LIPID LOWERING AND ANTIOXIDANT ACTIVITIES OF CASSIA TORA SEED EXTRACT IN ALLOXAN INDUCED DIABETIC RATS
Vishnu Kumar*, Farzana Mahdi*, Ashok Kumar Khanna**, Ranjana Singh***, Ramesh Chander*, Jitendra Kumar Saxena**, Abbas Ali Mahdi*** and Raj Kumar Singh***.
*Department of Biochemistry Era’s Lucknow Medical College& Hospital Lucknow, **Division of Biochemistry, Central Drug Research Institute, Lucknow, ***Department of Biochemistry, CSM Medical University Lucknow.
Running Title: Antidyslipidemic activity of Cassia tora seed extract in diabetic rats.
Address for Correspondence:
Dr Farzana Mahdi
Professor, Department of Biochemistry & Director (Academics)
Era’s Lucknow Medical College & Hospital Lucknow
Sarfarazganj, Hardoi Road, Lucknow, 226003 (India)
Phone: +91-522-3201150 (Off.); Fax +91-522-2409784
Mobile: +91-9839014878, +91-933592330
E-mail:,
Copy to-
Dr Vishnu Kumar
Assistant Professor, Departmant of Biochemistry,
Era’s Lucknow Medical College & Hospital Lucknow
Sarfarazganj, Hardoi Road, Lucknow, 226003 (India)
E-mail:
ABSTRACT
The antidyslipidemic activity of Cassia tora seed extract has been studied in alloxan induced diabetic rats. In this model, oral administration of root extract (500 mg/kg bw. p.o.) for 30 days resulted in significant decreased in the levels of blood glucose, plasma lipids and reactivated post heparin lipoprotein lipase activity in alloxan induced diabetic rats. Furthermore, the root extract (50-500 µg) when tested for its antioxidant activity, shown to inhibit the generation of super oxide anions and hydroxyl radicals, in both enzymic and non-enzymic systems in vitro. The results of the present study demonstrated antidyslipidemic and activities in Cassia tora seed which could help in prevention of diabetic- dyslipidemia and related complications.
KEY WORDS
Cassia tora seed extract, Anti-dyslipidemic agent, Natural Antioxidants, Post heparin lipolytic activity, Alloxan induced-diabetes. Hypoglycemic agent.
INTRODUCTION
Cassia tora Linn (Family- Caesalpiniaceae) commonly known Chakvat, Chakunda and Charota in Hindi, Foetid Cassia in English is an herbaceous foetid annual weed, almost on under shrub, up to 90 cm in height. It grows in tropical and Asian countries especially on way sides and waste places and on hills of low elevations up to 1,800 m as well as in plains. Different parts of the plant (Leaves, seed, and root) are claimed to be effective against a variety of ailments in indigenous medicine [1]. The leaves and seeds are acrid, thermogenic, laxative depurative, antiperiodic, liver tonic, antihelmintic, cardio tonic and are useful in ringworm, pruritis, leprosy, skin disease, jaundice, helminthiasis, flatulence, dyspepsia, intermittent fevers, constipation, ophthalmopathy, cough, bronchitis,cardiac disorders and haemorrhoids [2, 3].The leaves of C.tora are reported to have antirheumatic activity in folklore practice. Decoction of the leaves is used as laxative. The seeds of C. tora have been used in Chinese medicine as vision-improving, cardiotonic, hypolipidemic, aperients, antiasthnic and diuretic agent. Several polyharbal formulations are available in Chinese market for preventing the formation of atherosclerosis plaque[4].
Cardiovascular diseases are leading cause of death in both industrialized and developing nations [5]. Disorders of lipid metabolism following oxidative stress are the prime risk factors for initiation and progression of heart diseases [6]. The current therapies used for controlling hyperlipidemia; fibrates, stains and bile acid sequestraints are almost inefficient to regulate lipid metabolism. Furthermore, these drugs also cause a number of serious adverse effects in patients. Currently available treatment for hyperlipidemia in modern medicine, fibrats, statins or bile acids sequestraints and their combinations do not regulate lipid metabolism up to a appreciable mark, also have several adverse effects in patients [7]. Therefore, there is a need to develop safe and effective treatment modalities for hyperlipidemia. Furthermore medicinal plants play an important role in the treatment of lipid disorders, especially due to their lesser toxicity, side effects and cost effectiveness. Therefore, the research and development of lipid lowering drugs from natural products are the best option and also are in great demand. . In view of the above considerations, the present study was designed to investigate lipid lowering activity of Cassia tora seeds in hyperlipidemic rats.
MATERIALS AND METHODS
MATERIAL AND METHODS
Preparation of seed extract: Cassia tora seeds were collected from local area of Lucknow and identified taxonomically by Department of Pharmacology, Era’s Lucknow Medical College Lucknow. A voucher specimen (CT-005/10) was also submitted. Seeds were crushed and dried under shade. The powder (500g) was extracted with 95 % ethanol in a soxhlet extractor for 72 h, the extract was concentrated to dryness under reduced pressure and controlled temperature (50-60 °C), yielding 23g of reddish brown solid (crude extract). This was stored in refrigerator and used for in vivo and in vitro experiments [8].
