Antiparasitic activities of two sesquiterpenic lactones isolated from Acanthospermum hispidum D.C

Ethnopharmacological relevance

Aerial parts of Acanthospermum hispidum D.C. are often used by traditional healers in Benin for various diseases and especially for malaria.

Aim of the study

To identify active compounds from extracts of Acanthospermum hispidum D.CV. leaves previously shown to possess antimalarial properties and analyse in vivo activity and toxicity of crude extracts.

Materials and methods

Compounds were isolated from aerial part of Acanthospermum hispidum D.C. and structurally elucidated using extensive spectroscopic analysis. Antiplasmodial activity was evaluated in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity and in vivo against Plasmodium berghei berghei by the 4-day suppressive test. Selectivity of extract and purified compounds on Plasmodium parasites were evaluated by using MTT test on J774 macrophage like murine cells and WI38 human normal fibroblasts and also against two other parasites: Trypanosoma brucei brucei and Leishmania mexicana mexicana. Acute and sub-acute toxicities of a crude extract were evaluated on mice.

Results

Two known sesquiterpenic lactones were isolated: 1 (15-acetoxy-8β-[(2-methylbutyryloxy)]-14-oxo-4,5-cis-acanthospermolide) and 2 (9α-acetoxy-15-hydroxy-8β-(2-methylbutyryloxy)-14-oxo-4,5-trans-acanthospermolide). 1 and 2 showed in vitro antiplasmodial activity against the chloroquine-sensitive strain (3D7) with IC₅₀ of 2.9 ± 0.5 and 2.23 ± 0.09 μM respectively. Only 2 showed a high selectivity index (SI: 18.4) on Plasmodium compared to cytotoxicity against human fibroblasts cell line (WI38). 1 and 2 also showed interesting antiparasitic activities in vitro against Trypanosoma brucei brucei (IC₅₀ of 2.45 ± 0.49 and 6.36 ± 1.42 μM respectively) and Leishmania mexicana mexicana (IC₅₀ of 0.94 ± 0.05 and 2.54 ± 0.19 μM respectively). Furthermore, crude acidic water extract and fractions containing one of the two isolated compounds displayed a weak in vivo antimalarial activity against Plasmodium berghei berghei with a long half-life causing a delayed effect. In vivo acute (2000 mg/kg) and sub-acute (1000 mg/kg) toxicity tests on the crude acidic water extract did not show toxicity.

Conclusion

Crude acidic water extract, fractions and pure isolated compounds from Acanthospermum hispidum showed promising in vitro antiplasmodial activity. Despite our study did not show in vivo acute and subacute toxicities of the crude acidic water extract, its weak in vivo antimalarial activity and the in vitro cytotoxicity of pure compounds and enriched extracts containing 1 and 2 indicate that the aerial parts of Acanthospermum hispidum should be used with caution for malaria treatments.     

Bioactive styryllactones and alkaloid from flowers of Goniothalamus laoticus

Ethnopharmacological relevance

Goniothalamus laoticus (Annonaceae) is being used traditionally as a tonic and a febrifuge by the local people in the northeastern part of Thailand.

Aim of study

To investigate the Thai medicinal plant, Goniothalamus laoticus, for antiplasmodial, antimycobacterial and cytotoxicity activities.

Materials and methods

The flowers extracts of Goniothalamus laoticus were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The antiplasmodial, antimycobacterial and cytotoxicity evaluation of styryllactone derivatives and alkaloid were also performed.

Results

Ten compounds, cinnamic acid (1); dihydrochrysine (2); β-sitosterol (3); six styryllactones, (+)-3-acetylaltholactone (4), goniotriol (5), (+)-altholactone (6), (+)-goniofufurone (7), 9-deoxygoniopypyrone (8), howiinin A (9); and an aporphine alkaloid; (−)-nordicentrine (10) were isolated from flowers of Goniothalamus laoticus. Among these, compounds 1, 3–5, 8–10 are first isolated from the Goniothalamus laoticus. Besides, compound 10 is the first report from the Goniothalamus genus. The isolated compounds were evaluated in antiplasmodial, antimycobacterial and anticancer cell lines tests. Compounds 4–6 and 10 exhibited antiplasmodial activity against Plasmodium falciparum (IC₅₀ 2.6, 7.9, 2.6 and 0.3 μg/mL, respectively), while 5, 6, 9 and 10 showed antimycobacterial activity against Mycobacterium tuberculosis (MIC 100, 6.25, 6.25 and 12.5 μg/mL, respectively). In addition, compounds 4–10 showed cytotoxicity against cancer cells, KB, BC1, NCI-H187, and MCF-7 with IC₅₀ ranging from 0.4 to 22.7 μg/mL.

