人参生殖器官皂苷含量动态变化

为了明确从现蕾、开花到结实过程中的人参生殖器官中各单体皂苷含量的动态变化,应用HPLC法测定了人工栽培的五年生人参不同时期生殖器官中的人参单体皂苷Rb1、Rb2、Rb3、Rc、Rd、Re、Rg1和Rg3的含量。结果显示:从现蕾到果实成熟的过程中,人参单体皂苷Rb1、Rb2、Rb3、Rc、Rd、Re、Rg1和Rg3的含量的平均值分别为0.643%,0.189%,1.026%,1.014%,1.941%,8.381%,0.724%和0.041mg.g-1。从现蕾到果实成熟的过程中,人参单体皂苷Rb1含量的最高值在7月16日,单体皂苷Rb3、Rc、Rd和Rg1含量的最高值在7月11日,单体皂苷Rb2和Rg2含量的最高值在8月7日。     

西洋参总皂苷酶水解产物成分研究

西洋参总皂苷经β-糖苷酶催化水解,采用HPLC检测分析确定西洋参总皂苷中的主要原人参二醇型皂苷Rb1、Rd、Rc和Rb2已经完全被水解。水解产物通过反复硅胶柱层析和反向硅胶柱层析分离纯化得到7个皂苷,通过NMR谱图分析分别鉴定为人参皂苷compound K(1)、人参皂苷Mc(2)、人参皂苷Rg1(3)、人参皂苷Rg2(4)、人参皂苷Re(5)、人参皂苷F1(6)和拟人参皂苷F11(7)。β-糖苷酶催化西洋参总皂苷水解实验表明,西洋参中原人参二醇型皂苷的水解产物是人参皂苷compound K和人参皂苷Mc。     

土生曲霉转化三七中药材的研究

从土壤真菌中筛选出直接转化中药材三七化学成分的菌株YM31966,经鉴定该菌株为土生曲霉(Aspergillus terreus).以固态转化方式,结合化学提取分离方法,通过高效液相色谱、核磁共振及质谱等波谱检测,该菌株转化三七产物由三七皂苷nR2 、RX1和人参皂苷Rg1、Rd、Rh1、Rh4构成主体成分,而原三七成分Rb1、Rc、Re和R1、R3,R6等物质被分解.结果表明,土生曲霉是一株能转化中药材三七的微生物,它具有改变原三七化学成分,形成新化合物,以及提高某些原化合物成分含量的作用.     

人参皂苷与生态因子的相关性

环境条件影响中药材活性成分的形成和积累.利用各种数学统计分析方法探讨影响人参皂苷积累的生态因子,提高人参品质.人参样品采自人参道地产区(主产区)吉林、辽宁、黑龙江三省5年生栽培人参,同时采集采样点处的土壤样品.超高效液相(UPLC)色谱法分析了不同产区9种人参皂苷(Rg1、Re、Rf、Rg2、Rb1、Rc、Rb2、Rb3、Rd)的含量;利用“中药材产地适宜性分析地理信息系统”的生态因子空间数据库,获得采样区包括温度、水分、光照等10个生态因子数据;按土壤理化性质常规方法测定土壤样品中的有效硼、有效铁等微量元素和速效氮、速效钾等有效养分.对人参有效成分含量与土壤养分进行典型相关性分析发现,土壤中的有效硼、有效铁、速效氮与人参皂苷含量呈显著正相关,即适当提高土壤中有效硼、有效铁和速效氮的含量可以促进人参皂苷成分的积累,土壤水分与所测人参皂苷含量(Rb3除外)呈显著正相关,速效磷(P)、pH、速效锌(Zn)与各人参皂苷含量呈弱相关;人参皂苷与气候因子相关分析表明,温度(年活动积温、年平均气温、7月最高气温、7月平均气温、1月最低气温、1月平均气温)与人参皂苷含量呈显著负相关,其中与药典中人参含量测定项下的人参皂苷Rg1、Re、Rb1负相关尤为显著(r＞0.6),说明在一定温度范围内,人参皂苷是随着温度的降低而升高的,即适当低温有利于人参皂苷有效成分的积累;海拔与人参皂苷Rc、Rb2、Rb3含量呈显著正相关(r＞0.6),即相对较高的海拔可以促进这3种成分的积累;而年均降水量、年相对湿度和年均日照时数与人参皂苷相关不显著.通过主成分分析(PCA)、典型相关分析、排序等统计方法,考察不同产地样品中人参皂苷含量与生态因子间的相关性,研究结果揭示了温度在人参的主要活性成分-皂苷类形成中起决定性作用,在一定的温度范围内,温度越低越有利于人参皂苷的积累;阐明了土壤中的有效硼、有效铁、速效氮与人参皂苷含量成正相关.研究结果提示在人参实践生产中可以通过适当低温处理,增施硼、铁、氮肥等农艺措施来调控人参皂苷含量.     

