Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins

Combined with preparative high-performance liquid chromatography, high-speed counter-current chromatography was employed for isolation and purification of saponins from Gypsophila paniculata L. n-Hexane-n-butanol-methanol-0.02% TFA (1:9:1:9, v/v) was employed as solvent system and 210 nm was chosen as the wavelength of ultraviolet detection for the first time. The research tried to compare HSCCC with prep-HPLC, and further integrated their advantages to improve separation efficiency. Five known triterpene saponins were identified by 13C NMR and ESI-MS and their purities were all above 96%. The results demonstrated that adopted method was a feasible, economical and efficient technique for rapid preparative isolation of saponins.     

Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli

Escherichia coli K-12 was exposed to Quillaja saponaria saponins from various commercial firms (Sigma, Roth and Nor-feed) and to an extract of Yucca schidigera plant powder (DK Sarsaponin 30) at different concentrations (0·05–1·0% w/v). A concentration-dependent response was observed. Quillaja saponaria saponins from Sigma increased growth up to 0·1% (w/v) level, whereas Nor-feed and Roth saponins produced maximum growth at a much higher level (0·5 and 0·75%, w/v, respectively). These results suggest that quillaja saponins from various sources differ in their biological activity, although all three saponins had the same content of vanillin-sulphuric acid reactive moieties. The lyophilized water extract from the DK Sarsaponin powder showed maximum growth at 0·1% (w/v) level. The levels at which maximum growth was observed did not change on subjecting the quillaja or yucca saponins to heat treatment in an autoclave (121 °C for 30 min). All the saponins and the plant extract increased growth of Escherichia coli up to a certain concentration and thereafter decreased growth. In spite of the decreased growth at higher levels of saponins, it was higher compared to the control (without saponin) up to levels of 1% (w/v) for all saponins except Quillaja saponins from Sigma, for which the growth was lower at levels of 0·25% (w/v) and higher. Saponins have the potential to modulate microbial growth in natural and artificial fermenters.     

HSCCC-ELSD法分离纯化青葙子中的皂苷

连接有蒸发光散射检测器的高速逆流色谱仪首次成功的应用于制备和分离青葙子中的皂苷celosins A和B.二氯甲烷∶正丁醇∶甲醇∶水(4∶0.3∶3∶2)+0.5％冰醋酸作为洗脱溶剂系统.从半制备型HSCCC收集到的组分进行HPLC分析,可以得到:celosin A纯度为98.9％,celosin B的纯度为98.1％.这是高速逆流色谱仪首次被用于纯化青葙子中的皂苷,两个化合物的结构通过碳谱和质谱来确定.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin-rich fractions from different plant materials

Aims:  Investigation of the effects of saponin-rich fractions on rumen fermentation, methane production and the microbial community.
Methods and Results:  Saponins were extracted from Carduus , Sesbania and Knautia leaves and fenugreek seeds. Two levels of saponin-rich fractions with a substrate were incubated using the Hohenheim gas method. Methane was measured using an infrared-based methane analyser and microbial communities using quantitative PCR. On addition of saponin-rich fractions, methane and short-chain fatty acid production was not affected. The protozoal counts decreased by 10–39%. Sesbania saponins decreased methanogen population by 78%. Decrease in ruminal fungal population (20–60%) and increase in Fibrobacter succinogenes (21–45%) and Ruminococcus flavefaciens (23–40%) were observed.
Conclusions:  The saponins evaluated possessed anti-protozoal activity; however, this activity did not lead to methane reduction. Fenugreek saponins seemed to have potential for increasing rumen efficiency. The saponins altered the microbial community towards proliferation of fibre-degrading bacteria and inhibition of fungal population.
Significance and Impact of the Study:  The uni-directional relationship between protozoal numbers and methanogenesis, as affected by saponins, is not obligatory. All saponins might not hold promise for decreasing methane production from ruminants.     

Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC-MS/MS in dog plasma for pharmacokinetics of compound Danshen tablets

Compound Danshen tablets are composed of Panax notoginseng, Salvia miltiorrhiza and Borneol. The tablets are prescribed for treatment of cardiovascular diseases in China. The present study aimed at developing a specific and sensitive LC-MS/MS method to simultaneously determine three bioactive P. notoginseng saponins, i.e., notoginsenoside R1, ginsenoside Rg1 and Rb1, in dogs after a single oral administration of the compound tablets in order to obtain the clinically relevant saponin-related pharmacodynamics of the tablets in patients. The R1, Rg1 and Rb1 were extracted from dog plasma with acetone-methanol (80:20, v/v), separated by reversed phase liquid chromatography and determined by tandem mass spectrometry (LC-MS/MS) with positive electrospray ionization (ESI). The developed method reached lower limit of quantitation (LLOQ) at 0.10 ng/ml for the three saponins. The method was validated in terms of selectivity, matrix effects, linearity, precision and accuracy, and then was applied to a pharmacokinetic study of the three bioactive saponins simultaneously in dogs after a single oral administration of compound Danshen tablets at a clinical equivalent dose. The C(max) and AUC((0-∞)) for R1, Rg1 and Rb1 were 1.91, 3.34 and 28.6 ng/ml, and 7.5, 11.0, and 1712 (h ng/ml), respectively.     

Preparation of progenin III from total steroidal saponins of Dioscorea nipponica Makino using a crude enzyme from Aspergillus oryzae strain

Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.     

Simultaneous analysis of isomers of escin saponins in human plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study after oral administration

A rapid and sensitive bioassay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of four isomeric escin saponins (escin Ia, escin Ib, isoescin Ia and isoescin Ib) in human plasma has been developed and validated. Sample preparation of plasma after addition of telmisartan as internal standard (I.S.) involved solid-phase extraction (SPE) on C18 cartridges. Separation was based on reversed phase chromatography using gradient elution with methanol–acetonitrile (50:50, v/v) and 10 mM ammonium acetate solution (pH 6.8). MS/MS detection in the positive ion mode used multiple reaction monitoring of the transition at m/z 1113.8 → 807.6. Stability issues with the four saponins required the addition of formic acid to plasma samples prior to storage at ?80 °C and analysis within 30 days. The method was linear at concentrations up to 10 ng/mL with correlation coefficients > 0.996 for all analytes. The lower limit of quantitation (LLOQ) for all four saponins was 33 pg/mL. Intra- and inter-day precisions (as relative standard deviation) were all <15% and accuracies (as relative error) in the range ?5.3% to 6.1%. The method was successfully applied to a pharmacokinetic study of escins in healthy volunteers after oral administration of sodium aescinate tablets containing 60 mg escin saponins.     

巨尾桉叶中皂苷的提取分离及其抗氧化活性研究

开展了从巨尾桉叶中提取皂苷的工艺优化研究.考察了温度、时间,溶剂用量对皂苷得率的影响,并对粗提物进行纯化.结果表明,巨尾桉叶皂苷的优化条件是:提取温度61.8℃,提取时间5.02h,溶剂与巨尾桉叶的液料比为20.09∶1,皂苷的提取得率(g/g)为5.51％.提取物经大孔树脂-丙酮沉淀联用分离法纯化后皂苷含量由16.22％提高到50.35％.对粗提物、大孔树脂纯化物和大孔树脂吸附-丙酮沉淀纯化物进行抗氧化活性研究.结果表明,它们对DPPH自由基的最大清除率分别为83.02％、84.20％和85.62％,对过氧化氢的最大清除率分别为80.10％、55.02％和48.21％,对超氧阴离子自由基的最大清除率分别为16.62％、13.43％和10.01％.     

百合总皂苷提取工艺及抗抑郁活性研究

本文研究百合总皂苷最佳提取工艺,继而采用AB-8大孔吸附树脂富集纯化百合总皂苷,并测试其抗抑郁活性。采用正交实验,考察乙醇浓度、提取次数、固液比及提取时间四个因素对该工艺的影响;采用小鼠悬尾实验和小鼠强迫游泳实验评价百合总皂苷的抗抑郁活性。确立了百合总皂苷的最佳提取工艺为:提取时间3h,乙醇浓度80%,固液比1∶10,提取次数为3次。在最佳提取工艺条件下,总皂苷提取率为1.24%。经过AB-8大孔树脂富集纯化总皂苷的含量达到62%以上。药理实验表明,百合总皂苷中剂量、小剂量能明显缩短小鼠悬尾的不动时间和游泳时间(P<0.05或者P<0.01),百合富集纯化的总皂苷部位具有较好的抗抑郁活性。     

