三七细胞中SS、DS共超表达对皂苷合成的影响

为研究三七细胞中SS、DS共超表达对皂苷合成的影响,构建了三七SS超表达载体,并以已超表达了DS的愈伤组织为侵染材料,借助农杆菌LBA4404将SS导入并整合到三七基因组中,采用QRT-PCR法检测转基因株系中SS、DS的表达水平,香草醛-冰醋酸-高氯酸显色法检测皂苷含量。结果显示:三株同时转SS、DS细胞系(T-4、T-5、T-9)中,SS的相对表达量分别为仅转DS株系的5.66、7.37、7.46倍;DS的相对表达量分别为仅转DS株系的1.39、2、1.41倍;总皂苷含量为仅转DS株系的1.7倍,普通细胞的2.4倍;5种单体皂苷:R1、Rg1、Re、Rb1、Rd含量总和分别为仅转DS株系的1.33、1.61、1.71倍,普通细胞的5.62、6.81、7.26倍;此外,Re含量出现大幅增加,达到三七药材的5~6倍。表明SS、DS在皂苷生物合成途径中均起正调控作用,双基因共超表达能进一步提高皂苷含量,揭示途径中两个甚至更多基因间可能存在正协同调控效应。     

黑曲霉固体发酵三七药材的皂苷变化研究

为研究黑曲霉发酵三七药材过程中皂苷类成分的化学变化,利用黑曲霉对三七药材进行固体发酵,根据高效液相色谱-四级杆-飞行时间串联质谱（HPLC-Q-TOF-MS/MS）联用的检测结果,对三七发酵产物的总皂苷类成分进行分析和鉴定,并探讨了皂苷含量变化。结果表明,黑曲霉可以高效的转化三七药材中皂苷类成分。三七药材经黑曲霉发酵之后,人参皂苷Rb1几乎全部转化生成其他物质,在发酵产物中已未检出,三七皂苷R1和人参皂苷Rg1大幅度减少,三七皂苷R2、人参皂苷Rh1、Rd等含量明显增加,并且在三七发酵产物中检出了稀有人参皂苷F1、Rh4、Rg3、CK、三七皂苷Rh16和T5。     

RP-HPLC法测定三七养血胶囊中人参皂苷Rg_1、Re、Rb_1、Rd及三七皂苷R_1的含量

建立测定三七养血胶囊中人参皂苷Rg1、Re、Rb1、Rd及三七皂苷R1的含量测定方法。方法为:色谱柱为InertsilC8-3柱(5μm,4.6×250mm),流动相为乙腈-水,梯度洗脱,检测波长为203nm。结果表明:人参皂苷Rg1、Re、Rb1、Rd及三七皂苷R1的进样量分别在1.74～69.61μg(r=0.9997)、0.23～9.59μg(r=0.9998)、1.47～59.17μg(r=0.9998)、0.20～8.05μg(r=0.9997)、0.45～18.35μg(r=0.9999)范围内与峰面积积分值呈良好的线性关系;平均回收率(n=6)分别为0.57%、0.78%、1.08%、0.43%、1.24%。该方法灵敏准确、重复性好,专属性强,可用于本制剂的质量控制和评价。     

2017~2018年云南地区三七须根中6种皂甙含量监测结果分析

目的 了解云南地区三七须根中R1、Rg1、Re、Rb1、Rd、Rb3 6种皂甙含量, 为三七须根开发利用提供基础数据和科学依据。方法 按照《云南省食品安全风险监测方案》中样品采集要求, 在云南地区8个地区采集106件样品。样品参照《保健食品检验与评价技术规范》(2003)“保健食品中人参皂苷高效液相色谱法”进行监测。根据GB/T 19086-2008《地理标志产品 文山三七》进行评价。结果 6种皂甙含量中以R1+Rg1+Rb1为主。三七须根皂甙总量的合格率为60.4%, 优等品达标率为41.5%。文山地区三七须根合格率高于其他地区。结论 该云南地区三七须根中皂甙含量合格率较低, 应采取措施加以改进。     

可发酵三七等中药材的食用菌种筛选和皂苷生物转化产物的分析

研究了保加利亚乳杆菌、凝结芽孢杆菌、鼠李糖乳杆菌和冠突散囊菌等食品工业中常用的微生物菌种对三七之类的中药原料的生物转化,利用HPLC分析了不同菌种直接发酵对人参皂苷组成的影响。通过不同微生物菌种的发酵,三七、人参和西洋参所含的Rb1、Re等常见人参皂苷会分别转化为Rh1、C-K和Rd等稀有人参皂苷,产物中没有检测到Rh2等人参皂苷,说明菌种产生的糖苷酶具有相当的专一性,为进一步对特殊目标皂苷的开发利用提供了基础。     

