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.     

Activation of M3 muscarinic receptor and Ca²⁺ influx by crude fraction from Crotonis Fructus in isolated rabbit jejunum

Ethnopharmacological relevance

Crotonis Fructus is the mature fruit of Croton tiglium L. (Euphorbiaceae), which has been used in traditional Chinese medicine for the treatment of gastrointestinal (GI) diseases, such as constipation, abdominal pain, peptic ulcer, and intestinal inflammation for thousands of years. The aim of this study was to investigate the pharmacological effect of extracts and fractions from Crotonis Fructus on GI tract.

Materials and methods

The activities of methanol extract and fractions from Crotonis Fructus on the smooth muscle contractions were evaluated using isolated rabbit jejunum model.

Results

The results suggest that the n-BuOH and H₂O fractions showed spasmolytic activity, while the MeOH extract, PE and EtOAc fractions exerted spasmogenic effect. Moreover, bioassay-guided fractionation verified that the EtOAc fraction was more potent than others, followed by PE fraction and methanol extract. Additionally, atropine (10 μM), 4-DAMP (10 μM) and verapamil (0.1 μM) produced a significant inhibition of contractions caused by EtOAc fraction, while either hexamethonium (10 μM) or methoctramine (10 μM) was inactive. Additionally, a HPLC fingerprint of EtOAc fraction was appraised to ensure its chemical consistency and the main component has been identified as phorbol 12-acetate-13-tiglate.

Conclusions

These data indicate that the regulatory effect of EtOAc fraction on GI motility are medicated via the activation of M3 muscarinic receptor and Ca²⁺ influx through L-type Ca²⁺ channel. These provide a scientific basis for the traditional use of Crotonis Fructus in GI disorders.     

Identification and pharmacological characterization of the anti-inflammatory principal of the leaves of dwarf elder (Sambucus ebulus L.)

Aim of the study

The performed investigations aimed on the identification of the anti-inflammatory principal of extracts of leaves of Sambucus ebulus L. (dwarf elder) in order to rationalize the traditional use of this plant for the treatment of chronically inflammatory diseases.

Materials and methods

Dwarf elder leaf extract was subjected to activity guided fractionation using inhibition of TNFα induced expression of vascular cell adhesion molecule 1 (VCAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) as monitoring tool (positive control: parthenolide 10 μM, VCAM-1 expression (% of control): 5.35 ± 0.38%).

Results

Bio-guided isolation resulted in identification of ursolic acid as anti-inflammatory principal. Besides its inhibitory effects against TNFα induced expression of VCAM-1 (IC₅₀ 6.25 μM), ursolic acid inhibits also TNFα induced expression of ICAM-1 (IC₅₀ value between 3.13 and 6.25 μM) (positive control: parthenolide 10 μM, ICAM-1 expression (% of control): 38.89 ± 16.6%). Toxic effects of ursolic acid on HUVECs can be drastically reduced using an enriched extract instead of the pure compound.

Conclusions

Our findings suggest an additional mechanism of the anti-inflammatory activity of ursolic acid by demonstrating its ability to inhibit TNFα-stimulated expression of VCAM-1 and ICAM-1 and support the traditional use of extracts and preparations of Sambucus ebulus L., rich in ursolic acid, for the treatment of chronically inflammatory processes.     

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.     

Vascular effects and antioxidant activity of two Combretum species from Democratic Republic of Congo

Ethnopharmacological relevance

Combretum racemosum P. Beauv (Combretaceae) leaves (CrLv) and root bark (CrRB) and Combretum celastroides subsp. laxiflorum Welw (Combretaceae) leaves (ClLv) are used in Congolese traditional medicine for several therapeutic purposes, notably for the treatment of conditions consistent with hypertension. The present study aims to investigate the vasorelaxant and in vitro antioxidant activities of these plants polar extracts and to examine the in vivo antihypertensive effect of the extract which displays the most potent vasorelaxant effect.

