Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrome try（HPLC-ESI-MS/MS） method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng（RPG） and steamed Panax ginseng at high temperatures（SPGHT）. A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 oC. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains.

High-Performance Liquid Chromatographical Analysis of Ginsenosides in Panax ginseng,P.quinquef(?)lium and P.notoginseng

The compositions and contents of ginsenbsides in Panax ginseng,P.quinquefolium and P.notoginseng were determined and compared by reversed-phase High-Performance Liquid Chro- matography(HPLC).The method was performed on an Alltech Adsorbosphere HS C_(18) column,using 5×10^(-3)M NaH_2PO_4-H_3PO_4 buffer solution(pH 3.0)and acetonitrile-water(50:50)as gradient eluents. The baseline separation of ginsenosides Rb_1,Rb_2,Rb_1,Rc,Rd,Rf,Ro,and Re+Rg_1 was obtained in one analytical run.The ginsenosides are directly detected at 203 nm.The detection limit is 40μg at a signal to noise ratio of 3:1.The improved sample preparation and clean-up prior to injection with SEP-PAK C_(18)cartridge strongly reduced the front peaks caused by the impurities in the methanolic extracts of samples to afford a smooth baseline and clear background.The HPLC patterns of methanolic extracts mainly including the ginsenosides were found capable of serving as chemical fingerprints to differentiate the three species from each other.It was also found that there are no significant diffe- rences of the HPLC patterns between the wild Panax ginseng and the cultivated,the white and the red ginsengs,Chinese and Korean red ginsengs,and the tap roots of Panax ginseng collected in four consecutive months,only certain differences in contents of ginsenosides do exist.The contents of the nine major ginsenosides present in the rhizome,tap root and rootlet as well as the leaf of Panax quinquefolium were also determined and compared.

Transcriptome-Wide Identification and Functional Analysis of PgSQE08-01 Gene in Ginsenoside Biosynthesis in Panax ginseng C.A.Mey.

Panax ginseng C.A.Mey.is an important plant species used in traditional Chinese medicine,whose primary active ingredient is a ginsenoside.Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes.Nonetheless,the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites.Squalene epoxidase(SQE)is a key enzyme in the mevalonic acid pathway,which affects the biosynthesis of secondary metabolites such as terpenoid.Using ginseng transcriptome,expression,and ginsenoside content databases,this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng.We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis.Next,we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes,to obtain positive hairy-root clones.Thereafter,quantitative reverse transcriptionpolymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content,respectively.Then,we focused on the function of PgSQE08-01 gene,which was noted to be involved in ginsenoside biosynthesis.Thus,these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.

HPLC结合化学计量学分析研究人参和三七不同部位丙二酰基人参皂苷和中性皂苷的差异性

建立HPLC法同时测定人参和三七不同部位丙二酰基人参皂苷和中性皂苷的含量,并采用聚类分析和主成分分析的化学计量学方法分析丙二酰基人参皂苷和中性皂苷含量,以对人参和三七地上和地下药用部位进行分类。结果表明人参中性皂苷和丙二酰基人参皂苷在人参和三七不同部位中存在着显著的差异。在人参的地下部位中,丙二酰基人参皂苷的含量均略高于中性人参皂苷;在人参的地上部位,丙二酰基人参皂苷含量低于中性人参皂苷;然而丙二酰基人参皂苷不论在三七地下部位还是地上部位含量均较低。聚类分析和主成分分析结果显示,12批人参和三七样品分成4类,人参地下部位R1〜R4聚为一类,地上部位R5〜R6聚为一类;三七地下部位S1〜S4聚为一类,地上部位S5〜S6聚为一类。此方法灵敏度高,稳定可靠,可为人参和三七不同部位的准确鉴别提供科学依据。

Fingerprint analysis of different Panax herbal species by HPLC-UV method

Aim To establish a method for differentiating commercial samples of Panax species including notoginseng, cultivated ginseng （Chinese ginseng and Korean ginseng）, wild ginseng, red ginseng, three types of American ginsengs, and one American ginseng preparation with their HPLC fingerprints for assnrning the quality of different commercial samples of Panax species. Methods HPLC-UV method was used to establish their fingerprints, Zorbax Extend C18 （250 mm×4.6 mm, 5 μm） was used as the analytical column, and acetonitrile/KH2PO4 aqueous solution was used as the mobile phase with gradient elution. Results The fingerprints of different commercial samples of Panax species varied in their holistic chromatograms and some specific constituents. Conclusion This method is reliable, reproducible and simple, It could be applied in the routine authentication of different commercial samples of Panax species

Ocotillone-type Ginsenoside from Leaves of Panax ginseng

An ocotillone type ginsenoside, together with 2 known ginsenosides was isolated from leaves of Panax ginseng and identified as pseudoginsenoside RT 5 on the basis of chemical and physicochemical evidences. It has been so far the first example of ocotillone type ginsenoside discovered in Panax ginseng and its plausible biotransformation pathway also discussed.

