Antidiabetic Screening and Phytochemical Investigation of Selected Medicinal Plants

Drug discovery is the identification of novel active chemical compounds. The drug discovery is made through the observations of biological effects of new or existing natural products from micro organisms, plants etc. The drug discovery is also bound to therapeutic targets such as enzymes, receptors etc. Pharmacophore approaches have become one of the major tools in drug discovery. The ligand based and structure based methods have been developed for improved pharmacophore modeling. The drug target is the naturally existing cellular or molecular structure involved in the pathology of interest that the drug in development is meant to act on. The drug target may be a established target or new target. The process of finding a new drug against a chosen target for a particular disease usually involves high-throughput screening. Two major approaches exist for the finding of new bioactive chemical compound from natural sources. Screening the chemical compounds for biological activity and structure elucidation of chemical compounds by NMR, Mass spectroscopy The traditional herbal medications are become mainstream throughout the world. Since ancient times, plants have been source of medicines. Ayurveda and other Indian literature mention the use of plants in treatment of various human diseases. India has about 45000 plant species and among them several thousands have medicinal properties. Plants are major source of drugs and many of the currently available drugs have been derived directly or indirectly from them. OBJECTIVES: 1. To select the plants based on their ethnomedical uses and preparation of their extracts. 2. To screen the extracts for in vitro antioxidant activity. 3. To screen the extracts for in vitro antidiabetic activity. 4. To screen the plant extract for in vivo antidiabetic activity. 5. To isolate the chemical constituents from the plant extract and structure elucidation. MATERIALS AND METHODS: Plant Material: The whole plant of Actiniopteris radiata was collected from Nilgiri district, Tamil Nadu, India, in November 2007. The plant was identified by Dr. S. Rajan, Field Botanist, Survey of Medicinal Plants and Collection Unit, Emerald, Nilgiri (Voucher No: 135). A voucher specimen was deposited at Survey of Medicinal Plants and Collection Unit, Emerald, Nilgiri. Materials: Instruments: Melting points were determined using a Lab India melting point apparatus. UV-Visible spectrums were recorded using a Shimadzu UV-1700. IR spectrums were recorded on a Shimadzu FTIR-8400s. 1H (500 MHz) and 13C (100 MHz) spectrums were recorded on a BRUKER AV-400. EIMS was recorded by GC-MS on a P-POS/TOP MICRO, HITACHI. ESIMS spectrums were recorded on a HCT-Ultra PTM discovery, BRUKER. ELISA reader data recorded on a BIO - RAD 550. Chemicals: 2, 2 –diphenyl -1- picryl hydrazyl (DPPH) and 2, 21- azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt (ABTS) were procured from Sigma-Aldrich, California, USA. Rutin and p-nitroso dimethyl aniline (p-NDA) were procured from Acros Organics, New Jersy, USA. Naphthyl ethylene diamine dihydrochloride (NEDD) was procured from Roch – Light Ltd, Suffolk, UK. Nitro blue tetrazolium (NBT) was procured from S.D Fine Chem Ltd, Biosar, India. Glibenclamide was procured from Inga labs Ltd, Mumbai, India. Streptozotocin was procured from Hi media, Mumbai, India. All the other chemicals used were of analytical grade. Preparation of the plant extract: The plant was dried under shade for 7 days. The coarsely powdered plant material (500g) was packed in soxhlet apparatus. The packed plant material was extracted successively with petroleum ether, chloroform, ethyl acetate and ethanol for 18-20 hrs. These extracts were filtered and dried under vacuum. SUMMARY AND CONCLUSION: 1. The traditional herbal medications are become mainstream throughout the world. Since ancient times, plants have been source of medicines. Ayurveda and other Indian literature mention the use of plants in treatment of various human diseases. India has about 45000 plant species and among them several thousands have medicinal properties. Plants are major source of drugs and many of the currently available drugs have been derived directly or indirectly from them. 2. Since ancient times, plants have been used in the treatment of diabetes mellitus. There are many hypoglycemic plants and their active principles varies. Therefore considerable diversity in the mechanism of action. Some act by increasing the release of insulin and require a minimum of β-cells to exert their action. Other plant extracts or constituents act by modifying glucose metabolism. All are important since they are used to treat the different aspects of diabetes mellitus. 