Journal of Applied Pharmacy

INTRODUCTION

Diabetes mellitus (DM) may be a syndrome defined by chronic hyperglycemia and disturbances of saccharide, fat and macromolecule metabolism relating to absolute or relative deficiencies in endocrine secretion and/or endocrine action. There are a unit Associate in Nursing calculable 143 million folks within the world with diabetes and this variety will most ulikely double by the year 2030 [1]. In developing countries 80% of population are using traditional medicine in primary medical problems. By screening of anti-diabetic drugs, a large number of plant materials including phytoconstituents were found to possess potent anti-diabetic activity [2, 3]. Hemalatha et al. reported the pharmacologically tested anti-diabetic plant materials in steptozotocin induced diabetic animal model [4].

Andrographis echioides is a species of Andrographis commonly known as kalmegh in Hindi, belonging to the family Acanthaceae, is a dense herb found in plains and waste lands which fruits throughout the year. Andrographis echioides has been reported anti-Inflammatory, antimicrobial, anthelmintic, antioxidant and larvicidal activities [5-8]. Andrographis panniculata has been reported to possess anti-diabetic activity [9]. Pharmacological study on anti diabetic activity of Andrographis echioides has not been reported till now. Therefore the present study was aimed to investigate the antidiabetic effects of methanol extract of Andrographis echioides (MEAE) against streptozotocin (STZ)-induced diabetes in Wistar rats. The plant selected for this present work is locally available in the Erode district and has been used for long a time in local folklore medicine for the treatment of diabetes.

MATERIALS AND METHODS

Drugs and chemicals

STZ was from Sigma Chemical Co., USA. Glibenclamide was from Hoechst, India. All other reagents used were of analytical grade obtained commercially.

Collection and extraction

The fresh aerial parts of Andrographis echioides were collected from SKM Siddha and Ayurvedic medicines India Pvt. Ltd., Erode Dist., Tamilnadu, India,in the month of August 2013 and identified by GVS Murthy, Botanical Survey of India, Coimbatore, India. A voucher specimen has been deposited in the laboratory for future reference (BSI/SC/5/23/13-14/TECH.835). The aerial parts of the plant were shade dried and pulverized. The powder was defatted with petroleum ether. Later, it had been subjected to continuous hot extraction with 95% aqueous methanol in a Soxhlet apparatus. The extract (MEAE) was concentrated under vacuum and dried in desiccators (yield 71gm, 7.1%w/w). The dry extract was kept in vacuum desiccators until use. Preliminary phytochemical analysis [10] revealed the presence of flavonoids, alkaloids, and steroids in MEAE Plant material.

Animals

Adult male Wistar albino rats weighing 150–200 g were procured from Venkateshwara Enterprises, Bangalore, Karnataka, India and used throughout the study. All the animals were under the age of 8–12 weeks. They were housed in a very clean polypropylenecage and maintained under standard laboratory conditions (temperature 25 ± 2°C with dark/light cycle 12/12 h). They were fed with standard pellet diet and water ad libitum. The animals were acclimatized to laboratory conditions for one week before experiment. Experiments were performed complied with the rulings of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) New Delhi, India. (Registration No: 1135/a/07/CPCSEA) and the study was permitted by the Institutional Animal Ethics Committee (IAEC).

Acute toxicity

Acute toxicity studies were performed as per OECD-423 guidelines [11]. Male Wistar albino rats selected by random sampling technique were utilized during this study. The animals were fasted for 4h with free access to water only. The plant extract was administered orally at a dose of 5mg/kg initially and mortality if any was observed for 3 days. If mortality was ascertained in two out of three animals, then the dose administered was considered as toxic dose. However, if the mortality was ascertained in only one animal out of three animals then the identical dose was repeated again to confirm the toxic effect. If no mortality was ascertained, then higher (50,300 and 2000mg/kg) doses of extract were utilized for further toxicity studies.

