景洪哥纳香中苯乙烯内酯的UPLC-Q-TOF-MS分析

目的:采用超高效液相色谱与串联四级杆飞行时间质谱联用技术(UPLC-Q-TOF-MS)建立快速鉴景洪哥纳香中苯乙烯内酯类化合物的方法.方法:应用ACQU ITY UPLC BEH C18色谱柱,以0.1%甲酸水溶液(A)-乙腈(B)为流动相梯度洗脱,检测波长254 nm,电喷雾离子源,正离子模式下采集数据.结果:根据质谱信息推测了该植物氯仿提取物中12个苯乙烯内酯的结构.结论:本方法分离度好、灵敏度高、快速高效地鉴定了植物中的苯乙烯内酯,同时本文探讨了苯乙烯内酯类化合物的质谱裂解规律.     

野菊花的化学成分研究

目的:研究菊科植物野菊花的化学成分。方法:利用各种色谱技术进行分离,现代波谱技术鉴定化合物结构。结果:从野菊花中分离得到12个化合物,分别鉴定为金合欢素(1),麦黄酮(2),2′,4′-二羟基查尔酮(3),5-羟基-4′,7-甲氧基黄酮(4),7-羟基二氢黄酮(5),异鼠李黄素(6),5,6,7-三羟基-3′,4′,5′-三甲氧基黄酮(7),槲皮素(8),(3β,5α,6β,7β,14β)-eudesmen-3,5,6,11-tetrol(9),丁香脂素(10),鹅掌楸碱(11),5,4′-二羟基-7-甲氧基黄酮(12)。结论:化合物3~7,10~12均为首次从该种植物中分离得到,3,5,7,10,11为首次从该属植物中分离得到。     

美花石斛茎的淀粉粒数量、多糖含量与生物碱含量的相关性研究

目的:研究美花石斛茎的淀粉粒数量、多糖含量、生物碱含量之间的相关性.方法:显微计数法统计茎的淀粉粒数量,硫酸-蒽酮比色法测定多糖含量,溴甲酚绿比色法测定生物碱含量;Pearson积差相关分析、Kendall's等级相关分析、Spearman’s和谐系数分析研究三者的相关性.结果与结论:美花石斛茎的淀粉粒数量与其多糖含量有极显著的正相关性,而淀粉粒数量及多糖含量均与生物碱含量呈显著的负相关性.     

In vitro antitrypanosomal and antiplasmodial activities of crude extracts and essential oils of Ocimum gratissimum Linn from Benin and influence of vegetative stage

Ethnopharmacological relevance

Different parts of Ocimum gratissimum Linn are largely used in folk medicine for the treatment of many diseases, some of which related to parasitical infections as fevers and headaches. In order to validate their use and to clarify the plant part which possesses the best antiparasitic properties, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils and crude extracts from leaves, stems and seeds of Ocimum gratissimum as well as their cytotoxicity.

Materials and methods

The essential oils and ethanol crude extracts of leaves and stems of Ocimum gratissimum from Benin, were obtained in pre and full flowering stages. Seeds obtained only in full flowering stage, were also extracted. The oils were isolated by hydrodistillation and analyzed by GC/MS and GC/FID. Extracts and essential oils were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum. Cytotoxicity was evaluated in vitro against Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) through MTT assay to evaluate the selectivity and toxicity was assessed against Artemia salina Leach.

Results

The essential oils and non-volatile crude extracts of Ocimum gratissimum were more active on Trypanosoma brucei brucei than on Plasmodium falciparum (3D7). This activity varies according to the vegetative stage (pre and full flowering) and the plant part (seeds, stems and leaves) extracted. The best growth inhibition of Trypanosoma brucei brucei was observed with ethanol crude extracts of leaves (IC₅₀=1.66±0.48 μg/mL) and seeds (IC₅₀=1.29±0.42 μg/mL) in full flowering stage with good selectivity (SI>10). The chemical composition of the essential oil from aerial parts (47 compounds), characterized by the presence as main constituents of p-cymene, thymol, γ-terpinene, β-myrcene and α-thujene, depends on the vegetative stage. The oil contained some minor compounds such as myrcene (IC₅₀=2.24±0.27 μg/mL), citronellal (IC₅₀=2.76±1.55 μg/mL), limonene (IC₅₀=4.24±2.27 μg/mL), with good antitrypanosomal activities. These oils and crude extracts were not toxic against Artemia salina Leach and had a low cytotoxicity except leaves and seeds ethanol extracts obtained in full flowering which showed toxicity against CHO and WI38 cells.