不同林型、产地、参龄及坡向对林下参20 种单体皂苷含量的影响

采用同时测定20?种人参皂苷高效液相色谱法评价不同林型、产地、参龄及坡向对林下参皂苷含量的影响。结果表明,不同林型、产地、参龄及坡向林下参均含有14?种人参皂苷单体;与其他3?种林型比较,樟子松林型下栽培的林下参单体皂苷Rg1、Re、Rf、Rb1、Rb2、Rb3、Rg5含量与20?种单体皂苷加和值最高;与其他5?个产地相比,抚松县露水河镇林下参单体皂苷Rg1、Rf含量最高;3?种不同年生林下参比较,15?a生林下参单体皂苷Rg1、Rf、Rb1含量最高,5?a生林下参单体皂苷Compound?K、原人参二醇含量最高;3?种不同坡向相比,阳坡林下参单体皂苷Rb2、Rb3含量最高,阴坡林下参单体皂苷Rh2含量最高。不同林型、产地、参龄及坡向林下参皂苷种类相同但含量不同,5～15?a生林下参均达到2015年《中国药典》要求。     

[1]保加利亚乳杆菌发酵转化人参皂苷工艺研究

摘 要: 目的 以人参为原料, 通过保加利亚乳杆菌发酵提高人参皂苷含量。方法 利用单因素试验和响应面法优化发酵工艺, 并对发酵过程中原型人参皂苷生物转化可能途径进行分析。结果 在发酵培养基为MRS液体培养基的前提下, 最适发酵条件为发酵温度40℃, 发酵时间3 d, 接种量3%, 转化稀有人参皂苷含量在150 μg/mL。经对比发现, 原参中检测出Re、Rg1、Rb1、Rc、Rb2、Rd、Rh1 7种皂苷, 经过发酵后的人参中检测出Re、Rg1、Rb1、Rc、Rb2、Rh1、Rd、R-rg3、CK 9种皂苷。同时原参中的常规皂苷含量经发酵后有所下降, 稀有皂苷含量有所增加, 且多酚、黄酮含量增加, 总糖含量减少, 发酵过程中人参皂苷生物转化的可能途径与人参皂苷含量变化趋势一致。结论 保加利亚乳杆菌发酵人参能够有效将原型皂苷转化成稀有人参皂苷, 为人参的深加工奠定基础, 为人参发酵产品的开发和利用提供参考。     

产β-葡萄糖苷酶洋虫内共生真菌筛选、鉴定及联合转化人参皂苷作用机制

筛选、鉴定出洋虫成虫体内两株产β-葡萄糖苷酶真菌,两菌株联合发酵提高稀有皂苷转化率和发酵品质,对其动态发酵反应过程进行研究,明确人参皂苷的转化机制。通过MRS(七叶苷,柠檬酸铁)培养基筛选,18S rRNA基因扩增分别对两株产β-葡萄糖苷酶真菌进行鉴定,并与全组分人参皂苷发酵反应,利用LC-MS/MS对发酵产物进行分析。经鉴定两株产β-葡萄糖苷酶分别属于Chaetomium和Aspergillus属;WY1与人参主皂苷反应产物为Rd和Rh1;WY2与人参主皂苷反应产物为Rd、Rh1、Rg3和compound K;WY1/ WY2双菌联合发酵有效提高了Rh1、Rg3和compound K的转化率,其转化途径为Re→Rg1→Rh1;Rf→Rh1;Rb1/Rb2/Rc→Rd→Rg3/compound K,人参二醇类皂苷比人参三醇类皂苷更容易被转化且大部分被转化为Rg3。     

高效液相色谱法同时测定人参制剂中20 种人参皂苷方法的建立

为了更加有效评价人参制剂生产质量,建立了一种同时测定人参制剂中20 种人参皂苷的高效液相色谱方法。结果表明,20 种人参皂苷Rg1、Re、Rg2、Rg3、Rg5、Rf、F1、F2、Rc、Rd、Rb1、Rb2、Rb3、Rh2、compound K、20（R）-Rh1、Rk3、Rh4、原人参二醇及原人参三醇均得到良好分离,线性关系良好（R≥0.999 2）。该方法快捷简便、稳定可靠,能够精确全面检测分析人参皂苷含量,对于人参加工品及其制剂的质量控制更为全面准确可行。     

