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.     

Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells

Ethnopharmacological relevance

Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer.

Aim of the study

To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549.

Materials and methods

The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells.

Results

The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC₅₀ values of 0.26 equivalent/ml and 0.34 equivalent/ml, respectively (1 equivalent = amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15 equivalent/ml) inhibited mononuclear cell proliferation by 72.2 ± 2.7% and induced significant DNA damage both in tumor and mononuclear cells.

Conclusion

Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells.     

Suppression of interleukin (IL)-8 and human beta defensin-2 secretion in LPS-and/or IL-1β-stimulated airway epithelial A549 cells by a herbal formulation against respiratory infections (BNO 1030)

Aim of the study

A special ethanolic-aqueous extract from seven traditional medicinal plants (BNO 1030) has been used for several decades to treat recurrent infections of the respiratory tract. Considering the potential role of interleukin-8 (IL-8) and human beta defensin-2 (hBD-2) in inflammation, we investigated the effect of BNO 1030 on lipopolysaccharide (LPS) from Pseudomonas aeruginosa or IL-1β-induced inflammatory mediators in A549 human type II alveolar epithelial cells.

Materials and methods

A549 cells were stimulated with LPS (100 μg/ml) or IL-1β (50 ng/ml) in the presence of the preparation and the secretion of IL-8 and hBD-2 were measured after 18 h and 24 h in cell free supernatants using enzyme-linked immunosorbent assays (ELISA). Cell viability and cell growth was investigated by propidium iodide uptake and WST-1 assay, respectively.

Results

BNO 1030 inhibited the secretion of IL-8 and hBD-2 at non-cytotoxic concentrations (0.1-100 μg/ml; cell growth inhibitory concentration, 50% (IC₅₀) = 678 ± 87.6 μg/ml). Stimulation by IL-1β led to a 7-fold activation of IL-8 secretion, which was reduced by 37.7 ± 4.1% (p < 0.05) after incubation with 100 μg/ml BNO 1030. Inducible hBD-2 was suppressed by 91.8 ± 15.6% (p < 0.01) at the same concentration of BNO 1030 (IC₅₀ = 0.7 ± 0.1 μg/ml). The 2-fold increase of IL-8 secretion by LPS-stimulated cells was completely abolished at concentration of 50 μg/ml BNO 1030 (IC₅₀ = 5.7 ± 3.6 μg/ml).

Conclusion

BNO 1030 suppressed the secretion of IL-8 and hBD-2 in cultured epithelial A549 cells. These results support its use as a phytotherapeutic product prepared from traditional remedies in inflammatory diseases, especially those affecting the respiratory tract.     

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.     

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.     

Anti-cancer activity of compounds from Bauhinia strychnifolia stem

Ethnopharmacological relevance

The stem and root of Bauhinia strychnifolia Craib (Fabaceae family) have been traditionally used in Thailand to treat fever, alcoholic toxication, allergy and cancer. An EtOH extract of Bauhinia strychnifolia showed good inhibitory activity against several cancer cell lines including HT-29, HeLa, MCF-7 and KB. As there has been no previous reports on chemical constituents of Bauhinia strychnifolia, this study is aimed to isolate the pure compounds with anti-cancer activity.

Materials and methods

Five pure compounds were isolated from EtOH extract of Bauhinia strychnifolia stem using silica gel, dianion HP-20 and sephadex LH-20 column chromatography and were tested for their cytotoxic effects against HT-29, HeLa, MCF-7 and KB cell lines using the Sulforhodamine B (SRB) assay.

Results

Among five compounds, 3,5,7,3′,5′-pentahydroxyflavanonol-3-O-α-l-rhamnopyranoside (2) possessed very potent activity against KB (IC₅₀=0.00054 μg/mL), HT-29 (IC₅₀=0.00217 μg/mL), MCF-7 (IC₅₀=0.0585 μg/mL) and HeLa cells (IC₅₀=0.0692 μg/mL). 3,5,7-Trihydroxychromone-3-O-α-l-rhamnopyranoside (3) also showed good activity against HT-29 (IC₅₀=0.02366 μg/mL), KB (IC₅₀=0.0412 μg/mL) and MCF-7 (IC₅₀=0.297 μg/mL), respectively. The activity of 2 (IC₅₀=0.00054 μg/mL) against KB cell was ten times higher than that of the positive control, Camptothecin (anti-cancer drug, IC₅₀=0.0057 μg/mL). All compounds did not show any cytotoxicity with normal cells at the concentration of 1 μg/mL.