Conclusion

This finding showed that the styryllactone derivatives and alkaloid isolated from the flowers of Goniothalamus laoticus exhibited antiplasmodial activity against Plasmodium falciparum, antimycobacterail against Mycobacterium tuberculosis and cytotoxicity against four cancer cell lines.     

In vitro estrogenic activity of Asplenium trichomanes L. extracts and isolated compounds

Ethnopharmacological relevance

Asplenium trichomanes was used as an expectorant, anti-cough remedy, laxative, emmenagogue, abortifacient and for irregular menses.

Aim of the study

To investigate the in vitro estrogenic activity of Asplenium trichomanes extracts and isolated compounds and their ability to activate ERα and ERβ.

Materials and methods

Leaves infusion (IF), decoction (DC) and methanol extract (ME) were prepared. MCF7/EREluc cell line which expresses endogenous ERα, and SK-NBE cells transiently transfected with the estrogen receptors (ERα and ERβ) were used for the estrogenic activity assays. Phytochemical investigations were performed (CC, HPLC, etc.) and structure of isolated compounds were achieved on the basis of 1D and 2D NMR techniques and HR-MS spectrometry.

Results

IF and ME were active in our MCF7 model; selectivity for the ERβ receptor was observed in the SK-NBE test. Two new phenol derivatives, 4-vinyl-phenol-1-O-[α-l-rhamno(1 → 6)-β-d-glucopyranosyde] (1) and kaempferol-3-O-α-[2′acetyl]-arabinofuranosyl-7-O-α-l-rhamnopyranoside (2) were isolated with six known compounds (3–8). Compounds 2–4, 7 and 8 showed selectivity for the activation of the ERβ receptor although with a moderate activity compared with 17-β-estradiol.

Conclusion

Further investigations about the estrogenic effects of this plant are needed but our data can, at least in part, explain some of its traditional use as emmeagogue.     

Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents

Ethnopharmacological relevance

Arctium lappa and Tussilago farfara (Asteraceae) are two plant species used traditionally as antitubercular remedies. The aim of this study was (i) to screen Arctium lappa and Tussilago farfara extracts for activity against Mycobacterium tuberculosis and (ii) to isolate and identify the compound(s) responsible for this reputed anti-TB effect.

Materials and methods

The activity of extracts and isolated compounds was determined against Mycobacterium tuberculosis H₃₇R_v using a high throughput spot culture growth inhibition (HT-SPOTi) assay.

Results

The n-hexane extracts of both plants, the ethyl acetate extract of Tussilago farfara and the dichloromethane phase derived from the methanol extract of Arctium lappa displayed antitubercular activity (MIC 62.5 μg/mL). Further chemical investigation of Arctium lappa led to the isolation of n-nonacosane (1), taraxasterol acetate (2), taraxasterol (3), a (1:1) mixture of β sitosterol/stigmasterol (4), isololiolide (5), melitensin (6), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10). Compounds isolated from Tussilago farfara were identified as a (1:1) mixture of β sitosterol/stigmasterol (4), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10), loliolide (11), a (4:1) mixture of p-coumaric acid/4-hydroxybenzoic acid (12), p-coumaric acid (13). All compounds were identified following analyses of their physicochemical and spectroscopic data (MS, ¹H and ¹³C-NMR) and by comparison with published data. This is the first report of the isolation of n-nonacosane (1), isololiolide (5), melitensin (6) and kaempferol-3-O-glucoside (10) from Arctium lappa, and of loliolide (11) from Tussilago farfara. Amongst the isolated compounds, the best activity was observed for p-coumaric acid (13) (MIC 31.3 μg/mL or 190.9 μM) alone and in mixture with 4-hydroxybenzoic acid (12) (MIC 62.5 μg/mL).