HPLC法快速测定三七提取物中皂苷的含量

本文采用Kinetex核-壳技术色谱柱快速测定三七提取物中三七皂苷R1、人参皂苷Rg1和Rb1的含量。样品用甲醇超声波提取法提取,采用Phenomenex Kinetex C18100A(50mm×4.6mm,2.6μm)色谱柱,柱温常温,流动相乙腈:水,梯度洗脱,流速为1.0mL/min,检测波长为203nm,柱温为室温,对R1、Rg1和Rb1进行定量分析。结果表明:R1、Rg1和Rb1的线性良好,重复性试验中R1、Rg1和Rb1的相对标准偏差分别为3.2%、3.8%和2.6%,最低检出限为0.001μg;R1、Rg1和Rb1的加标回收率分别为99.3%、100.4%和100.7%。本方法快速、节省试剂,可用于三七提取物中三七皂苷R1、人参皂苷Rg1和Rb1的测定。     

β-Glycosidase-assisted bioconversion of ginsenosides in purified crude saponin and extracts from red ginseng (Panax ginseng C. A. Meyer)

Major ginsenosides in ginseng (Panax ginseng) and its products are highly glycosylated, hence poorly absorbed in the gastrointestinal tract. β-Glycosidase-assisted deglycosylation of pure ginsenosides was peformed to study bioconversion mechanisms. Ginsenoside standard compounds, crude saponin, and red ginseng extracts were incubated with β-glycosidase (0.05% w/v, 55°C). β-Glycosidase has a broad specificity for β-glycosidic bonds, hydrolyzing the β-(1→6), α-(1→6), and α-(1→2) glycosidic linkages. The final metabolite of protopanaxadiol ginsenosides was Rg3 while the metabolite of protopanaxatriol ginsenosides was Rh1. β-Glycosidase treatment of red ginseng extracts resulted in a decrease in the amounts of Rb1, Rc, Re, and Rg2 after 24 h, whereas levels of the less glycosylated Rd, Rb1, Rg, Rg3, Rg1, and Rh1 forms increased. When crude saponin was incubated with β-glycosidase for 24 h, levels of Rb1, Rc, Re, and Rg1 decreased while levels of Rd, Rg3, and Rh1 increased as deglycosylated ginsenosides.     

三七冻干粉及其活性组分对小鼠1型辅助性T细胞分化和活性的影响

目的:观察三七冻干粉及其有效成分对小鼠CD4+T细胞向1型辅助性T(Th1)细胞分化的影响,探究三七作为药食同源药物改善机体免疫力的可能作用机制。方法:提取C57BL/6小鼠脾细胞并分选出CD4+T细胞进行培养;分为空白对照组,三七冻干粉低、中、高剂量组(160、320、640 μg/mL)和三七总皂苷低、中、高剂量组(5、10、20 μg/mL),加入对应药物48 h后,采用CCK-8法筛选三七冻干粉及三七总皂苷的最佳给药浓度并检测二者对细胞活力的影响,用酶联免疫吸附试验(ELISA)检测上清液中 IFN-γ的变化。结果:与对照组相比,三七冻干粉组和三七总皂苷组的细胞因子IFN-γ水平,IFN-γ mRNA和T-bet mRNA表达水平均显著降低(P<0.05)。结论:三七冻干粉及其活性成分均能抑制CD4+T细胞向Th1细胞分化。     

人参-虫草双向固体发酵过程中人参皂苷含量变化研究

研究人参-冬虫夏草双向固体发酵过程中主要人参皂苷的组成和含量变化,为发酵产物的质量控制和开发利用提供理论依据。采用高效液相色谱-质谱联用（HPLC-MS）技术对发酵过程及产物中人参皂苷进行定性定量分析。结果表明,人参-虫草双向固体发酵产物中共鉴定出11种皂苷。其在200～1 500 ng/mL范围内线性关系良好（R2＞0.999）,加标回收率范围为95.13%～105.04%,相对标准偏差（RSD）为0.85%～2.36%,精密度、稳定性、重复性试验结果的RSD均＜2.6%,表明该检测方法精密度、准确度、稳定性及重复性良好。人参-虫草双向固体发酵过程中人参皂苷Rg1、Re、Rb1含量明显降低,人参皂苷Rh1、Rg2、Rd、Rg3、F2含量明显增加。     

