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Diosgenin has been widely used as a precursor in the pharmaceutical industry. The conventional diosgenin production method, direct acid hydrolysis, can cause severe environmental pollution and has low production efficiency. In this study, the β-glucosidase FBG1 was successfully over-expressed in Pichia pastoris. The engineered yeast was grown in 5- and 50-L bioreactors to a high-cell-density, and the resulting recombinant FBG1 without purification was directly used for trillin biocatalysis. A novel enzymatic biocatalysis method was established through efficient biocatalysis of trillin by recombinant FBG1 and a practical diosgenin purification process. Diosgenin production reached ∼1.7 mg/mL under optimized biocatalysis conditions with a corresponding ∼94 % trillin conversion. The production process was further scaled up from 2 mL to 20, 200, and 2000 mL working volumes, producing equivalent efficiency. This recombinant enzyme biocatalysis process resulted in ∼680 mg diosgenin when 1 g trillin was processed, together with ∼90 % environmental impact elimination and ∼36 % cost reduction. Enzymatic biocatalysis can convert trillin into a high-value-added pharmaceutical precursor of diosgenin with high eco-efficiency, cost-effectiveness, and sustainability is presented here for the first time and is a promising method for future applications in industrial processes.

A phytochemical study on the leaves of Crataegus pinnatifida Bge. var. major N.E.Br. was carried out, which finally led to the isolation of nineteen phenolic compounds (1-19). The structures of all compounds were established mainly by NMR and MS spectroscopic analysis as well as the necessary ECD experimental evidence, of which compounds 1-4 (crataegunins A-D) were identified as new phenylpropanoid-substituted epicatechins. HepG2 cells were induced by oleic acid and palmitic acid to establish the model of lipid metabolism disorder. All isolated compounds were used to intervene in the model, and the contents of triglyceride (TG) and total cholesterol (TC) were detected. Compound 2 could significantly reduce the content of TG, while compounds 2 and 11 both have good activity in reducing TC content.

Diosgenin is used widely to synthesize steroidal hormone drugs in the pharmaceutical industry. The conventional diosgenin production process, direct acid hydrolysis of the root of Dioscorea zingiberensis C. H. Wright (DZW), causes large amounts of wastewater and severe environmental pollution. To develop a clean and effective method, the endophytic fungus Fusarium sp. CPCC 400226 was screened for the first time for the microbial biotransformation of DZW in submerged fermentation (SmF). Statistical design and response surface methodology (RSM) were implemented to develop the diosgenin production process using the Fusarium strains. The environmental variables that significantly affected diosgenin yield were determined by the two-level Plackett-Burman design (PBD) with nine factors. PBD indicates that the fermentation period, culture temperature, and antifoam reagent addition are the most influential variables. These three variables were further optimized using the response surface design (RSD). A quadratic model was then built by the central composite design (CCD) to study the impact of interaction and quadratic effect on diosgenin yield. The values of the coefficient of determination for the PBD and CCD models were all over 0.95. P-values for both models were 0.0024 and

Three anthraquinone analogues (1–3) were isolated by phytochemical work on EtOAc-soluble ingredients extracted from the roots of Polygonatum odoratum. The structures of all isolates were elucidated by NMR, MS and CD experiments, of which 1 (polygodoquinone A) was identified as a new anthraquinone derivative. Specifically, 1 represents an unusual structure composed of a naphthoquinone derivative linked to an anthraquinone via a C-C bond. 1–3 exhibited remarkable influenza A virus inhibitory activity with IC50 values of 11.4, 11.0, and 2.3 μM, respectively, which were better than ribavirin as the positive control.

The taxonomic status of a novel bacterium, designated strain CPCC 100226T, isolated from a traditional Chinese medicinal herbal plant, Eucommia ulmoides Oliver, was characterized by using a polyphasic approach. The aerobic isolate formed pale white colonies on tryptic soy agar. Cells were Gram-stain-positive, rod-shaped, motile and endospore-forming. Chemotaxonomic investigations revealed the presence of meso-diaminopimelic acid as the diagnostic diamino acid, MK-7 as the predominant menaquinone, anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0 as the major fatty acids, and the strain had a phospholipid pattern of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and unidentified aminophospholipids. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was closely related to Paenibacillus aestuarii DSM 23861T with 95.1 % similarity. The G+C content of the genomic DNA was 47.9 mol%. On the basis of the genotypic and phenotypic data, the isolate is considered to represent a novel species of the genus Paenibacillus . The name proposed for this taxon is Paenibacillus eucommiae sp. nov. with CPCC 100226T (=DSM 26048T=KCTC 33054T) as the type strain.

