Identification of triterpenoid saponins from Anemone rivularis var. flore-minore and study on their antitumor activities / 中国药房

OBJECTIVE To study the triterpenoid saponins from Anemone rivularis var. flore-minore and their antitumor activities. METHODS The n-butanol extract of 70％ ethanol extract from rhizome of the plant was separated. The triterpenoid saponins were separated and purified by normal silica gel column chromatography ，reversed phase ODS column chromatography ， Sephadex LH- 20 gel column chromatography and semi-preparation high performance liquid chromatography. The structures of these saponins were identified by spectral analysis （NMR and MS ）and physical and chemical properties. MTT assay was used to test the proliferation inhibitory activity of the compounds against five kinds of human tumor cells （HL-60 cells，A549 cells，HepG2 cells，HeLa cells and U 87MG cells ）. The apoptosis inducing effect of compound 7 on U 87MG cells was evaluated by flow cytometric Annexin V-FITC/PI staining test. RESULTS：Sixteen triterpenoid saponins were obtained and identified as 3 β-O-β-D-xylopyranosyl-（1→2）-α-L-arabinopyranosyl-oleanolic acid-28-O-α-L-rhamnopyranosyl- （1→4） -β-D-glucopyranosyl-（1→6）-β-D-glucopyranoside（1），3β-O-L-arabinopyranosyl oleanolic acid- 28-O-β-D-glucopyranoside（2），saponin B （3）， 163.com oleanolic acid- 3β-O-β-D-glucopyranosyl-（1→2）-α-L-arabino- pyranoside（4），HN-saponin F （5），clematoside S （6），prosapogenin CP 4（7），cussonside B （8），pulsatilla saponin C （9）， clemastanoside D （10），3 β-O-β-D-glucopyranosyl-（1→2）-β-L-arabinopyranosyl-hederagenin-28-O-β-D-glucopyranoside（11）， ciwujianoside C 3（12），ciwujianoside A 1（13），huzhangoside D （14），kalopanaxsaponin B （15）and hederacolchiside E （16）. Compounds 3，4，6-9 displayed inhibitory activities on the proliferation of tumor cells to different extent ，and compound 7 had the strongest activity ；compound 7 induced the apoptosis of U 87MG cell so as to inhibit the proliferation of cancer cells in a time-dependent manner. CONCLUSIONS The obtained 16 saponins are all identified as oleanolane-type ，among which compound 1 is a new compound. The monodesmosidic saponins ，the sugar chain of which attached at C- 3 and a free carboxyl at C- 28， possess stronger antitumor activity than others.

New triterpenoid saponins from the leaves of Ilex chinensis and their hepatoprotective activity / 中国天然药物

Seven new triterpenoid saponins, including five ursane-type saponins, ilexchinenosides R-V (1-5), and two oleanane-type saponins, ilexchinenosides W-X (6-7), with four known triterpenoid saponins (8-11) were isolated from the leaves of Ilex chinensis. Their structures were elucidated by comprehensive spectroscopic 1D and 2D NMR and HR-ESI-MS data. Their sugar moieties were determined by HPLC analysis compared with standards after hydrolysis and derivatization. Compounds 1, 2, 4, 9 and 10 exhibited potential hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell injury in vitro.

Vascular mechanisms of monodesmosidic triterpene saponins isolated from Passiflora quadrangularis L / Mecanismos vasculares de saponinas triterpénicas monodesmosídicas aisladas de Passiflora quadrangularis L

