Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevance

Kaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer.

Aim of the study

In order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated.

Materials and methods

The impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively.

Results

In vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of V_max (15.276 ± 0.206 nmol/min) and lowest of K_i value (0.008 ± 0.002 μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low K_m value (1.599 ± 0.147 pmol). For in vivo studies, mice were orally treated with 250 mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity.

Conclusions

Kaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.     

In vitro inhibitory effect of West African medicinal and food plants on human cytochrome P450 3A subfamily

Aim of the study

In Africa, medicinal plants are used intensively and concomitantly with allopathic medicines in the treatment of opportunity diseases by many patients or by healthy person to prevent diseases. However, there is little information about the interactions between medicines and botanical products used currently in West Africa area. Therefore, the aim of the present investigation is to study the effect of some plant products on CYP3A4, CYP3A5 and CYP3A7, three individual enzymes of CYP3A subfamily, in vitro.

Materials and methods

Teas and ethanolic extracts of medicinal, food and co-administered plants were evaluated on CYP3A4, CYP3A5 and CYP3A7 individual enzymes in vitro using fluorometric assays.

Results

Extracts of adjuvant plants such as Aframomum cuspidatum, and Aframomum melegueta, as well as one medicinal plant (Harrisonia abyssinica) inhibited CYP3A4, CYP3A5 and CYP3A7 activity more than 90%. Phyllanthus amarus showed high inhibition of CYP3A5 and CYP3A7. Food plants (Solanum macrocarpon and Talinum triangulare) inhibited CYP3A4 and CYP3A5 less than 20%.

Conclusion

These results indicate that plants tested in this study affect in vitro the activity of the main three CYP3A subfamily enzymes. These active plants could interfere with the metabolism at phase I of conventional drugs in vivo as well act as pharmacoenhancers in herbal mixtures.     

Cytochrome P450 inhibitory potential and RP-HPLC standardization of trikatu—A Rasayana from Indian Ayurveda

Ethnopharmacological relevance

Trikatu is a very well known ‘Rasayana’ in Ayurveda and widely used as a polyherbal ayurvedic formulation in India. It consists of three well known plants, viz., Piper longum (PL), Piper nigrum (PN) and Zingiber officinale (ZO) in equal ratio. Trikatu has been prescribed for cough, cold, fever, asthma, respiratory problems and improvement of digestive disorders. The aim of the present study was to investigate the effect of individual ingredients of trikatu namely PL, PN, and ZO and formulations [Marketed formulation (MF) and laboratory formulation (LF)] on drug metabolizing enzymes (CYP3A4 and CYP2D6), to assess its herb–drug interaction potential through cytochrome P450 inhibition assays. Further this work was aimed to develop an RP-HPLC method for the identification and quantification of piperine and 6-gingerol in the crude drug trikatu.

Materials and methods

Enzyme inhibition effect of LF, MF, PL, PN and ZO was explored through CYP450–CO complex assay using rat liver microsomes (RLM) and a fluorescence screening method using individual isoenzymes (CYP3A4 and CYP2D6). The RP-HPLC method was developed for the identification and quantification of piperine and 6-gingerol in LF, MF and individual plant materials at the concentration of 1 mg/mL.

Results

RP-HPLC analysis confirmed the presence of piperine and 6-gingerol in LF and MF [Piperine: 7.89±2.12% (w/w) (MF), 6.70±2.13% (w/w) (LF)]; [6-gingerol: 5.3±1.21% (w/w) (MF), 4.95±2.34% (w/w) (LF)]. Inhibitory potential of MF and LF in CYP450–CO complex assay was found to be 37.54±3.12% (MF) and 35.12±2.31% (LF) and against CYP2D6 and CYP3A4 was estimated to be IC₅₀ 251.30±3.98 and 245.23±1.92 μg/mL and IC₅₀ 225.50±1.02 and 223.254±0.92 μg/mL respectively.

Conclusions

Different concentrations of the trikatu formulation and its individual components showed significantly (p<0.001) less inhibitory activity on individual isoenzymes as compared to the positive control. The crude drug exhibited inhibitory potential against the CYP450 enzymes in a concentration dependent manner. Outcome of the present study demonstrated that trikatu has less interaction potential with drug metabolizing enzymes.     

