Spatio-Temporal Expression Pattern of Six Novel Candidate Genes in Ginsenoside Biosynthesis from Panax ginseng C. A. Meyer

Abstract: To explore the mode of the spatio-temporal expression of six newly discovered ginsenoside biosynthesis candidate gene transcripts, both Northern blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were used to elucidate the mRNA expression levels of the transcripts in various tissues and organs of Panax ginseng C. A. Meyer during different growth development stages. The six gene transcripts were all differentially expressed in cultured callus, root, stem, leaf, and seed. The mRNA expression levels were significantly higher in four-year-old roots than in one-year-old roots, and results of semi-quantitative RT-PCR assays were in accordance with those of Northern blotting analyses. The results strongly suggest that all six genes were differentially expressed at root-specific developmental stages. In particular, when a quiescent early stage culture suspension of P. ginseng cells was exposed to the ginsenoside biosynthesis-promoting elicitor Aspergillus niger polysaccharide, the GBR6 gene transcript response showed time-dependent increments and was parallel with ginsenoside productivity (P < 0.01). Overexpressionof the GBR6 gene is likely to play a critically important role in the biosynthesis of ginsenosides. The results of the present study provided a background for the further elucidation of the structure and physiological function of these six candidate genes.

Optimizing SSR-PCR system of Panax ginseng by orthogonal design

An orthogonal design was used to optimize SSR-PCR amplification system using Panax ginseng genomic DNA as template. Four levels of five factors （DNA template, Taq DNA polymerase, Mg^2＋, primer, and dNTP） and annealing temperature have been tested separately in this system. The results demonstrated the reaction efficiency was affected by these factors. Based on the results, a stable, productive and reproducible PCR system and cycling program for amplifying a ginseng SSR locus were obtained： 20 μL system containing 1.0 U Taq DNA polymerase, 2.0 mmol·L^-1 Mg^2＋, 0.2 mmol·L^-1 dNTPs, 0.3 μmol·L^-1 SSR primer, 60 ng· μla^-1 DNA template, performed with a program of 94℃ for 5 min, 94℃ for 30 s, annealing at 56.3℃ for 30 s, 72℃ for 1 min, 37 cycles, finishing at 72℃ for 7 min, and storing at 4℃.

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.

Structure Analysis of Pectin SB_(1-1) from the Root of Panax ginseng

A water-soluble pectin SB_~1-1 was isolated and purified from the root of Panax ginseng C. A. Mey. The HPLC analysis indicates that SB_~1-1 is homogenous. Its molecular weight was estimated via gel filtration to be 10000. The GC analysis indicated that it contains the monosaccharides of GalA, Gal, Ara and Rha. Their molar ratio is 2.10∶1.00∶0.12∶0.13. Partial hydrolysis with acid, pectinase treatment, periodate oxidation, Smith degradation, methylation analyses, GC/MS analyses and NMR analyses were used for the structure analyses of SB_~1-1 . The results reveal that SB_~1-1 has a lower branched structure. The main chain is composed of GalA and Gal; the inner part is α-1,4-linked-GalA; the border is 1,4-linked-Gal. Some of the 1,4-linked-GalA and 1,4-linked-Gal residues are substituted at O6. On an average, there is one branch for every ten hexose residues. The side chain is composed of 1,6-linked-Gal and 1,3,6-linked-Gal. The nonreduced end is composed of Rha, Ara and Gal. The main glycosidic link of SB_~1-1 has an α configuration.

A new triterpene saponin from Panax japonicus C.A.Meyer var major(Burk.) C.Y.Wu et K.M.Feng

One new Iriterpene saponin was isolated from Panaxjaponicus C. A. Meyer var major （Burk.） C. Y. Wu et K. M. Feng, and established as oleanolic acid 3-O-[β-D-glucopyranosyl-（1 →2）-β-D-glucuronopyranosyl-6＇-O-n-butyl ester] which showed mod- erate antitumor activities against the A2780 cells and OVCAR-3 cells. Its structure was established by means of spectral data, particularly NMR, including HSQC and HMBC techniques.

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.

