effect of weight reduction drug chitocal contained gymnema … · medical journal of babylon-vol....

Medical Journal of Babylon-Vol. 9- No. 1 -2012 1021 -مجلة بابل الطبية- المجلد التاسع- العدد االول

Jabbar H.Yenzeel Al-Hilfy and Nusaibah Amer 200

Abstract Objective: The objective of this study was to investigate the effect of doses of Chitocal for 4 weeks on body

weight, lipid profiles, kidney functions and histological structure of kidney and intestine in male rats.

Methods: Twenty adult male albino rats (200 -215 gm) were divided into 2 groups: The first group was

considered as control group. The second group was treated orally with Chitocal (50 mg/kg b.w) by use of

intragastric tube. Different physiological parameters were performed including recording of the body weight

and measuring lipid profiles, creatinine and urea levels.

Results: Body weight gain, total cholesterol, triglyceride, LDL cholesterol, and VLDL cholesterol levels

were significantly (p< 0.05) reduced in Chitocal treated rats when compared with the control rats. HDL

cholesterol and urea levels were significantly (p< 0.05) increased in Chitocal treated rats, but creatinine level

was none significantly (P <0.05) increased when compared with the control rats. Histological examination

of Chitocal treated rats kidney’s showed congestion of blood vessels between renal tubules, aggregation of

inflammatory cells in lumen and wall of blood vessels, enlargement of renal tubules and aggregation of

inflammatory cells around glomerulus. In intestine tissue of Chitocal treated rats moderate increase of

monocytes in lamina propria and hyperplasia of goblet cells was observed, aggregation of inflammatory

cells in epithelium, adhesion of intestinal villi, hyperplasia in epithelium and aggregation of inflammatory

cells in lamina propria.

Conclusion: From the results of this study it can be conclude that the treatment with Chitocal produced a

significant reduction in body weight and lipid profiles, but it is incapable of improving the kidney functions.

Also there are histopathological effects on kidney and intestine tissues in treated rats.

تاثيرالعقارالمقلل للوزن شيتوكال الحاوي على مستخلص نبات الجيمينيما سلفستر في وزن الجسم ومستويات الدهون ووظائف االكلى والتركيب النسجي للكلية واالمعاء في ذكور الجرذان البيض.

الخالصةسم ومستويات الدهون ووظائف الكلى، وفي التركيب تم التحري عن تأثير تناول عقار الشيتوكال لمدة اربعة اسابيع في وزن الج

الى 022النسجي للكلى واالمعاء في ذكور الجرذان البيض.استعملت عشرون جرذا من ذكور الجرذان البيض البالغة تراوحت اوزانها مابين ملغرام / 22قار الشيتوكال مقدارها غرام قسمت الى مجموعتين: االولى عوملت كمجموعة سيطرة ، وتلقت المجموعة الثانية جرعة من ع 012

لكل كيلوغرام من وزن الجسم عن طريق الفم باستخدام انبوب المعدة.تم قياس عدة معايير فسلجية تضمنت وزن الجسم ومستويات الدهون رول والكليسيريدات ومستوى اليوريا والكرياتنين. اظهرت النتائج وجود انخفاض معنوي في معدل اكتساب وزن الجسم ومستويات الكوليستي

رة، في الثالثية والكوليستيرول واطئ الكثافة والكوليستيرول واطئ الكثافة جدا في دم الجرذان المعاملة بعقار الشيتوكال عند المقارنة بالسيط ند المقارنة بالسيطرة.حين لوحظ ارتفاع معنوي في مستويات الكوليستيرول عالي الكثافة واليوريا وارتفاع غيرمعنوي في مستوى الكرياتنين ع

اظهرت نتائج الفحص النسجي لنسيج كلى الجرذان المعاملة بالشيتوكال احتقان االوعية الدموية بين االنيبيبات الكلوية وتجمع الخاليا بيبية.وفي نسيج امعاء الجرذان االلتهابيه في جدار وتجويف االوعية الدموية، واتساع االنيبيبات البولية وتجمع الخاليا االلتهابيه حول اللمة الك

لتهابية المعاملة بالشيتوكال لوحظ زيادة معتدله في خاليا وحيدة النواة في الصفيحة االصيلة وفرط تنسج في الخاليا الكاسيه،وتجمع الخاليا اال

Effect of Weight Reduction Drug Chitocal Contained Gymnema

sylvester Extract on Body Weight, Lipid Profiles, Kidney

Function and Histological Structure of Kidney and Intestine in

Male Albino Rats

Jabbar H.Yenzeel Al-Hilfy Nusaibah Amer

Dept. of Biology, College of Science, University of Baghdad, Baghdad, Iraq.