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19)

Ethno pharmacological relevance

Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date.

Aim of the study

In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated.

Materials and methods

S-Mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects.

Results

Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K_i values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K_i = 39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K_i = 7.1 μg/ml or 20.6 μM).

Conclusions

It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).     

Inhibitory effects of curcumenol on human liver cytochrome P450 enzymes

Curcumenol, one of the major components of Zedoary turmeric oil, has been widely used to treat cancer and inflammation. As an antibiotic or anticancer drug, curcumenol is highly likely to be used in combination with various synthetic drugs in most cases, thus it is necessary to evaluate potential pharmacokinetic drug‐drug interactions induced by curcumenol. In this study, the inhibitory effects of curcumenol on seven CYP isoforms were investigated, and the results demonstrated that only CYP3A4 was strongly inhibited (IC₅₀ = 12.6 ± 1.3 μM). Kinetic analysis showed the inhibition type was competitive with K_i value of 10.8 μM. Time‐ and NADPH‐dependent inhibitions were also investigated to show curcumenol is not a mechanism‐based inhibitor. Employing these in vitro data and maximum plasma concentration of curcumenol in human predicted from beagle dog's in vivo pharmacokinetic data, the change in AUC of victim drugs was predicted to be 0.4%, which suggested that curcumenol may be safely used without inducing metabolic drug‐drug interaction through P450 inhibition. Nevertheless, due to the limited pharmacokinetic data available for curcumenol in humans, it is still not possible to evaluate its potential clinical effects on human patients from in vitro data. Thus, the magnitude of drug‐drug interaction (DDI) induced by curcumenol warrants further investigation. Copyright © 2010 John Wiley & Sons, Ltd.     

In vivo antimalarial activity of Keetia leucantha twigs extracts and in vitro antiplasmodial effect of their constituents

Ethnopharmacological relevance

The West African tree Keetia leucantha (Rubiaceae) is used in traditional medicine in Benin to treat malaria. The twigs dichloromethane extract was previously shown to inhibit in vitro Plasmodium falciparum growth with no cytotoxicity (>100 µg/ml on human normal fibroblasts).

Materials and methods

The dichloromethane and aqueous extracts of twigs of K. leucantha were evaluated in vivo against Plasmodium berghei NK 173 by the 4-day suppressive test and in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity. Bioguided fractionations were realized and compounds were structurally elucidated using extensive spectroscopic analysis.

Results

The in vivo antimalarial activity of K. leucantha dichloromethane and aqueous twigs extracts were assessed in mice at the dose of 200 mg/kg/day. Both extracts exhibited significant effect in inhibiting parasite growth by 56.8% and 53.0% (p<0.0001) on day 7-postinfection. An LC–MS analysis and bioguided fractionations on the twigs dichloromethane extract led to the isolation and structural determination of scopoletin (1), stigmasterol (2), three phenolic compounds: vanillin (3), hydroxybenzaldehyde (4) and ferulaldehyde (5), eight triterpenic esters (6–13), oleanolic acid and ursolic acid. The antiplasmodial activity of the mixture of the eight triterpenic esters showed an antiplasmodial activity of 1.66±0.54 µg/ml on the 3D7 strain, and the same range of activity was observed for isolated isomers mixtures.

Conclusions

This is the first report on the in vivo activity of K. leucantha extracts, the isolation of thirteen compounds and analysis of their antiplasmodial activity. The results obtained may partially justify the traditional use of K. leucantha to treat malaria in Benin.     

In vitro inhibition of metabolism but not transport of gliclazide and repaglinide by Cree medicinal plant extracts

Ethnopharmacological relevance

Interactions between conventional drug and traditional medicine therapies may potentially affect drug efficacy and increase the potential for adverse reactions. Cree traditional healing is holistic and patients may use medicinal plants simultaneously with the conventional drugs. However, there is limited information that these medicinal plants may interact with drugs and additional mechanistic information is required. In this study, extracts from traditionally used Cree botanicals were assessed for their potential interaction that could alter the disposition of two blood glucose lowering drugs, gliclazide (Diamicron) and repaglinide (Gluconorm) though inhibition of either metabolism or transport across cell membranes.

