In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract

Ethnopharmacological relevance

The aim of the study was to evaluate the in vitro antitrypanosomal and antileishmanial activity of crude extracts of 10 plant species traditionally used in Benin to treat parasitic infections.

Materials and methods

For each species, dichloromethane, methanol and aqueous extracts were tested. Their antitrypanosomal and antileishmanial activities were evaluated in vitro on Trypanosoma brucei brucei (strain 427) (Tbb) and on promastigotes of Leishmania mexicana mexicana (MHOM/BZ/84/BEL46) (Lmm).

Results

The best growth inhibition was observed with the dichloromethane extracts of aerial parts of Acanthospermum hispidum DC. (Asteraceae) (IC₅₀ = 14.5 μg/ml on Tbb and 11.1 μg/ml on Lmm), twigs of Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (IC₅₀ = 5.8 μg/ml on Tbb), aerial parts of Byrsocarpus coccineus Schumach. & Thonn (syn. Rourea coccinea (Schumach. & Thonn.) Hook.f.) (IC₅₀ = 14.7 μg/ml on Tbb) and aerial parts of Carpolobia lutea G.Don. (IC₅₀ = 18.3 μg/ml on Tbb). All these extracts had a low cytotoxicity. It is not the case for the methanolic and water extracts of roots of Anchomanes difformis (Blume) Engl. (IC₅₀ = 14.7 and 13.8 μg/ml on Tbb) which were toxic at the same concentration range on WI38, human cells. A bio-guided fractionation of the most active extract of Keetia leucantha allowed to identify oleanolic acid and ursolic acid as responsible for the observed activities.

Conclusion

Our study gives some justification for antiparasitic activity of some investigated plants.     

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.     

Xanthine oxidase inhibitory properties of Czech medicinal plants

Aim of the study

To investigate in vitro xanthine oxidase inhibitory properties of plants traditionally used in Czech Republic and Central-East Europe region for gout, arthritis or rheumatism treatment.

Materials and methods

Methylene chloride-methanolic and two ethanolic extracts of 27 plant species were screened for in vitro xanthine oxidase inhibitory activity using a spectrophotometric method.

Results

Around 50% of the species exhibited some degree of xanthine oxidase inhibitory properties at 200 μg/mL, showing a moderate correlation (r = 0.59) with total phenol content. The most active were methylene chloride-methanolic extracts of Populus nigra and Betula pendula, with IC₅₀ of 8.3 and 25.9 μg/mL, respectively, followed by 80% ethanolic extract of Caryophyllus aromaticus and Hypericum perforatum, both under 50 μg/mL.

Conclusions

Populus nigra and Betula pendula were identified as species with the highest xanthine oxidase inhibitory potential in our study. This correlates with the ethnobotanical data on their use in Central European folklore and provides the basis for further investigation on these plants.     

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaça as extractor liquid

Ethnopharmacological importance

Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated.

Aim of the study

Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaça as the extractor liquid.

Materials and methods

The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes.

Results

The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 μg/ml against S. aureus, and was inactive against Gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC₅₀ = 175 μg/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC₅₀ between 31 and 470 μg/ml, and the lyophilized Brazilian cachaça showed a CC₅₀ of 307 μg/ml. None of the extracts showed toxicity against human erythrocytes.

Conclusions

Among the plant species studied, C. americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases.     

Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolic-rich extract of Elephantopus mollis Kunth

Ethnopharmacological relevance

The decoction of the whole plant of Elephantopus mollis Kunth. is traditionally consumed to treat various free radical-mediated diseases including cancer and diabetes.

Aim of the study

This study was initiated to determine whether the most effective antioxidant compound isolated from the whole plant of Elephantopus mollis can also contribute to its claimed traditional values as anticancer and antidiabetes agents.

Materials and methods

An active antiradical phenolic compound (3,4-di-O-caffeoyl quinic acid) was isolated from the methanol extract (with the highest in polyphenolic content) and their antioxidant activities were compared using four different assays, that are DPPH, FRAP, metal chelating, and β-carotene bleaching tests. The compound was also evaluated for its cytotoxic activity, apoptotic induction and anti-glucosidase efficacies using methylene blue, DeadEnd™ assay and α-glucosidase assays, respectively.