Conclusions

Our study shows that ethanol crude extracts of leaves and seeds of Ocimum gratissimum in full flowering stage can be a good source of antitrypanosomal agents. This is the first report about the relation between the plant part extracted, the vegetative stage of the plant, the antitrypanosomal and antiplasmodial activities and the cytotoxicity of essential oils and non-volatile extracts of Ocimum gratissimum from Benin.     

野菊花的化学成分

目的:研究野菊花Chrysanthemum indicum的化学成分。方法:利用多种色谱方法进行分离,并用各种波谱学方法和色谱学方法进行鉴定。结果:共鉴定了13个化合物,刺槐素-7-O-β-D-葡萄糖苷(1),木犀草素(2),木犀草素-7-O-β-D-葡萄糖苷(3),蒙花苷(4),刺槐素7-O-(6″-O-α-L-鼠李糖基)-β-槐糖苷(5),绿原酸(6),丁香树脂醇4″-O-β-D-葡萄糖苷(7),5,7-二羟基色原酮(8),尿嘧啶(9),对羟基苯甲酸(10),4-O-β-D-葡萄糖氧基苯甲酸(11),boscialin(12),blumenol A(13)。结论:化合物5,7,8,11~13为首次从野菊花中分离得到。     

骆驼蓬总生物碱提取物溶解性能表征方法的探讨

目的:考察骆驼蓬总生物碱提取物在不同溶剂中的溶解性能,并对中药提取物溶解性的评价方法进行探讨。方法:分别采用沉淀法、指标成分测定法及粒径测定法测定骆驼蓬总生物碱提取物的溶解性及其在不同溶剂中的粒径分布情况。结果:骆驼蓬总生物碱提取物及其指标性成分骆驼蓬碱、去氢骆驼蓬碱在80%乙醇,95%乙醇及无水乙醇中均具有较好的溶解性,在60%乙醇中溶解性较差,在水中溶解性最差。骆驼蓬总生物碱提取物在不同溶剂中形成混悬液的粒径大小顺序为水95%乙醇无水乙醇80%乙醇60%乙醇。结论:骆驼蓬总生物碱提取物在80%以上的乙醇溶液中具有较好的溶解性。沉淀法和指标成分法测定结果具有一定的相关性;粒径测定法可提供提取物分散情况的信息;3种方法结合能较全面地反映中药提取物溶解性情况。     

杜仲雄花中三萜类化学成分研究

该研究采用正相硅胶柱色谱,反相柱色谱(RP-18),凝胶色谱(Sephadex LH-20)以及重结晶等多种分离纯化手段,从杜仲雄花中分离得到9个化合物,通过化合物的理化性质和光谱数据鉴定其结构分别为:3-oxo-12-en-ursane-28-O-α-Larabinofuranosyl(1→6)-β-D-glucopyranoside(1),2α,3β-dihydroxyurs-12-en-28-oic acid(28→1)-β-D-glucopyranosyl ester(2),熊果酸(3),α-香树脂醇(4),熊果醇(5),3-O-乙酰基熊果酸乙酸酯(6),3-O-乙酰基齐墩果酸(7),白桦脂酸(8),白桦脂醇(9)。其中化合物1为新化合物,化合物2,4~7为首次从该科植物中分离得到。采用MTT法对所分离的9个化合物进行抗肿瘤活性测试,结果表明仅有化合物3对K562和Hep G2细胞显示出一定的抗肿瘤活性,其余化合物对所选的2种细胞均无明显活性。     

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.     