Materials and methods

The effect of 17 extracts on metabolism was examined in a human liver microsome assay by HPLC and individual cytochrome P450s 2C9, 2C19, 2C8 and 3A4 in a microplate fluorometric assay. Gliclazide, rhaponticin and its aglycone derivative, rhapontigenin were also examined in the fluorometric assay. The effect on transport was examined with 11 extracts using the intestinal epithelial Caco-2 differentiated cell monolayer model at times up to 180 min.

Results

Both blood glucose lowering medications, gliclazide and repaglinide traversed the Caco-2 monolayer in a time-dependent manner that was not affected by the Cree plant extracts. Incubation of the Cree plant extracts inhibited CYP2C9, 2C19, 2C8 and 3A4-mediated metabolism, and the formation of four repaglinide metabolites: M4, m/z 451-A, m/z 451-B and the glucuronide of repaglinide in the human liver microsome assay. Gliclazide caused no significant inhibition. Likewise, rhaponticin had little effect on the enzymes causing changes of less than 10% with an exception of 17% inhibition of CYP2C19. By contrast, the aglycone rhapontigenin showed the greatest effects on all CYP-mediated metabolism. Its inhibition ranged from a mean of 58% CYP3A4 inhibition to 89% inhibition of CYP2C9. While rhaponticin and the aglycone did not show significant effects on repaglinide metabolism, they demonstrated inhibition of gliclazide metabolism. The aglycone significantly affected levels of gliclazide and its metabolites.

Conclusion

These studies demonstrate that the Cree plant extracts examined have the potential in vitro to cause drug interactions through effects on key metabolic enzymes.     

Inhibitory effects of cytochrome P450 enzymes CYP2C8, CYP2C9, CYP2C19 and CYP3A4 by Labisia pumila extracts

Ethnopharmacological relevance

Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug–herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated.

Aim of the study

To evaluate the in vitro inhibitory effects of various LP extracts (aqueous, ethanol, dichloromethane (DCM) and hexane) on cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP2C19 and CYP3A4 activities.

Materials and methods

Probe substrate-based high performance liquid chromatography (HPLC) methods were established for CYP2C9, CYP2C19 and CYP3A4 whereas a fluorescence-based enzyme assay was established for CYP2C8. The metabolite formations were examined after incubation of probe substrate with respective CYP isoform in the present or absent of LP extracts. The inhibitory effect of LP was characterized with kinetic parameters IC₅₀ and K_i values.

Results

LP extracts showed differential effect of CYP activities with the order of inhibitory potency as follows: dichloromethane>hexane>ethanol>aqueous. This differential effect was only observed in CYP2C isoforms but not CYP3A4. Both the hexane and DCM extracts exhibited moderate to potent inhibition towards CYP2C activities in different modes including non-competitive, competive and mixed-type. The DCM effect was notably strong for CYP2C8 and CYP2C9 showing K_i values of below 1 μg/ml. The selectivity of LP for CYP2C isoforms rather than CYP3A4 may be attributed to the presence of relatively small, lipophilic yet slightly polar compounds within the LP extracts.

Conclusions

The results of our study revealed that phytoconstituents contained in LP, particularly in hexane and dichloromethane extracts, were able to selectively inhibit CYP2C isoforms. The inactivation was characterized by low K_i values, in particular, in CYP2C8 and CYP2C9. These in vitro data indicate that LB preparations contain constituents that can potently inhibit CYP2C activities and suggest that this herb should be examined for potential pharmacokinetic drug interactions in vivo.     

The effect of Cree traditional medicinal teas on the activity of human cytochrome P450-mediated metabolism

Ethnopharmacological relevance

Rhododendron groenlandicum (Bog Labrador tea), Rhododendron tomentosum (Marsh Labrador tea) and Juniperus communis (Juniper) are used in medicinal teas by Canadian aboriginal cultures alone and in combination with conventional drug products. The safety of this combination had not been previously examined and this study was initiated to examine the potential of medicinal teas to inhibit the major human drug metabolizing enzyme, cytochrome P450 3A4 (CYP3A4).

Materials and methods

The decoctions of Rhododendron groenlandicum and Rhododendron tomentosum leaves and Juniperus communis berries were examined in a microtiter fluorometric assay to examine their potential to inhibit CYP-mediated metabolism.

