Antihyperglycemic Activity of Compounds Isolated from Indian Medicinal Plants
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Indian Journal of Experimental Biology Vol. 48, March 2010, pp. 294-298 Antihyperglycemic activity of compounds isolated from Indian medicinal plants Akanksha a, Arvind K Srivastava b & Rakesh Maurya a* aMedicinal and Process Chemistry Division, bDivision of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226 001, India Received 4 November 2009; revised 3 December 2009 Eleven antidiabetic Indian medicinal plants were investigated in streptozotocin induced diabetic rat model and provided scientific validation to prove their antihyperglycemic activity. Antidiabetic principles from five plants were isolated. All the compounds isolated were evaluated for antihyperglycemic activity in streptozotocin induced diabetic rat model and activities were compared with standard drug metformin. Some compounds were also screened in db/db mice. Two compounds (PP-1 and PP-2) inhibited significantly the activity of PTPase-1B in an in vitro system. This might be the underlying mechanism of antihyperglycemic activity of these compounds. Keywords : Antihyperglycemic activity, Normoglycemic rat model, PTPase-1B, STZ Diabetes mellitus is characterized by group of grandis, Tinospora cordifolia, Withania coagulans metabolic disorders. Deficiency or insensitivity of and Zingiber officinale, and isolated antidiabetic insulin causes glucose to accumulate in the blood, principals from F. bengalensis , P. pinnata , leading to various complications. When breakdown of P. marsupium , T. cordifolia and W. coagulans . glucose is stopped, body uses fat and protein for producing the energy. Due to this polydipsia, Materials and Methods polyuria, polyphagia, and excessive weight loss occur. Plant materials Authenticated leaves of Aegle High blood sugar harms organs and increases risk of marmelose (collected in the month of March from heart disease. Cardiovascular disease, retinopathy, Lucknow, CDRI plant code no. 4499), Coccinia neuropathy and nephropathy are the complications indica (collected in the month of July from Lucknow, associated with type 2 diabetes. Clinically diabetic CDRI plant code no. 4536), Dodecadenia grandiflora patients are characterized by marked increase in blood (collected in the month of October from Almora, glucose level followed by mild hyperlipidemia. CDRI plant code no. 4699) , Ocimum sanctum Effective treatment includes controlling hyperglycemia (collected in the month of December from Lucknow, as well as secondary complications. CDRI plant code no. 38) , Tectona grandis (collected For centuries, plants have been used to treat human in the month of February from Sirumalai, diseases. In Ayurveda various herbs are reported for Tiruchirapalli, CDRI plant code no. 4483) and fruits treating and preventing diabetes. Herbal drugs have of Pongamia pinnata (collected in the month of April lesser or no side effects and are less expensive as from Lucknow, CDRI plant code no. 3200) , Withania compared to synthetic drugs. Therefore, identification coagulans (collected in the month of November from of antihyperglycemic leads from the plants has Lucknow, CDRI plant code no. 4554) as well as aerial become more important. In the present study, We roots of Ficus bengalensis (collected in the month of investigated antihyperglycemic activity in the extracts November from Lucknow, CDRI plant code no. of Aegle marmelose, Coccinia indica, Dodecadenia 4617), heartwood of Pterocarpus marsupium grandiflora, Ficus bengalensis, Ocimum sanctum, (collected in the month of November from forest of Pongamia pinnata, Pterocarpus marsupium, Tectona Madhya Pradesh, CDRI plant code no. 4500) , stems ________________ of Tinospora cordifolia (collected in the month of *Correspondent author March from Palampur, CDRI plant code no. 4434) Telephone.: +91-(522)-2612411-18 Ext.4235 and rhizomes of Zingiber officinale (collected in the Fax: 091-(522)-2623405/2623938/2629504 E-mail: [email protected] month of January from Lucknow, CDRI plant code CDRI Comm. No. 7916 no. 4735) were taken for the study. AKANKSHA et al .: ANTIHYPERGLYCEMIC ACTIVITY OF INDIAN MEDICINAL PLANTS 295 Isolation of compounds Compound FB ( α-amyrin leading to isolation of compound WC (coagulin L). acetate) was obtained from the hexane fraction of the WC was further derivatized to WC-16 and WC-27, out ethanolic extract of the aerial roots of Ficus of which WC-1 is a new compound. WC (1 bengalensis 1 (Fig. 1). equivalent) was refluxed with hydroxylamine The ethanolic extract of fruits of Pongamia pinnata hydrochloride (31 equivalent) and crystallized sodium was triturated successively with n-hexane and acetate for 1 at 100 oC. The product oxime (WC-1) chloroform. The concentrated chloroform fraction was purified by column chromatography over flash was suspended in distilled water and then extracted silica gel using methanol:chloroform (1:4) eluate. with n-butanol (n-BuOH) saturated with water. The Compound WC-2, which is aglycon of WC, was chloroform fraction was subjected to repeated column formed by enzymatic