Antagonistic Effects of Sphingomonas and Pseudomonas aeruginosa on 4 Kinds of Pathogenic Bacteria of Ginseng

[Objectives]To explore effective biocontrol methods for diseases in the process of ginseng cultivation,and develop an efficient and environmentally friendly biocontrol agent.[Methods]In this study,2 strains were isolated from biogas slurry,and Cylindrocarpon destructans(XF),Fusarium solani(GF),Botrytis cinerea Pers(HM)and Alternaria panax Whetz(HB)were used as test materials.The strains were isolated and identified by dilution plate method,16S rDNA sequence identification method,confrontation culture method,filter paper method and ultraviolet spectrophotometer method,and the bacteriostatic activity and bacteriostatic rate were tested.[Results]Strain 15(Sphingomonas)and strain 19(Pseudomonas aeruginosa)were screened out through identification and analysis,and they grew stably within 8-10 d.The bacteriostatic rates of strain 15 against A.panax and B.cinerea were 47.37%and 43.40%,respectively,and the bacteriostatic rates of strain 19 against A.panax and B.cinerea were 62.30%and 63.27%,respectively.The bacteriostatic activity of the extract of strain 19 increased with the increase of OD_(600) value,and the bacteriostatic effect was optimal when the OD_(600) value was in the range of 0.8-1.0,up to 70%,so it had a strong biocontrol potential.[Conclusions]This experiment provides convenience for more effective inoculation,establishes a fast,simple and accurate method for the determination of the best bacteriostatic rate of P.aeruginosa culture solution to HM,and lays a foundation for large-scale culture of P.aeruginosa culture solution.Besides,it is expected to provide a theoretical basis for the efficient control of ginseng B.cinerea in field production,use it for the prevention and control of ginseng shoot diseases,and provide a reference for the efficient and diverse development of biocontrol agents for ginseng shoot diseases.

Active compounds in RenShenJian decoction ameliorate insulin resistance in vitro

Background:RenShenJian decoction,a combination of Pueraria lobata(Willd.)Ohwi and Panax ginseng C.A.Mey,has been used in China since the Song Dynasty(960-1279 C.E.)to relieve symptoms of diabetes mellitus.However,the key compounds in RenShenJian that ameliorate insulin resistance remain unclear.This study identified the anti-diabetic compounds in RenShenJian by rescuing the decreased function of adenosine 5’-monophosphate-activated protein kinase(AMPK),sirtuin 3(SIRT3),or glucose transporter isoform 4(GLUT4).Methods:After streptozotocin-induced diabetic mice were treated with RenShenJian,fasting blood glucose levels and protein expression of SIRT3,p-AMPK,and AMPK were determined.Compounds from RenShenJian in plasma were monitored using multiple responses by liquid chromatography-mass spectrometry.Additionally,two insulin-resistant cell models were incubated with compounds identified in RenShenJian in the blood.Glucose uptake was determined using the fluorescent analog 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol-4-yl)amino)-2-deoxyglucose.Protein expression levels of p-AMPK,AMPK,SIRT3,and GLUT4 were detected by western blotting.Results:RenShenJian decreased FBG levels and upregulated SIRT3 expression and AMPK phosphorylation in diabetic mice.Thirteen RenShenJian extracts were identified in the blood,11 of which increased the ratios of 2-(N-(7-nitrobenz-2-oxa-1,3-dia-xol 4-yl)amino)-2-deoxyglucose uptake in two insulin-resistant cell models.Nine extracts increased AMPK phosphorylation,nine increased SIRT3 expression,and six elevated GLUT4 expression in palmitate-induced HepG2 cells.Five extracts-puerarin,puerarin 6″-O-xyloside,genistein,ginsenoside Rb1,and ginsenoside Rd-simultaneously activated AMPK,SIRT3 and GLUT4.Conclusion:A series of compounds in RenShenJian that target AMPK,SIRT3,and/or GLUT4 was confirmed and indicate the chemical material basis of amelioration of insulin resistance by RenShenJian.

