20(S)-ginsenoside Rg3 enhances glucose-stimulated insulin secretion and activates AMPK

Although Panax ginseng has been widely used in oriental countries for pharmacological effects such as anti-diabetic, anti-inflammatory, adaptogenic and anti-fatigue activities, the active ingredient is not yet fully identified. In our preliminary studies, protopanaxadiol ginsenosides showed the insulin secretion-stimulating activity. In HIT-T15 cells, Rg3 enhanced the insulin secretion in a concentration dependent manner. This effect, however, was almost completely abolished in the presence of diazoxide (K+ channel opener) or nifedipine (Ca2+ channel blocker). Oral glucose tolerance test (OGTT) was also performed using ICR mice and Rg3 suppressed the blood glucose levels from rising by enhancing an insulin secretion at 30 min after administration. From these studies, we may conclude that Rg3 lowered the plasma glucose level by stimulating an insulin secretion and this action was presumably associated with ATP sensitive K+ channel. Next, to explore the hypothesis that ginsenoside Rg3 epimers may exhibit differential effects, glucose-stimulated insulin secretion activity and phosphorylation of AMP-activated protein kinase (AMPK) were compared between 20(S)- and 20(R)-ginsenoside Rg3. 5 microM of 20(S)-Rg3 enhanced the glucose-stimulated insulin secretion by 58% compared to the control, but 20(R)-Rg3 did not show any effect. In C2C12 myotubes, 20(S)- and 20(R)-Rg3 both markedly phosphorylated AMPK and acetyl-CoA carboxylase (ACC), although 20(R)-Rg3 showed a little less effect. Taken together, our results suggest that ginsenoside Rg3 epimers showed differential activities, and 20(S)-Rg3 epimer exhibited the higher pharmacological effects in insulin secretion and AMPK activation than 20(R)-Rg3. The novel characteristics of 20(S)-Rg3 may be a valuable candidate for anti-diabetic agent.     

20(S)-ginsenoside Rh2, a newly identified active ingredient of ginseng, inhibits NMDA receptors in cultured rat hippocampal neurons

Most herbal medicines that are orally administrated have been known to be metabolized before they are absorbed from the gastrointestinal tract. We, therefore, examined the effects of 20(S)-ginsenosides Rb1, Rg1 and Rg3, the three most commonly studied ginsenosides in the central nervous system, and their main metabolites on NMDA receptors using fura-2-based digital imaging and perforated whole-cell patch-clamp techniques. Among the nine ginsenosides tested, 20(S)-ginsenoside Rh2 (20(S)-Rh2) along with 20(S)-ginsenoside Rg3 (20(S)-Rg3) produced the highest inhibitory effect in cultured hippocampal neurons. Although 20(S)-Rg3 and 20(S)-Rh2 selectively targeted NMDA receptors with similar potency, they produced additive effects and seemed to modulate different NMDA receptor regulatory sites. As a competitive antagonist, 20(S)-Rh2 seems to inhibit the receptor via its interaction with polyamine-binding sites, and 20(S)-Rg3 does so using glycine-binding sites. Therefore, these results suggest that the treatment of 20(S)-Rh2, a newly identified active ingredient of ginseng, might be a novel preventive candidate in treating neurodegenerative disorders.     

Mutations of arginine 222 in pre-transmembrane domain I of mouse 5-HT(3A) receptor abolish 20(R)- but not 20(S)-ginsenoside Rg(3) inhibition of 5-HT-mediated ion currents

Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We previously investigated the structure-activity relationship of the ginsenoside Rg(3) stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg(3)) and 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(S)-Rg(3)) in regulating 5-HT(3A) receptor-mediated ion currents (I(5-HT)) expressed in Xenopus oocytes and found that 20(S)-Rg(3) rather than 20(R)-Rg(3) was more stronger inhibitor of I(5-HT). In the present study, we further investigated the effects of 20(R)-Rg(3) and 20(S)-Rg(3) on mouse 5-HT(3A) receptor channel activity after site-directed mutations of 5-HT(3A) receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT(3A) receptor was expressed in Xenopus oocytes, and I(5-HT) was measured using two-electrode voltage clamp technique. In wild-type, both 20(R)-Rg(3) and 20(S)-Rg(3) inhibited I(5-HT) with concentration-dependent and reversible manner. Induction of 5-HT(3A) receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC(50) values for I(5-HT) compared to wild-type but also abolished 20(R)-Rg(3)-induced inhibition of I(5-HT). Those mutations also shifted the IC(50) values by 20(S)-Rg(3) into right direction by 2- to 4-folds compared with wild-type. These results indicate that 5-HT(3A) receptor facilitation differentially affects 20(R)-Rg(3)- and 20(S)-Rg(3)-mediated 5-HT(3A) receptor channel regulation.     