Conclusions

The above results provide for the first time some scientific evidence to support, to some extent, the ethno-medicinal use of Arctium lappa and Tussilago farfara as traditional antitubercular remedies.     

The constituents of Cibotium barometz and their permeability in the human Caco-2 monolayer cell model

Ethnopharmacological relevance

Cibotium barometz (L.) J. Sm. (Dicksoniaceae) has been traditionally used as anti-inflammatory and anodyne.

Aim of the study

To investigate the constituents in the rhizomes of Cibotium barometz, and evaluate their permeability in the human Caco-2 model.

Materials and methods

The rhizomes extracts of Cibotium barometz were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The permeability of the main constituents was evaluated using human Caco-2 cell monolayer as a model system.

Results

Three unusual sesquiterpenes having 1-indanone nucleus (1, 3 and 4) and an unusual orthoester spiropyranosyl derivative of protocatechuic acid (2) were isolated from the rhizomes of Cibotium barometz. Among these, the bilateral permeation of 1, 3 and 4 in Caco-2 model was examined. The apparent permeability coefficients (P_app) of 1 was identical with those of propranolol, which is often used as reference standard of high permeability. The P_app values of 3 and 4 were in agreement with those of atenolol, which is often used as reference standard of poor permeability. The permeation rates of 1, 3 and 4 increased linearly as a function of time up to 180 min and with the concentration within the test range of 25–200 μM.

Conclusions

This is the first report on the presence of compounds 2 and 3 in this plant and 4 was a new compound. Compound 1 is assigned for a well-absorbed, and 2 and 3 are assigned for the poorly absorbed compounds in human intestine. A passive diffusion mechanism for transport of 1, 3 and 4 in Caco-2 model was proposed. The results provided some useful information for predicting the oral bioavailability of 1, 3 and 4.     

Antiparasitic compounds from Cornus florida L. with activities against Plasmodium falciparum and Leishmania tarentolae

Aim of the study

The objective of this study was to identify the antiplasmodial constituents from the bark of Cornus florida L., a plant traditionally used in North America for the treatment of malaria.

Methods and materials

Dried and powdered bark was extracted with 95% ethanol. The resultant extract was subjected to in vitro antiplasmodial-guided fractionation against Plasmodium falciparum (D10 strain). Antiplasmodial IC₅₀ values were calculated for pure compounds. Compounds were also assayed against Leishmania tarentolae, and rat skeletal myoblast L6 cells to assess antileishmanial activity and cytotoxicity, respectively.

Results

Antiplasmodial-guided fractionation afforded 8 compounds: betulinic acid (1), ursolic acid (2), β-sitosterol (3), ergosta-4,6,8,22-tetraene-3-one (4), 3β-O-acetyl betulinic acid (5), 3-epideoxyflindissol (6), 3β-O-cis-coumaroyl betulinic acid (7), 3β-O-trans-coumaroyl betulinic acid (8), of which, (6) is for the first time here isolated from a natural product and (4), (7) and (8) are reported for the first time from this genus. In vitro IC₅₀ values against P. falciparum for (4) (61.0 μM) (6) (128.0 μM), (7) (10.4 μM), (8) (15.3 μM) are reported for the first time. Antileishmanial IC₅₀ values are reported here for the first time for (4) (11.5 μM), (6) (1.8 μM), (7) (8.3 μM) and (8) (2.2 μM). Cytotoxicity against L6 cells is reported for all compounds.

Conclusions

The compounds isolated in this study, while displaying moderate in vitro antiplasmodial activity, do not fully support the historical importance of C. florida as an antimalarial remedy in North America. The traditional remedy may exert its well documented effects by mechanisms unrelated to direct antiplasmodial action. While not traditionally used to treat Leishmania, this work shows that several constituents of C. florida possess promising in vitro antileishmanial activity.     

Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum

Aim of the study

To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures.

Materials and methods

Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis.

Results

The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 μmol/kg), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside [2] (16.82 μmol/kg), kaempferol 3-O-neohesperidoside-7-O-α-rhamnopyranoside [3] (13.50 μmol/kg) or α-rhamnoisorobin [5] (23.13 μmol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 μmol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 μmol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 μmol/kg, s.c.; 66.1%).