Results

The compound acted as a greater primary antioxidant than its methanol extract, by having higher ferric reducing activity (EC₅₀ 2.18 ± 0.05 μg/ml), β-carotene bleaching activity (EC₅₀ 23.85 ± 0.65 μg/ml) and DPPH scavenging activity (EC₅₀ 68.91 ± 5.44 μg/ml), whereas the methanol extract exhibited higher secondary antioxidant activity as a metal chelator with lower EC₅₀ value (49.39 ± 3.68 μg/ml) than the compound. Cytotoxicity screening of this compound exhibited a remarkable dose-dependent inhibitory effect on NCI-H23 (human lung adenocarcinoma) cell lines (EC₅₀ 3.26 ± 0.35 μg/ml) and was found to be apoptotic in nature based on a clear indication of DNA fragmentation. This compound also displayed a concentration-dependent α-glucosidase inhibition with EC₅₀ 241.80 ± 14.29 μg/ml.

Conclusions

The findings indicate the major role of 3,4-di-O-caffeoyl quinic acid to antioxidant capacities of Elephantopus mollis extracts. The compound also exerted apoptosis-mediated cytotoxicity and α-glucosidase inhibitory effects and is thus a promising non toxic agent in treating cancer and type 2 diabetes mellitus.     

Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids

Ethnopharmacological relevance

The South African plant Sceletium tortuosum has been known for centuries for a variety of traditional uses, and, more recently, as a possible source of anti-anxiety or anti-depressant effects. A standardised extract Zembrin^® was used to test for pharmacological activities that might be relevant to the ethnopharmacological uses, and three of the main alkaloids were also tested.

Materials and methods

A standardised ethanolic extract was prepared from dried plant material, along with the purified alkaloids mesembrine, mesembrenone and mesembrenol. These were tested on a panel of receptors, enzymes and other drug targets, and for cytotoxic effects on mammalian cells.

Results

The extract was a potent blocker in 5-HT transporter binding assays (IC₅₀ 4.3 μg/ml) and had powerful inhibitory effects on phosphodiesterase 4 (PDE4) (IC₅₀ 8.5 μg/ml), but not other phosphodiesterases. There were no cytotoxic effects. Mesembrine was the most active alkaloid against the 5-HT transporter (K_i 1.4 nM), while mesembrenone was active against the 5-HT transporter and PDE4 (IC₅₀'s < 1 μM).

Conclusions

The activity of the Sceletium tortuosum extract on the 5-HT transporter and PDE4 may explain the clinical effects of preparations made from this plant. The activities relate to the presence of alkaloids, particularly mesembrine and mesembrenone.     

In vitro antiplasmodial activity and cytotoxicity of nine plants traditionally used in Gabon

Aim of the study

As part of a project to identify new compounds active on malarial parasites, we tested the in vitro antiplasmodial activity of nine plants traditionally used to treat malaria symptoms in Haut-Ogooué Province, South-East Gabon.

Materials and methods

Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activity on two chloroquine-resistant strains of Plasmodium falciparum (FCB and W2), based on lactate dehydrogenase activity. Cytotoxicity was assessed with the MTT test on MRC-5 human diploid embryonic lung cells.

Results

The methanolic extract of Staudtia gabonensis and the dichloromethane extract of Adhatoda latibracteata showed high antiplasmodial activity (IC₅₀ < 1 μg/ml) and low cytotoxicity, with selectivity indexes of about 58.25 and 16.43, respectively. The methanolic extract of Monodora myristica and the dichloromethane extract of Afromomum giganteum also showed promising activity (1 < IC₅₀ < 10 μg/ml) and low cytotoxicity, with selectivity indexes about 15.70 and 12.48, respectively. Dichloromethane extracts of Monodora myristica and Leonotis Africana showed moderate activity (10 < IC₅₀ < 40 μg/ml), with selectivity indexes about 6.07 and 28.89, respectively. Both extracts of Culcasia lancifolia had IC₅₀ values of 10-40 μg/ml but high cytotoxicity (selectivity indexes <2.77). The methanolic extract of Dorstenia klaineana had moderate antiplasmodial activity (IC₅₀ around 17 μg/ml) but strong cytotoxicity (0.43 μg/ml), giving a selectivity index of about 0.03.