人参皂苷降脂作用的研究

为了测定西洋参总皂苷及单体皂苷是否有降脂活性,通过体外实验分别测定脂肪分解活性、小肠刷状缘膜小囊吸收脂肪酸、三油酸甘油酯油酸的释放率。结果表明,西洋参茎叶总皂苷在0．5g／L浓度时,对胰脂肪酶活性的抑制率为90％;人参皂苷Rc,Rb1,Rb2对胰脂肪酶活性均显示很强的抑制作用,在0．5g／L浓度时抑制率分别为100％,96％,97％。西洋参总皂苷和人参皂苷Rc,Rh,Rb2可以通过抑制胰脂肪酶活性起到降脂作用。     

绵萆薢三萜皂苷类成分研究

为了解绵萆薢(Dioscorea spongiosa)的化学成分,从其70%乙醇水溶液提取物中分离鉴定了8个化合物,经理化性质和波谱数据分析分别鉴定为:20(S)-人参皂苷Rh1(1)、人参皂苷Rg1(2)、人参皂苷Re(3)、三七皂苷R1(4)、人参皂苷Rd(5)、人参皂苷Rb1(6)、常青藤皂苷元3-O-α-L-吡喃阿拉伯糖苷(7)和木通皂苷D(8)。化合物1、2、3、5和6为首次从该种植物中分离得到,化合物7和8为首次从薯蓣属植物中分离得到。     

RP-HPLC法测定伤科接骨片中两种有效成分的含量

目的:建立高效液相色谱法测定伤科接骨片中人参皂苷Rg1和人参皂苷Rb1的含量.方法:采用C18色谱柱(25cm×4.6mm,5μm),梯度洗脱,紫外检测器;检测波长205nm.结果:两种有效成分能够完全分离,在考察的浓度范围内线性关系良好(r大于0.999),回收率符合规定.结论:该方法准确,灵敏度高,可作为伤科接骨片中两种有效成分的含量检测方法.     

基于UPLC法测定离体大鼠及人源肠道菌对三七中人参皂苷代谢的差异

为探究人与大鼠肠道菌群对三七水煎液中三醇型人参皂苷Rg1、Re及二醇型人参皂苷Rb1、Rd体外代谢的差异性及发现其代谢产物原人参二醇PPD与原人参三醇PPT,实验利用UPLC方法测定三七水煎液分别与人、大鼠肠道菌群在厌氧条件下共培养24h后的孵育液中4种皂苷的含量及代谢产物PPD与PPT的含量。结果表明三七中含有三醇型人参皂苷Rg19.4500mg/g、Re1.8872mg/g,二醇型人参皂苷Rb18.5816mg/g、Rd1.9456mg/g。与人源肠道菌共培养后,三七中含有的二醇型、三醇型人参皂苷含量显著降低,重要的是,在培养液中检测到代谢产物PPD和PPT的存在,含量分别为0.2136mg/g及0.0344mg/g,与大鼠肠道菌共培养后,三七中含有的二醇型皂苷含量有轻微降低,而三醇型皂苷含量未见明显变化,但有少量PPT(0.0184mg/g)的生成。由此可见:在体外条件下,三七水煎液中人参皂苷会被人肠道菌群降解生成代谢产物PPD和PPT,而大鼠肠道菌群的降解产物却仅有PPT生成,二者存在种属差异。     