Conclusion

This is the first report of compounds 2 and 3 on anti-cancer activity and based on the anti-cancer activity of extracts and pure compounds isolated from Bauhinia strychnifolia stem, it might be suggested that this plant could be useful for treatment of cancer.     

Antiplasmodial activity of aporphine alkaloids and sesquiterpene lactones from Liriodendron tulipifera L.

Aim of the study

The objective of this study was to isolate and characterize the active constituents of the traditionally used antimalarial plant Liriodendron tulipifera by antiplasmodial-assay guided fractionation.

Materials and methods

Bark and leaves were extracted with solvents of increasing polarity. Fractions were generated using flash chromatography, counter current chromatography and preparative HPLC and subjected to in vitro antiplasmodial and cytotoxicity assays. Active fractions were subjected to further fractionation until pure compounds were isolated, for which the IC₅₀ values were calculated.

Results and discussion

Six known aporphine alkaloids, asimilobine (1), norushinsunine (2), norglaucine (3), liriodenine (4), anonaine (5) and oxoglaucine (6) were found to be responsible for the antiplasmodial activity of the bark. Leaves yielded two known sesquiterpene lactones, peroxyferolide (7) and lipiferolide (8) with antiplasmodial activity. The antiplasmodial activity of (2) (IC₅₀ = 29.6 μg/mL), (3) (IC₅₀ = 22.0 μg/mL), (6) (IC₅₀ = 9.1 μg/mL), (7) (IC₅₀ = 6.2 μg/mL) and (8) (IC₅₀ = 1.8 μg/mL) are reported for the first time.

Conclusion

This work supports the historical use of Liriodendron tulipifera as an antimalarial remedy of the United States and characterizes its antiplasmodial constituents.     

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.     

Antibacterial, antioxidant and fibroblast growth stimulation activity of crude extracts of Bridelia ferruginea leaf, a wound-healing plant of Nigeria

Aim of the study

Determination of pharmacological activity relevant to wound healing of Bridelia ferruginea leaf, a traditional medicine used to treat wounds in rural Nigeria.

Materials and methods

Aqueous and ethanolic leaf extracts were tested against bacterial species of relevance to wound infections: Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The ethanolic extracts were assessed for their ability to stimulate the growth of human dermal fibroblasts (FS5) and protect against damage induced by hydrogen peroxide. Antioxidant activity was also assessed using the DPPH assay.

Results

Both aqueous and ethanolic extracts had weak antibacterial activity (MIC > 470 μg/ml). A significant effect (p < 0.001) on the growth of FS5 fibroblasts was observed only at a concentration of 5 μg/ml (28% increase), above which the extracts appeared toxic to the cells. The ethanolic extract offered the highest protection against H₂O₂ damage to FS5 cells, comparable with catalase (82% at 250 μg/ml). The DPPH assay revealed antioxidant activity of the ethanolic leaf extract with IC₅₀ 12.5 ± 0.3 μg/ml comparable to l-ascorbic acid (7.3 ± 0.1 μg/ml).

Conclusion

The antibacterial, modest fibroblast stimulation activity and relatively strong antioxidant activity lend some support to the topical use of Bridelia ferruginea leaf for wound-healing in the traditional medicine of South-western Nigeria.     

Antiplasmodial, anti-inflammatory and cytotoxic activities of various plant extracts from the Mascarene Archipelago

Aim of the study

Antiplasmodial activity, inhibition of nitric oxide (NO) overproduction, and anti-proliferative activity were investigated in vitro to evaluate the bioactive potential of the traditional pharmacopoeia of the Mascarene Archipelago, which is known for its biodiversity and for the richness of its endemic flora.

Materials and methods

A total of 45 methanol (MeOH) and dichloromethane (DCM) extracts were prepared from 19 plant species collected on Réunion and Mauritius Islands. Ninety-six-well microplate assays were performed on chloroquine sensitive Plasmodium falciparum 3D7 strain, on LPS-stimulated Raw 264.7 murine macrophages and on A-549, DLD-1 and WS1 human cells. Activity was evaluated through spectrophotometric methods.

