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    Vol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 72 International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701 ______________________________________________________________________ Research Paper   In Vitro   Antioxidant and Antidiabetic activity of Asystasia gangetica   (Chinese Violet) Linn. (Acanthaceae) N. V. L. Suvarchala Reddy*, Sneha J. Anarthe and N. M. Raghavendra Gokaraju Rangaraju College of pharmacy, Bachupally, Hyderabad, A. P., India  __________________________________________________________________________________________ ABSTRACT The present investigation evaluates the in vitro  antioxidant and in vitro antidiabetic activity of methanolic extract of leaves of  Asystasia gangetica  in various models. Besides, total phenolic was tested using Folin Ciocalteau reagent. The DPPH radical scavenging of methanolic extract has IC 50  value 179.67 µg/ml  and reducing power of the extract was studied according to the reaction of Fe +3 to Fe +2 . The reducing power of the extract increased with the increasing amount of the concentration. The methanolic extract showed concentration dependant α -glucosidase (IC 50  -   325 µg/ml)   and α -amylase (IC 50 - 3.75 µg/ml) inhibitory activity. Hence α - glucosidase and α -amylase enzyme inhibition may be the possible mechanism for the plant to exert antidiabetic activity and considered as a potential candidate for the management of type-II diabetes mellitus. The in vitro  studies clearly indicate that the methanol extract of leaves of  Asystasia gangetica  has significant in vitro  antioxidant and α -glucosidase and α -amylase enzymes inhibitory activity. Key words:  Asystasia gangetica, in vitro  antidiabetic activity and in vitro  antioxidant activity   INTRODUCTION  Asystasia gangetica  (L).T. (Chinese violet) is a rapidly growing perennial shrubby herb mainly distributed in north India, which grows to 10 m height, at an altitude 300 m 1 neutralized in some waste areas 2 .   Leaves are opposite petioles, flowers are pale purple  blue to violet or lime white in colour, capsules are 2.5-3.5 cm long with wide base and the seeds are 5 mm in diameter. It is mainly used in mild hypoglycaemia, anticancer against epidermoid carcinoma of nasopharynx. The juice of the plant is also used as an anthelmintic 3 . It is used in swelling and rheumatism, as a remedy for gonorrhea and ear disease. It is used as folk remedy for the treatment of diabetes mellitus in parts of south India 4 . It is evaluated for anti-asthmatic activity 5 .  Asystasia gangetica  reported to contain biologically active substances such as   carbohydrates, proteins, alkaloids, tannins, steroidal aglycones, saponins, flavonoids and triterpenoids. Study was undertaken to investigate the in vitro  antioxidant and antidiabetic activity of leaves of     Asystasia gangetica.  EXPERIMENTAL Plant material and preparation of extract Leaves of  Asystasia gangetica  were collected  _  ____________________________ *Address for correspondence: E-mail: sneha.pharma@yahoo.co.in   from Siruvani forest Coimbatore district. The plant was authenticated by Dr. P. Venu joint director,  botanical survey of India, Tamil nadu (voucher specimen no BSI/SC/ 5/23/05-06/Tech-538). The air-dried leaves of  Asystasia gangetica  were pulverized and the powdered material was extracted with methanol (70 %) by cold maceration. The extract was concentrated on a rotary vacuum evaporator, which gave a greenish-brown yield (3.65% w/w). The  proximate phytochemical analysis of methanol extracts shows presence of flavonoids, proteins and carbohydrates, alkaloids, tannins, saponins 6 . Chemicals used Acarbose (Bicon Ltd) , α -glucosidase (SRL), maltose (Loba cheme), Glucose assay reagent (Agappe Diagnostics), α -amylase (SRL), and potato starch (S.D. Fine-Chem). DETERMINATION OF DPPH RADICAL SCAVENGING ACTIVITY 7   1 ml different conc. of extract solution and standard were taken in different vials. To this 5 ml of methanolic solution of DPPH was added, shaken well and mixture was incubated at 37 °C for 20 min. Measure the absorbance against methanol as blank at 517 nm. Take absorbance of the DPPH as control, Percent antiradical activity can be calculated by using following formula Control Abs- sample A % Antiradical activity = × 100 Control Abs.    