[email protected]

M J B

mailto:[email protected]



فيحة االصيله. ومن نتائج الدراسة في الطبقة الظهاريه، والتصاق الزغابات وفرط تنسج الطبقة الظهارية وتجمع الخاليا االلتهابية في الصيتضح ان عقار الشيتوكال له تاثير واضح في خفض وزن الجسم ومستويات الدهون ، لكن ليس له القدره على تعديل وظائف الكلى، كذلك

ظهرت له تاثيرات امراضيه نسيجية في نسيج الكلى واالمعاء في الجرذان.ــــــــــــــــ ــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــ ـــــــــــــــــ ـــــــــــــــــ ـــــــــــــــــ ـــــــــــــــــ ـــــــــــــــــــــــــ ــــــــــــــ ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــ

Introduction

besity refers to an abnormally

high proportion of total body fat.

Obesity increases the risk of type

2 diabetes, cardiovascular disease,

cancer, and premature death. The

excessive storage that creates obesity

eventually leads to the release of

excessive fatty acids from enhanced

lipolysis, which is stimulated by the

enhanced sympathetic state existing in

obesity [1]. The release of these

excessive free fatty acids then incites

lipotoxicity, as lipids and their

metabolites create oxidative stress to the

endoplasmic reticulum and mitochondria.

This affects adipose as well as non

adipose tissue [2]. Pharmacological

factor involved in obesity and diabetes

includes lipoprotein lipase, having a

central role in the metabolism of both

triglyceride‐rich particles and high

density lipoproteins (HDL) [3].

Approximately 80% of the world's

population currently uses herbal

medicines in healing different ailments

[4]. Chitocal is a highly effective weight

loss formula which acts as carbohydrate

blaster and fat absorption blocker.

Chitocal contains high density chitosan

which is a natural polysaccharide

comprising copolymers of glucosamine

and N-acetyl glucosamine, and can be

obtained by the partial deacetylation of

chitin, from crustacean shells, the second

most abundant natural polymer after

cellulose [5].

Chitosan is an extraordinary fat binder.

Chemically speaking, chitosan is an

amino polysaccharide that has the ability

to “bind” lipids in the stomach before

they are absorbed through the digestive

system into the blood stream. Not only

does chitosan attract and inhibit fats, it

offers an array of other desirable

physiological benefits that can foster

optimal health and longevity such as

hypocholesterolemic, antimicrobial, and

wound healing properties. Of equal

significance is the fact that when

chitosan is combined with other

compounds such as citric acid, ascorbic

acid and phytochemicals called indoles,

its action is enhanced, making it far more

valuable as both a fat binder and dietary

health aid. [6]

Chitocal contains Gymnema sylvestre

extract. Gymnema sylvestre R. belongs to

the family Asclepiadaceae. It is a woody

climber found in western India. The

active compound of this plant is a group

of acids termed as gymnemic acids.

Gymnemic acid molecules has the same

arrangement of glucose molecules and

has large molecular weight make it has

the affinity on the glucose receptor and

make competitive inhibition with glucose

on the receptor in small intestine,

Gymnema sylvestre is well recognized in

traditional medicine as a remedy for

diabetes mellitus and used in folk,

ayurvedic and homeopathic systems of

medicine, Gymnema leaves were also

used for stomach ailments, constipation,

water retention, and liver disease [7]. It

brings blood glucose homeostasis

through increased serum insulin levels

provided by repair or regeneration of the

endocrine pancreas [8].The G. sylvestre

leaf and callus extracts reduced blood

sugar and lipid profiles such as

cholesterol, triglyceride, HDL, LDL,

VLDL in alloxan-induced diabetic

Wistar rats [9].

