In vitro cytotoxic and anticlastogenic activities of saxifragifolin B and cyclamin isolated from Cyclamen persicum and Cyclamen libanoticum.

CONTEXT: The genus Cyclamen L. (Primulaceae) is rich in saponins known to have interesting biological activities. OBJECTIVE: To isolate saxifragifolin B and cyclamin, two triterpene saponins, from Cyclamen libanoticum Hildebr and Cyclamen persicum Mill, and to assess their cytotoxic, clastogenic/aneugenic, and anticlastogenic effects, as well as antioxidant potential. MATERIALS AND METHODS: Saxifragifolin B and cyclamin were tested for their cytotoxicity against SK-BR-3, HT-29, HepG2/3A, NCI-H1299, BXPC-3, 22RV1, and normal DMEM cell lines using WST-1 assay. Their clastogenic/aneugenic activities and anticlastogenic effects against the anticancer drug mitomycin C were assessed by the in vitro micronucleus assay in CHO cells. Their antioxidant capacities were determined using Fe(2+)-chelating and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays. RESULTS: Both saponins were described for the first time in Cyclamen libanoticum. They showed strong cytotoxic activities against the tested cancer cell lines. Saxifragifolin B was found to be 56- and 37-times more active than mitomycin C against breast adenocarcinoma (SK-BR-3) and lung carcinoma (NCI-H1299), respectively. Also, saxifragifolin B did not induce micronuclei formation and prevented cells from mitomycin C clastogenic effect. Cyclamin induced a significant increase of micronucleated cells after metabolic activation with S9 mix, and did not possess any anticlastogenic activity. Both molecules exhibited low antioxidant activities as compared to reference compounds. DISCUSSION AND CONCLUSIONS: This study showed the remarkable cytotoxic activity of saxifragifolin B, especially against breast adenocarcinoma and lung carcinoma and its chemoprotective activity against mitomycin C. Thus, saxifragifolin B could be suggested as a potential cytotoxic drug with a preventive effect against possible exposures to genotoxic agents.

HPLC analysis of Stephania rotunda extracts and correlation with antiplasmodial activity.

Stephania rotunda (Menispermaceae), a creeper commonly found in the mountainous areas of Cambodia, has been mainly used for the treatment of fever and malaria. Thus, the aim of this study is to investigate the chemical composition and antiplasmodial activity of different samples of S. rotunda and compare their antiplasmodial activity with their alkaloid content. Sixteen samples from different parts (roots, stem, and tuber) of S. rotunda were collected from four regions of Cambodia (Battambang, Pailin, Siem Reap, and Kampot). Reversed-phase HPLC was used to determine the content of three bioactive alkaloids (cepharanthine, tetrahydropalmatine, and xylopinine). These three alkaloids have been found in all samples from Battambang and Pailin (samples I-IX), whereas only tetrahydropalmatine was present in samples from Siem Reap and Kampot (samples X-XVI). The analyzed extracts were evaluated for their antiplasmodial activity on W2 strain of Plasmodium falciparum. Among them, 13 extracts were significantly active with inhibitory concentration 50 (IC(50) ) from 1.2 to 3.7 µg/mL and 2 extracts were moderately active (IC(50) = 6.1 and 10 µg/mL, respectively), whereas sample XI was not active (IC(50) = 19.6 µg/mL). A comparison between antiplasmodial activity and concentration of the three bioactive alkaloids in S. rotunda extracts has been realized.

DNA-damaging, mutagenic, and clastogenic activities of gentiopicroside isolated from Cephalaria kotschyi roots.

Gentiopicroside (1) is the major secoiridoid glucoside constituent of Cephalaria kotschyi roots. The mutagenicity, DNA-damaging capacities, and clastogenicity of this molecule were evaluated by the Salmonella typhimurium mutagenicity assay (Ames test) on tester strains TA97a, TA98, TA100, and TA102, the alkaline comet assay, and the micronucleus assay on CHO cells. All tests were performed with and without the metabolization mixture, S9 mix. In the Ames test, the mutagenicity of 1 was limited to TA102 without S9 mix (2.3 rev microg(-1)). The genotoxicity was more evident without S9 mix (0.78 OTMchi(2) units microg(-1) mL) than with the metabolic mixture (0.16 OTMchi(2) units microg(-1) mL) with the comet assay. Similarly, the clastogenicity without S9 mix was 0.99 MNC microg(-1) mL and 0.38 MNC microg(-1) mL with S9 mix in the micronucleus assay. The interaction of 1 with DNA is probably through the involvement of oxidative DNA lesions.