芦笋皂苷的提取纯化及其糖基组成

芦笋皂苷是以芦笋下脚料为原料提取出来的一种糖甙化合物。目的是充分利用再生资源,变废为宝,同时解决芦笋下脚料废弃物所造成的环境污染问题。实验探讨了乙醇浓度、液料比、温度、时间等工艺条件对芦笋皂苷提取率的影响,筛选出最佳提取工艺:乙醇浓度95 %、液料比(V/W)为6∶1、温度90℃、时间4h。从新鲜芦笋下脚料、芦笋干品提取皂苷的平均得率分别为1.70 %、4.01%。芦笋皂苷经氧化铝柱层析分离,以40%乙醇的溶液为洗脱剂,洗出曲线为单一对称峰。采用UV、IR、HPLC等色谱对芦笋皂苷的结构特点、糖基组成进行初步分析。结果显示芦笋皂苷具有呋喃甾烷的结构特征,其糖基由木糖(Xyl)、岩藻糖(Fuc)、阿拉伯糖(Ara)组成,摩尔比为Xyl∶Fuc∶Ara =1 0∶0 13∶19 4 2 ,平均分子质量(Mw)为185 0 0。     

Phytochemical complementarities among endophyte-infected tall fescue, reed canarygrass, birdsfoot trefoil and alfalfa affect cattle foraging

We determined whether plant diversity and sequence of plant ingestion affected foraging when cattle chose from plants that varied in concentrations of alkaloids, tannins and saponins. We hypothesized cattle that ate high-alkaloid grasses (endophyte-infected tall fescue (TF) or reed canarygrass (RCG)) would prefer forages high in tannins (birdsfoot trefoil, BFT+) or saponins (alfalfa, ALF+), because tannins and saponins can bind to alkaloids, presumably reducing their absorption. We further hypothesized that forages with tannins or saponins consumed before, rather than after, foraging on high-alkaloid grasses would promote greater use of those grasses presumably by binding to alkaloids, thereby reducing their absorption. In Phase 1, cattle (n = 32) grazed on either high (+) or low (-) alkaloid grass (TF or RCG) pastures for 30 min each morning at 0600 h and were then offered a choice of BFT+, BFT-, ALF+ and ALF- for 60 min each day for 12 days. In Phase 2, cattle (n = 32) were first offered a choice of BFT+ or ALF+ for 30 min at 0600 h and then placed on grass (TF+ or -, or RCG+ or -) pastures for 60 min for 12 days. In both phases, we had four spatial replications of four treatments with 2 per calves assigned to each of the 16 replications per treatment combinations. Scan samples of individuals at 2-min intervals were used to determine incidence of foraging on each plant species (%). Cattle grazed more on RCG than on TF in Phases 1 (62% v. 27%; P = 0.0015) and 2 (71% v. 32%; P = 0.0005). In Phase 1, cattle that first foraged on RCG+ or TF- subsequently preferred ALF over BFT, whereas cattle offered RCG- or TF+ foraged on ALF and BFT equally. Foraging by cattle on RCG was cyclic during Phase 1, whereas cattle foraging on TF markedly decreased incidence of use of TF from 41% to only 16% by the end of the 12-day trial (P = 0.0029). Contrary to the cyclic (RCG) or steadily declining (TF) use of grasses in Phase 1, cattle steadily and dramatically increased foraging on both RCG and TF throughout Phase 2, when they first grazed BFT+ or ALF+ followed by high-alkaloid grasses (P = 0.0159). Our findings suggest that in plant species the sequence of ingestion influenced foraging behavior of cattle and that secondary compounds influenced those responses.     

Steroidal saponin production in callus cultures of Solanum aculeatissimum Jacq.

