The microbiological transformation of steroidal saponins by Curvularia lunata

The microbiological transformation of polyphyllin I (compound I), polyphyllin III (compound II), polyphyllin V (compound III) and polyphyllin VI (compound IV) by Curvularia lunata into their corresponding subsaponins, for example, diosgenin-3-O-α-l-arabinofuranosyl (1→4)-β-d-glucopyranoside (compound V), diosgenin-3-O-α-l-rhamnopyranosyl (1→4)-β-d-glucopyranoside (compound VI), diosgenin-3-O-β-d-glucopyranoside (compound VII) and pennogenin-3-O-β-d-glucopyranoside (compound VIII), were studied in this paper. Curvularia lunata is able to hydrolyze terminal rhamnosyls that are linked by 1→2 C- bond to sugar residues of steroidal saponins at C-3 position with high activity and regioselectivity.     

Structural characterization of steroidal saponins by electrospray ionization and fast-atom bombardment tandem mass spectrometry

The structural characterization of four steroidal saponin compounds involving two and three sugar groups, namely spirostanol saponins and furostanol saponins, were investigated by positive ion fast-atom bombardment (FAB), electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques. Important structural information was obtained from collision-induced dissociation (CID) and FAB-MS spectra with different liquid matrices. It was found that a characteristic fragmentation involving the loss of 144 Da arising from the cleavage of the E-ring was observed when there was no sugar chain at the C-26 position. When a glucoside group was substituted at the C-26 position, this C-26 sugar moiety was preferentially eliminated. All of these compounds produced a major product ion with a stable skeleton structure at m/z 255. The results of this paper can assist structural analysis of mixtures of steroidal saponins.     

Hepatoprotective and hepatotoxic actions of oleanolic acid-type triterpenoidal glucuronides on rat primary hepatocyte cultures

The protective effects of oleanolic acid-type saponins and their derivatives on in vitro immunological liver injury of primary cultured rat hepatocytes were studied. A known antihepatotoxic saponin (chikusetsusaponin IVa, 1) showed hepatoprotective activity in this model. Although a rhamnosyl derivative (2) of 1 similarly showed hepatoprotective activity, its prosapogenin (5) did not show any hepatoprotective activity. On the contrary, 5 exhibited cytotoxicity toward liver cells. In the absence of antiserum, monodesmosyl saponins showed hepatotoxicity, while the bisdesmosyl saponins except for 1, did not show such hepatotoxicity. In order to clarify the effects of the sugar residues at C-3 and C-28 responsible for hepatoprotective and hepatotoxic actions, oleanolic acid 3-O-glucuronide (2a) and oleanolic acid 28-O-glucoside (2b) were prepared and tested. 2b showed neither hepatoprotective action nor hepatotoxicity. In contrast, 2a was effective at 90 microM on hepatoprotection, although it showed strong hepatotoxicity. Oleanolic acid (2c) itself showed both hepatoprotective action and weak hepatotoxicity. Therefore, the hepatoprotective activity of these types of saponins could represent a balance between hepatoprotective action and hepatotoxicity.     

Synthesis and structure–activity relationship study of cytotoxic lupane-type 3β-O-monodesmosidic saponins with an extended C-28 side chain

A concise synthesis of lupane triterpenes with an elongated carbon chain at the C-28 position, as well as saponins containing d-mannose, l-arabinose, and l-rhamnose moieties at the C-3 position is described. The overall synthesis of the new triterpenes involved seven linear steps starting from natural betulin: selective protection of a hydroxyl group, oxidation, elongation of the carbon chain by Grignard reaction, and deoxygenation. O-Glycosides were obtained by glycosylation of triterpenes with classical Schmidt's donors. Additionally, all new compounds were evaluated in vitro for their cytotoxic activities. Several triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against human cancer cell lines. The therapeutical index of active triterpenes is very high, since almost none of them were cytotoxic for normal BJ fibroblasts. These results open the way to the synthesis of various lupane-type saponin derivatives as potentially bioactive compounds.     

Structural analysis of complex saponins of Balanites aegyptiaca by 800 MHz 1H NMR spectroscopy

The main saponin (1) present in the mesocarp of Balanites aegyptiaca fruit is a mixture of 22R and 22S epimers of 26-(O-beta-D-glucopyranosyl)-3-beta-[4-O-(beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranosyloxy]-22,26-dihydroxyfurost-5-ene. This structure differs from a previously reported saponin isolated from this source by the site of attachment of the rhamnosyl residue, and presumably represents a structural revision of the latter. The main saponin (2) present in the kernel is a xylopyranosyl derivative of 1. The use of high-field NMR enabled the practically complete assignment of 1H and 13C chemical shifts of these complex saponins, existing as a mixture of C-22 epimers. Moreover, the work represents a new approach to structural elucidation of saponins: direct preparative-scale HPLC-RID of crude extracts followed by high-field NMR investigations supported by ESI-MSn.     