Conclusions

Most extracts of nine selected plants traditionally used to treat malaria in Gabon had interesting antiplasmodial activity in vitro. This supports continued investigations of traditional medicines in the search for new antimalarial agents. The compounds responsible for the observed antiplasmodial effects are under investigation.     

Anti-inflammatory, antioxidant, anti-tyrosinase and phenolic contents of four Podocarpus species used in traditional medicine in South Africa

Ethnopharmacological relevance

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest complaints, arthritis, rheumatism, venereal diseases and distemper in dogs.

Aims of the study

To investigate the antioxidant, anti-inflammatory and anti-tyrosinase activities of four Podocarpus species, Podocarpus elongatus, Podocarpus falcatus, Podocarpus henkelii and Podocarpus latifolius, used in traditional medicine in South Africa. Phytochemical analysis to determine the phenolic contents was also carried out.

Materials and methods

DPPH, FRAP and β-carotene-linoleic acid assays were used to determine the antioxidant/radical scavenging activities of these species. Anti-inflammatory activity of these species was assayed against two cyclooxygenase enzymes (COX-1 and COX-2). Tyrosinase inhibition activity was analysed using the modified dopachrome method with l-DOPA as the substrate. Phenolics were quantitatively determined using spectrophotometric methods.

Results

Stems of Podocarpus latifolius exhibited the lowest EC₅₀ (0.84 μg/ml) inhibition against DPPH. The percentage antioxidant activity based on the bleaching rate of β-carotene ranged from 96% to 99%. High ferric reducing power was observed in all the extracts. For COX-1, the lowest EC₅₀ value was exhibited by stem extracts of Podocarpus elongatus (5.02 μg/ml) and leaf extract of Podocarpus latifolius showed the lowest EC₅₀ against COX-2 (5.13 μg/ml). All extracts inhibited tyrosinase activity in a dose-dependent manner with stem extract of Podocarpus elongatus being the most potent with an EC₅₀ value of 0.14 mg/ml. The total phenolic content ranged from 2.38 to 6.94 mg of GAE/g dry sample.

Conclusion

The significant pharmacological activities observed support the use of these species in traditional medicine and may also be candidates in the search for modern pharmaceuticals in medicine, food and cosmetic industries.     

Antiprotozoal activities of traditional medicinal plants from the Garhwal region of North West Himalaya, India

Ethnopharmacological relevance

In a search for new plant-derived biologically active compounds against protozoan parasites, an ethnopharmacological study was carried out to evaluate extracts from selected 17 traditional medicinal plants which were used by healers from the Garhwal region of North West Himalaya for the treatment of protozoal infections and fever including malaria.

Materials and methods

In vitro activity against erythrocytic stages of Plasmodium falciparum was determined using a modified [3H]-hypoxanthine incorporation assay with the chloroquine- and pyrimethamine-resistant K1 strain. Activity against Trypanosoma brucei rhodesiense was performed on the STIB 900 strain and activity against Trypanosoma cruzi on infected rat skeletal myoblasts (L6 cells) seeded in 96-well microtitre plates while amastigotes of Leishmania donovani strain MHOM/ET/67/L82 were used to assess activity against Leishmania donovani. Cytotoxicity assays were performed against rat skeletal myoblasts (L6-cells).

Results and conclusions

Extracts of Artemisia roxburghiana, Roylea cinerea, Leucas cephalotes, Nepeta hindostana and Viola canescens showed good antiplasmodial activity (IC50 < 5 μg/ml). The chloroform extract of Artemisia roxburghiana was the most active (IC50 value of 0.42 μg/ml) and the most selective (SI = 78) extract for Plasmodium falciparum among all plants extracts examined. The chloroform extract of Leucas cephalotes and the petroleum ether extract of Viola canescens exhibited substantial activities against Leishmania donovani with IC50 values of 3.61 μg/ml (SI = 8) and 0.40 μg/ml (SI = 30), respectively. The petroleum ether extract of Viola canescens exhibited activity against Trypanosoma cruzi with an IC50 value of 1.86 μg/ml (SI = 7). Methanol and water extracts from all plants under investigation were found inactive against all parasites tested. These results support investigation of components of traditional medicines as potential new antiprotozoal agents. On the other hand since herbalism has become the main stream throughout the world, investigation demonstrates that these non-polar plant extracts of six of the plants examined in this study could play an important role in herbal formulations for the treatment of vector borne protozoal diseases.     

