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.     

Prophylactic and curative activities of extracts from Warburgia ugandensis Sprague (Canellaceae) and Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae) against Plasmodium knowlesi and Plasmodium berghei

Ethnopharmacological relevance

This work reports the anti-plasmodial activities of Warburgia ugandensis and Zanthoxylum usambarense commonly used as phytomedicines against malaria by some Kenyan communities.

Aim of study

To determine the anti-plasmodial activities of extracts from Warburgia ugandensis and Zanthoxylum usambarense against Plasmodium knowlesi and Plasmodium berghei.

Materials and methods

Eight plant extracts were screened for in vitro anti-plasmodial activity against Plasmodium knowlesi, in a 96-well plate incubated at 37 °C on a RPMI culture medium supplemented with baboon serum. Of the eight, three were investigated for prophylactic and curative activities in BALB/c mice against drug-sensitive Plasmodium berghei in a 4-day test at a dose rate of 200 mg/kg/day.

Results

Inhibitory concentrations (IC₅₀) values of between 3.14 and 75 μg/ml, up to 69% chemosuppression of parasites growth and over 80% survivorship of treated mice were observed.

Conclusion

The two medicinal plants, Warburgia ugandensis and Zanthoxylum usambarense possess bioactive compounds against malaria parasites and could be exploited for further development into malaria therapy.     

Ethnopharmacology guided screening of traditional Indian herbs for selective inhibition of Plasmodium specific lactate dehydrogenase

Ethnopharmacological relevance

Medicinal plants traditionally used to treat malaria can provide quality leads towards identifying novel anti-malarial drugs. Here we combined this approach with target based drug discovery and explored Plasmodium specific lactate dehydrogenase (LDH) inhibitory activity of 8 Indian plants which are ethnically used to treat malaria.

Methods

LDH from Indian Plasmodium falciparum and Plasmodium vivax strains, were cloned and expressed in Escherichia coli, followed by purification of recombinant enzymes (rPfLDH and rPvLDH respectively). Extracts of 8 plants in different organic and aqueous solvents, were screened for their inhibitory activity on rPfLDH, rPvLDH and mammalian LDHs. Phyllanthus amarus aqueous extract was further tested for in vitro parasiticidal activity.

Results

Aqueous extract of Phyllanthus amarus Schum. and Thonn. and chloroform extract of Murraya koenigii (L.) Spreng. exhibited profound and exclusive inhibitory effect on Plasmodium falciparum LDH (IC₅₀=11.2 μg/ml±0.4) and Plasmodium vivax LDH (IC₅₀=6.0 μg/ml±0.6) respectively. Moreover, Phyllanthus amarus aqueous extract also demonstrated antiplasmodial activity in vitro, on Chloroquine sensitive and resistant strains of Plasmodium falciparum (IC₅₀=7.1 μg/ml±0.5 and 6.9 μg/ml±0.7 respectively).

Conclusion

Target specific screening of traditional herbs used in malaria treatment has proffered Phyllanthus amarus and Murraya koenigii extracts as hits which can optimistically provide novel antimalarial drugs.     

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 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.     

Antiplasmodial activity of sesquiterpene lactones and a sucrose ester from Vernonia guineensis Benth. (Asteraceae)

Ethnopharmacological relevance

Aqueous preparations of Vernonia guineensis Benth. (Asteraceae) are used in Cameroonian folk medicine as a general stimulant and to treat various illnesses and conditions including malaria, bacterial infections and helminthic infestations.

Materials and methods

Ten gram samples of the leaf and tuber powders of Vernonia guineensis were extracted separately using dichloromethane, methanol and distilled water. The extracts were dried in vacuo and used in bioassays. These extracts and three compounds previously isolated from Vernonia guineensis [vernopicrin (1), vernomelitensin (2) and pentaisovalerylsucrose (3)] were screened for antiplasmodial activity against chloroquine (CQ)-sensitive (Hb3) and CQ-resistant (Dd2) Plasmodium falciparum lines.

Results

Crude extracts and pure compounds from Vernonia guineensis showed antiplasmodial activity against both Hb3 and Dd2. The IC₅₀ values of extracts ranged from 1.64 to 27.2 µg/ml for Hb3 and 1.82–30.0 µg/ml for Dd2; those for compounds 1, 2 and 3 ranged from 0.47 to 1.62 µg/ml (1364–1774 nM) for Hb3 and 0.57–1.50 µg/ml (1644–2332 nM) for Dd2. None of the crude extracts or pure compounds was observed to exert toxic effects on the erythrocytes used to cultivate the Plasmodium falciparum lines.

