Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells

Sutherlandia frutescens is a well-known South African herbal remedy traditionally used for stomach problems, internal cancers, diabetes, various inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The influence of crude Sutherlandia frutescens extracts (prepared with 70% ethanol) was investigated on cell numbers, morphology, and gene expression profiles in a MCF-7 human breast adenocarcinoma cell line. Time-dependent (24, 34, 48 and 72 h) and dose-dependent (0.5-2.5 mg/ml) studies were conducted utilizing spectrophotometrical analysis with crystal violet as DNA stain. A statistically significant decrease to 50% of malignant cell numbers was observed after 24 h of exposure to 1.5 mg/ml Sutherlandia frutescens extract when compared to vehicle-treated controls. Morphological characteristics of apoptosis including cytoplasmic shrinking, membrane blebbing and apoptotic bodies were observed after 24h of exposure. A preliminary global gene expression profile was obtained by means of microarray analysis and revealed valuable information about the molecular mechanisms and signal transduction associated with 70% ethanolic Sutherlandia frutescens extracts.     

The apoptosis inducing effects of Sutherlandia spp. extracts on an oesophageal cancer cell line

Aim of study

Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the “cancer bush”, is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line.

Materials and methods

Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs).

Results

Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5 mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs.

Conclusions

S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents.     

Differential signaling involved in Sutherlandia frutescens-induced cell death in MCF-7 and MCF-12A cells

Ethnopharmacological relevance

The scientific study of natural products traditionally used in anticancer preparations has yielded several therapeutically relevant compounds. One of these traditional preparations with potentially beneficial properties is aqueous extracts of Sutherlandia frutescens, a shrub indigenous to the Western Cape region of South Africa. The aims of this study were to evaluate in vitro efficacy of these preparations on the MCF-7 breast adenocarcinoma and MCF-12A non-tumorigenic cell lines in terms of cell proliferation, cell morphology and possible induction of cell death.

Materials and methods

Crystal violet staining was used to evaluate cell proliferation, light-and fluorescence microscopy were used to investigate both intracellular and extracellular morphological features of apoptosis and autophagy (e.g. membrane blebbing, condensed chromatin and intracellular lysosomes), while flow cytometry quantified cell cycle changes and induction of apoptosis through analysis of the flip-flop translocation of phosphatidylserine.

Results

Crystal violet staining showed a time- and dose specific response to aqueous Sutherlandia frutescens extracts, revealing exposure to 1 mg/ml aqueous extract for 48 h to be ideal for comparing the differential effects of Sutherlandia frutescens in the MCF-7 and MCF-12A cell lines. Microscopy showed distinct morphological changes with hallmarks of apoptosis being observed in both cell lines. Flow cytometry revealed a decrease in actively cycling cells in both cell lines, and a 4.36% increase in phosphatidylserine translocation in the MCF-7 cell line, indicative of apoptosis induction, while fluorescence microscopy showed evidence of the induction of autophagy.

Conclusions

Analyses revealed the carcinogenic MCF-7 cell line to be more susceptible to the cytostatic and cytotoxic effects of aqueous extracts of Sutherlandia frutescens when compared to the non-tumorigenic MCF-12A cell line, thus warranting further research into the exact cellular mechanisms involved and the possible synergistic activities of Sutherlandia frutescens ingredients.     

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.     

黄芩苷对非白念珠菌生物膜抑制作用的研究

目的:研究黄芩苷对光滑念珠菌、近平滑念珠菌、克柔念珠菌、季也蒙念珠菌等非白念珠菌生物膜的影响.方法:96孔微量培养板上构建4种非白念珠菌生物膜;微量稀释法检测黄芩苷对4种非白念珠菌最低抑菌浓度(MIC);XTT减低法评价黄芩苷对4种非白念珠菌生物膜SMIC及菌细胞黏附性的影响.结果:4种非白念珠菌在96孔板中可形成成熟的生物膜;黄芩苷对4种非白念珠菌MIC分别是125,250,125,62.5 mg·L~(-1);黄芩苷对4种非白念珠菌生物膜的SMIC50分别是＞1 000,500,125,250 mg·L~(-1);SMIC80均大于或等于1 000 mg·L~(-1).黄芩苷对4种非白念珠菌的黏附有一定的抑制效应.结论:黄芩苷对近平滑念珠菌、克柔念珠菌、季也蒙念珠菌生物膜有较强的抑制作用.     