Research progress on chemical diversity of saponins in Panax ginseng

Saponins,the major bioactive components of Panax ginseng C.A.Mey.,are gradually emerging as research hotspots owing to the possession of various pharmacological activities.This review updates the ginsenosides list from P.ginseng and the steam-processed ginseng(red ginseng and black ginseng)up to 271 by June of 2024,encompassing 243 saponins from different parts of P.ginseng(roots,stems,leaves,flowers,berries,and seeds),103 from red ginseng,and 65 from black ginseng,respectively.Among 271 saponins,there are a total of 249(1–249)dammarane type(with a–z subtypes)tetracyclic triterpene saponins reported from each part of P.ginseng and steam-processed ginseng,two(250–251)lanostane type tetracyclic triterpene saponins identified from red ginseng,18(252–269)oleanane type pentacyclic triterpenoid saponins discovered from each part of P.ginseng and steam-processed ginseng,and two(270–271)ursane type pentacyclic triterpenoid saponins reported from red ginseng.Overall,this review expounds on the chemical diversity of ginsenosides in various aspects,such as chemical structure,spatial distribution and subtype comparison,processed products,and transformation.This facilitates more indepth research on ginsenosides and contributes to the future development of ginseng.

超临界CO_2反相微乳萃取人参中人参皂苷的研究

目的提高超临界CO2萃取人参中人参皂苷的萃取率。方法采用二-(2-乙基己基)磺基琥珀酸钠(AOT)/乙醇/水/超临界CO2反相微乳对人参皂苷的萃取进行研究。结果在萃取压力25MPa、温度45℃、时间4h、CO2流量为2L/h条件下,超临界CO2反相微乳萃取的人参皂苷萃取率是乙醇/水/超临界CO2萃取的3.2倍。人参皂苷的萃取率随加入的水量、萃取压力的增大而增大,随AOT浓度、萃取温度的升高先增大后减小。萃取人参皂苷时,采用适量水先浸泡物料与萃取过程中加入水相比,人参皂苷的萃取率要提高30%。结论结合实验结果与理论探讨,超临界CO2反相微乳萃取人参皂苷的机制主要是其形成的极性水池增大了对人参皂苷的溶解度。

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。

人参RAPD产物的限制性内切酶消化

为了鉴定随机扩增产物的同源性以及更有效地利用DNA分子标记研究人参种质资源，我们首次将毛细管PCR扩增的RAPD反应产物进行限制性内切酶消化并有效的进行了酶切位点分析，结果证明PCR-RFLP是筛选人参特异DNA分子标记的一种简单易行的新方法，可为药用植物种质资源研究提供另一有用的工具。

酶法修饰人参茎叶总皂苷及其HPLC图谱研究

目的研究生物酶制剂对人参茎叶总皂苷的修饰及其修饰前后HPLC图谱的变化。方法利用复酶制剂A对人参茎叶总皂苷进行酶法修饰,采用TLC法鉴别修饰前后的变化,并比较修饰前后的HPLC图谱的变化。结果在设定的积分事件下,复酶制剂A修饰人参茎叶总皂苷后色谱特征峰由34个减至16个,其中有13个特征色谱峰相对峰面积和相对峰高均明显降低,21个色谱峰检测不到相应信号,产生4个明显增加或新色谱峰。结论复酶制剂A能够有效地修饰人参茎叶总皂苷,采用HPLC图谱能够很好的监测其变化。

基于GC-MS法的不同品种人参中脂肪酸成分及含量分析

[目的]研究不同品种人参的脂肪酸成分及相对含量,为人参的良种选育工作提供一项重要指标。[方法]采用索式提取法提取,GC-MS法测定相对含量。[结果]鉴定出20种脂肪酸,其中主要为(Z,Z)-9,12-十八碳二烯酸、棕榈酸和(Z,Z,Z)-9,12,15-十八碳三烯酸;不同品种人参脂肪酸单体的含量存在差异,主要为①(Z,Z)-9,12-十八碳二烯酸含量大马牙47.896%、二马牙50.901%、圆膀圆芦50.254%、长脖49.456%,其中二马牙含量最高;②棕榈酸含量大马牙12.370%、二马牙12.535%、圆膀圆芦12.614%、长脖11.983%,圆膀圆芦含量最高;③(Z,Z,Z)-9,12,15-十八碳三烯酸含量大马牙10.407%、二马牙12.487%、圆膀圆芦11.936%、长脖12.026%,二马牙含量最高。[结论]用该法可鉴别出不同品种的人参,有望成为人参品种选育的一项重要手段。