糖苷酶在人参皂苷转化中的应用

人参皂苷是人参中的主要活性成分。人参皂苷中含量较高的主要成分如Rb1、Rb2、Rc、Rd、Rg1和Re均是在人参皂苷的苷元原人参二醇(APPD)或苷元原人参三醇(APPT)上加上不同数量的葡萄糖基、阿拉伯糖基、木糖基或鼠李糖基等糖基形成的。这些主要人参皂苷脱去部分或全部的糖基的产物具有更强的生物活性及更好的人体吸收率。去除糖基的产物如Rg3、Rh2、化合物K(C-K)、F2、Rh1、Rg1、APPD、APPT在天然人参中不存在或含量极低,因此也被称为稀有人参皂苷。稀有人参皂苷可以通过糖苷酶水解主要人参皂苷获得。已报道的具备人参皂苷水解活力的糖苷酶有β-葡萄糖苷酶、α-L-阿拉伯吡喃糖苷酶、α-L-阿拉伯呋喃糖苷酶、β-半乳糖苷酶及β-木糖苷酶。我们简要综述近5年来糖苷酶用于制备稀有人参皂苷的研究进展。     

西洋参茎叶化学成分及生物利用度的研究

应用多种色谱技术进行分离纯化,从西洋参茎叶中分离得到10个化合物,经理化性质和光谱数据分析鉴定分别为:拟人参皂苷RT4(1)、拟人参皂苷RT5(2)、24(R)-Ocotillol苷元(3)、20(S)-人参皂苷Rh1(4)、20(S)-人参皂苷Rg1(5)、20(S)-人参皂苷Rg2(6)、20(S)-人参皂苷Rh2(7)、20(R)-人参皂苷Rh2(8)、20(S)-人参皂苷Rg3(9)、拟人参皂苷F11(10)。化合物1和3为首次从西洋参茎叶中分离得到。首次建立和认证了20(S)-人参皂苷Rg3肌内注射的生物利用度的测定方法,采用本文方法测定犬肌注20(S)-人参皂苷Rg3的生物利用度为96.7%,为20(S)-人参皂苷Rg3的新药开发提供了临床前药代动力学依据。     

Transformation of Ginsenosides Rb1 and Re from <Emphasis Type="Italic">Panax ginseng</Emphasis> by Food Microorganisms

Ginsenosides Rb1 and Re, respectively belonging to the major protopanaxadiol and protopanaxatriol ginsenosides, were transformed using cell-free extracts from food microorganisms. Rb1 was transformed into compound K via Rd and F2 by Bifidobacterium sp. Int57, Bif. sp. SJ32, Aspergillus niger and A.␣usamii. Lactobacillus delbrueckii, and Leuconostoc paramesenteroides transformed Rb1 into Rh2 via Rd and F2. Bifidobacterium sp. SH5 transformed Rb1 into F2 via Rd. Re was transformed into Rh1 via Rg2 by Bif. sp. Int57 and Bif. sp. SJ32. A. niger transformed Re into Rh1 via Rg1. A. usamii transformed Re into Rg2. Transformation of Rb1 proceeded at a higher rate and needed less amount of enzymes than that of Re. Taken together, these processes would allow a specific bioconversion process possible to obtain specific ginsenosides using an appropriate combination of ginsenoside substrates and specific microbial enzymes.     