Results

Activity was attributed to plant extracts expressing IC₅₀ < 50 μg/ml for antiplasmodial response, IC₅₀ < 100 μg/ml for cytotoxicity, and IC₅₀ < 130 μg/ml for anti-inflammatory reaction. The majority of the extracts tested (69%) exhibited potency in at least one of these three types of activity. This is the first report describing promising antiplasmodial activity (IC₅₀ < 15 μg/ml) for Psiadia dentata DCM extract and Terminalia bentzoe MeOH bark extract. NO inhibition assay revealed seven interesting plants, described for the first time as anti-inflammatory: Aphloia theiformis, Buddleja salviifolia, Eupatorium riparium, Hiptage benghalensis, Psiadia arguta, Psiadia dentata, and Scutia commersonii. Finally, anti-proliferative activity was observed for two endemic species, Geniostoma borbonicum and Nuxia verticillata.

Conclusion

Using the criterion of endemism as part of the criteria for traditional medicinal use raises the chances of finding original active principles. In our case, 86% of the endemic plants tested displayed pharmacological interest.     

Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks

Ethnopharmacological relevance

The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation.

Aim of the study

There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells.

Materials and methods

The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells.

Results

Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI₅₀ = 28.03 ± 6.44 μg/ml) and A549 (GI₅₀ = 89.63 ± 2.12 μg/ml) cells (p value < 0.0001). The hexane extract was found to exhibit crucial antiproliferative effects on U87MG (GI₅₀ = 166.30 ± 30.50 μg/ml) (p value < 0.0001) but not on A549 cells. Neither plant extract possessed noticeable antiproliferative effects on the non-cancerous MRC5 cells (GI₅₀ > 300 μg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin &; Eosin (H &; E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis.

Conclusion

The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs.     

Comparative in vitro bioactivities of tea extracts from six species of Ardisia and their effect on growth inhibition of HepG2 cells

Aim of the study

Ardisia species, notably A. compressa, are used in some regions of the world as food or in traditional medicine for prevention and treatment of certain health conditions including liver disease. We investigated the chemical composition and relative anticancer potential of six Ardisia species [A. japonica (AJ), A. escallonioides (AES), A. mamillata (AM), A. compressa (AC), A. crenata (ACR), and A. elliptica (AE)].

Materials and methods

Antioxidant capacity, DNA human topoisomerase II catalytic inhibition, and cytotoxicity on human liver cancer cells (HepG2) were determined in vitro in tea extracts of the 6 Ardisia species evaluated. Selected pure phenolic compounds present in Ardisia species were also evaluated.

Results

AC showed the highest topoisomerase II catalytic inhibition (IC₅₀ = 12 μg/ml) and cytotoxicity (IC₅₀ = 117 μg/ml) against HepG2 cells, followed by ACR and AJ. Total polyphenols ranged from 21 to 72 mg equivalents of gallic acid (GA)/g solid extract (SE). LC–MS analysis revealed the presence of GA, quercetin derivatives, ardisenone, ardisiaquinone, ardisianone, bergenin, norbergenin, and embelin. However, neither total polyphenol concentration nor antioxidant capacity correlated with anticancer capacity. Significant HepG2 cytotoxicity was also achieved by bergenin (IC₅₀ = 18 μM) and embelin (IC₅₀ = 120 μM). AC, bergenin, embelin, and quercetin showed a tendency to accumulate cells in the G1 phase and reduced G2/M leading to apoptosis.

Conclusions

Although the mechanism is not entirely clear, AC, ACR, and AJ are the Ardisia species with the greatest anticancer potential against liver cancer cells in vitro and deserve further investigation.     

Screening of plants used in Danish folk medicine to treat depression and anxiety for affinity to the serotonin transporter and inhibition of MAO-A

Ethnopharmacological relevance

A number of plant species are used in Danish folk medicine for treatment of depression and anxiety.

Materials and methods

Aqueous and ethanolic extracts of 17 plant species were tested for affinity to the serotonin transporter and for inhibition of MAO-A—both targets for antidepressive treatment.