Preparation of Root Extract:
C. tora seeds were collected from local area of Lucknow and identified taxonomically by Department of Pharmacology, Era’s Lucknow Medical College, Lucknow. A voucher specimen (HRS-001/06) was also submitted. Roots were crushed and dried under shade. The powder (500g) was extracted with 95%, ethanol, (being highly polar, so as to extract the maximum phytoconstituents present in the roots) in a soxhlet extractor for 72 hrs (20). The extract was concentrated to dryness under reduced pressure and controlled temperature (40-45°C), yielding 50g of reddish brown solid (crude extract). The extract was stored in refrigerator and used for in vivo and in vitro experiments.
Antidyslipidemic activity in Alloxan induced diabetic rats: Animal study was performed with the approval of Animal Care Committee of Division of Laboratory Animal; Central Drug Research Institute, Lucknow, India and confirmed to the guideline for Care and Use of Laboratory Animals of the institute. Male adult rats of Charles Foster strain (200-225g) bred in the animal house of the Institute were used. The animals were kept in controlled conditions; temperature 25 -26˚C, relative humidity 60-70% and 12/12 hrs light / dark cycle (light from 08:00 AM to 08:00PM), provide with standard pellet diet (Lipton India Ltd.), and water adlibitum. One group of normal rats without treatment with alloxan was used to serve as control. In remaining animals, diabetes was induced by a single intraperitonial injection of alloxan monohydrate 150 mg/Kg b.w. After three days of injection, diabetes was confirmed by glucometer. The rats with serum glucose level 280-320 mg/dl were taken for the study (21).
Experimental design: The rats were divided in four groups having six animals in each as follows: Group 1: control rats (on normal saline); Group 2, Alloxan treated diabetic rats (on normal saline); Group 3, Alloxan treated diabetic rats + C. tora seeds (500mg/kg b.w); Group 4, Alloxan treated diabetic rats + glibenclamide (600μg/kg b.w). After 30 days of feeding rats were fasted overnight, anaesthetized with thiopental solution, and injected (ip) with 0.1ml/kg bw of 10mg/ml solution of heparin. After 15 min blood was withdrawn from the retro-orbital plexus and collected in EDTA coated tubes. The blood was used for the estimation of glucose level (21), simultaneously plasma was separated and used for the estimations of Total Cholesterol: TC (22), Phospholipids: PL (23), Triglyceride: TG (24) Free Fatty Acids: FFA (25) Plasma posts heparin lipolytic activity (26) by standard spectrophotometeric methods. Plasma level of lipid peroxide was estimated as ThioBarbituric Acid Reactive Substances: TBARS (27).
In vitro Anti oxidant activity:
Enzymic and non enzymic generation of superoxide anions: The effect of C. tora seed extract on the generation of superoxide anions (O2–) in vitro, in an enzymic system of xanthine-xanthine oxidase was investigated (28). Xanthine oxidase activity in system (A) containing xanthine and different concentrations of C. tora seeds extract (50-500µg) added with 0.03 U/ml of xanthine oxidase in phosphate buffer, was assayed spectrophotometrically at 295 nm. The change in optical density corresponding to amount of uric acid formed was compared with reaction mixture which did not include with their test substance. The influence of C. tora seeds extract on nitro blue tetrazolium (NBT) reduction by O2– anions, was measured in a reaction mixture (B) containing xanthine oxidase and NBT in absence or presence of extract (50-500 µg). After incubation, the amount of formazone formed was measured at 560 nm on spectrophotometer. Another system employed for non-enzymic generation of O2– anions was comprised of phenazine methosulphate, NADH and NBT (29). After 90 sec incubation in absence or presence of test extract 50-500 µg, the amount of formazone formed was read at 560 nm against respective reagent blank.
Enzymic and non enzymic generation of hydroxyl radical: C. tora seed extract (50–500 µg) was tested against the formation of hydroxyl radicals (OH–) in vitro in an enzymic system composed of sodium salicylate, FeSO4, hypoxanthine and xanthine oxidase, assayed for 2, 3-dihydroxybenzoate formed by OH– mediated hydroxylation of salicylate on spectrophotometer at 510 nm (30). In another set of experiment, OH– was generated non-enzymically by FeSO4, sodium ascorbate, H2O2 and deoxyribose. After reaction in the absence or presence of C. tora seeds extract (50–500 µg), incubation mixture was assayed for malondialdehyde formed (31).
Statistical Analysis: One way analysis of variance (ANOVA- New man’s student test) was performed by comparison of values for alloxan treated group with control, alloxan and drug treated groups with alloxan. All hypothesis testing were two tailed. p<0.05 was considered statistically significant and the results were expressed as mean +SD. The statistical analysis was carried out by the Graph pad INSTAT 3.0 software (32). Similarly, the generations of oxygen free radicals with different concentrations of Hibiscus root extract were compared with that of their formation without extract. The values were tested for significance at P 0.05.