Results

The decoctions showed progressive inhibition towards CYP3A4 the longer the leaves or berries were brewed. R. Rhododendron groenlandicum and Juniperus communis may have the potential to inhibit CYP3A4-mediated metabolism.

Conclusions

The findings of this study with these traditional medicines are significant in that they provide mechanistic support that these products have the potential to affect the safety and efficacy of other health and medicinal products. As this study only examined CYP3A4, it is possible that these medicinals contain substances that could also affect other metabolic enzymes.     

In vitro antimicrobial activity of extracts and compounds of some selected medicinal plants from Cameroon

Aim of the study

Seven extracts and eight compounds from four selected Cameroonian medicinal plants, Solanecio mannii Hook f. (Asteraceae), Monodora myristica Dunal (Annonaceae), Albizia gummifera (J.F. Gmel) C.A. Smith (Fabaceae/Mimosoideae) and Glyphaea brevis (Spreng) Monachino (Tiliaceae), traditionally used for the treatment of hepatitis, parasites and other infectious diseases, were tested in vitro for their antimicrobial activity against Gram-positive (5 species) and Gram-negative (4 species) bacteria species and pathogenic yeasts (2 Candida species), to establish whether or not they have antimicrobial activity and to validate scientifically their use in traditional medicine.

Materials and methods

The agar disc diffusion and the microbroth dilution methods were used to determine the zone of inhibition between the edge of the filter paper and the edge of the inhibition area (IZ) and the minimal inhibitory concentration (MIC) respectively.

Results

The most active extracts against Candida albicans and Candida krusei were respectively the cyclohexane extract from the fruits of Monodora myristica and the ethyl acetate extract from the stem bark of Albizia gummifera (MIC = 6.3 μg/ml for both extracts). The lowest MIC value (1.6 μg/ml) for purified compounds was obtained on Candida albicans with a mixture of linear aliphatic primary alcohols (n-C24H50O to n-C30H62O), with n-hexacosanol (1b) as major compound and mixture of fatty acid esters of diunsaturated linear 1,2-diols (6).

Conclusion

These results afford ground informations for the potential use of the crude extracts of these species as well as of some of the isolated compounds in bacterial and fungal infections.     

In vitro estrogenic activity of Asplenium trichomanes L. extracts and isolated compounds

Ethnopharmacological relevance

Asplenium trichomanes was used as an expectorant, anti-cough remedy, laxative, emmenagogue, abortifacient and for irregular menses.

Aim of the study

To investigate the in vitro estrogenic activity of Asplenium trichomanes extracts and isolated compounds and their ability to activate ERα and ERβ.

Materials and methods

Leaves infusion (IF), decoction (DC) and methanol extract (ME) were prepared. MCF7/EREluc cell line which expresses endogenous ERα, and SK-NBE cells transiently transfected with the estrogen receptors (ERα and ERβ) were used for the estrogenic activity assays. Phytochemical investigations were performed (CC, HPLC, etc.) and structure of isolated compounds were achieved on the basis of 1D and 2D NMR techniques and HR-MS spectrometry.

Results

IF and ME were active in our MCF7 model; selectivity for the ERβ receptor was observed in the SK-NBE test. Two new phenol derivatives, 4-vinyl-phenol-1-O-[α-l-rhamno(1 → 6)-β-d-glucopyranosyde] (1) and kaempferol-3-O-α-[2′acetyl]-arabinofuranosyl-7-O-α-l-rhamnopyranoside (2) were isolated with six known compounds (3–8). Compounds 2–4, 7 and 8 showed selectivity for the activation of the ERβ receptor although with a moderate activity compared with 17-β-estradiol.

Conclusion

Further investigations about the estrogenic effects of this plant are needed but our data can, at least in part, explain some of its traditional use as emmeagogue.     

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes

Ethnopharmacological relevance

The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes.

Material and methods

A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression.

Results

The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Cl_int) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment.

Conclusion

We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.     

In vitro antibacterial effects of Cinnamomum extracts on common bacteria found in wound infections with emphasis on methicillin-resistant Staphylococcus aureus

Ethnopharmacological relevance

Cinnamomum species have been widely used in many traditional systems of medicine around the world. In the Malaysian traditional system of medicine, the leaves, stem bark and stem wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum have been used to treat wound infections. To study the antibacterial effects of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum against common bacteria found in wound infections with primary focus on methicillin-resistant Staphylococcus aureus (MRSA).