In this work, a simple and rapid high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) method was first developed for the quantitation of toosendanin, the major constituent of the dried fruit of Melia toosendan Sieb. Et Zucc. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 μm) by isocratic elution using 33 % acetonitrile and 67 % water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL min−1. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 °C. The established method was well validated. Satisfactory linearity was achieved (r 2 > 0.9997) in a relatively wide concentration range (5–500 μg mL−1). The intra- and inter-day precisions, repeatability, and stability of the method were good with relative standard deviations (RSDs) of 1.05, 2.23, 2.39, and 2.03 %, respectively. The method also showed excellent accuracy with recovery rates of 97.42–101.87 %. Particularly, CAD showed much better sensitivity (LOQ 4 μg mL−1) than evaporative light scattering detector (LOQ 100 μg mL−1) for toosendanin’s determination. The established method was further applied in the quantitation of toosendanin in 39 batches of raw and stir-fried toosendan fructus. The HPLC-CAD method was rapid and accurate, and could be used for the routine analysis and quality control of toosendan fructus and its preparations.

In this study, endophytic fungi were isolated from Dioscorea zingiberensis C.H. Wright (DZW), and a novel clean process to prepare diosgenin from DZW was developed. A total of 123 strains of endophytic fungi were isolated from different plant tissues of DZW. Among them, the strain Fusarium sp. (CPCC 400709) showed the best activity of hydrolyzing steroidal saponins in DZW into diosgenin. Thus, this strain was used to prepare diosgenin from DZW by solid-state fermentation. The fermentation parameters were optimized using response surface methodology, and a high yield of diosgenin (2.16%) was obtained at 14.5% ammonium sulfate, an inoculum size of 12.3%, and 22 days of fermentation. Furthermore, the highest diosgenin yield (2.79%) was obtained by co-fermentation with Fusarium sp. (CPCC 400709) and Curvularia lunata (CPCC 400737), which was 98.9% of that obtained by β-glucosidase pretreated acid hydrolysis (2.82%). This process is acid-free and wastewater-free, and shows promise as an effective and clean way to prepare diosgenin for use in industrial applications from DZW.

Three new minor oleanane triterpenoid saponins, cylindrosides B (1), C (2), and D (3), were isolated from the seed of Cylindrokelupha dalatensis using chromatographic method. Their structures were established on the basis of the chemical and spectroscopic evidences. They displayed significant antitumor activity in vitro against HL60 cancer cell lines and IC50 values were 7.15 ± 0.63, 10.07 ± 0.97, and 4.74 ± 0.57 μM, respectively, by MTT method.

In this study seven strains of the genus Arthrobacter were screened by biotransformation to discover glycosylating patterns on steroid saponins. A strain of Arthrobacter nitroguajacolicus (CPCC 203516) was found to have the ability of fructosylation. Crude enzyme of the strain was extracted for the further study of conversion characteristics and patterns. Sucrose was used as a non-activated sugar donor, and fifteen steroidal saponins were involved. Nine furostan saponins of the substrates were converted, and ten products were isolated and identified. Based on the HR-ESI-MS, 1D, and 2D NMR spectral data, one fructosyl was added to furostan saponins at C6-OH of 26-O-β- d -glucopyranosyl by A. nitroguajacolicus for all nine products. One product was distinguished by an additional fructosyl at the position of C6-OH on the first added fructosyl. Spirostan saponins of the substrates could not be converted. Steroidal saponins embracing a fructosyl are quite rare according to other reports based on similar studies. This study successfully converted furostan saponins into new compounds.