Isolation and identification of antiplasmodial sesquiterpene lactones from Oncosiphon piluliferum

Oncosiphon piluliferum (Asteraceae) is used traditionally to treat a variety of ailments, mainly fevers. This prompted the screening of this plant for antiplasmodial properties. The dichloromethane extract of the aerial parts of the plant showed activity in vitro against the chloroquine-sensitive (IC(50) 2.6microg/ml) and the chloroquine-resistant (IC(50) 3.1microg/ml) strains of Plasmodium falciparum. Through conventional chromatographic techniques and bioassay-guided fractionation, sesquiterpene lactones of the germacranolide and eudesmanolide type displaying significant in vitro antiplasmodial activity (IC(50) values ranging from 0.4 to 4.4microg/ml) were isolated and identified by spectroscopic data. In addition, the cytotoxic effects of the active compounds against Chinese Hamster Ovarian (CHO) cells were evaluated and the compounds were found to be toxic to mammalian cells at similar concentrations. Structure-activity relationships were assessed.     

Cytotoxic and antibacterial triterpenoids derivatives from Clematis ganpiniana

Ethnopharmacological relevance

The roots and rhizomes of Clematis are commonly used as an analgesic, abirritative, antibacterial, antiphlogistic, anticancer and diuretic agent. The Naxi people traditionally used Clematis ganpiniana's (Lévl. Et Vant.) as a diuretic agent, an anti-inflammatory and anticancer remedy.

Aim of study

To investigate the cytotoxic and antibacterial components from Clematis ganpiniana.

Materials and methods

The aboveground part of Clematis ganpiniana was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. Their cytotoxic, apoptosis and antibacterial activities of purified components were also performed.

Results

By bioassay-guided fractionation techniques and chemical characterization, four triterpene glycosides were isolated and their cytotoxicity against cancer cells and antibacterial activity were tested. They showed significant inhibitory activities against MCF-7, MDA-MB-231 with IC50 value of 0.7–16.5 μg/ml, and significant apoptosis for MCF-7 and MDA-MB-231. Moreover, compound 4 showed weak wide-spectrum antibacterial activity.

Conclusion

These results provide promising baseline information for the potential use of Clematis ganpiniana as well as some of the isolated compounds in the treatment of cancer and infectious disease.     

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

Aim of the study

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

Materials and methods

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

Results and discussion

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

Conclusion

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

川山橙枝叶的化学成分研究

对川山橙的化学成分进行研究,通过采用硅胶柱、ODS、Sephadex LH-20等柱色谱方法进行分离纯化,根据理化性质与波谱数据鉴定了14个化合物的结构,分别为11-hydroxytabersonine(1)、venalstonidine(2),sandine(3),齐墩果酸(4),熊果酸(5),白桦脂醇(6),(+)松脂醇(7),(-)丁香脂素(8),8-羟基-松脂素(9),(-)-latifolin(10),甲基条叶蓟素(11),马钱素(12),对苯二甲酸二丁酯(13),β-谷甾醇(14),其中化合物4 ～ 14为首次从该植物中分离得到.     

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.     

川山橙枝叶的化学成分研究

对川山橙的化学成分进行研究,通过采用硅胶柱、ODS、Sephadex LH-20等柱色谱方法进行分离纯化,根据理化性质与波谱数据鉴定了14个化合物的结构,分别为11-hydroxytabersonine(1),venalstonidine (2),sandine(3),齐墩果酸(4),熊果酸(5),白桦脂醇(6),(+)松脂醇(7),(-)丁香脂素(8),8-羟基-松脂素(9),(-)-latifolin (10),甲基条叶蓟素(11),马钱素(12),对苯二甲酸二丁酯(13),β-谷甾醇(14),其中化合物4～14为首次从该植物中分离得到.     

Antiplasmodial activity of root extract and fractions of Croton zambesicus

Aim of the study

Antiplasmodial activity of root extract and fractions of Croton zambesicus were evaluated to ascertain the folkloric claim of its antimalarial activity and elucidate its antiplasmodial mechanism of action.

