Hepatoprotective activity of Phyllanthus amarus Schum. et. Thonn. extract in ethanol treated rats: in vitro and in vivo studies

The present study was undertaken to investigate the protective effect and possible mechanism of aqueous extract from Phyllanthus amarus Schum. et. Thonn. (PA) on ethanol-induced rat hepatic injury. In the in vitro study, PA (1-4 mg/ml) increased %MTT reduction assay and decreased the release of transaminases (AST and ALT) in rat primary cultured hepatocytes being treated with ethanol. Hepatotoxic parameters studied in vivo included serum transaminases (AST and ALT), serum triglyceride (STG), hepatic triglyceride (HTG), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1beta), together with histopathological examination. In acute toxicity study, single dose of PA (25, 50 and 75 mg/kg, p.o.) or SL (silymarin, a reference hepatoprotective agent, 5 mg/kg), 24h before ethanol (5 g/kg, p.o.) lowered the ethanol-induced levels of transaminases (AST and/or ALT). The 75 mg/kg PA dose gave the best result similar to SL. Treatment of rats with PA (75 mg/(kg day), p.o.) or SL (5 g/(kg day), p.o.) for 7 days after 21 days with ethanol (4 g/(kg day), p.o.) enhanced liver cell recovery by bringing the levels of AST, ALT, HTG and TNF-alpha back to normal. Histopathological observations confirmed the beneficial roles of PA and SL against ethanol-induced liver injury in rats. Possible mechanism may involve their antioxidant activity.     

Evaluation of in vivo antihypertensive and in vitro vasodepressor activities of the leaf extract of Syzygium guineense (Willd) D.C

The aim of this work was to evaluate the antihypertensive activity of the hydroalcohol extract of the leaves of Syzygium guineense (Willd) D.C. (Myrtaceae) in a 1-kidney-1-clip rat model and its vasorelaxant effect on isolated aorta. The extract reduced blood pressure in a dose and time dependent fashion. Following 3 days of treatment, single oral daily doses of 50, 100 and 150 mg/kg caused an overall reduction (p < 0.05) of systolic blood pressure by 6.9, 34.0 and 40.8 mmHg, respectively. The diastolic blood pressure was, however, significantly reduced (p < 0.05) by 100 mg/kg (10.3 mmHg) and 150 mg/kg (18.4 mmHg) doses only. The mean blood pressure was reduced by 5.0, 18.3 and 25.9 mmHg by the respective doses. The extract also caused a dose-dependent relaxation of aorta precontracted with KCl at a concentration of 5-70 mg/mL, with a maximum relaxation of 56.22% achieved at 70 mg/mL concentration. The relaxation mechanism was found to be independent of the endothelium system, muscarinic receptors, histamine receptors, ATP dependent K(+) channels, cyclooxygenase enzymes and cGMP/NO pathway. The findings suggest that the extract had an antihypertensive effect most likely caused by dilation of the blood vessels, a confirmation for the folkloric use of the plant.     

The anti-inflammatory activities of an extract and compounds isolated from Platycladus orientalis (Linnaeus) Franco in vitro and ex vivo

Ethnopharmacological relevance

As a Chinese traditional herbal medicine, leaves of Platycladus orientalis (Linnaeus) Franco (LPO) are used to treat coughs, excessive mucus secretion, chronic bronchitis, bronchiectasis, and asthma, etc. The experiments were carried out to investigate their anti-inflammatory properties and mechanisms, which could support the Chinese traditional uses of treating inflammatory airway diseases.

Materials and methods

The anti-inflammatory activities of the chloroform fraction (CHL) and pure compounds of LPO were evaluated for their abilities to inhibit pro-inflammatory enzymes in vitro, and production of tumor necrosis factor-α (TNF-α) and nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Furthermore, the arachidonic acid metabolites, stimulated by calcium ionophore A23187, were also determined by HPLC.

