): 683-690 687


started to develop odours. The Tachinids (Diptera)
appeared on the 2nd, 3rd and the 4th days on the chicken,
fish and the beef respectively and kept revisiting up to
the ninth day on all three carrion while there were few on
the pork. The low numbers of insects on the pork may
have been due to the fact that the part used (pig feet) was
not comparatively succulent and fleshy and did not have
much body fluids to attract and allow oviposition by
these dipterans. All the flies either laid eggs or
oviposited on the carrion (Gullan and Cranston, 2010).
The larvae later left the carrion especially the chicken
and the fish and pupated in the ground.
As the ammonia smell from the fish, chicken and
the beef minimized, coleopteran adults and larvae,
mainly Dermestids and Clerids, became abundant from
the second week onwards feeding on the keratin and the
maggots. A single clerid species belonging to the genus
Necrobia was recovered from the fish, beef and chicken
carrion. Adults were observed to feed on dipteran larvae
as well as on carrion (Braack, 1987). The pork
experienced slow decay possibly due to its high fat
content that melted slowly down the whole carrion in the
scotching sun. The flies and ants that attempted to feed
on the pork carrion were trapped by the sticky nature of
the melting fat and subsequently died.
In the fourth week, the final stage of dry decay
was observed; the larvae and adult of the beetle families,
Scarabaeidae, Histeridae and Dermestidae and
Scolytidae were abundant on the fish, beef and the
chicken. Oecophylla longinoda (Hymenoptera:
Formicidae) however made short the work of the fish.
They were mainly attracted by the abundance of
maggots. The beetles and ants dominated the carrion
especially the fish.
Insect Abundance
During the first week of the set-up, the fish
carrion attracted the highest number of insects dominated
by the Dipterans, the first insect order to normally invade
carrion. This was probably due to the high water content
688
Kyerematen et al., 2012
as well as the freshness and also the strong odour emitted
by the rotten fish. A drop of about 33% (114 insects)
was observed in the second week and increased slightly
to 243 insects in the third week. This was attributed to
the fact that some of the flies had laid their eggs or l
oviposited and left. Also, the beetles that appeared
afterwards may have fed on the larvae of the flies
(Nuorteva, 1970). The invasion of the carrion by
O. longinoda was also a major contributory factor to
fluctuation in the insect population. They dominated in
the third week because there was an abundance of food
i.e. maggots and larvae of flies and beetles. This food
resource was depleting by the fourth week and so lower
insect numbers were recorded on the fish carrion.
Insect composition recorded on the chicken in
the first week was dominated by flies (Dipterans) due to
the tenderness and the strong odour of the chicken
carrion. Concomitantly, blow flies (Calliphoridae) and
flesh flies (Sarcophagidae) colonize carrion to find a
suitable site for the development of their offspring
(Carter et al., 2007). The insect population dropped
slightly in the second week and remained constant
throughout the third week. It was observed that, the
tender nature of the chicken was conserved due to a
downpour in the later part of the second week. This was
helpful in retaining much of the water content thereby
constantly attracting the flies to the carrion. In the fourth
week, the insect population dropped sharply to 99 insects
when the chicken started drying up and at this stage it
attracted a new wave of insect fauna mainly the
Coleopterans and the Formicids. This is consistent with
(Wastis 1972) study on arthropod succession patterns.
The total number of insects attracted to the beef
carrion lagged behind those of fish and chicken, with the
singular exception of the fourth week. The pork did not
experience much decay due to its hard skin and high
content of fat. Houseflies and blowflies were the most
abundant in the first six days. The Dermestes frischii and
the Hister sp., became predominant from the seventh day
Journal of Research in Biology (2012) 2(7): 683-690
Kyerematen et al., 2012

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