G. sylvestre leaf extract when orally

administered to experimentally induced

hyperlipidaemic rats, reduced the

elevated serum triglyceride (TG), total

cholesterol (TC), very low-density

lipoprotein (VLDL)-and low-density

lipoprotein (LDL)-cholesterol in a dose-

dependent manner. Gymnema sylvestre

O



R. was investigated in normal and obese

streptozotocin induced diabetic rats and

was found to produced significant

reduction in glucose level, lipid profile

and body weight [10]. The aim of the

present study was to investigate the

effects of daily oral consumption of

chetocal for 4 weeks on body weight,

kidney function, lipid profiles and

histological structures of kidney and

intestine in rats to show the preventive

and side effects of Chitocal.

Materials and Methods

Animals and experimental design

Twenty adult 12 weeks old, male

albino rats (Rattus norvegicus), weighing

200-215 gm were obtained from animal’s

house of the College of Science,

University of Baghdad, Iraq. They were

housed in standard plastic cages. The

animals were kept in a well ventilated

room, temperature of 24- 28°C with 12

hrs natural light and 12 hrs darkness. The

rats had free access to tap water and dry

rat pellets obtained from local market ad

libitum. The rats were allowed to

acclimatize for ten days and then divided

into 2 groups; (10 per group) as follows:

control group treated with normal saline ,

and Chitocal treated group which

received 50mg/kg b.w. Chitocal

intragastrically for 4 weeks. (Chitocal

produced by the Arab Co. for Gelatin and

Pharma. for Al-Debeiky Pharma.Egypt:

(Compositions: Citosan H.D. 500mg,

Ascorbic Acid 100mg, and Gymnema

sylvestre Extract 50mg ).

Collection of blood samples and

biochemical analysis

At the end of the experiment, blood

samples were taken by cardiac puncture

and blood was collected in clean EDTA

tubes, then plasma was separated by

centrifugation (3000 rpm for 15 min.)

and stored at -20°C. Triglycerides level

was estimated by use of Randox kit

according to [9]. Cholesterol level was

estimated by Randox kit according to

Allain et al. 1974 [11]. The HDL

Cholesterol (HDL‐C) was estimated by

BioMaghreb kit according to Demacherp

1980 [12]. VLDL composition and LDL

cholesterol can be calculated with

reasonable accuracy by the Friedewald

formula [13]. Kits of creatinine, and urea

were purchased from Spinreact, S.A.

Ctra. Spain. Creatinine was determined

by kinetic method described by [14],

determination of urea was according to

the enzymatic method of [15].

Histological examination

Animals were killed and small piece of

kidney and intestine tissues taken from

experimental animals were fixed in 10%

neutral formalin, alcohol-dehydrated,

paraffin-embedded and the section to

mean thickness of 4 μm. The histological

examination was evaluated by assessing

the morphological changes with

Hematoxylin and Eosin (H&E) stains

[16].

Statistical analysis

Data are expressed as the mean ± SE.

The statistical significance was carried

out using one- way analysis of variance

test followed by Duncan’s Multiple

Range Test (SPSS statistical software

package) [17]. A possibility of P value

(p< 0.05) was considered as significant

differences between means.

Results and Discussion

Changes in body weight

From table 1, it is shown that, there

was a significant (p< 0.05) decrease in

the mean body weight of the Chitocal

treated rats when compared with normal

control groups. The results revealed that

Chitocal decreased body weight gain and

had an anti-obesity potential. These

findings are in agreement with other

investigators [9]. Studies reveal the

ability of Gymnema sylvestre to support

enzyme activity for glucose utilization

and to moderate glucose uptake into the

intestines [9].

Gymnemic acid isolated from the leaves

of Gymnema sylvestre has been reported

to inhibit the intestinal absorption of



glucose and oleic acid and reported to

exert beneficial effect in diabetes and

obesity [18]. Gymnemate promoted

weight loss in fatty rat model which

exhibits progressive overweight,

hyperlipidemia and hyperglycemia. [19].