Results

For the first time, the assays of eicosanoids in intact cells showed that the CHL, hinokiol, and acacetin had significant inhibitory effects on 5-hydroxy-eicosa-tetra-enoic acid (5-HETE) and leukotriene B₄ (LTB4) formations. And cell-free enzyme assays (5-lipoxygenase, leukotriene A₄-hydrolase, cyclooxgenase-2) demonstrated the potent inhibitory effects of the CHL, hinokiol and acacetin on 5-lipoxygenase (5-LOX). Then, the inhibitions of the CHL, hinokiol on NO biosynthesis and the inhibitions of the CHL, 8(14),15-pimaradien-3β,18-diol, and hinokiol on TNF-α release were also confirmed in the RAW264.7 murine macrophages.

Conclusion

The data indicate that the inhibitory effects of the CHL and its components (hinokiol and acacetin) on 5-LOX contribute to the anti-inflammatory activity of LPO. Moreover, the CHL and its components also show beneficial effects on NO and TNF-α production. Consequently, these results provide a rationale for LPO's traditional applications in the treatment of inflammatory airway diseases.     

In vitro anti-prostate cancer and ex vivo antiangiogenic activity of Vernonia guineensis Benth. (Asteraceae) tuber extracts

Ethnopharmacological relevance

Prostate cancer is a major problem worldwide and affects most men above the age of forty-five. Vernonia guineensis Benth. (Asteraceae) root decoction is used in folk medicine in Cameroon to treat a number of ailments including prostate cancer. The aim of this study was to provide a preliminary validation of the use of Vernonia guineensis Benth. extracts to treat prostate cancer by evaluating the in vitro activity of its crude extracts and isolated molecules on prostate cancer cells lines and effect on angiogenesis which is essential for growth and metastases of prostate cancer.

Materials and methods

Aqueous, dichloromethane and methanol extracts of Vernonia guineensis Benth. tubers were tested for activity against three prostate cancer cell lines (PC-3, DU-145 and AT3B-1). The dichloromethane extract was subjected to bioactivity guided fractionation. Anti-proliferation, clonogenic and antiangiogenic activity of the crude extracts and isolated compound were tested. The WST-1 assay was used for the anti-proliferation activity meanwhile the standard clonogenic test and the rat ring aorta assay were carried out to determine the clonogenic and antiangiogenic activity of tested products respectively.

Results

The aqueous and methanol extracts of Vernonia guineensis Benth. demonstrated weak activity against prostate cancer cell lines in vitro with IC₅₀ > 100 μg/mL. The dichloromethane extract was more potent with IC₅₀ of 56.233 ± 3.630 μg/ml and 67.316 ± 2.452 μg/ml against the DU-145 and PC-3 cell lines respectively. Activity guided fractionation of this extract yielded a Pentaisovalerylsucrose (1) isolated for the first time from a natural source to the best of our knowledge. Compound 1 demonstrated in vitro activity against the human prostate cancer cell lines PC-3 and DU-145 with IC₅₀ of 5.701 ± 0.142 μM and 4.275 ± 0.710 μM, respectively. The IC₅₀ of the compound was 5.763 ± 0.425 μM against AT3B-1, a rat prostate cancer cell line expressing P-glycoprotein which is linked to drug resistance in most metastatic cancers. Compared to compound 1, Paclitaxel and Docetaxel were active against AT3B-1 at 2.641 ± 1.253 μM and 0.613 ± 0.251 μM. Paclitaxel showed IC₅₀ values of 0.004 ± 0.002 μM and 0.003 ± 0.001 μM against DU-145 and PC-3 prostate cancer cell lines respectively. Docetaxel showed IC₅₀ values of 0.002 ± 0.001 μM and 0.004 ± 0.001 μM against DU-145 and PC-3 prostate cancer cell lines respectively.

Conclusion

The in vitro anti-prostate cancer and the antiangiogenic activity of Vernonia guineensis Benth. extracts and isolated compound support the use of the tubers of this plant for the treatment of prostate cancer.     