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.     

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 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.     

巫山淫羊藿离体胚培养的研究

目的:研究药用植物巫山淫羊藿组织培养技术,为工厂化育苗提供科学根据。方法:以巫山淫羊藿的种胚为外植体,采用MS培养基,附加不同浓度2,4-D,6-BA,IBA,NAA进行正交试验。结果:诱导愈伤组织的最优培养基为:MS+2,4-D2 mg.L-1+IBA 2 mg.L-1+NAA 0.5 mg.L-1;愈伤组织分化的最优培养基为:MS+6-BA 1 mg.L-1+NAA 0.5 mg.L-1+IBA 1 mg.L-1;诱导芽的最优培养基为:MS+IBA 2 mg.L-1+6-BA 0.5 mg.L-1;芽增殖的最佳培养基为:MS+6-BA 1.0 mg.L-1+NAA 0.5 mg.L-1。结论:通过诱导愈伤组织途径和丛生芽途径,建立了巫山淫羊藿种胚外植体诱导和培养方法,达到快速繁殖的目的。     

In vitro and in vivo antimalarial and cytotoxic activity of five plants used in congolese traditional medicine

Aim of the study

The in vitro antiplasmodial activity and cytotoxicity of methanolic and dichloromethane extracts from five Congolese plants were evaluated. The plants were selected following an ethnobotanical survey conducted in D.R. Congo and focusing on plants used traditionally to treat malaria. The in vivo antimalarial activity of aqueous and methanolic extracts active in vitro was also determined in mice infected by Plasmodium berghei berghei.

Materials and methods

The growth inhibition of Plasmodium falciparum strains was evaluated using the measurement of lactate dehydrogenase activity. The extracts (aqueous, CH₃OH, EtOH and CH₂Cl₂) were prepared by maceration and tested in vitro against the 3D7 (chloroquine sensitive) and W2 (chloroquine resistant) strains of Plasmodium falciparum and against the human normal fetal lung fibroblasts WI-38 to determine the selectivity index. Some extracts were also used at the dose of 300 mg/kg to evaluate their activity in mice infected since 4 days by Plasmodium berghei.

Results

Two plants presented a very high activity (IC₅₀ < 3 μg/ml). These plants were Strychnos icaja roots bark (MeOH and CH₂Cl₂) and Physalis angulata leaves (MeOH and CH₂Cl₂). One plant (Anisopappus chinensis whole plant, MeOH and CH₂Cl₂) presented a high activity (IC50 < 15 μg/ml). The extracts of Anisopappus chinensis and Physalis angulata showed also a good inhibition of parasitemia in vivo. Flavonoids, phenolic acids and terpenes were identified in these plants by a general phytochemical screening method.

Conclusion

Three plants showed a very interesting antiplasmodial activity (Anisopappus chinensis, Physalis angulata and Strychnos icaja) and one of them showed a good selectivity index (>10, Anisopappus chinensis). Anisopappus chinensis and Physalis angulata were also active in vivo.     

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana

Ethnopharmacological relevance

Tridax procumbens is an active herb against leishmaniasis.

Aim of the study

Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World.

Materials and methods

Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT.

Results

We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC₅₀) of 3 μg/ml, while oxylipin 1 exhibited the highest inhibition at IC₅₀ = 0.478 μg/ml.

Conclusions

In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.     

In vitro antigonococcal activity of Coscinium fenestratum stem extract

Ethnopharmacological relevance

Gonorrhea is a sexually transmitted disease (STD), which originates from bacteria, Neisseria gonorrhoeae. It is still one of the major health problems worldwide in both men and women. Many medicinal plants have been recorded in Thai folk medicine for relieving STD but there is no scientific report of these plants for antigonococcal activity.