Materials and methods

The crude extracts from the leaves, stem-bark and stem-wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum were obtained using sequential extraction with hexane, ethylacetate, methanol and water. The volatile oils were obtained by hydro-distillation. The antibacterial activities of extracts were investigated using disk diffusion assays and broth microdilution assays.

Results

The volatile oils obtained from the stem-bark of Cinnamomum altissimum, Cinnamomum porrectum and Cinnamomum impressicostatum have shown significant antibacterial activity against a wide range of Gram positive and Gram negative bacteria including MRSA. A few test extracts have shown better activity against MRSA as compared to methicillin sensitive Staphylococcus aureus (MSSA). Amongst all the test extracts, Cinnamomum impressicostatum stem-bark water extract produced the largest inhibition zone of 21.0 mm against MRSA while its inhibition zone against MSSA was only 8.5 mm. The minimum inhibitory concentration (MIC) of this extract against MRSA was 19.5 μg mL⁻¹ and the corresponding minimum bactericidal concentration (MBC) was 39.0 μg mL⁻¹.

Conclusions

This study has scientifically validated the traditional use of Cinnamomum species in treating wound infections. Of high scientific interest was the observation that the antibacterial effect of Cinnamomum impressicostatum stem-bark crude water extract against MRSA was significantly higher than its effect against MSSA, suggesting that the extract contains a compound(s) with higher specific neutralising activity against the drug resistance markers of MRSA.     

In vitro antimicrobial, anthelmintic and cyclooxygenase-inhibitory activities and phytochemical analysis of Leucosidea sericea

Ethnopharmacological relevance

Leucosidea sericea is used as a vermifuge and in the treatment of ophthalmia by various tribes in southern African countries.

Aim of the study

The study aimed at screening leaves and stems of Leucosidea sericea for pharmacological activity and validating the plant's traditional use. A general phytochemical screening was also carried out.

Materials and methods

Petroleum ether (PE), dichloromethane (DCM), ethanol (EtOH) and water extracts of the plant parts were investigated for antimicrobial, anthelmintic and cyclooxygenase (COX) inhibitory activities. Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) and Candida albicans were used for the antimicrobial evaluation. Caenorhabditis elegans was used for the anthelmintic assay using the microdilution technique. Cyclooxygenase-1 and -2 (COX-1 and -2) were used to evaluate the anti-inflammatory potential of the plant extracts. Phytochemical analysis for phenolic compounds, including gallotannins, condensed tannins and flavonoids was done using 50% methanol extracts of the leaves and stems employing spectrophotometric methods.

Results

The leaf extracts exhibited broad spectrum antibacterial activity ranging from 0.025 to 6.25 mg/ml. The most noteworthy minimum inhibitory concentration (MIC) of 0.025 mg/ml was exhibited by PE and DCM leaf extracts against Bacillus subtilis and Staphylococcus aureus, respectively. In the anthelmintic assay, the best minimum lethal concentration (MLC) value of 0.26 mg/ml was observed for the DCM and EtOH leaf extracts. Both leaf and stem organic solvent extracts exhibited high to moderate inhibition against COX-1 and -2 at a screening concentration of 250 μg/ml. At lower concentrations, the extracts displayed a dose-dependent inhibition, with the lowest IC₅₀ values of 0.06 μg/ml (COX-1) and 12.66 μg/ml (COX-2) exhibited by the PE extract of the leaves. Generally, the leaf extracts exhibited better pharmacological activities and contained higher amounts of phenolic compounds than the stem extracts. Alkaloids and saponins were only detected in the leaf and stem extracts, respectively.

Conclusion

The reported results support the local use of Leucosidea sericea against eye infections and as a vermifuge. The pharmacological activities exhibited by the leaf extracts are probably due to their higher phenolic levels.     

