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 R(c) by human intestinal bacteria and its related antiallergic activity

When ginsenoside R(c) was anaerobically incubated with human fecal microflora, all specimens metabolized ginsenoside R(c) to compound K and protopanaxadiol. The main metabolite was compound K. Among the bacteria isolated from human fecal microflora, most bacteria, such as Bacteroides sp., Eubacterium sp., and Bifidobacterium sp. potently transformed ginsenoside R(c) to compound K. Bifidobacterium K-103 and Eubacterium A-44 transformed it to compound K via ginsenoside R(d) and Bacteroides HJ-15 and Bifidobacterium K-506 metabolized to compound K via ginsenoside Mb, which was isolated as a new metabolite (M.W. 940[+Na]). Among ginsenoside R(c) and its metabolites, compound K exhibited the most potent antiallergic activity on the IgE-induced RBL cell line as well as potent cytotoxic activity against tumor cell lines.     

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.     

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.     

Constitutive beta-glucosidases hydrolyzing ginsenoside Rb1 and Rb2 from human intestinal bacteria

When ginsenoside Rb1 and Rb2 were anaerobically incubated with human intestinal microflora, these ginsenosides were metabolized to 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (compound K) and 20(S)-protopanaxadiol. Several kinds of intestinal bacteria hydrolyzed these ginsenosides. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp., which more potently hydrolyzed gentiobiose than sophorose, metabolized ginsenoside Rb1 to compound K via ginsenoside Rd rather than gypenoside XVII. However, Fusobacterium K-60, which more potently hydrolyzed sophorose than gentiobiose, metabolized to compound K via gypenoside XVII. Ginsenoside Rb2 was also metabolized to compound K via ginsenoside Rd or compound O by human intestinal microflora. Eubacterium sp., Streptococcus sp. and Bifidobacterium sp. metabolized ginsenoside Rb2 to compound K via ginsenoside Rd rather than compound O. Fusobacterium K-60 metabolized ginsenoside Rb2 to compound K via compound O.     

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.     

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.     

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.     

Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer

Three new dammarane-type triterpene ginsenosides, together with six known ginsenosides, were isolated from the leaves of Panax ginseng C.A. Meyer. The new saponins were named as ginsenoside Rh??, ginsenoside Rh??, and ginsenoside Rh??. Their structures were elucidated as (20S)-3β,6α,12β,20-tetrahydroxydammara-25-ene-24-one 20-O-β-d-glucopyranoside (1), (20S)-3β,12β,20,24,25-pentahydroxydammarane 20-O-β-d-glucopyranoside (2), and (20S,23E)-3β,12β,20,25-tetrahydroxydammara-23-ene 20-O-β-d-glucopyranoside (3) on the basis of 1D and 2D NMR experiments and mass spectra. The known ginsenosides were identified as ginsenoside M(?cd), ginsenoside Rg?, ginsenoside Rb?, gypenoside XVII, gypenoside IX, and 20-(E)-ginsenoside F?.     

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。     

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.     

A ginsenoside metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, triggers apoptosis in activated rat hepatic stellate cells via caspase-3 activation

We investigated the apoptotic effects of the protopanaxadiol ginsenosides, Rb (1) and Rb (2), and their intestinal bacterial metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1), and of the protopanaxatriol ginsenoside, Rg (1), and its intestinal bacterial metabolite, 20(S)-protopanaxatriol, in activated rat hepatic stellate cells (HSCs) transformed by Simian virus 40 (T-HSC/Cl-6). As HSCs play a central role in liver fibrosis, agents that selectively induce apoptosis of HSCs could be used to treat this disease. Apoptosis was measured using cell viability tests, DNA fragmentation analysis, and immunoblot analysis of poly(ADP-ribose) polymerase cleavage. M1 (40 microM for 24 h) significantly induced apoptosis in activated rat HSCs. M1 induced apoptosis in a dose-dependent manner as shown by DNA fragmentation, an increased population of cells in the sub-G1 phase, and reduced mitochondrial transmembrane potential. M1 induced caspase-3 activity in a dose- and time-dependent manner. A specific inhibitor of caspase-3 prevented induction of apoptosis by M1 as shown by DNA fragmentation analysis. It is concluded that M1 induces apoptosis in T-HSC/Cl-6 cells via caspase-3 activation.     

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.     

Ginsenoside 20(S)-protopanaxadiol inhibits the proliferation and invasion of human fibrosarcoma HT1080 cells

Ginsenoside 20(S)-protopanaxadiol, one of metabolites of ginseng saponins, has been well characterized to possess the pleiotropic anticancer capabilities in several cancer cell lines. The object of this study was to investigate the effects of ginsenoside 20(S)-protopanaxadiol on the invasion in vitro and the expression of matrix metalloproteinase-2 in human fibrosarcoma HT1080 cells in absence of cytotoxicity. Our results showed that ginsenoside 20(S)-protopanaxadiol exerted a concentration-dependent inhibitory effect on the proliferation of HT1080 cells (IC50 was 76.78+/-2.24 microM, 48 hr). Treatment with 20(S)-protopanaxadiol significantly declined the invasive capacity of HT1080 cells compared to the control cells (P<0.01) in the in vitro invasion assay. Further analysis with gelatin zymography and western blotting revealed that both the activity and the expression of matrix metalloproteinase-2 decreased dramatically in a concentration-dependent manner (P<0.01). Taken together, these results indicated that ginsenoside 20(S)-protopanaxadiol is able to inhibit the invasiveness of HT1080 cells significantly in vitro and this action may be primarily due to down-regulating the expression of matrix metalloproteinase-2. Ginsenoside 20(S)-protopanaxadiol, a metabolite of ginseng, may be applied as a potential therapeutic agent in the prevention and treatment of cancer.     