Current production of diosgenin mainly depends on the acid hydrolysis of steroidal saponins from Dioscorea plants, and, in China especially, Dioscorea zingiberensis C. H. Wright (DZW) is used. The experimental results we obtained demonstrated that furostanol saponins, as the main constituents in DZW, were prone to generate 25-spirosta-3,5-diene as side product during the acid hydrolysis process, while spirostanol saponins hardly generated 25-spirosta-3,5-diene. This 25-spirosta-3,5-diene was the key reason leading to the low yield of diosgenin from DZW by acid hydrolysis. Effective conversion of furostanol saponins into spirostanol saponins can avoid the generation of 25-spirosta-3,5-diene so as to increase the yield of diosgenin, suggesting the importance of this preliminary conversion for improving the yield of diosgenin from DZW by acid hydrolysis. The conversion of furostanol saponins into spirostanol saponins can be performed by either enzymatic hydrolysis or spontaneous fermentation, whereas enzymatic hydrolysis is more controllable compared with spontaneous fermentation.

A Gram-positive bacterium originating from the surface-sterilized leaf of Paris polyphylla var. yunnanensis (Franch.) was characterized by using a polyphasic approach. The isolate formed yellow, smooth, circular colonies on nutrient agar with 0.2 % starch (NSA). Cells were non-motile, non-sporulating, irregular rods or cocci. Strain CPCC 203535T had the highest 16S rRNA gene sequence similarity to the type strain of Ornithinimicrobium kibberense (96.9 %) and formed the deepest branch in the genus Ornithinimicrobium in the neighbour-joining (NJ) phylogenetic tree based on 16S rRNA gene sequences. The major menaquinones of strain CPCC 203535T were MK-8(H4), MK-8(H2) and MK-8. The peptidoglycan contained ornithine as the diagnostic diamino acid. The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI) and unknown lipid (UL). The major fatty acids iso-C14 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0 were consistent with the fatty acid patterns reported for members of the genus Ornithinimicrobium . The DNA G+C composition is 71.4 mol%. The results of physiological and biochemical tests allowed phenotypic differentiation of strain CPCC 203535T from its closest phylogenetic species in the genus Ornithinimicrobium . Strain CPCC 203535T represents a novel species of the genus Ornithinimicrobium , for which the name Ornithinimicrobium flavum sp. nov. is proposed, with CPCC 203535T (=NBRC 109452 T=KCTC 29164T) as the type strain.

Three new oleanane-type triterpenoid saponins named celosins H (1), I (2), and J (3) were isolated from the seeds of Celosia argentea L. Their structures were characterized as 3-O-β-d-xylopyranosyl-(1 → 3)-β-d-glucuronopyranosyl-polygalagenin 28-O-β-d-glucopyranosyl ester, 3-O-β-d-glucuronopyranosyl-medicagenic acid 28-O-β-d-xylcopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranosyl ester, and 3-O-β-d-glucuronopyranosyl-medicagenic acid 28-O-α-l-arabinopyranosyl-(1 → 3)-[β-d-xylcopyranosyl-(1 → 4)]-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranosyl ester by NMR, MS, and chemical evidences, respectively. In our opinion, celosins H–J could be used as chemical markers for the quality control of C. argentea seeds.

Twelve flavonoid glycosides were involved in the biotransformation of the glycosyl moieties by Curvularia lunata 3.4381, and the products were analyzed by UPLC/PDA-Q-TOF-MS(E). Curvularia lunata displayed hydrolyzing activities on the terminal Rha or Glc units of some flavonoid glycosides. Terminal Rha with a 1 → 2 linkage of isorhamnetin-3-O-neohesperidoside and typhaneoside could be hydrolyzed by Curvularia lunata, but terminal Rha with a 1 → 6 linkage of rutin, typhaneoside, and quercetin-3-O-apiosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-glucoside could not be hydrolyzed. Curvularia lunata could also hydrolyze the Glc of icariin, floramanoside B, and naringin. This is the first report of the hydrolysis of glycosyl units of flavonoid glycosides by Curvularia lunata. A new way to convert naringin to naringenin was found in this research.

