同时测定不同人参加工产品中20 种人参皂苷的UPLC-PDA方法开发和验证

建立一种简单、有效、精密和准确的超高效液相色谱方法评价不同人参加工产品的质量,同时快速测定20 种人参皂苷Rg1、Re、Rf、20(S)-Rg2、20(R)-Rg2、Rb1、Rc、Ra1、Rb2、Rb3、Rd、Rk3、F2、20(S)-Rg3、20(R)-Rg3、Compound K（CK）、Rg5、20(S)-Rh2、20(R)-Rh2和protopanaxadiol（PPD）。采用二极管阵列检测器和ACQUITY UPLC BEH-C18（2.1 mm×50 mm,1.7 μm）色谱柱,以乙腈-水为流动相,流速0.3 mL/min,柱温30 ℃,梯度洗脱。20 种人参皂苷在31 min内可达到良好的分离,考察方法的线性范围、回收率、日内和日间精密度。在本方法条件下,线性关系良好,相关系数R2均大于0.998,日内相对标准偏差不大于4.65%,日间相对标准偏差不大于4.88%,回收率为85.71%～108.50%。方法检出限为0.81～3.10 μg/mL,方法定量限为2.88～10.00 μg/mL。本方法快速、可靠,已成功用于不同人参加工产品包括保鲜参、红参和白参中20 种人参皂苷的分析检测,有效揭示不同人参加工产品中人参皂苷含量水平的显著变化,可用于鲜人参及其加工产品中活性化合物的分析和质量控制。     

同时测定不同人参加工产品中20种人参皂苷的UPLC-PDA方法开发和验证（英文）

建立一种简单、有效、精密和准确的超高效液相色谱方法评价不同人参加工产品的质量,同时快速测定20种人参皂苷Rg1、Re、Rf、20(S)-Rg2、20(R)-Rg2、Rb1、Rc、Ra1、Rb2、Rb3、Rd、Rk3、F2、20(S)-Rg3、20(R)-Rg3、Compound K(CK)、Rg5、20(S)-Rh2、20(R)-Rh2和protopanaxadiol(PPD)。采用二极管阵列检测器和ACQUITY UPLC BEH-C18(2.1 mm×50 mm,1.7μm)色谱柱,以乙腈-水为流动相,流速0.3 mL/min,柱温30℃,梯度洗脱。20种人参皂苷在31 min内可达到良好的分离,考察方法的线性范围、回收率、日内和日间精密度。在本方法条件下,线性关系良好,相关系数R2均大于0.998,日内相对标准偏差不大于4.65%,日间相对标准偏差不大于4.88%,回收率为85.71%～108.50%。方法检出限为0.81～3.10μg/m L,方法定量限为2.88～10.00μg/m L。本方法快速、可靠,已成功用于不同人参加工产品包括保鲜参、红参和白参中20种人参皂苷的分析检测,有效揭示不同人参加工产品中人参皂苷含量水平的显著变化,可用于鲜人参及其加工产品中活性化合物的分析和质量控制。     

天然柠檬汁催化人参须根粉制备20（S,R）-Rg3和Rg5工艺研究

利用多功能改进型索氏提取器,研究天然柠檬汁催化人参须根粉转化制备稀有人参皂苷20（S,R）-Rg3、Rg5的方法。以乙醇浓度、提取时间和提取筒温度为影响因素,人参皂苷20（S,R）-Rg3、Rg5总得率为指标进行正交试验,对提取工艺进行优化。结果表明,最佳提取条件为提取时间4 h、乙醇浓度30%、提取筒温度75℃。在此条件下,人参皂苷20（S,R）-Rg3、Rg5总得率为3.68%。多功能改进型索氏提取器的梯度提取制备工艺可显著缩短人参皂苷的提取时间,简化提取过程,并有效提高人参皂苷20（S,R）-Rg3、Rg5的总得率。     