In vitro antifungal activity of extract and plumbagin from the stem bark of Diospyros crassiflora Hiern (Ebenaceae)

In this study the methanol/dichloromethane (1:1) extract and plumbagin isolated from extract of stem barks of Diospyros crassiflora were tested for their antifungal activity against 12 strains of yeast pathogens and filamentous fungi: Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Cryptococcus neoformans, Aspergillus niger, Aspergillus flavus, Alternaria sp., Cladosporium sp., Geotrichum candidum, Fusarium sp. and Penicillium sp. The growth of all fungi strains tested was inhibited by the extract and plumbagin. The diameter of inhibition zones varied from 12 to 18 mm and from 21 to 35 mm for the extract and plumbagin, respectively. The MIC values ranged from 12.5 to 25 mg/mL for the extract and 0.78-3.12 microg/mL for plumbagin. It is therefore suggested that extracts from the stem bark of Diospyros crassiflora could be used traditionally in the treatment of fungal infections. Compared with ketoconazole used as a standard antifungal, plumbagin could be considered as a promising antifungal agent.     

In vitro anti-beta-secretase and dual anti-cholinesterase activities of Camellia sinensis L. (tea) relevant to treatment of dementia

The primary target of licensed drugs for the treatment of Alzheimer's disease is the inhibition of the enzyme acetylcholinesterase, although preventing beta-amyloidosis is a prime target for drugs in development. The in vitro dual anti-cholinesterase and beta-secretase activities of Camellia sinensis L. extract (tea) is reported. Green and black tea inhibited human acetylcholinesterase (AChE) with IC(50) values of 0.03 mg/mL and 0.06 mg/mL respectively, and human butyrylcholinesterase (BuChE) with IC(50) values 0.05 mg/mL. Green tea at a final assay concentration of 0.03 mg/mL inhibited beta-secretase by 38%. These novel findings suggest that tea infusions contain biologically active principles, perhaps acting synergistically, that may be used to retard the progression of the disease assuming that these principles, yet to be identified, reach the brain.     

In vitro inhibitory activities of plants used in Lebanon traditional medicine against angiotensin converting enzyme (ACE) and digestive enzymes related to diabetes

AIM OF THE STUDY: In recent years the use of medicinal plants has been growing worldwide and this is particularly true in Lebanon. In the present investigation we report the inhibitory activity against digestive enzymes related to diabetes and angiotensin converting enzyme (ACE) of extracts of nine plant species collected in Lebanon, where they have a traditional use against diabetes. MATERIALS AND METHODS: In order to evaluate if the extraction procedure could influence the activity we decided to perform different extractions with methanol, n-hexane and chloroform of Calamintha origanifolia, Satureja thymbra, Prangos asperula, Sideritis perfoliata, Asperula glomerata, Hyssopus officinalis, Erythraea centaurium, Marrubium radiatum and Salvia acetabulosa and test each of them. RESULTS AND CONCLUSIONS: Marrubium radiatum and Salvia acetabulosa methanol extracts exerted the highest activity against alpha-amylase (IC(50) 61.1 and 91.2mug/ml, respectively) and alpha-glucosidase (IC(50) 68.8 and 76.9mug/ml, respectively). The same extracts exhibited a strong inhibitory activity against ACE with IC(50) of 72.7 and 52.7mug/ml, respectively. The results support the traditional use of some the species examined.     

In vitro activities of Citrus medica L. cv. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer's disease

The present study showed for the first time the in vitro properties (antioxidant, hypoglycaemic and anticholinesterase) of Citrus medica L. cv. Diamante which belongs to the Rutaceae family. The n-hexane extract of Diamante citron peel is characterized by the presence of monoterpenes and sesquiterpenes. The most abundant constituents were two monoterpenes: limonene and gamma-terpinene. The extract showed significant antioxidant activity that was carried out using different assays (DPPH test, beta-carotene bleaching test and bovine brain peroxidation assay). Oxidative damage, caused by the action of free radicals, may initiate and promote the progression of a number of chronic diseases, including diabetes and Alzheimer's disease. Diamante citron peel extract showed hypoglycaemic activity and an anticholinesterase effect.     

