Antihyperglycemic effect of orthosiphon stamineus benth leaves extract and its bioassay-guided fractions

Preliminary investigations were carried out to evaluate the antidiabetic effects of the leaves of O. stamineus extracted serially with solvents of increasing polarity (petroleum ether, chloroform, methanol and water); bioassay-guided purification of plant extracts using the subcutaneous glucose tolerance test (SbGTT) was also carried out. Only the chloroform extract, given at 1 g/kg body weight (b.w.), significantly reduced (P < 0.05) the blood glucose level of rats loaded subcutaneously with 150 mg/kg (b.w.) glucose. The active chloroform extract of?O. stamineus was separated into five fractions using a dry flash column chromatography method. Out of the five fractions tested, only chloroform fraction 2 (C?2), at the dose of 1 g/kg (b.w.) significantly inhibited (P < 0.05) blood glucose levels in SbGTT. Active C?2 was split into two sub-fractions C?2-A and C?2-B, using a dry flash column chromatography method. The activities C?2-A and C?2-B were investigated using SbGTT, and the active sub-fraction was then further studied for anti-diabetic effects in a streptozotocin-induced diabetic rat model. The results clearly indicate that C?2-B fraction exhibited a blood glucose lowering effect in fasted treated normal rats after glucose-loading of 150 mg/kg (b.w.). In the acute streptozotocin-induced diabetic rat model, C?2-B did not exhibit a hypoglycemic effect on blood glucose levels up to 7 hours after treatment. Thus, it appears that C?2-B functions similarly to metformin, which has no hypoglycemic effect but demonstrates an antihyperglycemic effect only in normogycemic models. The effect of C?2-B may have no direct stimulatory effects on insulin secretion or on blood glucose levels in diabetic animal models. Verification of the active compound(s) within the active fraction (C?2-B) indicated the presence of terpenoids and, flavonoids, including sinensitin.     

Bioactivity-guided isolation of ethyl-p-methoxycinnamate, an anti-inflammatory constituent, from Kaempferia galanga L. extracts

This study evaluated the anti-inflammatory effect of Kaempferia galanga (KG) using an activity-guided approach. KG rhizomes were serially extracted with petroleum ether, chloroform, methanol and water. These extracts (2 g/kg each) were tested for their ability to inhibit carrageenan-induced rat paw edema. The chloroform extract was found to exert the highest inhibition (42.9%) compared to control (p < 0.001), hence it was further fractionated by washing serially with hexane, hexane-chloroform (1:1) and chloroform. The chloroform fraction (1 g/kg) showed the highest inhibitory effect (51.9%, (p < 0.001), on carrageenan-induced edema. This chloroform fraction was further fractionated with hexane-chloroform (1:3) and chloroform, and of the two fractions, the hexane-chloroform sub-fraction was the most effective in inhibiting edema (53.7%, p < 0.001). GC-MS analysis of the active sub-fraction identified ethyl-p-methoxycinnamate (EPMC) as the major component, which was re-crystallized. EPMC dose-dependently inhibited carrageenan-induced edema with an MIC of 100 mg/kg. Moreover, in an in vitro study, EPMC non-selectively inhibited the activities of cyclooxygenases 1 and 2, with IC?? values of 1.12 μM and 0.83 μM respectively. These results validate the anti-inflammatory activity of KG which may be exerted by the inhibition of cyclooxygenases 1 and 2. EPMC isolated from this plant may be the active anti-inflammatory agent.     

Antiangiogenic Activity and Bioassay‐guided Isolation of Aqueous Extract of Orthosiphon stamineus

Orthosiphon stamineus Benth. (Family: Lamiaceae), a famous traditional medicinal plant in tropical and sub‐tropical region, is being used for treating various ailments including inflammatory conditions. Since anti‐inflammation and antiangiogenesis involve cyclooxygenase‐II pathway, the present study is aimed to carry out antiangiogenic studies and bioassay‐guided isolation on aqueous extract of the plant. After antiangiogenic activity studies on the extract, fractions were prepared by partitioning the extract with hexane, chloroform and ethyl acetate, which were then evaluated for antiangiogenic activity. The most active, hexane fraction (HF) upon activity‐guided isolation gave three compounds, 1 (betulinic acid), 2 (oleanolic acid) and 3 (ursolic acid). The total contents of these three compounds were determined in the extract and its fractions using HPLC. The extract and HF exhibited 20% and 80% antiangiogenic activity, respectively, whereas, chloroform and ethyl acetate fractions were inactive. The three isolated compounds exhibited 100% antiangiogenic activity. Median effective concentrations (EC_50s) of HF, 1, 2 and 3 were found to be 84, 32, 34 and 38 μg/mL, respectively. The results of this study indicate that hexane fraction and the isolated compounds have promising antiangiogenic activity. Additionally, the isolated compounds may be sued as analytical markers to standardize extracts and formulations prepared from O. stamineus.