Background: Passiflora quadrangularis L. has antihypertensive and anxiolytic properties observed in experimental models. Objectives: The aim of this work was to establish the vascular effects exerted by two known monodesmosidic triterpene saponins, 3-O-ß-D-glucopyranosyloleanolic acid (Compound 1) (not previously described for this plant) and, 3-O-[ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl] oleanolic acid (Compound 2), isolated from the ethanolic extract of Passiflora quadrangularis L. leaves. Methods: The structural elucidation was achieved by Nuclear Magnetic Resonance (NMR) experiments and High-Resolution Mass Spectrometry (HRMS). Aortic rings from Wistar rats, previously stimulated with phenylephrine (PE, 1µM) and washed, were exposed to cumulatively concentrations of compound 1 and compound 2 (10 to 400 µM). Ethanolic extract from leaves of P. quadrangularis L. (10 to 320 µg/ mL) and clonidine (1nM to 100µM) were also used for comparison. Concentration response curves of compounds 1 and 2 were examined in presence and absence of: endothelium, the alpha-2 antagonist yohimbine (1 and 100 µM), the alpha non selective antagonist phentolamine (1µM), the alpha-1 antagonist prazosin (1µM) and the calcium channel blocker verapamil (10 and 100 µM). In addition, cumulatively response curve of acetylcholine (ACh, 10nM to 10µM) and sodium nitroprusside (SNP, 1nM to 100µM) were assayed in rings precontracted with compounds 1 and 2 (400 µM). Results: Compounds 1 and 2 elicited a vasoconstriction response in intact aorta rings in similar way (pEC50: 3.92±0.01 and 4.09±0.01, respectively), effect that did not change in denuded rings (pEC50: 3.90±0.01 and 4.11±0.01). The potency order (pEC50) of compounds 1 and 2 decreased according to the following: verapamil (3.53±0.01 and 3.90±0.02; p<0.05) < yohimbine (3.65±0.01 and 3.94±0.02; p<0.05) < prazosin (3.86±0.01 and 4.30±0.02) < phentolamine (4.05±0.02 and 4.05±0.01). SNP but not ACh, was able to decrease the vasopressor effect of compounds 1 and 2 (pIC50: 8.61±0.01 and 8.24 ± 0.15, respectively). Conclusions: Compounds 1 and 2 are key metabolites responsible for the ex vivo vasoconstrictor response induced by P. quadrangularis L. Activation of voltage-dependent calcium channels and/or α2-adrenergic receptors stimulation could be mechanisms implicated.

Antecedentes: Passiflora quadrangularis L. tiene propiedades antihipertensivas y ansiolíticas observadas en modelos animales. Objetivos: El objetivo de este trabajo fue establecer los efectos vasculares ejercidos por dos conocidas saponinas triterpénicas monodesmosídicas: el ácido 3-O-ß-D glucopiranosiloleanólico (Compuesto 1) (no descrito previamente para esta especie vegetal) y el ácido 3-O-[ß-D-glucopiranosil- (1→2)-ß-D-glucopiranosil]oleanólico (Compuesto 2), aisladas del extracto etanólico de las hojas de Passiflora quadrangularis L. Métodos: La elucidación estructural se llevó a cabo mediante experimentos de Resonancia Magnética Nuclear (NMR) y determinaciones de Espectrometría de Masas de Alta Resolución (HRMS). Los anillos aórticos de ratas Wistar, previamente estimulados con fenilefrina (PE, 1 µM) y lavados, fueron expuestos a concentraciones acumulativas del compuesto 1 y compuesto 2 (10 a 400 µM). El extracto etanólico de las hojas de P. quadrangularis L. (10 a 320 µg / ml) y clonidina (1 nM a 100 µM) se utilizaron para la comparación. Las curvas de concentración respuesta de los compuestos 1 y 2 se examinaron en presencia y ausencia de: endotelio, el antagonista alfa-2 yohimbina (1 y 100 µM), el antagonista alfa no selectivo fentolamina (1 µM), el antagonista alfa-1 prazosina (1 µM) y el bloqueador de canales de calcio verapamilo (10 y 100 µM). Además, la curva de concentraciones acumulativas de acetilcolina (ACh, 10 nM a 10 µM) y nitroprusiato de sodio (SNP, 1 nM a 100 µM) se ensayó en anillos pre-contraídos con los compuestos 1 y 2 (400 µM). Resultados: Los compuestos 1 y 2 provocaron una respuesta de vasoconstricción en los anillos de aorta intactos de manera similar (pEC50: 3.92 ± 0.01 y 4.09 ± 0.01, respectivamente), este efecto no cambió en los anillos denudados (pEC50: 3.90 ± 0.01 y 4.11 ± 0.01). El orden de potencia (pEC50) de los compuestos 1 y 2 disminuyó de la siguiente manera: verapamilo (3.53 ± 0.01 y 3.90 ± 0.02; p <0.05) < yohimbina (3.65 ± 0.01 y 3.94 ± 0.02; p <0.05) < prazosina (3.86 ± 0.01 y 4.30 ± 0.02)