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。     

[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。     

人参果浆中人参皂苷Re的提取和生物转化

为了充分利用人参资源,本文以加工厂废弃的人参果浆为原料,分析人参果浆中的皂苷含量和组成,并从中提取人参皂苷Re,以人参皂苷Re为底物,采用人参自身酶为催化剂,生物转化得到人参皂苷Rg2组。结果表明,人参加工厂废弃果浆的干品中,皂苷含量为6.21%(W/W),其中人参皂苷Re的含量为55.1%(W/W)。从果浆的干品中提取纯化得到了人参皂苷Re,得率为2.4%(W/W)。人参皂苷Re生物转化制备得到人参皂苷Rg2组,得率为65%(W/W)。经高效液相色谱(HPLC)及超高效液相-四级杆飞行时间串联质谱(UPLC-Q-TOF-MS)分析得出,人参皂苷Rg2组由20(S)-Rg2、20(R)-Rg2、Rg4和Rg6组成,本论文为人参加工厂废弃果浆的综合利用提供了理论依据。     

人参皂苷Rg2立体异构体对皮层神经元抗凋亡作用的研究

先前的研究已经证明人参皂苷Rg2(ginsenoside-Rg2,Rg2)对人类健康有许多益处,包括抗休克,预防或治疗冠心病和预防多梗塞性痴呆等。另外,Rg2包含2个立体异构体:20(R)-人参皂苷Rg2[20(R)-Rg2]和20(S)-人参皂苷Rg2[20(S)-Rg2]。然而,目前Rg2对脑缺血再灌注损伤(cerebral ischemia and reperfusion injury,CIRI)诱导神经元凋亡的影响以及这种影响是否具有立体异构性尚未见报道。因此,研究使用大脑皮层神经元建立常用于体外模拟CIRI的氧糖剥离/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)的细胞模型来探索Rg2立体异构体对OGD/R诱导的皮层神经元凋亡的影响。结果显示与模型组相比,20(R)-Rg2和20(S)-Rg2的3种剂量(20、40、80μmol/L)预处理均显著增加细胞存活率(P0.05),显著降低胱天蛋白酶-3(Caspase-3)的活性(P0.05)和凋亡率(P0.05);40、80μmol/L剂量组均显著提高缺氧诱导因子-1α(hypoxiainducible factor,HIF-1α)-促红细胞生成素(erythropoietin,EPO)信号通路活化(P0.05)。20(R)-Rg2的这种抗凋亡作用显著大于20(S)-Rg2。这些结果表明20(R)-Rg2和20(S)-Rg2均能降低由OGD/R诱导的皮层神经元凋亡,并且20(R)-Rg2具有更好的预防作用。20(R)-Rg2有潜力被用作预防CIRI的功能性食品中的有效成分之一。     

二步法制备稀有人参皂苷Rh1组异构体

本论文以制备人参皂苷Rh1为目标,选择三醇类人参皂苷Re为底物,采用酶转化和金属离子催化联用的二步法催化转化,研究了各步骤中的催化反应条件,并对产物进行了纯化和组成分析。结果表明:Absidia sp. P39r菌株产酶能催化转化Re生成Rg1,确定其最佳反应条件为:缓冲液pH5.0,反应温度40 ℃,底物浓度1.2%,反应时间16 h,乙醇浓度10%,在此反应条件下得到的Rg1质量分数最高,为70.5%。然后以Rg1为底物,确定在乙醇-水体系下Fe³⁺的催化反应产物20(S,R)-Rh1的质量分数最高,催化反应条件优化结果为:乙醇浓度50%,反应温度50 ℃,底物浓度1.7%,Fe³⁺溶液反应浓度1.4 mol/L,反应时间14 h,20(S,R)-Rh1的质量分数高达61.83%,Rk3、Rh4的质量分数之和为27.34%。在上述条件下将20 g人参皂苷Re与酶液反应,反应结束后用AB-8大孔吸附树脂分离干燥得到含有Rg1的产物14.1 g。再取10.2 g反应得到的Rg1与Fe³⁺溶液反应,干燥后最终得到的人参皂苷Rh1组异构体质量为8.18 g,得率为80.2%,其中20(S)-Rh1,20(R)-Rh1,Rk3和Rh4的含量分别为37.71%,24.12%,7.27%,20.07%。     