 Oral glucose tolerance test (OGTT)

III, and IV received MEAE at the doses of 200, 500, and 800 mg/kg b.w., p.o., respectively. Group V received glibenclamide 1 mg/kg b.w. p.o. 30 min after these treatments, all groups received glucose (4 g/ kgb.w.) orally. Blood was withdrawn from the tail vein justprior to and 30, 60, and 120 min after the oral glucose administration [12, 13]. Blood glucose levels were measured using glucoseoxidase-peroxidase reactive strips and a portable glucometer (Accu Sure blood glucose monitoring system).

Induction of experimental diabetes mellitus

The rats were rendered diabetic by a single intraperitoneal dose of 55 mg/kg b.w. STZ freshly dissolved in ice cold 0.1 M citrate buffer (pH 4.5). After 72 h, fasting blood glucose (FBG) levels were measured and only those animals showing blood glucose level = 225 mg/dl were used for the subsequent investigation. The day on which hyperglycemia had been confirmed was designated as day 0[14, 15].

Treatment schedule and estimation of FBG level

Normal and hyperglycemic rats were divided into seven groups (n = 6) receiving the following treatment [16]:

Group I: Non-diabetic or normal control, received the vehicle (distilled water) 5 ml/kg b.w., p.o. (NC).

Group II: Non-diabetic control, received MEAE 500 mg/ kgb.w., p.o.

Group III: Diabetic control, received the vehicle (distilled water) 5 ml/kg b.w., p.o. (DC).

Group IV: Diabetic treatment, received MEAE 200 mg/ kg b.w., p.o.

Group V: Diabetic treatment, received MEAE 500 mg/kg b.w., p.o.

Group VI: Diabetic treatment, received MEAE 800 mg/ kg b.w., p.o.

Group VII: Diabetic treatment, received glibenclamide 1 mg/kg b.w., p.o.

The above treatment was continued daily for 21 days. Fasting blood glucose concentrations were measured with a portable glucometer (Accu Sure blood glucose monitoring system) at days 0, 7, 14, and 21.

Body weight

The body weights of rats of each group were recorded on 1st, 7th, and 15th day of MEAE treatment.

Estimation of serum biochemical parameters

After twenty one days treatment, blood samples were drawn from overnight fasted rats by retroorbital veinpuncture technique from light-ether anesthetized animals. The nonheparinized blood was allowed to coagulate before being centrifuged (4000 rpm for 20 min) and the blood serum separated. Blood serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), glycosylated hemoglobin (HbA1C), aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) were estimated enzymatically using commercially available reagent kits (Erba Diagnostics and Span diagnostics Ltd.).

Statistical analysis

The data were expressed as mean ±standard error of mean (SEM). Statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s posthoc test of significance using Graph Pad (Instat) software version 4.0. P values of < 0.05 were considered as statistically significant.

RESULTS AND DISCUSSION

Pharmacological treatment with of diabetes is based on oral hypoglycaemic agents and insulin. However, long term treatment with these drugs is expensive and poses life threatening adverse effects. Management of diabetes without any side effects continues to be a challenge to the medical community [17]. Despite considerable progress made in the conventional anti-diabetic management strategies, the search continues for plant-based products for the managemment of diabetes, which are deemed safe [18]. Phytotherapy has been extremely accepted worldwide in the health care system for DM.  In this study, the hypoglycemic activity of MEAE was evaluated in STZ-induced diabetic rats. Here, STZ at the dose of 55 mg/kg, i.p., was used to induce hyperglycemia after performing a pilot study for optimized dose to elevate blood glucose >250 mg/dl. The use of lower dose of STZ (55 mg/kg) produced an incomplete destruction of pancreatic ß cells although the rats become permanently diabetic [19].

Acute toxicity

The MEAE did not show any toxic effect or death up to the dose of 2000 mg/kg, b.w., p.o. in mice.

 Oral glucose tolerance (OGTT)

MEAE exhibited significant reduction in blood sugar in diabetic rats at the doses of 200, 500, and 800 mg/ kg. Though MEAE at 200 and 500 mg/kg reduced the hyperglycemia significantly as compared to the DC group, it did not restore the FBG level to that of the NC group, while with MEAE at 800 mg/kg the blood sugar levels of diabetic rats were reduced to NC group level. Blood glucose levels in normal rats treated with MEAE 500 at mg/kg were insignificant from that of the NC group, indicating that MEAE maintained glucose homeostasis. The hypoglycaemic action of MEAE could also be because of promotion of insulin release from existing ß cells of the islets of Langerhans. The plasma glucose lowering activity was compared with that of glibenclamide, the reference oral hypoglycemic that has been used for several years to treat diabetes, to stimulate pancreatic ß cells [20].