Research progress on natural pentacyclic triterpenoids and derivatives in anti-cardiovascular and cerebrovascular diseases / 中草药

Cardiovascular and cerebrovascular diseases are the leading cause of death among adult populations in China. The incidence and mortality rate shows an upward trend, and age of onset is younger trend. It is necessary to treat cardiovascular and cerebrovascular diseases with Chinese materia medica. Natural products have been the important and productive source of leading compounds for the development of anti-cardiovascular and cerebrovascular drugs. Naturally occurring triterpenoid saponins are generally plant-derived secondary metabolites with various valuable pharmacological properties. Pentacyclic triterpenoids of which show obvious anti-inflammatory, antitumor and antiviral effects. In recent years, increasing researches focused on the anticardiovascular and cerebrovascular mechanisms and modifications to improve the pharmacological activity and druggability of pentacyclic triterpenes. Based on previous studies, the literatures and patents of nearly 10 years on pentacyclic triterpenoids and their derivatives have been consulted. The research progress of anti-cardio-cerebrovascular activities of four types of compounds, oleanane, ursane, lupane, and friedelane, related to pentacyclic triterpenoids is reviewed, which provides a basis and reference for its further development and utilization in the treatment of cardiovascular and cerebrovascular diseases.

Study on triterpenoid glycosides from roots of Clematis manshurica / 中草药

Objective: To study the chemical constituents from the roots of Clematis manshurica. Methods: Ten compounds were separated and purified by silica gel, Sephadex LH-20, and preparative HPLC. The structures were identified by physicochemical properties and spectral analyses. Results: A total of ten oleanane-type triterpenoid saponins were isolated and identified as clematomandshurica saponin L (1), clematichinenoside A (2), clematochinenoside F (3), clematernoside A (4), clematichinenoside B (5), clematichinenoside C (6), clematomandshurica saponin B (7), clematomandshurica saponin D (8), clematomandshurica saponin C (9), and huzhangoside B (10), respectively. Conclusion: Compound 1 is a new oleanane-type triterpenoid glycoside, and compounds 2-6 are isolated from this plant for the first time.

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation / 中国中药杂志

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

Chemical constituents from Isodon coetsa distributed in Guizhou / 中草药

Objective: To investigate chemical constituents from Isodon coetsa distributed in Guizhou. Methods: Chemical constituents were isolated and purified by chromatography with silica gel, Sephadex LH-20, and semi-preparative HPLC, and their structures were identified by analysis of spectroscopic evidences and physicochemical properties as well as relevant references. Results: A total of 19 compounds were isolated from plant material extracted with 95% aqueous methanol, which were elucidated as β-sitosterol (1), stigmasterol (2), β-daucosterol (3), methyl linolenat (4), 9,12-octadecadienoic acid-2-hydroxy-1,3-propanedinyl ester (5), ursolic acid (6), oleanoic acid (7), 3β-hydroxy-urs-20-en-28-oic acid (8), betulinic acid (9), kurarinone (10), maslinic acid (11), 2α-hydroxy ursolic acid (12), 2α,3α-dihydroxy-urs-12-en-28-oic acid (13), 2α,3α,24-trihydrxyolean-12-en-28-oic acid (14), 2α,3β,24- trihydroxyolean-12-en-28-oic acid (15), formononetin (16), emodin (17), uracil (18), and sucrose (19). Conclusion: Compounds 2, 4, 5, 9, 11, 13-19 are isolated from this plant for first time, and compounds 11, 17, 19 are isolated from the genus of Isodon for the first time.