Conclusions

Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.     

Isolation and identification of antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Ethnopharmacological relevance

Muntingia calabura (Elaeocarpaceae) is one of the most common roadside trees in Malaysia. Its leaves, barks, flowers and roots have been used as a folk remedy for the treatment of fever, incipient cold, liver disease, as well as an antiseptic agent in Southeast Asia. The aim of this study is to isolate and identify the antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Materials and methods

Antibacterial and cytotoxic activities were determined by micro-broth dilution and MTT assays, respectively. Seven fractions (F1–F7), three flavones and a chalcone were isolated from the active EtOAc extract using bioassay-guided screening. The structures of four compounds were elucidated by spectroscopic methods and compared with published data. The compounds were further tested for their antibacterial and cytotoxic activities.

Results

Three flavones and a chalcone [5,7-dihydroxy-3,8-dimethoxyflavone (1), 2′,4′-dihydroxychalcone (2), 5-hydroxy-3,7-dimethoxyflavone (3) and 3,5,7-trihydroxy-8-methoxyflavone (4)] were isolated from the active fraction F5 of EtOAc extract. Compounds 1 and 3 were isolated for the first time from Muntingia calabura L. Antibacterial activity indicates that compound 2 exhibited the most significant activity with MIC value of 50 μg/mL and 100 μg/mL against MSSA and MRSA, respectively. Cytotoxic activity indicates that compounds 2 and 3 exhibited very strong activity against HL60 with IC₅₀ values of 3.43 μg/mL and 3.34 μg/mL, respectively.

Conclusion

The antibacterial activity of the leaves of Muntingia calabura L. is ascribable to the active compound 2 while the cytotoxic activity is ascribable to the active compounds 2 and 3.     

4-Methylthio-butanyl derivatives from the seeds of Raphanus sativus and their biological evaluation on anti-inflammatory and antitumor activities

Ethnopharmacological relevance

Raphanus sativus seeds (Brassicaceae) known as Raphani Semen have long been used as anti-cancer and/or anti-inflammatory agents in Korean traditional medicine. This study was designed to isolate the bioactive constituents from the seed extracts of Raphanus sativus and evaluate their anti-inflammatory and antitumor activities.

Material and methods

Bioassay-guided fractionation and chemical investigation of a methanolic extract of the seeds of Raphanus sativus led to the isolation and identification of seven 4-methylthio-butanyl derivatives. Structural elucidation of the isolated compounds was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques (¹H, ¹³C, COSY, HMQC and HMBC experiments) and mass spectrometry.

Results

The isolated compounds were characterized as in the following: three new 4-methylthio-butanyl derivatives, sinapoyl desulfoglucoraphenin (1), (E)-5-(methylsulfinyl)pent-4-enoxylimidic acid methyl ester (2), and (S)-5-((methylsulfinyl)methyl)pyrrolidine-2-thione (3), together with four known compounds, 5-(methylsulfinyl)-4-pentenenitrile (4), 5-(methylsulfinyl)-pentanenitrile (5), sulforaphene (6), and sulforaphane (7). Full NMR data assignments of the three known compounds 4–6 were also reported for the first time. We evaluated the anti-neuroinflammatory effect of 1–7 in lipopolysaccharide-stimulated murine microglia BV2 cells. Compound 1 significantly inhibited nitrite oxide production with IC₅₀ values of 45.36 μM. Moreover, it also reduced the protein expression of inducible nitric oxide synthase. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and all of them showed antiproliferative activity against the HCT-15 cell, with IC₅₀ values of 8.49–23.97 μM.

Conclusions

4-Methylthio-butanyl derivatives were one of the main compositions of Raphanus sativus seeds, and activities demonstrated by the isolated compounds support the ethnopharmacological use of Raphanus sativus seeds (Brassicaceae) as anti-cancer and/or anti-inflammatory agents.     

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita

Aim of the study

Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita.

Materials and methods

A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively.

Results

The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin (1), jaceosidin (2), cirsilineol (3), cirsimaritin (4), hispidulin (5), quercetin (6), 6-methoxytricin (7), acacetin (8), and apigenin (9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol (3), 6-methoxytricin (7) and apigenin (9) significantly inhibited T cell proliferation and activation in the bioassays.

