Triterpene glycosides of Lupinus angustifolius

Investigation of whole seeds of Lupinus angustifolius L. (Leguminosae) yielded the two triterpenoid saponins with branched monosaccharide chain 3 beta,21 beta,22 beta,24-tetrahydroxyolean-12-en-3-O-alpha-L-rhamnopyranosyl-(1-->3)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranoside (3) and 3 beta,21 beta,22 beta,24-tetrahydroxyolean-12-en-3-O-alpha-L-rhamnopyranosyl-(1-->3)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21-O-alpha-L-rhamnopyranoside (4) along with the known compounds soyasaponin I (1) and 3 beta,21 beta,22 beta,24-tetrahydroxyolean-12-en-3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-21-O-alpha-L-rhamnopyranoside (2). The structures of the compounds were elucidated using hydrolysis, FAB-MS and extensive NMR experiments. Compounds 2-4 showed moderate antifungal activity against Candida albicans with MIC values of 25, 25 and 30 microg/ml, respectively. Only soyasaponin I was found weakly hemolytic (HC(50) >500 microg/ml).     

Triterpene saponins and flavonoids in the seeds of Trifolium species

Seeds of 57 species of the genus Trifolium have been studied for the occurrence and concentration of soyasapogenol B glycosides and flavonoids. It was shown that all tested species contained soyasaponin I and in some species astragaloside VIII and/or 22-O-glucoside and 22-O-diglucoside of soyasaponin I were also present. Total concentration of saponins ranged from trace amounts up to 10 mg/g(DM). It was suggested that soyasapogenol B glycosides could be recognized as chemotaxonomic character of Fabaceae family. All but three tested species contained flavonoids. The majority of species contained quercetin as a sole flavonoid or in the mixture with a number of unidentified flavonoid components. Concentration of quercetine in some species was at the level of about 3 mg/g(DM). This high quercetin concentration and soyasaponin occurrence makes the seeds of some Trifolium species a potential source of health beneficial phytochemicals, to be used in human nutrition.     

Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max

Triterpene saponins are a diverse group of compounds with a structure consisting of a triterpene aglycone and sugars. Identification of the sugar-transferase involved in triterpene saponin biosynthesis is difficult due to the structural complexity of triterpene saponin. Two glycosyltransferases from Glycine max, designated as GmSGT2 and GmSGT3, were identified and characterized. In vitro analysis revealed that GmSGT2 transfers a galactosyl group from UDP-galactose to soyasapogenol B monoglucuronide, and that GmSGT3 transfers a rhamnosyl group from UDP-rhamnose to soyasaponin III. These results suggest that soyasaponin I is biosynthesized from soyasapogenol B by successive sugar transfer reactions.     

Evaluation of cytotoxicity and immune modulatory activities of soyasaponin Ab: An in vitro and in vivo study

To improve the immune efficacy of protein subunit vaccines, novel adjuvants are needed to elicit a suitable protective immune response and to promote long term immunologic memory. In this work, soyasaponin Ab, a major constituent among group A soyasaponins in soybeans was purified and prepared from soy hypocotyls. The immunomodulatory effects of soyasaponin Ab both in vitro and in vivo were investigated, and its pro-immunomodulatory molecular mechanism was also studied. For in vitro assays, with mouse macrophage cell line RAW264.7 as the studying model, both cytotoxicity and immune stimulatory activity were investigated to evaluate the potential of soyasaponin Ab as the vaccine adjuvant. The results indicated that soyasaponin Ab could be significantly safer than Quillaja saponins (QS). Soyasaponin Ab showed no toxicities over the tested concentration ranges compared to QS. Soyasaponin Ab was proved able to promote releases of inflammatory cytokines like TNFα and IL-1β in a dose-dependent manner. Furthermore, NF-κB signalling was also activated by soyasaponin Ab effectively. In addition, with TLR4 gene expression of RAW264.7 cell inhibited by RNA interference, immune stimulatory effects by soyasaponin Ab dropped down significantly. On the other hand, the in vivo experiment results showed that anti-ovalbumin (OVA) IgG, IgG1, IgG2a, IgG2b were significantly enhanced by the soyasaponin Ab and QS groups (p < 0.05 or p < 0.01). The results suggested that compared to QS, soyasaponin Ab may represent a viable candidate for effective vaccine adjuvant. TLR4 receptor dependent pathway may be involved in immune stimulatory effects of soyasaponin Ab.     

2070-DTI, a topoisomerase inhibitor produced by Streptomyces sp. strain No. 2070

A novel inhibitor of topoisomerase II designated as 2070-DTI was isolated from the culture filtrate of Streptomyces sp. strain No. 2070. The structure was determined to be that of the known soyasaponin I on the basis of spectroscopic methods (NMR and MS). 2070-DTI strongly inhibited the decatenation activity of human placenta topoisomerase II in a noncompetitive manner, and weakly inhibited or was inert towards the relaxation activities of various topoisomerase I's and DNA-related enzymes. 2070-DTI is an inhibitor belonging to the cleavable complex-nonforming type without DNA intercalation.     