Two actinobacterial strains, CPCC 203464T and CPCC 203448, isolated from surface-sterilized stems of medicinal plants were subjected to a polyphasic taxonomic study. These two aerobic organisms formed pale yellow colonies on tryptic soy agar (TSA). Cells were Gram-stain-positive, non-acid-fast, non-motile, rod- or coccoid-like elements. Comparative 16S rRNA gene sequence analysis indicated that strains CPCC 203464T and CPCC 203448 were most closely related to the type strains of the species of the genus Williamsia . Chemotaxonomic properties such as containing meso-diaminopimelic acid in the cell wall, arabinose, galactose and ribose being the whole-cell hydrolysate sugars, phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylinositol (PI) as the phospholipids, and C16 : 0, 10-methyl C18 : 0, C18 : 1ω9c, C16 : 1ω7c and/or iso-C15 : 0 2-OH as major fatty acids supported the affiliation of strains CPCC 203464T and CPCC 203448 to the genus Williamsia . The DNA–DNA hybridization values in combination with differentiating chemotaxonomic and physiological characteristics strongly suggested that these two isolates should be classified as representatives of a novel species of the genus Williamsia . The name Williamsia sterculiae sp. nov. is proposed, with strain CPCC 203464T ( = DSM 45741T = KCTC 29118T) as the type strain.

A Gram-stain-positive, rod-shaped actinobacterium, designated strain I10A-01402T, was isolated from surface-sterilized roots of a medicinal plant, Perilla frutescens, collected in a suburb of Beijing, China. Chemotaxonomically, the strain contained ll-diaminopimelic acid as the diagnostic diamino acid and MK-8(H4) as the predominant menaquinone. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The major fatty acids were C17 : 1ω9c, C18 : 1ω9c, C17 : 0, C16 : 0 and iso-C16 : 0. The genomic DNA G+C content was 70.4 mol%. 16S rRNA gene sequence analysis indicated that strain I10A-01402T belonged to the genus Nocardioides . Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolate formed a robust cluster with Nocardioides ginsengisegetis Gsoil 485T, N. koreensis MSL-09T and N. alkalitolerans KSL-1T. On the basis of the evidence from our polyphasic taxonomic study, a novel species, Nocardioides perillae sp. nov., is proposed. The type strain is I10A-01402T ( = CPCC 203382T = DSM 24552T = KCTC 29022T).

Ginsenoside Rh2, one of the characteristic components in red ginseng (roots steamed at 98-100 °C without peeling), is a rare ginsenoside with potential bioactivity. The preparation of ginsenoside Rh2 was studied by using acid hydrolysis, alkaline hydrolysis, synthesis and biotransformation techniques. Microbial and enzymatic transformations were regarded as the optimization methods of all these techniques, owing to their high specificity, yield and productivity. The pharmacological activities and pharmacokinetics of ginsenoside Rh2 and its derivatives were extensively researched. In this paper, their preparation and activities have been reviewed, which can provide information for the research and development of ginsenoside Rh2.

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.

Eleven new furostanol saponins, typaspidosides B-L (1-11), one new spirostanol saponin, typaspidoside M (12), and five known spirostanol saponins, 25S-atropuroside (13), neoaspidistrin (14), (25S)-pratioside D1 (15), 25S-aspidistrin (16) and 25S-neosibiricoside (17) were isolated from the rhizomes of Aspidistra typica Baill. The structures of the new compounds were established using 1D and 2D NMR (1H-1H COSY, HMQC, HMBC and ROESY) spectroscopy, high resolution mass spectrometry, and chemical methods. The aglycones of 1-3 (unusual furostanol saponins with opened E ring type), 9 and 10 (the methoxyl substituent at C-23 position) were found, identified from natural products for the first time. Moreover, the anti-HIV activities of the isolated steroidal glycosides were assessed, and compounds 13, 14, 16 and 17 exhibited high active against HIV-1.

AIM: To investigate the chemical constituents from the fresh fruits of Momordica charantia L. METHODS: Chemical constituents were isolated and purified by chromatography on silica gel and octadecylsilanized (ODS) silica gel, and the structures were determined by spectral data and chemical evidence. RESULTS: A new C30 sterol glycoside, named 25-isopropenylchole-5, (6)-ene-3-O--D-glucopyranoside (1), was isolated from the fresh fruits of M. charantia L. CONCLUSION: Compound 1 is a new C30 sterol glycoside.

