Reference ID: 00076

TITLE
EFFECTS OF NIGELLA SATIVA SEEDS ON LIPID PROFILE AND
PLASMA GLUCOSE LEVEL IN HIGH FAT DIET FED DIABETIC RAT
MODELS

INTRODUCTION

India is the diabetes capital of the world; every fifth diabetic in the world
is an Indian [1]. The increase in the prevalence of type 2 diabetes is
closely linked to the upsurge in obesity. About 90% of type 2 diabetes is
attributable to excess weight. Approximately 197 million people
worldwide have impaired glucose tolerance, most commonly because of
obesity and the associated metabolic syndrome [2]. A scientific
investigation of traditional herbal remedies for diabetes may provide
valuable leads for the development of alternative drugs and strategies.
Alternatives are clearly needed for better management of diabetes
because of high cost and poor availability of current therapies for many
rural populations, particularly in developing countries [3].

Nigella sativa (NS) (Kalonji, Mungrela) an annual herbaceous plant of the

Ranunculaceae family, is a commonly used spice in Indian food and has
been used for medicinal purposes in Asian and African countries [4]. Its
seeds have been used for many years for therapeutic purposes, including
their potential anti-diabetic properties. In India NS is cultivated or
occasionally found as a weed Punjab, Himachal Pradesh, Bihar and
Assam.
Studies have shown that NS extracts have shown promise as an anti-
diabetic agent in streptozotocin-induced diabetic rats by exerting a
therapeutic and protective effect on the pancreas, by decreasing oxidative
stress and preserving B-cell integrity [5]. NS also increased Serum
Insulin, and decreased the diabetes induced hyper-glycaemia. [5][6][7]
Studies have also shown significant improvement with reference to lipid
profile [8] [9].

This research has been planned to study the effects of NS seeds on lipid
profile and plasma glucose level in diabetic rat models that have been fed
with high fat diet. These parameters will also be studied in experimental
animals treated with metformin. This would help to compare the
effectiveness of alternative preparation-NS with that of metformin. To
best of our knowledge no such research has been undertaken till date,
there fore such a project becomes relevant.
 OBJECTIVES
1. To measure Blood glucose before and 7 days after starting NS
treatment in experimental animals.
2. To evaluate quantitatively Lipid Profile before and 7 days after
starting NS treatment.
3. To measure weight and abdominal circumference (AC) before and at
the end of treatment with NS.
4. To measure above parameters in experimental animals before and
after treatment with Metformin.
5. The above readings will be analyzed to study and compare the effect
of NS treatment and Metformin on lipid profile and blood glucose
level in diabetic rat models fed with high fat diet.

MATERIAL AND METHODS

PROCUREMENT OF SEEDS
N. sativa seeds will be obtained from NBRI (National Botanical Research
Institute), Lucknow and authenticated by a botanist at NBRI.

PREPARATION OF PLANT EXTRACT

Ethanollic extract of NS seeds will be prepared by maceration.
50 mg seeds will be soaked in 150 ml Ethanol (analytical grade) for 7
days with frequent stirring with a sterile glass rod. After 7 days, extract
will be filtered using Whatman Filter paper- No. 1 and the filtrate
concentrated using Rotary Evaporator and stored at 4˚C till further use.
Dose of NS will be used as per earlier studies [9].

ANIMALS
Male Wistar rats (200-250 gm) will be obtained from CDRI, Lucknow and
kept in the institutional Animal House under 12 hour day and night
cycle and standard conditions. Food and water will be provided ad
libitum.
All experiments will be performed after approval from Institutional Ethics
Committee and as per the guidelines of Animal Care by CPCSEA.
Streptozotocin will be obtained from Sigma Aldrich, USA.

Induction of Type 2 Diabetes Mellitus

Streptozotocin induced hypoglycemia has been described as a useful
experimental model to study the activity of hypoglycemic agents [11].In
this study also DM will be induced using streptozotocin.
After overnight fasting, diabetes will be induced by single intra-peritoneal
injection of streptozotocin 50mg/Kg. The animals will be allowed to drink
5% glucose solution overnight to overcome drug-induced hypoglycemia.
After a week’s time for development of diabetes, diabetic rats (blood
glucose above 250 mg/dl) will be used for further study.
High Fat Diet
The diabetic rats will be fed a high fat diet (HFD) for 4 weeks.
HFD will be composed of 300 gm concentrates, 350 corn, 300 gm beef
tallow, 50 gm vitamins, minerals and fibers. [12]
Calculations of HFD was 20% crude protein, 35% fat, 40 % CHO (starch
35%, 5% sucrose) 5% vitamins and minerals and fibers. Metabolic energy
of this diet is 5130 Kcal /kg. 61% of this energy will be from fat.