Material and methods

The vasorelaxant effect of CrLv, CrRB and ClLv methanolic extracts was studied on rat aorta rings pre-contracted with phenylephrine (PE, 1 μM) in the presence or absence of the endothelium. In some experiments, prior to the addition of the extract, rings were incubated for 30 min with either L-N^G-nitroarginine methyl ester (L-NAME; 100 μM), a nitric oxide synthase (NOS) inhibitor, indomethacin (10 μM), a cyclooxygenase inhibitor, or 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM), a guanylate cyclase inhibitor. The antioxidant activity was determined by the measurement of the scavenging ability of extracts towards the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Blood pressure was measured on normotensive Wistar rats and spontaneously hypertensive rats (SHR) treated orally with a daily dose (40 mg/kg) of the CILv extract for 5 weeks. Tested extracts have been characterised by TLC profiles targeted at flavonoids.

Results

All tested extracts showed an important DPPH scavenging activity, ranging from 0.6 to 1.1 quercetin-equivalents. They caused a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1 μM). The responses to CrRB and CrLv methanolic extracts reached 74.0±5.1% and 62.2±8.6% at a cumulative concentration of 50 μg/ml, respectively. The ClLv (10 μg/ml) extract was more active and, in the same conditions, relaxed aortic rings by 90.3±5.8%. The vasorelaxant activity of all extracts disappeared or was significantly attenuated by removal of the endothelium or after pretreatment with L-NAME or ODQ. Indomethacin only inhibited the activity of CrLv and CrRB extracts. The ClLv extract was able to lower the systolic blood pressure in SHR rats by 7% after a 5-week treatment.

Conclusions

The present study shows that methanolic extracts from ClLv, CrRB and CrLv have an antioxidant activity and an endothelium-dependent vasorelaxant effect. ClLv induces the vasorelaxant effect through the NO-cGMP pathway while CrLv and CrRB extracts also act via a prostanoid pathway. ClLv extract demonstrated a modest but significant antihypertensive activity in SHR rats.     

Vasorelaxant and antihypertensive effects of methanolic extracts from Hymenocardia acida Tul

Ethnopharmacological relevance

In Congolese traditional medicine, decoctions of Hymenocardia acida root bark (HaRB) and trunk bark (HaTrB) are used for the treatment of conditions assumed to be hypertension. In this work, we propose to study the vasorelaxant effect of HaRB and HaTrB methanolic extracts on isolated rat thoracic aorta, to characterize the group of molecules responsible for the observed vasorelaxant activity, to evaluate the in vitro antioxidant activity of these extracts and to determine the antihypertensive activity of the HaRB extract on spontaneously hypertensive rats (SHR).

Materials and methods

The vasorelaxant effect of the HaRB and HaTrB methanolic extracts was studied on endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 1 μM). The mechanism of this vasorelaxant effect was investigated on endothelium-denuded vessels and on endothelium-intact aortic rings in the presence of three inhibitors: l-N^G-nitroarginine methyl ester (100 μM), indomethacin (10 μM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 μM). To determine the nature of the compounds responsible for the vasorelaxant activity, we carried out a fractionation of the extracts and a thiolysis of the most active fraction followed by a liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) analysis. The extracts antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) colorimetric assay. In vivo anti-hypertensive activity of the HaRB extract was conducted on SHR.

Results

HaRB and HaTrB methanolic extracts produced a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1 μM). The vasorelaxant responses obtained were 95.3±1.5% (5 μg/ml) and 100.6±3.0% (1 μg/ml), respectively. The effect was markedly attenuated by removal of endothelium or pretreatment of aortic rings with all inhibitors except indomethacin. The LC/ESI-MS analysis of the thiolysis products indicated that the fraction which caused the most important vasorelaxation (97.9±2.5% at 3 μg/ml) was a mixture of procyanidins and prodelphinidins, with a predominance of procyanidins. Both extracts and all fractions from HaRB extract showed a DPPH scavenging activity, ranging from 0.4 to 0.8 quercetin-equivalents. The HaRB methanolic extract reduced the systolic blood pressure in SHR (from 214±3 mmHg to 194±4 mmHg) after a 5-week treatment.