Therapeutic potential of 20(S)-ginsenoside Rg(3) against streptozotocin-induced diabetic renal damage in rats

The inhibitors of advanced glycation endproduct and oxidative stress, as well as N-methyl-d-aspartate (NMDA) receptor antagonists have received considerable interest because of their close association with renoprotective effects. The therapeutic potential of 20(S)-ginsenoside Rg(3) (20(S)-Rg(3)), isolated from Panax ginseng, against streptozotocin-induced diabetic renal damage, was investigated in this study. The diabetic rats received 5, 10, and 20 mg/kg body weight/day of 20(S)-Rg(3) orally via gavage for fifteen consecutive days. The physiological abnormalities such as increases in water intake and urine volume of diabetic rats were significantly decreased by the 20 mg/kg body weight of 20(S)-Rg(3) administration. The elevated serum glucose, glycosylated protein, and thiobarbituric acid-reactive substance levels in diabetic rats were also significantly reduced by the 20(S)-Rg(3) administrations. Moreover, the renal dysfunction of diabetic rats was significantly ameliorated by the 20(S)-Rg(3) administrations in a dose-dependent manner. These beneficial effects on diabetic renal damage were related to the inhibitory effect of 20(S)-Rg(3) against NMDA receptor-mediated nitrosative stress.     

Metabolism of 20(S)- and 20(R)-ginsenoside Rg3 by human intestinal bacteria and its relation to in vitro biological activities

When ginsenoside Rg3 was anaerobically incubated with human fecal microflora, all specimens metabolized ginsenoside Rg3 to ginsenoside Rh2 and protopanaxadiol. The main metabolite was ginsenoside Rh2. 20(S)-ginsenoside Rg3 was quickly transformed to 20(S)-ginsenoside Rh2 or 20(S)-protopanaxadiol in an amount 19-fold that compared with the transformation of 20(R)-ginsenoside Rg3 to 20(R)-ginsenoside Rh2 or 20(R)-protopanaxadiol. Among the bacteria isolated from human fecal microflora, Bacteroides sp., Eubacterium sp., and Bifidobacterium sp. metabolized ginsenoside Rg3 to protopanaxadiol via ginsenoside Rh2. However, Fusobacterium sp. metabolized ginsenoside Rg3 to ginsenoside Rh2 alone. Among ginsenoside Rg3 and its metabolites, 20(S)-protopanaxadiol and 20(S)-ginsenoside Rh2 exhibited the most potent cytotoxicity against tumor cell lines, 20(S)- and 20(R)-protopanaxadiols potently inhibited the growth of Helicobacter pylori, and 20(S)-ginsenoside Rh2 inhibited H+/K+ ATPase of rat stomach.     

Stereospecific effects of ginsenoside Rg3 epimers on swine coronary artery contractions

Previous reports have shown that ginseng saponins, the active ingredients of Panax ginseng, induce relaxation of hormone- or high K(+)-induced blood vessel contraction. We recently demonstrated that 20(R)- and 20(S)-ginsenoside Rg(3) epimers regulate ion channel activities in a stereospecific manner. Here, we examined whether ginsenoside Rg(3) epimers also exhibit differential effects on swine coronary artery contractions induced by high K(+) or 5-HT. We found that treatment with 20(S)- but not 20(R)-ginsenoside Rg(3) caused a potent concentration-dependent, endothelium-independent relaxation of coronary artery contraction induced by 25 mM KCl. However, treatment with both 20(S)- and 20(R)-ginsenoside Rg(3) induced a significant, concentration-dependent relaxation of 3 microM 5-HT-induced coronary artery contractions in intact samples, while only 20(S)-ginsenoside Rg(3) inhibited coronary artery contraction in endothelium-denuded arteries. 20(S)- but not 20(R)-ginsenoside Rg(3) inhibited L-type Ca(2+) channel currents in a dose- and voltage-dependent manner. These results indicate that 20(S)- and 20(R)-ginsenoside Rg(3) epimers might exhibit stereospecific relaxation effects on swine coronary artery contractions caused by high K(+) and 5-HT receptor activation.     