 Callus was induced from the epicotyl of S. aculeatissimum, and the relation between culture conditions and the production of steroidal saponins in the callus was studied. The results indicated that the callus produced the steroidal saponins aculeatiside A and B. The highest production of steroidal saponins occurred at the middle of the log phase. Optimal conditions for the production of steroidal saponins were culturing on MS basal medium supplemented with the combination of 0.05 ppm or 0.1 ppm NAA and 10 ppm BA, and fructose as a carbon source, in the dark at 25  °C. Under these optimal conditions, the callus produced 0.8% (per dry weight) steroidal saponins, or 0.32% aculeatiside A and 0.48% aculeatiside B. Received: 24 December 1999 / Revision received: 21 February 2000 / Accepted: 16 May 2000     

Effects of Gypsophila saponins on bacterial growth kinetics and on selection of subterranean clover rhizosphere bacteria

Plant secondary metabolites, such as saponins, have a considerable impact in agriculture because of their allelopathic effects. They also affect the growth of soil microorganisms, especially fungi. We investigated the influence of saponins on rhizosphere bacteria in vitro and in soil conditions. The effects of gypsophila saponins on the growth kinetics of rhizosphere bacteria were studied by monitoring the absorbance of the cultures in microtiter plates. Gypsophila saponins (1%) increased the lag phase of bacterial growth. The impact of gypsophila saponins on subterranean clover rhizosphere was also investigated in a pot experiment. The addition of gypsophila saponins did not modify clover biomass but significantly increased (twofold with 1% saponins) the weight of adhering soil. The number of culturable heterotrophic bacteria of the clover rhizosphere was not affected by the addition of gypsophila saponins. Nevertheless, the phenotypical characterization of the dominant Gram-negative strains of the clover rhizosphere, using the Biolog system, showed qualitative and quantitative differences induced by 1% saponins. With the addition of saponins, the populations of Chryseomonas spp. and Acinetobacter spp., the two dominant culturable genera of control clover, were no longer detectable or were significantly decreased, while that of Aquaspirillum dispar increased and Aquaspirillum spp. became the major genus. Aquaspirillum dispar and Aquaspirillum spp. were also the dominant rhizosphere bacteria of Gypsophila paniculata, which greatly accumulates these saponins in its roots. These results suggest that saponins may control rhizosphere bacteria in soil through rhizodeposition mechanisms.     

Effects of antinutritional factors on plasma lipoprotein levels in Japanese flounder Paralichthys olivaceus

This study examined the effects of four types of antinutritional factor (phytic acid, stachyose, soy saponins and soy isoflavones) on lipoprotein levels in plasma of Japanese flounder Paralichthys olivaceus. A basal diet was prepared with fish meal as primary protein source, the other diets were supplemented with 0·2, 0·4 or 0·8% phytic acid, 0·4, 0·8 or 1·5% stachyose, 0·1, 0·35 or 0·7% soy saponins and 0·10, 0·35 or 0·70% soy isoflavones, by dry mass, in place of white flour in the basal diet. Total cholesterol (TC) and triglyceride (TG) levels in plasma of P. olivaceus were not affected by phytic acid or stachyose. In general, addition of 0·2-0·8% phytic acid or 0·4-1·5% stachyose decreased plasma high-density lipoprotein cholesterol (HDL-C) levels, increased plasma low-density lipoprotein cholesterol (LDL-C) levels, thereby increasing the LDL-C:HDL-C ratio. By contrast, supplementation with 0·35-0·7% soy saponins generally depressed plasma TC levels and the LDL-C:HDL-C ratio. Supplementation with 0·35-0·7% soy isoflavones, however, increased plasma TC and TG levels. These results indicate that soy saponins may be partly responsible for the cholesterol-lowering effects of soybean meal.     

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.     

Hemolytic and cytotoxic properties of saponin purified from Holothuria leucospilota sea cucumber

Background:

Holothuroids (sea cucumbers) are members of the phylum echinodermata, which produce saponins. Saponins exhibit a wide spectrum of pharmacological and biological activities. In this study, we isolated the crude saponins from the body wall of the dominant Iranian species of sea cucumber, Holothuria leucospilota (H. leucospilota). The purpose of this study was to confirm the presence of saponins in the Persian Gulf H. leucospilota and study the hemolytic and cytotoxic activities of these compounds.

Methods:

The body wall of sea cucumber was dried and powdered and the crude saponins were isolated using various solvents. The crude saponins were further purified by column chromatography using HP-20 resin. The foam test, Thin Layer Chromatography (TLC), hemolytic assay, and Fourier Transform Infrared Spectroscopy (FTIR) confirmed the presence of saponins. Cytotoxicity was analyzed using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay on A549 cells, a human lung cancer cell line.