Values are mean ± SEM (n = 6).^aP < 0.05 and ^bP < 0.01 when compared with STZ control group; ^cP < 0.05 and ^dP < 0.01 when compared with normal control group.

In insulin-deficient diabetes, the concentration of serum fatty acids is elevated as a result of free fatty acid out flow from fat depots, wherever the balance of the free fatty acid esterification-TG lipolysis cycle is displaced in favour of lipolysis.  High-density lipoprotein (HDL) is an antiatherogenic lipoprotein. It transports cholesterol from peripheral tissues into the liver and thereby acts as a protective factor against coronary cardiovascular disease. The amount of HDLC, which increased after MEAE administration, may be due to the increase in the activity of lecithin cholesterol acyl transferase (LCAT), which can contribute to the regulation of blood lipids [25]. Oral administration of MEAE reduced the elevated serum lipids such as TC, LDLC, and TGs toward normal in diabetic rats. Elevation of serum biomarker enzymes such as SGOT, SGPT, and SALP was observed in diabetic rats indicating impaired liver function, which was obviously due to hepatocellular necrosis. It has been reported that liver necrosis occurred in STZ-induced diabetic rats [26]. Therefore, increase in the activities of AST, ALT, and ALP gives an indication on the hepatotoxic effect of STZ. Twenty-one days of treatment with MEAE restored all the above-mentioned serum hepatic biochemical parameters toward the normal values in a dose-dependent manner, thereby alleviating liver injury caused by STZ-induced diabetes.

CONCLUSION

In this study, administration of MEAE to STZ-induced hyperglycaemic rats incontestable distinguished reduction in blood glucose level, standardization of serum biochemical profiles including lipid contents, comparing to STZ control rats. Therefore, it can be concluded that the MEAE is remarkably effective against STZ-induced diabetes in Wistar rats thereby validating its ethnomedicinal usage. From the ascertained oral hypoglycemic activity of MEAE in STZ-induced diabetic rats, it can be further inferred that Andrographis echioideswill function a motivating candidate in complementary and alternative medicine for the effective management of diabetes.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

REFERENCES

1.       Jatin, J., Hemant, B., Shruti, S., Hemendra, G., Surabhi, S., Ch V Rao. (2014). Anti-diabetic activity of methanolic extract of calotropis gigantea seeds on STZ induced diabetic rats. Int J Pharm Pharm Sci 6: 254-257.

2.       Patel, DK., Kumar, R., Laloo, D., Hemalatha, S. (2012). Diabetes mellitus: An overview on its pharmacological aspects and reported medicinal plants having anti-diabetic activity. Asian Pac J Trop Biomed 2: 411-420

3.       Patel, DK., Laloo, D., Kumar, R., Hemalatha, S. (2011). Pedalium murex Linn-An overview of its phyto Pharmacological aspects. Asian Pac J Trop Biomed 4: 748-755.

4.       Patel, DK., Kumar, R., Laloo, D., Hemalatha, S. (2012). Natural medicines from plant source used for therapy of diabetes mellitus: An overview of its pharmacological aspects. Asian Pac J Trop Dis 2: 239-250.

5.       De-Yang, S., Shin-Hun, J., Ping-Chung, K., Guan-Jhong, H., Yu-Yi, C., Amooru, GD. (2013). Chemical Constituents from Andrographis echioides and their anti-inflammatory activity. Int J Mol Sci 14: 496-514.

6.       Padma., Sarojini Devi., Manjunatha, B., Philip, G., Venkata Raju, R. (2012). Preliminary phytochemical screening and anthelmintic activity of Andrographis echioides Nees. J Pharm Res 5: 4801-4803.

7.       Premkumar, P., Priya, J., Suriyavathana, M. (2010). Evaluation of antioxidant potential of Andrographis echioides and Boerhavia diffusa. Int J Curr Res 3: 59-62.