Biosynthesis analysis of hederagenin pathway and its construction in yeast cells / 中国中药杂志

Hederagenin is an effective constituent of many medical plants, such as Clematidis Radix, and has a wide range of applications in anti-tumor, anti-inflammatory, antidepressant, hepatoprotective antibacterial, et al. In order to obtain the efficient production of yeast cells for hederagenin,we successfully cloned and screened out a P450 gene MdMA02 from Malus×domestica which can catalyze oleanolic acid C-23 oxidation with our developed plug and play platform. Its amino acid homology is only 32% as compared to characterized CYP72A68v2. By transforming MdMA02 to the oleanolic acid-producing strain BY-OA, a hederagenin-producing strain was constructed and hederagenin's titer could achieve 101 mg·L⁻¹ using high cell density fermentation, which was 337 times higher than in shake flasks culturing. This study provides a basis for further research on promoting the creation of oleanane-type pentacyclic triterpenoids biosynthetic pathway analysis and relative cell factories construction.

Chemical constituents from flowers of Gentiana dahurica / 中草药

Objective: To investigate the chemical constituents of the flowers of Gentiana dahurica. Methods: All compounds were isolated and purified by silica gel, Sephadex LH-20, ODS, and MCI column chromatography. Their structures were determined by physicochemical properties and spectral data. Results: Twenty compounds were isolated from the flowers of G. dahurica. Among them, eight triterpenoids were identified as roburic acid (1), 3β-acetoxy-28-hydroxy-12-ene-oleanane (2), 28-hydroxy-α-amyrin (3), 28-hydroxy-β-amyrin (4), α-amyrin (5), β-amyrin (6), ursolic acid (7), and oleanolic acid (8). Twelve flavonoids were identified as 1-hydroxy-3,7,8-trimethoxyxanthone (9), kaempferol (10), naringenin (11), apigenin (12), luteolin (13), (2S)-naringenin-7-O-β-D- glucopyranoside (14), apigenin-7-O-β-D-glucopyranoside (15), luteolin-7-O-β-D-glucopyranoside (16), isoorientin (17), isovitexin (18), saponarin (19), and lutonarin (20). Conclusion: Seventeen compounds 1-11, 13-16, 19, and 20 are found from flowers of G. dahurica for the first time; Compounds 2-7, 9-11, 13-16, 19, and 20 are isolated from this species for the first time.

Research progress in structure characteristic, spectral regularity, and bioactivity of barrigenol type triterpenoids / 中草药

Barrigenol type triterpenoids which distribute widely in many plants have the mother structure of oleanane skeleton with polyhydroxy substitution. There are always acyl and glucose substitutes on the hydroxyl groups which lead to the variety biological activity of barrigenol type triterpenoids. Large amounts of research have revealed that the barrigenol type triterpenoids have strong cytotoxic activity against many human cancer cell lines and could improve learning and memory of rats that maybe can prospect for antitumor uses and neurodegenerative disease. Combined with the literature, we summarize the structure elucidation, spectral regularity, and bioactivity of barrigenol type triterpenoids for the future development and application of this kind of compounds.

Four new triterpenoid saponins from Polygala crotalarioides / 中草药

Objective: To study the chemical constituents of Polygala crotalarioides. Methods: The compounds were isolated by silica gel, RP-18, Sephadex LH-20 column chromatography, and semi-pre HPLC. Their structures were elucidated on the basis of spectral analysis and chemical evidence. Results: Four oleanane-type saponins were isolated from the roots of P. crotalarioides. Their structures were elucidated as 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-β-D-fucopyranoside (1), 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-D-glucopyranosyl-(1→3)]-[4-O-(E/Z)-4″-mehoxycinnamoyl]-β-D-fucopyranoside (2), 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-D-glucopyranosyl-(1→3)]-[4-O-(E/Z)-3″, 4″-dimethoxycinnamoyl]-β-D-fucopyranoside (3), and 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-D-glucopyranosyl-(1→3)]-[4-O-(E/Z)-3″, 4″, 5″-trimethoxy-cinnamoyl]-β-D-fucopyranoside (4), respectively. Conclusion: Compounds 1-4 are new compounds named crotalarioside C, (E/Z)-crotalariosides D, E, and F, respectively. Among them, compounds 2-4 are cis-trans-isomers, which can not be separated by the experimental technique because of the configuration.