Conclusions

The methanolic extracts of Hymenocardia acida root and trunk bark have vasorelaxant activity. The vasorelaxant effect observed is endothelium-dependent and seems mainly mediated through the NO-cGMP pathway. The COX pathway is not involved. The vasorelaxant activity appears to be due to polymeric procyanidins and prodelphinidins. These extracts also have an antioxidant effect. The extract of Hymenocardia acida root bark shows a significant but weak antihypertensive activity in SHR.     

Hepatoprotective effects of lychee (Litchi chinensis Sonn.): a combination of antioxidant and anti-apoptotic activities

Aim of the study

Gimjeng and Chakapat lychee (Litchi chinensis Sonn.) were evaluated for hepatoprotective activity on CCl₄-induced hepatotoxicity in rats.

Materials and methods

Fruit pulp extracts of the lychees were examined for vitamin C, phenolic contents, anti-lipid peroxidation activity and hepatoprotective effect. Male Wistar albino rats were intraperitoneally injected (ip) with CCl₄ (2 ml/kg), then were orally administered (po) with silymarin (100 mg/kg), and Gimjeng or Chakapat extracts (100 and 500 mg/kg). After ten days, the rats were sacrificed and their livers were examined histopathologically and immunohistochemically. Their serum glutamate-pyruvate transaminase, glutamate-oxalate transaminase, and alkaline phosphatase activities were analyzed. Apoptotic activity of the livers was assessed quantitatively.

Results

The Gimjeng and Chakapat extracts showed the contents of vitamin C (1.2 ± 0.6 and 4.3 ± 0.1 mg/100 g) and phenolics like trans-cinnamic acid and pelargonidin-3-O-glucoside (9.80 ± 0.21 and 19.56 ± 0.4 mg GAE/g extract, respectively), and trolox equivalent antioxidant capacity (TEAC) values (11.64 and 9.09 g/mg trolox), respectively. The Gimjeng as compared to the Chakapat demonstrated a better antioxidant activity as revealed by anti-lipid peroxidation activity with the TEAC values. Administration of CCl₄ in rats elevated the serum GPT, GOT, and ALP level whereas silymarin, Gimjeng and Chakapat extracts prevented these increases significantly. Significant decrease of apoptotic cells together with restoration of morphological changes confirmed the hepatoprotective effect in the CCl₄-induced rats pretreated with the extracts.

Conclusion

Antioxidant properties of the Gimjeng and Chakapat lychees as evidenced by the vitamin C and phenolic compounds, anti-lipid peroxidation and anti-apoptosis could explain the hepatoprotective effects in CCl₄-induced hepatotoxicity.     

Antioxidant activities of aqueous leaf extracts of Toona sinensis on free radical-induced endothelial cell damage

Ethnopharmacological relavence

In Taiwan, Toona sinensis (Toona sinensis) is well known as a traditional Chinese medicine, while the underlying pharmacological mechanisms of this drug are still a matter of debate.

Materials and methods

The purpose of this study was to evaluate the protective effects of non-cytotoxic concentrations of aqueous leaf extracts of Toona sinensis (TS extracts; 50-100 μg/mL) and gallic acid (5 μg/mL), a major component of these extracts, against AAPH-induced oxidative cell damage in human umbilical vein endothelial cells (ECs).

Results

Exposure of ECs to AAPH (15 mM) decreased cell viability from 100% to 43%. However, ECs were pre-incubated with TS extracts prior to AAPH induction resulted in increased resistance to oxidative stress and cell viability in a dose-dependent manner. An increase in ECs-derived PGI₂ and IL-1β in response to AAPH exposure was positively correlated with cytotoxicity and negatively with TS extracts concentrations. In addition, gallic acid also suppressed PGI₂ and IL-1β production in AAPH-induced ECs. Notably, TS extracts/gallic acid treatment significantly inhibited ROS generation, MDA formation, SOD/catalase activity, and Bax/Bcl-2 dysregulation in AAPH-stimulated ECs. Pretreatment of ECs with TS extracts/gallic acid also suppressed AAPH-induced cell surface expression and secretion of VCAM-1, ICAM-1 and E-selectin, which was associated with abridged adhesion of U937 leukocytes to ECs. Moreover, TS extracts/gallic acid treatment significantly inhibited the AAPH-mediated up regulation of PAI-1 and down regulation of t-PA in ECs, which may decrease fibrinolytic activity.

