Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C₁₈ RP) thin-layer chromatography (TLC) and C₁₈ RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C₁₈ RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C₁₈ RP-TLC but not on the C₁₈ RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C₁₈ RP be performed before HPLC analysis.     

Die Glykoside der Wurzeln von Glossostelma carsoni (N. E. BR.) BULLOCK. Glykoside und Aglykone, 297. Mitteilung

Pentacyclic triterpenes, sterols, cardenolides and pregnane derivatives could be identified in extracts from roots of Glossostelma carsoni (N.E. BR .) Bullock. From the first group, the following substances could be isolated in crystalline form: β-amyrin, lupeol, their 3-O-acetyl derivatives, and 3-O-isovaleryl-β-amyrin. The sterols were isolated in the form of two mixed but crystalline fractions. The less polar fraction was shown to be a mixture of β-sitosterol, campesterol, stigmasterol, and cholesterol (stereoisomers are not excluded); the more polar fraction is probably a mixture of monohydroxy derivatives of the four above mentioned sterols.     

Anti-leptospiral activities of an endemic plant Glyptopetalum calocarpum (Kurz.) Prain used as a medicinal plant by Nicobarese of Andaman and Nicobar Islands

Leaves of an endemic plant Glyptopetalum calocarpum are used by Nicobarese tribes of Andaman and Nicobar Islands, India, to prepare traditional medicine for treating fever. In the present investigation, pharmacologically active compounds were isolated from this plant and their antimicrobial efficacy was evaluated against the leptospiral strains. The anti-leptospiral activity of six plant-derived compounds was determined by both microdilution and macrodilution methods. Two out of six compounds, namely lupenone and stigmasterol, showed anti-leptospiral activity. The minimum inhibitory concentrations of the two compounds tested against pathogenic leptospiral strains belonging to 10 serovars were in the range of 100–200 μg/mL. The range of minimum bactericidal concentrations was 400–800 μg/mL. Compounds lupenone, stigmasterol, lupeol, β-amyrin and β-amyrin acetate had negligible or no haemolytic activity, exhibiting IC₅₀ values of greater than 5 mg/mL. Further in vivo studies are needed to investigate the pharmacological and toxicological properties of G. calocarpum before it can be considered as a new anti-leptospiral agent.     

A new fatty aldol ester from the aerial part of Mimosa invisa (Mimosaceae)

A new aldol ester named 17-O-triacontanoylheptadecanal (1) was isolated from the aerial part of Mimosa invisa (Mimosaceae) together with eight known compounds identified as β-sitosterol (2), α-amyrine (3), lupeol (4), 4'-O-methylepinumisoflavone (5), alpinumisoflavone (6), betulinic acid (7), 3-O-β-D-glucopyranoside of sitosterol (8) and epirobinetinidol (9). The structures of compounds were determined on the basis of NMR and mass spectrometry data as well as by comparing the data reported in the literatures. The antimicrobial activities of the crude extract and compounds 1 and 9 were investigated against seven microbial species. The natural products showed moderate activities compared to that of the crude extract.     

Phytochemical Studies of Berberis vulgaris

This study presents a phytochemical investigation of the fruits of Berberis vulgaris Linn. The isolation and structure elucidation of four compounds are discussed. The terpenoids lupeol (1) and oleanolic acid (2) and the steroids stigmasterol (3) and stigmasterol glucoside (4) are isolated for the first time from this plant. The structure and stereochemistry at various asymmetric centers were established by different spectroscopic techniques.     

Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots

The aim of this study was to explore the extract/fractions and compounds of Diospyros lotus against various Gram-positive and Gram-negative bacteria strain. The results showed marked susceptibility of extract and its fractions against test pathogens. Among them, chloroform fraction was most dominant and effective against all tested bacteria. The chloroform fraction was subjected to column chromatography which led to the isolation of lupeol (1), 7-methyljuglone (2), β-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin (6) and 8-hydroxyisodiospyrin (7). Among the isolated compounds, betulinic acid (5) showed significant activity against most of the tested pathogen. In conclusion, our study validated the traditional uses of the plant in the treatment of infectious diseases which was also strongly supported by the isolated compound, betulinic acid (5).     

