Transformation of ginseng saponins to ginsenoside rh<Subscript>2</Subscript> by acids and human intestinal bacteria and biological activities of their transformants

When ginseng water extract was incubated at 60 degrees C in acidic conditions, its protopanaxadiol ginsenosides were transformed to ginsenoside Rg3 and delta20-ginsenoside Rg3. However, protopanaxadiol glycoside ginsenosides Rb1, Rb2 and Rc isolated from ginseng were mostly not transformed to ginsenoside Rg3 by the incubation in neutral condition. The transformation of these ginsenosides to ginsenoside Rg3 and delta20-ginsenoside Rg3 was increased by increasing incubation temperature and time in acidic condition: the optimal incubation time and temperature for this transformation was 5 h and 60 degrees C resepectively. The transformed ginsenoside Rg3 and delta20-ginsenoside Rg3 were metabolized to ginsenoside Rh2 and delta20-ginsenoside Rh2, respectively, by human fecal microflora. Among the bacteria isolated from human fecal microflora, Bacteroides sp., Bifidobacterium sp. and Fusobacterium sp. potently transformed ginsenoside Rg3 to ginsenoside Rh2. Acid-treated ginseng (AG) extract, fermented AG extract, ginsenoside Rh2 and protopanaxadiol showed potent cytotoxicity against tumor cell lines. AG extract, fermented AG extract and protopanaxadiol potently inhibited the growth of Helicobacter pylori.     

Metabolism of ginsenoside Re by human intestinal microflora and its estrogenic effect

To understand the relationship between the metabolism and biological activity of ginsenoside Re, a main protopanaxatriol saponin in Panax ginseng C. A. MEYER, its metabolic pathway and estrogenic effect by human intestinal microflora were investigated. All human fecal specimens metabolized ginsenoside Re, mainly to ginsenoside Rh1 and ginsenoside F1, via ginsenoside Rg1, with protopanaxadiol as a minor component. Almost all isolated ginsenoside Re-metabolizing intestinal bacteria (GHIB) also metabolized ginsenoside Re, mainly to ginsenosides Rh1 and F1, via ginsenoside Rg1. Alpha-Rhamnosidase and beta-glucosidase, partially purified from the most potent GHIB, Bacteroides JY-6, hydrolyzed ginsenoside Re and ginsenoside Rg1, respectively; however, they did not hydrolyze ginsenosides Rh1 and F1. These findings suggest that the ginsenosides Rh1 and/or F1 may not be suitable substrates of intestinal bacteria, particularly Bacteroides JY-6. The estrogenic effects of ginsenoside Re and its main metabolites, ginsenosides Rg1 and Rh1, were also investigated. Ginsenoside Rh1 showed the greatest estrogenic effect in human breast carcinoma MCF-7 cells. Based on these findings, the estrogenic effect of ginsenoside Re may be expressed by intestinal microflora.     

Ginsenosides Rg3 and Rh2 inhibit the activation of AP-1 and protein kinase A pathway in lipopolysaccharide/interferon-gamma-stimulated BV-2 microglial cells

The anti-inflammatory effect of ginsenosides Rg3 and Rh2, which improves ischemic brain injury induced by middle cerebral artery occlusion, was investigated in lipopolysaccharide (LPS) and IFN-gamma-induced murine BV-2 microglial cells. Ginsenoside Rh2 inhibited the production of NO, with an IC50 value of 17 microM. The inhibitory effect of Rh2 on NO correlates with the decreased protein and mRNA expression of an inducible NO synthase (iNOS) gene. Additionally, ginsenoside Rh2 inhibited the expression of COX-2, pro-inflammatory TNF-alpha and IL-1beta in BV-2 cells induced by LPS/IFN-gamma, while it increased the expression of the anti-inflammatory cytokine IL-10. Electrophoretic mobility shift assays revealed that ginsenoside Rh2 significantly inhibited the LPS/IFN-gamma-induced AP-1 DNA binding activity, while it enhanced the protein binding to CRE sequences. However, it did not affect NF-kappaB binding activity. Thus, the anti-inflammatory effect of Rh2 appears to depend on the AP-1 and protein kinase A (PKA) pathway. The anti-inflammatory effect of ginsenoside Rg3 against LPS/IFN-gamma-activated BV-2 cells was less potent than that of ginsenoside Rh2. These findings suggest that the in vivo anti-ischemic effect of ginsenoside Rg3 may originate from ginsenoside Rh2, which is a main metabolite of ginsenoside Rg3 by intestinal microflora, and that of ginsenoside Rh2 may be due to its anti-inflammatory effect in brain microglia.     