Five commercial enzyme preparations were screened for hydrolysis of the glucuronic acid units of glycyrrhizin (GL) and baicalin. Two preparations hydrolyzing GL to glycyrrhetic acid (GA) and four enzyme preparations hydrolyzing baicalin to baicalein were obtained. One enzyme preparation with the ability to hydrolyze both GL and baicalin, namely Rapidase Pineapple, was purified by anion exchange, cation exchange and molecular sieve chromatography. The results of purification indicated that the enzymes containing the glycyrrhizin-β-d-glucuronidase (GBDG) and baicalin-β-d-glucuronidase (BBDG) activities were distinct, with different substrate specificities, molecular weights and enzymatic characteristics. GBDB hydrolyzed GL to GA, but had no detectable activity on baicalin, and BBDG hydrolyzed baicalin to baicalein, but could not hydrolyze GL. However, both GBDG and BBDG could hydrolyze the artificial substrate p-nitrophenyl- β-d-glucuronide (pNPGA).

Ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/ESI-Q-TOF-MS/MS) was applied to separate and identify triterpenoid saponins in crude extract from the stem bark of Albizia julibrissin Durazz. The molecular weights were determined by comparing quasi-molecular ions [M+NH(4)](+) in positive mode and [M-H](-) and [M-2H](2-) ions in negative mode. The MS/MS spectra of the [M-H](-) ions for saponins provided a wealth of structural information related to aglycone skeletons, sugar types and linked sequence. On the basis of the fragmentation behavior of known saponins isolated before, saponins from this plant were identified, even though references were not available. As a result, a total of twenty-eight saponins in the crude extract were identified, which all had a common basic skeleton of the triterpene oleanolic acid and eight of them were new compounds.

Aim To study the hydroxylation in glycyrrhetinic acid (GA) by biocatalysis, and evaluate the antibacterial activities of the derivatives. Methods The hydroxylation was performed via incubating GA with a fungus, Cunninghamella blakesleeana (C. blakesleeana) AS 3.970 at 30 °C for 5 days. Results GA was converted into two major derivatives and four minor derivatives. The two major derivatives were isolated and elucidated as 3-oxo-7β-hydroxy-glycyrrhetinic acid (GA-1) and 7β-hydroxy-glycyrrhetinic acid (GA-2) based on their spectral data, and other derivatives (GA-3–GA-6) were identified by HR-ESI-MS. The antibacterial activities of substrate and two major derivatives were evaluated on four bacteria strains and one yeast strain. Conclusion Cunninghamella blakesleeana AS 3.970 has the capability of hydroxylation for GA. The two major derivatives and GA were found to have considerable activities against drug-resistant Enterococcus faecalis.

It is known that the sugar chain linked to steroidal frame plays an important role in physiological and pharmacological activities. In the previous research, we found and confirmed that the terminal C3- O -α-1,2-rhamnosyl moiety linked to the C-3 of steroidal saponin is the key group of platelet aggregation and cytotoxicity. In order to make a complete approach for the structure–activity relationship, we have tried to find the specific enzymes modifying the structure of C3-sugar chain. In the present paper, we describe a novel enzyme from, Klerzyme-150 (K-150), which is specifically capable of hydrolyzing the α-1,4-glycosyl residues at C-3 postion of steroidal saponins. 15 steroidal saponins with different monosaccharides at C3-sugar chains were chosen as substrates to investigate the substrate specificity of K-150. The results showed, based on TLC, HPLC and spectra data analyses, that all products were determined as secondary saponins with the α-1, 4-glycosyl residues removed, which indicated that the enzyme exhibited strict regioselectivity and stereoselectivity. The novel enzyme was purified from K-150 to apparent homogeneity and its structure was identified as rhamnogalacturonan lyase A (Rgl A). The molecular mass of the purified enzyme was 52.08 kDa.

Two new spirostanol saponins, kingianoside I (1) and kingianoside K (2), corresponding to (3β,23S,25R)-23-hydroxy-12-oxospirost-5-en-3-yl 4-O-β-D-glucopyranosyl-β-D-galactopyranoside (1) and (3β,25R)-7-oxospirost-5-en-3-yl α-L-arabinofuranosyl-(14)-[6-deoxy-α-L-mannopyranosyl-(12)]-β-D-glucopyranoside (2), along with 13 known compounds, daucosterol, (25R)-kingianoside G, (25RS)-kingianoside A, pratioside D1, (25RS)-pratioside D1, (25S)-kingianoside C, kingianoside C, ginsenoside Rb1, saponins Tb and Pb, dioscin, gracillin, and saponin Pa, were isolated from the processed rhizomes of Polygonatum kingianum. The structures of the new compounds were elucidated by detailed spectroscopic analyses, including 1D- and 2D-NMR techniques, and chemical methods. Compound 2 contains a novel unusual spirostanol saponin aglycone. Ginsenoside Rb1 and saponin Tb were isolated for the first time from the genus Polygonatum. The 13 known compounds were detected for the first time in the processed Polygonatum kingianum.