Morphological Characterization,Chemical Components,and Biofunctional Activities of Panax ginseng,Panax quinquefolium,and Panax notoginseng Roots: A Comparative Study

The pharmacological activities of Panax ginseng, Panax quinquefolium, and Panax notoginseng as herbal medicinal resources were reviewed to evaluate their biofunctional effects. P. ginseng contains relatively many kinds of ginsenoside that possess pharmacological activity compared with P. quinquefolium and P. notoginseng. However, the ratio of panaxatriol and panaxadiol of ginsenoside in P. notoginseng is higher than those of P. ginseng and P. quinquefolium. The free sugar content of P. ginseng is similar to P. quinquefolium but is different from P. notoginseng. The total free amino acid content is highest in P. ginseng and lowest in P. notoginseng. P. ginseng has the highest antioxidative effects and P. ginseng and P. quinquefoilum possess more potent cytotoxicities on A549 and SK-OV-3 cell lines than P. notoginseng. Particularly, red ginseng of P. ginseng shows the highest antidiabetic effects, suggesting that it possesses activity to enhance insulin secretion as well as to prevent a destruction of pancreatic islet cells.     

正交试验优化超声辅助法提取三七有效成分工艺

研究超声辅助法提取三七有效成分的工艺。通过单因素试验研究乙醇溶液体积分数、料液比、超声波频率和提取时间对三七人参皂苷Rg1提取率的影响。在单因素试验基础上对4个因素安排L9(34)正交试验,得出影响三七有效成分人参皂苷Rg1提取率的先后次序为：乙醇体积分数＞提取时间＞料液比＞超声频率。选出最佳提取工艺为：乙醇体积分数70%、料液比1:12(g/mL)、超声频率59kHz、提取时间60min。在此最佳工艺下,得到三七干燥后的人参皂苷Rg1含量为1.85%。     

Manipulation of ginsenoside heterogeneity in cell cultures of Panax notoginseng by addition of jasmonates

Manipulation of the heterogeneity of ginsenosides with different pharmacological activities was attempted by adding methyl jasmonate (MJA) or dihydro-methyl jasmonate (HMJA) to suspension cultures of a high-saponin-producing cell line of Panax notoginseng. Compared with HMJA, MJA not only increased ginsenoside content to a large degree but also significantly altered the ratio of Rb to Rg groups of ginsenosides. The ginsenoside Rd was not detected in either control cultures (without MJA addition) or when HMJA was added, but interestingly was detected upon supplementation of MJA. By increasing the concentration of MJA added from 0 to 500 microM, the ratio of Rb to Rg groups also increased from 0.49+/-0.06 to 2.49+/-0.06 (on day 15). The cells exhibited their maximal ginsenoside content when 200 microM MJA was added. The maximal Rb1 content was 3.15+/-0.08 mg/100 mg DW (day 15), which was about ninefold that of the control. The maximal Rg1 and Re contents were 0.90+/-0.01 (day 12) and 0.80+/-0.01 (day 15) mg/100 mg DW, respectively, which were about twofold that of the control. The information obtained will be useful for the exploitation of the diversity of biopharmaceutical molecules and for the large-scale bioprocessing of heterogeneous products.     

Effects of steaming the root of Panax notoginseng on chemical composition and anticancer activities

The root of Panax notoginseng has been shown to change its saponin composition upon steaming. This study examines the effects of different steaming times and temperature on notoginseng root for saponin composition and anticancer activities. Steaming decreased the content of notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, R2, Rb3 and Rd, but increased the content of Rh1, Rg2, 20R-Rg2, Rg3 and Rh2. Steaming significantly influenced the transformation of Rg3. The amount of ginsenoside Rg3, an anticancer compound, was 5.23-fold greater in root steamed for 2 h at 120 °C than at 100 °C, and 3.22-fold greater when steamed for 4 h than for 1 h at 120 °C. For anticancer effects, the extract of steamed root significantly inhibited proliferation of SW-480 human colorectal cancer cells. The IC₅₀ of the steamed extract for 1, 2, 4 and 6 h at 120 °C was 259.2, 131.4, 123.7 and 127.1 μg/mL, respectively; the effect of unsteamed extract was low. Flow cytometric analysis demonstrated that the apoptotic cell induction rates of SW-480 cells were 56.3% and 64.4% with 150.0 and 200.0 μg/mL extract steamed for 6 h. Compared with Rg1 and Rb1, only Rg3 had a significant antiproliferative effect.