Vol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 73 International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701 REDUCING POWER ASSAY 8   1ml of different concentrations of extract solution was mixed with 2.5 ml phosphate buffer and 2.5 ml of potassium ferricyanide. The mixture was incubated at 50 ºC for 20 minutes 2.5 ml of TCA was added to the mixture, which was then centrifuged at 3000 rpm for 10 minutes 2.5 ml of upper layer solution was taken and mixed with 2.5 ml distilled water and 0.5 ml of ferric chloride solution and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.  IN VITRO  INHIBITION α  -GLUCOSIDASE 9   The enzyme α - glucosidase inhibitory activity is determined by incubating solution (0.1 ml) of an enzyme preparation with 0.2 M Tris buffer, pH 8.0 (1.0ml) containing various concentrations of extract at 37 ºC for 60 minutes by using glucose as working standard. The reaction mixture is heated for two minutes in boiling water bath to stop the reaction. The amount of liberated glucose is measured by glucose oxidation method. (Prashanth D, 2001) (Assay condition 37ºC±0.1ºC, pH-8.0; O.D at 540 nm). ( Enzyme activity of control – Enzyme activity of extract) % inhibition =------------------------------------------------------ × 100 Enzyme activity of control  IN VITRO  INHIBITION OF α  - AMYLASE 10 A starch solution (0.1% w/v) was obtained by stirring 0.1g of potato starch in 100ml of 16 mM of sodium acetate buffer. The enzyme solution was  prepared by mixing 27.5mg of α -amylase in 100 ml of distilled water. The colorimetric reagent is  prepared by mixing sodium potassium tartarate solution and 3, 5 di nitro salicylic acid solution 96 mM. Both control (Acarbose) and plant extracts were added with starch solution and left to react with α -amylase solution under alkaline conditions at 25 ºC. The reaction was measured over 3 minutes. The generation of maltose was quantified by the reduction of 3, 5 dinitro salicylic acid to 3-amino-5- nitro salicylic acid. This reaction is detectable at 540 nm. (Temperature 25ºC±0.1 ºC, pH 4.8; O.D. at 540 nm). (Maltose) test % Reaction = --------------------------- × 100 (Maltose) control % Inhibition = 100- % reaction ± SD TOTAL PHENOLIC CONTENT 11   Total phenolic content of  Asystasia gangetica  extract was measured by Folin Ciocalteau reagent method. In this method, the blue colour formed due to the polyphenol present in the extract was measured at 760 nm using UV spectrophotometer and results were expressed as g/100g of gallic acid equivalent. RESULTS AND DISCUSSION The methanolic extract demonstrates potent antioxidant activity in different in vitro  models. The DPPH radical scavenging of methanolic extract has IC 50  value 179.6 µg/ml  which is compared with ascorbic acid as a standard (Table 1). In addition to this the methanolic extract also possesses potent reducing power which is compared with butylated hydroxyl anisole (Table 2). The methanolic extract found to contain a noticeable amount of total phenol. The total phenolic content of  Asystasia gangetica  was found to be 0.902 mg/ml, which play major role in controlling antioxidants 12  . The result of this study shows that the methanolic extract can be used as easily accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical industry. The  in vitro   α -glucosidase inhibitory studies demonstrated that methanolic extract of  Asystasia gangetica   had α -glucosidase inhibitory activity. The  percentage inhibition at 0.2, 0.4, 0.6, 0.8 and 1 mg/ml concentration showed a concentration dependant reduction in percentage inhibition (Table 3). Thus the highest concentration of 1 mg/ml tested showed maximum inhibition of nearly 91.9 %. The  percentage inhibition varied from 46-91 %. The 50 % inhibitory concentration of methanolic extract of  Asystasia gangetica  was found to be 325 µg/ml which is compared with the standard acarbose having IC 50  value 0.65 µg/ml.  