Conclusion

In Cameroonian folk medicine, Vernonia guineensis may be used to treat malaria in part due to the antiplasmodial activity of sesquiterpene lactones (1, 2), a sucrose ester (3) and perhaps other compounds present in crude plant extracts. Exploring the safety and antiplasmodial efficacy of these compounds in vivo requires further study.     

Antiprotozoal screening and cytotoxicity of extracts and fractions from the leaves,stem bark and root bark of Alstonia congensis

Ethnopharmacological relevance

To evaluate the antiprotozoal activity and cytotoxicity of extracts and fractions from the leaves, root bark and stem bark of Alstonia congensis (Apocynaceae), used in traditional medicine against parasitic diseases.

Materials and methods

The aqueous and 80% MeOH extracts, and a series of fractions and subfractions from the leaves, stem and root bark of Alstonia congensis were tested in vitro for their antiprotozoal activity against Trypanosoma brucei brucei, Trypanosoma cruzi, Lesihamania infantum and the chloroquine and pyrimethamine-resistant K1 strain of Plasmodium falciparum. Their cytotoxicity on MRC-5 cells (human lung fibroblasts) was evaluated as well.

Results

The aqueous and 80% MeOH extracts and a series of subfractions of each plant part exhibited pronounced antiprotozoal activity against the K1 strain of Plasmodium falciparum with IC₅₀ values ranging from 2 to 5 µg/ml, and good activity against Trypanosoma brucei brucei and Trypanosoma cruzi with IC₅₀ values ranging between 5 and 10 µg/ml. The residual 80% MeOH extract from the leaves, and the total alkaloid extract from stem and root bark were the only subfractions active against Leishmania infantum with IC₅₀ values <10 µg/ml. None of the samples from the root bark was cytotoxic against MRC-cell lines (CC₅₀>64 µg/ml). In general, the aqueous extract (traditional decoction) showed the highest selectivity, especially against Plasmodium falciparum.

Conclusion

These results can partly support and justify the traditional use of these plant parts of Alstonina congensis as raw materials for the preparation of traditional remedies to treat parasitic diseases such as malaria and trypanosomiasis.     

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

Ethnopharmacological relevance

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

Aim of the study

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

Materials and methods

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

Results

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

Conclusions

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

The bioactivity of novel furanoterpenoids isolated from Siphonochilus aethiopicus

Aim of the study

This study investigated the medicinal plant Siphonochilus aethiopicus (Zingiberaceae) for antiplasmodial activity.

Materials and methods

The ethyl acetate extract of Siphonochilus aethiopicus rhizomes was fractionated using solid phase extraction (SPE) and purified by high performance liquid chromatography. Structure elucidation was performed with nuclear magnetic resonance and mass spectrometry. The in vitro cytotoxicity and antiplasmodial activity was determined. In vivo schizontocidal activity was performed in a malaria mouse-model. Additional in vitro testing was done against Staphylococcus aureus, Klebsiella pneumoniae and Mycobacterium tuberculosis.

Results and discussion

The ethyl acetate extract showed in vitro activity against the chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains of Plasmodium falciparum with IC₅₀-values of 2.9 μg/ml and 1.4 μg/ml, respectively. Bioassay-guided fractionation led to the isolation of three novel furanoterpenoids with moderate in vitro antiplasmodial activity. The crude extract showed very good in vivo activity. The compounds and crude extract were more active against the CQR strain than the CQS strain of Plasmodium falciparum. The SPE fractions were more active than the isolated compounds. The compounds did not show good activity against the micro-organisms tested. No in vitro cytotoxicity was observed.

Conclusion

This study provides evidence of antiplasmodial compounds present in Siphonochilus aethiopicus.     

Screening of antiplasmodial properties among some traditionally used Iranian plants

Ethnopharmacological relevance

An investigation of plants was undertaken through interviews and literature surveys on plants used to treat malaria or cancer or microbial diseases in Iran.

Aim of study

In vitro and in vivo antiplasmodial tests were carried out on selected plants traditionally used in Iran.

Materials and methods

Thirty-two plants were extracted with methanol and tested for their in vitro (pLDH assay) activity against Plasmodium falciparum, in vivo activity against Plasmodium berghei and assessed for any cytotoxicity against the human cancer cell line MCF7 and the normal cell MDBK.