Conclusions

Therefore, Toona sinensis may possess antioxidant properties that protect endothelial cells from oxidative stress. Our results also support the traditional use of Toona sinensis in the treatment of free radical-related diseases and atherosclerosis.     

Screening of Caesalpinia bonduc leaves for antipsoriatic activity

Ethnopharmacological relevance

Leaves of Caesalpinia bonduc (L.) Roxb. (Caesalpiniaceae) have been used by traditional Siddha healer of Malabar region for psoriasis treatment.

Aim of the study

To evaluate the Caesalpinia bonduc decoction (CBD), Caesalpinia bonduc hydroalcoholic extract (CBHA) for antipsoriatic activity.

Materials and methods

Mouse tail test for psoriasis was used for the evaluation of antipsoriatic activity. Extracts were tested at a dose of 500 mg/kg b.w. and fractions at 250 mg/kg b.w. in Swiss albino mice. Parameters studied in the mouse tail test were changes in epidermal thickness and percentage orthokeratotic values. In vitro antiproliferant assay on HaCaT cell lines and in vitro lipoxygenase inhibition were also carried out.

Results

Butanol fraction of Caesalpinia bonduc hydroalcoholic extract (CBHAB) and water fraction of Caesalpinia bonduc hydroalcoholic extract (CBHAW) produced significant orthokeratosis (p < 0.001). In relative epidermal thickness, a significant (p < 0.05) reduction with respect to control was observed in groups treated with retinoic acid, CBD, butanol fraction of Caesalpinia bonduc decoction (CBDB), water fraction of Caesalpinia bonduc hydroalcoholic extract (CBHAW). Maximum antiproliferant activity was shown by CBHA (IC₅₀, 77.5 ± 12.7 μg/ml). In lipoxygenase inhibition assay, water fraction of Caesalpinia bonduc decoction (CBDW) showed maximum activity with an IC₅₀ value of 164.71 ± 4.57 μg/ml.

Conclusions

Among all the tested samples only CBHAW showed good activity in the mouse tail test, antiproliferant activity in HaCaT cells and lipoxygenase inhibition assay. Other extracts and fractions showed varying degrees of activity. The present study supports the traditional use of Caesalpinia bonduc leaves for psoriasis treatment.     

Acetylene compound isolated from Atractylodes japonica stimulates the contractility of rat distal colon via inhibiting the nitrergic-purinergic relaxation

Aim of the study

Our previous research has showed that rhizome of Atractylodes japonica Koidz (Compositae) exhibits an increase in the spontaneous contractility of distal colon in rats. The aims of this study are to identify the phytochemical(s), which stimulate(s) the colonic contractility, contained in Atractylodes japonica and to evaluate the pharmacological mechanism responsible for the colonic muscle contraction.

Materials and methods

Based on the stimulatory activity-guided fractionation on the isometric contraction of rat distal colonic strips, atractylodiol (ATD) and diacetyl-atractylodiol (DATD) were isolated from the CHCl₃ fractions of Atractylodes japonica.

Results

ATD and DATD dose-dependently increased both tension and amplitude of distal colon longitudinal muscle (DCLM), but they stimulated only amplitude in the distal colon circular muscle. The ED₅₀ values of ATD and DATD to stimulate the amplitude of DCLM were revealed as 9.1 × 10⁻⁹ M and 1.8 × 10⁻⁸ M, respectively. l-NAME (0.1 mM) significantly increased the ADT (1 μM)-induced contraction of DCLM, whereas SNAP (0.1 mM) markedly reduced the stimulatory effects of ATD on DCLM contractility. The combined effects of SNAP and atropine (0.5 μM) on the ATD-induced contraction of DCLM were similar to the inhibitory effects of SNAP alone. Suramin (0.1 mM) significantly enhanced the increase of ATD-induced DCLM contraction, whereas ADPβS (0.1 mM) markedly abolished the stimulatory effects of ATD on the spontaneous contractility of DCLM.