The effects of glycine and L-arginine on heat stability of ginsenoside Rb1

To identify the effects of amino acids on the heat stability of ginsenoside Rb(1) (Rb(1)), Rb(1) was heat-processed at 120 degrees C with or without glycine or L-arginine. Rb(1) was changed into 20(S)-Rg(3), 20(R)-Rg(3), Rk(1), and Rg(5) by heat-processing through glycosyl elimination and epimerization of carbon-20 by SN1 reaction. Similarly, Rb(1) was changed into 20(S)-Rg(3), 20(R)-Rg(3), Rk(1), and Rg(5) when it was heat-processed with the same amount of glycine, but the generated amount of 20(S)-Rg(3) was higher than when Rb(1) was heat-processed without amino acids, and a significant increase in Maillard reaction products (MRPs) was noted. On the other hand, there were no structural changes in Rb(1) and the generation of MRPs when Rb(1) was heat-processed with the same amount of L-arginine. The improved heat stability of Rb(1) brought about by the addition of L-arginine was thought to be closely related to its characteristics of interfering with nonenzymatic glycation and forming hydrogen bonds with Rb(1).     

鳕鱼肉酶解产物对小鼠抗疲劳和体内抗氧化作用

目的：研究鳕鱼肉酶解产物的抗疲劳和体内抗氧化作用。方法：以阿拉斯加狭鳕鱼肉为原料,经菠萝蛋白酶和风味蛋白酶分步酶解,通过离心、喷雾干燥等工艺制得鳕鱼肉酶解产物。测定酶解产物的氨基酸组成;给小鼠灌胃不同剂量的酶解产物,测定小鼠力竭游泳时间,肝糖原含量,血清中乳酸、尿素氮、丙二醛含量以及超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶活力。结果：酶解产物能显著延长小鼠的游泳时间,与空白对照组相比,低、中、高剂量组能够降低小鼠体内乳酸、血尿素氮和丙二醛含量,提高小鼠体内肝糖原含量,且能够提高小鼠体内超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶的活力。结论：酶解产物具有较好的抗疲劳和体内抗氧化作用。     

Hepatoprotective effect of 20(S)-ginsenosides Rg3 and its metabolite 20(S)-ginsenoside Rh2 on tert-butyl hydroperoxide-induced liver injury

To evaluate the hepatoprotective effect of Red Ginseng (RG), we isolated a main constituent 20(S)-ginsenoside Rg3 from RG, and its metabolite 20(S)-ginsenoside Rh2 by human intestinal microflora, and investigated their hepatoprotective activities in tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity of HepG2 cells and mice. When HepG2 cells were treated with t-BHP, its cytotoxicity was significantly increased. 20(S)-Ginsenoside Rh2 potently protected its cytotoxicity, but 20(S)-ginsenoside Rg3 weakly protected it. Intraperitoneally and orally administered 20(S)-ginsenoside Rh2 to t-BHP-injured mice significantly inhibited the increase of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. Orally administered 20(S)-ginsenoside Rg3 also showed the inhibition against the increase of ALT and AST of t-BHP-induced mice. However, intraperitoneally administered 20(S)-ginsenoside Rg3 could not inhibit the elevation of serum ALT and AST activities. These results suggest that 20(S)-ginsenoside Rg3 a main component of RG may be a prodrug for hepatotoxicity.     