Material and method

The crude ethanolic root extract (27–81 mg/kg) and gradient fractions ( n- hexane, chloroform, ethyl acetate and methanol; 54 mg/kg) of Croton zambesicus were investigated for antiplasmodial activity against chloroquine - sensitive Plasmodium berghei infections in mice. The antiplasmodial activity during early and established infections as well as the prophylactic activity were investigated. Chloroquine (5 mg/kg) and pyrimethamine (1.2 mg/kg) were used as positive controls. Thin films made from tail blood of each mouse were used to assess the level of parasitaemia of the mice. Oxidant generation potentials of the crude extract and fractions was also evaluated to elucidate their mechanism of action.

Results

The crude root extract (27 – 81 mg/kg) demonstrated significant (P < 0.01–0.001) schizonticidal activity during early and established infections and also had prophylactic activity. The activity was comparable to that of the standard drug used (chloroquine 5 mg/kg, pyrimethamine 1.2 mg/kg). Methanol, ethyl acetate and chloroform fractions had comparative in vivo antiplasmodial activity and oxidant generation potentials.

Conclusion

The antiplasmodial activity of this root extract and fractions which is likely to be through peroxidation confirms the folkloric use of this plant.     

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.     

Chemical composition,cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin

Ethnopharmacological relevance

Cymbopogon species are largely used in folk medicine for the treatment of many diseases some of which related to parasitical diseases as fevers and headaches. As part of our research on antiparasitic essential oils from Beninese plants, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils of four Cymbopogon species used in traditional medicine as well as their cytotoxicity.

Materials and methods

The essential oils of four Cymbopogon species Cymbopogon citratus (I), Cymbopogon giganteus (II), Cymbopogon nardus (III) and Cymbopogon schoenantus (IV) from Benin obtained by hydrodistillation were analysed by GC/MS and GC/FID and were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum respectively for antitrypanosomal and antiplasmodial activities. Cytotoxicity was evaluated in vitro against Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) through MTT assay to evaluate the selectivity.

Results

All tested oils showed a strong antitrypanosomal activity with a good selectivity. Sample II was the most active against Trypanosoma brucei brucei and could be considered as a good candidate. It was less active against Plasmodium falciparum. Samples II, III and IV had low or no cytotoxicity, but the essential oil of Cymbopogon citratus (I), was toxic against CHO cells and moderately toxic against WI38 cells and needs further toxicological studies. Sample I (29 compounds) was characterised by the presence as main constituents of geranial, neral, β-pinene and cis-geraniol; sample II (53 compounds) by trans-p-mentha-1(7),8-dien-2-ol, trans-carveol, trans-p-mentha-2,8-dienol, cis-p-mentha-2,8-dienol, cis-p-mentha-1(7),8-dien-2-ol, limonene, cis-carveol and cis-carvone; sample III (28 compounds) by β-citronellal, nerol, β-citronellol, elemol and limonene and sample IV (41 compounds) by piperitone, (+)-2-carene, limonene, elemol and β-eudesmol.

Conclusions

Our study shows that essential oils of Cymbopogon genus can be a good source of antitrypanosomal agents. This is the first report on the activity of these essential oils against Trypanosoma brucei brucei, Plasmodium falciparum and analysis of their cytotoxicity.     

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.     

Antimycobacterial terpenoids from Juniperus communis L. (Cuppressaceae)

Ethnopharmacological relevance

Juniperus communis is a plant which has been reported as a traditional cure for tuberculosis (TB) and other respiratory diseases.

Aim of the study

The aim of this study was to isolate and identify the constituents responsible for the activity of the n-hexane extract of Juniperus communis roots against Mycobacterium tuberculosis H₃₇Rv and Juniperus communis aerial parts against Mycobacterium aurum. Subsequently, it was to evaluate the activity of the pure isolated compounds against (i) drug-resistant Mycobacterium tuberculosis variants, (ii) non-replicating Mycobacterium tuberculosis and (iii) a range of non-tuberculous mycobacteria (NTM).