Chemical constituents in Akebiae Fructus based on UFLC-Q-TOF/MS / 中草药

Objective: To identify the chemical constituents in Akebiae Fructus by UFLC-Q-TOF/MS method. Methods: The separation was performed on an Acquity UPLC BEH C18 Column (100 mm × 2.1 mm, 1.7 μm), with a mobile phase using 0.1% formic acid-acetonitrile and water containing 0.1% formic acid (B) for gradient elution. The flow rate was 0.3 mL/min, the temperature of column was 40°C with injection volume of 1 μL. TOF/MS and electrospray ion (ESI) source were applied for the qualitative analysis under the negative ion mode, and the full mass scan range was m/z 100-1500. Results: According to MS principle, twenty-five triterpenoids were identified from the methanol extract of Akebiae Fructus and the chemical structures of other nine unknown compounds were deduced. Conclusion: UFLC-Q-TOF/MS method could identify the main chemical constituents in Akebiae Fructus rapidly and accurately, which lays a foundation for the quality control of Akebiae Fructus.

Resíduos madeireiros do alburno de pau-rainha (Brosimum rubescens): investigação de metabólitos secundários e alguns aspectos tecnológicos / Residues of sapwood of "pau-rainha" (Brosimum rubescens): investigation of secondary metabolites and some technological aspects

Estudos fitoquímicos prévios com resíduos do cerne de pau-rainha (Brosimum rubescens), identificaram um alto teor de xantiletina, uma cumarina com potencial biológico. Dando prosseguimento aos estudos com serragens desta espécie, este estudo relata a densidade básica bem como o isolamento e identificação do triterpeno 3ß-acetoxi-olean-12-eno-28-al e do ß- sitosterol nos extratos hexânico e metanólico do alburno da planta. A estrutura do triterpeno foi determinada com base nos espectros de RMN em 1D (¹H e 13C) e 2D (HSQC e HMBC) além de comparação com dados da literatura. A densidade básica encontrada para o alburno foi de 0,58 g cm-3 que, embora seja inferior a do cerne, poderá ser utilizada na confecção de vários produtos, inclusive em técnicas de marchetaria.

Previous phytochemical studies on residues of pau-rainha's heartwood (Brosimum rubescens) showed a high content of xanthyletin, a coumarin with biological potential. Continuing our studies with sawdust of this species, this work relates the basic density, isolation and identification of the triterpene 3ß-acetoxy-olean-12-ene-28-al and ß-sitosterol in n-hexane and MeOH extracts of the plant´s sapwood. The structure of the triterpene was determined on the basis of NMR spectra in 1D (¹H and 13C) and 2D (HSQC and HMBC) and comparison with literature data. The basic density found for sapwood was 0.58 g cm-3, that even so is inferior the one of heartwood, could be used in the confection of some products, also in marquetry techniques.

Chemical constituents from Astragalus annularis Forssk. and A. trimestris L., Fabaceae / Constituintes químicos de Astragalus annularis Forssk. and A. trimestris L., Fabaceae

The phytochemical investigation of the aerial parts of Astragalus annularis Forssk. and A. trimestris L., Fabaceae, growing in Egypt, resulted in the isolation of eight compounds, for the first time from these two species. These compounds were identified by chemical methods, NMR spectroscopy, UV spectroscopy as well as MS analysis. The antimicrobial activity of the isolated compounds were tested against Staphylococcus aureus, Esherichia coli and Candida albicans.

A investigação química das partes aéreas de Astragalus annularis Forssk. e A. trimestris L., Fabaceae, do Egito, resultou no isolamento de oito compostos, pela primeira vez nestas duas espécies. Estes compostos foram identificados por métodos químicos, espectroscopia de RMN, espectroscopia UV, bem como análise de EM. A atividade antimicrobiana dos compostos isolados foi testada contra Staphylococcus aureus, Escherichia coli e Candida albicans.