Antimalarial and safety evaluation of extracts from Toddalia asiatica (L) Lam. (Rutaceae)

Ethnopharmacological relevance

Toddalia asiatica (L) Lam. (Rutaceae) is a medicinal plant traditionally used in Kenya by many communities for the treatment of malaria and other ailments. All parts of the plant are claimed to have medicinal value, but the root bark in particular is believed to be more potent. Decoctions or infusions of the roots are taken orally to treat malaria, fever and stomach ache.

Aim of the study

To evaluate antimalarial activity of aqueous and organic extracts prepared from Toddalia asiatica and determine in vitro and in vivo safety of the extracts.

Materials and methods

Aqueous, ethyl acetate, hexane and methanol extracts were obtained from Toddalia asiatica root bark, fruits and leaves. In vitro antiplasmodial activity was done using chloroquine-sensitive (D6) and chloroquine-resistant (W2) Plasmodium falciparum strains and the concentration causing 50% inhibition of radioisotope incorporation (IC₅₀) was determined. In vivo assay was done by administering mice infected with Plasmodium berghei four consecutive daily doses of the extracts through oral route following Peters 4-Day suppressive test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Quinine hydrochloride was used as positive control while double distilled water or 20% Tween-80 was used as a negative control. In vivo acute toxicity was determined in mice using standard procedures. In vitro cytotoxicity assay was carried out using actively dividing sub-confluent Vero cells.

Results

Inhibitory concentrations of ethyl acetate extract of Toddalia asiatica fruits showed high activity against chloroquine resistant (W2) strains of Plasmodium falciparum (IC₅₀=1.87 μg/ml), followed by root bark aqueous extract (IC₅₀=2.43 μg/ml). Tested in vivo against Plasmodium berghei, the fruit ethyl acetate extract (500 mg/kg) and root bark aqueous extract (250 mg/kg) reduced malaria parasitaemia by 81.34% and 56.8% respectively. Higher doses were found to be less effective in vivo. Acute toxicity and cytotoxictiy of the tested extracts, with the exception of hexane extract from the roots, showed LD₅₀>1000 mg/kg and CC₅₀>100 μg/ml respectively.

Conclusions

The results obtained contribute to the validation of traditional use of Toddalia asiatica and provides in vivo and safety data of the plant extracts tested for the first time. Ethyl acetate extract of the fruits was active against chloroquine resistant Plasmodium falciparum as well as against Plasmodium berghei. These findings confirm the suitability of Toddalia asiatica as a good candidate for further tests to obtain a prototype for antimalarial medicine.     

Actions of ethnobotanically selected Cree anti-diabetic plants on human cytochrome P450 isoforms and flavin-containing monooxygenase 3

Aim of the study

Cree traditional medicine is commonly used concomitantly with prescribed drugs to treat health problems related to type II diabetes (T2D) that is endemic in the Cree population. However, the safety of traditional Cree medicines with respect to drug metabolism is unknown.

Materials and methods

Seventeen anti-diabetic plant extracts were screened for their potential inhibition of 11 isoforms of the drug-metabolizing cytochrome P450s (CYPs), and flavin-containing monooxygenase 3 (FMO3) in fluorometric plate reader assays. Comparative analyses were conducted to determine if particular extracts were more inhibitory, or if particular enzymes were more inhibited.

Results

Many anti-diabetic plant extracts inhibited the CYPs, with CYP2C and 3A isoforms being most prone to inhibition. The order of inhibition for the enzymes by the Cree plant extracts was: 2C19 > 3A7 > 3A5 > 3A4 > 2C9 > 2C8 > FMO3 > 1A2 > 2E1 > 19 > 2D6 > 2B6. Extracts from Rhododendron groenlandicum, Sorbus decora, and Kalmia angustifolia were identified as having strong inhibition towards many CYP isoforms.

Conclusion

These findings demonstrate that extracts from most plant species examined have the potential to affect CYP2C- and 3A4-mediated metabolism, and have the potential to affect the bioavailability and pharmacokinetics of conventional and traditional medicines during concomitant use.     