Ethnopharmacological survey and in vitro evaluation of wound-healing plants used in South-western Nigeria

Ethnopharmacological relevance

Traditional healers in Nigeria employ a range of plant preparations as wound healing agents. Despite the use of local plants in wound healing, there is only scant literature on the wound healing properties of these plants to support the continued therapeutic application of these herbal remedies.

Aim of the study

To document plants commonly used to treat wounds in South-western Nigeria and to test the scientific basis of such claims using relevant in vitro tests.

Materials and methods

Structured questionnaires were used to determine which plant preparations are in common use, via interviews with Yoruba traditional healers. Aqueous and ethanolic extracts of the nine most common plants cited by the healers were collected, identified and tested using relevant in vitro wound healing assays. Minimum inhibitory concentrations (MIC) were determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Antioxidant activity was measured by DPPH assay and fibroblast proliferation determined by neutral red assay.

Results

A total of 20 traditional healers from South-western Nigeria were involved in the study. Thirty-six plant species were recorded with their local names and parts used in the traditional wound healing preparations. Ethanolic extracts of nine species most frequently cited by the healers exhibited strong antioxidant activities (3.8-31.3 μg/ml) comparable to ascorbic acid (7.3 μg/ml). Crude extracts of the selected plants also inhibited the growth of bacteria with MIC values 0.3-7.6 mg/ml. Ethanol extracts of Bridelia ferruginea Benth. (1-30 μg/ml) and Parkia biglobosa Jacq. (15-30 μg/ml) influenced the proliferation of dermal fibroblasts significantly (p < 0.05). Extracts from the remaining seven plants either had no effect on fibroblast proliferation or were cytotoxic.

Conclusion

Traditional use of many wound-healing plants from Nigeria can be rationalised by activity determined in relevant in vitro investigations of ethanol and aqueous extracts. These results support the traditional selection of these plants in South-western Nigeria for wound healing.     

Traditional herbal antimalarial therapy in Kilifi district, Kenya

Aim of study

To identify plant species used by the traditional health practitioners (THPs) in treatment of malaria, carry out cytotoxicity and efficacy evaluation of the identified plants and to evaluate combination effects.

Materials and methods

Thirteen plants were selected through interviews with traditional healers. In vitro antiplasmodial testing was done by measuring ability of the test sample to inhibit the incorporation of radio-labelled hypoxanthine into the malaria parasite. The extracts were tested singly and then in combination using the standard fixed ratio analysis to evaluate synergism. In vivo bioassay was done in mice using Peter's 4-days suppressive test and cytotoxicity evaluated in vitro using Vero E6 cells.

Results

Of the plants tested in vitro, 25% were highly active (IC₅₀ < 10 μg/ml), 46% moderately active (IC₅₀ 10-50 μg/ml), 16% had weak activity of 50-100 μg/ml while 13% were not active IC₅₀ >100 μg/ml. Methanolic extracts of Azadirachta indica, Premna chrysoclada and Uvaria acuminata were the most active (IC₅₀ < 10 μg/ml) against both the chloroquine (CQ) sensitive (D6) and the CQ resistant (W2) Plasmodium falciparum clones. When tested in vivo in a mouse model, Azadirachta indica, Rhus natalensis and Grewia plagiophylla depicted the highest percent parasite clearance and chemo suppression of 89%, 82% and 78%, respectively. Evaluating effect of combining some of these extracts with one another against a multi-drug resistant Plasmodium falciparum (W2) clone revealed synergism among some combinations. The highest synergy was between Uvaria acuminata and Premna chrysoclada. The interaction between Grewia plagiophylla and Combretum illairii was largely antagonistic. Impressive cytotoxicity results were obtained with most of the plants tested revealing high selectivity indices an indication of enabling achievement of therapeutic doses at safe concentrations. Uvaria acuminata was, however, toxic to the cultured cells. Mild cytotoxicity was also observed in Hoslundia opposita and Lannea schweinfurthii (CC₅₀ 37 and 76 μg/ml, respectively).

Conclusions

This study identified plants with low IC₅₀ values, high percent chemo suppression and low cytotoxicity thus potential sources for novel antiplasmodial agents. The findings remotely justify use of combined medicinal plants in traditional medicine practices as synergy among some plant species was demonstrated.     

