Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: gene expression studies.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

Anti-inflammatory effect of the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden, the Tiger Milk mushroom.

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) is used as a traditional medicine to relieve cough, asthma and chronic hepatitis. The traditional uses of the sclerotium are presumably related to its anti-inflammatory effect. The present study was carried out to evaluate the anti-inflammatory activity of the sclerotial powder of L. rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) cultivar TM02. METHODS: The anti-acute inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 was investigated using carrageenan-induced paw edema test while the inhibition of transudative and proliferative phases of chronic inflammation were studied by cotton pellet induced granuloma model. Sprague Dawley rats were used in both studies. The anti-inflammatory activity was also measured by inhibition of lipopolysaccharide induced TNF-alpha production in RAW 264.7 macrophage cells. RESULTS: Cold water extract (CWE), hot water extract (HWE) and methanol extract (ME) of the sclerotial powder of L. rhinocerotis cultivar TM02 possessed anti-acute inflammatory activity as was measured by carrageenan-induced paw edema test, with CWE being the most potent. The acute anti-inflammatory activity of the cold water extract (CWE) was mainly contributed by its high molecular weight (HMW) fraction isolated by Sephadex G50 gel filtration chromatography. Its protein component was very potent in the inhibition of TNF-alpha production with an IC50 of 0.76 µg/ml. CWE at 200 mg/kg did not inhibit transudative and proliferative phase of chronic inflammation as shown by using the cotton pellet induced granuloma model. CONCLUSIONS: Our results suggested that most of the bioactive substance(s) contributed to the acute-inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 appear to be in the CWE, particularly its HMW fraction. The anti-inflammatory activity of CWE was mainly contributed by the protein component of the high molecular weight (HMW) fraction and it exhibited strong inhibitory effect on TNF-alpha production but the possibility of synergistic effect between HMW, MMW and LMW fractions cannot be excluded. Future studies will provide new insights into the anti-inflammatory activity of the sclerotial powder.

Effect of Mucuna pruriens Seed Extract Pretreatment on the Responses of Spontaneously Beating Rat Atria and Aortic Ring to Naja sputatrix (Javan Spitting Cobra) Venom.

Mucuna pruriens Linn. (velvet bean) has been used by native Nigerians as a prophylactic for snakebite. Rats pretreated with M. pruriens seed extract (MPE) have been shown to protect against the lethal and cardiovascular depressant effects of Naja sputatrix (Javan spitting cobra) venoms, and the protective effect involved immunological neutralization of the venom toxins. To investigate further the mechanism of the protective effect of MPE pretreatment against cobra venom toxicity, the actions of Naja sputatrix venom on spontaneously beating rat atria and aortic rings isolated from both MPE pretreated and untreated rats were studied. Our results showed that the MPE pretreatment conferred protection against cobra venom-induced depression of atrial contractility and atrial rate in the isolated atrial preparations, but it had no effect on the venom-induced contractile response of aortic ring preparation. These observations suggested that the protective effect of MPE pretreatment against cobra venom toxicity involves a direct protective action of MPE on the heart function, in addition to the known immunological neutralization mechanism, and that the protective effect does not involve action on blood vessel contraction. The results also suggest that M. pruriens seed may contain novel cardioprotective agent with potential therapeutic value.

Spasmolytic effect of citral and extracts of Cymbopogon citratus on isolated rabbit ileum.

Cymbopogon citratus, commonly known as lemongrass, has been shown to have antioxidant, antimicrobial and chemo-protective properties. Citral, a monoterpenoid, is the major constituent of C. citratus that gives off a lemony scent and is postulated to be responsible for most of its actions. In addition, C. citratus has been traditionally used to treat gastrointestinal discomforts, however, the scientific evidence for this is still lacking. Thus, the aim of the present study was to investigate the effect of the extracts of various parts of C. citratus (leaves, stems and roots) and citral on the visceral smooth muscle activity of rabbit ileum. The effect of the test substances were tested on the spontaneous contraction, acetylcholine (ACh)- and KCl-induced contractions. Citral at doses between 0.061 mM to 15.6 mM and the extract of leaves at doses between 0.001 mg/mL to 1 mg/mL significantly reduced the spontaneous, ACh- and KCl-induced ileal contractions. When the ileum was incubated in K(+)-rich-Ca(2+)-free Tyrode's solution, it showed only minute contractions. However, the strength of contraction was increased with the addition of increasing concentrations of CaCl(2). The presence of citral almost abolished the effect of adding CaCl(2), while the leaf extract shifted the calcium concentration-response curve to the right, suggesting a calcium antagonistic effect. These results were similar to that elicited by verapamil, a known calcium channel blocker. In addition, the spasmolytic effect of citral was observed to be reduced by the nitric oxide synthase inhibitor, L-NAME. In conclusion, citral and the leaf extract of C. citratus exhibited spasmolytic activity and it appeared that they may act as calcium antagonists. Furthermore, the relaxant effect of citral, but not that of the leaf extract may be mediated by nitric oxide suggesting the presence of other chemical components in the leaf extract other than citral.