Aim To study of structural modification in steroidal saponin by biotransformation technology. Methods Structural modification was carried out of glycosyl residues of dichotomin at C-3 position by Pectinex BE XXL (PBX). Cytotoxicity against HL-60 cells of all products was investigated. Results The PBX could hydrolyze the glycosyl residues of dichotomin one-by-one to produce nine transformed compounds. The transformed product (mixture) and four secondary spirostanosides among them exhibited cytotoxicity. Compounds P5, P6 and P7 with α-1, 2 end-rhamnosyl residues showed a potential cytotoxic effect on HL-60 cells; P8 with α-1, 4 end-rhamnosyl residue had a weak effect on HL-60 cells. Dichotomin, secondary furostanosides and aglycone were not cytotoxic. Conclusion PBX has various hydrolyzation to the glycosidic bonds of dichotomin. Nine obtained transformed compounds widen steroidal saponin library. Meanwhile, screening activity affords data for further pharmacological study.

Two novel furostanol saponins were isolated from the fresh tubers of Ophiopogon japonicus. Comprehensive spectroscopic analysis allowed the chemical structures of the compounds to be assigned as (25R)-26-[(O-beta-d-glucopyranosyl-(1 --> 2)-beta-d-glucopyranosyl)]-22alpha-hydroxyfurost-5-ene-3-O-beta-d-xylopyranosyl-(1 --> 4)-O-[alpha-l-rhamnopyranosyl-(1 --> 2)]-beta-d-glucopyranoside (1, ophiopogonin F) and (25R)-26-[(O-beta-d-glucopyranosyl-(1 --> 6)-beta-d-glucopyranosyl)]-22alpha-hydroxyfurost-5-ene-3-O-beta-d-xylopyranosyl-(1 --> 4)-O-[alpha-l-rhamnopyranosyl-(1 --> 2)]-beta-d-glucopyranoside (2, ophiopogonin G). The rare furostanol saponins with two glucosyl residues at C-26 position were isolated from the natural source for the first time.

A new triterpenoid compound (1) and a known compound (2) were isolated from the product of biotransformation of glycyrrhizic acid by Aspergillus niger. On the basis of the 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC and NOESY) and MS spectrometry, their structures were established as 7beta, 15alpha-dihydroxy-3,11-dioxo-oleana-12-en-30-oic acid (1) and 15alpha-hydroxy-3,11-dione-oleana-12-en-30-oic acid (2), respectively.

A mixture of a pair of stereoisomeric new spirostanol saponins (1a and 1b) and a new cholestane saponin (2) were isolated from the rhizome of Paris pollyphylla Smith var. yunnanensis. Their structures were elucidated as (25R)-spirost-5-en-3beta, 7beta-diol-3-O-alpha-L-arabinofuranosyl-(1 --> 4)-[alpha-L-rhamnopyranosyl-(1 --> 2)]-beta-D-glucopyranoside (1a), (25R)-spirost-5-en-3beta, 7alpha-diol-3-O-alpha-L-arabinofuranosyl-(1 --> 4)-[alpha-L-rhamnopyranosyl-(1 --> 2)]-beta-D-glucopyranoside (1b) and 26-O-beta-D-glucopyranosyl-(25R)-Delta(5(6), 17(20))-dien-16, 22-dione-cholestan-3beta, 26-diol-3-O-alpha-L-arabinofuranosyl-(1 --> 4)-[alpha-L-rhamnopyranosyl-(1 --> 2)]-beta-D-glucopyranoside (2) by a combination of HR-ESI-MS, FAB-MS, 1D and 2D NMR techniques (including (1)H-NMR, (13)C-NMR, (1)H--(1)H COSY, HSQC, HMBC and NOESY).

The galactosylation of furostanosides and spirostanosides were investigated by using β-galactosidase from Lactobacillus bulgaricus L3 as a catalyst and lactose as a sugar donor. Four novel galactosylated furostanoside products (compounds 1-4) from compound F, compound G, compound I, and compound H were obtained and their structures were identified by HR-ESI-MS, 1D and 2D NMR spectra. The result showed that galactosyl moiety was found to be added to the 6-OH of the 26-O-glucosyl in these four furostanoside substrates.

