Mohd. Zaini Asmawi

ANTI-INFLAMMATORY ACTIVITIES OF EMBLICA OFFICINALIS GAERTN LEAF EXTRACTS

Abstract— Emblica officinalis Gaertn, a tree growing in subtropical and tropical parts of China, India, Indonesia and the Malay Peninsula, has been used for anti‐inflammatory and antipyretic treatments of rural populations in these areas. In the present study, we examined the effects of Emblica officinalis extracts on carrageenan‐ and dextran‐induced rat hind paw oedema. Anti‐inflammatory activity was found in the water fraction of methanol extract of the plant leaves. The effects of the same fraction were tested on the synthesis of mediators of inflammation such as leukotriene B4 (LTB4), platelet‐activating factor (PAF) and thromboxane B2 (TXB2), and on LTB4‐ and N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (FMLP)‐induced migration of human polymorphonuclear leucocytes (PMNs) in‐vitro. The water fraction of the methanol extract inhibited migration of human PMNs in relatively low concentrations. It did not inhibit LTB4 or PAF synthesis in human PMNs or TXB2 synthesis in human platelets during clotting, suggesting that the mechanism of the anti‐inflammatory action found in the rat paw model does not involve inhibition of the synthesis of the measured lipid mediators.

Studies on diuretic and hypouricemic effects of Orthosiphon stamineus methanol extracts in rats

AIM OF THE STUDY Orthosiphon stamineus (Labiatae) is a traditional folk medicine widely used in Southeast Asia for the treatment of several kidney disorders, gout and as a diuretic. This study was conducted to examine the diuretic and hypouricemic effects of Orthosiphon stamineus leaf extracts. MATERIALS AND METHODS The diuretic effect of different methanol extracts was examined by treating different groups of Sprague-Dawley rats with single (2g/kg) oral doses of methanol and methanol:water (1:1) extracts. Hydrochlorothiazide (10mg/kg) was used as positive control in acute study. Methanol and methanol water (1:1) extracts at 0.5 g/kg were administered for a period of 7 consecutive days. Cumulative urine volume and electrolytes (Na+ and K+) concentrations at different time intervals were measured. On the other hand, hypouricemic activity of methanol:water extract (1:1) was experimented using different oral single doses (0.25, 0.5, 1 and 2g/kg). Allopurinol was used as positive control. Uric acid concentration in serum was analyzed by using RP-HPLC at 280 nm. RESULTS Sodium and potassium excretion increased significantly (p<0.05 and <0.01) in the first 8h of treatment with a single dose (2g/kg) of the extracts in a pattern comparable to that of the known diuretic hydrochlorothiazide. Meanwhile, repeated administration of 0.5 g/kg methanol:water (1:1) extract showed a significant increase in urine volume (from day 3 to day 7) (p<0.01) and electrolytes excretion (Na+ and K+) from day 2 to day 7 (p<0.05 and <0.01). On the other hand, 0.5, 1 and 2g/kg of methanol:water (1:1) extract and the standard allopurinol reduced the serum urate level in hyperuricemic rats at hour 6. CONCLUSION These results provided an evidence of the high tendency of methanol:water (1:1) extract of Orthosiphon stamineus towards diuretic and hypouricemic effects in rats.

Effects of Flavonoids on Prostaglandin E2 Production and on COX-2 and mPGES-1 Expressions in Activated Macrophages