Evaluation of the sub-acute toxicity of the sclerotium of Lignosus rhinocerus (Cooke), the Tiger Milk mushroom.

ETHNOPHARMACOLOGICAL RELEVANCE: Lignosus rhinocerus (known locally as 'Tiger Milk mushroom') is the most important medicinal mushroom used by the indigenous communities of Malaysia to treat fever, cough, asthma, cancer, food poisoning and as a general tonic. The sclerotium of the mushroom is the part with medicinal value. Lignosus rhinocerus was hitherto unexploited commercially because of limited supply. Recently, the mushroom was successfully cultivated. MATERIALS AND METHODS: Sprague Dawley rats (5 rats/group/sex) were fed orally with 250, 500 and 1,000 mg/kg TM02, 1,000 mg/kg TM03 as well as 1,000 mg/kg wild type Lignosus rhinocerus sclerotial powder. Sclerotial powder was orally administered once daily and consecutively for 28 days. Body weight of each animal was measured and any gross behavioral change was observed daily. Hematological and clinical biochemical parameters as well as histopathological analysis were carried out on 29th day. RESULTS: The results showed that oral administration of the sclerotial powder at daily dose of up to 1,000 mg/kg had no adverse effect on the growth rate, hematological and clinical biochemical parameters (including renal and liver function parameters). Histological studies showed that the treatments did not induce any pathological changes in the liver, kidney, heart, spleen and lung of the animals. CONCLUSION: In conclusion, our results show that there was no treatment-related sub-acute toxicity in rats following 28-days oral administration of 250, 500 and 1,000 mg/kg TM02, 1,000 mg/kg TM03 as well as 1,000 mg/kg wild type Lignosus rhinocerus sclerotial powder. As the highest tested dose of 1,000 mg/kg was not associated with any toxicity concern, the NOAEL dose is higher than 1,000 mg/kg.

Mucuna pruriens Linn. seed extract pretreatment protects against cardiorespiratory and neuromuscular depressant effects of Naja sputatrix (Javan spitting cobra) venom in rats.

Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.

The protective effect of Mucuna pruriens seeds against snake venom poisoning.

ETHNOPHARMACOLOGICAL RELEVANCE: The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite. AIM OF THE STUDY: To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms. MATERIALS AND METHODS: Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization. RESULTS: In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested. CONCLUSIONS: The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.

Phenylacetic acids were detected in the plasma and urine of rats administered with low-dose mulberry leaf extract.

The use of a high quercetin dose to demonstrate its absorption and bioavailability does not reflect the real dietary situation because quercetin glycosides are usually present in small amounts in the human diet. This study aimed to demonstrate the absorption and bioavailability of quercetin in mulberry leaves that represents a more physiologic dietary situation. Mulberry leaf ethanol extract was prepared similar to tea infusion, which is the way the tea leaves are generally prepared for consumption. Accordingly, rats were fed by oral intubation the mulberry leaf ethanol extract (15 g%/rat per day) or pure rutin (135 microg/rat per day) for 2 weeks. The control group received a similar volume of the vehicle, 10% ethanol. There was a significant increase in total antioxidant activity (TAA) in the urine and feces of the antioxidants-fed rats. Phenylacetic acid, a microbial metabolite of quercetin, was detected in the urine of the test animals, and quercetin was present in the fecal samples. By using an in situ intestinal preparation, 3-hydroxyphenylacetic acid, another microbial metabolite of quercetin, was detected in the plasma when the duodenal segment was instilled with 2 mg of rutin. This microbial metabolite retained 50% of the TAA of quercetin. The results of this study indicate that in a more realistic dietary situation, an increase in TAA in the body after consumption of quercetin-containing foods is contributed mainly by the microbial metabolites.