Effects of tanshinone IIA, a major component of Salvia miltiorrhiza, on platelet aggregation in healthy newborn piglets

Ethnopharmacological relevance

Tanshinone IIA (STS), an active ingredient of the Chinese herb Danshen (Salvia miltiorrhiza) for angina and stroke in adults, has been reported to inhibit platelet function. However, its effect on platelet and underlying mechanism remain largely unknown, particularly in neonates.

Materials and Methods

To investigate the effect of STS on the platelet aggregation and its interaction with various platelet activation pathways, platelet aggregatory function was studied in whole blood stimulated by collagen (2-10 μg/ml) ex vivo in newborn piglets receiving intravenous STS (0.1-10 mg/kg, n = 8) and in vitro in whole blood from newborn piglets (n = 6) incubated with STS (0.1-100 μg/ml). The respective morphological changes of platelets were also examined by scanning electron microscopy. Plasma levels of nitrite/nitrate (NOx) and thromboxane B₂ (TxB₂), matrix metalloproteinase (MMP)-2 and -9 activities were also examined. To further delineate the mechanistic pathway, the effect of STS on endothelial microparticles release from cultured human umbilical vein endothelial cells (HUVECs) was quantified by flow cytometry.

Results

STS impaired the ex vivo, but not in vitro, collagen-stimulated platelet aggregation. Infusion of STS elevated the plasma level of TxB₂ at 10 mg/kg. However, STS had no effect on NOx level. Incubating cultured HUVECs with STS (1 and 10 μg/ml) caused a significant release of endothelial microparticles. Morphologically, STS elicited platelet activation in vivo, but not in vitro.

Conclusions

STS impairs the ex vivo whole blood platelet aggregatory function by activating platelet in vivo in healthy newborn piglets. It implies that STS may elicit its effects by stimulating endothelial microparticles production and eicosanoid metabolism pathway.     

The antimicrobial,antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea

Ethnopharmacological importance

Many Bauhinia species, including those indigenous to South Africa, are used in traditional medicine across the world for treating ailments such as gastrointestinal tract (GIT) disorders, diabetes, infectious diseases and inflammation.

Aims

Several relevant aspects of different fractions of leaf extracts of Bauhinia bowkeri (BAB), Bauhinia galpinii (BAG), Bauhinia petersiana (BAP), and Bauhinia variegata (BAV) used in South African traditional medicine to alleviate diarrhoea related symptoms were evaluated.

Materials and Methods

The antioxidative activities of the extracts were determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging and ferric reducing antioxidant power (FRAP) methods. In vitro antimicrobial activities of the extracts were determined against bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) and clinical isolates of the opportunistic fungal strains (Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans) using a serial dilution microplate method. The polyphenolic contents were quantified using standard methods, and anti-inflammatory activities of the crude extracts were determined using the cyclooxygenase and soybean 15-lipoxygenase enzyme inhibitory assays. The safety of the extracts was evaluated by determining the cytotoxicity against Vero cell lines.

Results

The acidified 70% acetone crude extract and their fractions had good antiradical potency against the DPPH and ABTS radicals. The methanol soluble portions of the butanol fractions were more potent (EC₅₀ ranges from 0.64±0.05 to 1.51±0.07 and 0.88±0.18 to 1.49±0.09 μg/ml against DPPH and ABTS radical respectively) compared to the standard, trolox and ascorbic acid (EC₅₀ ranges from 1.47±0.24 to 1.70±0.27 μg/ml) for both DPPH and ABTS. The crude extracts contained variable quantities of phenolic content. The crude extracts and their fractions had weak to good antimicrobial activities, inhibiting the growth of the organisms at concentrations ranging from 39 to 2500 μg/ml. The BAG crude extract and its fractions were the most active against the fungi (MICs ranging from 39 to 625 μg/ml) while the BAB extract and its fractions were the least active with the MICs ranging between 39 and 2500 μg/ml. Aspergillus fumigatus was the least susceptible fungus while Cryptococcus neoformans was the most susceptible.The phenolic-rich crude extracts of BAB, BAG, and BAP had moderate to good dose-dependent cyclooxygenase-1 enzyme inhibitory activity with inhibitions between 22.8% and 71.4%. The extracts were however, inactive against cyclooxygenase-2. The extracts had some level of cytotoxicity towards Vero cell lines, reducing cell viability to less than 10% at concentrations more than 50 μg/ml.