Hypotensive effect of aqueous extract of Averrhoa carambola L. (Oxalidaceae) in rats: an in vivo and in vitro approach

Aim of the study

Averrhoa carambola L. (Oxalidaceae) leaves are used in Brazilian traditional medicine to treat hypertension. This study was conducted to evaluate the hypotensive effect of the aqueous extract of Averrhoa carambola (AEAc) and its underlying mechanisms in the isolated rat aorta.

Materials and methods

The effect of AEAc on the mean arterial pressure (MAP) was determined in vivo in anesthetized rats. In vitro, thoracic aortic rings were isolated and suspended in organ baths, and the effects of AEAc were studied by means of isometric tension recording experiments. In HPLC analysis, the fingerprint chromatogram of AEAc was established.

Results

In normotensive rats, AEAc (12.5-50.0 mg/kg, i.v.) induced dose-dependent hypotension. In vitro, AEAc caused a depression in the E_max response to phenylephrine without a change in sensibility. Also, in a depolarized Ca²⁺-free medium, AEAc inhibited CaCl₂-induced contractions and caused a concentration-dependent rightward shift of the response curves, indicating that AEAc inhibited the contractile mechanisms involving extracellular Ca²⁺ influx.

Conclusions

These results demonstrate the hypotensive effects of AEAc, and these effects may, in part, be due to the inhibition of Ca²⁺, which supports previous claims of its traditional use.     

Evaluation of mutagenic and antimutagenic activities of neem (Azadirachta indica) seed oil in the in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Aim of the study

The possible mutagenic and antimutagenic activity of neem oil (NO) and its DMSO extract (NDE) were, examined in the Ames Salmonella/microsome mutagenicity test and the mouse bone marrow micronucleus assay.

Materials and methods

Eight different strains of Salmonella typhimurium were, used to study the genotoxicity of neem oil both in the presence and absence of Aroclor-1254 induced rat liver homogenate (S9). Two-dose treatment protocol was, employed to study the cytogenetic activity in micronucleus assay. Similarly, the antimutagenic activity of neem oil and NDE was studied against mitomycin (MMC) and 7,12-dimethylbenz[a]anthracene (DMBA) in the above two test systems.

Results

Neem oil was non-mutagenic in all the eight tester strains of Salmonella typhimurium both in the presence and absence of S9 mix. In the present study, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in neem oil treated groups over the negative control (DMSO) group of animals, indicating the non-clastogenic activity of neem oil in the micronucleus test. Neem oil showed good antimutagenic activity against DMBA induced mutagenicity compared to its DMSO extract. However, neem oil showed comparatively less antimutagenicity against MMC in the Ames assay. In vivo anticlastogenic assays shows that neem oil exhibited better activity against DMBA induced clastogenicity.

Conclusion

These results indicate non-mutagenic activity of neem oil and significant antimutagenic activity of neem oil suggesting its pharmacological importance for the prevention of cancer.     

Anti-diarrhoea and analgesic activities of the methanol extract and its fractions of Jasminum amplexicaule Buch.-Ham. (Oleaceae)

Jasminum amplexicaule Buch.-Ham. (Oleaceae) has been commonly used in the traditional medicine in dysentery, diarrhoea and bellyache in China. In the present work, the methanol extract of Jasminum amplexicaule and different fractions of this extract were studied for anti-diarrhoea and analgesic activities. The anti-diarrhoea activities were investigated using castor oil-induced, magnesium sulphate-induced diarrhoea models, antienteropooling assay and gastrointestinal motility models in mice. The analgesic activities were studied using hot-plate, writhing and formalin models in mice. At the doses of 100, 200 and 400mg/kg, the methanol extract (ME) showed significant and dose-dependent anti-diarrhoea and analgesic activity in these models. The chloroform fraction (CHF), ethyl acetate fraction (EAF) and the residual methanol fraction (RMF) exhibited similar activity using a dose of 200mg/kg in these models. The pharmacological activities of the n-butanol fraction (BUF) were lesser than the ME extract and other fractions. These results may support the fact that this plant is traditionally used to cure diarrhoea and pain.     

