酶转化三七茎叶总皂苷制备稀有人参皂苷C-K

为了提高三七茎叶总皂苷的利用率和C-K酶转化得率,采用Aspergillus niger sp. G8菌所产酶与Aspergillus niger sp. G4菌所产酶的等体积混合酶(以下简称G8-G4混合酶),研究了酶转化三七茎叶总皂苷进而制备C-K的方法。结果表明,市销的三七茎叶总皂苷中,主要成分是人参二醇类皂苷,其中含量最多的皂苷是Rb3、C-Mx1、Rc和Fc,质量分数分别为26. 38%、14. 28%、12. 73%、10. 25%。G8-G4混合酶的最佳反应条件为45℃、p H 5. 0、底物质量浓度25 g/L、反应24 h。三七茎叶总皂苷经G8-G4混合酶转化后得到4种产物为C-K、C-Mx、Fc、R7,其中C-K的转化得率为32. 7%。     

新型C18核壳结构色谱柱快速分析人参中的 3种皂苷

目的建立高效液相色谱法(high performance liquid chromatography,HPLC)测定人参中人参皂苷Rg_1、Re和Rb_1 3种人参皂苷的含量。方法采用月旭Boltimate~(TM)C_(18)新型核壳色谱柱对3种人参皂苷进行分离,以乙腈-1.0%磷酸水溶液为流动相进行梯度洗脱,采用HPLC进行检测。结果 3种人参皂苷在月旭Boltimate~(TM)C_(18)新型核壳色谱柱能够快速分离,且分离效果较好。3种人参皂苷的在0.4~4.0μg质量浓度范围内呈良好的线性关系,线性相关系数(R~2)分别为0.9998,0.9996和0.9997;Rg_1、Re和Rb_1检出限分别为0.003、0.001和0.002μg/kg;加标回收率为98.3%~101.3%,相对标准偏差RSD分别为0.8%、1.1%和1.5%(n=6)。结论本方法具有快速、柱压低及节省溶剂等优势,可用于人参提取物中人参皂苷的测定。     

液相色谱-四级杆-飞行时间质谱检测人参奶粉中的皂苷类成分

人参山羊奶粉是一种由人参超微粉与奶粉混合而成的乳制品,生产过程中常需要精确控制其中的皂苷成分。本文建立了一种人参奶粉中皂苷类成分的提取分离方法,并结合液相色谱-四级杆-飞行时间质谱(LC-Q-TOF-MS)技术对其中的皂苷单体成分进行了鉴定和定量分析。该提取方法回收率(101.1%~105.3%)和精密度(1.28%)高,稳定性好。通过二级质谱碎裂技术鉴定了奶粉中20种皂苷单体成分,并定量测定了Rg1,Re,Rb1,Rb2,Rc,Rd六种皂苷单体含量。干法制备的奶粉中总皂苷含量(3.44±0.51 mg/g)高于湿法制备获得的人参奶粉(2.38±0.14mg/g)。皂苷单体的测定结果与总皂苷一致,两种奶粉中皂苷单体的测定结果为:Rg1(11.22±3.1μg/g v.s. 8.03±2.9μg/g),Re (22.99±5.5μg/g v.s. 18.91±0.38μg/g),Rb1(7.43±0.41μg/g v.s. 4.83±0.14μg/g),Rb2(65.29±5.32μg/g v.s. 58.21±3.15μg/g),Rc(11.64±1.02μg/g v.s. 8.80±0.92μg/g),Rd(10.09±0.69μg/g v.s.7.81±0.52μg/g)。湿法制备得到的人参奶粉的均匀度较好,两种奶粉提取物中质谱检测未发现明显的皂苷降解峰。本方法可以为含有人参成分的食品分析检测和质量控制提供技术方案。     

不同种类人参及其各部位中皂苷组成和比例的研究

研究了不同种类人参中皂苷含量 ,以及人参不同部位中的皂苷成分及比例。总皂苷含量测定结果为 ,三七参根中含 14 .2 % ,西洋参须中含 10 .1% ,人参根中含 4.4% ,人参头中含 8.6% ,人参皮中含 6.7% ,人参叶中含 7.7% ,人参须中含 9.9%。对西洋参须、三七参根及吉林产的人参头、人参皮、人参叶、人参根、人参须中的总皂苷作TLC检测及薄层扫描表明 ,三七参根中Rg1含量最高 ,西洋参须中Rb1含量最高 ,人参根中Rd的含量可以忽略 ,人参叶中含有特征皂苷F2 ;它们的Rb1/Rg1值分别为 ,西洋参须中 2 2 .2 ,三七参根中 0 .6,人参头中 4.3 ,人参皮中 6.7,人参叶中 0 .2 ,人参根中 2 .4,人参须中 11.9。     