Ceramide and Cerebroside from the stem bark of Ficus mucuso (Moraceae)

Two new sphingolipids mucusamide (1) and mucusoside (2) have been isolated from methanol soluble part of the stem bark of Ficus mucuso WELW., together with fifteen known secondary metabolites including cellobiosylsterol (3), β-sitosterol (4), stigmasterol (5), β-sitosterol 3-O-β-D-glucopyranoside (6), lupeol acetate (7), ursolic acid (8), procatechuic acid (9), 2-methyl-5,7-dihydroxychromone 8-C-β-D-glucoside (10), apigenin (11), (-)-epicatechin (12), (+)-catechin (13), N-benzoyl-L-phenylalanilol (14), α-acetylamino-phenylpropyl α-benzoylamino-phenylpropionate (15), asperphenamate (16) and bejaminamide (17). Structures of compounds 1 and 2 were elucidated by spectroscopic analysis and chemical methods.     

Antioxidant activity and total phenols from the methanolic extract of Miconia albicans (Sw.) Triana leaves

Miconia is one of the largest genus of the Melastomataceae, with approximately 1,000 species. Studies aiming to describe the diverse biological activities of the Miconia species have shown promising results, such as analgesic, antimicrobial and trypanocidal properties. M. albicans leaves were dried, powdered and extracted to afford chloroformic and methanolic extracts. Total phenolic contents in the methanolic extract were determined according to modified Folin-Ciocalteu method. The antioxidant activity was measured using AAPH and DPPH radical assays. Chemical analysis was performed with the n-butanol fraction of the methanolic extract and the chloroformic extract, using different chromatographic techniques (CC, HPLC). The structural elucidation of compounds was performed using 500 MHz NMR and HPLC methods. The methanolic extract showed a high level of total phenolic contents; the results with antioxidant assays showed that the methanolic extract, the n-butanolic fraction and the isolated flavonoids from M. albicans had a significant scavenging capacity against AAPH and DPPH. Quercetin, quercetin-3-O-glucoside, rutin, 3-(E)-p-coumaroyl-α-amyrin was isolated from the n-butanolic fraction and α-amyrin, epi-betulinic acid, ursolic acid, epi-ursolic acid from the chloroformic extract. The results presented in this study demonstrate that M. albicans is a promising species in the search for biologically active compounds.     

Constituents of Perltrophe Japonica (Thunb.) Bremk

A naturally occurring derivatives of pyridine, 3,5-pyridinedicarboxamide ( 1 ), lupeol ( 2 ), a steroidal mixture [stigmasterol ( 3a ) and β-sitosterol ( 3b )], steroidal glycosides [stigmasteryl-glucoside ( 4a ) and β-sitosteryl glucoside ( 4b )] and allantoin ( 5 ) were isolated and characterized from the aerial parts of Peritrophe japonica. Their structures were determined by spectral analysis.     

Isolation and characterization of triterpenes from the leaves of Orthosiphon stamineus

Orthosiphon stamineus, Benth, belonging to the family Lamiaceae, is a medicinal plant growing wild in tropical countries. Seven triterpenes, ursolic acid, oleanolic acid, betulinic acid, hydroxybetulinic acid, maslinic acid, α-amyrin and β-amyrin have been isolated from the leaves of Orthosiphon stamineus. The structures of these compounds have been established by spectroscopic methods. α-Amyrin was isolated from this plant for the first time.     