Antioxidant activity of bisbenzylisoquinoline alkaloids from Stephania rotunda: cepharanthine and fangchinoline.

In the present study, we determined the antioxidant activity of cepharanthine and fangchinoline from Stephania rotunda by performing different in vitro antioxidant assays, including 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, N,N- dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging, superoxide anion (O2*-) radical scavenging, hydrogen peroxide scavenging, total antioxidant activity, reducing power, and ferrous ion (Fe2+) chelating activities. Cepharanthine and fangchinoline showed 94.6 and 93.3% inhibition on lipid peroxidation of linoleic acid emulsion at 30 microg/mL concentration, respectively. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol, and trolox indicated inhibitions of 83.3, 92.2, 72.4, and 81.3% on peroxidation of linoleic acid emulsion at the same concentration (30 microg/mL), respectively. According to the results, cepharanthine and fangchinoline have effective antioxidant and radical scavenging activity.

Cytotoxic activity of alkaloids isolated from Stephania rotunda [corrected].

Three major alkaloids: cepharanthine (1), tetrahydropalmatine (2) and xylopinine (3) isolated from Stephania rotunda tuber were investigated for their cytotoxic activity in a panel of human cancer cells (HT29, LS174T, SW620 and HepG2) using MTT assay. In the present study, cepharanthine (1) exerted potent cytotoxicity against colon and hepatoma cancer cell lines with IC(50) values between 2.4 and 5.3 microM while tetrahydropalmatine (2) and xylopinine (3) displayed weak cytotoxicity. In addition, the mutagenic activity of cepharanthine (1) was investigated using a modified liquid incubation technique of the Salmonella/microsomal assay. This alkaloid (1) was found to be non-mutagenic for doses up to 8.2 microM.

Alpha-hederin potentiates 5-FU antitumor activity in human colon adenocarcinoma cells.

The aim of this study was to investigate the ability of alpha-hederin to improve the efficacy of widely prescribed 5-fluorouracil (5-FU) in a human colon adenocarcinoma model. Drug combinations of alpha-hederin and 5-FU using both fixed-concentration and combination index methods were performed in vitro in HT-29 cells. The results showed that alpha-hederin at sub-IC(50) cytotoxic concentrations enhanced 5-FU cytotoxicity about 3.3-fold (p < 0.001). Simultaneous combination of alpha-hederin and 5-FU at their IC(50) ratio showed either a synergistic effect at a moderate cytotoxic range (25% of cell growth inhibition) or an antagonistic effect at a high level of growth inhibition. The data indicate therefore that it is possible to optimize colorectal cancer cell sensitivity to 5-FU with alpha-hederin.

Cytotoxic steroidal saponins from the flowers of Allium leucanthum.

Allium leucanthum C. Koch is an endemic Caucasian species that grows in Georgia. The flowers are used in traditional medicine. Phytochemical investigation allowed the isolation of seven spirostanol type saponins from the flowers. Their structures were elucidated on the base of NMR and HRESIMS spectrometry data. A new compound, which we have named leucospiroside A (5), has been identified as (25R)-5alpha-spirostane-2alpha,3beta,6beta-triol 3-O-beta-glucopyranosyl-(1-->3)-beta-glucopyranosyl-(1-->2)-[beta-glucopyranosyl-(1-->3)]-beta-glucopyranosyl-(1-->4)-beta-galactopyranoside. The six others were known substances, but are described in this plant for the first time. The crude extract, spirostanol and furostanol fractions, as well as isolated compounds, were evaluated for their in vitro cytotoxic activity. Compounds 1-3 and 5 were found to be the most active, with relatively similar IC50 values ranging from 3.7 to 5.8 microM for a lung cancer cell line (A549) and 5.6 to 8.2 microM for a colon cancer cell line (DLD-1).