HPLC法同时测定人参北芪片中人参皂苷Rg1和人参皂苷Re的含量

目的建立测定人参北芪片中人参的有效成分人参皂苷Rg1和人参皂苷Re的高效液相色谱（HPLC）测定方法。方法采用ODS-2HYPERSIL柱（250mm×4.6mm5μm）;乙腈-0.1%磷酸溶液（20∶80）为流动相;检测波长为203nm;流速1.0ml/min;柱温35℃。结果人参皂苷Rg1在0.2024～5.0600μg具有良好的线性关系（r=0.9995）,平均回收率为99.0%,RSD为1.04%（n=9）;人参皂苷Re在0.2152～5.3800μg的范围具有良好的线性关系（r=0.9998）,平均回收率为98.2%,RSD为0.70%（n=9）。结论本法快速、准确,可同时测定人参北芪片中人参皂苷Rg1、人参皂苷Re的含量。     

Inhibitory effects of Korean red ginseng and its genuine constituents ginsenosides Rg3, Rf, and Rh2 in mouse passive cutaneous anaphylaxis reaction and contact dermatitis models

The inhibitory effects of the Korean red ginseng (steamed root of Panax ginseng C.A. MEYER, family Araliaceae) saponin fraction (KRGS) and its constituents ginsenosides Rg3, Rf, and Rh2 in mouse passive cutaneous anaphylaxis (PCA) and contact dermatitis models were measured. Orally administered KRGS and its genuine ginsenosides potently inhibited the PCA reaction induced by IgE. However, when these ginsenosides were intraperitoneally administered, ginsenoside Rh2 showed the most potent inhibition. The ginsenoside Rh2 also the most potently inhibited the beta-hexosaminidase release from RBL-2H3 cells induced by IgE with antigen. KRGS administered topically at a dose of 0.1% suppressed ear swelling in an oxazolone-induced mouse contact dermatitis model by 38.8%. Its constituents ginsenosides Rg3, Rf, and Rh2 at a concentration of 0.05% also potently suppressed mouse ear swelling by 47.5%, 34.8%, and 49.9% at 16 d, respectively. These ginsenosides also significantly reduced mRNA expression levels of cyclooxygenase (COX)-2, interleukin (IL)-1beta, tumor necrosis factor-alpha and interferon-gamma induced by oxazolone applied to mouse ears. However, the ginsenosides, except for ginsenoside Rh2, almost did not notably reduce IL-4 levels. The ginsenoside Rh2 also potently inhibited COX-2 and inducible NO synthetase protein expression in liphopolysaccharide-stimulated RAW264.7 cells. Based on these findings, KRGS and its ginsenosides are suggested to improve atopic and contact dermatitis by regulating expression of cytokines.     

人参皂苷Rg3抗肝癌作用研究进展

2018年GLOBOCAN数据显示,肝癌发病例数位居恶性肿瘤第4位,死亡例数位居第2位。中晚期肝癌患者推荐中医中药辅助肝癌的治疗。人参皂苷Rg3是一种四环三萜达玛烷型稀有人参皂苷,存在20（R）和20（S）两种对映异构体。以人参皂苷Rg3单体为主要成分的抗癌新药参一胶囊于2003年上市,主要应用于肺癌、肝癌的辅助治疗。人参皂苷Rg3体内外研究均证实其有良好的抗肝癌活性。人参皂苷Rg3抗肝癌的作用机制是多方面的,包括诱导肝癌细胞凋亡、调控自噬逆转耐药、抑制细胞侵袭及转移、抑制血管生成。本文就人参皂苷Rg3治疗肝癌的临床前及临床研究进展进行综述,以期对后续研究提供参考。     

人参皂苷Rg3与Rh2的研究进展

人参是我国传统的名贵中草药材,人参皂苷是人参的主要有效成分。人参皂苷Rg3,Rh2具有抗疲劳、舒张血管、提高免疫力、抗肿瘤等药理作用。综述了人参皂苷Rg3,Rh2的药理作用及制备方法。     

The effects of ginsenoside Re and its metabolite, ginsenoside Rh1, on 12-O-tetradecanoylphorbol 13-acetate- and oxazolone-induced mouse dermatitis models

The effects of the main constituent ginsenoside Re in ginseng and its metabolite, ginsenoside Rh1, were investigated in 12-O-tetradecanoylphorbol 13-acetate (TPA)- and oxazolone-induced mouse ear dermatitis models. Ginsenoside Rh1 potently suppressed the TPA- and oxazolone-induced swellings as well as mRNA expression levels of cyclooxygenase-2, IL-1beta and TNF-alpha, although these were only weakly inhibited by ginsenoside Re.     