Conclusion

The biological activities observed in Bauhinia species provide a scientific basis for the use of the plants in traditional medicines to treat diseases with multi-factorial pathogenesis such as diarrhoea, with each aspect of activity contributing to the ultimate therapeutic benefit of the plants. However, the use of the phenolic-rich extracts of these plants to treat diarrhoea or any other ailments in traditional medicine needs to be monitored closely because of potential toxic effects and selective inhibition of COX-1 with the associated GIT injury.     

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 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).     

An aqueous birch leaf extract of Betula pendula inhibits the growth and cell division of inflammatory lymphocytes

Ethnopharmacological relevance

Leaf extracts of Betula pendula have been traditionally used for the treatment of patients with rheumatoid arthritis (RA) or osteoarthritis.

Aim of the study

We investigated the anti-proliferative capacity of an aqueous leaf extract of Betula pendula (BPE) on human primary lymphocytes in vitro, because activated lymphocytes play a major role in the initiation and maintenance of RA.

Materials and methods

Lymphocyte proliferation and cell division was measured by the activity of mitochondrial dehydrogenases and by using the membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE), respectively. Apoptosis was analyzed by surface staining of phosphatidylserine and intracellular activation of effector caspases 3 and 7 in comparison to the drug methotrexate using flow cytometric and photometrical analysis. In addition, the impact of the extract on cell cycle distribution was investigated by propidium iodide staining of DNA. For the bioassays BPE concentrations of 10-160 μg/mL were investigated. A phytochemical analysis, using LC-MS and HPLC, was conducted to identify the polyphenolic constituents of the birch leaf extract.

Results

Leaf extracts of Betula pendula inhibited the growth and cell division (CD8⁺: 40 μg/mL: 45%; 80 μg/mL: 60%; 160 μg/mL: 87%) (CD4⁺: 40 μg/mL: 33%; 80 μg/mL: 54%; 160 μg/mL: 79%) of activated, but not of resting T lymphocytes in a significant dose-dependent manner. The inhibition of lymphocyte proliferation due to apoptosis induction (compared to untreated control: 40 μg/mL: 163%; 80 μg/mL: 240%; 160 μg/mL: 348%) and cell cycle arrest was comparable to that of methotrexate. LC-MS analyses showed that the extract contains different quercetin-glycosides.

Conclusion

Our results give a rational basis for the use of Betula pendula leaf extract for the treatment of immune disorders, like rheumatoid arthritis, by diminishing proliferating inflammatory lymphocytes.     

Cytochrome P450 inhibitory potential of Triphala--a Rasayana from Ayurveda

Ethnopharmacological relevance

‘Triphala’ is one of the age-old, most commonly used polyherbal preparation from Ayurveda as Rasayana drug.

Aim of the study

This study was aimed at evaluating the effect of ‘Triphala’ on drug modulating enzymes to assess its safety through its potential to interact with co-administered drugs.

Materials and methods

The cytochrome P450 inhibitory effect of ‘triphala’ formulation was investigated on rat liver microsomes using CYP450-CO complex assay and on individual isoform such as CYP3A4 and 2D6 using fluorescence screening. RP-HPLC method was developed to standardize ‘triphala’ and its individual components using gallic acid as analytical marker compound.

Results

RP-HPLC analysis demonstrated the presence of gallic acid (4.30 ± 2.09 mg/g) in the formulation. The formulation showed 23% inhibition of the rat liver microsomes through CYP450-CO complex assay which is comparatively less when compared with the individual components. Further, the effect of standardized formulation dissolved in ethanol showed CYP3A4 and CYP2D6 inhibitory activity at the IC₅₀ values of 119.65 ± 1.91 μg/ml and 105.03 ± 0.98 μg/ml respectively. Gallic acid was also found to inhibit both the isoforms at the IC₅₀ values of 87.24 ± 1.11 μg/ml and 92.03 ± 0.38 μg/ml respectively.

