|Year : 2014 | Volume
| Issue : 1 | Page : 34-38
Lack of hypoglycemic activity in total flavonoid fraction of Wrightia tinctoria on alloxan induced hyperglycemia
AK Shukla, Papiya Bigoniya
Department of Pharmacology and Pharmacognosy, Radharaman College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh, India
|Date of Web Publication||16-Jul-2014|
Radharaman College of Pharmacy, Ratibad, Bhopal-462002, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The objective of this study was to investigate the hypoglycemic activity of Wrightia tinctoria (Roxb) (R. Br. Linn., Family- Apocynaceae) total flavonoid (WTTF) fraction on alloxan induced hyperglycemia. Materials and Methods: Total flavonoid fraction from WTTF seed was separated and LD 50 calculated as 1006.93 mg/kg, i.p., following Organization for Economic Co-operation and Development guideline 423. The effect of total flavonoid fraction was observed in three different dose levels 50, 75, and 100 mg/kg. Hypoglycemic activity was carried out using alloxan induced hyperglycemia model. The parameters observed were body weight, blood glucose, total cholesterol, triglycerides (TG), low density lipoprotein, high density lipoprotein, urea and creatinine along with relative organ weight of liver, kidney, and pancreas. Results: Total flavonoid fraction of W. tinctoria did not have any significant effect on reducing glucose level. WTTF had significantly reduced creatinine (P < 0.001), TG (P < 0.05) level and relative weight of liver in diabetic rat at the dose of 100 mg/kg. Conclusion: All the findings indicate absence of antidiabetic activity in W. tinctoria seed but possible presence of liver and kidney protective property.
Keywords: Alloxan monohydrate, flavonoid, seed, Wrightia tinctoria
|How to cite this article:|
Shukla A K, Bigoniya P. Lack of hypoglycemic activity in total flavonoid fraction of Wrightia tinctoria on alloxan induced hyperglycemia. J Pharm Negative Results 2014;5:34-8
|How to cite this URL:|
Shukla A K, Bigoniya P. Lack of hypoglycemic activity in total flavonoid fraction of Wrightia tinctoria on alloxan induced hyperglycemia. J Pharm Negative Results [serial online] 2014 [cited 2020 Jun 2];5:34-8. Available from: http://www.pnrjournal.com/text.asp?2014/5/1/34/136793
| Introduction|| |
Worldwide diabetes mellitus (DM) has reached epidemic proportions as per the World Health Organization, which is strongly related to lifestyle and economic changes. Over the next decade the number of diabetic patient worldwide will exceed the figure of 200 million. By the year 2025, India is predicted to have the most number of people with DM in the world.  Diabetes requires continuing medical care to prevent acute complications and the risk of long-term complications. Many diabetic people turn to complementary therapies to control the chronic nature and threat to quality of life eventually reducing the complications.  For a long time plants based herbal medicines have been the major source of drugs for treatment of DM in Ayurveda and other ancient systems of medicine, as plant products are generally considered to be less toxic and free from side-effects compared with modern synthetic drugs. , Ethnobotanical information refers to suggestive antidiabetic potential in about 800 medicinal plants and many herbs have been reported to possess hypoglycemic activity in the literature. ,
Wrightia tinctoria (Roxb.) R.Br. is a small deciduous tree of the family Apocynaceae distributed in Central India, Burma and Timor. This plant is extensively used in the Indian system of medicine. Fresh leaves are pungent and are chewed for relief from toothache. Bark and seeds are antidysenteric, carminative, astringent, aphrodisiac and diuretic, used in flatulence, stomach pain and bilious affections.  W. tinctoria has reported antipsorasis,  antinociceptive,  wound healing effect,  hepatoprotective activity,  antiulcer and immunomodulatory effect.  The methanolic, ethanolic and ethyl acetate extract of W. tinctoria showed rich presence of alkaloid, glycoside, triterpene, tannins and phenolic compounds. , Until date, no pharmacological evidence for effectiveness of W. tinctoria phenolic and flavonoid content has been reported. Flavonoids contain wide range of biological activity and lot of research has been carried out on their potential role in treating diabetes and other diseases. Flavonoids are capable of improving, stabilizing and long sustaining the insulin secretion, human islets and pancreatic cell respectively.  This study was performed to assess the antidiabetic efficacy of W. tinctoria seed to explore the hypoglycemic effect of total flavonoid fraction.
| Materials and methods|| |
0Collection and identification of plant material
Wrightia tinctoria seeds were purchased from local market of Bhopal, Madhya Pradesh, India. The plant was identified and authenticated by Dr. Tariq Hussain, Head, Herbarium Department, National Botanical Research Institute, Lucknow, Uttar Pradesh. A voucher specimen number was allotted (No. 97314/2009) and kept for future reference.