稀有人参皂苷C-K转化菌株的筛选鉴定及其转化特性

以原人参二醇型皂苷混合物为底物对10种霉菌进行筛选,发现3.26号菌株能够将高含量的原人参二醇型皂苷Rb1、Rb2、Rc等转化为具有抗肿瘤活性的稀有人参皂苷Compound K(C-K)。经形态学和ITS序列分析鉴定,该菌株属于附球霉属(Epicoccum)真菌。从该菌株培养液中分离出人参皂苷水解酶粗酶E-I,确定其最适pH值和最适温度分别为pH 5.0和40℃。用EI分别转化人参皂苷Rb1和Rb2、Rc,发现它既能水解人参皂苷Rb1,也能水解Rb2和Rc,但是对Rb1的水解活性更强。上述结果说明:3.26号菌株产生的糖苷酶具有广泛的底物专一性,但是对葡萄糖苷键的专一性更高;该菌的生物转化途径为人参皂苷Rb1、Rb2、Rc→Rd→F2→C-K。     

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。     

Changes in ginsenosides and antioxidant activity of Korean ginseng (Panax ginseng C.A. Meyer) with Heating Temperature and Pressure

This study was carried out to investigate the changes of ginsenoside compositions and antioxidant activity of fresh ginseng induced by thermal processing at different temperatures (25, 100, 121, and 150°C), pressure (0.1, 10, 20, and 30 MPa), and soaking solvents (water and ethanol). The levels of ginsenosides were similar trend with the pressure of 0.1–30 MPa, while there were significantly differences in heated ginseng with heating temperature and soaking solvent. When water and ethanol was used, the ginsenoside compositions significantly changed at 100 and 121°C, respectively, and it was rapidly decreased at 150°C. After heating, the level of 3 ginsenosides (Re, Rf, and Rg1) decreased and that of 5 other ginsenosides [Rb1, Rb2, Rb3, Rc, and Rg2(S)] increased up to 121°C compare to raw ginseng. Ginsenoside F2, F4, Rg2(R), Rk3, Rh4, Rg3(S), Rg3(R), Rk1, and Rg5, which was absent in raw ginseng, was detected in heated ginseng. Especially, ginsenoside Rg3(S), Rg3(R), Rk1, and Rg5 were remarkably produced after thermal processing. After heating, the phenolic compounds (1.43–11.62 mg/g), 50% inhibition concentration (IC₅₀) value (1.48–3.11 mg/g), and ABTS radical scavenging activity (0.66–9.09 mg AA eq/g) of heated ginseng were increased.     

抗抑郁20(S)-原人参二醇型人参皂苷成分分析

本文研究了抗抑郁活性三七叶总皂苷提取物中的20(S)-原人参二醇(PPD)型人参皂苷成分。采用对照品以及文献对比相结合的方法,初步分析该活性提取物的人参皂苷成分;采用高效液相色谱法,色谱条件为:甲醇-0.2%磷酸溶液(70:30)为流动相,检测波长203 nm,柱温为30℃,同时测定提取物中人参皂苷Rb_1、Rc、Rb_2、Rb_3,三七皂苷Fc这5个代表性的PPD型人参皂苷活性成分的含量。结果鉴别出Rb_1、Rc、Fc、Rb_2、Rb_3等15个成分,其中13个为PPD型人参皂苷。HPLC含量测定中,Rb_1、Rc、Fc、Rb_2、Rb_3分离良好,分离度1.5以上,平均加样回收率分别为98.92±2.67%(RSD=2.73%)、99.51±1.33%(RSD=1.34%)、100.09±1.45%(RSD=1.45%)、100.53±2.24(RSD=2.22%)、100.12±1.74%(RSD=1.74%)。本研究表明抗抑郁活性三七叶总皂苷主要是PPD型人参皂苷成分,其中Rb_1、Rc、Fc、Rb_2、Rb_3的总含量高达38.67±0.18%。     