RESULTS
Effect of C. tora seeds extract in alloxan induced hyperglycemia: The acute administration of alloxan caused marked increase in their plasma levels of blood glucose 260% , TC 57% , TG 84% , PL 45 %, FFA 49% and lipid peroxide 229% following decrease in PHLA by 31 %. However, treatment with C. tora seeds extract caused reversal in these levels of blood glucose by 30 %, TC by 24.0%, PL by 17%, TG by 23.0%, FFA by 10.0%.,lipid peroxide by 36% and reactivation of PHLA by 17 %. The anti-diabetic and lipid lowering activities of C. tora seeds extract was comparatively less to that of Glibenclamide (Table-1).
Effect of C. tora seeds extract on generation of super oxide anions:
The data in Table -2 showed that enzymic oxidation of xanthine to uric acid (A) as well as the generation of O2– anions in xanthine–xanthine oxidase system, as measured by reduction of NBT to Formazone (B) were inhibited to varying extents by seed extract in a concentration dependent manner and this effect was maximum by 55% and 51% respectively at 500 µg/ml of test sample. The seed extract also trapped the O2– anions generated by non enzymic system of NADH – Phenozine–Methosulphate and were responsible for reduction of NBT in the reaction mixture. The effect was dose dependent and was highest by 58% at 500 µg/ml of test substance.
Effect of C. tora seeds on generation of hydroxyl radicals
The data in Table -2 also showed that C. tora seeds extract inhibited the formation of OH – by enzymic system of hypoxanthine–xanthine oxidase and Fe++. Addition of extract (50-500 µg) inhibited the OH- mediated formation of 2, 3 dihydroxybenzoate in concentration dependant manner which was 50% at 500 µg/ml of test extract. Furthermore, this preparation , when added with reaction mixture containing Fe2+ –Sodium ascorbate- H2O2 employed for nonenzymic generation of OH- inhibited fragmentation of deoxyribose into MDA and this effect was maximum by 48 % at peak concentration (500 µg/ml) of seed
extract.
NEW AS PER RAMESH SIR CASSIA TORA
References:
- Warrier P K.Indian Medicinal Plants, a compendium of 500 species 2001; 2: 26.
- Pawar H A, D’mello PM .Cassia tora Linn:An Overview. Int J Pharmaceutical Sci Res. 2011; 2(9): 2286-2291.
- Chaurasia B, Dhakad R S, Dhakar V K, Jain P K.Preliminary Phytochemical and pharmacological (Antidiabetic) screening of Cassia tora Linn. Int. J. of Pharm. Life Sci.2011; 2(5):759-766 759.
- Kee C H.The pharmacology of Chinese Herbs.CRC Press.BocaRaton ,2001; pp.103.
- Gupta R, Joshi P, Mohan V, Reddy KS, Yusuf S.(2008) Epidemiology and causation of coronary heart disease and stroke in India. Heart .2008; 94:16-26.
- Mohammed K., Ali K.M., Narayan V,Tandon N. Diabetes and coronary heart disease: Current perspectives. Indian J Med Res.2010; 130: 584-597.
- Chattopadhyaya R, Pathak D, Jindal DP.Antihyperlipidemic agents. A review. Indian drugs.1996; 33: 85-97.
- Satyavati, G V. Guggulipid:A promising hypolipedemic agent from gum guggul (Commiphora wightii).In Economic and Medicinal Plant Research, Vol.V. Plants and Traditional Medicine, Academic Press, New York, 1991; pp. 47-82.
Discussion:
In the present study, seeds of C. tora were tested for its anti-diabetic, anti-dyslipidemic and anti-oxidant activities in alloxan induced diabetic rats. Alloxan caused reversible damage to insulin-producing β-cells found in the pancreas, and that is why this animal model have been used for primary screening of test drugs for antidiabetic activity (33). We have found that intoxication with alloxan caused increased levels of plasma glucose in rats and their reversal by the treatment with C. tora seeds extract. Furthermore seed extract also reduced lipid peroxide levels in above diabetic rats following inhibition of ROS generation in vitro.
The level of serum lipids is usually raised in diabetes and represents a risk factor for cardiovascular disease (34). Furthermore, hyperglycemia and dyslipidemia induced metabolic alterations caused a variety of pathophysiological changes also impose marked oxidative stress in diabetic patients. The increase in non enzymic glycosylation occurs, accompanied with glucose oxidation and these reactions are catalyzed by Cu+2 and Fe+2 resulting in the formation of O2- and OH- radicals which further accelerates the risk of dyslipidemia and cardiac disease in them (35). Thus to overcome these ailments, a drug having multifold properties such as antidiabetic, lipid lowering and antioxidant activities altogether are in great demand. Though there are various approaches to reduce the ill effects of diabetes and its secondary complications, herbal formulations are preferred due to lesser side effects and its low cost. The phytochemical studies showed that C. tora seeds extract contains a variety of Sterols, Carbohydrates and glycosides, Tannins, Flavonoids(36).
We have reported that hypoglycemic and hypolipidemic action of glibenclamide is mediated through activation of PHLA, LPL, and lecithin cholesterol acyl transferase activities, inhibition of hepatic cholesterol biosynthesis, and increased faecal bile acid excretion [9]. The same mechanisms may also interplay to cause the hypolipidemic effect of C. tora seed extract.