后熟阶段的人参(Panax ginseng C.A.Mey)种子对脱水和贮存温度的反应

完成形态后熟和生理后熟期中的人参（PanaxyinsenyC．A．Mey）湿润种子的含水量由47％降至9．4％和5．8％，形成于籽后保存于室温，0～5℃和-196℃条件下1～3年。保存后的形态后熟于籽再低温处理，继续完成生理后熟。结果显示：完成形态后熟的种子在1～5℃保存1年后，种子胚根能萌发，但胚轴、胚芽不能正常生长，保存3年，种子丧失生活力；生理后熟期中的种子在0～5℃保存1年尚能正常出苗，出苗率为44．0％，保存3年，种胚已不能正常发育。但是在-196℃保存3年后，出苗率同保存在0～5C1年的相近．仍为43．1％。说明完成形态后熟种子还没有产生足够的脱水耐性，而经低温层积处理于生理后熟期中的种子就具有了这种耐性，并能进行低温短期或超低温长期保存。

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.

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℃.

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.

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.

Identification of anti-inflammatory components in Panax ginseng of Sijunzi Decoction based on spectrum-effect relationship

Objective: This study aimed to identify the main medicinal active components of Panax ginseng(P. ginseng) in the compatibility environment of clinical application. For this purpose, the anti-inflammatory ingredients of P. ginseng were investigated based on its therapeutic effect in Sijunzi Decoction(SJD) which is a widely used traditional Chinese formula.Methods: The fingerprints of 10 batches of SJD consisting of different sources of P. ginseng were established by UPLC technique to investigate the chemical components. At the same time, the antiinflammatory effects of these components were evaluated by dextran sulfate sodium-induced ulcerative colitis mouse model. Grey relational analysis was applied to explore the correlation degree between fingerprints and anti-inflammatory effects in SJD. Lipopolysaccharide-stimulated RAW264.7 murine macrophages were established to evaluate the anti-inflammatory action of the screened effective substances of P. ginseng.Results: According to grey relational analysis, notoginsenoside R1, ginsenoside Rg2 and ginsenoside Rb3of P. ginseng were the major anti-inflammatory contributions in SJD. They had been proven to be closely associated with the anti-inflammatory process of SJD and displayed a close effect compared with SJD by LPS-stimulated RAW264.7 murine macrophages.Conclusion: Our work provides a general strategy for exploring the pharmacological ingredients of P. ginseng in traditional Chinese formulas which is beneficial for establishing the quality standards of traditional herbs in traditional Chinese medicine prescription based on their clinical therapeutic effect.

Identification of microRNA and analysis of target genes in Panax ginseng

Objective: Ginsenosides, polysaccharides and phenols, the main active ingredients in Panax ginseng, are not different significantly in content between 3 and 5 years old of ginsengs called Yuan ginseng and more than ten years old ones called Shizhu ginseng. The responsible chemical compounds cannot fully explain difference in efficacy between them. According to reports in Lonicerae Japonicae Flos(Jinyinhua in Chinese) and Glycyrrhizae Radix et Rhizoma(Gancao in Chinese), microRNA may play a role in efficacy,so we identified microRNAs in P. ginseng at the different growth years and analyzed their target genes.Methods: Using high-throughput sequencing, the RNA-Seq, small RNA-Seq and degradome databases of P. ginseng were constructed. The differentially expressed microRNAs was identified by qRT-PCR.Results: A total of 63,875 unigenes and 24,154,579 small RNA clean reads were obtained from the roots of P. ginseng. From these small RNAs, 71 miRNA families were identified by bioinformatics target prediction software, including 34 conserved miRNAs, 37 non-conserved miRNA families, as well as 179 target genes of 17 known miRNAs. Through degradome sequencing and computation, we finally verified 13 targets of eight miRNAs involved in transcription, energy metabolism, biological stress and disease resistance, suggesting the significance of miRNAs in the development of P. ginseng. Consistently, major miRNA targets exhibited tissue specificity and complexity in expression patterns.Conclusion: Differential expression microRNAs were found in different growth years of ginsengs(Shizhu ginseng and Yuan ginseng), and the regulatory roles and functional annotations of miRNA targets in P. ginseng need further investigation.

Effects of Raphani Semen on anti-fatigue and pharmacokinetics of Panax ginseng

Objective: To explain the phenomenon that Panax ginseng is not compatible with Raphani Semen based on pharmacodynamics and pharmacokinetics.Methods: The forced swimming time and biochemical parameters such as blood lactate(BLA), serum urea nitrogen(SUN), and hepatic glycogen(GLU) were determined for anti-fatigue experiment. The UPLCMS/MS was used to analyze the pharmacokinetics of Rg1, Re, Rb1, and Rd after orally administration of P. ginseng and P. ginseng combined with Raphani Semen to rats. Pharmacokinetic differences of four ginsenosides between single uses of P. ginseng and combined with Raphani Semen were investigated.Results: The results showed that Raphani Semen tended to significantly reduce the anti-fatigue activity of P. ginseng. Co-administration of P. ginseng and Raphani Semen had significant effects on the pharmacokinetics of the four ginsenosides in rats compared to that observed with P. ginseng extract alone. The AUC0–12 hvalues of the four ginsenosides in PG group were higher than the corresponding values in the PR group. It can be inferred that Raphani Semen decreased the blood exposure of the four ginsenosides in rats when it combined with P. ginseng.Conclusion: The anti-fatigue activity and pharmacokinetic results showed that Raphani Semen may reduce the pharmacological actions of P. ginseng.