Conclusion

The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.     

Antimicrobial activity of the crude extract,fractions and compounds from stem bark of Ficus ovata (Moraceae)

Aim of the study

This study was designed to investigate the antimicrobial activities of the methanol extracts from the stem bark of Ficus ovata (FOB), fractions (FOB1–6) and compounds isolated following bio-guided fractionation [3-friedelanone (1), taraxeryl acetate (2), betulinic acid (3), oleanoïc acid (4), 2-hydroxyisoprunetin (5), 6,7-(2-isopropenyl furo)-5,2,4-trihydroxyisoflavone (6), Cajanin (7) and protocatechuic acid (8)].

Materials and Methods

The micro-dilution method was used for the determination of the minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC) against fungi (two species), Gram-positive (three species) and Gram-negative bacteria (five species).

Results

The results of the MIC determinations indicated that the crude extract (FOB), fractions FOB2 and FOB4 as well as compound 5 were active on the entire studied organisms. Other samples showed selective activity, fractions FOB1, FOB3 and FOB5 being active against 50% of the tested microbial species while FOB6 was active on 40%. Compounds 8, 6, 2 and 7 prevented the growth of 80%, 70%, 50% and 20% of the organisms respectively. The lowest MIC value (156g/ml) observed with the crude extract was recorded on Streptococcus faecalis, Candida albicans and Microsporum audouinii. The corresponding value for fractions (39 μg/ml) was noted with FOB4 against Staphylococcus aureus, while that of the tested compounds (10 μg/ml) was observed with compound 8 on Microsporum audouinii. The results of the MMC determination suggested that the cidal effect of most of the tested samples on the studied microorganisms could be expected.

Conclusions

The overall results provided evidence that the studied plant extract, as well as some of the isolated compounds might be potential sources of new antimicrobial drug.     

Bioactivity-guided isolation of polyacetylenes with inhibitory activity against NO production in LPS-activated RAW264.7 macrophages from the rhizomes of Atractylodes macrocephala

Ethnopharmacological relevance

The rhizome of Atractylodes macrocephala (Compositae) is one of the most well-known traditional Chinese medicine in China, Japan and Korea, which has a long history of use for the treatment of splenic asthenia, edema, anorexia, and excessive perspiration, etc. As active compounds of anti-inflammatory activity of this medicinal plant have not been fully elucidated, the aim of this study was to isolate and identify the active constituents inhibiting nitric oxide (NO) production from the rhizomes of A. macrocephala.

Materials and methods

Inhibitory activity against NO production in lipopolysaccharide-activated RAW264.7 macrophages was evaluated by Griess reaction. Fifteen polyacetylenes were isolated from the active ethyl acetate extract using activity-guided screening. The structures of all compounds were elucidated by spectroscopic methods and comparison with published data. The compounds were further tested for their inhibitory activity against NO production.

Results

Seven new polyacetylenes, named atractylodemaynes A–G (1–7), along with eight known ones (8–15) were isolated. Compound 14 was isolated for the first time from the rhizomes of A. macrocephala. The study showed that the tested compounds exhibited inhibitory activity against NO production in a dose-dependent manner. Among them, compounds 10, 11 and 12 had relatively stronger inhibitory effect with IC₅₀ values of 28, 23 and 19 μM, respectively.

Conclusion

The results demonstrated that the polyacetylenes might greatly contribute to the anti-inflammatory activity of the rhizomes of A. macrocephala.     

In vitro relaxant and spasmolytic effects of constituents from Viburnum prunifolium and HPLC quantification of the bioactive isolated iridoids

Ethnopharmacological relevance

Viburnum prunifolium is a North America shrub used in ethnomedicine because of its spasmolytic, sedative, and anti-asthmatic properties.

Aim of the study

Contrasting results were reported in past literature about the active principles of this plant. Our aim was to clarify this matter by evaluating the relaxant and spasmolytic activities of the main constituents obtained from the drug.

Materials and methods

The pharmacological assays were carried out on rabbit jejunum spontaneous contractions and on guinea-pig carbachol-precontracted trachea.