Conclusions

Various concentrations of the formulation and its individual components showed significantly less inhibitory activity (p < 0.001) on individual isoforms when compared with the positive control. Assessment on the in vitro effect of ‘triphala’ on drug modulating enzymes has important implications for predicting the likelihood of herb-drug interactions if these are administered concomitantly.     

Potent antiplasmodial extracts from Cameroonian Annonaceae

Aim of the study

In a search for new antimalarial leads, we have carried out a preliminary ethnopharmacological study with the aim of evaluating the in vitro antiplasmodial activity of extracts from thirteen Annonaceae species growing in Cameroon, and of assessing the acute toxicity of promising fractions in Swiss albino mice.

Materials and methods

Plants were selected on the basis of an ethnobotanical survey carried out in four sites in centre and south regions of Cameroon (Yaoundé neighbourhoods, Kon-Yambetta, Ngobayang and Mbalmayo) on Annonaceae plants locally used to treat malaria and related symptoms. The choice of the sites was mainly based on environmental factors enabling mosquito breeding, cosmopolitan areas regrouping people from different cultural origins, areas with limited access to health centers, and areas with people relying exclusively on traditional medical practices. Collected materials were extracted by maceration in 95% ethanol. The crude extract was partitioned using organic solvents and the fractions afforded were evaluated for antiplasmodial activity in culture against the W2 strain of Plasmodium falciparum. Promising fractions (methanol fractions) were assessed for their acute toxicity in Swiss albino mice.

Results

From the results achieved, 37 (31.3%) out of 118 extracts tested exhibited antiplasmodial activity, with IC₅₀ values ranging from 1.07 μg/ml to 9.03 μg/ml. Of the active extracts, 29 (78.4%) were methanol fractions, 21 (72.4%) of which inhibited the parasites with IC₅₀ < 5 μg/ml. The promising fractions proved to be safe through oral administration in mice.

Conclusions

The activities and toxicity profiles of methanol fractions indicate that they deserve to be further investigated in detail for antimalarial lead discovery.     

Relaxant effects of Artemisia ludoviciana on isolated rat smooth muscle tissues

Ethnopharmacological relevance

Artemisia ludoviciana spp. mexicana (Willd. Ex.) Spring D.D. Keck (Asteraceae), known as “estafiate” is employed for the treatment of diarrhea, dysentery, parasites, abdominal pain, vomiting, stomach ache, and also as antispasmodic agent. The aim of the present study was to evaluate the relaxant effect of hexanic (HEAl), dichloromethanic (DEAl) and methanolic (MEAl) extracts on isolated trachea, ileum and aorta rat rings, and to establish the tracheo-relaxant mode of action of DEAl.

Materials and methods

All extracts were investigated based on their capacity of to inhibit the rat ileum spontaneous contraction, to relax contraction induced by noradrenaline (0.1 μM) on endothelium-intact and endothelium-denuded thoracic aorta rat rings, and also to inhibit contraction provoked by carbachol (1 μM) on rat trachea.

Results

Organic extracts had no spasmolytic action on ileum strips compared to positive control (papaverine, p < 0.05). On the other hand, all extracts induced a significant concentration- and partial endothelium-dependent vasorelaxant activity. Extracts also showed significant relaxant effect on pre-contracted tracheal tissue in a concentration-dependent manner. In last two experiments, DEAl was the most potent and efficient extract; however, it was less potent than papaverine and theophylline, used as positive controls (p < 0.05). In tracheal preparation, DEAl shifted to the right, in a parallel manner, the concentration-response curves induced by carbachol (p < 0.05). Also, DEAl induced a significant relaxant effect on the contraction produced by potassium chloride (KCl, 80 mM). Pre-incubation with 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 10 μM), indomethacin (10 μM), N_ω-nitro-l-arginine methyl ester (l-NAME, 10 μM), glibenclamide (10 μM) and 2-aminopyridine (2-AP, 100 μM) did not modify the DEAl-relaxant curves.

Conclusions

Functional experiments suggest that the most active extract, DEAl, induced its relaxant effect by possible muscarinic receptors antagonism and calcium channel blockade in tracheal rings. On the other hand, significant vasorelaxant activity showed by DEAl is partially endothelium-dependent. Finally, spasmolytic activity induced by the extracts in the rat ileum was not significant, which suggests that the antidiarrheic effect of the plant is related to antimicrobial and antiparasitic properties previously described.