Prostaglandin E2 (PGE2) has a central role in inflammation and both cyclooxygenase-2 (COX-2) and prostaglandin E synthases are critical enzymes in its synthesis. In inflammation, bacterial products and cytokines enhance the expression of COX-2 and inducible microsomal prostaglandin E synthase-1 (mPGES-1) which are functionally coupled to result in increased PGE2 formation in macrophages and tissue cells. In the present study, we systematically investigated the effects of 26 naturally occurring flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. Twelve flavonoids, i.e., flavone, luteolin-7-glucoside, kaempferol, isorhamnetin, morin, quercetin, naringenin, taxifolin, pelargonidin, daidzein, genistein, and genistin effectively inhibited lipopolysaccharide (LPS)-induced PGE2 production. Four flavonoids (flavone, isorhamnetin, daidzein, and genistein) inhibited significantly LPS-induced COX-2 expression, while mPGES-1 expression was downregulated by kaempferol and isorhamnetin. The present study characterizes the effects of flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. The results add to our knowledge of the anti-inflammatory actions of flavonoids and introduce kaempferol and isorhamnetin as compounds capable of downregulating the expression of mPGES-1.

An investigation of the anti-inflammatory and analgesic effects of Orthosiphon stamineus leaf extract.

Anti-inflammatory and analgesic activities of a standardized Orthosiphon stamineus methanol:water (50:50 vol/vol) leaf extract (SEOS) were evaluated in animal models. Oral administration of SEOS at doses of 500 and 1,000 mg/kg significantly reduced the hind paw edema in rats at 3 and 5 hours after carrageenan administration (P < .01 and P < .01; P < .01 and P < .05, respectively). SEOS (1,000 mg/kg, p.o.) also produced significant (P < .05) analgesic activity in both the acetic acid-induced writhing test and the formalin-induced licking test (late phase) in mice and rats, respectively. However, SEOS showed no effect on the tail flick and hot plate tests in mice. The results of the present study support the proposal that O. stamineus has anti-inflammatory and non-narcotic analgesic activities. These findings justify the traditional use of the plant for treating pain and inflammation.

Antioxidant and hepatoprotective effects of Orthosiphon stamineus Benth. standardized extract.

Orthosiphon stamineus (OS), Benth. (Lamiaceae) is widely used in Malaysia for treatments of various kidney and liver ailments. In the experiment, DPPH* radicals scavenging, Fe(3+)-induced lipid peroxidation inhibiting activities and trolox equivalent antioxidant capacity (TEAC) of methanol/water extract of Orthosiphon stamineus (SEOS) were determined. The results indicated that SEOS exhibited antioxidant, lipid peroxidation inhibition and free radical scavenging activities. The hepatoprotective activity of the SEOS was studied using CCl(4)-induced liver toxicity in rats. The activity was assessed by monitoring liver function tests through the measurement of alanine transaminase (ALT) and aspartate transaminase (AST). Furthermore, hepatic tissues were also subjected to histopathological studies. Pretreatment of SEOS (125, 250, 500 and 1000 mg/kg p.o.) dose-dependently reduced the necrotic changes in rat liver and inhibited the increase of serum ALT and AST activities. The results of the present study indicated that the hepatoprotective effect of Orthosiphon stamineus might be ascribable to its antioxidant and free radical scavenging property.

Toxicity evaluation of a standardised 50% ethanol extract of Orthosiphon stamineus.

AIM OF THE STUDY The present investigation was carried out to evaluate the safety of standardised 50% ethanol extract of Orthosiphon stamineus plant by determining its potential toxicity after acute and subchronic administration in rats. MATERIALS AND METHODS For acute toxicity study, up and down method (limit dose) was adapted. A single dose of 5000 mg/kg of the standardised 50% ethanol extract of O. stamineus was given orally to 5 healthy Sprague-Dawley (SD) female adult rats. The rats were observed for mortality and clinical signs for 3 h and then periodically for 14 days. While in the subchronic toxicity study, the extract was administered orally at doses of 1250, 2500 and 5000 mg/kg per day for 28 days to female and male SD rats, respectively. The animals were sacrificed, followed by examination of their organs and blood serum. RESULTS In the acute toxicity study, standardised 50% ethanol extract of O. stamineus at a dose of 5000 mg/kg caused neither visible signs of toxicity nor mortality. All five rats survived until the end of observation period. While in subchronic toxicity, administration of the standardised 50% ethanol extract of O. stamineus at 1250, 2500, and 5000 mg/kg for 28 days did not produce any mortality and there were no significant differences in the general condition, growth, organ weights, hematological parameters, clinical chemistry values, or gross and microscopic appearance of the organs from the treatment groups as compared to the control group. CONCLUSIONS Standardised 50% ethanol extract of O. stamineus did not cause any death nor did it cause abnormalities in necropsy and histopathology findings. There were no acute or subchronic toxicity observed and this extract could be devoid of any toxic risk. The NOAEL for the standardised 50% ethanol extract of O. stamineus is 5000 mg/kg per day for 28 days.