3. The selected plant Actiniopteris radiata was collected from Nilgiri district, Tamilnadu and authenticated. The dried plant material was subjected to successive extraction with petroleum ether, chloroform, ethyl acetate and ethanol by soxhlet method. The extracts were concentrated under reduced pressure and controlled temperature. 4. The phytochemical studies of the extracts gave positive test for the flavanoids, carbohydrates, hydrocarbons, fatty acids, sterols, steroids, steroidal glycoside and unknown glycoside. Determination of water soluble extract, alcohol soluble extract, total ash, acid insoluble ash and water soluble ash were carried out. The quantitative phytochemical estimations total phenol content and total flavonoid content of the extracts were estimated. 5. Qualitative and quantitative determinations of ethyl acetate extract and fractions were done by HPTLC method. 6. In vitro antioxidant studies DPPH, ABTS, Nitric oxide, Super oxide and Deoxy ribose were carried out. The ethyl acetate extract shown potent in vitro antioxidant activity. The ethyl acetate extract of Actiniopteris radiata was selected for in vivo anti-diabetic activity based on the in vitro antioxidant activity. 7. In vitro antidiabetic activity was performed by alpha glucosidase inhibition activity. The successive extracts of Actiniopteris radiata and fractions of ethyl acetate extract were screened for alpha glucosidase inhibition activity. The results was compared with the values of standard (acarbose). The ethyl acetate extract has shown significant antidiabetic activity and fraction 5, 6, 7 shown moderate antidiabetic activity. The ethyl acetate extract was selected for in vivo antidiabetic activity based on the results of in vitro antidiabetic activity. 8. Wistar rats of either sex weighing 180-220 g (6 to 8 weeks) with no prior drug treatment were used for in vivo anti-diabetic activity. The acute toxicity study of the ethyl acetate extract was determined according to the O E C D guidelines No.425. Female Wistar rats weighing 180-220 g (6 to 8 weeks) were used for this study. The test samples in a single dose of 400 mg/kg b.w, 800 mg/kg b.w and 2 g/kg b.w were given orally. The animals were observed for 24 hours and monitored for 14 days to record general behaviour and mortality. No mortality was observed till the end of the study. 9. Streptozotocin was used to induce diabetes. Streptozotocin 55 mg/kg b.w was administered intraperitoneally. The rats with blood glucose level above 200 mg/dl were considered diabetic and used in the experiment. 10. The oral glucose tolerance test was performed in overnight fasted normal rats. Zero hour blood sugar was determined in overnight fasted rats. After 30 min of drug treatment, the rats were fed with 2 g/kg b.w glucose and blood glucose was determined after 30, 60, 120 and 180 min of the glucose load. Blood glucose concentration was estimated by GODPOD method. The ethanol and ethyl acetate extract have shown significant antidiabetic activity. 11. Blood was withdrawn from the retroorbital sinus under ether anaesthesia. The serum was separated immediately by centrifugation and analysed for glucose, cholesterol, triglyceride, HDL cholesterol and LDL cholesterol. 12. The ethyl acetate extract was selected for in vivo anti-diabetic activity. The ethyl acetate extract was administered at a dose of 100, 200 and 400 mg/kg b.w for 7 days. The ethyl acetate extract treatment reduces the glucose, triglycerides, cholesterol, LDL cholesterol and increases the HDL cholesterol. The ethyl acetate extract at 400 mg/kg b.w shown significant antidiabetic activity. The results are compared with the standard. 13. The ethyl acetate extract was subjected to column chromatography and isolated the active constituents. Two new compounds were isolated and characterised by TLC, IR, UV spectral analysis, NMR and Mass spectra. Compound 1 is 2-(3, 4-O – Diglucos cinnamoyl) – 4 – hydroxyl furan and compound 2 is 1-Heptaloyl, 8-hexyl, 3-(O – diglucos), 10 – methyl, 9. 10 – dihydro naphthalene