Pharmacokinetics and acetylcholine releasing effects of ginsenoside Rg1 in hippocampus of beta-amyloid model rats

This study aimed at investigating the pharmacokinetics (PK) and acetylcholine (Ach) releasing effects of ginsenoside Rg1 in hippocampus (HIP) of soluble oligomeric assemblies of amyloid beta (ADDLs) induced Alzheimer’s disease (AD) model rats. Extracellular fluid was collected by microdialysis technique. The concentrations of Rg1 and Ach in dialysates were detected by liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. The results showed, after administration, Rg1 was soon detected in the HIP of model rats and extracellular Ach concentrations in the HIP were significantly increased.     

Induction of apoptosis by ginsenoside Rk1 in SK-MEL-2-human melanoma

Ginsenosides are active compounds isolated from Panax ginseng Meyer. Among these ginsenosides, less polar ginsenosides such as ginsenoside Rg3 and ginsenoside Rh2 have been demonstrated to have tumor inhibitory effects because of their cytotoxicity. In this study, we evaluated the apoptotic effects of ginsenoside Rk1 in SK-MEL-2 human melanoma. Ginsenoside Rk1 isolated from red ginseng is one of the novel ginsenosides that shows strong cytotoxicity compared to ginsenoside Rg3 in dose- and time-dependent manners. The results of DNA fragmentation, 4′,6-diamidino-2-phenylindole staining, and flow cytometric analysis are corroborated that ginsenoside Rk1 induced apoptosis in SK-MEL-2 cells. Western blot analysis revealed up-regulation of Fas, FasL, and Bax protein expression and down-regulation of procaspase-8, procaspase-3, mutant p53 and Bcl-2 protein expression. These findings suggest that ginsenoside Rk1 might be a promising compound to induce apoptosis through both extrinsic and intrinsic pathways in SK-MEL-2 cells.     

HPLC-ELSD法测定丹鳖胶囊中人参皂苷Rg1的含量

目的用HPLC-ELSD法测定丹鳖胶囊中人参皂苷Rg1的含量。方法 HPLC-ELSD法,色谱柱为Purospher STAR RP-18e(250mm×4.6mm,5μm),流动相采用水-乙腈梯度变化。检测器为蒸发光散射检测器,漂移管温度105℃,N2流速1.9L/min。结果人参皂苷Rg1进样量在2～10μg范围内线性关系良好(r=0.9995),平均加样回收率为99.5%,RSD为1.63%。结论本法简便、准确,可作为该制剂的质量控制方法。     

HPLC法测定升脉康颗粒中人参皂苷Rg_1的含量

目的建立升脉康颗粒中人参皂苷Rg1含量的测定方法。方法甲醇提取,蒸干,加水溶解,水饱和的正丁醇萃取,正丁醇液以2%氢氧化钠溶液碱洗及水洗后蒸干,甲醇溶解后进样测定。采用高效液相色谱法(HPLC),十八烷基硅烷键合硅胶为填充剂;乙腈-水(23:77)为流动相;检测波长203 nm。理论塔板数按人参皂苷Rg1峰计算应不低于3000。结果通过方法学的系统考察和三批样品的含量测定,建立了本制剂中人参皂苷Rg1的含测方法。人参皂苷Rg1在0.096~0.960 mg.mL-1范围内峰面积与其浓度呈良好的线性关系。回归方程Y=5 964 600X+160 410,r=1.0。测得三批样品的含量范围为4.951~5.398 mg袋/,平均回收率为97.8%,变异系数RSD为1.36%。结论本法灵敏度高,重现性好,专属性强,是控制本品内在质量的较理想含测方法。     

HPLC-ELSD法同时测定复方三七漱口液中三七皂苷R_1、人参皂苷Rg_1、Rb_1的含量

目的建立同时测定复方三七漱口液中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的HPLCELSD法。方法采用DIKMA Diamonsil C18(2)(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-水梯度洗脱,流速1.0 mL/min,柱温30℃,蒸发光散射检测器漂移管温度47℃,载气流速1.5 L/min。结果三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1分别在0.41¹.53、1.551.53、1.55⁵.83、1.585.83、1.58⁵.92μg呈良好线性关系;复方三七漱口液中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的平均回收率(n=9)分别为99.31%、100.30%、99.98%,RSD分别为1.60%、0.56%、0.97%。结论该方法简便、准确,专属性、重复性好,可用于复方三七漱口液的质量控制。     

人参首乌胶囊中人参皂苷Rg1的含量测定

目的建立人参首乌胶囊中人参皂苷Rg1的含量测定方法。方法采用高效液相色谱（HPLC）法。固定相为kromasailC18柱,流动相为乙腈-0.05%磷酸溶液（19：81）,紫外检测波长203nm。结果人参皂苷Rg1在0.0524～0.524mg/ml范围内线性关系良好,r=0.9999（n=5）;平均加样回收率为96.86%（n=9）,RSD=1.19%。结论该方法简便、快捷、准确,可用于人参首乌胶囊中人参皂苷Rg1的含量测定。     