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism

BackgroundIn the present study, we tested a 50% ethanolic extract of Orthosiphon stamineus plants and its isolated bioactive compound with respect to their α-glucosidase and α-amylase inhibitory activities.MethodsBioactive flavonoid sinensetin was isolated from 50% ethanolic extract of Orthosiphon stamineus. The structure of this pure compound was determined on the NMR data and the α-glucosidase and α-amylase inhibitory activities of isolated sinensetin and 50% ethanolic extract of Orthosiphon stamineus were evaluated.ResultsIn vitro studies of a 50% ethanolic extract of O. stamineus and the isolated sinensetin compound showed inhibitory activity on α-glucosidase (IC50: 4.63 and 0.66 mg/ml, respectively) and α-amylase (IC50: 36.70 mg/ml and 1.13 mg/ml, respectively). Inhibition of these enzymes provides a strong biochemical basis for the management of type 2 diabetes via the control of glucose absorption.ConclusionAlpha-glucosidase and α-amylase inhibition could the mechanisms through which the 50% ethanolic extract of O. stamineus and sinensetin exert their antidiabetic activity, indicating that it could have potential use in the management of non-insulin-dependent diabetes.

HPLC and Anti-Inflammatory Studies of the Flavonoid Rich Chloroform Extract Fraction of Orthosiphon Stamineus Leaves

The aim of the present study was to verify the anti-inflammatory activity of Orthosiphon stamineus leaf extracts and to identify the active compound(s) contributing to its anti-inflammatory activity using a developed HPLC method. Active chloroform extract of O. stamineus was fractionated into three fractions using a dry flash column chromatography method. These three fractions were investigated for anti-peritoneal capillary permeability, in vitro nitric oxide scavenging activity, anti-inflammatory and nitric oxide (NO) inhibition using carrageenan-induced hind paw edema method. The flavonoid rich chloroform extract fraction (CF2) [containing sinensetin (2.86% w/w), eupatorin (5.05% w/w) and 3’-hydroxy-5,6,7,4’-tetramethoxyflavone (1.101% w/w)], significantly reduced rat hind paw edema, NO and decreased dye leakage to peritoneal cavity at p < 0.05. IC50 of in vitro NO scavenging of CF2 was 0.3 mg/mL. These results suggest that the anti-inflammatory properties of these CF2 may possibly be due to the presence of flavonoid compounds capable of affecting the NO pathway.

