Anti-diabetic activity of Ocimum sanctum L . roots and isolation of new phytoconstituents using two-dimensional nuclear magnetic resonance spectroscopy

In view of alleged antidiabetic potential, effect of methanolic extract of Ocimum sanctum L. (Lamiaceae) roots on fasting blood sugar levels and serum biochemical analysis in streptozotocin-induced diabetic rats were investigated. The resulted extract had shown significant protection and lowered the blood glucose levels to normal in glucose tolerance test. In long term treatment of streptozotocin-induced diabetic rats, the degree of protection was determined by measuring blood glucose, triglycerides, total cholesterol and serum insulin levels. Phytochemical investigation of the roots resulted in the isolation of three new active constituents characterized as urs-12-en-3β,6β,20β-triol-28-oic acid (2), 1 ́ ́-menthyl-2glucopyranosyloxybenzoate (4) and n-decanoyl-β-D-glucopyranosyl-(2a→1b)-β-D-glucopyranosyl(2b→1c)-β-D-glucopyranosyl-(2c→1d)-β-D-glucopyranosyl-2d-2 ́-hydroxybenzoate (5), along with known compounds of ursolic acid (1) and palmityl glucoside (3). The structures of the isolated compounds were established by proton nuclear magnetic resonance (1H-NMR), carbon-13 NMR ( 13 C NMR), fast atom bombardment (FAB) mass, 1 H1 H correlation spectroscopy (COSY) and heteronuclear multiple-bond correlation (HMBC) spectral techniques.

Though there are few reports on anti-diabetic activity of O. sanctum leaf and fruit extracts (Grover et al., 2002;Hannan et al., 2006;Chattopadhyay, 1993), till now no reports have been published to authenticate the activity of roots of this plant in the management of diabetes mellitus in streptozotocin-induced conditions on Male Wister Rat.The stem and leaves of holy basil contain a volatile oil composed of limonene, borneol, α-copaene, βcaryophyllene and elemol; phenolic compounds (rosmarinic acid, apigenin, cirsimaritin, isothymusin), flavonoids (orientin, vicenin) and aromatic compounds (methyl chavicol, methyl eugenol) (Vani et al., 2009).The roots of O. sanctum possesses ocimol, galactose, arabinose, β-sitosterol and ocimic acid (List and Horhammer, 1977).This study was aimed at describing the anti-diabetic activity of O. sanctum roots and identifying new chemical constituents isolated from the roots by spectral data analyses.

MATERIALS AND METHODS
Streptozotocin was purchased from Sigma-Aldrich Co., USA.Glucose, total cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides were assayed using kits from Ranbaxy Diagnostics, New Delhi, India and the One Touch glucometer (Accu-chek sensor) of Roche Diagnostics, Germany.Blood glucose level was estimated by glucose oxidase method, using a commercial diagnostic kit from Span diagnostic Ltd. (Surat, India).Insulin was estimated quantitatively by Enzyme-linked immunosorbent assay (ELISA) kit from Mercodia (Uppsala, Sweden).Other chemicals used were of analytical grade.Double distilled water was used in all assay procedures.Melting points were determined on a Perfit melting point apparatus (Ambala, India) and were uncorrected.Infrared (IR) spectra were recorded using KBr pellets, with a Jasco FT/IR-5000 Spectrometer (FTS 135,Hongkong).Ultraviolet (UV) spectra were measured with a Lambda Bio 20 spectrophotometer (Perkin Elmer, Schwerzenbach, Switzerland) in methanol.Proton ( 1 H) and carbon-13 ( 13 C) nuclear magnetic resonance (NMR) spectra were recorded using Bruker ARX-400 NMR Spectrometer (Rheinstetten, Germany), with tetramethylsilane (TMS) as internal standard.The chemical shifts were measured in δ values (ppm).Fast atom bombardment (FAB) mass, 1 H-1 H correlation spectroscopy (COSY) and heteronuclear multiple-bond correlation (HMBC) spectra were obtained using a JEOL-JMS-DX 303 Spectrometer (Peabody, MA, USA).Column chromatography was performed on silica gel (Qualigens, Mumbai, India), 60-120 mesh.Thin layer chromatography (TLC) was run on silica gel G (Qualigens) and spots were visualized by exposure to iodine vapors, UV radiation and by spraying with ceric sulphate solution.