hydrolysis of compound WC by chromatography furnishing compounds PP-1 using culture of Aspergillus ochraceus . (pongamol) and PP-2 (karanjin) 2-3. Animals Male albino rats of Sprague–Dawley Compound PM (pteroside) was isolated from n- strain (body weight 140 ± 20 g) was selected for the BuOH fraction of the aqueous extract of heartwood of study. C57BL/KsJ-db/db mice were taken as type 2 Pterocarpus marsupium through repeated flash diabetes model. chromatography 4. Assessment of antihyperglycemic activity in The n-BuOH fraction of the ethanolic extract of the normoglycemic rat model Male albino rats of stem of Tinospora cordifolia was subjected to Sprague Dawley strain were selected for this study repeated column chromatography affording having body weight 140±20g. After 14-16 h compound TC (Tinosporaside) 5. starvation fasting blood glucose of each animal was The aqueous extract of W. coagulans was subjected measured and the animals showing blood glucose to extensive column chromatographic procedure, level between 60 to 80 mg/dl were finally selected Fig. 1 Isolated compounds and derivatives 296 INDIAN J EXP BIOL, MARCH 2010 and divided into groups of 5 animals each. Rats of time 0 min. (baseline), followed by 30, 60, 90 and 120 experimental group were orally administered the min after an oral glucose load of 3.0 gm/kg of body suspension of the desired compounds at a dose of 100 weight. Quantitative glucose tolerance of each animal mg/kg body weight prepared in 1.0% gum acacia. was calculated by area under curve (AUC) method Suspension of the standard drug metformin was given using Prism Software. Comparing the AUC of at 100 mg/kg dose level. Animals of control group experimental and control groups determined the received vehicle (1.0% gum acacia). An oral sucrose percentage antihyperglycemic activity 6. load of 10 g/kg body weight was given to rats of all Protein tyrosine phosphatase-1B assay Protein group post 30 min administration of the test tyrosine phosphatase-1B inhibitory activity of the sample/vehicle. Blood glucose level was measured at compounds was determined by comparing the activity 30, 60, 90 and 120 min post administration of of the enzyme in the control, with a sample containing sucrose 6. pure compound. The assay was performed by adding Assessment of antihyperglycemic activity in compound to the reaction mixture containing 10 mM streptozotocin (STZ) induced diabetic rat pNPP in 50 mM HEPES buffer ( pH 7.0) with 1 mM model Male albino rats of Sprague Dawley strain DTT, 2 mM EDTA and defined unit of enzyme were selected for this study having body weight protein. The reaction was terminated after 10 min of o 140±20g. Streptozotocin was dissolved in 100 m M incubation at 37 C by the addition of 0.1N NaOH and citrate buffer, pH 4.5, and calculated amount of the the absorbance was determined at 405 nm. A molar −1 −1 fresh solution was injected to overnight fasted rats (60 extinction coefficient of 1.78×104 M cm was mg/kg) intraperitoneally. Blood glucose was checked utilized to calculate the concentration of the 48 h later by glucometer by using glucostrips and p-nitrophenolate ion produced in the reaction mixture. animals showing blood glucose values between 144 IC 50 and K i values were determined by measuring the and 270 mg/dl were selected and divided into groups inhibitory activity of compounds at different 3 of 5 animals each. Rats of experimental groups were concentrations . administered suspension of the desired test samples Results orally (made in 1.0% gum acacia) at a dose of 100 We have investigated eleven Indian medicinal mg/kg body weight. Animals of control group were plants for their antihyperglycemic activity (Table 1). given an equal amount of 1.0% gum acacia. A sucrose Ethanolic extracts of Pongamia pinnata fruits, load of 2.5 g/kg of body weight was given after 30 Tinospora cordifolia stems, Ficus bengalensis aerial min of drug administration. After 30 min of post- roots and aqueous extracts of Pterocarpus marsupium sucrose load, blood glucose level was again checked heartwood, Withania coagulans fruits were further at 1, 2, 3, 4, 5 and at 6 h, respectively. Comparing the taken for isolation of active compounds. AUC of experimental and control groups determined 6 Compound FB isolated from the hexane fraction of the percent antihyperglycemic activity . ethanolic extracts of F. bengalensis aerial roots Assessment of antihyperglycemic activity in Type 2 exhibited 19.4% blood glucose lowering in diabetes model (C57BL/KsJ-db/db mice)Hyper- normoglycemic model at the dose of 100 mg/kg. glycemic animals were divided into four groups having Administration of compound FB in sucrose five animals each. Animals of one group were regarded challenged STZ-induced diabetic rats at the dose of as control group (orally administered 1% gum acacia) 50 mg/kg caused fall in their blood glucose by 35.6% and other groups were treated as experimental groups (P<0.01), comparable to metformin (37.8%, P<0.01). In (treated with suspension of the desired test substances db/db mice compound FB significantly lowered blood at a dose of 50 mg/kg body weight).