Assessment of extracts from mistletoe (Viscum album) for herb-drug interaction by inhibition and induction of cytochrome P450 activities

Three commercially available extracts from mistletoe (Viscum album L.) grown on ash tree (abnobaVISCUM^® Fraxini 20 mg), on fir (abnobaVISCUM^® Abietis 20 mg), and on pine (abnobaVISCUM^® Pini 20 mg) were tested in vitro for their potential to interfere with the major drug metabolizing cytochromes P450 by hepatocyte viability, by inhibition of cytochromes P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, and by the induction of cytochromes P450 1A2, 2B6, 2C9, 2E1 and 3A4. As the three extracts are produced from mistletoe plants belonging to three different subspecies of Viscum album L. they have explicit differences in the content and spectrum of various active ingredients, e.g. mistletoe specific lectins. Cytotoxic effects on liver cells were observed for abnobaVISCUM^® Fraxini with a high lectin content with an EC₅₀ value of 2.56 µg/mL, for abnobaVISCUM^® Abietis with a moderate lectin content with an EC₅₀ value of 5.79 µg/mL and for abnobaVISCUM^® Pini with a low lectin content with an EC₅₀ value of 30.86 µg/mL. The induction of cytochromes P450 was tested on human liver cells from three donors. Inhibition of cytochromes P450 was carried out on human liver microsomes. No or minor induction and inhibition was observed for all three extracts. The data indicate no or minor potential for herb–drug interactions by interference with cytochromes P450 by any of the three mistletoe extracts. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts and fractions from the leaves,root-bark and stem-bark of Triclisia gilletii

Ethnopharmacological Relevance

To evaluate the in vitro antiplasmodial activity and cytotoxicity, and the in vivo activity of extracts and fractions from the leaves, root-bark and stem-bark of Triclisia gilletii (De Wild) Staner (Menispermaceae), used in traditional medicine against malaria.

Materials and Methods

The aqueous and 80% MeOH extracts, and a series of fractions and subfractions from the leaves, stem and root-bark of Triclisia gilletii were tested in vitro for their antiplasmodial activity against a Congolese-sensitive strain of Plasmodium falciparum, against the chloroquine and pyrimethamine-resistant K1 strain of Plasmodium falciparum, for cytotoxicity against MRC-5 cells, and in vivo in mice infected with Plasmodium berghei berghei.

Results

Many samples from the three plant parts exhibited pronounced activity against the Congolese chloroquine-sensitive strain of Plasmodium falciparum with some IC₅₀ values <0.02 µg/ml, and against the K1 strain, with some IC₅₀ <0.25; the selectivity was higher against the Congolese strain. At oral doses of 200 and 400 mg/kg body weight in infected mice, the aqueous, 80% methanol and total alkaloid extracts from the three plant parts produced more than 65% and 75% chemosuppression, respectively. The antiplasmodial activity of these three plant parts of Triclisia gilletii can at least in part be attributed to bisbenzylisoquinoline alkaloids, and supports its use for the treatment of uncomplicated malaria in traditional medicine.     

The effect of Salvia divinorum and Mitragyna speciosa extracts,fraction and major constituents on place aversion and place preference in rats

Ethnopharmacological relevance

Consumer use of botanicals has increased despite, in many instances, the paucity of research demonstrating efficacy or identifying liabilities. This research employed the place preference/aversion paradigm to characterize the psychoactive properties of Salvia divinorum extract (10, 30, 100 mg/kg), salvinorin A (0.1, 0.3, 1.0 mg/kg), Mitragyna speciosa MeOH extract (50, 100, 300 mg/kg), Mitragyna speciosa alkaloid-enriched fraction (12.5, 25, 75 mg/kg) and mitragynine (5, 10, 30 mg/kg) in rats.

Material and methods

Following apparatus habituation and baseline preference scores, male Sprague-Dawley rats were given eight counter-balanced drug versus vehicle conditioning trials followed by a preference test conducted under drug-free states. S(+)-amphetamine (1 mg/kg) served as the positive control (in Exp. 2) and haloperidol (0.8, 1.0 mg/kg) served as the negative control in both studies.

Results

Rats displayed place aversion to both Salvia divinorum and salvinorin A that exceeded that of haloperidol. Rats showed place preference to mitragynine that was similar to that of S(+)-amphetamine. This CPP effect was much less pronounced with the Mitragyna speciosa extract and its fraction.

Conclusions

These findings suggest that both botanicals possess liabilities, albeit somewhat different, that warrant caution in their use.     

In vitro effects of Sutherlandia frutescens water extracts on cell numbers,morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2–10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P < 0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G₁ apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.