The weight reducing effect may be due to

inhibition of lipogenesis in rats [20].The

possible mechanism of action may be

due to the hydrolysis of triglycerides and

activation of insulin by the enzyme

lipoprotein lipase [21]. Gymnema helps

to promote weight control by its ability to

reduce the cravings for sweetsand control

blood sugar levels [22]. A peptide

isolated from Gymnema, gurmarin, has

also been shown to block the sweet taste

of glucose and sucrose in animal models

[22]. Gurmarin temporarily binds to the

sweet and bitter receptors on the tongue,

thereby blocking the taste sensation and

reducing sweet cravings [22]. Gurmarin,

another constituent of the leaves, and

gymnemic acid have been shown to

block the ability to taste sweets in

humans [23].

Lipid profile:

Table 1, shows the effect of Chitocal

on the levels of plasma lipid profile.

Chitocal produced a significant (p< 0.05)

decrease in plasma cholesterol,

triglycerides, VLDL and LDL-

cholesterol levels compared with normal

rats, but plasma HDL-cholesterol level

slightly increase when compared with

normal rats. These findings are in

agreement with other studies. Preuss et

al. [24] showed a significant lowering of

cholesterol with Gymnema sylvestre

ingestion in hypertensive rats fed a high

sucrose diet, whereas the placebo group

showed a significant increase in

cholesterol levels.

The total cholesterol was decreased,

moreover LDL+VLDL (low-density and

very-low-density lipoprotein) cholesterol

decreased, the proportion of HDL (high-

density lipoprotein) cholesterol to the

total cholesterol was increased and the

serum triglyceride was decreased in fatty

rat exhibits hyperlipidemia after treated

with Gymnema sylvestre [19]. The HDL-

cholesterol is involved in transport of

cholesterol from peripheral tissue to liver

and thereby acts as protective factors.

This indicates that Gymnema sylvestre

may help to increase transport of

peripheral tissue cholesterol to liver and

thereby decrease blood level cholesterol.

The possible mechanism of action of

Gymnema sylvestre for increased HDL

may be due to increase in lecithin activity

[25]. The possible mechanism of action

in reduction of LDL- cholesterol may be

oxidation of LDL by enzymes. Gymnema

sylvestre is reported to produce

hypolipidemic effect by inhibition of

intestinal absorption of fatty acid in rats

[18].

According to animal studies, the leaves

are also noted for lowering serum

cholesterol and triglycerides [26]. While

studies have shown that a water-soluble

acidic fraction of the leaves provides

hypolipidemic actions, the specific

constituent in the leaves responsible for

this action has not been clearly identified.

Some researchers have suggested

gymnemic acid as one possible candidate

[27]. Antihyperlipidemic activity which

may be due to presence of flavonoids,

phenol, tannis (phenolic compounds) and

tritterpenoids found in the preliminary

phytochemichal screening [28]. The

possible mechanism for decreased lipid

levels could be either insulin releasing

effect of Gymnema sylvestre active

compounds or insulin sensitizing activity,

because insulin has been proved to

inhibit the activity of the hormone

sensitive lipase in adipose tissue and

suppresses the release of lipids [21].

Chitosan able to chelate many

substances like lipid and uric acid), the

mechanism of conjugation mediated by

high positive charge in the surface of

chitosan molecule. The activity of

chelation depends on the highly reactive

amino group which is able to chelate

lipids and many transitional ions. [29]



There are several proposed mechanisms

for cholesterol reduction by chitosan.

The latest findings in this field consider

more than one hypothesis. The

entrapment caused by a viscous

polysaccharide solution is thought to

reduce the absorption of fat and

cholesterol in the diet. On the other hand,

the presence of the amino group in its

structure determines the electrostatic

force between chitosan and anion

substances, such as fatty acids and bile

acids [30].