Study on Antitumor Plant Gymnosporia Trilocularis

The alcoholic extract of Gymnosporia trilocularis Hay. (Celastraceae) was found to exhibit antitumor activities against P388 lymphocytic leukemia and KB cell culture systems. From this extract β-amyrin ( 1 ), β-sitosterol ( 2 ) and its monoglucoside ( 6 ), stigmasterol ( 3 ) and its monoglucoside ( 7 ), campesterol ( 4 ) and its monoglucoside ( 8 ), maniladiol ( 5 ), dulcitol ( 9 ) and six yet unidentified principles ( 10–15 ) were isolated. The stereochemistry of C-16-OH of maniladiol ( 5 ) was confirmed spectroscopically by ¹H nmr to be equatorial. Of all the isolated compounds, the mixture of three steroid monoglucosides ( 6:7:8 =28:1.1:1) exhibited cytotoxicity (ED₅₀=2.6×10⁰ mcg/ml) and 5 showed only marginal effect (ED₅₀=2.1×10¹ mcg/ml) in KB test system.     

Bioactive phenolic amides from Celtis africana

Nine compounds have been isolated for the first time from Celtis africana, namely trans-N-coumaroyltyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), lauric acid (4), oleic acid (5), palmitic acid (6), lupeol (7), β-sitosterol (8) and oleanolic acid (9), respectively. Their structures have been elucidated by different spectroscopic techniques. The isolated compounds were screened for their antioxidant, anti-inflammatory and acetylcholinestrease enzyme inhibitory activities. Compounds 1-3 showed significant antioxidant and anti-inflammatory activities and weak to moderate acetylcholinestrease enzyme inhibition activity.     

A new dibenzofuran and other constituents from Ligularia caloxantha, a Chinese medicinal plant

A new dibenzofuran named 1,2,4-trimethyl-7,8-dimethoxy-dibenzofuran (1), together with seven known compounds, euparin (2), 2,5-diacetyl-6-hydroxy-benzofuran (3), 2-acetyl-5,6-dimethoxy-benzofuran (4), gummosogenin (5), lupeol (6), stigmasterol (7) and (E)-2,5-dihydroxy-cinnamic acid (8), were isolated from the roots of Ligularia caloxantha, a Chinese medicinal plant. The structures of the compounds were elucidated by spectroscopic methods.     

Long-chain alkanoic acid esters of lupeol from Dorstenia harmsiana Engl. (Moraceae)

In addition to lupeol (1a), three long-chain alkanoic acid esters of lupeol, in which two were new, were isolated from the hexane and ethyl acetate twigs extract of Dorstenia harmsiana Engl. (Moraceae). The structures of the new compounds were elucidated on the basis of 1D and 2D NMR experiments. Some isolated compounds were evaluated for their antimicrobial activities. The lupeol and its three long-chain alkanoic acid esters showed antifungal and antibacterial activities.     

GC-MS Identification and GC-FID Quantitation of Terpenoids in Ononidis spinosae Radix

A GC-MS method has been developed for the qualitative analysis of sterol and triterpene (terpenoid) constituents of Ononis spinosa L. (spiny restharrow) root without derivatization. β-Sitosterol, campesterol, stigmasterol, stigmastan-3,5-diene sterol compounds and the triterpene derivatives β-amyrin and α-onocerin were identified. A validated GC-FID quantitative method was developed for measuring β-sitosterol, the main sterol component, in various extracts of this plant, obtained with organic solvents and by supercritical fluid extraction. The extracts were cleaned by saponifying and then the β-sitosterol was quantified in the non-saponifiable fractions by GC-FID with an internal standard. In addition, the relative concentrations of the other terpenoids were also determined. The β-sitosterol content in the non-saponifiable solvent extraction fractions was 0.19–5.5%, that of the supercritical fractions were 4.8–9.2%, depending on the experimental conditions. The hexane and the pilot scale SFE extracts were considered as main sources of terpenoids (71.8%; 93.3%, respectively).     

Caesaldecan, a cassane diterpenoid from the leaves of Caesalpinia decapetala

A new cassane diterpenoid, caesaldecan, was isolated from Caesalpinia decapetala with eight known compounds, spathulenol, 4,5-epoxy-8(14)-caryophyllene, squalene, lupeol, trans-resveratrol, quercetin, astragalin, and stigmasterol. The (1)H- and (13)C-NMR spectra of the new compound were completely assigned by using a combination of 2D NMR techniques, namely, (1)H-(1)H COSY, HMQC, HMBC, and ROESY.     