A novel saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta

We isolated a soybean saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (sdn1) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The sdn1 gene was successfully expressed in Trichoderma viride under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-D-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates.     

2070-DTI,A Topoisomerase Inhibitor Produced by Streptomyces sp. Strain No. 2070

A novel inhibitor of topoisomerase II designated as 2070-DTI was isolated from the culture filtrate of Streptomyces sp. strain No. 2070. The structure was determined to be that of the known soyasaponin I on the basis of spectroscopic methods (NMR and MS). 2070-DTI strongly inhibited the decatenation activity of human placenta topoisomerase II in a noncompetitive manner, and weakly inhibited or was inert towards the relaxation activities of various topoisomerase I's and DNA-related enzymes. 2070-DTI is an inhibitor belonging to the cleavable complex-nonforming type without DNA intercalation.     

Analysis and quantitative determination of group B saponins in processed soybean products

Pure analytical standards for the major saponins present in processed soy products, the group B saponins (soyasaponins I, II, III and IV), were isolated in mg quantities by a combination of processing, precipitation/re-solubilisation, TLC and preparative HPLC. These standards were determined to be pure by LC-ESI/MS analysis and NMR. The standards were used to perfect a facile analytical HPLC method using spectrometric detection to determine the percent composition of the group B soyasaponins in various products from processing of soybean.     

Triterpenoid saponins from Argania spinosa.

Five new oleanane saponins named arganine A, B, D, E and F and two known saponins: arganine C and mi-saponin A were isolated from the kernel of Argania spinosa. The structures of these saponins were elucidated by using 1H NMR, 1H-1H COSY NMR, 13C NMR, FAB mass spectrometry and chemical evidence.     

Triterpenoid saponins from the stem bark of Caryocar villosum

Five triterpenoid saponins, caryocarosides II-22 (3), III-22 (4), II-23 (5), III-23 (6), and II-24 (7), have been isolated from the methanol extract of the stem bark of Caryocar villosum, along with two known saponins (1-2). The seven saponins are glucuronides of hederagenin (II) or bayogenin (III). Caryocaroside II-24 (7) is an unusual galloyl ester saponin acylated on the sugar chain attached to C-28, the 3-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-beta-D-glucuronopyranosyl hederagenin-28-O-[2-O-galloyl-beta-D-glucopyranosyl] ester. The structures of the saponins were established on the basis of extensive NMR ((13)C, (1)H, COSY, TOCSY, HSQC, HMBC and ROESY) and ESI-MS studies. The cytotoxic activity of saponins 2 and 3 was evaluated in vitro against human keratinocytes. The DOPA-oxidase inhibition and the lipolytic activities were evaluated ex vivo using an explant of human adipose tissue.     

Isolation and Structural Elucidation of DDMP-Conjugated Soyasaponins as Genuine Saponins from Soybean Seeds

The composition and the structures of native “group B saponin” in soybean seeds were reinvestigated. Five kinds of saponins named soyasaponins αg, βg, βa, γg, and γa, according to elution order from HPLC, were isolated and the structures were characterized as 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) attaching through an acetal linkage to the C-22 hydroxyl of the aglycones of soyasaponins V, I, II, III, and IV, respectively, by UV, IR, MS, and NMR. DDMP-conjugated saponins were detected as major saponin constituents by extraction under mild conditions, and soyasaponins I–V were not detected. Therefore it was strongly suggested that these DDMP-conjugated saponins were genuine saponins in the intact soybeans.     

Molluscicidal saponins from Phytolacca icosandra

Five monodesmosidic serjanic acid saponins and a monodesmosidic spergulagenic acid saponin were isolated from an aqueous extract of the berries of Phytolacca icosandra. A methanol extract of the berries furnished three bidesmosidic serjanic acid glycosides. Their structures were established by spectroscopic (ES-MS, 1H NMR, COSY, HSQC, HMBC, 13C NMR) and chemical methods. The molluscicidal, spermicidal and haemolytic properties of the saponins were investigated.     

New acylated triterpenoid saponins from Maesa lanceolata

Ten new acylated triterpenoid saponins were isolated from the leaves of Maesa lanceolata. For their structure elucidation extensive use was made of homo- and heteronuclear 2D NMR techniques such as COSY, NOESY, HSQC and HMBC. All saponins identified contained the same tetraglycosidic side chain, but the triterpenoid moiety showed a variable esterification pattern. Monoester, diester and triester derivatives were present. Maesasaponin I was a 21-monoester derivative, i.e. ?3 beta-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl- (1-->3)]-[beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl+ ++?-21 beta-angeloyloxy-13 beta, 28-oxidoolean-16 alpha, 22 alpha, 28 alpha-triol. Maesasaponins III, IV3, V3 and VI2 had an additional acetyl, propanoyl, n-butanoyl and angeloyl substituent, respectively, in position 22. Maesasaponins II, IV2, V2, VI3 and VII1 were characterised as the 16-acetyl derivatives of maesasaponins I, III, IV3, V3 and VI2, respectively. Structures of saponins previously reported in M. lanceolata had to be revised.     