超高效液相色谱法检测6种人参皂苷含量

采用超声提取药食同源植物(人参、西洋参、三七)中原人参三醇皂苷(Rg1、Re、Rf)和原人参二醇皂苷(Rb1、Rc、Rd),建立了超高效液相色谱(UPLC)检测方法。以50%甲醇溶液为提取剂,料液比1∶80(g∶m L),超声时间30 min。采用乙腈和0.05%磷酸水为流动相,梯度洗脱,检测波长为203 nm。该方法在质量浓度5~1 000μg/m L范围内线性良好,6种皂苷的最低检出限在22.5~51.0 mg/kg之间,平均回收率98.1%~105.5%。该方法准确、灵敏度高、重现性好、省时快捷,适合日常、大批量样品的检测。用此方法测定市售人参、西洋参及三七样品,结果表明3类样品中原人参二醇类皂苷含量高于三醇类皂苷,三七样品中Rg1的含量比人参和西洋参高,西洋参中Re含量高于人参和三七样品,而Rf仅在人参样品中检测到。     

RP-HPLC法分析测定人参、西洋参、三七中的皂苷类化合物

利用反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)技术对人参、西洋参和三七不同方法提取物中的8种皂苷进行定量分析。采用乙醇回流、温浸法和超声辅助提取法提取3种药材中的有效成分,并结合高效液相色谱技术进行分析。结果表明:不同提取方法比较,超声辅助法所提取的3种药材中皂苷含量较高;3种五加科人参属的药材中,三七提取物中主要皂苷含量较高,人参提取物中的皂苷种类较多。     

高效液相色谱法测定人参保健酒中11种人参皂苷含量

该研究建立了高效液相色谱法(HPLC)同时测定人参保健酒中11种人参皂苷(人参皂苷Rg1、Re、Rf、Ra2、Rb1、Rc、Ra1、Ro、Rb2、Rb3、Rd)的方法。采用Agilent SB C₁₈色谱柱(4.6 mm×250 mm,5μm),以乙腈-0.1%磷酸为流动相梯度洗脱,流速1.0 m L/min,柱温28℃,人参皂苷的检测波长203 nm,进样量10μL的条件进行测定。结果表明,11种人参皂苷在各自浓度范围内线性关系良好(R2≥0.999 9),检出限在1.52～3.03 mg/L,定量限在4.59～9.18 mg/L,该方法精密度试验、稳定性试验、重复性试验结果相对标准偏差(RSD)均小于5%,平均加标回收率95.39%～101.96%,RSD为0.07%～2.46%。该方法操作简单,准确度高,重复性好,可用于人参保健酒中11种人参皂苷含量测定。     

HPLC测定参苓白术丸中人参皂苷的含量

目的建立高效液相色谱(HPLC)测定参苓白术丸中人参皂苷含量的方法。方法采用Thermo ODS C_(18)色谱柱,以乙腈为流动相A,以水为流动相B,梯度洗脱,流速为1.0 ml/min,检测波长为203 nm。结果人参皂苷Rg_1在0.0402～0.804 mg/ml范围内线性关系良好,r=0.9995(RSD=1.6%,n=5),平均回收率为94.2%(RSD=1.0%,n=5);人参皂苷Re在0.04038～0.8076 mg/ml范围内线性关系良好,r=0.9997(RSD=1.3%,n=5),平均回收率为92.6%(RSD=1.5%,n=6);人参皂苷Rb_1在0.04072～0.8144 mg/ml范围内线性关系良好,r=0.9999(RSD=1.4%,n=5),平均回收率为97.2%(RSD=1.3%,n=6)。结论方法专属性好、准确、灵敏,可用于参苓白术丸中人参成分的定量检测。     

