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。     

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。     

超高效液相色谱-四极杆静电场轨道阱高分辨质谱法测定米炒人参炮制前后皂苷成分及其裂解规律的研究

目的 建立超高效液相色谱-四极杆静电场轨道阱高分辨质谱法（ultra performance liquid chromatography-quadrupole-orbitrap-mass spectrometry, UPLC-Q-Orbitrap-MS）检测分析方法。方法 采用Supelco C18色谱柱,以乙腈-0.1%甲酸水溶液梯度洗脱,应用电喷雾离子源（ESI electro-spray ionization）,负离子全扫描模式采集一、二级质谱数据,扫描范围为150~2000 m/z。结合质谱数据库及相关文献信息,运用X Calibur2.2软件对米炒人参中皂苷类成分进行鉴定。以6种人参皂苷Re、Rg1、Rb1、Rc、Rb2、Rb3进行模拟炮制,确定皂苷类成分裂解产物,明确皂苷成分的裂解规律。结果 从人参中检测出14个成分,鉴定出13种人参皂苷成分;米炒人参中检测出23个成分,鉴定出20种人参皂苷成分。通过比较人参米炒前后的皂苷类成分,发现米炒人参中存在人参中未检测到的8种稀有人参皂苷20(S)-Rg2、20(S)-Rh1、20(R)-Rh1、F2、20(S)-Rg3、20(R)-Rg3、20(S)-Rs3、20(R)-Rs3。模拟炮制结果表明,人参皂苷Re脱去C-20糖基,转化为稀有人参皂苷20(S)-Rg2;人参皂苷Rg1脱去C-20位糖基,转化为稀有人参皂苷20(S)-Rh1、20(R)-Rh1;人参皂苷Rb1、Rb2、Rb3、Rc脱去C-20或C-3位糖基,转化为稀有人参皂苷20(S)-Rg3、20(R)-Rg3或F2。结论 人参经米炒后,稀有人参皂苷成分增加,产生的稀有皂苷为原型皂苷发生苷键裂解而获得,模拟炮制可作为其裂解规律研究的有效方法。     

基于酿酒酵母体系的人参皂苷抗衰老活性筛选及评价

以酿酒酵母作为模式生物对人参皂苷进行抗衰老活性筛选并对其活性展开初步探讨。基于模式菌酿酒酵母（Saccharomyces cerevisiae BY4742）的生长曲线筛选出人参皂苷的最适给药浓度,在此基础上,根据酵母的生长曲线和超氧化物歧化酶SOD活性筛选八种人参皂苷单体Rb1、Rb2、Rg1、Rg2、Rg3、Rh1、Rh2、Rd的抗衰老作用,找出效果最佳的一种单体皂苷,再通过抗氧化指标检测及细胞形态变化分析,初步探究人参皂苷的抗衰老作用。同时提取酿酒酵母蛋白进行蛋白组学分析,确定具有显著差异的蛋白,结合GO富集分析相关生物学分析,对差异蛋白的功能通路、属性和代谢通路等进行分析研究。结果表明,人参皂苷的最适给药浓度是180 μg/mL,常用的八种单体皂苷中人参皂苷Rg1具有明显的抗衰老作用,能延缓酵母进入衰亡期。人参皂苷Rg1可以不同程度提高酵母细胞内的抗氧化酶:超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）活性,减少活性氧（ROS）含量和丙二醛（MDA）含量。蛋白组学分析结果显示人参皂苷延缓酿酒酵母衰老可能与14个显著差异蛋白有关,同时与细胞代谢有密切关系。     

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。     

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。     

天冬氨酸降解人参二醇组皂苷及其美拉德反应产物的抗氧化活性

以天冬氨酸为催化剂,在高温蒸煮条件下,研究了天冬氨酸对人参二醇组皂苷的降解和美拉德反应的影响 以及美拉德反应产物的抗氧化活性。结果表明：天冬氨酸能使人参二醇组皂苷降解成稀有皂苷20S-Rg3、20R-Rg3、 Rk1和Rg5,其转化途径为人参二醇组皂苷→20S-Rg3/20R-Rg3→Rk1/Rg5。人参皂苷20R-Rg3和Rg5的质量浓度随着 温度的升高而升高,20S-Rg3和Rk1的质量浓度随着温度的升高而降低。抗氧化实验结果表明：随着蒸煮温度的升 高,天冬氨酸与人参二醇组皂苷反应产物对1,1-二苯基-2-三硝基苯肼自由基的清除作用明显增强（P＜0.01）,其抗 氧化作用的主要物质为天冬氨酸与人参皂苷上水解产生的糖发生反应生成的美拉德反应产物。本研究对开发绿色环 保型人参稀有皂苷保健食品具有参考价值和意义。     