黄秋葵提取物抗疲劳的实验研究

目的观察黄秋葵水提物对实验动物的抗疲劳作用,探讨黄秋葵水提物抗疲劳作用的机制。方法以观察小鼠负重力竭游泳时间探讨黄秋葵提取物抗疲劳作用;测定小鼠游泳前后血清尿素的变化、小鼠肝糖原含量、小鼠血乳酸等探讨抗疲劳机制。结果黄秋葵提取物能明显延长小鼠负重游泳时(P<0.05),减少小鼠血清尿素产生(P<0.01),减少小鼠血乳酸含量(P<0.05),对小鼠肝糖原含量无明显影响。结论黄秋葵提取物具有良好的抗疲劳作用。该作用可能与提高小鼠代谢能力和增强应激能力有关。     

人参皂甙Rg3对乳腺癌MCF-7线粒体凋亡途径的影响

目的探讨20(R)–人参皂甙Rg3(SPG-Rg3)对人乳腺癌MCF-7细胞的诱导凋亡作用及其可能机制。方法人乳腺癌细胞MCF-7细胞株分为空白对照组、实验对照组及SPG-Rg3多个浓度组。利用MTT法观察人参皂甙Rg3对MCF-7细胞生长的抑制作用,并计算出IC-50,进一步确定其有效浓度;流式细胞术检测人参皂甙Rg3作用后MCF-7细胞周期的变化;利用AnnexinV-EGFP/PI双染法,检测人参皂甙Rg3诱导MCF-7细胞凋亡情况;免疫细胞化学染色检测MCF-7细胞凋亡与Fas、FasL蛋白表达的关系。结果 SPG-Rg3作用48h的IC-50为244.54μg/mL。流式细胞仪检测Rg3使MCF-7的S期细胞比率明显增加(P<0.01),凋亡率明显增加(P<0.01)。免疫细胞化学显示Rg3能使MCF-7细胞胞浆内细胞色素C增加(P<0.01)。结论 SPG-Rg3能诱导乳腺癌MCF-7细胞凋亡,其机制可能与诱导线粒体释放细胞色素C有关。     

Two new dammarane triterpene glycosides from the rhizomes of Panax notoginseng

Two new dammarane triterpene glycosides named notoginsenosides Rw 1 (1) and Rw 2 (2) were isolated from the rhizomes of Panax notoginseng, together with 20 known compounds including protopanaxadiol (3), protopanaxatriol (4), ginsenosides Rb1 (5), Rd (6), Re (7), Rg1 (8), Rg2 (9), 20-(S)-Rg3 (10), 20-(R)-Rg(3) (11), Rh1 (12), Rh4 (13), Rf (14), 20-O-glucopyranosyl Rf (15), notoginsenosides R1 (16), R2 (17), T5 (18), S (19), T (20), and Fa (21), and koryoginsenoside R1 (22). Based on FABMS, HRFABMS, IR, (1)H, 13C, and 2D-NMR (HSQC, HMBC, and COSY) spectral data, the structures of the new compounds were elucidated as 6-O-beta-d-xylopyranosyl-20-O-beta-d-xylopyranosyl-(1 --> 6)-beta-d-glucopyranosyldammar-24-ene-3beta,6alpha,12beta,20(S)tetraol (1) and 6-O-beta-d-xylopyranosyl-(1 --> 2)-beta-d-glucopyranosyldammar-22-ene-(trans)-3beta,6alpha,12beta,20(S), 25-pentaol (2). Compounds 3, 4, 13, and 22 were isolated from P. notoginseng for the first time.     

海洋生药尖海龙的抗疲劳作用研究

研究海洋生药尖海龙的抗疲劳作用,分析了尖海龙的营养成分;用尖海龙提取的总脂肪和酶水解液进行抗疲劳动物实验。结果表明,尖海龙总脂肪、酶水解液都能显著延长小鼠游泳时间（较空白对照P<0.05）,减少运动引起的血乳酸增加和加速运动后血乳酸含量的降低（P<0.05）,增加小鼠肌糖原和肝糖原的储备（P<0.05）。证明尖海龙的总脂肪和酶水解液都具有抗疲劳作用。实验结果同时证实,尖海龙整体生药的抗疲劳作用更为有效（较空白对照,P<0.01）。     

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.     