Plant material
O. sanctum roots were procured from Coimbatore, Tamilnadu (India) and authenticated by Dr. H.B Singh, Scientist F and Head, RHMD, National Institute of Science Communication and Information Resources, New Delhi, India.A voucher specimen (no.RHMD/08-09/990/21) was deposited in the Raw Materials Herbarium and Museum, NISCAIR, New Delhi, India.

Experimental animals
Wister albino rats (150 -250 g) were procured from the Central Animal Facility, Jamia Hamdard and maintained under controlled condition of illumination (12 h light / 12 h darkness) and temperature 20-25°C.They were housed under ideal laboratory conditions, maintained on standard pellet diet (Lipton rat feed, Ltd; Pune) and water ad libitum throughout the experimental period.Animals were acclimatized to the conditions before the start of the experiments.The experimental study was approved by the Institutional Animal Ethics Committee (IAEC) of Jamia Hamdard, New Delhi, India.All the extracts and the standard drugs were administered orally.

Experimental design
Initial screening of the extract for the hypoglycemic activity was performed in normal healthy rats.The antidiabetic affect was studied in diabetic animals by two methods: (i) By studying the effect of different doses of the aqueous extract on fasting blood glucose (FBG) levels of sub and mild diabetic rats during glucose tolerance test.(ii) By giving the most effective dose of extract (500 mg/kg) daily once for 21 days to streptozotocin (STZ)-induced severely diabetic rats and observing the changes in fasting blood glucose (FBG) and lipid profile.

Biochemical estimations
Blood glucose was estimated using one touch glucometer for regular checkup and kit was used for weekly estimations.Blood glucose, total cholesterol (TC), high density lipoprotein cholesterol (HDLc) and triglyceride (TG) levels in serum were measured spectrophotometrically by prescribed methods (Buccolo and David, 1973).Low density lipoprotein cholesterol (LDLc) was calculated from the above measurement using Friedewald formula.Serum insulin level, thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) were evaluated using the commercially available kits.

Extraction and isolation
O. sanctum roots (2.5 kg) was dried at 45°C, coarsely powdered and extracted exhaustively with methanol in a soxhlet apparatus.The methanolic extract was dried on a steam bath under reduced pressure to get dark brown viscous mass (230 g, 9.2% yield).The viscous mass was dissolved in small quantity of methanol and adsorbed on silica gel (60 -120 mesh) for preparation of slurry.Afterwards, it was dried, packed and chromatographed over silica gel column packed in petroleum ether.The column was eluted successively with various combinations of petroleum ether, chloroform and methanol in increasing polarity (e.g., petroleum ether, petroleum ether-chloroform in the ratio of 9:1, 3:1, 1:1 and 1:3 v/v, chloroform, chloroform-methanol in the ratio of 99:1, 98:2, 95:5, 9:1, 3:1, 1:1, 1:3 v/v, and methanol).

Fasting blood glucose level
There was a significant elevation in fasting blood glucose level after a single dose of streptozotocin compared to control.However, this parameter approached the control level after supplementation of methanolic extract of O. sanctum roots (Tables 1 and 2).

Serum lipid profile
Treatment with extracts of O. sanctum roots in higher doses of (500 mg/kg) lowered the elevated levels of these cholesterols (TCh, LDL-C and VLDL-C) and triglycerides significantly (P<0.01).However, the HDLcholesterol levels were significantly (P<0.01)increased The data are expressed in mean ± SEM; n = 6 in each group.## (P <0.01) compared with the corresponding value for normal control animals (group I).*(P <0.05), **(P <0.01) compared with the corresponding value for diabetic control animals (group II).ns: Not significant.when compared with diabetic control.On the other hand, glimepiride treatment also significantly reduced elevated levels of all these cholesterols (TCh, LDL-C and VLDL-C) and triglycerides.Atherogenic index was calculated for diabetic control group, which was found to be significantly higher than normal control rats.The extract treatment resulted in a significant decrease in the atherogenic index found to be significantly increased by the STZ treatment (Table 3).