Metabolomics analysis reveals the renal protective effect of Panax ginseng C. A. Mey in type 1 diabetic rats

The dry root and rhizome of Panax ginseng C. A. Mey has garnered much interest owing to its medicinal properties against diabetes and cardiovascular diseases. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS)-based metabolomics approach was used to illustrate the therapeutic mechanisms of ginseng extract on the serum and urinary metabolic profiles in streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. Pharmacological and renal parameters in response to the administration of ginseng were also evaluated. In total, 16 serum endogenous metabolites and 14 urine endogenous metabolites, including pyruvic acid, indoleacetic acid, and phenylacetylglycine, were identified as potential biomarkers for diabetes. Pathway enrichment and network analysis revealed that the biomarkers modulated by ginseng were primarily involved in phenylalanine and pyruvate metabolism, as well as in arginine biosynthesis. Moreover, the levels of several renal injury-related biomarkers in T1DM rats were significantly restored following treatment with ginseng. The administration of the extract helped maintain tissue structure integrity and ameliorated renal injury. The findings suggest that the regulatory effect of ginseng extract on T1DM involves metabolic management of diabetic rats, which subsequently attenuates T1DM-induced early renal dysfunction.

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.

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.

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。

高架十字迷路课题中人参根和茎叶皂苷抗焦虑效果的研究

目的 研究人参根和茎叶皂苷抗焦虑效果。方法 高架十字迷路法。结果 中枢兴奋药咖啡因 40～ 80 m g/kg ig给药能使小鼠在开放通路中连续停留时间缩短 ,在封闭通路中停留时间延长。而中枢抑制药安定 1.5～ 3.0mg/ kg却能明显地延长小鼠在开放通路中的停留时间。人参根和茎叶皂苷 5 0～ 2 0 0 m g/ kg ig给药后同样地使小鼠在开放通路中连续停留时间延长 ,并且能使小鼠在开放路中往返次数增加。

人参茎叶挥发油中倍半萜烯化合物的分离与鉴定

对人参茎叶挥发油的有效成分进行分离纯化 ,利用薄层层析 (TL C)、红外光谱 (IR)、气相色谱 (GC)、气相色谱 -质谱 (GC/MS)联机技术从该挥发油中分离鉴定 6个倍半萜烯类化合物 .

植物激素对人参毛状根生长和皂甙含量的影响

就植物激素IAA、IBA、NAA、2,4 D和6 BA对人参(PanaxginsengC.A.Meyer)毛状根生长及皂甙含量的影响进行了研究。结果表明,4种生长素在适宜的浓度下均可不同程度地促进人参毛状根的生长以及皂甙的积累,同时能影响单体皂甙的分布。NAA和IBA能显著促进毛状根的生长,其中0.500mg LIBA能显著促进毛状根生长和总皂甙的积累。细胞分裂素6 BA在较低浓度时虽然对生长无明显的促进作用,但对皂甙积累有利,同时显著促进单体皂甙Rb1的积累,增大Rb1在总甙中所占的比例。

复方人参注射液粉末X-射线衍射指纹图谱研究

目的 :建立复方人参注射液的粉末X 射线衍射指纹图谱。方法 :采用粉末X 射线衍射衍射法测定药材、中间体和注射液的衍射图谱 ,对所得到的图谱进行特征峰指认和对比分析 ,获得各自的标准指纹图谱。结果 :10个批次的药材、中间体和注射液均有非常好的重现性 ,相似度达 85 %以上。结论 :粉末X 射线衍射方法可用于复方人参注射液的质量控制。

人参农家类型遗传多样性的ISSR分析

目的探讨人参农家类型的遗传多样性和亲缘关系,为人参的栽培和选育种提供遗传学依据。方法采用ISSR分子标记分析5种人参农家类型7个居群120个样本的遗传多样性。结果人参农家类型有较丰富的遗传多样性,平均多态位点百分率为48.85%;不同农家类型的遗传多样性水平有差异,和其他农家类型相比,长脖和竹节芦的遗传变异较小,且两者之间的相似性系数高达97%;不同产地的同一人参农家类型间也存在很大的遗传差异。结论研究表明人参农家类型的遗传差异主要存在于各类型内部,而且可能更多地存在于同一类型的不同居群内部。为促进人参新品种的选育,有必要在现有的栽培群体中补充不同产地的同一类型的种质资源。

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。

人参总皂苷诱导K562细胞凋亡的实验研究

目的 研究人参总皂苷诱导白血病细胞凋亡及其机制 ,为进一步开发人参总皂苷提供实验依据。方法 采用细胞体外培养、图象分析技术、流式细胞术、形态学观察与免疫细胞化学等方法 ,研究人参总皂苷诱导 K5 6 2细胞凋亡。结果 人参总皂苷对 K5 6 2细胞有增殖抑制作用 ,并可诱导 K5 6 2细胞凋亡明显增加 ,同时 K5 6 2细胞癌基因产物 C- MYC,BCL- 2表达明显降低。结论 人参总皂苷能诱导 K5 6 2细胞凋亡 ,其机制可能与调控癌基因产物的表达有关。