Bowdenol, a new 2,3-dihydrobenzofuran constituent from Bowdichia virgilioides

From the stem bark of Bowdichia virgilioides, lupeol, lupeol acetate, sitosterol, stigmasterol and methyl-3-[2-(1-hydroxymethylvinyl)-2,3-dihydrobenzo[b]furan-5-yl]-(E)-2-propenoate have been isolated. The latter is a new compound and it has been named as bowdenol. Their structures were elucidated with the aid of spectroscopic techniques, mostly 1 and 2D NMR. The biogenetic pathway for bowdenol has been suggested.     

Epunctanone,a New Benzophenone,and Further Secondary Metabolites from Garcinia epunctata Stapf (Guttiferae)

A new polyprenylated benzophenone, named epunctanone ( 1 ), was isolated from the stem bark of Garcinia epunctata Stapf , together with eight known compounds, 7‐epiisogarcinol ( 2 ), 2,6‐dimethoxy‐p‐benzoquinone ( 3 ), friedelin ( 4 ), lupeol ( 5 ), 16β‐hydroxylupeol ( 6 ), betulin ( 7 ), stigmasterol ( 8 ), and rheediaxanthone A ( 9 ). The structure of epunctanone ( 1 ) was established by detailed analysis of its spectroscopic data, especially 1D‐ and 2D‐NMR, and HR‐MS data. All these compounds were evaluated for their antimicrobial and anti‐protozoan activities. They were also assayed to determine if any of the compounds were nonpeptide agonist ligands for nematodal G‐protein‐coupled receptors, which would be an indication of potential antinematodal activity. Among the isolated compounds, 7‐epiisogarcinol ( 2 ) was the most active against Candida albicans.     

Chemical constituents and in vitro antioxidant activity of Phyllanthus wightianus

The whole plant of Phyllanthus wightianus (PW) was investigated for the antioxidant effects of three successive extracts: hexane (PWHE), chloroform (PWCE) and methanol (PWME), using standard in vitro models. The PWME exhibited a strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radicals and nitric oxide radical inhibition activity, due to possessing the highest content of tannins. The free radical scavenging effect of PWME was comparable with that of reference antioxidants. The extracts were subjected to isolation of their compounds: isomeric sterol mixture (1) [stigmasterol (1a), compesterol (1b) and β-sitosterol (1c)], fredilin (2), lupeol (3), gallic acid (4), bergenin (5), geraniin (6), corilagin (7) and ellagic acid (8) were established through the use of column chromatographic methods and spectral data. The percentage of tannins was also determined and estimated using the HPLC method. The data suggest that tannins are the active antioxidant compounds of P. wightianus. This study provides proof for the ethnomedical claims and reported biological activities of this plant. The plant therefore has very good therapeutic potential.     

Allelopathic activity and chemical constituents of extracts from roots of Euphorbia heterophylla L

Euphorbia heterophylla L. is regarded as a major weed worldwide. Its high aggressiveness in agricultural environment prompted us to investigate the allelopatic activity and chemical constitution of extracts from roots of this plant. Hexane extract showed low phytotoxic activity. Methanol extract at 2.0 mg mL^?1 inhibited 100% of germination, root and shoot growth of the indicator plants Sorghum bicolor and Lactuca sativa. β-sitosterol, stigmasterol, and esters of lupeol, germanicol, taraxasterol, pseudotaraxasterol, α-amyrin and β-amyrin were isolated from the hexane extract and their structures elucidated on the basis of MS and ¹H, ¹³C and DEPT-135 NMR data. GC-MS analysis of the derivatized methanol extract allowed for identifying a series of allelopathic organic acids potentially involved in allelopathic interactions of E. heterophylla. This is the first study on the allelopathic activity of extracts and identification of metabolites from roots of E. heterophylla.