缢蛏水解物的抗疲劳作用

目的研究缢蛏水解物(HSC)的抗疲劳作用及机制。方法采用不同浓度HSC溶液灌胃给药,30 d后测定小鼠负重游泳时间及血清尿素氮含量、血浆乳酸浓度、血清乳酸脱氢酶活性、血红蛋白浓度、肝糖原与肌糖原含量等生化指标。结果 HSC可明显延长小鼠负重游泳时间,显著降低运动后小鼠的血清尿素氮含量和血乳酸水平,提高了血红蛋白浓度、肝糖原与肌糖原水平及乳酸脱氢酶的活性。结论 HSC具有良好的抗疲劳作用。     

Modifications of aliphatic side chain of 20(S)-ginsenoside RG3 cause an enhancement or loss of brain Na+ channel current inhibitions

A line of evidence has shown that ginsenoside Rg3 (Rg3) could be one of bioactive ligands in brain Na+ channel regulations. Rg3 exists as stereoisomer of 20(R)- or 20(S)-form. Rg3 consists of three different parts; steroid- like backbone structure, carbohydrate portion, and aliphatic side chain [-CH2CH2CH=C(CH3)2], which is coupled to the carbon-20 of backbone structure. In the previous report, we demonstrated that 20(S)- but not 20(R)-Rg3 and carbohydrate portion of Rg3 play important roles in rat brain NaV1.2 channel regulations. However, little is known about the role of aliphatic side chain coupled to the carbon-20 in brain Na+ channel regulations. In the present study, we prepared Rg3 derivatives by modifying the aliphatic side chain of Rg3, remaining with backbone structure and carbohydrate portion intact, and examined the effects of Rg3 derivatives on Na+ channel activity. We found that reduction of double bond in aliphatic side chain of Rg3 exhibited agonistic actions in Na+ channel current inhibitions by shifting concentration-response curve to leftward by three-fold, whereas deletion, hydroxylation, or oxygenation of aliphatic side chain caused an attenuation or loss of Na+ channel current inhibitions. These results provide evidences that the aliphatic side chain of Rg3 is also involved in Na+ channel regulations and further show a possibility that the aliphatic side chain of Rg3 could be the target of chemical modifications for abolishment or potentiation of Rg3 actions in Na+ channel regulations.     

人参皂苷Rg3立体异构体差异抑制糖尿病调控基因蛋白-PXR机制的分子动力学研究

目的：为进一步了解Rg3对映体的立体化学选择性,对PXR配体结合结构域（PXR ligand-binding domain,PXR-LBD）中20（R/S）-Rg3的结合模式进行建模。方法：使用计算对接,分子动力学（molecular dynamics,MD）和基本动力学分析（essential dynamics analysis,EDA）等技术手段进行建模。结果：PXR中20（S）-Rg3的MM / PB-SA估计结合能大于20（R）-Rg3。两种配合物的RMSFs表明,20（S）-Rg3结合的LBD的迁移率比20（R）型对映体的迁移率降低得多。EDA预测和两个复合物的叠加三维结构都表明20（S）-Rg3在PXR中的结合比20（R）-Rg3更可能与生物学结果一致。结论：以上结果表明,基于目前的模拟结果,PXR中20（S）-Rg3的结合模式比20（R）-Rg3更有可能与生物实验结果吻合。     

炙甘草汤的化学成分研究

目的：对炙甘草汤中的化学成分进行系统研究。方法：利用硅胶柱色谱柱层析和重结晶等方法分离纯化,依据理化性质和波谱数据鉴定化学结构。结果：从炙甘草汤的乙酸乙酯部位得到甘草素（Ⅰ）、异甘草素（Ⅱ）、芒柄花素（Ⅲ）、白桦脂酸（Ⅳ）、香草酸（Ⅴ）,正丁醇部位得到20（R）-人参皂苷Rg3（Ⅵ）。结论：以上化合物均为首次从炙甘草汤中分得,其中化合物Ⅰ～Ⅲ来源于甘草;化合物Ⅳ来源于大枣;化合物Ⅵ来源于人参,化合物Ⅴ未见从复方单味药中分离的报道。     

The angiosuppressive effects of 20(R)- ginsenoside Rg3

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.     

八种海洋生药抗疲劳作用的初步研究

本文通过实验探讨了海洋生物药尖海龙、三斑海马、牡蛎、龟板、刺参、海星黄、螺旋藻、海带的抗疲劳作用。结果表明,这八种生药均能不同程度地延长小鼠负重游泳时间,有效降低游泳后血乳酸含量（P＜0．05或P＜0．01）,证明有推迟运动性疲劳出现和促进疲劳恢复的积极作用。比较实验结果表现,尖海龙抗疲劳作用效果最好。