Isolation of total flavonoid from Wrightia tinctoria seed
Ground seed was extracted in a soxhlet extractor for 18 h with methanol and filtered through Whatman fiter paper. The mixture was concentrated under reduced pressure using Rotatory Evaporator (Jyoti Scientific, India) at temperature not >50°C and dried. The obtained methanolic extract was dissolved in aqueous acetone (75%), filtered, evaporated under reduced pressure and dried which gave positive result for flavonoid. The percentage yield of total flavonoid was 4.40% w/w, designated as WTTF.
Laboratory bred Swiss Albino mice (20-25 g) and Wistar albino rats (120-150 g) of both sex were maintained under standard laboratory conditions at 22°C ± 2°C, relative humidity 50% ±15% and photoperiod (12 h dark and light), were used for the experiment. Commercial pellet diet (Hindustan Lever, India) and water were provided ad libitum. Ethical Committee approval was obtained from Institutional Animal Ethical Committee (approved body of "committee for the purpose of control and supervision of experiments on animals" Chennai, India) of Radharaman College of Pharmacy, Bhopal, before carrying out the experiments (IAEC/RCP/July 2011/05).
Acute toxicity studies (LD 50 )
LD 50 was determined according to the guidelines of Organization for Economic Co-operation and Development (OECD) following the up and down method (OECD Guideline No. 423) and fixed dose method (OECD Guideline No. 420). Based on these guidelines, a limit test was performed at 1000 mg/kg (i.p.) for WTTF, which showed 40.00% mortality. A main test was performed to determine the exact LD 50 value following OECD up and down method. 
Induction of diabetes in rats
Diabetes was induced on overnight fasted rats by a single dose administration of freshly prepared alloxan monohydrate (150 mg/kg, ip.) in normal saline.  Blood glucose (BG) level was measured by using one-touch glucometer and diabetes was confirmed after 72 h of alloxanization. Rats with fasting BG level >150 mg/dL were considered to be diabetic and were selected for studies.
Animals were randomly divided into six groups with six rats in each.
- Group I: Vehicle control group (0.2 ml/100 g body weight, i.p): 1% tween 80 solution in water for injection
- Group II: Diabetic control
- Group III: Metformin (20 mg/kg body weight, p.o.)
- Group IV: WTTF (50 mg/kg body weight, i.p)
- Group V: WTTF (75 mg/kg body weight, i.p)
- Group VI: WTTF (100 mg/kg body weight, i.p).
During the study period, body weight and BG level were recorded at 1 st , 7 th 14 th and 21 st day. On the 21 st day animals were sacrificed and organs (liver, pancreas, and kidney) were isolated and kept in ice cold saline solution, blotted and weighed. Blood samples collected by heart puncture, serum was separated after coagulating at 37°C for 30 min and centrifuged at 3000 rpm for 10 min.  Serum was analyzed for various biochemical parameters total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), urea, and creatinine.
Serum content of glucose, TC, TG, HDL, urea and creatinine were estimated in Span Auto analyzer (India) using commercial kit (Span Diagnostics Pvt. Ltd., India). Serum LDL cholesterol content was calculated with formula using serum concentration values of TC, TG, and HDL cholesterol.
Experimental data were analyzed using one-way ANOVA followed by Turkey-Kramer multiple comparison test. P < 0.05 were considered statistically significant. GraphPad Prism version 3.02 (GraphPad Software, Inc., USA) was used for statistical calculations.
| Results|| |
LD 50 was calculated as 1006.93 mg/kg from graphical representation. A dose range of 50, 75 and 100 mg/kg was selected for WTTF to evaluate the pharmacological activities.
Effect on body weight
Body weight gain in vehicle control group after 21 days was 6.14%. Diabetes prevents the weight gain on 14 th day and 21 th day by 7.38% and 8.94% respectively. Standard drug metformin treated group and WTTF (50, 75 and 150 mg/kg) treated groups showed decrease in body weight gain by 2.41%, 7.05%, 8.82% and 6.37% on 21 th respectively [Table 1].