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。     

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。     

保健酒中人参皂甙含量的测定

应用高效液相色谱法和香草醛比色法分别测定保健酒中人参皂甙的含量。高效液相色谱法采用线性梯度洗脱同时测定6种主要人参皂甙Rg1、Re、Rb1、Rc、Rb2、Rd。比色法以香草醛-高氯酸为显色剂,人参皂甙Re为对照品,测定人参总皂甙的含量。两种方法均简便、准确、灵敏度高、重复性好,适用于保健酒中人参皂甙的测定。     

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法测定复方保健酒中人参皂苷和西红花苷的含量

采用高效液相色谱仪建立测定复方保健酒中人参皂苷Rg₁、人参皂苷Rb₁、人参皂苷Rb₂、人参皂苷Rc、西红花苷I、西红花苷II含量的方法。结果表明,最佳测定条件为采用Agilent SB-Aq色谱柱(4.6 mm×250 mm,5μm),以乙腈-水为流动相梯度洗脱,流速1.0 mL/min,柱温30℃,人参皂苷的检测波长203 nm,西红花苷的检测波长为440 nm,进样量10μL。人参皂苷Rg₁、人参皂苷Rb₁、人参皂苷Rb₂、人参皂苷Rc、西红花苷I、西红花苷II在各自浓度范围内线性关系良好(R≥0.999 9),平均加标回收率97.83%～101.72%,精密度试验结果相对标准偏差(RSD)0.43%～1.85%。实验结果表明该方法操作简单,精密度和准确度高,重复性好,可用于复方保健酒中人参皂苷和西红花苷含量测定。     

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。     

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.     

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。     

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.     

Retention of Ginsenosides in Dried Ginseng Root: Comparison of Drying Methods

North American ginseng (Panax quinquefolius) has a long history of use and is currently a commercially reliable natural health commodity. Ginsenosides or triterpene saponins are generally regarded as bioactive constituents for several observed health effects associated with ginseng. North American ginseng was dried using 3 different drying techniques to assess the ginsenoside content of prepared extracts. Drying methods included freeze‐drying (FD), air‐drying (AD), and vacuum microwave‐drying (VMD) of ginseng root. High‐performance liquid chromatography (HPLC) analysis showed that FD ginseng processing gave greater (P≥ 0.05) amounts of the fingerprint ginsenosides Rg1 (28 ± 0.9 mg/g, dry weight) and Re (45 ± 0.1) compared with AD (Rg1 19 ± 0.7, Re 29 ± 0.1) and VMD (Rg1 22 ± 0.8, Re 24 ± 0.1); whereas, VMD produced greater amounts of Rb1 (83 ± 0.1) and Rd (13 ± 0.0) than FD (Rb1 62 ± 0.1, Rd 9 ± 0.1) and AD (Rb1 69 ± 0.1, Rd 5 ± 0.0), respectively. Total ginsenoside content was similar for FD and VMD and was the lowest (P≥ 0.05) for AD. Electrospray mass spectrometry (ESI‐MS) analysis showed a total of 12 compounds detected in FD ginseng compared with 10 compounds in ginseng dried by both VMD and AD. Our results support the fact that FD and VMD drying methods of North American ginseng can improve both extraction efficiency and actual retention of individual ginsenoside in root material.     

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.     

Use of Lactobacillus rossiae DC05 for bioconversion of the major ginsenosides Rb1 and Re into the pharmacologically active ginsenosides C-K and Rg2

Rb1 and Re are the major ginsenosides in protopanaxadiol and protopanaxatriol with contents of 38.89 and 13.34%, respectively. β-Glucosidase-producing food grade Lactobacillus rossiae DC05 was isolated from kimchi using esculin-MRS agar and an enzyme of L. rossiae DC05 was used for bioconversion of the major ginsenosides Rb1 and Re. Strain DC05 showed strong activity in converting ginsenosides Rb1 and Re into the minor ginsenosides compound-K and Rg2, respectively. Within 4 days, 100% of ginsenoside Rb1 was decomposed and converted into C-K, while 85% of Re was decomposed and converted into Rg2 after 6 days of incubation. The biosynthesis rate of ginsenoside C-K was 72.88%, and the biosynthesis rate of Rg2 was 53.94%. Strain DC05 hydrolyzed ginsenosides Rb1 and Re along the pathway Rb1→Rd→F2→CK and the pathway Re→Rg2, respectively. The optimum temperature and pH of the enzyme were 30°C and 7.0, respectively.