Ginsenoside Rh2 reduces ischemic brain injury in rats

Ginseng was incubated under mildly acidic conditions and its inhibitory effect on a rat ischemia-reperfusion model was investigated. When ginseng was treated with 0.1% hydrochloric acid at 60 degrees C, its protopanaxadiol saponins were transformed to diasteromeric ginsenoside Rg3 and Delta20-ginsenoside Rg3. When the transformed ginseng extract, of which the main component was ginsenosides Rg3, was treated with human intestinal microflora, the main metabolite was ginsenoside Rh2. Orally administered acid-treated ginseng (AG) extract and ginsenoside Rh2 potently protect ischemia-reperfusion brain injury. The ginsenoside Rh2 also inhibited prostaglandin-E2 synthesis in lipopolysaccharide-stimulated RAW264.7 cells, but showed no in vitro antioxidant activity. These results suggest that AG and ginsenoside Rh2 can improve ischemic brain injury.     

Ginsenoside Rh2 induces apoptosisvia activation of caspase-1 and -3 and up-regulation of bax in human neuroblastoma

In human neuroblastoma SK-N-BE(2) cells undergoing apoptotic death induced by ginsenoside Rh2, a dammarane glycoside that was isolated from Panax ginseng C. A. Meyer, caspase-1 and caspase-3 were activated. The expression of Bax was increased in the cells treated with ginsenoside Rh2, whereas Bcl-2 expression was not altered. Treatment with caspase-1 inhibitor, Ac-YVAD-CMK, or caspase-3 inhibitor, Z-DEVD-FMK, partially inhibited ginsenoside Rh2-induced cell death but almost suppressed the cleavage of the 116 kDa PARP into a 85 kDa fragment. When the levels of p53 were examined in this process, p53 accumulated rapidly in the cells treated early with ginsenoside Rh2. These results suggest that activation of caspase-1 and -3 and the up-regulation of Bax are required in order for apoptotic death of SK-N-BE(2) cells to be induced by ginsenoside Rh2, and p53 plays an important role in the pathways to promote apoptosis.     

Pharmacokinetic study of ginsenoside Re with pure ginsenoside Re and ginseng berry extracts in mouse using ultra performance liquid chromatography/mass spectrometric method

Ginsenoside Re is the major ginsenoside in ginseng berry(GB) extract and its pharmacokinetics were studied following the intravenous and oral administration of pure Re or ginseng berry extract in mouse with doses of 10 and 50 mg/kg using ultra performance liquid chromatography mass spectrometric (UPLC/MS) method which can simultaneously determine ginsenoside Re, Rg1 and Rh1 in mouse serum. The serum samples were pretreated by protein precipitation and chromatographic separation was performed on AQUITY UPLC BEH C₁₈ column using gradient elution with the mobile phase of 5 mM ammonium formate and acetonitrile. Analytes and digoxin (I.S.) were analyzed and identified using an electrospray negative ionization mass spectrometry in the selected ion monitoring mode with the linear concentration range of 5.0–5000 ng/mL and lower limits of detection (LLOD) under 2.5 ng/mL. Ginsenoside Re was rapidly cleared from the body with a short half-life (0.2 ± 0.03 h for male and 0.5 ± 0.08 h for female mice after i.v.) and oral absorption was generally poor (F% 0.19–0.28). Notably, GB extract showed a superior oral absorption of ginsenoside Re (F% 0.33–0.75) at equivalent ginsenoside Re dose to pure ginsenoside Re, indicating that GB extract might be a good form for ginsenoside Re intake.     

壳聚糖包覆的人参皂苷Rg3脂质体的制备及质量评价

目的制备壳聚糖包覆的人参皂苷Rg3脂质体,并进行质量评价。方法采用薄膜分散法、逆相蒸发法、注入法制备脂质体,用壳聚糖包覆;以形态、粒径、包封率为指标筛选制备方法。结果薄膜分散法制备的脂质体外形规则、光滑、平均粒径为10.4μm,包封率为46.96%;壳聚糖包覆后外形圆整,平均粒径为11.1μm,包封率为54.79%。结论采用薄膜分散法制备脂质体并用壳聚糖包覆,质量合格。     

人参皂苷Rg1的微生物转化研究

论文首次报道了利用49种微生物菌株对人参皂苷Rg1进行生物转化研究,发现小型丝状真菌黑曲霉(Aspergillus niger 3．1858)与蓝色梨头霉(Absidia coerulea 3．3538)具有转化能力。经正交试验优化转化条件,放大培养,制各得到产物MT1与MT2,经化学与波谱技术鉴定,MT1与MT2为同一产物即人参皂苷Rh1。运用TLCS方法探索了真菌体系中底物人参皂苷Rg1与转化产物人参皂苷Rh1的生物转化动力学曲线。