人参、西洋参的基因组DNA的RAPD分析

用Operon公司的E．F．C组引物对北京和吉林左家特区的栽培人参和西洋参四大居群各10个样本进行RAPD分析，筛选出18个引物，可以检测到102个多态性位点，其中67．6％表现出多态性。在北京栽培人参、西洋参。吉林左家特区栽培人参、西洋参中分别检测到74，86，74，9O个标记位点，在各居群中均有丰富的多样性存在。西洋参的遗传多态性明显高于人参。聚类分析结果表明，同种不同居群的样本首先聚在一起，种间遗传分化明显大于地理环境造成的分化。由OPF02，OPF04，OPF07，OPF20，OPG12，OPG13，OPG14扩增的指纹图谱中可以找到不同种，不同居群的标记带型及居群内部的丰富遗传多样性。

人参毛状根及愈伤组织中ATP动态变化的生物发光法测定

ATP content of cultured callus and hairy root introducted by Ri plasmid of \%Panax ginseng \%C. A. Mey was detected by bioluminescence. The result showed that ATP content in hairy root is obviously higher than that in callus within a growth cycle. The highest ATP content in hairy root is 26.6×10\+\{-12\}mmol/g (fresh weight), while ATP in callus is 2.68×10\+\{-12\}mmol/g (fresh weight). Total saponin content in hairy root reaches 2.486% (dry weight), while the saponin in callus is 1.105% (dry weight). The hairy root inducted by Ri plasmid has vigorous ability in secondary metabolism for ginseng saponin synthesis.

人参总皂苷体外定向诱导CD34^+细胞分化为粒系血细胞的研究

目的研究人参总皂苷(TSPG)协同造血生长因子对CD34+细胞体外定向诱导分化为粒系血细胞的影响。方法应用阴性磁性分选策略,以StemsepTM系统从正常人骨髓细胞中分离CD34+造血干/祖细胞(HSC/HPC),通过甲基纤维素半固体培养法检测TSPG诱导CD34+HSC/HPC向粒系祖细胞集落(CFU-GM)增殖与分化的能力;在SCF+IL-3+GM-CSF+G-CSF(SIGG)液体培养体系中加入不同剂量的TSPG,检测对CD34+HSC/HPC增殖形成CFU-GM的影响以及CD33+细胞比例的变化。结果TSPG(10～50μg/mL)均能提高CD34+细胞形成CFU-GM的集落产率,以TSPG20μg/mL效果最为明显;不同质量浓度的TSPG协同造血生长因子在液体培养体系中诱导CD34+细胞2周,观察粒系血细胞的分化,TSPG10～70μg/mL均可不同程度地提高细胞总数、CFU-GM扩增倍数及CD33+细胞比例,TSPG20μg/mL是液体培养诱导CD34+细胞向粒系分化的最佳质量浓度。结论TSPG协同造血生长因子能促进CD34+细胞定向诱导分化为粒系血细胞。

人参根际土壤微生物PCR-DGGE反应体系优化

[目的]优选人参根际土壤微生物的PCR-DGGE反应体系。[方法]以种植1年的人参土壤为材料,利用PCR-DGGE技术,对反应体系中的几种重要参数不同梯度进行了优化研究,包括模板、dNTPs、引物及Mg2+的用量。[结果]最适宜的反应体系为:模板DNA浓度为0.8μg/μl,dNTPs浓度为0.2 mmol/L,引物浓度为0.4μmol/L,Mg2+浓度为1.5 mmol/L。[结论]该方法简便快捷,为进一步研究人参根际土壤微生物多样性奠定了基础。

Preparation and antiviral effects of polysaccharides from Panax japonicus

Water-soluble polysaccharides were prepared from Panax japonicus by hot water extraction and ethanol precipitation.The polysaccharides were further purified by ion exchange chromatography to obtain neutral and acidic polysaccharides.The neutral polysaccharide fraction mainly contained Glc(90.2%),which was a glucan fraction.The acidic polysaccharide fraction mainly contained GalA(43.6%),Gal(21.7%),and Ara(15.4%),with a degree of methyl-esterification of 20.3%,which was a pectic polysaccharide.The acidic polysaccharide of Panax japonicus could effectively inhibit the replication of human seasonal influenza virus H1N1 and canine influenza virus H3N2 in MDCK cells and A549 cells and significantly reduce the virus titer in infected cells.It also effectively inhibited the number of infected cells of the SARS-CoV-2 South Africa strain and the Omicron strain.The acid polysaccharide of Panax japonicus showed good efficacy against influenza virus and COVID-19 infection,which could be used as a potential antiviral candidate drug molecule in the future.

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.