Ultra high pressure extraction (UHPE) of ginsenosides from Korean Panax ginseng powder

To elucidate the potential of ultra high pressure (UHP) processing on ginseng, effect of UHP on extraction yield, crude saponin content, and ginsenoside contents of ginseng powder was investigated. Ginseng slurries (70, 80, and 90% moisture content) were put into a retortable pouch then hermetically sealed. These mixtures were pressurized at room temperature up to 600 MPa for 5–15 min. UHP ginseng showed relatively higher extraction yield (312.2–387.1 mg) and amounts of crude saponins (19.3–32.6 mg/g ginseng) than control ginseng (189.9 and 17.5 mg/g ginseng, respectively). Correlation coefficient between extraction yield and crude saponin content was relatively low (R²=0.2908). In high performance liquid chromatography (HPLC) analysis, amounts of measured total ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) increased with UHP processing but pressure level and pressing time did not proportionally influence the ginsenosides content. This work shows a potential of UHP processing on extraction of ginseng powder and provides basic information on UHP extraction of ginseng powder.     

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.     

Ginsenoside Composition and Antiproliferative Activities of Explosively Puffed Ginseng (Panax ginseng C.A. Meyer)

ABSTRACT: The puffing process was evaluated as an alternative to the steaming process for producing a biologically more active ginseng product, like red ginseng, from raw ginseng. A puffing treatment of dried raw ginseng roots induced an overall increase in crude saponin content. As puffing pressure increased, the content of ginsenoside Re, Rg1, Rb1, Rc, and Rb2 decreased, while ginsenoside Rg3 increased significantly as compared to raw ginseng. The content of ginsenoside Rg3 in puffed ginseng at a pressure of 490 kPa was similar to that of red ginseng. Cancer cell lines (HeLa, MCF-7, and HepG2) showed that antiproliferative effects of saponin extract of puffed ginseng increased with an increase in puffing pressure. Ginseng explosively puffed at 490 kPa had similar saponin constituents and antiproliferative effects as those of red ginseng. Practical Application: The puffing process could provide an alternative mean to produce functional ginseng products, along with a reduction in processing time as compared to traditional red ginseng processing by steam.     

Chemical conversion of ginsenosides in puffed red ginseng

The effects of puffing process on chemical conversion of ginsenosides, extraction yields and crude saponin contents in red ginseng were investigated. To reach a maximum extraction yield, puffed red ginseng took only 8 h, while non-puffed red ginseng required at least 20 h showing extraction yields of 45.7 g solid extract/100 g sample and 44.5 g solid extract/100 g sample, respectively. Extraction yield increased slightly with increasing puffing pressure. Puffed red ginseng showed higher crude saponin contents (201.0-219.0 mg/g extract) than non-puffed one (161.7-189.0 mg/g extract). As the puffing pressure increased, minor ginsenosides (Rg3, F2, Rk1 and Rg5) increased but the contents of major ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) decreased. These results indicated that a puffing process may provide an effective method to reduce the extraction time, improve the extraction yield and increase the crude saponin content of red ginseng.     

Biotransformation of ginsenoside Rd in the ginseng extraction residue by fermentation with lingzhi (Ganoderma lucidum)

Ginseng and lingzhi (Ganoderma lucidum) both are valuable traditional Chinese medicines and have been extensively utilised in functional foods and traditional medicines in many Asian countries. However, massive quantity of ginseng residue is produced after extraction of ginseng which still contains a lot of bioactive compounds such as ginsenosides. The goal of this study was to reuse the American ginseng extraction residue as the fermentation medium of G. lucidum to produce bioactive ginsenoside enriched biotransformation products. The changes of ginsenosides in the fermentation products were analysed during fermentation. Our results showed that after 30 days of fermentation, ginsenoside Rg1, Rd, and compound K (CK) significantly increased, especially Rd, while other ginsenosides (Re, Rb1 and Rc) decreased during fermentation. Ginsenoside Rd is the major ginsenoside in the final fermentation product. Furthermore, the biotransformation of ginsenosides was the major reaction in this fermentation process.