In vitro anti-Helicobacter pylori potential of methanol extract of Allium ascalonicum Linn. (Liliaceae) leaf: susceptibility and effect on urease activity

The crude methanol extract of the leaf of Allium ascalonicum was screened in vitro against fi ve strains of Helicobacter pylori (Hp) (ATCC 24376, UCH 97001, UCH 97009, UCH 98026 and UCH 99039) for antibacterial activity by the agar diffusion method in Mueller-Hinton agar supplemented with de fi brinated horse blood. All the strains were inhibited by the extract to varying degrees. The minimum inhibitory concentrations (MICs) of the extract against all the tested strains ranged from 6.25 to 12.5 mg/mL. The effects of increasing concentrations of the extract on the urease activity of three of the Helicobacter pylori strains were investigated further. The results showed that increasing the concentration of the extract decreased the urease activity of all the strains tested. Phytochemical screening of the plant showed that it contains alkaloids, cardiac glycosides and saponins. The anti-Hp activity observed is discussed in relation to the chemical constituents reportedly isolated from these plants and their traditional uses. The result of this work suggests that Allium ascalonicum has some therapeutic potential against Helicobacter pylori infection, which could be explored for patients with gastroduodenal disorders.     

In vitro hepatotoxicity of Petasites hybridus extract (Ze 339) depends on the concentration,the cytochrome activity of the cell system,and the species used

Ze 339, a CO₂ extract prepared from the leaves of Petasites hybridus, possesses antispasmodic and anti‐inflammatory effects and is proven to be effective in the treatment of allergic rhinitis. To study possible hepatotoxic effects of Ze 339, its main constituents and metabolites, a series of in vitro investigations were performed. Furthermore, different reconstituted fractions of extract (petasins and fatty acid fraction) were examined in three in vitro test systems using hepatocytes: Two human cell lines, with lower and higher activity of cytochrome P450 enzymes (HepG2, HepaRG) as well as a rodent cell line with high cytochrome P450 activity (H‐4‐II‐E), were used. Metabolic activity, assessed by the WST‐1 assay, was chosen as indicator of cytotoxicity. To assess potential bioactivation of Ze 339 compounds, metabolic experiments using S9 fractions from rats, dogs, and humans and isolated cytochromes (human/rat) were performed, and the formation of reactive metabolites was assessed by measuring cellular concentrations of glutathione and glutathione disulphide. Our data revealed that the cytotoxicity of Ze 339, its single constituents, and main metabolites depends on the concentration, the cytochrome activity of the cell system, and the species used.     

In vitro protective effects of Withania somnifera (L.) dunal root extract against hydrogen peroxide and β‐amyloid(1–42)‐induced cytotoxicity in differentiated PC12 cells

Withania somnifera L. Dunal (Solanaceae), also known as ‘ashwagandha’ in Sanskrit and as ‘Indian ginseng’, is used widely in Ayurvedic medicine as a nerve tonic and memory enhancer, with antiaging, antistress, immunomodulatory and antioxidant properties. There is a paucity of data on the potential neuroprotective effects of W. somnifera root, as traditionally used, against H₂O₂‐ and Aβ_(1–42)‐induced cytotoxicity which are current targets for novel approaches to treat dementia, especially dementia of the Alzheimer's type (AD). In this study, an aqueous extract prepared from the dried roots of W. somnifera was assessed for potential protective effects against H₂O₂‐ and Aβ_(1–42)‐aggregated fibril cytotoxicity by an MTT assay using a differentiated rat pheochromocytoma PC12 cell line. The results suggest that pretreatments of differentiated PC12 cells with aqueous extracts of W. somnifera root significantly protect differentiated PC12 cells against both H₂O₂‐ and Aβ_(1–42)‐induced cytotoxicity, in a concentration dependent manner. To investigate the compounds that could explain the observed effects, the W. somnifera extract was analysed by liquid chromatography–serial mass spectrometry and numerous withanolide derivatives, including withaferin A, were detected. These results demonstrate the neuroprotective properties of an aqueous extract of W. somnifera root and may provide some explanation for the putative ethnopharmacological uses of W. somnifera for cognitive and other neurodegenerative disorders that are associated with oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.