Results

Extracts from four plants, Buxus hyrcana Pojark. (Buxaceae), Erodium oxyrrhnchum M. Bieb. (Geraniaceae), Glycyrrhiza glabra L. (Fabaceae) and Ferula oopoda (Boiss and Bushe) Boiss. (Apiaceae) were found to have significant antiplasmodial activity (IC₅₀ ranging from 4.7 to 26.6 μg/ml). These findings lend support to the use of Buxus hyrcana and Glycyrrhiza glabra in traditional medicine. The chloroformic fraction also was active against K1 and 3D7 strains. The chloroformic fraction was studied at 10 mg per kg body weight mouse per day. This fraction reduced parasitaemia by 86.1% compared to untreated control mice.

Conclusion

Glycyrrhiza glabra showed antiplasmodial activity and has selectivity for Plasmodium falciparum and Plasmodium berghei when tested on mammalian cells. This is the first report that mentioned in vitro and in vivo antiplasmodial activity of Glycyrrhiza glabra.     

Evaluation of 13 selected medicinal plants from Burkina Faso for their antiplasmodial properties

Aim of the study

The aim of this study was to evaluate the antiplasmodial properties of 13 plants used against malaria in traditional medicine in Burkina Faso.

Materials and methods

In vitro antiplasmodial activity of dichloromethane, methanol and aqueous crude extracts obtained from vegetal samples collected in Burkina Faso was first evaluated on the Plasmodium falciparum 3D7 chloroquine-sensitive strain using a colorimetric method.

Results

Thirteen extracts obtained from 8 different species were found to exhibit antiplasmodial activity (IC₅₀ < 50 μg/ml). Five species demonstrated a moderate activity (15 μg/ml < IC₅₀ < 50 μg/ml): Boswellia dalzielii (leaves), Waltheria indica (roots and aerial parts), Bergia suffruticosa (whole plant), Vitellaria paradoxa (bark) and Jatropha gossypiifolia (leaves). The best results were obtained with extracts from the Dicoma tomentosa whole plant, from Psorospermum senegalense leaves and from Gardenia sokotensis leaves. These extracts found to display promising antiplasmodial activity, with IC₅₀ values ranging from 7.0 to 14.0 μg/ml.The most active plant extracts were then tested for in vitro activity on the Plasmodium falciparum W2 chloroquine-resistant strain and also for in vitro cytotoxicity on normal human fibroblasts (WI-38) in order to determine the selectivity index.

Conclusions

Dicoma tomentosa (Asteraceae) and Psorospermum senegalense (Clusiaceae) appeared to be the best candidates for further investigation of their antiplasmodial properties, reported for the first time by this study.     

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.     

Antimalarial and safety evaluation of Pluchea lanceolata (DC.) Oliv. & Hiern: In-vitro and in-vivo study

Ethnopharmacological relevance

Many of the effective therapeutic strategies have been derived from ethnopharmacologically used natural products. Pluchea lanceolata is an herb employed in Indian folk medicine for malaria like fever but it lacks proper pharmacological intervention.

Aim of the study

To evaluate antimalarial and safety profile of Pluchea lanceolata: an in-vitro, in-vivo for its ethnopharmacological validation.

Materials and methods

Methanol, butanol, ethyl acetate, chloroform, hexane extracts and its isolate, taraxasterol acetate (TxAc) were obtained from air dried aerial part of Pluchea lanceolata. These were tested in-vitro against chloroquine-sensitive strain of Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase activity. The most potent hexane extract and TxAc were further validated for in-vivo antimalarial and safety evaluation. TxAc, a pentacyclic-triterpene isolated from the most active fraction was further evaluated with special emphasis on inflammatory mediators involved in malaria pathogenesis. Murine malaria was induced by intra-peritoneal injection of Plasmodium berghei infected red blood cells to the male Swiss inbred mice. Mice were orally treated following Peters 4-Day suppression test. In-vivo antimalarial efficacy was examined by evaluating the parasitaemia, percent survival, mean survival time, blood glucose, haemoglobin and pro-inflammatory mediators involved in malaria pathogenesis.

Results

Hexane extract and TxAc showed promising antimalarial activity in-vitro and in-vivo condition. TxAc attributed in inhibition of the pro-inflammatory cytokines as well as afford to significant increase in the blood glucose and haemoglobin level when compared with vehicle treated infected mice. We have not observed the synergistic action of combinations of chloroquine and TxAc from our experimental results. In-vitro and in-vivo safety evaluation study revealed that hexane extract is non toxic at higher concentration.