Synergistic antidiabetic activity of Vernonia amygdalina and Azadirachta indica: biochemical effects and possible mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE A decoction from a combination of herbs is commonly used in Traditional African Medicine for the management of chronic ailments. In Nigeria, the leaves of Vernonia amygdalina Del. (VA) and Azadirachta indica A. Juss (AI) are used traditionally as a remedy against diabetes mellitus for which empirical evidence attests to its efficacy. AIM OF THE STUDY To evaluate the synergistic antidiabetic action of VA and AI, the biochemical effects and possible mechanism in streptozotocin-induced diabetic rat (SDR) models. MATERIALS AND METHODS Ethanolic extracts of VA and AI were co-administered (200 mg/kg, 50:50) to non-diabetic rats (NDRs) and SDRs for 28 days. Blood glucose and body weight were monitored during this period, and at end of treatment, serum glucose, insulin, triiodothyronine (T3), tetraiodothyronine (T4) and α-amylase activity were studied. Glucose and activities of antioxidant enzymes, e.g., catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), were estimated in hepatocytes, along with the impact on the histology of the liver and pancreas. Medium acting insulin, HU (5 IU/kg, s.c.) was used as a positive control. RESULTS The study reveals that compared with single extracts, the combined extract (VA/AI) promptly lowered blood glucose and maintained a relatively steady level over the study period, in tandem with HU. During this period, body weight gain successively increased. In SDRs, fasting blood glucose at days 0 and 28 was raised by 4.33 and 3.16 fold, respectively, and the serum glucose was raised by 7.70 fold vs. normal control (P<0.05). The discrepancies in the individual effects of VA and AI on hepatic glucose and α-amylase activity were also restored. In NDRs, VA/AI lowered blood and serum glucose (1.14 and 1.94 fold, respectively), although to a lesser extent when compared with HU. Furthermore, VA/AI was found to lower serum insulin, T3 and T4 by 1.66, 1.57 and 2.16 fold, respectively, in SDR (P<0.05). This was similar to HU, which demonstrated 1.79 and 1.68 fold reduction of insulin and T3, respectively (P<0.05), but had no effect on T4. Conversely, in NDRs, VA/AI caused 1.32, 4.93 and 1.04 fold increase in insulin, T3 and T4, respectively, reciprocal to its effect on blood and serum glucose. Oxidative stress in SDR, characterised by decreased GPx and CAT activities, was ameliorated, as the activities of the enzymes and SOD increased following a 28-day treatment with VA/AI (P<0.05). The features of diabetic pathology, indicated in the histology of the liver and pancreas, were reversed. However, the extent of recovery was partial with VA, better with AI, and distinct and total with VA/AI, compared with a null effect by HU. CONCLUSION Taken together, our results contribute towards validation of enhanced antidiabetic efficacy of VA and AI when combined. This synergy may be exerted by oxidative stress attenuation, insulin mimetic action and β-cell regeneration.

Hypoglycemic and anti–hyperglycemic study of Gynura procumbens leaf extracts

OBJECTIVE To study the antidiabetic activity of Gynura procumbens (G. procumbens) used in the traditional management of diabetes in Southern Asia. METHODS G. procumbens leaves were extracted sequentially with graded percentage of ethanol in water (95%, 75%, 50%, 25% and 0%), and the extracts were tested for antidiabetic activity using acute (7 h), subcutaneous glucose tolerance test and sub-chronic (14 d) test in non-diabetic and streptozotocin-induced diabetic rats. The extracts were further subjected to phytochemical studies. RESULTS In acute dose (1 g/kg), the extracts significantly lowered fasting blood glucose (FBG) in streptozotocin-induced diabetic rats (P<0.05). However, the FBG-lowering effect of the 25% extract compared to the other extracts, was rapid (47% after 2 h) and the highest: 53%, 53% and 60% in the 3rd, 5th, and 7th h, respectively (P<0.05), comparable only to the effect of metformin. Furthermore, the extracts suppressed peak FBG in subcutaneous glucose tolerance test, but only the 0% and 25% extracts, and metformin sustained the decrease until the 90th min (P<0.05). Moreover, in the 14 days study, the 25% extract exerted the highest FBG-lowering effect, namely 49.38% and 65.43% on days 7 and 14, respectively (P<0.05), similar to the effect of metformin (46.26% and 65.42%). Total flavanoid and phenolic contents in the extracts were found to decrease with increase in polarity of extraction solvents. The composition of reference compounds (chlorogenic acid, rutin, astragalin and kaempferol-3-O-rutinoside) followed a similar trend. CONCLUSIONS G. procumbens contains antidiabetic principles, most extracted in 25% ethanol. Interaction among active components appears to determine the antidiabetic efficacy, achieved likely by a metformin-like mechanism.