Determination of Saponins in Leaf of Panax Ginseng C. A. Mey. by High Performance Liquid Chromatography

A high performance liquid chromatography（HPLC） with UV detection was established for simultaneous determination of saponins in the leaf of Panax ginseng C. A. Mey. Nine ginsenosides（Rbl, Rb2, Rb3, Rc, Rd, F1, F2, F3, F5） and notoginsenoside Fe（NFe） were studied. Among the saponins, the ginsenosides F1, F2, F3, F5 and NFe were determined by HPLC-UV method for the first time. The determination of the ginsenosides via the HPLC-UV method was performed on a reversed-phase C18 column with gradient elution in 40 min. The linearity, precision, accuracy, and detection limit for determining the saponins were studied and the samples from different areas in China were analyzed. The HPLC-ESI-MS was used to identify the saponins. The results indicate that the HPLC-UV provided a good accuracy, reproducibility and sensitivity for the determination of the ten saponins.

HPLC法测定益真健脑丸中人参皂苷Rg_(1)、Re、Rb_(1)含量

目的采用HPLC对益真健脑丸中西洋参药材3种主要成分人参皂苷Rg_(1)、Re、Rb_(1)进行含量测定。方法采用C18色谱柱,流动相为乙腈(A)-0.1%磷酸溶液(B),梯度洗脱,检测波长203nm,流速1.0mL·min^(-1),柱温40℃。结果人参皂苷Rg_(1)、Re、Rb_(1)分别在2.00~20.00μg·m L^(-1)、4.00~40.00μg·m L^(-1)、10.00~100.00μg·m L^(-1)浓度范围内线性关系良好。结论该方法简单、稳定,可用于益真健脑丸中3种人参皂苷的含量测定。

Penicillium sp.YJM-2013 induces ginsenosides biosynthesis in Panax ginseng adventitious roots by inducing plant resistance responses

Objective:Fusarium oxysporum is a common pathogenic fungus in ginseng cultivation.Both pathogens and antagonistic fungi have been reported to induce plant resistance responses,thereby promoting the accumulation of secondary metabolites.The purpose of this experiment is to compare the advantages of one of the two fungi,in order to screen out more effective elicitors.The mechanism of fungal elicitor-induced plant resistance response is supplemented.Methods:A gradient dilution and the dural culture were carried out to screen strains.The test strain was identified by morphology and 18 s rDNA.The effect of different concentrations(0,50,100,200,400 mg/L)ofPenicillium sp.YJM-2013 and F.oxysporum on fresh weight and ginsenosides accumulation were tested.Signal molecules transduction,expression of transcription factors and functional genes were investigated to study the induction mechanism of fungal elicitors.Results:Antagonistic fungi ofF.oxysporum was identified as Penicillium sp.YJM-2013,which reduced root biomass.The total ginsenosides content of Panax ginseng adventitious roots reached the maximum(48.95±0.97 mg/g)treated with Penicillium sp.YJM-2013 at 200 mg/L,higher than control by 2.59-fold,in which protopanoxadiol-type ginsenosides(PPD)were increased by 4.57 times.Moreover,Penicillium sp.YJM-2013 activated defense signaling molecules,up-regulated the expression of PgWRKY 1,2,3,5,7,9 and functional genes in ginsenosides synthesis.Conclusion:Compared with the pathogenic fungi F.oxysporum,antagonistic fungi Penicillium sp.YJM-2013 was more conducive to the accumulation of ginsenosides in P.ginseng adventitious roots.Penicillium sp.YJM-2013 promoted the accumulation of ginsenosides by intensifying the generation of signal molecules,activating the expression of transcription factors and functional genes.

A new panaxadiol from the acid hydrolysate of Panax ginseng

A new panaxadiol （compound 1） was obtained from the acid hydrolysate of the total ginsenosides of Panax ginseng C. A. Meyer （Araliaceae）. On the basis of spectroscopic data and single-crystal X-ray diffraction data, its chemical structure was elucidated to be dammar-（E）-20（22）-ene-3β,12β,25 -tfiol.

Ginsenoside-Rg_6, a Novel Triterpenoid Saponin from the Stem-Leaves of Panax ginseng C. A. Mey.