Simultaneous Determination and Analysis of Major Ginsenosides in Wild American Ginseng Grown in Tennessee

Ginsenosides are the major constituent that is responsible for the health effects of American ginseng. The ginsenoside profile of wild American ginseng is ultimately the result of germplasm, climate, geography, vegetation species, water, and soil conditions. This is the first report to address the ginsenoside profile of wild American ginseng grown in Tennessee (TN), the third leading state for production of wild American ginseng. In the present study, ten major ginsenosides in wild American ginseng roots grown in TN, including Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rg3, were determined simultaneously. The chemotypic differences among TN wild ginseng, cultivated American ginseng, and Asian ginseng were assessed based on the widely used markers of ginsenoside profiling, including the top three ginsenosides, ratios of PPD/PPT, Rg1/Rb1, Rg1/Re, and Rb2/Rc. Our findings showed marked variation in ginsenoside profile for TN wild ginseng populations. Nevertheless, TN wild ginseng has significant higher ginsenoside content and more ginsenoside diversity than the cultivated ginseng. The total ginsenoside content in TN wild ginseng, as well as ginsenosides Rg1 and Re, increases with the age of the roots. Marked chemotypic differences between TN wild ginseng and cultivated American ginseng were observed based on the chemotypic markers. Surprisingly, we found that TN wild ginseng is close to Asian ginseng with regard to these characteristics in chemical composition. This study verified an accessible method to scientifically elucidate the difference in chemical constituents to distinguish wild from the cultivated American ginseng. This work is critical for the ecological and biological assessments of wild American ginseng so as to facilitate long‐term sustainability of the wild population.     

Simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma by liquid chromatography/electrospray ionization mass spectrometric method and its application to pharmacokinetic study of 'SHENMAI' injection

A sensitive and rapid liquid chromatography-mass spectrometric method for the simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma was developed and validated. Chromatographic separation was performed on a C18 column using a step gradient program with the mobile phase of 0.5mmol/L ammonium chloride solution and acetonitrile. The analytes and I.S. were detected using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. The method was linear over the investigated concentration range with a good correlation coefficient higher than 0.997. The lower limits of detection (LLOD) of these analytes were all lower than 2.0ng/mL. The intra- and inter-day precisions were all no more than 7.5% and accuracies were within the range of 97.5-107.0%. The validated method was successfully applied to investigate the pharmacokinetics of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat after intravenous administration of 'SHENMAI' injection.     

Ginsenoside Re impacts on biotransformation products of ginsenoside Rb1 by Cellulosimicrobium cellulans sp. 21 and its mechanisms

Ginsenoside Rg3, a known anti-cancer agent, is usually prepared by enzyme-mediated and acid hydrolysis of ginsenoside Rb1 and Rd. In this study, we used the bacterium Cellulosimicrobium cellulans sp. 21 to transform Rb1 into Rg3. When Rb1 was used as the sole substrate, the transformation products included Rg3, Rh2, C-K and PPD. However, when Rb1 and Re were mixed, the yield of Rg3 was significantly higher, indicating that Re attenuates the activity of β-1,2-glucosidase secreted by C. cellulans sp. 21. β-1,2-glucosidase hydrolyzes the β-1,2-glucose moiety at the C-3 position of Rb1, but Re dose not modify enzymes that produce Rg3 by hydrolyzing glucose at the C-20 position in aglycon. We also tested the inhibitory effects from various ginsenosides on β-1,2-glucosidase, and discovered that sugar chains played key roles in inhibiting β-1,2 glucosidase activity, whereas aglycones of protopanaxadiol and protopanaxatriol had little inhibitory effects. Some sugar chains with different linkages, such as C-20, C-3 and C-6, exhibited different inhibitory effects. Overall, our findings demonstrate that a combination of substrates, in addition to microorganism-secreted enzymes, can be used for selective biotransformation. This approach provides a novel strategy for natural product preparations via microbial transformation.     

A novel ginsenosidase from an Aspergillus strain hydrolyzing 6-O-multi-glycosides of protopanaxatriol-type ginsenosides, named ginsenosidase type IV