Results

Cumulative concentration (1–100 μg/ml) of Viburnum prunifolium methanolic extract (MeOH extract), its purified fractions soluble in ethylacetate (EtOAc fraction) and in n-butanol (BuOH fraction), and the iridoid glucosides (2 × 10⁻⁵ to 4 × 10⁻⁴ M): 2′-O-acetyldihydropenstemide (1), 2′-O-trans-p-coumaroyldihydropenstemide (2), 2′-O-acetylpatrinoside (3), and patrinoside (4), isolated from EtOAc fraction (1 and 2) and BuOH fraction (3 and 4), induced both relaxant effect of rabbit jejunum spontaneous contractions and spasmolytic effect on guinea-pig carbachol (5.5 × 10⁻⁷ M)-precontracted trachea. Propranolol (10⁻⁶ M) antagonised all Viburnum prunifolium tested components relaxant and spasmolytic effects. At non-relaxing concentrations (0.5 μg/ml), MeOH extract and its fractions induced a potentiating effect of isoprenaline cumulative concentrations also in both isolated tissues.

Conclusion

In both tissues, the order of potency was EtOAc fraction > BuOH fraction > MeOH extract and 1 > 2 > 3 > 4 suggesting that the major iridoids of EtOAc fraction may be considered among the most active compounds.HPLC analysis of the bioactive iridoids indicates that 1 and 2 are present for 7.38% and 14.90% in EtOAc fraction, and 3 and 4 for 18.47% and 8.86% in BuOH fraction. By comparing the values of EC₅₀ of the fractions and compounds isolated from them, we may assume that the iridoids play a significant role in the biological activity of the corresponding fractions.     

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.     

Monoterpene bisindole alkaloids,from the African medicinal plant Tabernaemontana elegans, induce apoptosis in HCT116 human colon carcinoma cells

Ethnopharmacological relevance

Tabernaemontana elegans is a medicinal plant used in African traditional medicine to treat several ailments including cancer. The aims of the present study were to identify anti-cancer compounds, namely apoptosis inducers, from Tabernaemontana elegans, and hence to validate its usage in traditional medicine.

Methods and materials

Six alkaloids, including four monomeric indole (1–3, and 6) and two bisindole (4 and 5) alkaloids, were isolated from the methanolic extract of Tabernaemontana elegans roots. The structures of these compounds were characterized by 1D and 2D NMR spectroscopic and mass spectrometric data. Compounds 1−6 along with compound 7, previously isolated from the leaves of the same species, were evaluated for in vitro cytotoxicity against HCT116 human colon carcinoma cells by the MTS metabolism assay. The cytotoxicity of the most promising compounds was corroborated by Guava-ViaCount flow cytometry assays. Selected compounds were next studied for apoptosis induction activity in HCT116 cells, by evaluation of nuclear morphology following Hoechst staining, and by caspase-3 like activity assays.

Results

Among the tested compounds (1−7), the bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were found to be cytotoxic to HCT116 cells at 20 µM, with compound 5 being more cytotoxic than the positive control 5-Fluorouracil (5-FU), at a similar dose. In fact, even at 0.5 µM, compound 5 was more potent than 5-FU. Compounds 4 and 5 induced characteristic patterns of apoptosis in HCT116 cancer cells including, cell shrinkage, condensation, fragmentation of the nucleus, blebbing of the plasma membrane and chromatin condensation. Further, general caspase-3-like activity was increased in cells exposed to compounds 4 and 5, corroborating the nuclear morphology evaluation assays.

Conclusions

Bisindole alkaloids tabernaelegantine C (4) and tabernaelegantinine B (5) were characterized as potent apoptosis inducers in HCT116 human colon carcinoma cells and as possible lead/scaffolds for the development of anti-cancer drugs. This study substantiates the usage of Tabernaemontana elegans in traditional medicine to treat cancer.     

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana

Ethnopharmacological relevance

Tridax procumbens is an active herb against leishmaniasis.

Aim of the study

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

Materials and methods

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

Results

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

Conclusions

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

Antimicrobial activity of the methanolic extract and compounds from Morus mesozygia stem bark

Aim of the study

This study was aimed at investigating the antimicrobial activity of the methanolic extract (MMB) and compounds isolated from the stem bark of Morus mesozygia, namely 3β-acetoxyurs-12-en-11-one (1), moracin Q (2), moracin T (3), artocarpesin (4), cycloartocarpesin (5), moracin R (6), moracin U (8), moracin C (9), and moracin M (10).