Compound 1, a triterpenoid saponin from Ardisia gigantifolia Stapf. showing potential anti-tumor activity, was transformed into three derivatives (2-4) by Aspergillus avenaceus 3.4454. Among them, compounds 2 and 3 are new compounds. Their structures were elucidated on the basis of 1D NMR, 2D NMR, HR-ESI-MS, and optical rotation data. Compounds 1-3 were evaluated for their cytotoxicity against human hepatocellular carcinoma and normal liver cells by cell counting kit 8 colorimetric assay. Compound 3 displayed better cytotoxicity against Bel-7402 and HepG2 cell lines and much weaker cytotoxicity against normal liver L02 cell than that of positive control (epirubicin hydrochloride).

Phytochemical investigation of the fresh tubers of Ophiopogon japonicus led to the isolation of two new furostanol saponins (1 and 2) together with two known steroidal saponins (3 and 4). Comprehensive spectroscopic analysis allowed the chemical structures of two new compounds to be elucidated as (25R)-26-O-[β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl]-5-ene-furost-1β,3β,22α,26-tetraol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 4)]-β-d-glucopyranoside (1, ophiopogonin P) and (25R)-26-O-[β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl]-5-ene-furost-1β,3β,22α,26-tetraol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 4)]-β-d-glucopyranoside (2, ophiopogonin Q). Furostanol saponins with the disaccharide chain linked at C-26 hydroxy group of the aglycone have been rarely reported from natural sources.

Five new steroidal glycosides, timosaponin J ( 1), timosaponin K ( 2), (25 S)-karatavioside C ( 5), timosaponin L ( 6), and (25 S)-officinalisnin-I ( 8), together with eight known steroidal saponins, timosaponin E (1) ( 3), purpureagitosid ( 4), timosaponin BII ( 7), timosaponin B III ( 9), anemarrhenasaponin I ( 10), anemarrhenasaponin III ( 11), anemarrhenasaponin A (2) ( 12), and timosaponin A III ( 13), were isolated from the rhizomes of Anemarrhena asphodeloides. Their structures were elucidated on the basis of spectroscopic and chemical evidence. The aglycones of compounds 1 and 2 are new aglycones. Compounds 1- 13 were evaluated for their platelet aggregation activities, and compound 13 exhibited the strongest inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation.

This paper was designed to develop a novel method via ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-i¬‚ight tandem mass spectrometry (UPLC-ESI-QTOF-MSE) for the analysis and identification of saponins from extract of Ziziphi spinosae Semen (ZSS). The possible fragmentation pathways were proposed on the basis of MSE data. In results, 13 saponins, including two unknown compounds, were identified and they were divided into two groups on the basis of mass spectral analysis. Compound 1~6, which were of keto-dammarane type triterpene saponin, underwent McLafferty-type (or McLafferty) rearrangement, and compound 7~13, which were of dammarane type triterpene saponin, underwent retro Diels-Alder (RDA) cleavage. The results testified that the developed method was useful to rapidly and simultaneously identify the constituents in ZSS. In this paper, the principles of McLafferty-type (or McLafferty) rearrangement were firstly utilized to elucidate the proposed fragmentation pathways of saponins from ZSS. Key words: Ziziphi spinosae semen, ultra-performance liquid chromatography (UPLC), quadrupole time-of-i¬‚ight tandem mass spectrometry (QTOF-MSE), retro Diels-Alder cleavage, McLafferty-type rearrangement.

Background Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation. Objective To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation. Methods and results Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways. Conclusion We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.