Herein, a novel ginsenosidase, named ginsenosidase type IV, hydrolyzing 6-O-multi-glycosides of protopanaxatrioltype ginsenosides (PPT), such as Re, R1, Rf, and Rg2, was isolated from the Aspergillus sp. 39g strain, purified, and characterized. Ginsenosidase type IV was able to hydrolyze the 6-O-alpha-L-(1-->2)-rhamnoside of Re and the 6-O-beta-D- (1-->2)-xyloside of R1 into ginsenoside Rg1. Subsequently, it could hydrolyze the 6-O-beta-D-glucoside of Rg1 into F1. Similarly, it was able to hydrolyze the 6-O-alpha-L-(1-->2)- rhamnoside of Rg2 and the 6-O-beta-D-(1-->2)-glucoside of Rf into Rh1, and then further hydrolyze Rh1 into its aglycone. However, ginsenosidase type IV could not hydrolyze the 3-O- or 20-O-glycosides of protopanaxadioltype ginsenosides (PPD), such as Rb1, Rb2, Rb3, Rc, and Rd. These exhibited properties are significantly different from those of glycosidases described in Enzyme Nomenclature by the NC-IUBMB. The optimal temperature and pH for ginsenosidase type IV were 40°C and 6.0, respectively. The activity of ginsenosidase type IV was slightly improved by the Mg(2+) ion, and inhibited by Cu(2+) and Fe(2+) ions. The molecular mass of the enzyme, based on SDS-PAGE, was noted as being approximately 56 kDa.     

Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡中Bcl-2、Bax表达的影响

目的：探讨人参皂甙Rb1、Rg1在肾缺血／再灌注血清诱导HK-2细胞凋亡中对Bol-2、Bax表达的影响。方法：制备家兔肾缺血／再灌注血清（SIR）和对照组血清（SC）用于HK-2细胞培养,TUNEL法检测细胞凋亡。实验分组：对照组、缺血／再灌注组、Rb1干预组、Rg1干预组,培养24h后免疫细胞化学法检测Bcl-2、Bax的表达。结果：与缺血／再灌注组比较,Rb1干预组和Rg1干预组Bax的表达明显下降（P〈0．01）,Bcl-2／Bax比值增大。结论：人参皂甙Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡具有保护作用。     

黑根霉对人参皂苷Re的生物转化

利用菌种黑根霉Rhizopus sp.对人参皂苷Re进行生物转化,并对人参皂苷Re及其发酵产物进行HPLC系统分析比较,经液相色谱-质谱分析得出人参皂苷Re转化率为92.16%,并制备出人参皂苷Re发酵产物中峰值升高的成分,转化后的人参皂苷发酵产物中化合物1确定为人参皂苷Rg2,化合物2为Rg2的同分异构体,得率为10.13%;化合物3和化合物4确定为人参皂苷Rg5/Rk1,得率为29.23%。从结果初步推测得出人参皂苷Re被黑根霉转化为人参皂苷Rg2的机理,人参皂苷Re转化成人参皂苷Rg5/Rk1的机理还有待于进一步研究。     

Ginsenoside content and variation among and within American ginseng (Panax quinquefolius L.) populations

The contents of five ginsenosides (Rg1, Re, Rb1, Rc and Rd) were measured in American ginseng roots collected from 10 populations grown in Maryland. Ginsenoside contents and compositions varied significantly among populations and protopanaxatriol (Rg1 and Re) ginsenosides were inversely correlated within root samples and among populations. The most abundant ginsenoside within a root and by population was either Rg1 or Re, followed by Rb1. Ginseng populations surveyed grouped into two chemotypes based on the relative compositions of Rg1 and Re. Four populations, including the control population in which plants were grown from TN and WI seed sources, contained roots with the recognized chemotype for American ginseng of low Rg1 composition relative to Re. The remaining 6 populations possessed roots with a distinctive chemotype of high relative Rg1 to Re compositions. Chemotype did not vary by production type (wild versus cultivated) and roots within a population rarely exhibited chemotypes different from the overall population chemotype. These results provide support for recent evidence that relative Rg1 to Re ginsenoside contents in American ginseng roots vary by region and that these differences are likely influenced more by genotype than environmental factors. Because the physiological and medicinal effects of different ginsenosides differ and can even be oppositional, our findings indicate the need for fingerprinting ginseng samples for regulation and recommended usage. Also, the High Rg1/Low Re chemotype discovered in MD could potentially be used therapeutically for coronary health based on recent evidence of the positive effects of Rg1 on vascular growth.