A convenient synthesis of the core trisaccharide of the N-glycans

A convenient synthesis of the core trisaccharide of the N-glycans was described. Orthogonal one-pot glycosylation of three monosaccharide building blocks was performed to furnish β-glucosyl chitobiose, which was then transformed to β-mannosyl chitobiose by intramolecular epimerization of the C-2 position of the β-glucoside. The key glucosyl donor 7c with differentiated 2,3-OH was prepared following the 4,6-O-benzylidene-protected 1,2-orthoester strategy.     

Starfish saponins VI - unique 22,23-epoxysteroidal cyclic glycosides,minor constituents from Echinaster sepoeitus

On the basis of comparative ¹H? and ¹³C-n.m.r. data, the structures of the minor saponins from the starfish Echinaster sepositus have been elucidated to be 2-4. They closely resemble 1, the major saponin from the same starfish, and, in addition to the cyolic trisaccharide moiety bridging C-3 and C-6 of the steroid, include epoxide functionalities in the steroidal side-chains.     

Steroidal saponins from the bulbs of Allium karataviense.

Chemical examination of the bulbs of Allium karataviense led to the isolation of five new spirostanol saponins (7-11) and a new furostanol saponin (12), together with a known steroidal sapogenin (1) and five known saponins (2-6). The structures of the new saponins were determined by detailed analysis of their spectral data, including two-dimensional NMR spectroscopy. The steroidal saponins produced by A. karataviense, except for 5 and 6, were found to be based upon (25R)-5 alpha-spirostane-2 alpha,3 beta,5,6 beta-tetrol (alliogenin) and contain a beta-D-glucopyranosyl moiety with the formation of an O-glycoside linkage to C-2 of the polyhydroxylated steroidal skeleton as the common structural feature. The isolated compounds were evaluated for cytostatic activity against human promyelocytic leukemia HL-60 cells.     

Characterization of steroidal saponins in crude extract from Dioscorea nipponica Makino by liquid chromatography tandem multi-stage mass spectrometry

The liquid chromatography-electrospray ionization-tandem multi-stage mass spectrometry (LC-ESI-MSⁿ) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C₁₈ column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [M − H]⁻ ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [M − H]⁻ ions at m/z 1063, 1045, 901 and 1047, respectively, were observed during the LC-ESI(−)-MS analysis, and three groups of them except the pair of isomers with the [M − H]⁻ ions at m/z 1047 could be differentiated by LC-ESI(−)-MS³. Furthermore, the ESI-MSⁿ fragmentation behaviors of the [M + Li]⁺ ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [M − H]⁻ ion at m/z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.     

柘树根多糖的分离纯化及结构表征

以柘树[Cudrania tricuspidata(Carr.) Bur.]的根为材料, 经热水抽提、木瓜蛋白酶-Sevag法除蛋白、乙醇沉淀和DEAE-Sephadex A-50凝胶柱层析分离纯化, 得到一种水溶性的柘树根多糖(CPS-0). 采用HPLC、糖基组成分析、甲基化分析、GC、GC-MS、NMR(1H NMR, 13C NMR及HMQC)、元素分析、UV和IR等技术对CPS-0的纯度、性质、组成和结构进行表征. 结果表明, CPS-0仅含葡萄糖, 分子量为4.6×103, 主链由1,4-连接的α-D-葡萄糖残基组成, 其侧链由末端及1,4-连接的葡萄糖残基构成, 取代于主链分支点葡萄糖的6位, 平均每10个葡萄糖残基组成的重复单元中含有1个分支.     

A New Furostanol Saponin from the Water-extract of Dioscorea nipponica Mak., the Raw Material of the Traditional Chinese Herbal Medicine Wei Ao Xin

26-O-β-D-Glucopyranosyl-furost-5(6),20(22)-dien-3β,26-diol 1, a new furostanol saponin, was isolated from the water-extract of Dioscorea nipponica Mak., the raw material of a traditional Chinese herbal medicine Wei Ao Xin. The structure of 1 was determined on the basis of its spectral data especially by NMR spectroscopy. The result provides the first example of naturally occurring furostanol saponins with a single saccharide chain at the C-26 position.     