Aim of the study

This study was conducted to evaluate antigonococcal activity of 22 Thai medicinal plants selected from the plants popularly used in Thai folk medicine for treatment of gonorrhea.

Materials and methods

This study was performed by disc diffusion and agar dilution methods against Neisseria gonorrhoeae. Active compound was investigated by bioautographic assay.

Results

Among the selected plants, Coscinium fenestratum (Gaertn.) Colebr. extract showed the most effective activity against Neisseria gonorrhoeae ATCC 49226 with MIC value of 47.39 μg/ml. Bioautographic assay revealed that berberine was the active compound of Coscinium fenestratum against Neisseria gonorrhoeae. The average MIC values of purified berberine against Neisseria gonorrhoeae ATCC 49226 and 11 clinical isolates were 13.51 and 17.66 μg/ml, respectively while average MIC value of the crude extract of Coscinium fenestratum against all clinical isolates was about 56.39 μg/ml. There was no acute toxicity detected at the dose of 5 g of Coscinium fenestratum crude extract per kilogram.

Conclusions

These results provide theoretical support for ethnopharmacological relevance of antigonococcal activity of Coscinium fenestratum and its active compound.     

In vitro and in vivo safety evaluation of Acer tegmentosum

Ethnopharmacological relevance

Acer tegmentosum, which contains salidroside and tyrosol, has been used for the treatment of hepatic disorders in eastern Asia. However, little is known about its safety.

Aim of the study

To determine the safety of Acer tegmentosum, we evaluated its acute oral toxicity and genotoxicity profiles.

Materials and methods

Salidroside and tyrosol present in Acer tegmentosum were quantified using high-performance liquid chromatography. Acute oral toxicity testing of Acer tegmentosum was performed in rats. Genotoxicity of Acer tegmentosum was assessed by bacterial reverse mutation, chromosomal aberration, and bone marrow micronucleus tests. All the tests were conducted in accordance with the good laboratory practices.

Results

The amounts of salidroside and tyrosol in Acer tegmentosum were found to be 85.01±1.21 mg/g and 3.12±0.04 mg/g, respectively. In the bacterial reverse mutation test, Acer tegmentosum increased the number of revertant Salmonella typhimurium TA98 colonies, regardless of metabolic activation by S9 mixture. In contrast, Acer tegmentosum application did not significantly increase the number of chromosomal aberrations in Chinese hamster ovary (CHO)-K1 cells and micronucleated polychromatic erythrocytes in mice. In the acute oral toxicity test, the median lethal dose (LD₅₀) of Acer tegmentosum was found to be >2000 mg/kg in rats.

Conclusion

Take together, Acer tegmentosum exhibits mutagenicity, which was evident from the bacterial reverse mutation test. Further studies are needed to identify the components responsible for such an effect and the underlying mechanisms.     

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.     

In vitro cytotoxic activity of nine plants used in Mayan traditional medicine

Ethnopharmacological relevance

Plants have been used in folk medicine by Mayan ancient people from the Yucatan Peninsula, Mexico, to treat some diseases considered as cancer diseases such as chronic wounds or tumors.

Aim of the study

We collected a selection of nine plants in order to investigate their cytotoxic activity against cancer cell lines.

Materials and methods

Methanolic extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on four cancer cell lines; nasopharynx carcinoma (KB), laryngeal carcinoma (Hep-2), cervix adenocarcinoma (HeLa), and cervix squamous carcinoma cells (SiHa) and one normal cell line; canine kidney (MDCK).

Results

All species exhibited some degree of cytotoxic activity. The root bark extract of Hamelia patens exhibited the highest cytotoxic activity on HeLa cells with a CC₅₀ of 13 μg/mL and selectivity index of 13.3, higher than docetaxel. Gossypium schottii and Dioon spinulosum showed similar good cytotoxic activity and selectivity index on HeLa and Hep-2 cells, respectively.

