人参皂苷Rb1转化菌株筛选及转化产物分析

【背景】许多微生物能够对皂苷类化合物进行生物转化,因此,通过微生物对皂苷类化合物不同位置结构的修饰能获得高活性的皂苷成分。【目的】从分离纯化的菌株中筛选能将人参皂苷Rb₁转化为药理活性较高的稀有人参皂苷。【方法】从三七根际土壤及三七茎中分离纯化了36株真菌,首先利用产β-葡萄糖苷酶的方法对菌株进行皂苷转化活性初筛,再以人参皂苷Rb₁为底物进行皂苷转化活性复筛,通过薄层色谱(thinlayerchromatography,TLC)、高效液相色谱(high performance liquid chromatography, HPLC)和质谱(mass spectrometry, MS)等方法对转化产物进行分析。【结果】筛选出一株对人参皂苷Rb₁具有较高转化活性的菌株F17,通过形态学观察及对内转录间隔区(internaltranscribedspacer,ITS)序列分析,菌株F17被鉴定为拟盘多毛孢属菌(Pestalotiopsis biciliata)。P. biciliata可将人参皂苷Rb₁转...     

专一转化人参二醇类皂苷Rb1为Rd的真菌菌株的筛选

于2009年的7－10月间在辽宁省的桓仁,吉林省的集安、靖宇、抚松等药材产区采集人参及人参根际土壤样品45份。通过真菌分离和培养,共获得真菌菌株105株,经形态学鉴定分属于15属48种。通过活性筛选,得到具有转化人参总皂苷活性的菌株25株,其中菌株SR87和SR105对人参皂苷Rb1具有专一转化活性。通过TLC和HPLC检测,其转化产物为人参皂苷Rd。经形态学鉴定,确定阳性菌株SR87为莫勒接霉Zygorhynchus moelleri,SR105为灰绿犁头霉Absidia glauca。这两株真菌均有较高的转化潜力,可以应用于制备人参皂苷Rd。     

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.     

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。     

稀有人参皂苷IH901酶法转化与制备研究

本研究利用酶制剂蜗牛酶,酶法转化三七二醇组皂苷制备稀有人参皂苷IH901,正交实验优化酶解条件,建立酶法转化工艺.结果表明:超声法提取三七总皂苷正交实验优化条件为用75%乙醇溶液,15倍溶剂用量,超声波提取210 min作为最佳条件,三七总皂苷得率为12.21%;酶法转化二醇组人参皂苷制备稀有人参皂苷IH901,正交实验优化的条件为物料比为6/1、反应时间9 h、反应温度为45℃、pH值为3.0,酶解得率为54.24%;经硅胶柱分离获得IH901单体化合物,HPLC测定纯度达98%.酶法转化制备皂苷IH901的工艺方法简便,切实可行,可为中试生产提供参考.     

糖苷酶转化人参皂苷研究进展

人参皂苷为人参主要的药理活性组成部分,通过水解二醇系人参皂苷的糖苷配基是制备稀有人参皂的常用方法。酶法转化因其底物高度专一、条件温和、副产物少等潜在优势而被作为结构修饰和生理研究的主要技术手段。本文主要对糖苷酶转化人参皂苷研究进展进行了综述,为其工业化生产高活性皂苷提供理论依据。     

HPLC法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2

目的:建立高效液相色谱法同时测定人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2的方法.方法:采用ODSC18(4.6 mm×150 mm)色谱柱,流动相乙腈-0.05%磷酸水,梯度洗脱,流速1 Ml/min,检测波长203 nm,柱温35 ℃.结果:人参皂苷Rb1、Rc、Rd、Rg3、CK和Rh2分离效果良好,线性关...     