Conclusions

The present results demonstrate that acetylene compounds, ATD and DATD, are the effective phytochemical of Atractylodes japonica to stimulate the motility of distal colon in rats, and ATD possibly enhances the spontaneous contractility of distal colon through inhibiting the mechanism of nitrergic-purinergic relaxation.     

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.     

In vitro antiplasmodial activity of indole alkaloids from the stem bark of Geissospermum vellosii

Ethnopharmacological relevance

The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the antiplasmodial constituents have not yet been described. As part of our ongoing investigations of new bioactive compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of isolated fractions and purified alkaloids from Geissospermum vellosii.

Materials and methods

Indole alkaloids were isolated and identified from a methanolic crude extract of Geissospermum vellosii bark using a combination of high performance counter current chromatography, mass spectrometry and nuclear magnetic resonance technologies. The methanolic extract, the crude alkaloid fractions and the purified compounds were tested for in vitro antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10).

Results

An indole alkaloid (4) along with four known indole alkaloids, geissolosimine (1), geissospermine (2), geissoschizoline (3), and vellosiminol (5) were isolated and structure elucidated. The antiplasmodial activity (IC₅₀) of the methanolic crude extract was 2.22 μg/mL, while for the isolated compounds it ranged from 0.96 μM to 13.96 μM except for (5) which showed a low activity (157 μM). Geissolosimine (1) showed the highest antiplasmodial activity (0.96 μM).

Conclusions

This study provides evidence to support the use of Geissospermum vellosii as an antimalarial agent, as used by the native populations. Geissolosimine (1) is a lead molecular structure for possible antimalarial drug development.     

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.     

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.     

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.     

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.     

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.     

Proliferative effects of five traditional Nigerian medicinal plant extracts on human breast and bone cancer cell lines

Ethnopharmacological relevance

The medicinal plants Hunteria umbellata (HUL), Cola lepidota (CCL), Persea americana leaf (PAL), Root bark of Persea americana (RPA) and Plukenetia conophora (PCL) are used in Nigerian traditional medicine for the treatment of cancer and cancer related diseases.

Aim of the study

To scientifically evaluate the cell proliferative and apoptotic effects of the plants extracts using breast and osteocarcinoma cell lines, and also to identify the possible components via LC-MS to have a kind of chemical fingerprint.

Materials and methods

The antiproliferative and apoptotic effects of methanolic extracts (10 μg/ml) of the five medicinal plants were subjected to in vitro evaluation using four cancer cell lines (breast-MCF-7 and BT-20; Osteocarcinoma-MG-63 and Saos-2) measured by flow cytometry. Non-tumorigenic controls MCF-12A and primary isolated osteoblasts (POB) were chosen to eliminate negative influence on healthy tissue.

Results

Of the five extracts RPA demonstrated a significant (P < 0.05) anti-proliferative activity against estrogen receptor positive breast cancer cell lines (MCF-7). The proliferative phase was decreased by 18%, whereas, a significant increase in cell proliferation (about 27%) was observed for RPA at a concentration of 10 μg/ml. PCL, CCL, HUL and PAL did not show marked inhibition of the proliferation of cell line MCF-7.

Conclusion

These results give suggestive evidence that the plant extracts exhibit some correlation between the claimed ethnomedicinal uses and the cell proliferative activity. RPA extract includes chemical compounds with estrogen-like activity and validates its potential use as anticancer agent, particularly against breast carcinoma; provided important information potentially helpful in drug designing and discovery. Further studies will involve the isolation of anti tumour compounds in RPA by LC-MS and detailed mechanism of anticancer activities.