Creatinine and urea: Chitocal produced a significant (p<

0.05) increase in plasma levels of urea

and none significant (P < 0.05) increase

in plasma levels of creatinine, when

compared with normal control groups as

shown in Table 1. No study available

reported the effect of Chitocal on kidney

function parameters, but the result of

histological examination in this study

may support these results because they

showed negative effects of Chitocal on

kidney tissue, and this was affect kidney

function. The treatment with Gymnema

sylvestre extract found to decrease serum

creatinine and urea levels, and this may

be correlated with decrease in glucose

level by Gymnema sylvestre [21], and the

increases in these levels in our finding

may be due to the other composition of

Chitocal.

Table 1 Body weight and levels of plasma lipid, creatinine, and urea in rats treated with

50 mg/kg b.w. Chitocal.

Groups

Parameters

Normal control group

Mean ±S.E

Chitocal treated group

(treated with Chitocal

50 mg/kg b.w.)

Mean ±S.E

Initial body weight. g 205.8 ± 6.57 204.4±8.26

Final body weight. g 274±7.77 226±6.55*

Cholesterol mg /dl 91±2.64 75.8±0.97*

Triglyceride mg /dl 78±7.51 58±2.54*

HDL cholesterol mg /dl 41.62±1.33 48.02±1.37*

LDL cholesterol mg /dl 33.92±1.53 16.18±2.44*

VLDL cholesterol mg /dl 15.6±1.50 11.6±0.50*

Creatinine mg /dl 0.476±0.076 0.64±0.074

Urea mg /dl 28.8±3.96 40.6±2.96*

Values are expressed as mean ± S.E of 10 animals.

* Values are statistically significant P

<0.05 when compared with normal control.

Histological examination

The present study showed some

histopathologicl effects in kidney and

intestine tissues, histological examination

of the normal control kidney, tissues

showed normal histology (Fig. 1).

Kidney of Chitocal treated rats showed

congestion of blood vessels between

renal tubules, aggregation of

inflammatory cells (especially

monocytes) in lumen and wall of blood

vessels (Fig. 2), enlargement of renal

tubules and aggregation of inflammatory

cells around glomerulus (Fig. 3). Also

the results showed normal histology of

the normal control intestine tissue



(Fig.4).In intestine tissue belongs to

Chitocal treated rats moderate increase of

monocytes in lamina propria and

hyperplasia of goblet cells was observed

(Fig.5), aggregation of inflammatory

cells in epithelium, adhesion of intestinal

villi , hyperplasia in epithelium and

aggregation of inflammatory cells in

lamina propria (Fig.6).

These results may be due to the effect

of secondary metabolites result from

biodegradation of Chitocal components

which may have side effects on kidney

and intestinal tissues. The major role of

kidney is exertion of drug metabolites

and this may be the cause of these

negative effects. Muzzarelli et al

.propose a spontaneous formation of

insoluble chitosan salts from bile acids

whose hydrophobic nature should permit

the collection of cholesterol and lipids

via hydrophobic interaction [30], and this

may be affect the surface epithelium of

intestine and cause the adhesion of villi

and hyperplasia of goblet cells. From

these results it can be conclude there are

negative side effects of Chitocal on

histology of kidney and intestine, and

further studies are needed in this respect.

Figure 1 Section of kidney tissue belongs to normal control rat showing normal histology

of the kidney (H&E) 400X.

Figure 2 Section of kidney tissue belongs to rat treated with Chitocal showing

congestion of blood vessels between renal tubules ( ) aggregation of inflammatory

cells (especially monocytes) in lumen and wall of blood vessels ( ) (H&E) 400X.



Figure 3 Section of kidney tissue belongs to rat treated with Chitocal showing

enlargement of renal tubules ( ) aggregation of inflammatory cells around glomerulus

( ) (H&E) 400X.

Figure 4 Section of intestine tissue belongs to normal control rat showing normal

histology of the intestine (H&E) 400X.

Figure 5 Section of intestine tissue belongs to rat treated with Chitocal showing

moderate increase of monocytes in lamina propria ( ) hyperplasia of goblet cells

( ) (H&E) 400X.



Figure 6 Section of intestine tissue belongs to rat treated with Chitocal showing

aggregation of inflammatory cells in epithelium ( ) adhesion of intestinal villi and

hyperplasia in epithelium ( ) aggregation of inflammatory cells in lamina propria

( ) (H&E) 400X.

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