Teaching of pharmacology in Universiti Malaya and the other medical schools in Malaysia -- a historical perspective.

Traditional pharmacology teaching has focused more on drug instead of therapeutics, such that although pharmacological knowledge is acquired, practical skills in prescribing remain weak. In Malaysia many new medical schools (both public and private) have been set up in the last 12 years due to a change in government policy, resulting in a wide spectrum of medical curricula. Universiti Malaya (UM) being the oldest medical school in Malaysia was deep set in its traditional way of teaching-learning, since its inception in 1962, until a visit from the General Medical Council of the United Kingdom in 1984 triggered off a change of tide. Since then the medical curriculum in UM has undergone two major revisions. The first revised curriculum (1988) aimed to inject more clinical relevance into basic science teaching, through introducing clinical lectures and skills in the paraclinical year. Professional behaviour was also addressed. The second revised curriculum (1998) sought to improve further the integration of knowledge as well as to produce a holistic doctor, viewing the patient as a person instead of a clinical entity. The teaching-learning of pharmacology has gradually moved from factual regurgitation to more clinical reasoning, from lab-based to more patient-oriented approach. As more new medical schools are being set up in Malaysia, exchange of experience in this area of learning will hopefully help us find a happy medium between "the old is best" and "the new is better" type approach so that a pedagogically sound and yet logistically practical curriculum can be found in our local setting, to help produce doctors with good prescribing practice.

Vascular effects of Siberian ginseng (Eleutherococcus senticosus): endothelium-dependent NO- and EDHF-mediated relaxation depending on vessel size.

Siberian ginseng (SG) has been widely and historically consumed as a health food product for the improvement of self well-being, but whether vascular relaxation may contribute to such a therapeutic health effect has not been studied. We therefore investigated the vasorelaxant effect of the aqueous extract of the roots of SG (Eleutherococcus senticosus Maxim) using several in vitro vascular rings prepared from dog carotid artery, rat aorta and rat mesenteric artery. SG extract (0.04-0.8 mg/ml) caused concentration-dependent relaxation in dog carotid arterial rings pre-contracted with 100 microM phenylephrine (PE), and the relaxation was primarily endothelium-dependent. Treatment with 100 microM L-NOARG (a nitric oxide synthase inhibitor) either prevented or totally reverted SG-induced relaxation, suggesting that the endothelium-dependent relaxation was mediated by NO. Similar endothelium-dependent vascular relaxant responses were also obtained with rat aortic and mesenteric arterial rings, except that it occurred over a relatively higher concentration range of SG (0.5-2.0 mg/ml). When tested in the presence of 300 microM L-NAME, the vasorelaxant effect of SG was inhibited totally in rat aorta but only partially in rat mesenteric artery. The relaxation to SG that was insensitive to L-NAME in rat mesenteric arterial rings was eliminated when the rings (both proximal and distal ends) were pre-treated with a combination of 300 microM L-NAME and 15 mM KCl indicating the involvement of endothelium-derived hyperpolarizing factor (EDHF). This vasorelaxant response of the SG extract was inhibited partially by atropine (1 microM), completely by TEA (5 mM), but not by indomethacin (1 microM) or propranolol (10 microM). SG up to 2 mg/ml had no effect on KCl-induced contraction in any of the vascular rings studied. When compared with carbachol-induced (CCh) relaxation, SG resembles CCh in that the sensitivity to L-NAME inhibition is dependent on vascular size, i.e. aorta >proximal end of mesenteric artery >distal end of mesenteric artery. However, SG exhibited different potencies to relaxation while CCh showed similar potency (EC(50) of about 0.2 microM) in all three vascular segments. In conclusion, we have demonstrated that the vascular effect of SG is endothelium-dependent and mediated by NO and/or EDHF depending on the vessel size. Other vasorelaxation pathways, such as inhibition of K(+)-channels and activation of muscarinic receptors, may also be involved.