Conclusion

Present study further validates the ancient Indian traditional knowledge and use of Pluchea lanceolata as an antimalarial agent. Study confirms the suitability of Pluchea lanceolata as a candidate for further studies to obtain a prototype for antimalarial medicine.     

Investigation of plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts

Aim of the study

To investigate the activities of the 217 plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts.

Materials and methods

Plant extracts were prepared by the method of maceration using solvents of different polarities. The growth inhibition of chloroquine-resistant Plasmodium falciparum strain (FcB1) was determined by measuring the radioactivity of the tritiated hypoxanthine incorporated. Activity against Leishmania (Leishmania) chagasi and Trypanosoma cruzi was measured by the MTT colorimetric assay. The antifungal tests were carried out by using the CLSI method. The active extracts were tested also by cytotoxicity assay using NIH-3T3 cells of mammalian fibroblasts.

Results

Two hundred and seventeen extracts of plants were tested against Plasmodium falciparum. The eleven active extracts, belonging to eight plant species were evaluated against L. (L.) chagasi, Trypanosoma cruzi, yeasts and in NIH-3T3 cells. The results found in these biological models are consistent with the ethnopharmacological data of these plants. The ethyl acetate extract of Diospyros hispida root showed IC₅₀ values of 1 μg/mL against Plasmodium falciparum. This extract demonstrated no toxicity against mammalian cells, resulting in a significant selectivity index (SI) of 435.8. The dichloromethane extract of Calophyllum brasiliense root wood was active against Cryptococcus gattii LMGO 01 with MIC of 1.95 μg/mL; and Candida albicans ATCC 10231 and Candida krusei LMGO 174, both with MIC of 7.81 μg/mL. The same extract was also active against Plasmodium falciparum and L. (L.) chagasi with IC₅₀ of 6.7 and 27.6 μg/mL respectively. The ethyl acetate extract of Spiranthera odoratissima leaves was active against Cryptococcus gattii LMGO 01 with MIC of 31.25 μg/mL, and against Plasmodium falciparum with IC₅₀ of 9.2 μg/mL and Trypanosoma cruzi with IC₅₀ of 56.3 μg/mL.

Conclusion

The active extracts for protozoans and human pathogenic yeasts are considered promising to continue the search for the identification and development of leading compounds.     

New antiplasmodial alkaloids from Stephania rotunda

Ethnopharmacological relevance

Stephania rotunda Lour. (Menispermaceae) is a creeper growing in many countries of Asia and commonly found in the mountainous areas of Cambodia. As a folk medicine, it has been mainly used for the treatment of fever and malaria. The pharmacological activity is mostly due to alkaloids. Thus the aim of this study is to isolate new bioactive alkaloids from Stephania rotunda and to evaluate their in vitro antiplasmodial activity.

Materials and methods

Alkaloids were isolated and identified from dichloromethane and aqueous extracts using a combination of flash chromatography, high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The purified compounds were tested for in vitro antiplasmodial activity on chloroquine-resistant W2 strain of Plasmodium falciparum.

Results

A new aporphine alkaloid named vireakine (2) along with two known alkaloids stephanine (1) and pseudopalmatine (8), described for the first time in Stephania rotunda, and together five known alkaloids tetrahydropalmatine (3), xylopinine (4), roemerine (5), cepharanthine (6) and palmatine (7) were isolated and identified. The structure of the new alkaloid was established on the basis of 1D and 2D NMR experiments and mass spectrometry. The compounds were evaluated for their in vitro antiplasmodial and cytotoxic activities. All tested compounds showed significant antiplasmodial activities with IC₅₀ ranged from 1.2 μM to 52.3 μM with a good selectivity index for pseudopalmatine with IC₅₀ of 2.8 μM against W2 strain of Plasmodium falciparum and IC₅₀>25 μM on K562S cells.

Conclusions

This study provides evidence to support the use of Stephania rotunda for the treatment of malaria and/or fever by the healers. Alkaloids of the tuber exhibited antiplasmodial activity and particularly cepharanthine and pseudopalmatine.     