A novel dammarane-type triterpene oligoglycoside, named ginsenoside-Rg6 3, was isolated from the stem-leaves of Panax ginseng C. A. Mey., together with two known ones, 20(S)-ginsenoside-Rg2 1 and 20(R)-ginsenoside-Rg2 2. On the basis of chemical and physicochemical evidence , the structure of ginsenoside-Rg6 have been elucidated as 6-O-(-L-rhamnosyl-(1?2)-(-D-glucopyranosyl-dammarane-(E)-20(22), 24-diene-3(, 6(, 12(-triol.

A New Dammarane Glycoside, Ginsenoside Ⅲ from the Flower-buds of Panax ginseng C.A. Meyer

From the dried flower-buds of Panax ginseng C.A. Meyer, a new ndnor dammaranetype triterpene saponin named ginsenoside III was iso1ated. On the basis of spectral and chemical evidence, the structure of the new saponin was elucidated as 3 -O- [β-D -glucopyranosyl (1→2 ) - βD- glucopyranosyl] - 20-O-β-D-glucopyranosyl 3 β, 12β- 20(S) -trihydroxydammar- 25 - en- 24-one.

Pharmacological activities and herb-drug interactions of Panax ginseng and its chemical components:a review

OBJECTIVE Panax ginseng C.A.Meyer(ginseng)is a well-known medicinal plant worldwide and a key ingredient in many commercially-available health products.It is used as a tonic for invigoration and for tification in times of fatigue and debility or declining capacity for work and concentration.Previous in-house study has surveyed over three hundred ginseng and ginseng products(including P.ginseng,P.quinquefolius,P.notoginseng,P.pseudoginseng)available in Singapore.This review presents an overview of the pharmacological activities and herb-drug interactions of P.ginseng and its ginsenosides.METHODS Literature searches of PubMed and ScienceDirect were done to identify pharmacological activities and herb-drug interactions of P.ginseng,its extracts and its chemical components,including ginsenosides.Studies of whole plant extracts include both White ginseng and Red ginseng.The studies for the pharmacological activities of whole plant extract were limited to those published from 2009 to 2015.There was no restriction on the time frame of other studies.Terms such as″P.ginseng″,″Ginsenosides″were searched.Studies found included in vitro assays,in vivo animal studies,human clinical trials as well as individual case reports.RESULTS A total of 112 studies were found on whole plant extracts and 257 studies on its individual components.Whole plant extracts of ginseng were found to possess over fifty different pharmacological activities,while its individual components exhibit parts of this spectrum.P.ginseng was found to interact with drugs such as 5-fluorouracil,irinotecan,mitomycin C,docetaxel,cisplatin,alcohol,midazolam,warfarin,phenelzine,raltegravir and imatinib.CONCLUSION P.ginseng and its components exhibit a wide range of pharmacological activities and interact with some drugs.There remain much opportunities for future research.

Characterization of Panax ginseng UDP- Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rhl in Metabolically Engineered Yeasts

Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

Minor Saponins from the Leaves of Panax ginseng C.A. Meyer

Five minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20（R）-protopanaxatriol （1）, daucosterin （2）, 3β, 12β-dihydroxy-dammar-20 （22）, 24-diene-3-O-β-D-glucopyranoside （3）, 20 （R）-protopanaxadiol-3-O-β-D-glucopyranoside （4） and ginsenoside-Rh2 （5）, respectively, on the basis of spectral analyses and chemical evidence. The two new saponins, 3 and 4, were named as ginsenoside-Rh3 and 20（R）-ginsenoside-Rh2.Nine other major saponins obtained simultaneously were identical with ginsenoside-Rh1（6）,-Rg3 （7）, -Rg2 （8）, -Rg1 （9）,-Re（10）,-Rd （11）, -Rc （12）, -Rb2（13） and Rb1 （14）, respectively.