Bioassay-guided isolation and UHPLC-DAD-ESI-MS/MS quantification of potential anti-inflammatory phenolic compounds from flowers of Inula montana L.

ETHNOPHARMACOLOGICAL RELEVANCE: Flowers of Inula montana L. (Asteraceae), commonly known as "Arnica de Provence", are used in the traditional medicine of Provence in France with the same indication as Arnica montana, for the relief of bruises, as an anti-inflammatory agent. AIMS OF THE STUDY: The aim of our study is to evaluate its anti-inflammatory properties and to justify its traditional uses. Its potential valorization is evaluated in order to propose Inula montana as an alternative to Arnica montana. MATERIALS AND METHODS: Bio-guided fractionation of ethanolic extract allowed the isolation of compounds responsible of the inhibition of NO production. The fractionation was realized using chromatographic techniques and structure elucidation was conducted by ESI-MS and NMR spectral data. Anti-inflammatory effect of ethanolic extract, different fractions and isolated pure compounds was studied in vitro on immortalized mouse macrophages RAW 264.7. An analytical UHPLC-DAD-ESI-MS/MS method was developed for the identification of these compounds in the herbal drug. This UHPLC-DAD method was validated and was used to compare the phenolic profile and content in plant material from the two collection sites: Bonnieux and Merindol. RESULTS: Eleven compounds were identified by UHPLC-MS. Chlorogenic acid (1), Luteolin (2), Nepetin (3), 3,5-O-Dicaffeoylquinic acid (4), 1,5-O-Dicaffeoylquinic acid (5), Nepitrin (6), Hispiduloside (7) and Jaceosid (8) were isolated and identified by NMR. Compounds 9, 10 and 11 were confirmed to be 6-Hydroxykaempferol 3,7-dimethyl ether, Hispidulin and Chrysosplenol C, respectively by comparing retention times and MS/MS data with those of the authentic substances. Six compounds: 1 and 4-8 are reported for the first time in Inula montana L. Compounds 2-8 showed promising anti-inflammatory activity with the release of NO with IC50 value < 7 µM. The UHPLC-DAD method of quantification of three major bioactive compounds (1, 3 and 5) was validated. CONCLUSION: Flowers extracts and isolated compounds present promising anti-inflammatory activity which provides a scientific basis for the traditional use of Inula montana and may be proposed in the same indications as Arnica montana. The developed and validated simple, accurate and rapid UHPLC method can be used for the quality control of the herbal drug.

Apoptosis and cytolysis induced by giganteosides and hederacolchisides in HL-60 cells.

UNLABELLED: The viability, cytolysis and apoptosis-mediated cellular death induced by giganteosides D and E (Gig-D and Gig-E) and hederacolchisides A and A1 (Hcol-A and Hcol-A1) were analysed in HL-60 cells. MATERIALS AND METHODS: The end-point metabolic (WST1) and lactate dehydrogenase (LDH) assays were used. Cell cycle analysis and apoptosis were measured by flow cytometry, DNA laddering and caspase-3 analyses. RESULTS: the HL-60 cell line was more sensitive to Hcol-A1 and Gig-D (IC50 3-5 microM) than to Gig-E and Hcol-A (IC50 8-13 microM; WST1 assay). This was related to LDH release. The induction of apoptosis could be detected without caspase-3 activation after 24 h of treatment. DNA fragmentation could be detected only with Gig-D. With Hcol-A1 and Gig-D, an accumulation of cells in the S-phase and an increase of cells in sub-G1 peak were observed. By the annexinV-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (AAD) assay, the majority of cells were in late apoptosis with Gig-D, and in necrosis with Hcol-A1. CONCLUSION: Hcol-A1 is more cytotoxic than Gig-D, followed by Gig-E and finally Hcol-A. This is related to a membrane permeabilization effect, leading to cytolysis.

Antimalarial activity of alkaloids isolated from Stephania rotunda.

Stephania rotunda (Menispermaceae) is used in traditional medicine for the treatment of fever. Four major alkaloids: dehydroroemerine, tetrahydropalmatine, xylopinine, cepharanthine as well as aqueous extract (SA), dichloromethane extracts (SD1 and SD2) from this plant were tested against Plasmodium falciparum W2 in vitro. Dehydroroemerine, cepharanthine and SD1 were the most active against W2 with IC(50) of 0.36, 0.61microM and 0.7microg/mL, respectively. Their IC(50) on human monocytic THP1 cells were 10.8, 10.3microM and >250microg/mL, respectively. Cepharanthine, SD1 and SA were selected for in vivo antimalarial test against Plasmodium berghei in mice. The results of SD1 and SA at dose of 150mg/kg showed a decrease of 89 and 74% of parasitaemia by intra-peritoneal injection and 62.5 and 46.5% of parasitaemia by oral administration, respectively. The result of cepharanthine at dose of 10mg/kg showed a decrease of 47% of parasitaemia by intra-peritoneal injection and 50% of parasitaemia by oral administration. Drug interaction of chloroquine and major alkaloids indicates that cepharanthine-chloroquine and tetrahydropalmatine-xylopinine associations are synergistic. These results are in agreement with the use of this plant in the treatment of malaria. This is the first report on in vivo antimalarial investigation for Stephania rotunda.

In vitro antimicrobial activity of plants used in Cambodian traditional medicine.

The purpose of the present study was to screen 27 plant species used in the traditional medicine of Cambodia for in vitro antibacterial and antifungal activities. Thirty-three methanolic extracts were tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis and Candida albicans. Screened by disk diffusion assay, the extracts showed antimicrobial activity especially on Gram-positive bacteria. None of the crude methanolic extracts showed activity against P. aeruginosa. Twenty-five selected extracts were evaluated using a micro-dilution test. Harrisonia perforata (roots) and Hymenodictyon excelsum (bark) exhibited a bactericidal effect against S. aureus at a concentration of 500 microg/ml. Azadirachta indica (bark), Harrisonia perforata (roots and stem) and Shorea obtusa (roots) exhibited a bactericidal effect against M. smegmatis at 250 microg/ml.

New sesquiterpene acid and inositol derivatives from Inula montana L.

A phytochemical investigation of the ethanol extract of leaves and flowers of Inula montana L. led to the isolation of one new sesquiterpene acid called Eldarin (1) and four new inositol derivatives, Myoinositol,1,5-diangelate-4,6-diacetate (2), Myoinositol,1,6-diangelate-4,5-diacetate (3), Myoinositol-1-angelate-4,5-diacetate-6-(2-methylbutirate) (4), Myoinositol-1-angelate-4,5-diacetate-6-isovalerate (5) isolated for the first time, along with eleven known compounds described for the first time in Inula montana, 1ß-Hydroxyarbusculin A (6), Artemorin (7), Santamarin (8), Chrysosplenol C (9), 6-Hydroxykaempferol 3,7-dimethyl ether (10), Reynosin (11), Calenduladiol-3-palmitate (12), Costunolide (13), 4-Hydroxy-3,5-dimethoxybenzenemethanol (14), 9ß-Hydroxycostunolide (15) and Hispidulin (16). Structural elucidation has been carried out by spectral methods, such as 1D and 2D NMR, IR, UV and HR-ESI-MS. These compounds have been tested in vitro for anti-inflammatory and cytotoxic activity on macrophages RAW 264.7. As a result, compounds 2, 3, 7, 13, 14, 15 and 16 showed a release of NO with IC50 value <30µM on macrophages.

Screening of selected indigenous plants of Cambodia for antiplasmodial activity.

The in vitro antiplasmodial activity of 117 aqueous, methanol and dichloromethane extracts derived from different parts of 28 indigenous wild plant species was studied. These plants are commonly used in Cambodian traditional medicine. The plant extracts were tested for in vitro activity against a chloroquine resistant Plasmodium falciparum strain (W2). Nine extracts were moderately active with IC(50) values ranging between 5 and 10 microg/ml, 17 extracts were active with IC(50) values ranging between 1 and 5 microg/ml. These 26 extracts derived from eight plants belong to six families. The most active extracts were dichloromethane and came from Stephania rotunda and Brucea javanica with IC(50) values of 1 microg/ml and a selectivity index > or = 25. It is interesting to note that some aqueous extracts were as active as dichloromethane extracts especially aqueous extracts of Stephania rotunda, Brucea javanica, Phyllanthus urinaria and Eurycoma longifolia with IC(50) values of < or = 4 microg/ml. These results are in agreement with statements of healers on traditional uses of these plants for the treatment of malaria and/or fever. In this study, we report the antiplasmodial potential activity of eight plant species from Cambodia. Among them four are tested for the first time.

Chemical Composition, Antioxidant and Cytotoxic Activities of Roots and Fruits of Berberis libanotica.

Fourteen compounds belonging to different chemical classes were characterized in the roots and fruits extracts from Berberis libanotica, using the same HPLC-DAD-MS method. Thirteen were reported, for the first time, from the fruits whereas the roots contained mostly alkaloids of which 3 out of 5 are reported for the first time. Their structures were established on the basis of MS data as gallic acid (1), chlorogenic acid (2), delphinidin (3), oxyacanthine (4), rutin (5), hyperoside (6), berbamine (7), isoquercitrin (8), quercitrin (9), jatrorrhizine (10), palmatine (11), berberine (12), quercetin (13) and luteolin (14). Extracts containing compounds 4 and 7 showed significant cytotoxicity against the HT29 cell line with an IC50 of 12.2-26.1 µg/mL. Fruits extracts, due mostly to compounds 1 and 2, showed potent antioxidant activities with an EC50 of 0.0025-0.019 mg/mL.

Acute and chronic antiinflammatory effects of Hedera colchica in rats.

Hedera helix and Hedera colchica are the members of Araliaceae family. In the present study, we tested the possible antiinflammatory effects of crude (CSE) and purified (SPE) extracts of Hedera colchica in carrageenan- and cotton pellet-induced acute and chronic inflammation models in rats. Both CSE and SPE of Hedera colchica were found to have antiinflammatory effects. The most potent drug was found as indomethacin (90%) in the 4 h measurements, while SPE (83%) and CSE (55%) of Hedera colchica in 100 mg/kg doses were found to have quite potent effects in acute phase of inflammation, in respect to control values. For testing chronic antiinflammatory (antiproliferative) effects, cotton pellet granuloma test was conducted. SPE was found as the most potent drug in chronic phase of inflammation with an effect of 64.32%. Indomethacin was found more potent than the CSE of Hedera colchica, and potency was found as 61.2 and 58%, respectively.

HPLC analysis and cytotoxic activity of Vernonia cinerea.

The extracts of five Cambodian medicinal plants (Aganosma marginata, Dracaena cambodiana, Harrisonia perforata, Hymenodictyon excelsum and Vernonia cinerea) were evaluated in vitro for their cytotoxic activity against HT29 colon adenocarcinoma cells and HepG2 hepatoma cells, using the MTT assay. Among these five plants, Vernonia cinerea displayed potent cytotoxicity. One main sesquiterpene lactone, 8alpha-tigloyloxy-hirsutinolide-13-O-acetate was isolated from the whole plant of V. cinerea. This compound was active against both cancer cell lines (IC50 = 3.50 microM for HT29 and IC50 = 4.27 microM for HepG2). To quantify this compound in the plant, an analytical high-performance liquid chromatography (HPLC) method was developed and validated.

Chionanthus virginicus L.: phytochemical analysis and quality control of herbal drug and herbal preparations.

Root barks of Chionanthus virginicus L. are used in homeopathic medicines in the treatment of icterus and hepatitis. The objective of this study is to identify novel secoiridoids and lignans and to develop a simple and reliable HPLC method for the determination of oleuropein, phillyrin, total secoiridoids and total lignans for quality control and stability studies of C. virginicus herbal drug and preparations. Secoiridoids and lignans were purified by preparative HPLC. Compounds previously described were identified by HPLC according to their retention times and UV spectra. Structures of new compounds were determined by NMR. Two compounds namely excelside B and acetoxypinoresinol-4"-O-beta-D-glucoside are described for the first time in the drug. HPLC separation was performed on Symmetry C18 (Waters) by gradient elution using acetonitrile and 0.2% aqueous phosphoric acid. The method was validated for specificity, linearity, precision, accuracy, limits of detection and quantification for simultaneous determination of secoiridoids and lignans in herbal drug and herbal preparations as mother tinctures. The proposed HPLC method is linear in the range studied (r2 > or = 0.9989) for all the analytes. The method is precise with intra- and inter-day variations of less than 4%. The mean recoveries of the analytes range from 99.65 to 102.81%. The method is successfully applied to the quantification of nine compounds belonging to secoiridoids and lignans and for the stability studies of these compounds. The study allowed completing the phytochemical knowledge of C. virginicus. This simple developed assay could be used as tools for routine quality control of C. virginicus herbal drug and herbal medicinal products.

HPLC determination of majdine in Vinca herbacea.

A reliable HPLC method coupled with DAD detection was developed and validated for determination of majdine in Vinca herbacea. The chromatographic separation was carried out on a Symmetry C18 column (250 mm x 4.6 mm, 5 microm, Waters) with an isocratic solvent system of 25 mM potassium phosphate buffer (pH = 3.0)-acetonitrile. UV detection was performed at 225 nm. Good linear behavior over the investigated concentration range was observed with the value of r2 > 0.9978. The method was reproducible with intra- and inter-day variations of less than 4.38%. The proposed method was linear, accurate, precise and specific. The validated method was successfully applied to quantify majdine in various parts of V. herbacea, which was collected during the flowering months of April and May. The results indicated that the developed HPLC method could be used for the quality control of V. herbacea and for the standardization of its extracts in majdine.

New triterpenoid saponins from Leontice smirnowii.

Three new triterpene saponins, leonticins I (1), J (2) and L (3) were isolated from the tubers of Leontice smirnowii. On the basis of spectroscopic methods, including 2D NMR experiments (DEPT, gs-COSY, gs-HMQC, gs-HMBC and gs-HSQC-TOCSY), mass spectrometry (HR-ESI-MS) and chemical degradation, the structures of the new compounds were elucidated as 3-O-ß-D-glucopyranosyl-(1 → 3)-[ß-D-xylopyranosyl-1 → 2)]-α-L-arabinopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (1), 3-O-[ß-D-xylopyranosyl-(1 → 3)-ß-D-galactopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 3)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (2) and 3-O-[ß-D-xylopyranosyl-(1 → 3)-ß-D-galactopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 3)]-[ß-D-xylopyranosyl-(1 → 2)]-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (3), respectively. The aglycone 3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid was observed for the first time in Leontice species.

Simultaneous HPLC determination of three bioactive alkaloids in the Asian medicinal plant Stephania rotunda.

A reliable high-performance liquid chromatography (HPLC) method coupled with photodiode array detection has been developed and validated for the determination of three major alkaloids: cepharanthine, tetrahydropalmatine and xylopinine in Stephania rotunda Lour. (Menispermaceae) collected in Cambodia. The chromatographic separation was carried out on a Symmetry C8 column (250 mm x 4.6 mm, 5 microm, Waters), with an isocratic solvent system of 25 mM potassium phosphate buffer (pH 3.5) - acetonitrile. UV detection was performed at 282 nm. Good linear behavior over the investigated concentration ranges was observed with values of r2 > 0.9964 for all the analytes. The method was reproducible with intra- and inter-day variations of less than 3.91%. The mean recoveries of the analytes ranged from 95.7 to 104.6%. The proposed method was linear, accurate, precise and specific. The validated method was successfully applied to quantify the three alkaloids in various parts of Stephania rotunda and in tubers collected from different Cambodian regions. The results indicated that the developed HPLC method could be used for the quality control of S. rotunda.