Structural characterization of a glucan from Angelica sinesis (Oliv) Diels

Angelica sinesis (Oliv) Diels is a traditional Chinese drug used clinically in the treatment of gynecological diseases. Its polysaccharide fraction been reported to have several immunomodulating, antitumor, antiradiation activities^[1] and a recent paper showed it could obviously act on the hemopoietic system^[2]. Haruki Y. et al. have elucidated the chemical structure of a α(1→4) linked D-glucan from Angelica acutiloba^[3]. We now describe the structure of a water soluble glucan, AS-1, which was isolated and purified from the water extract of Angelica sinesis (Oliv) Diels. It is only composed of D-glucose and its mean molecular weight is estimated to be 5.3×10⁵. The absorption peak of 846cm^-1 on IR, the chemical shift of δ 99.98 ppm for C-1,and the 168 Hz of ¹J_C-H suggested that the D-glucose residues were α-linked. Methylation analysis (Table 1),periodate oxidation, Smith degradation, ¹H NMR and ¹³C NMR (Table 2) showed that it is α(l→6) linked D-glucan to which are attached two glucosyl side chains, Glc-(l→3)-Glc→1 and Glc-(1→4)-Glc→1, both at 3-O of the glucosyl residues of every 14th glucose units of the main chain. The structure was further proved by using HMBC,HMQC and DQF-COSY techniques. The details of its pharmacological results will be published elsewhere.     

Qualitative and Quantitative Analysis of Microbial Transformation of Steroidal Saponins in Dioscorea zingiberensis

Diosgenin is an important starting material in the steroidal hormone industry. In this paper diosgenin was obtained by biotransformation of steroidal saponins in Dioscorea zingiberensis with the fungus Aspergillus oryzae. In the pre- and post-biotransformed samples, five main steroidal saponins and the aglycone (diosgenin) were identified by evaporative light scattering detector. An LC-UV quantification method for them was established by using C18 column and a mobile phase of aqueous acetonitrile. By means of this method, a good linearity (r > 0.9995) and recovery of the analytes (97.5–101.3%) were obtained for all the compounds. During biotransformation, the sugar chains of saponins were hydrolyzed and the content of the hydrolyzed product (diosgenin) increased 16 times after 84 h fermentation.     

Structure and antitumor activity of the less-branched derivatives of an alkali-soluble glucan isolated from Omphalia lapidescens. (Studies on fungal polysaccharide. XXXVIII).

The structure and antitumor activity of Smith-type degradation products (OL-2-I, OL-2-II and OL-2-III) of an alkali-soluble glucan, OL-2, isolated from a crude fungal drug "Leiwan" (Omphalia lapidescens) were investigated. Methylation analysis suggested that OL-2-I was a (1----3)-beta-D-glucan with approximately one branch at every three main chain glucosyl units at each C-6 position; OL-2-II was a (1----3)-beta-D-glucan with approximately one branch at twenty four main chain glucosyl units at each C-6 position (number of all main chain glucosyl units is on average). OL-2-I, OL-2-II and OL-2-III which were Smith-type degradation products of OL-2, showed potent antitumor activity against the solid form of sarcoma 180 in ICR mice. These results indicated that the degree of beta-linked branching at position 6 was remarkably related to the antitumor activity.     

Asparinins, asparosides, curillins, curillosides and shavatarins: structural clarification with the isolation of shatavarin V, a new steroidal saponin from the root of Asparagus racemosus

A new steroidal saponin, shatavarin V, (3-O-{[α-l-rhamnopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosyl}-(25S)-5β-spirostan-3β-ol), was isolated from the roots of Asparagus racemosus by RP-HPLC, and its structure determined by 1D and 2D NMR studies. This data permits clarification of the structures reported for several known saponins: asparinins A and B; asparosides A and B; curillin H; curillosides G and H and shavatarins I and IV.     

Chromatographic behaviour of steroidal saponins studied by high-performance liquid chromatography-mass spectrometry

The chromatographic behaviour of steroidal saponins found in Anemarrhena asphodeloides, Asparagus officinalis, Convallaria majalis, Digitalis purpurea and Ruscus aculeatus was studied by HPLC-MS using a C-18 reversed-phase column and aqueous acetonitrile or aqueous methanol mobile phase gradients, with or without the addition of 1% acetic acid. The behaviour was compared to that of triterpene saponins found in Aesculus hippocastanum, Centella asiatica, Panax notoginseng and Potentilla tormentilla. Inclusion of methanol in the mobile phase under acidic conditions was found to cause furostanol saponins hydroxylated at C-22 to chromatograph as broad peaks, whereas the peak shapes of the spirostanol saponins and triterpene saponins studied remained acceptable. In aqueous methanol mobile phases without the addition of acid, furostanol saponins chromatographed with good peak shape, but each C-22 hydroxylated furostanol saponin was accompanied by a second chromatographic peak identified as its C-22 methyl ether. Methanolic extracts analysed in non-acidified aqueous acetonitrile mobile phases also resolved pairs of C-22 hydroxy and C-22 methoxy furostanol saponins. The C-22 methyl ether of deglucoruscoside was found to convert to deglucoruscoside during chromatography in acidified aqueous acetonitrile, or by dissolving in water. Poor chromatography of furostanol saponins in acidified aqueous methanol is due to the interconversion of the C-22 hydroxy and C-22 methoxy forms. It is recommended that initial analysis of saponins by HPLC-MS using a C-18 stationary phase is performed using acidified aqueous acetonitrile mobile phase gradients. The existence of naturally-occurring furostanol saponins methoxylated at C-22 can be investigated with aqueous acetonitrile mobile phases and avoiding methanol in the extraction solvent.     