Materials and methods

The antimycobacterial activity of Juniperus communis extracts, fractions and constituents was determined against Mycobacterium tuberculosis H₃₇Rv, and against rifampicin-, isoniazid-, streptomycin- and moxifloxacin-resistant variants, using the microplate broth Alamar Blue assay (MABA) method. Isolated constituents were tested against non-replicating Mycobacterium tuberculosis H₃₇Rv, using the low oxygen recovery assay (LORA), and against NTM (Mycobacterium aurum, Mycobacterium phlei, Mycobacterium fortuitum and Mycobacterium smegmatis), using a broth microdilution method. Cytotoxicty studies were performed using mammalian Vero cells.

Results

The antimycobacterial activity of Juniperus communis was attributed to a sesquiterpene identified as longifolene (1) and two diterpenes, characterised as totarol (2) and trans-communic acid (3). All compounds were identified following analysis of their spectroscopic data (1D- and 2D-NMR, MS) and by comparison with the literature and commercial authentic standards when available. Revised assignments for 3 are reported. Totarol showed the best activity against Mycobacterium tuberculosis H₃₇Rv (MIC of 73.7 μM). It was also most active against the isoniazid-, streptomycin-, and moxifloxacin-resistant variants (MIC of 38.4, 83.4 and 60 μM, respectively). Longifolene and totarol were most active against the rifampicin-resistant variant (MICs of 24 and 20.2 μM, respectively). Totarol showed the best activity in the LORA assay (MIC of 81.3 μM) and against all NTM species (MICs in the range of 7–14 μM). Trans-communic acid showed good activity against Mycobacterium aurum (MIC of 13.2 μM). The low selectivity indices (SI) obtained following cytotoxicity studies indicated that the isolated terpenoids were relatively toxic towards mammalian cells. This is the first report of the isolation of (1) and (2) from Juniperus communis roots, and of (3) from the aerial parts. The antimycobacterial activity of (1) and (3), and the activity of (2) against Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium phlei, is reported for the first time. The effect of totarol on drug-resistant variants and non-replicating Mycobacterium tuberculosis has never been published.

Conclusions

The presence of antimycobacterial terpenoids in Juniperus communis aerial parts and roots justifies, to some extent, the ethnomedicinal use of this species as a traditional anti-TB remedy.     

Bioactive Lignans from Zanthoxylum alatum Roxb. stem bark with cytotoxic potential

Ethanopharmacological relevance

Zanthoxylum alatum is used in traditional medicinal systems for number of disorders like cholera, diabetes, cough, diarrhea, fever, headache, microbial infections, toothache, inflammation and cancer. The aim of the present study was to evaluate Zanthoxylum alatum stem bark for its cytotoxic potential and to isolate the bioactive constitiuents.

Material and methods

Cytotoxicity of the different extracts and isolated compounds was studied on lung carcinoma cell line (A549) and pancreatic carcinoma cell line (MIA-PaCa) using MTT assay. Isolation of compounds from most active extract (petroleum ether) was done on silica gel column. Structure elucidation was done by using various spectrophotometric techniques like UV, IR, ¹H NMR, ¹³C NMR and mass spectroscopy. The type of cell death caused by most active compound C was explored by fluorescence microscopy using the acridine orange/ethidium bromide method.

Result

Petroleum ether extract of plant has shown significant cytotoxic potential. Three lignans sesamin (A), kobusin (B), and 4'O demethyl magnolin (C) has been isolated. All lignans showed cytotoxic activities in different ranges. Compound C was the novel bioactive compound from a plant source and found to be most active. In apoptosis study, treatment caused typical apoptotic morphological changes. It enhances the apoptosis at IC₅₀ dose (21.72 µg/mL) however showing necrotic cell death at higher dose after 24 h on MIA-PaCa cell lines.

Conclusion

Petroleum ether extract (60–80 °C) of Zanthoxylum alatum has cytotoxic potential. The lignans isolated from the petroleum ether extract were responsible for the cytotoxic potential of the extract. 4'O demethyl magnolin was novel compound from Zanthoxylum alatum. Hence the Zanthoxylum alatum can be further explored for the development of anticancer drug.