Conclusions

Hamelia patens, Dioon spinulosum and Gossypium schottii demonstrated promising cytotoxic activity and have been selected for future bio-guided fractionation and isolation of active cytotoxic compounds.     

In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica

Ethnopharmacological relevance

Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure.

Aim of the study

The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms.

Materials and methods

High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6β-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC₅₀ and K_i, to characterize modulatory effects.

Results

CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K_i = 39.1 μg/ml) and dichloromethane (K_i = 26.6 μg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC₅₀'s of 123.3 μg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC₅₀'s of 117.9 μg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K_i = 9.1 μg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K_i's ranged from 17.2 to 84.4 μg/ml). On the other hand, the IC₅₀ values for asiaticoside were high (1070.2 μg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity.

Conclusion

Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug–herb interactions through CYP2C9 inhibition.     

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.     

In vitro antiplasmodial activity of plants used in Benin in traditional medicine to treat malaria

Aim of the study

The aim of the study was to evaluate the in vitro antiplasmodial activity of crude extracts of 12 plant species traditionally used in Benin for the treatment of malaria in order to validate their use.

Materials and methods

For each species, dichloromethane, methanol and total aqueous extracts were tested. The antiplasmodial activity of extracts was evaluated using the measurement of the plasmodial lactate dehydrogenase activity on chloroquine-sensitive (3D7) and resistant (W2) strains of Plasmodium falciparum. The selectivity of the different extracts was evaluated using the MTT test on J774 macrophage-like murine cells and WI38 human normal fibroblasts.

Results

The best growth inhibition of both strains of Plasmodium falciparum was observed with the dichloromethane extracts of Acanthospermum hispidum DC. (Asteraceae) (IC₅₀ = 7.5 μg/ml on 3D7 and 4.8 μg/ml on W2), Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (Rubiaceae) leaves and twigs (IC₅₀ = 13.8 and 11.3 μg/ml on 3D7 and IC₅₀ = 26.5 and 15.8 μg/ml on W2, respectively), Carpolobia lutea G.Don. (Polygalaceae) (IC₅₀ = 19.4 μg/ml on 3D7 and 8.1 μg/ml on W2) and Strychnos spinosa Lam. (Loganiaceae) leaves (IC₅₀ = 15.6 μg/ml on 3D7 and 8.9 μg/ml on W2). All these extracts had a low cytotoxicity.

Conclusion

Our study gives some justifications for the traditional uses of some investigated plants.     

In vitro genotoxicity of Melaleuca alternifolia essential oil in human lymphocytes

Ethnopharmacological relevance

The volatile essential oil derived from the plant Melaleuca alternifolia, also called tea tree oil (TTO), is largely employed for its antimicrobial properties against several human pathogens. It is used in many topical formulations to treat cutaneous infections.

Aim of the study

Since very few studies have been done on the safety and toxicity of the crude Melaleuca alternifolia essential oil, current investigation evaluates the possible genotoxic effects of TTO in human lymphocyte cultures.

Material and methods

The composition of current TTO sample was determined by GC/MS and NMR. The level of cytotoxicity in TTO treated cultures was determined by decrease of mitotic index when compared to that in negative control. The genotoxic potential of TTO was assessed by the in vitro mammalian cell micronucleus and the chromosome aberrations (CA) tests.

Results

Twenty-seven compounds were identified, accounting for 98.9% of the constituents. Terpinen-4-ol (42.8%), γ-terpinene (20.4%), p-cymene (9.6%), α-terpinene (7.9%), 1,8-cineole (3%), α-terpineol (2.8%) and α-pinene (2.4%) were the major compounds of the oil sample. None of the tested TTO concentrations (95 μg/ml, 182 μg/ml and 365 μg/ml) caused a significant increase in the observed frequencies of micronuclei when compared to those in the untreated cultures (negative control). Additionally, no significant differences regarding the frequencies of CA were observed among the tested TTO concentrations and the negative control.

Conclusions

Results demonstrate that TTO, in the tested concentrations, is not genotoxic in in vitro mammalian cells.