HPLC法测定人参、西洋参和三七不同部位中人参皂苷的含量

目的:为了探讨人参、西洋参和三七中人参皂苷的资源含量,以确保人参、西洋参和三七中人参皂苷资源充分被利用,为开发以人参皂苷为主的创新药物提供科学数据。方法:采用高效液相色谱法对人参、西洋参和三七不同部位中人参皂苷的含量进行测定。色谱条件为:Agilent 1100 Series 高效液相色谱仪;色谱柱为德国 Nucleosil-C_(18)(4.6 mm×150 mm,5μm);流动相为水-乙腈梯度洗脱,流速1.5 mL·min^(-1);A为水,B为乙腈;梯度洗脱程序为:0～16 min,56%B;16～20 min,56%→100%B;20～38 min,100%B;38～45 min,100%→56%B;45～60 min,56%B;60～70 min,56%B。所有组分均70 min 内出完。检测波长203 nm,柱温35℃,灵敏度为0.02AUFS。线性关系考察r=0.9994;精密度试验 RSD=0.26%,平均回收率为99.88%,重现性试验 RSD=2.0%,分离度为R=3.042。结果:人参须根含有较高的人参皂苷 Rb_1(1.082%),人参茎叶则含有较高的人参皂苷 Rc(1.002%)、Re(3.430%)和 Rg_1(1.303%);西洋参根含有较高的人参皂苷 Rb_1(2.213%)和 Re(0.9188%),西洋参芦头中含有较高的人参皂苷 Rb_1(2.840%)和Re(1.224%);三七根含有较高的人参皂苷 Rb_1(2.163%)和 Rg_1(2.633%),三七芦头中含有较高的人参皂苷 Rb_1(4.376%)和 Rg_1(4.145%)。结论:高效液相色谱法分离、分析人参皂苷效果好、准确、迅速、简便,也可作为评价人参属植物质量的有效分析方法。建议对人参、西洋参和三七中含量较高的人参皂苷进行提取分离,直接用于创新药物的开发。

反相HPLC法测定药用人参中多种人参皂甙的含量

采用HPLC法对不同产地药用人参中多种人参皂甙（GinsenosideRf、Re、Rd、Rb1、Rb2、Rg1、Rg2、Re）进行定量分析，流动相分别选用乙腈-0．05mol／L磷酸二氢钠（30∶70）和（20∶80），流速1ml／min，紫外检测波长203nm，色谱柱：YMCParkA-312ODS（6min×150mmid），柱温35℃，回收率均≥96.90％，RSD＜0.56％。该法操作简便，精密度高，重视性好，为评价药用人参的品质提供了参考依据。

不同产地西洋参皂甙成分的HPLC分析

以西洋参的主要皂甙成分人参皂甙Re和Rb1为标准对照品 ,建立西洋参药材的HPLC定量分析技术 ,并参考人参皂甙Rc,Rd及Rg2 的相对峰面积进行主成分分析。色谱条件为 :C18柱 (5 μm ,3.9× 15 0mm) ,乙腈 :水流动相 ,二元梯度洗脱 ,检测波长 2 0 3nm。结果表明 ,就皂甙成分的组成与含量而言通过人参皂甙Re和Rb1的含量测定和皂甙的主成分分析 ,不同产地的西洋参药材皂甙成分存在一定的差别。吉林省靖宇县产的西洋参与进口品最为接近。

HPLC-MS/MS法测定10批参须中人参皂苷Rg_1、Re和Rb_1的含量

目的建立同时检测参须中人参皂苷Rg1、Re和Rb1含量的HPLC-MS/MS方法,测定10批参须中人参皂苷Rg1、Re和Rb1的含量。方法采用液质联用(HPLC-MS/MS)法,色谱柱为SHIMADZU VP-ODS(4.6 mm×150 mm,5μm),流动相为乙腈-0.1%甲酸水,梯度洗脱(0~3 min,60%乙腈;3~5 min,90%乙腈),流速0.5 m L·min-1,柱温40℃。质谱条件:正离子检测模式,用于定量分析的离子对分别为:人参皂苷Rg1 m/z 823.3→643.6,人参皂苷Re m/z 969.7→789.6和人参皂苷Rb1 m/z1131.5→365.3。结果人参皂苷Rg1在0.43~27.6μg·m L^(-1)与峰面积线性关系良好,r=0.9950,平均回收率为94.0%(RSD=1.5%);人参皂苷Re在0.46~29.6μg·m L^(-1)与峰面积线性关系良好,r=0.9990,平均回收率为95.3%(RSD=1.1%);人参皂苷Rb1在0.41~26.4μg·m L^(-1)与峰面积线性关系良好,r=0.9980,平均回收率为93.4%(RSD=1.2%)。结论该方法简便、准确、快速,可用于参须中人参皂苷Rg1、Re和Rb1的含量检测。

三七及其混淆品的HPLC指纹图谱鉴定

目的 建立三七及其混淆品的指纹图谱鉴定方法。方法 采用 HPL C方法 ,测定三七、人参、西洋参、姜状三七、三七不同部位及其 2种混淆品的指纹图谱。结果 三七与人参、西洋参、姜状三七及其 2种混淆品的指纹图谱有明显区别 ,三七不同部位也有差异。结论 HPL C皂苷指纹图谱鉴别三七及其混淆品具有重复性好、特征性强、方法简便等特点 。