Effect on liver, kidney and pancreas weight
Liver kidney and pancreas weight were determined on 21 st day after sacrifice of animals. The increase in weight of liver and pancreas was highly significant (P < 0.001) in diabetic animals compared with vehicle control group. Metformin significantly reduced the liver (P < 0.001) and pancreas (P < 0.01) weight compared with diabetic control group. WTTF at dose of 100 mg/kg dose treatment significantly (P < 0.001) decreased liver weight in diabetic animals [Table 1].
|Table 1: Effects of WTTF treatment on body weight of alloxan induced diabetic rats |
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Effect on serum blood glucose
Fasting BG levels were determined on day 1 st and every 7 th day for 3 weeks [Table 2]. BG in the diabetic group increased gradually from the 1 st day to the termination of experimental period (P < 0.001) in comparison to vehicle control group. WTTF in dose of 50, 75 and 150 mg/kg has nonsignificant effect on BG level. Metformin treated groups significantly suppressed (P < 0.001) serum BG level after 14 th day to the termination of experimental protocol.
|Table 2: Effects of WTTF treatment on blood glucose level of alloxan induced diabetic rats |
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Effect on biochemical parameters
Alloxan induced diabetes has significantly (P < 0.001) increased the serum level of TC, TG, LDL and urea level in animals compared to vehicle control group. Effect of WTTF at 50, 75 and 150 mg/kg dose on serum TC, LDL, HDL and Urea was nonsignificant. WTTF had significantly reduced creatinine (P < 0.001) and TG (P < 0.05) level in diabetic rat at the dose of 100 mg/kg [Table 3].
|Table 3: Effects of WTTF treatment on serum biochemical parameter of alloxan induced diabetic rats |
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| Discussion|| |
In this study, hypoglycemic effect of total flavonoid isolated from W. tinctoria seed was evaluated by assessing body weight change, relative organ weight, BG level, and serum lipid parameters. Obesity is mostly associated with DM, indicating weight control as an important aspect of diabetes management. Our results showed that all the animals treated with WTTF have lost weight during the study period. The weight loss was highest in the diabetic control group while metformin treated group showed minimum weight loss. Metformin has effective reduced the elevated liver and pancreas weight in diabetic animals. WTTF had no effect on pancreas though the liver weight was significantly normalized. Bigoniya and Rana (20 10) reported significant reduction of serum glutamate oxaloacetate transaminase level without effecting serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase level by the alcoholic extract of W. tinctoria bark signifying its hepatoprotective effect. The triterpenoidal fraction of bark is reported to have hepatoprotective against carbon tetrachloride induced acute rat liver toxicity.  This indicates protective effect of WTTF on liver either by reducing oxidative damage or by normalizing liver lipid metabolizing and turnover mechanism.
The diabetic control group animals showed fasting BG level above 250 mg/dl, which has remained higher throughout the study period of 21 days. Metformin (20 mg/kg) was used as a standard drug, showed decrease in BG level on 7 th day onwards. The WTTF in all treated doses showed nonsignificant change in BG level throughout the study period. On long-term prolonged use W. tinctoria bark showed increase in lipid profile along with increased body weight, hemopoiesis and glucose suggesting its anabolic effect.  Methanolic extract of W. tinctoria bark with rich flavonoid content has potential antianemic and hematopoietic effect. 
Kedar and Chakrabarti have reported that the cholesterol, TG and SGPT levels are increased in case hyperglycemia. Deficiency of insulin caused the increase in the level of these enzymes in liver and serum of diabetic animals. It was also reported that the elevated level of enzymes in the liver and serum decreases significantly with the treatment of antidiabetic drugs like phenformin, metformin with improved body weight gain. 
In this study TC, TG, LDL, urea, and creatinine was increased in diabetic group and metformin effectively control all these disturbed parameters. WTTF treated group showed nonsignificant change in serum lipid parameter except decrease in TG and creatinine level. Extract induced decrease in TG level may be due to its effectiveness against diabetes induced fatty liver. Phytoconstituents like flavonoids are known to possess hepatoprotective activity. ,
Kidney maintains optimum chemical composition of body fluid by acidification of urine and removal of metabolic wastes such as urea, uric acid, creatinine, and ions. During renal diseases, the concentration of these metabolites increases in blood.  In this study, the diabetic animals showed significantly higher level of serum creatinine, which has been reduced to normal by WTTF at 100 mg/kg dose treatment. This effect may be due to the diuretic action of W. tintoria ethanolic extract as reported by Bigoniya et al., 2008. Normalization of creatinine level by WTTF indicates that it has protective effect on the kidney and further studies are required to explore effect of WTTF on kidney. The WTTF in all treated doses showed absence of antidiabetic effect indicating no effect on β-cells of the pancreas or not related to increased utilization of glucose by cells. On many cases beneficial flavonoids are less effective due to poor solubility, decreased bioavailability, first pass metabolism and intestinal degradation. 
| Conclusion|| |
The effect of W. tinctoria seed flavonoid fraction was not significant on hyperglycemia and other disturbed biochemical parameter induced by alloxan, but it has significant effect on normalization of serum creatinine level and lowering of TG and relative weight of liver indicating possible presence of kidney and liver protective property.