The in vitro   α -amylase inhibitory studies demonstrated that methanolic extract of  Asystasia gangetica   had α -amylase inhibitory activity. The  percentage inhibition at 1, 2, 3, 4 and 5 mg/ml concentration showed a concentration dependant reduction in percentage inhibition (Table 4). Thus the highest concentration of 5 mg/ml tested showed maximum inhibition of nearly 72.1%. The percentage inhibition varied from 4-72 %. The 50 % inhibitory concentration of methanolic extract was found to be 3.75 µg/ml  which is compared with standard drug acarbose having 50 % inhibitory concentration 92 µg/ml.  Thus, data presented here indicate that methanolic extract of  Asystasia gangetica  possesses significant in vitro  antidiabetic activity. The mechanism by which  Asystasia gangetica  exerted action may be due to its action on carbohydrate  binding regions of α - glucosidase enzyme, α -   amylase, endoglucanases that catalyse hydrolysis of the internal α -1, 4 glucosidic linkages in starch and other related polysaccharides have also been targets for the suppression of postprandial hyperglycemia . This enzyme is responsible in hydrolyzing dietary starch into maltose which then breaks down to glucose prior to absorption. Since α -amylases play an important role in starch break down in human beings and animals, the presence of such inhibitors in food stuffs    Vol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 74 International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701 may be responsible for impaired starch digestion 13, 14 .   α -amylase inhibitor may be of value as novel therapeutic dietetic agents. 15 In conclusion, data  presented here rationalize the methanolic extract have  potential to emerge as new remedy for treatment of type-II diabetes mellitus. Acarbose- like drugs, that inhibit α -glucosidase present in the epithelium of small intestine, have been demonstrated to decrease post- prandial hyperglycemia, 16 and improve impaired glucose metabolism without promoting insulin secretion in NIDDM patients 17 . These medications are most useful for people who have just been diagnosed with type-II diabetes and who have blood glucose levels slightly above the level considered serious for diabetes. They are also useful for people taking sulfonylurea medication, who need an additional medication to keep their blood glucose level within safe range. Therefore, the retardation and delay of carbohydrate absorption with a plant-based α -glucosidase inhibitor offers a prospective therapeutic approach for the management of type-II diabetes mellitus  18 . Table 1 : Anti-radical activity of ascorbic acid and methanolic extract of  Asystasia gangetica leaves. S. no. Control (mcg) Mean   SEM % Antiradical activity ASC 10 0.3411±0.00043 59.98 ASC 20 0.2508±0.000026 70.51 ASC 30 0.2273±0.00011 73.28 ASC 40 0.1954±0.00045 76.99 ASC 60 0.1949±0.04303 74.20 ASC 80 0.1705±0.04194 83.02 AGM 100 0.2471±0.000116 63.94 AGM 250 0.2365±0.000693 69.57 AGM 500 0.3066±0.00066 70.95 ASC-Ascorbic acid, methanolic extract of  Asystasia gangetica, Absorbance of blank = 0.8512 Table 2 : Reducing power assay of BHA and methanolic extract of  Asystasia gangetica  leaves. S. no. Control (mcg) Mean   SEM BHA 10 0.0716  0.0017 BHA 30 0.3476  0.0014 BHA 40 0.5516  0.0014 BHA 50 1.01  0.00118 AGM 25 0.04  0.0011 AGM 50 0.0369  0.00176 AGM 100 0.0386  0.00033 AGM 200 0.0346  0.00033 AGM- methanolic extract of  Asystasia gangetica , BHA- Butylated