Serum insulin, TBARS and GSH levels
The experimental animals showed a marked reduction in serum insulin levels and tissue glutathione, with increase in the level of TBARS.When compared with diabetic control rats, administration of the methanolic extract of O. sanctum roots (500 mg/kg) elevated serum insulin levels, but the level of significance was found to be P < 0.05 (Table 4).

DISCUSSION
The present study dealt with two dimensions for antidiabetogenic effects of the methanolic extract of O. sanctum roots in separate manner.In one dimension, the fasting blood glucose levels at different doses were measured.In the other dimension, antihyperlipidemic potency was studied as there was a close correlation between hyperglycemia and hyperlipidemia (Cho et al., 2002).Here, we selected the STZ-induced hyperglycemia as an experimental model because it was one of the best models to study the effect of the antidiabetogenic agent (Carter et al., 1971).The specific doses of the extract used and duration of the treatment adopted here were selected by trial and error where good promising results were noted without any metabolic toxicity induction.Supplementation of the separate extract of the plants parts resulted a significant correction of fasting blood glucose (FBG) level with respect to STZ-induced diabetic group and this recovery was more effective when treatment of extracts of O. sanctum roots in higher doses of 500 mg/kg was used, which primarily focused on the antidiabetic activity of the plant products.Meanwhile, the actual mechanism of such antidiabetogenic activity is not clear from this study but following possible dimensions may be enlighten.The aforementioned parameters recovered showed a more potent correction after extract of O. sanctum roots treatment and this was equal to control, which may be due to the insulinotropic effect of this extract.O. sanctum treated animals showed a significant increase in the levels of GSH and significant decrease in the levels of TBARS elevation.The reduction in lipid peroxide levels might be due to the electron and H + donating capacity of O. sanctum, causing termination of lipid peroxidation chain reaction or interacting with cell membranes, improving their fluidity, thereby protecting them against lipid peroxidation.
The O. sanctum has beneficial effects on blood glucose levels in STZ-induced diabetes, as well as in improving hyperlipidemia due to diabetes.The active ingredient(s) present here may recover the disorders in carbohydrate metabolism noted in diabetic state by stimulating existing β-cell or by increasing the rate of β-cell regeneration or by modulating intracellular glucose utilization.

Conclusion
The present work described the anti-diabetic activity of the roots of Ocimum sanctum along with the isolation of new phytoconstituents, which may be useful in the medicinal properties of the drug.
).The mass spectrum showed an ion peak arising at m/z 443 [M-COOH] + , suggesting the presence of carboxylic functions in the molecule.The ion peaks formed at m/z 223 and 265 were generated due to retro Diels Alder fragmentation, and indicated the vinylic linkage at C-12, two hydroxyl groups in rings A and B and one hydroxyl in ring D or E.Moreover, the ion peaks produced at m/z 206 [223-H 2 O] + , 191 [206-Me] + , 188 [206-H 2 O] + and 173 [188-Me] + supported the location of two hydroxyl groups in rings A and B. The ion peak arose at m/z 219 [265-COOH] + further supported the existence of the carboxylic functions in ring D/E.The 1

Table 1 .
Hypoglycemic effect of single dose treatment of O. sanctum (roots) extracts on blood glucose levels in STZ-induced diabetic Wistar rat.

Table 2 .
Hypoglycemic effect of 21 days treatment of O. sanctum (roots) extracts on blood glucose levels in STZ-induced diabetic Wistar rat.
The data are expressed in mean SEM; n = 6 in each group.##(P <0.01) compared with the corresponding value for normal control animals (group I).* (P <0.05), ** (P <0.01) compared with the corresponding value for diabetic control animals (group II).

Table 3 .
Effect of O. sanctum (roots)extracts on lipid profile in STZ-induced diabetic Wistar rat.
The data are expressed in mean SEM; n=6 in each group.#(P<0.05);## (P <0.01) compared with the corresponding value for normal control animals (group I).**(P <0.01) compared with the corresponding value for diabetic control animals (group II).

Table 4 .
Effect of O. sanctum (roots)extracts on insulin, TBARS and GSH levels in STZ-induced diabetic Wistar rat.

Table 5 .
1 H and 13 C NMR spectral values of Compound 2.

Table 6 .
1 H and 13 C NMR spectral values of compound 5.