Metabolomic investigation of the ethnopharmacological use of Artemisia afra with NMR spectroscopy and multivariate data analysis

Ethnopharmacological relevance

Artemisia afra has been used as an infusion to treat malaria throughout the southern parts of Africa, in much the same way as the antimalarial plant Artemisia annua in China. The antiplasmodial activity of purified components from an apolar fraction of Artemisia afra has been shown in the past. No data on the efficacy of the tea infusion prepared from Artemisia afra are however available.

Objective

To investigate the antiplasmodial activity of various extracts of Artemisia afra including an ethnopharmacological prepared sample. To identify polar metabolites in Artemisia afra and Artemisia annua and by using multivariate data analysis investigate the metabolic differences between these species.

Materials and methods

The antiplasmodial activity of Artemisia afra and Artemisia annua extracts were tested for activity against Plasmodiam falciparum 3D7 (chloroquine-sensitive strain) with chloroquine, quinine and artemisinin as positive controls. Hydrophilic metabolites in Artemisia afra and Artemisia annua were identified directly from the crude extracts through 1D- and 2D-NMR spectra. The NMR spectra were also used to differentiate between the two species using principal component analysis (PCA) for quality control purposes.

Results

The apolar fractions of both Artemisia afra and Artemisia annua showed activity against P. falciparum while activity was only found in the tea infusion of Artemisia annua. Metabolomic studies using 1D- and 2D-NMR spectroscopy identified 24 semi-polar components in Artemisia afra including three new phenylpropanoids for this species: caffeic acid, chlorogenic acid and 3,5-dicaffeoyl quinic acid. PCA analysis conducted on the samples yielded good separation between the polar extracts of Artemisia afra and Artemisia annua.

Conclusion

These findings shows that there are no in vitro activity in the tea infusion of Artemisia afra and lists the identified metabolites causing the metabolic differences between Artemisia afra and Artemisia annua for quality control purposes.     

Medicinal plants from the Yanesha (Peru): Evaluation of the leishmanicidal and antimalarial activity of selected extracts

Aim of the study

Ninety-four ethanolic extracts of plants used medicinally by the Yanesha, an Amazonian Peruvian ethnic group, for affections related to leishmaniasis and malaria were screened in vitro against Leishmania amazonensis amastigotes and against a Plasmodium falciparum chloroquine resistant strain.

Materials and methods

The viability of Leishmania amazonensis amastigote stages was assessed by the reduction of tetrazolium salt (MTT) while the impact on Plasmodium falciparum was determined by measuring the incorporation of radio-labelled hypoxanthine.

Results and conclusions

Six plant species displayed good activity against Plasmodium falciparum chloroquine resistant strain (IC₅₀ < 10 μg/ml): a Monimiaceae, Siparuna aspera (Ruiz & Pavon), A. DC., two Zingiberaceae, Renealmia thyrsoidea (Ruiz & Pavon) Poepp. & Endl. and Renealmia alpinia (Rottb.), two Piperaceae (Piper aduncum L. and Piper sp.) and the leaves of Jacaranda copaia (Aubl.) D. Don (Bignoniaceae).Eight species displayed interesting leishmanicidal activities (IC₅₀ < 10 μg/ml): Carica papaya L. (Caricaceae), Piper dennisii Trel (Piperaceae), Hedychium coronarium J. König (Zingiberaceae), Cestrum racemosum Ruiz & Pav. (Solanaceae), Renealmia alpinia (Rottb.) Zingiberaceae, Lantana sp. (Verbenaceae), Hyptis lacustris A. St.-Hil. ex Benth. (Lamiaceae) and Calea montana Klat. (Asteraceae). Most of them are used against skin affections by Yanesha people.Results are discussed herein, according to the traditional use of the plants and compared with data obtained from the literature.     

HPLC-based activity profiling for antiplasmodial compounds in the traditional Indonesian medicinal plant Carica papaya L

Ethnopharmacological relevance

Leaf decoctions of Carica papaya have been traditionally used in some parts of Indonesia to treat and prevent malaria. Leaf extracts and fraction have been previously shown to possess antiplasmodial activity in vitro and in vivo.

Materials and methods

Antiplasmodial activity of extracts was confirmed and the active fractions in the extract were identified by HPLC-based activity profiling, a gradient HPLC fractionation of a single injection of the extract, followed by offline bioassay of the obtained microfractions. For preparative isolation of compounds, an alkaloidal fraction was obtained via adsorption on cationic ion exchange resin. Active compounds were purified by HPLC–MS and MPLC–ELSD. Structures were established by HR-ESI-MS and NMR spectroscopy. For compounds 5 and 7 absolute configuration was confirmed by comparison of experimental and calculated electronic circular dichroism (ECD) spectroscopy data, and by X-ray crystallography. Compounds were tested for bioactivity in vitro against four parasites (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum), and in the Plasmodium berghei mouse model.