两个新的20（S）-原人参二醇油酸酯衍生物

由20（S）-原人参二醇制得两个未见文献报道的新的20（S）-原人参二醇油酸酯衍生物，命名为12-油酰基-20(S)-原人参二醇（1）和3，12-二油酰基-20(S)-原人参二醇（2）。     

RP-HPLC法同时测定人参提取物转化产物中20(S)、20(R)-人参皂苷Rg2和20(S)、20(R)-人参皂苷Rh1的含量

目的建立同时测定人参提取物转化产物中20(S)、20(R)-人参皂苷Rg2和20(S)、20(R)-人参皂苷Rh1含量的方法。方法采用RP-HPLC法。色谱柱为迪马C18柱(4.6 mm×250 mm,5μm),流动相为乙腈-水梯度洗脱,检测波长为203 nm,流速为1.0 mL.min-1,柱温为30℃。结果人参提取物转化产物中20(S)、20(R)-人参皂苷Rg2和20(S)、20(R)-人参皂苷Rh1质量浓度分别在4.0~80 mg.L-1、2.8~56 mg.L-1、3.2~64 mg.L-1、2.4~48 mg.L-1内与峰面积呈良好的线性关系,相关系数分别为0.999 8、0.999 8、0.999 1、0.999 7,回收率分别为100.3%、98.1%、98.0%、99.9%,回收率的RSD值分别为3.1%、2.7%、4.7%、4.0%。结论方法准确、可靠、重现性好,为人参提取物转化产物的质量控制提供参考依据。     

Inhibitory effect of ginsenoside Rg5 and its metabolite ginsenoside Rh3 in an oxazolone-induced mouse chronic dermatitis model

The effect of a main constituent ginsenoside Rg5 isolated from red ginseng and its metabolite ginsenoside Rh3 in a chronic dermatitis model was investigated. Ginsenosides Rg5 and Rh3 suppressed swelling of oxazolone-induced mouse ear contact dermatitis. These ginsenosides also reduced mRNA expressions of cyclooxygenase-2, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. The inhibition of ginsenoside Rh3 was more potent than that of ginsenoside Rg5. These findings suggest that ginsenoside Rh3 metabolized from ginsenoside Rg5 may improve chronic dermatitis or psoriasis by the regulation of IL-1beta and TNF-alpha produced by macrophage cells and of IFN-gamma produced by Th cells.     

20(R)-人参皂苷Rg3人体药代动力学研究

目的　研究 2 0 (R) 人参皂苷Rg3(GRg3)人体药代动力学。 方法　高效液相色谱 -紫外检测法。结果8名健康志愿者单剂量口服 3 2mg·kg-1GRg3 ,其药时曲线符合口服吸收有滞后时间的二房室模型 ,Tmax为 (0 6 6± 0 10 )h ,Cmax为 (16± 6 )ng·mL-1,T1/ 2α为 (0 46± 0 12 )h ,T1/ 2 β为 (4 9± 1 1)h ,T1/ 2 (Ka) 为 (0 2 8± 0 0 4)h ,AUC0 -∞ 为 (77± 2 6 )ng·mL-1·h ;6名健康志愿者单剂量口服 0 8mg·kg-1GRg3 ,由于血药浓度低 ,可测数据点少 ,未进行模型模拟 ;两组给药剂量与相应Cmax实测值比较 ,二者成正比关系。结论　本品口服吸收快 ,消除也较快 ,但血药浓度很低。在所试剂量范围内 ,GRg3属一级动力学吸收、消除过程。     

Natural products from ginseng inhibit [3H]batrachotoxinin A 20-alpha-benzoate binding to Na+ channels in mammalian brain

A [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) binding assay was used to investigate the interaction of two ginseng aglycones (20(S)protopanaxadiol and 20(S)protopanaxatriol) and Rh(2) (a monoglucoside of 20(S)protopanaxadiol) with voltage-gated sodium channels in mouse brain. All compounds inhibited the binding of [(3)H]BTX-B and IC(50)s were established at 42 microM (20(S)protopanaxadiol), 79 microM (20(S)protopanaxatriol) and 162 microM (Rh(2)). Scatchard analysis confirmed that 20(S)protopanaxadiol and Rh-2 reduced the B(max) of [(3)H]BTX-B binding while Rh(2) also increased the K(d). At IC(50) concentrations and above, 20(S)protopanaxadiol and Rh(2) increased the dissociation of the [(3)H]BTX-B:sodium channel complex above that produced by a saturating concentration of veratridine, but failed to reduce the rate of association of [(3)H]BTX-B with sodium channels. Reversal of the inhibition of [(3)H]BTX-B binding by 20(S)protopanaxadiol and Rh(2) occurred slowly. We conclude that the 20(S)protopanaxadiol and the less potent inhibitor Rh(2) destabilize BTX-B-activated sodium channels through non-covalent allosteric modification of neurotoxin binding site 2.