This study compared the pharmacokinetics of albiflorin (ALB) and paeoniflorin (PAE), respectively, after oral administration of ALB, PAE, Radix Paeoniae alba (RPA) extract, and Danggui-Shaoyao-San (DSS) extract to rats on separate occasions. Analytes were detected simultaneously with liquid chromatography-tandem mass spectrometry. Noncompartmental pharmacokinetic parameters were calculated. After oral administration of RPA and DSS extract to rats, ALB reached maximum concentrations of 4637 ± 2774 ng/ml (0.40 ± 0.14 h) and 226 ± 122 ng/ml (0.35 ± 0.14 h) and PAE reached maximum concentrations of 2132 ± 560 ng/ml (0.40 ± 0.14 h) and 143 ± 65 ng/ml (0.45 ± 0.11 h), respectively. Compared to the AUC(0 - t) value (1122 ± 351 and 722 ± 158 ng h/ml for ALB and PAE, respectively) after administration of monomers, larger AUC(0 - t) value of ALB (4755 ± 2560 ng h/ml) and PAE (2259 ± 910 ng h/ml) after administration of RPA extract and smaller AUC(0 - t) value of ALB (411 ± 118 ng h/ml) and PAE (242 ± 126 ng h/ml) after administration of DSS extract were obtained. The C(max), AUC, and K(el) of ALB and PAE were remarkably increased (P 0.05, 0.01 or 0.005) during oral administration of RPA extract in comparison to that of DSS extract.

Objectives Total steroidal saponins extracted from the rhizome of Paris polyphylla (TSSP) have been used in China for the treatment of abnormal uterine bleeding. The aim of this study was to analyse the structure–activity relationship of steroidal saponins purified from P. polyphylla Sm. var. yunnanensis on rat myometrial contractions, and investigate the synergism among themselves as well as with known inherent agonists, such as Prostaglandin F2α (PGF-2α). Methods In this study, 22 steroidal saponins purified from TSSP were screened for their contractile activity in isolated uterine strips from estrogen-primed rats. Key findings It was shown that spirostanol glycosides exhibited inducible or inhibitory activity in rat uterine contraction based on the difference of their structures, which was not only attributed in part to the number, the length and the position of sugar side chains attached by a glycoside, but also related to the structure of the aglycone. Furthermore, synergistic actions were observed among pennogenin or diosgenin glycosides as well as with the known inherent agonist PGF-2α, indicating they may share, at least in part, similar pathways with PGF-2α in stimulating myometrial contractions. Finally, the contractile response of rat myometrium to spirostanol glycosides was significantly enhanced with advancing pregnancy. Conclusions Together, these data support the possibility that some spirostanol glycosides may represent a new type of contractile agonist for the uterus and their synergism may be responsible for the therapeutic effect of TSSP on abnormal uterine bleeding.

Further studies on the fresh rhizomes of Polygonatum kingianum led to the isolation of one new spirostanol saponin (25R)-kingianoside G (1), and two pairs mixture of 25R and 25S stereoisomeric spirostanol saponins (25R, S)-pratioside D1 (2a, 2b) and (25R, S)-kingianoside A (3a, 3b), among them 2b and 3b were new spirostanol saponins, together with another two known compounds, disporopsin (4) and daucosterol (5). The structures of the new saponins were determined by detailed analysis of their 1D and 2D NMR spectra, and chemical evidences.

Effects of four Si-Wu-Tang (SWT)'s constituents, fructose (Fru), paeoniflorin (Pae), ferulic acid (FA), tetramethyl pyrazine (TP), and their combination on irradiated mice as model of anaemia were investigated, with the purpose of further understanding the relationship between SWT's constituents and activities. Similarly to SWT, oral administration of Fru, Pae, FA, TP and their combination, to some extent, all showed effects of increasing the number of peripheral leukocyte and increasing four types of progenitor cells in bone marrow, including colony-forming unit-granulocyte-macrophage (CFU-GM), colony-forming unit-mature erythroid (CFU-E), colony-forming unit-immature erythroid (BFU-E) and colony-forming unit-multipotential (CFU-mix). Pae and FA showed significant body weight reducing effect, which were largely abolished when they were combined with Fru and TP. The SWT, Fru and combination significantly increased the thymus index while Pae significantly decreased it. Both SWT and TP significantly increased the spleen index but the combination did not. The results suggested that multiple constituents contribute to the promoting effect of SWT on hematopoiesis. Although being a very common compound in plants, the Fru has a special contribution to SWT's effect, which cannot be neglected. It may be an important active constituent that is responsible for SWT's promoting effect on hematopoiesis and immunity. Another suggestion is that when being combined, some effect of one constituent, sometimes is unexpected side effect, may be abolished by other. This may reflect the advantage of multiple constituent characteristics possessed by most TCMs.