Ligand fishing from Dioscorea nipponica extract using human serum albumin functionalized magnetic nanoparticles

Dioscorea nipponica and the preparations made from it have been used for long to prevent and treat coronary heart disease in traditional Chinese medicine. A group of steroidal saponins present in the plant are believed to be the active ingredients. It has been a challenge to study the individual saponins separately due to the similarities in their chemical and physical properties. In this work, human serum albumin (HSA) functionalized magnetic nanoparticles (MNPs) were used to isolate and identify saponin ligands that bind to HSA from D. nipponica extract. Electrospray ionization mass spectrometry (ESI-MS) was used for compound identification and semi-quantification. Three saponins, i.e. dioscin, gracillin, and pseudo-protodioscin showed affinity to HSA-MNPs and thus isolated effectively from the extract. The other two saponins detected in the extract (i.e. protodioscin and 26-O-β-d-glucopyranosyl-3β,20α,26-triol-25(R)-Δ^5,22-dienofurostan-3-O-α-l-rhamnopyranosyl (1→2)-[α-l-rhamnopyranosyl (1 → 4)]-β-d-glucopyranoside) exhibited no affinity at all. Among the three saponins fished out, dioscin bound to HSA much stronger than gracillin and pseudo-protodioscin did. The results indicated that affinity interaction between HSA immobilized on MNPs and small molecule compounds were highly dependent on chemical structures and, potentially, medicinal usefulness. The present work demonstrates a facile and effective way to isolate and identify ligands of receptors from medicinal plants.     

Facile construction of 1,2-cis glucosidic linkage using sequential oxidation-reduction route for synthesis of an ER processing α-glucosidase I substrate

The fluorescence-labeled hexasaccharide (Glcα1-2Glcα1-3Glcα1-3Manα1-2Manα1-2Manα) was synthesized as a substrate for the processing enzyme α-glucosidase I. To construct the 1,2-cis glucosidic linkages, we employed an α stereoselective coupling using the mannosyl donor by assisted neighboring-group participation, followed by conversion of the stereochemistry of the C-2 hydroxyl group in the mannose residue using sequential oxidation of C-2 hydroxyl group to a 2-keto group and stereoselective reduction of the hydroxyl group to the gluco-configuration to provide the corresponding α-glucoside derivative. Using this strategy, the three consecutive α-glucosidic linkages were easily obtained in a stereoselective manner. Finally, the Dansyl labeled hexasaccharide derivative was used to measure the activity of processing α-glucosidase I.     

Synthesis of terminal disaccharide unit of Klebsiella pneumoniae ssp. R20

Synthesis of the terminal disaccharide unit of a novel α-(1→2) linked heptoglycan of K. pneumoniae ssp. strain R20 from methyl α-d-mannopyranoside has been presented. Central to the strategy is the application of Sharpless asymmetric dihydroxylation to introduce a new center at C-6 position of mannopyranoside. The coupling of two heptoglycans (12 and 13) was accomplished by a Lewis acid catalyst.     

桑叶多糖SJB的结构分析和蛋白酪氨酸磷酸酯酶PTP1B抑制活性

通过DEAE-纤维素和凝胶过滤柱色谱对桑叶碱提粗多糖进行分级分离, 获得均一多糖SJB, 进行结构鉴定. 采用蛋白酪氨酸磷酸酯酶PTP1B体外模型对SJB进行降血糖活性测定. 结果表明: SJB的相对分子质量为5.4×104, 由鼠李糖、阿拉伯糖、葡萄糖、半乳糖、半乳糖醛酸组成的酸性杂多糖; 主链由1,2-、1,2,4-连接的鼠李糖和1,4-、1,3,4-连接的半乳糖醛酸组成; 侧链包括末端、1,5-、1,3,5-连接的阿拉伯糖; 末端、1,4-连接的葡萄糖以及末端、1,3-、1,4-、1,6-连接的半乳糖, 主要通过鼠李糖的O4位和半乳糖醛酸的O3位与主链相连. 该多糖为首次从桑叶中获得的酸性杂多糖. 20 μg/mL SJB对PTP1B的抑制率为31.7%.