Materials and Methods

The liquid microdilution assay was used in the determination of the minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC), against nine bacterial and two fungal species.

Results

The results of the MIC determination showed that the compounds 3, 4, 8 and 9 were able to prevent the growth of all tested microbial species. All other samples showed selective activities. Their inhibitory effects were noted on 90.9% studied organisms for the crude extract, 81.8% for compound 6, 72.7% for compound 10, 63.6% for compound 1, 54.5% for compound 5, and 45.5% for compound 2. The lowest MIC value of 39 μg/ml was obtained with the crude extract against Escherichia coli. The corresponding value for compounds (5 μg/ml) was registered with compound 9 on Shigella dysenteriae and compound 3 on E. coli, S. dysenteriae, Pseudomonas aeruginosa, Salmonella typhi and Bacillus cereus. The lowest MIC value (39 μg/ml) observed with the crude extract (on E. coli) was only eightfold greater than that of gentamycin used as reference antibiotic (RA) while the corresponding value (5 μg/ml) recorded with compounds 3 and 9 was equal to that of RA on the corresponding microorganisms.

Conclusions

The obtained results highlighted the interesting antimicrobial potency of M. mesozygia as well as that of the studied compounds, and provided scientific basis for the traditional use of this species.     

Anti-HIV-1 activities of compounds isolated from the medicinal plant Rhus chinensis

Aim of the study

Previously, we reported that the petroleum ether fraction, RC-1, and EtOAc fraction, RC-2, of the medicinal plant Rhus chinensis showed potent anti-HIV-1 activities. To address anti-HIV-1 constituents of RC-1 and RC-2, 17 compounds were isolated. Anti-HIV-1 activities and possible action mechanisms of these compounds were investigated.

Methods

The syncytial formation induced by HIV-1 was determined under the inverted microscope, cellular toxicity and protection assay were assessed by MTT method, reduction of p24 antigen expression level and RT activity were measured by ELISA, and inhibition of recombinant HIV-1 PR was monitored by the fluorescent signal.

Results

The compounds 1 and 13 inhibited HIV-1-induced syncytium formation potently with TI value of 42.31 and 19.07, respectively. Compounds 4, 5, 6, 9 and 10 were less potent with TI value of 8.94, 8.22, 4.14, 5.11 and 5.34, respectively. Compound 1, a benzofuranone-type compound, previously reported as a novel anti-HIV-1 agent, might target late-steps of HIV-1 life cycle. Compound 13 inhibited HIV-1 replication with EC₅₀ of 7.16 μg/ml and might target at/before integration step.

Conclusion

These compounds might contribute to anti-HIV-1 activities extracts of the medicinal plant Rhus chinensis.     

Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease

Aim of the study

Parkinson's disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of Banisteriopsis caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism.

Materials and methods

The aqueous extract of Banisteriopsis caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron^®. Structure elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl transferase enzymes, as well as antioxidant and cytotoxic activities of both Banisteriopsis caapi extract and isolated compounds was evaluated.

Results

An examination of the aqueous extracts of Banisteriopsis caapi cultivar Da Vine yielded two new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing “azepino[1,2-a]tetrahydro-β-carboline” unique carbon framework. One additional new natural tetrahydronorharmine (4), four known β-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two known proanthocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and a new disaccharide β-d-fructofuranosyl-(2 → 5)-fructopyranose (14) together with known sacharose (15) and β-d-glucose (16) were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10–13, 17, 18) were also prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant human brain monoamine oxidase (MAO)-A and -B enzymes (IC₅₀ 2.5 and 2.0 nM, and 25 and 20 μM, respectively), and (−)-epicatechin (8) and (−)-procyanidin B2 (9) showed potent antioxidant and moderate MAO-B inhibitory activities (IC₅₀ < 0.13 and 0.57 μg/mL, and 65 and 35 μM). HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7–9) were present in high concentrations in dried bark of large branch. Analysis of regular/commercial Banisteriopsis caapi dried stems showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency.

Conclusion

Collectively, these results give additional basis to the existing claim of Banisteriopsis caapi stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders.