Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Investigation of Antiparasitic Activity of 10 European Tree Bark Extracts on Toxoplasma gondii and Bioguided Identification of Triterpenes in Alnus glutinosa Barks.

Toxoplasmosis is a worldwide parasitosis that affects one-third of the population. People at risk, such as immunocompromised patients (AIDS, chemotherapy treatment) or fetuses (maternal-fetal transmission) can develop severe forms of the disease. The antiparasitic activity of extracts of different polarities (n-heptane, MeOH, MeOH/H2O) of 10 tree species endemic to temperate regions was investigated against Toxoplasma gondii infection in vitro. Our results showed that the n-heptane extract of the black alder (Alnus glutinosa) exhibited a significant antiparasitic activity without any cytotoxicity at the tested concentrations, with an IC50 of up to 25.08 µg/mL and a selectivity index higher than 3.99. The chemical profiling of this extract revealed triterpenes as major constituents. The ability of commercially available triterpene (betulin, betulinic acid, and betulone) to inhibit the growth of T. gondii was evaluated and showed growth inhibition rates of 44%, 49%, and 99% at 10 µM, respectively.

The Three Pillars of Natural Product Dereplication. Alkaloids from the Bulbs of Urceolina peruviana (C. Presl) J.F. Macbr. as a Preliminary Test Case.

The role and importance of the identification of natural products are discussed in the perspective of the study of secondary metabolites. The rapid identification of already reported compounds, or structural dereplication, is recognized as a key element in natural product chemistry. The biological taxonomy of metabolite producing organisms, the knowledge of metabolite molecular structures, and the availability of metabolite spectroscopic signatures are considered as the three pillars of structural dereplication. The role and the construction of databases is illustrated by references to the KNApSAcK, UNPD, CSEARCH, and COCONUT databases, and by the importance of calculated taxonomic and spectroscopic data as substitutes for missing or lost original ones. Two NMR-based tools, the PNMRNP database that derives from UNPD, and KnapsackSearch, a database generator that provides taxonomically focused libraries of compounds, are proposed to the community of natural product chemists. The study of the alkaloids from Urceolina peruviana, a plant from the Andes used in traditional medicine for antibacterial and anticancer actions, has given the opportunity to test different approaches to dereplication, favoring the use of publicly available data sources.

In Vitro and In Vivo Activity of Anogeissus leiocarpa Bark Extract and Isolated Metabolites against Toxoplasma gondii.

Toxoplasma gondii, belonging to the Apicomplexa phylum, is a cosmopolitan protozoan parasite that affects at least 30% of the world's population. In West Africa, the leaves and bark of the tree species Anogeissus leiocarpa (DC.) Guill. & Perr. are used against zoonosis in traditional medicine and play a key role in controlling diseases induced by Apicomplexans such as malaria. In this study, extracts, fractions, and pure compounds obtained from an ethanol extract of the bark of A. leiocarpa were evaluated against T. gondii infection in vitro and in vivo. The crude bark extract showed significant activity on tachyzoites from the T. gondii RH strain (IC50 = 59.30 µg/mL). The crude bark extract without tannins and pure trachelosperogenin E purified by centrifugal partition chromatography showed the highest activity (IC50s = 12.83 and 26.63 µg/mL, respectively) with satisfying selectivity indexes of 9.61 and 9.75, respectively. The crude bark extract without tannins and pure trachelosperogenin E were able to significantly inhibit host cell invasion by the parasite in vitro, while the crude bark extract without tannins was able to increase mice survival in our murine model of chronic toxoplasmosis. These results provide new biological data for natural compounds that could enhance the current panoply of treatments against toxoplasmosis.

Cytotoxicity of Labruscol, a New Resveratrol Dimer Produced by Grapevine Cell Suspensions, on Human Skin Melanoma Cancer Cell Line HT-144.

A new resveratrol dimer (1) called labruscol, has been purified by centrifugal partition chromatography of a crude ethyl acetate stilbene extract obtained from elicited grapevine cell suspensions of Vitis labrusca L. cultured in a 14-liter stirred bioreactor. One dimensional (1D) and two dimensional (2D) nuclear magnetic resonance (NMR) analyses including ¹H, 13C, heteronuclear single-quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY) as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) were used to characterize this compound and to unambiguously identify it as a new stilbene dimer, though its relative stereochemistry remained unsolved. Labruscol was recovered as a pure compound (>93%) in sufficient amounts (41 mg) to allow assessment of its biological activity (cell viability, cell invasion and apoptotic activity) on two different cell lines, including one human skin melanoma cancer cell line HT-144 and a healthy human dermal fibroblast (HDF) line. This compound induced almost 100% of cell viability inhibition in the cancer line at a dose of 100 µM within 72 h of treatment. However, at all tested concentrations and treatment times, resveratrol displayed an inhibition of the cancer line viability higher than that of labruscol in the presence of fetal bovine serum. Both compounds also showed differential activities on healthy and cancer cell lines. Finally, labruscol at a concentration of 1.2 µM was shown to reduce cell invasion by 40%, although no similar activity was observed with resveratrol. The cytotoxic activity of this newly-identified dimer is discussed.

13C NMR and LC-MS Profiling of Stilbenes from Elicited Grapevine Hairy Root Cultures.

Resveratrol and related oligostilbenes are defense molecules produced by grapevine in response to stresses including various elicitors or signal molecules. Together with their prominent role in planta, these compounds have been the center of much attention in recent decades due to their pharmacological properties. The cost-effective production of resveratrol derivatives such as viniferins or more structurally complex stilbene oligomers remains a challenging task. In this study, the chemical diversity of stilbenes produced by Vitis vinifera Pinot Noir hairy roots was investigated after elicitation for 4 days with a mixture of methyl jasmonate (100 µM) and cyclodextrins (50 mM). Two crude extracts obtained from the culture medium and from the hairy roots were fractionated by centrifugal partition chromatography. The fractions were chemically investigated by two complementary identification approaches involving a 13C NMR-based dereplication method and liquid chromatography coupled to mass spectrometry (LC-MS). In total, groups of 21 and 18 molecules, including flavonoids and stilbenes, were detected in the culture medium and root extracts, respectively. These included resveratrol monomers, dimers, trimers, and a tetramer, thus highlighting the ability of elicited hairy root culture systems to synthesize a wide diversity of secondary metabolites of pharmaceutical significance. The main compounds were unambiguously identified as trans-resveratrol, ε-viniferin, trans-piceatannol, pallidol, scirpusin A, eriodictyol, naringenin, vitisin B, and maackin.

Intensified Separation of Steviol Glycosides from a Crude Aqueous Extract of Stevia rebaudiana Leaves Using Centrifugal Partition Chromatography.

Aqueous extracts of Stevia rebaudiana leaves have been approved since 2008 by the Joint Expert Committee for Food Additives as sugar substitutes in many food and beverages in Western and Far East Asian countries. The compounds responsible for the natural sweetness of Stevia leaves include a diversity of diterpenoid glycosides derived from a steviol skeleton. These steviol glycosides also exhibit a low calorific value as well as promising therapeutic applications, particularly for the treatment of sugar metabolism disturbances. In this work, centrifugal partition chromatography is proposed as an efficient technical alternative to purify steviol glycosides from crude aqueous extracts of Stevia leaves on a multigram scale. Two different commercial instruments, including an ASCPC250® and a FCPE300® made of columns containing 1890 and 231 twin-cells, respectively, were evaluated and compared. All experiments were performed with a polar biphasic solvent system composed of ethyl acetate, n-butanol, and water in a gradient elution mode. When using the 1890 partition cell centrifugal partition chromatography column of 250 mL, 42 mg of stevioside, 68 mg of dulcoside A, and 172 mg of rebaudioside A, three major constituents of the initial extract were obtained from 1 g of the initial mixture at purities of 81%, 83%, and 99%, respectively. The productivity was further improved by intensifying the procedure on the 231 partition cell centrifugal partition chromatography column of 303 mL with the sample mass loading increased up to 5 g, resulting in the recovery of 1.2 g of stevioside, 100 mg of dulcoside A, and 1.1 g of rebaudioside A at purities of 79%, 62%, and 98%, respectively. The structures of the isolated compounds were validated by HPLC-UV, ESI-MS, (1)H, and (13)C NMR analyses. Altogether, the results demonstrate that the column design (i.e., the partition cell number) is an important aspect to be considered for a larger scale centrifugal partition chromatography isolation of Stevia-derived natural sweeteners.

Identification of natural metabolites in mixture: a pattern recognition strategy based on (13)C NMR.

Because of their highly complex metabolite profile, the chemical characterization of bioactive natural extracts usually requires time-consuming multistep purification procedures to achieve the structural elucidation of pure individual metabolites. The aim of the present work was to develop a dereplication strategy for the identification of natural metabolites directly within mixtures. Exploiting the polarity range of metabolites, the principle was to rapidly fractionate a multigram quantity of a crude extract by centrifugal partition extraction (CPE). The obtained fractions of simplified chemical composition were subsequently analyzed by (13)C NMR. After automatic collection and alignment of (13)C signals across spectra, hierarchical clustering analysis (HCA) was performed for pattern recognition. As a result, strong correlations between (13)C signals of a single structure within the mixtures of the fraction series were visualized as chemical shift clusters. Each cluster was finally assigned to a molecular structure with the help of a locally built (13)C NMR chemical shift database. The proof of principle of this strategy was achieved on a simple model mixture of commercially available plant secondary metabolites and then applied to a bark extract of the African tree Anogeissus leiocarpus Guill. & Perr. (Combretaceae). Starting from 5 g of this genuine extract, the fraction series was generated by CPE in only 95 min. (13)C NMR analyses of all fractions followed by pattern recognition of (13)C chemical shifts resulted in the unambiguous identification of seven major compounds, namely, sericoside, trachelosperogenin E, ellagic acid, an epimer mixture of (+)-gallocatechin and (-)-epigallocatechin, 3,3'-di-O-methylellagic acid 4'-O-xylopyranoside, and 3,4,3'-tri-O-methylflavellagic acid 4'-O-glucopyranoside.

New biphasic solvent system based on cyclopentyl methyl ether for the purification of a non-polar synthetic peptide by pH-zone refining centrifugal partition chromatography.

A new type 1 ternary biphasic system composed of cyclopentyl methyl ether, dimethylformamide and water was developed, characterized and successfully used for the purification of a lipophilic, protected peptide by pH-zone refining centrifugal partition chromatography. The protected peptide is an 8-mer, key intermediate in bivalirudin (Angiomax®) synthesis and shows a very low solubility in the solvents usually used in liquid chromatography. All ionic groups, except the N-terminal end of the peptide, are protected by a benzyl group. The purification of this peptide was achieved with a purity of about 99.04% and a recovery of 94% using the new ternary biphasic system cyclopentyl methyl ether/dimethylformamide/water (49:40:11, v/v) in the descending pH-zone refining mode with triethylamine (28 mM) as the retainer and methanesulfonic acid (18 mM) as the eluter.

Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts.

Sophorolipids represent an important class of natural surfactants with a variety of environmental, cosmetic, and pharmaceutical applications. Despite their promising physicochemical and biological properties, the use of sophorolipids is hampered by the lack of information regarding their individual structure-activity relationships. The major difficulty in isolating pure sophorolipids arises from the high complexity of crude fermentation media composition and from their strong structural similarities. In this work, a centrifugal partition chromatography method was developed in an original gradient elution mode for the separation of sophorolipids produced by the yeast Candida bombicola. Experiments were realized by using three sets of solvent systems composed of n-heptane, ethyl acetate, n-butanol, methanol, and water in different proportions. The separation was performed at 5 mL/min in the ascending mode by increasing progressively the polarity of the organic mobile phase. In these conditions, more than 80% of the sophorolipids present in the initial crude fermentation extract were eluted successively from the most hydrophobic lactone forms to the most hydrophilic acid forms. The structures of the isolated sophorolipids were further elucidated by HPLC and NMR analyses.

Anti-tumoral Effect of Thymelaea hirsuta L. Extracts in Colorectal Cancer Cells.

BACKGROUND: Conventional chemotherapeutic treatment of colorectal cancer has low efficiency because of its high toxicity. Several studies identified natural compounds as potential antitumor agents by inducing cancer cell cycle arrest or apoptosis and exhibiting a potential synergy in drug combination therapy. Natural compounds derived from plants represent an important source of pharmacologic agents toward several diseases. For example, the Tunisian Thymelaeaceae plants are used in folk medicine for the treatment of different pathologies such as diabetes and hypertension. OBJECTIVE: The Thymelaea hirsuta L. extracts were evaluated for their anti-tumoral activities and their adjuvant potential that could be used in conventional colorectal cancer therapy. METHODS: Fractionation of total methanolic extract from the plant leaves provided 4 fractions using vacuum liquid chromatography. The cytotoxic activities of these fractions were tested toward colorectal cancer cells. RESULTS: Ethyl acetate fraction (E2 fraction) induced cell cycle arrest and apoptosis by activating caspase-3. E2 fraction inhibited cell invasion by reducing integrin α5 expression and FAK phosphorylation. Moreover, E2 fraction potentialized colorectal cancer cells to 5-FU treatment. CONCLUSION: The selected plant Thymelaea hirsuta is the source of natural compounds that inhibited cell growth and invasion and induced cell cycle arrest in colorectal cancer cells. The most interesting result was their potential synergy in 5-FU combination treatment. Further analysis will identify the active compounds and confirm their role in chemotherapeutic treatment by sensitizing colorectal cancer cell to anti-cancer drugs.

Anti-Toxoplasma gondii effect of lupane-type triterpenes from the bark of black alder (Alnus glutinosa) and identification of a potential target by reverse docking.

Toxoplasmosis is a worldwide parasitosis that is generally benign. The infestation may pose a risk to immunocompromized patients and to fetuses when pregnant women have recently seroconverted. Current treatments have numerous side effects and chemoresistance is emerging, hence the need to find new anti-Toxoplasma gondii substances. This study focuses on the antiparasitic potential of lupane-type pentacyclic triterpenes isolated from the bark of black alder (Alnus glutinosa), as well as the hypothesis of their macromolecular target by an original method of reverse docking. Among the isolated triterpenes, betulone was the most active compound with an IC50 of 2.7 ± 1.2 µM, a CC50 greater than 80 µM, and a selectivity index of over 29.6. An additional study of the anti-T. gondii potential of commercially available compounds (betulonic acid methyl ester and betulonic acid) showed the important role of the C3 ketone function and the C28 oxidation level on the lupane-type triterpene in the antiparasitic activity since their IC50 and CC50 were similar to that of betulone. Finally, the most active compounds were subjected to the AMIDE reverse docking workflow. A dataset of 87 T. gondii proteins from the Protein Data Bank was created. It identified calcium-dependent protein kinase CDPK3 as the most likely target of betulin derivatives.

TITLE: Potentiel anti-Toxoplasma gondii de triterpènes de type lupane de l'écorce de l'aulne glutineux, Alnus glutinosa, et identification d'une cible potentielle par docking inverse. ABSTRACT: La toxoplasmose est une parasitose mondiale, généralement bénigne. Les personnes à risque sont les patients immunodéprimés et les fœtus chez les femmes enceintes nouvellement séroconverties. Les traitements actuels ont de nombreux effets secondaires et des phénomènes de chimiorésistance apparaissent, d'où la nécessité de trouver de nouvelles substances actives contre T. gondii. Cette étude porte sur le potentiel antiparasitaire des triterpènes pentacycliques de type lupane isolés de l'écorce de l'aulne glutineux (Alnus glutinosa) et formule une hypothèse quant à leur cible protéique par l'utilisation d'une méthode originale de docking inverse. Parmi les triterpènes isolés, la bétulone s'est révélée être la plus active avec une CI50 de 2,7 µM ± 1,2 µM, une CC50 supérieure à 80 µM et un indice de sélectivité supérieur à 29,6. L'étude complémentaire du potentiel anti-T. gondii de composés disponibles commercialement et analogues à la bétulone (acide bétulonique et methyl ester de l'acide bétulonique) a montré le rôle important de la fonction cétone en C3 et du degré d'oxydation de la position 28 du squelette triterpénique de type lupane dans l'activité antiparasitaire puisque leurs CI50 et CC50 étaient similaires aux valeurs rencontrées pour la bétulone. Enfin, les composés les plus actifs ont été soumis au flux de travail de docking inverse d'AMIDE. Un ensemble de 87 protéines de T. gondii de la Protein Data Bank a été créé. La protéine kinase calcium dépendante CDPK3 a été identifiée comme la cible la plus probable des dérivés de la bétuline.

Anti-radical flavonol glycosides from the aerial parts of Cleome chelidonii L.f.

Eleven previously undescribed flavonoid glycosides, named cleomesides C-M, along with five known compounds, were isolated from the aerial parts of Cleome chelidonii L.f. (Cleomaceae). All flavonol glycosides were esterified derivatives of 3,7-O-diglycosides of quercetin or kaempferol. Their structures were elucidated by analysis of the 1D and 2D NMR spectra, HR-ESI-MS data, UV spectra, optical rotation and by comparison with literature data. The DPPH radical scavenging properties of the flavonoid glycosides were studied in order to appreciate the effect of the glycoside parts and of the ester groups on this activity compared with the quercetin and kaempferol aglycones. An acetate at position 3 of rhamnose linked to C-7 of flavonol, gave compounds with the strongest antiradical activity. An aromatic ester group at position 6 of terminal glucose of diglycoside chain linked to C-3 of flavonol did not seem to influence the antiradical activity.

Schinus terebinthifolius countercurrent chromatography (Part III): Method transfer from small countercurrent chromatography column to preparative centrifugal partition chromatography ones as a part of method development.

Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are support free liquid-liquid chromatography techniques sharing the same basic principles and features. Method transfer has previously been demonstrated for both techniques but never from one to another. This study aimed to show such a feasibility using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. Heptane - ethyl acetate - methanol -water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3ß-masticadienolic acids as target compounds. The optimized separation methodology previously described in Part I and II, was scaled up from an analytical hydrodynamic CCC column (17.4mL) to preparative hydrostatic CPC instruments (250mL and 303mL) as a part of method development. Flow-rate and sample loading were further optimized on CPC. Mobile phase linear velocity is suggested as a transfer invariant parameter if the CPC column contains sufficient number of partition cells.

Bioactivity-guided identification of antimicrobial metabolites in Alnus glutinosa bark and optimization of oregonin purification by Centrifugal Partition Chromatography.

Barks from conifers and broadleaved trees constitute abundant wastes generated from wood harvesting and logging activities. Extracts of such residues obtained from Alnus trees have been reported as interesting resources with potent antibacterial activities. The present study aims to determine the antimicrobial activity of a crude methanol extract prepared from the bark of Alnus glutinosa against a panel of 22 bacteria and yeasts and to optimize a purification method enabling the high production of the most active substances. Fractionation of the crude extract was performed by Centrifugal Partition Chromatography (CPC) using a three-phase solvent system composed of n-heptane, methyl-ter-butyl ether, acetonitrile and water. The major known compounds contained in the fractions produced by CPC were chemically profiled by (13)C NMR dereplication, resulting in the unambiguous identification of oregonin, hirsutanonol, betulinic acid, and alusenone 1a. The antibacterial evaluation of the fractions by bioautography on Staphylococcus aureus revealed that oregonin, in addition to being the major metabolite of the crude extract (∼32% w/w), was the most active with an antibacterial inhibitory effect comparable to antibiotics. The purification of oregonin was optimized at the laboratory-scale by CPC. A single injection of 3.7g of crude extract resulted in a recovery of 72% (850mg) of the available oregonin at purity higher than 94%.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described.

Triterpenoid saponins from the roots of Gypsophila trichotoma Wender.

Eleven triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wender. (G. trichotoma Wender. var. trichotoma) (Caryophyllaceae), together with one known compound. The structures were established on the basis of extensive NMR analysis ((1)H, (13)C NMR, COSY, TOCSY, ROESY, HSQC, and HMBC), completed by analysis of HR-ESI-MS and ESI-MS(n). The saponins have the commonly found gypsogenin as the aglycone substituted at C-3 with trisaccharide and at C-28 with oligosaccharide through a fucose residue, as saponins isolated from Gypsophila perfoliata L. originated from China. The oligosaccharide attached to C-28 is substituted with acetyl and (or) sulfate groups. Тhe cytotoxicity of the saponin extract from G. trichotoma was evaluated against a rat alveolar macrophage-like cell line NR8383 and human leukemia cell lines U937 and BV-173. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.

Triterpene saponins from Antonia ovata leaves.

Six pentacyclic triterpenoid saponins, named antoniosides E-J along with two known alkaloids, were isolated from the leaves of Antonia ovata. Their structures were determined by the extensive use of 1D and 2D-NMR experiments along with HRESIMS analysis and acid hydrolysis. All isolated saponins contained the same pentasaccharide chain: 3-O-[ß-D-glucopyranosyl-(1→2)]-[ß-D-glucopyranosyl-(1→4)]-[ß-D-glucopyranosyl-(1→3)-α-L-arabinopyranosyl(1→6)]-ß-D-glucopyranoside, linked at C-3 of esterified derivatives of polyhydroxyoleanene triterpenoids (theasapogenol A and 15α-hydroxy-theasapogenol A). Isolated compounds were evaluated for their cytotoxic activity against KB cell line by a WST-1 assay, and the IC(50) values ranged from 3.3 to 5.3 µM.

Cytotoxic triterpenoid saponins from the stem bark of Antonia ovata.

Phytochemical investigation of the MeOH extract of the stem bark of Antonia ovata led to the isolation of four triterpenoid saponins, along with eleven known compounds. Their structures were established by extensive 1D and 2D NMR, as well as HR-MS analysis and acid hydrolysis. All isolated saponins contained the same tetrasaccharide chain O-beta-d-xylopyranosyl-(1-->2)-O-beta-d-glucopyranosyl-(1-->3)-O-[beta-d-glucopyranosyl-(1-->2)]-beta-d-glucuropyranoside linked to C-3 of esterified derivatives of R(1)-barrigenol, A(1)-barrigenol, barringtogenol C, or camelliagenin. Biological evaluation of the compounds against KB cell line revealed a potent cytotoxic activity with IC(50) values ranging from 3.1 to 6.6microM. The known compounds were found to be inactive at 10microg/ml concentration.