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。     

稀有人参皂苷compound K转化菌株的筛选及转化条件优化

从22种植物病原真菌中筛选出6种能够转化原人参二醇型皂苷为稀有人参皂苷compound K（C-K）的真菌,其中转化效率最高的1.91号真菌经形态学鉴定为卵形孢霉属（Oospora Wallr.）真菌。1.91号真菌生物转化人参皂苷Rb1和Rb2的途径分别为:Rb1→Rd→F2→C-K,Rb2→C-O→C-Y。1.91号真菌生物转化人参皂苷Rb1为C-K的最佳条件为:最佳底物添加时间是孢子预培养12h后,转化最适pH5.0～6.0,转化最适温度是45℃。本研究为稀有人参皂苷C-K的工业制备奠定了一定的基础。      

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.     

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.     

人参酶解物中的皂苷成分及其对髓源抑制性细胞的作用研究

本文建立了同时测定人参酶解物中12种人参皂苷的高效液相色谱检测方法,探索人参提取物及酶解物主要皂苷成分差异,并以酶解前后的人参提取物作为对象,探讨其对髓源抑制性细胞(Myeloid derived suppressed cell,MDSC)的影响。人参提取物和酶解物的总皂苷含量差异不明显,而酶解物中12种人参皂苷和稀有皂苷含量(Rh1、F1、F2、Rg3、CK和Rh2)均显著高于人参提取物;稀有皂苷含量增加了4.48倍,尤其是酶解后转化生成了大量的F2和少量的F1、CK和Rh2等稀有皂苷。人参提取物及酶解物均能显著抑制MSC2细胞增殖和降低结肠癌荷瘤小鼠脾脏中的MDSC细胞比例。其中人参酶解物效果更佳,相对人参提取物,对MSC2细胞增殖抑制率提高30.00%和MDSC细胞比例降低40.50%。人参酶解物含有丰富的稀有皂苷,具有更高的生物活性,能够有效改善肿瘤微环境,从而加强了抗肿瘤能力。     

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.     

Enhancement of low molecular weight ginsenosides from low-quality ginseng through ultra-high-pressure and fermentation processes

This study aimed to analyze the conversion pattern of high to low molecular weight ginsenosides in low-quality ginseng during lactic acid fermentation associated with an ultra-high-pressure process. It was found that the relative quantities of various low molecular weight ginsenosides were increased by 20 min of pressure treatment at 500 MPa following fermentation with Bifidobacterium longum. Specifically, after ultra-high-pressure extraction, the triol-type, low molecular weight Rg2 was the most abundant ginsenoside, at 1.213 mg/g. However, when low-quality ginseng was fermented, the concentrations of diol-type, low molecular weight ginsenosides (e.g., Compound-K (CK), Rh2, and Rg3) largely increased to 1.52, 1.241, and 0.947 mg/g, respectively. These data indicate that high molecular weight ginsenosides in ginseng could be broken down by two different hydrolysis mechanisms. In the fermentation process, the β-1,2 and β-1,4 glycosidic bonds in high molecular weight ginsenosides such as Re, Rc, and Rb1 were hydrolyzed to diol-type, low molecular weight ginsenosides by the β-glucosidase enzyme of the lactic acid bacterium. Meanwhile, the physical energy of the ultra-high-pressure process specifically hydrolyzed relatively weak bonds of the sugars in high molecular weight ginsenosides such as Re to form the low molecular weight ginsenoside Rg2. Rg2, Rg3, Rh2, and CK increased to 2.043, 1.742, 1.834, and 2.415 mg/g, respectively, possibly due to a synergistic effect of combining both processes. Therefore, low molecular weight ginsenosides with higher biological activities than high molecular weight ginsenosides can be selectively obtained from low-quality ginseng using both fermentation and ultra-high-pressure processes.     

高效液相色谱法测定人参酒中人参单体皂苷的研究

建立以NH2基键合相色谱柱、甲醇-异丙醇（60：40V/V）为流动相、配合二极管阵列检测器测定人参酒中单体皂苷Rg1、Re、Rb1的方法。三种皂苷的检出限分别为120ng、167ng和297ng，实际测定了不同批号和不同品种人参酒样品中人参单体皂苦的含量。样品预处理简便、快捷、回收率高，分析方法灵敏、准确、重现性好，测定结果令人满意。