hydroxyl anisole as standard. Table 3 : Inhibitory activity of methanolic extract of  Asystasia gangetica  and standard drug acarbose against α -glucosidase Sample Concentration (µg/ml)  % inhibition (±SD )  IC 50   Acarbose 0.2 35.0±1.087 0.65µg/ml  0.4 42.47±1.12 0.6 47.9±0.73 0.8 58.43±1.23 1.0 62.2±0.43  Asystasia gangetica  200 46.81±0.381 325 µg/ml  400 60.13±1.011 600 76.77±2.057 800 89.58±0.233 1000 91.19±0.806 Data expressed as mean ± SD, n=6. Table 4 : Inhibitory activity of methanolic extract of  Asystasia gangetica  and standard drug acarbose against α -amylase. Sample Concentration (µg/m l) % inhibition (±SD )  IC 50   Acarbose 50 40.3±6.929 92 µg/ml  100 52.0±3.323 150 64.38±1.202 200 76.95±0.565 250 89.265±1.279    Vol. 1 (2) Oct – Dec 2010 www.ijrpbsonline.com 75 International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701  Asystasia gangetica  100 4.05±1.131 3.75 µg/ml 200 14.4±0.353 300 25.95±0.636 400 63.3±0.1414 500 72.1±2.545 Data expressed as mean ± SD, n=6. REFERENCES: 1.   Smith, Clifford W. Impact of alien plants on Hawaii’s native biota in Hawaii is terrestrial ecostims: preservation and management, co-operative national park resources studies unit, university of Hawaii, Manoa, 1985; 180. 2.   Sykes WR. Contributions of the flora of nine New Zealand Department of scientific and industrial. Res Bull 1970;37:200. 3.   Kirtikar KR, Basu BD. Medicinal plants in India, vol.-I, Pullaiah Regency publication, New Delhi, 1998; 1892. 4.   Guha Bakshi DN, Sensarma P, Pal DC. A lexion of medicinal plants in India, vol.-I, line drawings, (Vol. I). Naya Praheash publisher, Calcutta, India, 1999; 552. 5.   Akah PA, Ezike AC, Nwafor SV, Okoli CO, Enwerem NM. Evaluation of the anti-asthmatic  property of  Asystasia gangetica  leaf extracts . J Ethnopharmacol. 2003;89:25-36. 6.   Kokate CK. Handbook of Practical Pharmacognosy, Vallabh Prakashan, 4 th  edition, 1994;58-60. 7.   Tepe B, Sokmen M, Akpulat HA, Sokmen A. Screening of the antioxidant potentials of six salvia species from turkey. Food Chem 2006;95:200-4. 8.   Kim HY, Yokozawa T, Cho EJ, Cheigh HS, Chung HY.  In vitro  and in vivo  antioxidant effects of mustard leaf (Brassica juncea). Phytother Res 2003;17:465-71. 9.   Prashanth D, Amit A, Samiulla DS, Asha MK, Padmaja R. α -glucosidase Inhibitory activity of Mangifera indica bark. Fitoter 2001;72:686-8. 10.   Conforti F, Scatti G, Loizzo MR, Sacchetti GA, Poli F, Menichini F.  In vitro  antioxidant effect and inhibition of α -amylases of two varieties of  Amaranthus caudatus  seeds. Bio Pharm Bull 2005;28(6):1098-02. 11.   Ilhani G, Muniro I, Asian A. Determination of antioxidant activity of lichen cetraria islandica (L) Ach. J Ethnopharmacol 1997;57:21-7. 12.   Yerra R, Senthil K, Gupta M, Muzumdar UK. Studies on invitro antioxidant activities of methanolic extract of Mucuna Pruriens seeds (Fabaceae). European Bulletin of Drug Research 2005;13,31-39. 13.   Marshall JJ. Hypothesized that negatively charged residues of pilaic acid from membrane. Am Chem Soc Symposium Series 1975;15:244-66. 14.   Jaffe WG, Lette CLV. Heat labile growth inhibiting factors in bens (Phaseolus vulgaris). J  Nutr 1968;94,203-10. 15.   Plus W, Keup U. Influence of an alpha-amylase inhibitor (Bay d 7791). Diabetolgia 1973;9:97-101. 16.   Sima AAF, Chakrabarti S. Long term suppression of post prandial hyperglycemia with acarbose retards the development of neuropathies in the BB/W-rat. Diabetologia 2004;35:325-330. 17.   Carrascosa JM, Molero JC, Fermin Y, Martinez C, Andres A, Satrustegui J. Effect of chronic treatment with acarbose on glucose and lipid metabolism in obese diabetic wistar rats . Diabetes Obes Metab 2001;3:240-248. 18.   McCue P, Kwon YII, Shetty K. Anti diabetic and anti-hypertensive potential of sprouted and solid-state bioprocessed soyabean. Asia Pac J Clin Nutr    2005;14:145-152.
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