| References|| |
|1.||Sridhar GR. Diabetes in India: Snapshot of a panorama. Curr Sci 2002;83:791. |
|2.||Israili ZH, Hernández-Hernández R, Valasco M. The future of antihypertensive treatment. Am J Ther 2007;14:121-34. |
|3.||Akhtar FM, Ali MR. Antihyperglycemic activity of polysaccharide from Lyceum barbarum. J Pak Med Assoc 1984;34:239-44. |
|4.||Brinker F. Herb Contraindications and Drug Interactions. 2 nd ed. USA: Eclectic Medical Publications; 1998. |
|5.||Ilango K, Chitra V. Antidiabetic and antioxidant activity of Limoni acidissima Linn. in alloxan induced rats. Der Pharmacia Lettre 2009;1:117-25. |
|6.||Pepato MT, Baviera AM, Vendramini RC, Perez Mda P, Kettelhut Ido C, Brunetti IL. Cissus sicyoides (princess vine) in the long-term treatment of streptozotocin-diabetic rats. Biotechnol Appl Biochem 2003;37:15-20. |
|7.||Kirtikar KR, Basu BD. Indian Medicinal Plants. 2 nd reprint ed. Delhi: Jayyed Press; 1975. |
|8.||Mitra SK, Seshadri SJ, Venkataranganna MV, Gopumadhvan S. Reversal of parakeratosis, a feature of psoriasis by Wrightia tinctoria (in emulsion) histological evaluation based on mouse tail. Indian J Dermatol 1998;43:102-4. |
|9.||Reddy YS, Venkatesh S, Ravichandran T, Murugan V, Suresh B. Antinociceptive activity of Wrightia tinctoria bark. Fitoterapia 2002;73:421-3. |
|10.||Veerapur VP, Palkar MB, Srinivasa H, Kumar MS, Patra S, Rao PG, et al. The effect of ethanol extract of Wrightia tinctoria bark on wound healing in rats. J Nat Remedies 2004;4:155-9. |
|11.||Chandrashekhar VM, Haseeb TS, Habbu PV, Nagappa AN. Hepatoprotective activity of Wrightia tinctoria (Roxb) in rats. Indian Drugs 2004;41:336-70. |
|12.||Bigoniya P, Rana AC, Agrawal GP. Evaluation of the antiulcer activity of hydroalcoholic extract of Wrightia tinctoria bark in experimentally induced acute gastric ulcers on rat. Niger J Nat Prot Med 2006;10:36-40. |
|13.||Bigoniya P, Shukla A, Agrawal GP, Rana AC. Pharmacological screening of Wrightia tinctoria barks hydro-alcoholic extract. Asian J Exp Sci 2008;2:235-44. |
|14.||Bigoniya P, Singh S, Singh CS, Shukla A. Anti-anemic potential estimation on mice and characterization of flavonoids using HPTLC in W. tinctoria bark fraction. J Nat Pharm 2013;4:47-56. |
|15.||Mohan SN. Role of various flavonoids: Hypotheses on novel approach to treat diabetes. J Med Hyp Ideas 2014;8:1-6. |
|16.||OECD. Guideline for the Testing of Chemicals, Guidance Document on Acute Oral Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment. Environment Directorate, Paris; 2000. |
|17.||Etuk EU, Muhammed BJ. Evidence based analysis of chemical method of induction of diabetes mellitus in experimental rats. Asian J Exp Bio Sci 2010;1:139-42. |
|18.||Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother 2010;1:87-93. |
|19.||Bigoniya P, Rana AC. Protective effect of Wrightia tinctoria bark triterpenoidal fraction on CCl 4 induced acute rat liver toxicity. Iran J Pharmacol Ther 2010;9:55-62. |
|20.||Bigoniya P, Rana AC. Effect of subacute exposure of Wrigtia tinctoria bark extract on hematological, biochemical and antioxidant enzyme parameters of rat. Pharmacogn Mag 2009;5:372-80. |
|21.||Kedar P, Chakrabarti CH. Effects of jambolan seed treatment on blood sugar, lipids and urea in streptozotocin induced diabetes in rabbits. Indian J Physiol Pharmacol 1983;27:135-40. |
|22.||Baek NL, Kim YS, Kyung JS, Park KH. Isolation of anti-hepatotoxic agent from the roots of Astragalus membranaceous. Korean J Pharmacog 1996;27:111-6. |
|23.||Oh H, Kim DH, Cho JH, Kim YC. Hepatoprotective and free radical scavenging activities of phenolic petrosins and flavonoids isolated from Equisetum arvense. J Ethnopharmacol 2004;95:421-4. |
|24.||Virdi J, Sivakami S, Shahani S, Suthar AC, Banavalikar MM, Biyani MK. Antihyperglycemic effects of three extracts from Momordica charantia. J Ethnopharmacol 2003;88:107-11. |
[Table 1], [Table 2], [Table 3]