8.       Sankaran, R., Arulsamy, J., Rajarathinavelu, N. (2012). Synergistic effect of Andrographis echioides and Cadaba trifoliata leaf extracts against larvae of dengue mosquito Aedes aegypti L. Asian Pac J Trop Biomed S1588-S1591.

9.       Yaman, N., Hanani, E., Ruray, D., Santi Purna, S. (2013). The effect of Andrographis paniculata extract capsules on blood glucose level in patients with type 2 diabetes mellitus. Int J Curr Res 5: 578-581.

10.    Harborne, JB. (1998). Phytochemical Methods, A Guide to Modern Techniques of Plant Analysis. New Delhi: Springer (India) Pvt. Ltd.

11.    Ecobichon, DJ. (1997). The basis of Toxicology testing. 1^st Edn., CRC Press, New York, ISBN:10: 0849385547.

12.    Schoenfelder, T., Cirimbelli, TM., Citadini-Zanette, V. (2006). Acute effect of Trema micrantha (Ulmaceae) on serum glucose levels in normal and diabetic rats. J Ethnopharmcol 107: 456-459.

13.    Barik, R., Jain, S., Qwatra, D., Joshi, A., Tripathi, GS., Goyal, R. (2008). Antidiabetic activity of aqueous extract of Ichnocarpus frutescens in streptozotocin-nicotinamide induced type-II diabetes in rats. Ind J Pharmacol 40: 19-22.

14.    Li, T., Liu, J., Zhang, X., Ji, G. (2007). Antidiabetic activity of lipophilic (-) epigallocatechin-3-gallate derivative under its role of a-glucosidase inhibition. Biomed Pharmacother 20: 1-6.

15.    Schmatz, R., Schetinger, MR., Spanevello, RM., Mazzanti, CM., Stefanello, N., Maldonado, PA. (2009). Effects of resveratrol on nucleotide degrading enzymes in streptozotocin-induced diabetic rats. Life Sci 84: 345-350.

16.    Mohammadi., Naik, PR. (2008). Evaluation of hypoglycaemic effect of Morus alba in an animal model. Ind J Pharmacol 40: 15-18.

17.    Titas, D., Rajesh, R., Murugananthan, G., Sahil, T., Nandakumar, K. (2013). Hypoglycaemic effects of alcoholic root extract of Borassus flabellifer (Linn.) in normal and diabetic rats. Pak J Pharm Sci 26: 673-679.

18.    Farah, G., Zafar, HI., Badiaâ, L. (2010). Hypoglycemic and hypolipidemic effects of an aqueous extract of chamaerops humilis leaves in obese, hyperglycaemic and hyperlipidemic meriones shawi rats. Pak J Pharm Sci 23: 212-219.

19.    Aybar, MJ., Sánchez Riera, AN., Grau, A., Sánchez, SS. (2001). Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats. J Ethnopharmacol 74: 125-132.

20.    Stephen, ND. (2001). Insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas. In: Laurence, LB., John, SL., Keith, LP., eds. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. New York: McGraw-Hill, pp1613–1647.

21.    Swanston-Flatt, SK., Day, C., Bailey, CJ., Flatt, PR. (1990). Traditional plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetologia 33: 462-464.

22.    Shirwaikar, A., Rajendran, K., Dinesh Kumar, C., Bodla, R. (2004). Antidiabetic activity of aqueous leaf extract of Annona squamosa in streptozotocin-nicotinamide type 2 diabetic rats. J Ethnopharmacol 91: 171-175.

23.    Arvind, K., Pradeep, R., Deepa, R., Mohan, V. (2002). Diabetes and coronary artery diseases. Ind J Med Res 116: 163-176.

24.    Murali, B., Goyal, RK. (2002). Effect of chronic treatment with losartan on streptozotocin induced diabetic rats. Ind J Exp Biol 40: 31-34.

25.    Patil, UK., Saraf, S., Dixit, VK. (2004). Hypolipidemic activity of seeds of Cassia tora Linn. J Ethnopharmacol 90: 249-252.