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。     

人参皂苷CK的研究进展

人参皂苷coumpound K(CK)是人参中原人参二醇型皂苷在人体肠道内的主要代谢产物,属于稀有人参皂苷。人参皂苷CK独特的生物活性已经引起了人们广泛关注,针对它的科学研究也日益增多。因此,有必要介绍近年来人参皂苷CK药理活性和制备方法方面的研究进展。     

外源人参皂苷对人参种子萌发和幼根抗氧化酶活性的影响

研究不同浓度外源人参皂苷(人参总皂苷,人参二醇组皂苷,人参三醇组皂苷, Rb族,Rb3,Re共4种皂苷混合物和两种单体皂苷)对人参种子萌发,幼苗根长、鲜重,幼根中抗氧化酶活性和MDA含量的影响.结果表明:所测试人参皂苷对人参种子萌发、人参幼苗根长生长和幼根鲜重增加均具有抑制化感效应,且抑制程度均随处理浓度的升高而增强;对人参幼根中抗氧化酶活性方面,不同浓度人参总皂苷,人参二醇组皂苷,人参三醇组皂苷处理后,人参根系中SOD,POD和CAT活性均有明显提高,呈现出各酶活性随浓度升高而逐渐增强的效应;人参皂苷Rb族处理后,SOD活性在低中浓度处理时,与对照差别不大,中高浓度处理后低于对照,POD活性在中高浓度处理后显著提高,高浓度处理后活性降幅较大难以恢复到对照水平,CAT活性均低于对照;人参皂苷Rb3处理后,SOD活性均低于对照水平,POD活性在低浓度处理时与对照相当,中高浓度处理后显著低于对照水平,CAT活性逐渐降低,在低中浓度处理时略高于对照,高浓度处理后低于对照水平;人参皂苷Re处理后,SOD和POD活性均显著低于对照.人参幼根中MDA含量均随着处理浓度的增加而升高.     

生物合成稀有人参皂苷的研究进展*

人参皂苷是我国传统中药人参的主要活性物质,稀有人参皂苷相较人参皂苷具有更好的生物活性,也更利于身体吸收,具有镇静催眠、促进细胞分化增殖、抗肿瘤、降血糖、提升免疫力等作用。然而,稀有人参皂苷结构复杂且在人参中含量极低,限制了其开发利用。随着生物技术的发展,利用生物法合成稀有人参皂苷成为本领域的研究热点。因此,对近年来生物合成稀有人参皂苷研究进行汇总梳理,总结稀有人参皂苷的主要种类结构及近年来生物转化法和异源合成法合成稀有人参皂苷的研究进展,生物转化法汇总了以人参皂苷为底物的转化生物,异源合成法总结人参皂苷的生物合成途径及形成结构多样化人参皂苷的酶。对生物合成稀有人参皂苷存在的问题进行了讨论,同时展望了其前景以及未来研究方向,以期为从事人参研究者提供更多生物线索和制备策略。     

Conversion of major ginsenoside Rb1 to 20(S)-ginsenoside Rg3 by Microbacterium sp. GS514

Ginseng saponin, the most important secondary metabolite in ginseng, has various pharmacological activities. Many studies have been directed towards converting major ginsenosides to the more active minor ginsenoside, Rg3. Due to the difficulty in preparing ginsenoside Rg3 enzymatically, the compound has been mainly produced by either acid treatment or heating. A microbial strain GS514 was isolated from soil around ginseng roots in a field and used for enzymatic preparation of the ginsenoside Rg3. Blast results of the 16S rRNA gene sequence of the strain GS514 established that the strain GS514 belonged to the genus Microbacterium. Its 16S rRNA gene sequence showed 98.7%, 98.4% and 96.1% identity with those of M. esteraromaticum, M. arabinogalactanolyticum and M. lacticum. Strain GS514 showed a strong ability to convert ginsenoside Rb1 or Rd into Rg3. Enzymatic production of Rg3 occurred by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1 showing the biotransformation pathway: Rb1-->Rd-->Rg3.     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