Results

An ethanolic extract of aerial parts of Borago officinalis had affinity to the serotonin transporter. Ten extracts, from eight plants, had IC₅₀ values below 25 μg/ml extract in the MAO-A assay. The most active extracts in the MAO-A assay were the ethanol extract of seeds of Trigonella foenum-graecum (IC₅₀ 4 μg/ml); ethanol extract of leaves of Apium graveolens (IC₅₀ 5 μg/ml) and the water extract of aerial parts of Calluna vulgaris (IC₅₀ 8 μg/ml).

Conclusions

Besides Borago officinalis, which toxicity profile excludes it from further development as an herbal drug, none of the plants had potential as serotonin reuptake inhibitors. Several plants had MAO-A inhibitory activity.     

Water extract of Rheum officinale Baill. induces apoptosis in human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines

Ethnopharmacological relevance

Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study.

Aim of the study

The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways.

Materials and methods

Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay.

Results

Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC₅₀ values 620 ± 12.7 and 515 ± 10.1 μg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells.

Conclusions

This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.     

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.     

In vitro xanthine oxidase inhibitory activity of the fractions of Erythrina stricta Roxb.

Aim of the study

To assay the in vitro xanthine oxidase inhibitory activity of the various fractions of the hydromethanolic extract of the leaves of Erythrina stricta and to determine its enzyme inhibition mechanism.

Materials and methods

Xanthine oxidase inhibitory activity was assayed spectrophotometrically under aerobic conditions and the degree of enzyme inhibition was determined by measuring the increase in absorbance at 295 nm associated with uric acid formation. Enzyme kinetics was carried out using Lineweaver-Burk plots using xanthine as the substrate.

Results

Among the fractions tested, the chloroform fraction exhibited highest potency (IC₅₀ 21.2 ± 1.6 μg/ml) followed by the pet–ether (IC₅₀ 30.2 ± 2.2 μg/ml), ethyl acetate (IC₅₀ 44.9 ± 1.4 μg/ml) and residual (IC₅₀ 100 ± 3.3 μg/ml) fractions. The IC₅₀ value of allopurinol used, as the standard was 6.1 ± 0.3 μg/ml. Enzyme inhibition mechanism indicated that the mode of inhibition was of a mixed type.

Conclusion

These results suggest that the use of Erythrina stricta for the treatment of gout could be attributed to its xanthine oxidase inhibitory activity.     

In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid

Ethnopharmacological relevance

Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far.

Aim of the study

To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer.

Material and methods

Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231.

Results

The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC₅₀ of 50 μg/ml and 30 μg/ml for 24 h, 28 μg/ml and 22 μg/ml for 48 h and 25 μg/ml and 20 μg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC₅₀ value of 7.5 μM and 7.0 μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts.

Conclusion

Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.     

Characterization of the anticancer properties of monoglycosidic cardenolides isolated from Nerium oleander and Streptocaulon tomentosum

Aim of the study

For identification of the active constituents we investigated the anticancer activity of cardenolides from Streptocaulon tomentosum Wight & Arn. (Asclepiadaceae) and from Nerium oleander L. (Apocynaceae) which are both used against cancer in the traditional medicine in their region of origin.

Material, methods and results

The antiproliferative activity of cardenolides isolated from roots of Streptocaulon tomentosum (IC₅₀ < 1-15.3 μM after 2 days in MCF7) and of cardenolide containing fractions from the cold aqueous extract of Nerium oleander leaves (“Breastin”, mean IC₅₀ 0.85 μg/ml in a panel of 36 human tumor cell lines), their influence on the cellular viability and on the cell cycle (block at the G2/M-phase or at the S-phase in tumor cells, respectively) were determined using different cell lines. The murine cell line L929 and normal non-tumor cells were not affected. Bioactivity guided fractionation of Breastin resulted in the isolation of the monoglycosidic cardenolides oleandrine, oleandrigeninsarmentoside, neritaloside, odoroside H, and odoroside A (IC₅₀-values between 0.010 and 0.071 μg/ml).

Conclusions

The observed anticancer activities of extracts and isolated cardenolides are in agreement with the ethnomedicinal use of Streptocaulon tomentosum and Nerium oleander. The most active anticancer compounds from both species are monoglycosidic cardenolides possessing the 3β,14β-dihydroxy-5β-card-20(22)-enolide structure with or without an acetoxy group at C-16. The results indicate that the cytotoxic effects are induced by the inhibition of the plasma membrane bound Na⁺/K⁺-ATPase.     

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.