Triterpenoid saponins from Albizia boromoensis Aubrév. & Pellegr

As part of our search of new bioactive saponins from Cameroonian medicinal plants, phytochemical investigation of the roots of Albizia boromoensis led to the isolation of four new oleanane-type saponins, named boromoenosides A–D (1–4). Their structures were established by direct interpretation of their spectral data, mainly HRESIMS, 1D NMR (¹H, ¹³C NMR, and DEPT) and 2D NMR (COSY, NOESY, HSQC and HMBC), and by comparison with the literature data. All isolated saponins were assayed for their cytotoxicity against U-87 MG human glioblastoma cell lines and TG1 glioblastoma stem-like cells with no positive activity detected.     

Utility of coupled-HSQC experiments in the intact structural elucidation of three complex saponins from Blighia sapida

The structures of three complex saponins from the fruit pods of Blighia sapida have been elucidated and their ¹H and ¹³C NMR spectra assigned employing a variety of one- and two-dimensional NMR techniques without degradative chemistry. The saponins have either four or six monosaccharide units linked to a triterpene aglycone. High-resolution, proton-coupled-HSQC spectra were important for determining both the identities of the intact monosaccharide units and coupling constants in strongly coupled proton spin systems. These NMR experiments will prove crucial as the complexity of saponin structures reaches the limit that can be determined solely by NMR.     

Triterpenoidal saponins from Gleditsia sinensis

Six bisdesmosidic triterpenoidal saponins, gleditsiosides H-K and gleditsia saponins C' and E', were isolated from the anomalous fruits of Gleditsia sinensis. Their structures were established by a combination of extensive NMR (DEPT, DQF-COSY, HETCOR, HOHAHA, HMBC and ROESY) studies and chemical degradation.     

Triterpenoid saponins from Impatiens siculifer

Triterpenoid saponins, impatienosides A-G, together with 12 known saponins, were isolated from the whole plants of Impatiens siculifer. Their structures were established on the basis of extensive 1D and 2D NMR and MS analyses coupled with chemical degradation. Cytotoxic activities of the isolated saponins were evaluated against three human cancer cell lines: human myeloid leukemia HL-60 cells, human stomach KATO-III adenocarcinoma, and human lung A549 adenocarcinoma.     

Bacopaside I and II: two pseudojujubogenin glycosides from Bacopa monniera

Two saponins, designated as bacopaside I and II, have been isolated from Bacopa monniera Wettst. and their structures have been elucidated as 3-O-alpha-L-arabinofuranosyl-(1-->2)-[6-O-sulphonyl-beta-D-glucopyranosyl-(1-->3)]-alpha-L-arabinopyranosyl pseudojujubogenin (1) and 3-O-alpha-L-arabinofuranosyl-(1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl pseudojujubogenin (2) mainly on the basis of 2D NMR and other spectral analyses.     

Steroidal saponins from the tuber of Ophiopogon japonicus

Eight novel steroidal saponins, ophiopogonins H-O (1-8), along with seven known steroidal saponins (9-15) were isolated from the tubers of Ophiopogon japonicus. The structures of these new compounds were determined by detailed spectroscopic analysis, including extensive 1D and 2D NMR data, and the analysis of hydrolytic reaction products. For the first time, rare furostanol saponins with disaccharide moiety linked at position C-26 of the aglycone were reported to be isolated from a natural source.     

Preparative separation of four triterpene saponins from radix astragali by high-speed counter-current chromatography coupled with evaporative light scattering detection

Four triterpene saponins, including astragaloside IV, astragaloside II, astragaloside I and acetylastragaloside I, were successfully isolated and separated by high-speed counter-current chromatography coupled with evaporative light scattering detection from Radix Astragali using stepwise elution with a pair of solvent systems composed of n-hexane:ethyl acetate:ethanol:water in volume ratios of 1:0.6:0.6:1 and 1:1:1:1 (by volume). The isolation produced 26.5 mg astragaloside IV, 28.2 mg astragaloside II, 48.7 mg astragaloside I and 17.6 mg acetylastragaloside I with purities of 97.6, 96.4, 98.8 and 96.8%, respectively, determined by high-performance liquid chromatography from 250 mg crude extract. The chemical structures of the isolated compounds were identified by UV, NMR and MS, and confirmed by authentic standards.