Biotransformation of Saponins by Endophytes Isolated from Panax notoginseng

The biotransformation of the major saponins in Panax notoginseng, including the ginsenosides Rg1, Rh1, Rb1, and Re, by endophytes isolated from P. notoginseng was studied. One hundred and thirty‐six endophytes were isolated and screened for their biotransformational abilities. The results showed that five of the tested endophytes were able to transform these saponins. These five strains were identified based on their ITS or 16S rDNA sequences, which revealed that they belonged to the genera Fusarium, Nodulisporium, Brevundimonas, and Bacillus genera. Ten transformed products were isolated and identified, including a new compound 6‐O‐[α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐glucopyranosyl]‐20‐O‐β‐D ‐glucopyranosyldammarane‐3,6,12,20,24,25‐hexaol ( 3 ), and nine known compounds, compound K ( 1 ), ginsenoside F2 ( 2 ), vinaginsenoside R13 ( 4 ), vinaginsenoside R22 ( 5 ), pseudo‐ginsenoside RT4 ( 6 ), (20S)‐protopanaxatriol ( 7 ), ginsenoside Rg1 ( 8 ), vinaginsenoside R15 ( 9 ), and (20S)‐3‐O‐β‐D ‐glucopyranosyl‐6‐O‐β‐D ‐glucopyranosylprotopanaxatriol ( 10 ). This is the first study on the biotransformation of chemical components in P. notoginseng by endophytes isolated from the same plant.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

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.     

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。     

Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice

Ginsenoside, one of the active ingredients of Panax ginseng, has a variety of physiological and pharmacological actions in various organs. However, little is known about the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker potentials by ginsenoside in the interstitial cells of Cajal (ICCs) using the whole-cell patch clamp technique in the current clamp mode. Among ginsenosides, we investigated the effects of ginsenoside Rb1, Rg3 and Rf. While externally applied Rb1 and Rg3 had no effects on pacemaker potentials, Rf caused membrane depolarization. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker potentials and inhibited the Rf-induced membrane depolarization. Membrane depolarization induced by Rf was not inhibited by intracellular application of guanosine 5′-[β-thio]diphosphate trilithium salt. Pretreatment with a Ca²⁺-free solution, thapsigargin, a Ca²⁺-ATPase inhibitor of the endoplasmic reticulum, U-73122, a phospholipase C inhibitor, or 2-APB, an IP3 receptor inhibitor, abolished the generation of pacemaker potentials and suppressed Rfinduced actions. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block Rf-induced effects on pacemaker potentials. These results suggest that ginsenoside Rf modulates the pacemaker activities of ICCs and therby regulates intestinal motility.     

Generation of ginsenosides Rg3 and Rh2 from North American ginseng

Rg3 and Rh2 ginsenosides are primarily found in Korean red ginseng root (Panax ginseng C.A. Meyer) and valued for their bioactive properties. We quantified both Rh2 and Rg3 ginseng leaf and Rg3 from root extracts derived from North American ginseng (Panax quinquefolius). Quantification was obtained by application of HPLC with ion fragments detected using ESI-MS. Ginseng leaf contained 11.3+/-0.5 mg/g Rh2 and 7.5+/-0.9 mg/g Rg3 in concentrated extracts compared to 10.6+/-0.4 mg/g Rg3 in ginseng root. No detectable Rh2 was found in root extracts by HPLC, although it was detectable by ESI-MS analysis. Ginsenosides Rg3 and Rh2 were detected following hot water reflux extraction, but not from tissues extracted with 80% aqueous ethanol at room temperature. Therefore ginsenosides Rg3 and Rh2 are not naturally present in North American ginseng, but are products of a thermal process. Using ESI-MS analysis, it was found that formation of Rg3 and Rh2, among other compounds, were a function of heating time and were breakdown products of the more abundant ginsenosides Rb1 and Rc. Our findings that heat processed North American ginseng leaf is an excellent source of Rh2 ginsenoside is an important discovery considering that ginseng leaf material is obtainable throughout the entire plant cycle for recovery of valuable ginsenosides for pharmaceutical use.