Results:

The foam test, hemolytic assay, and TLC supported the presence of saponin compounds in the 80% ethanol fraction of H. leucospilota. The infrared (IR) spectrum of the extract showed hydroxyl (-OH), alkyl (C-H), ether (C-O) and ester (–C=O) absorption characteristic of teriterpenoid saponins. The C-O-C absorption indicated glycoside linkages to the sapogenins. The crude saponin extracted from sea cucumber was cytotoxic to A549 cells.

Conclusion:

The 80% ethanol fraction of saponin isolated from H. leucospilota exhibited hemolytic activity and offers promise as an anti-cancer candidate.Key Words: Sea cucumber, Holothuria leucospilota, Saponin, Hemolytic assay, Cytotoxicity assay     

Effect of dietary saponins from Quillaja saponaria L. on fatty acid composition and cholesterol content in muscle Longissimus dorsi of lambs

The purpose of this study was to evaluate the effects of increasing levels of saponins from Quillaja saponaria on fatty acid (FA) composition and cholesterol content in muscle Longissimus dorsi of lambs. A total of 24 Barbarine lambs were assigned to four dietary treatments: control diet (C) consisting of oat hay ad libitum and 400 g of concentrate (80% barley, 17.5% soybean meal and 2.5% vitamin and mineral supplement); C diet plus 30 ppm of Q. saponaria L. (QS30); C diet plus 60 ppm of Quillaja (QS60); C diet plus 90 ppm of Quillaja (QS90). Saponin supplementation reduced the concentration of C14:1 cis-9 (P = 0.001) and of its desaturation index (P = 0.002). None of the FA intermediates of ruminal biohydrogenation (BH) was affected by Quillaja saponin supplementation (P > 0.05). The concentration of C20:4n-6 was higher in the meat of animals receiving 60 ppm of Quillaja than C and QS30 groups. Supplementing 60 ppm of Quillaja reduced the ratio between α-linolenic and linoleic acids compared with the C group (P = 0.023). We did not find any significant effect of Quillaja saponins on muscle cholesterol level. Further investigations are necessary to assess the metabolic fate of saponins in the rumen and to understand whether there is an effect of saponin on Δ9-desaturase enzyme activity, ruminal BH and cholesterol metabolism in ruminants. Supplementing up to 90 ppm of Quillaja saponins did not produce detrimental effects on the overall meat FA profile.     

HPLC测定中药百合中2个甾体皂苷的含量

首次建立百合中2个甾体皂苷的高效液相色谱含量测定方法.采用Shim-pack VP-ODS (4.6 mm×250mm,5μm)色谱柱,甲醇-水梯度洗脱,流速1.0 mL/min,检测波长205 nm.化合物1和2的线性范围及相关系数分别为:1.03 ～10.30ug(r=0.9998)和1.26～12.56 μg(r=0.9999);平均回收率分别为100.59％和99.97％.该方法操作简便,结果准确,重现性好,可用于百合中2个甾体皂苷的含量测定,为建立和完善中药百合药材的质量控制方法提供依据.     

基于绿色低共熔溶剂法高效提取鸡骨草中的黄酮和皂苷

为了研究鸡骨草中总黄酮和总皂苷的最佳提取工艺,通过单因素试验和星点设计-响应面法,对低共熔溶剂的性质(如种类、组分摩尔比、含水量)、提取温度、提取时间、液料比等多个因素进行考察。结果显示,总黄酮的最佳提取条件为:摩尔比1∶2、含水量30%的氯化胆碱/乙二醇作溶剂,提取温度80℃,提取时间40min,液料比15∶1(mL/g);总皂苷的最佳提取条件为∶摩尔比1∶4、含水量25%的氯化胆碱/乳酸作溶剂,提取温度80℃,提取时间64min,液料比56∶1(mL/g)。在最佳条件下,总黄酮和总皂苷的提取率较传统提取溶剂分别提高了33.3%和96.4%。本研究为鸡骨草中黄酮和皂苷的高效、安全提取提供了一条新思路。