Diabetic rats will be divided into groups of 6 each and treated as follows:
i. Group 1: Diabetic control will be given vehicle orally.
ii. Group 2: NS ethanollic extract 300mg/kg body weight orally for 7
days.
iii. Group3: NS ethanollic extract 600mg/kg body weight orally for 7
days.
iv. Group 4: Tab Metformin 100mg/kg/day for 7 days.

In all four groups 1ml of blood will be withdrawn from tail vein at Day 0
and Day 7 for analysis of Lipid Profile and Blood Glucose using Auto-
analyzer.
AC [13] will be assessed on the largest zone of the rat abdomen using a
plastic non extensible measuring tape with an accuracy of 0.1 cm. AC
corresponds to visceral fat mass in rodents.

STATISTICAL ANALYSIS
Lipid profile and blood glucose, body weight and AC parameters in the
different groups will be compared using ANOVA.
Relationships between body weight, AC and response to drug treatment
would be measured using Pearson’s Test.
All analyses will be done using SPSS 17.0 Version. P <0.05 will be
considered as significant.

IMPLICATIONS
So far very few studies have been carried out to evaluate the effect of NS
on lipid profile and blood glucose level in STZ-diabetic models. To the
best of our knowledge, no such study has been carried out to evaluate
these parameters on high fat diet fed STZ-diabetic rats. Considering the
added risk of obesity and hyper-lipidaemia in diabetics, such a study
becomes worth-while.
It will help in learning research methodology in the fields of diabetes and
obesity.
It is positively hoped that this study will go a long way in devising
appropriate dietary intervention and treatment in patients with co-
existing DM and obesity, which is a growing problem in India.

REFERENCES
1) Shashank R Joshi, Rakesh M Parikh. India - Diabetes Capital of the World : Now
Heading Towards Hypertension. JAPI 2007;55:323-24.
2) Parvez Hossain,Bisher Kawar and Meguid El Nahas. Obesity and Diabetes in the
Developing World — A Growing Challenge. N Engl J Med 2007; 356:213-215.
3) Nalamolu Koteswara Rao and Srinivas Nammi. Antidiabetic and renoprotective
effects of the chloroform extract of Terminalia chebula Retz. seeds in
streptozotocin-induced diabetic rats. BMC Complement Altern Med. 2006; 6: 17.
4) Pandey B. P.A textbook of Botany- Angiosperms, 2001 pg 916.
5) Kapoor S. Emerging clinical and therapeutic applications of Nigella sativa in
gastroenterology. World J Gastroenterology 2009 May; 15(17):2170-1.
6) Pari L, Sankaranarayanan C. Beneficial effects of thymoquinone on hepatic key
enzymes in streptozotocin-nicotinamide induced diabetic rats. Life Sci. 2009 Dec
16;85(23-26):830-4.
7) Kapoor S. Emerging clinical and therapeutic applications of Nigella sativa in
gastroenterology. World J Gastroenterology 2009 May; 15(17):2170-1.
8) Najmi A, Nasiruddin M, Khan RA, Haque SF. Effect of Nigella sativa oil on
various clinical and biochemical parameters of insulin resistance syndrome. Int
J
Diabetes Dev Ctries. 2008 Jan;28(1):11-4.
9) Kaleem M, Kirmani D, Asif M, Ahmed Q, Bano B. Biochemical effects of Nigella
sativa L seeds in diabetic rats. Indian J Exp Biol 2006 Sep;44(9):745-8.
10) Bailey CJ. Biguanides and NIDDM. Diabetes Care 1992;15:755–772.
11) Amin KA, Kamel HH, Abd Eltawab MA. Protective effect of Garcinia against renal
oxidative stress and biomarkers induced by high fat and sucrose diet. Lipids in
Health Dis 2011 Jan 14;10(1):6.
12) Kanter M, Meral I, Yener Z, Ozbek H, Demir H. Partial regeneration/proliferation
of the beta-cells in the islets of Langerhans by Nigella sativa L. in streptozotocin-
induced diabetic rats. Tohoku J Exp Med 2003 Dec; 201(4):213-9.
13) Visceral fat mass determination in rodent: validation of dual-energy x-ray
absorptiometry and anthropometric techniques in fat and lean rats Maude
Gerbaix, Lore Metz, Emeline Ringot, Daniel Courteix.