Results

Profiling indicated flavonoids and alkaloids in the active time windows. A total of nine compounds were isolated. Four were known flavonols – manghaslin, clitorin, rutin, and nicotiflorin. Five compounds isolated from the alkaloidal fraction were piperidine alkaloids. Compounds 5 and 6 were inactive carpamic acid and methyl carpamate, while three alkaloids 7–9 showed high antiplasmodial activity and low cytotoxicity. When tested in the Plasmodium berghei mouse model, carpaine (7) did not increase the survival time of animals.

Conclusions

The antiplasmodial activity of papaya leaves could be linked to alkaloids. Among these, carpaine was highly active and selective in vitro. The high in vitro activity could not be substantiated with the in vivo murine model. Further investigations are needed to clarify the divergence between our negative in vivo results for carpaine, and previous reports of in vivo activity with papaya leaf extracts.     

In vivo antimalarial activities of Enantia polycarpa stem bark against Plasmodium berghei berghei in mice

Ethnopharmacological relevance

Enantia polycarpa (PC) Engl. Et Diels (Annonaceae) is used in traditional medicine as an antimalarial remedy in Southern Nigeria.

Aim of the study

The antimalarial activities of ethanolic stem bark extracts of Enantia polycarpa was studied in vivo, in mice infected with Plasmodium berghei berghei.

Materials and methods

The ethanolic stem bark extract of Enantia polycarpa was administered at doses ranging from 200 to 600 mg/kg/day to Plasmodium berghei infected mice in both early and established models of antiplasmodial studies.

Results

The extract of Enantia polycarpa exhibited promising antimalarial activity against both early and established infections. At a dose of 600 mg/kg the extract achieved a 75.8% and 72% chemosuppression of parasitaemia in the study of acute and established infections, respectively. The extract also prolonged mean survival time of Plasmodium berghei infected mice during the study of established infection. The mean survival time of mice administered Enantia polycarpa extract at 600 mg/kg/day (27 days) was significantly longer than infected/untreated control (12 days). For the acute toxicity study the extract had an intraperitoneal LD₅₀ of 186 mg/kg but caused no mortality when administered orally at doses as high as 2,000 and 4,000 mg/kg.

Conclusions

Collectively, the results indicate that Enantia polycarpa is safe when administered orally and possesses promising antimalarial activity, thus supporting its use in traditional medicine for the treatment of malaria.     

A new protoberberine alkaloid from Meconopsis simplicifolia (D. Don) Walpers with potent antimalarial activity against a multidrug resistant Plasmodium falciparum strain

Ethnopharmacological relevance

The aerial components of Meconopsis simplicifolia (D. Don) Walpers are indicated in Bhutanese traditional medicine for treating malaria, coughs and colds, and the infections of the liver, lung and blood. This study is to validate the ethnopharmacological uses of this plant and also identify potent antimalarial drug leads through bioassays of its crude extracts and phytochemical constituents.

Materials and methods

Meconopsis simplicifolia (D. Don) Walpers was collected from Bhutan and its crude MeOH extract was subjected to acid-base fractionation. Through repeated extractions, separations and spectroscopic analysis, the alkaloids obtained were identified and tested for their antimalarial and cytotoxicity activities.

Results

Phytochemical studies resulted in the isolation of one new protoberberine type alkaloid which we named as simplicifolianine and five known alkaloids: protopine, norsanguinarine, dihydrosanguinarine, 6-methoxydihydrosanguinarine and oxysanguinarine. Among the five of the alkaloids tested, simplicifolianine showed the most potent antiplasmodial activities against the Plasmodium falciparum strains, TM4/8.2 (chloroquine–antifolate sensitive strain) and K1CB1 (multidrug resistant strain) with IC₅₀ values of 0.78 μg/mL and 1.29 μg/mL, respectively. The compounds tested did not show any significant cytotoxicity activities against human oral carcinoma KB cells and normal Vero cells of African kidney epithelial cells.

Conclusions

This study validated the traditional uses of the plant for the treatment of malaria and identified a new alkaloid, simplicifolianine as a potential antimalarial drug lead.