Hepatic Effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, the Signature Component of Traditional Chinese Medicine Heshouwu: Advances and Prospects.

Traditional Chinese medicine Heshouwu, named Polygoni Multiflori Radix in Pharmacopoeia of the People's Republic of China (PPRC, 2020), is derived from the root tuber of Polygonum multiflorum Thunb., Heshouwu or processed Heshouwu is well known for its function in reducing lipids and nourishing the liver. However, increasing cases of Heshouwu-induced hepatotoxicity were reported in recent years. Researchers have begun to study the paradoxical effects of Heshouwu on the liver. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), an abundant functional component of Heshouwu, shows various biological activities, among which its effect on the liver is worthy of attention. This paper reviews the current studies of TSG on hepatoprotection and hepatotoxicity, and summarizes the doses, experimental models, effects, and mechanisms of action involved in TSG's hepatoprotection and hepatotoxicity, aiming to provide insight for future study of TSG and understanding the effects of Heshouwu on the liver. Emerging evidence suggests that TSG ameliorates both pathological liver injury and chemical-induced liver injury by modulating lipid metabolism, inhibiting the inflammatory response and oxidative stress in the liver. However, with the reports of clinical cases of Heshouwu induced liver injury, it has been found that long-term exposure to a high dose of TSG cause hepatocyte or hepatic tissue damage. Moreover, TSG may cause hepatotoxicity by affecting the transport and metabolism of other possible hepatoxic compounds in Heshouwu. Studies indicate that trans-TSG can be isomerized into cis-TSG under illumination, and cis-TSG had a less detrimental dose to liver function than trans- TSG in LPS-treated rats. In brief, TSG has protective effects on the liver, but liver injury usually occurs under highdose TSG or is idiosyncratic TSG-induced liver injury.

Inhibition of CYP3A4 enhances aloe-emodin induced hepatocyte injury.

Aloe-emodin (AE) is a natural hydroxyanthraquinone derivative that was found in many medicinal plants and ethnic medicines. AE showed a wide array of pharmacological activities including anticancer, antifungal, laxative, antiviral, and antibacterial effects. However, increasing number of published studies have shown that AE may have some hepatotoxicity effects but the mechanism is not fully understood. Studies have shown that the liver injury induced by some free hydroxyanthraquinone compounds is associated with the inhibition of some metabolic enzymes. In this study, the CYP3A4 and CYP3A1 were found to be the main metabolic enzymes of AE in human and rat liver microsomes respectively. And AE was metabolized by liver microsomes to produce hydroxyl metabolites and rhein. When CYP3A4 was knocked down in L02 and HepaRG cells, the cytotoxicity of AE was increased significantly. Furthermore, AE increased the rates of apoptosis of L02 and HepaRG cells, accompanied by Ca2+ elevation, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) overproduction. The mRNA expression of heme oxygenase-1 in L02 and HepaRG cells increased significantly in the high-dose of AE (40 µmol/L) group, and the mRNA expression of quinone oxidoreductase-1 was activated by AE in all concentrations. Taken together, the inhibition of CYP3A4 enhances the hepatocyte injury of AE. AE can induce mitochondrial injury and the imbalance of oxidative stress of hepatocytes, which results in hepatocyte apoptosis.

Exploration of components and mechanisms of Polygoni Multiflori Radix-induced hepatotoxicity using siRNA -mediated CYP3A4 or UGT1A1 knockdown liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygoni Multiflori Radix, the dried root of Polygonum multiflorum Thunb., and its processed products have been used as restoratives for centuries in China. However, the reports of Polygoni Multiflori Radix-induced liver injury (PMR-ILI) have received wide attention in recent years, and the components and mechanism of PMR-ILI are not completely clear yet. Our previous studies found that the PMR-ILI was related to the down-regulation of some drug metabolism enzymes (DME). AIM OF THE STUDY: To explore the effect of the inhibition of CYP3A4 or UGT1A1 on PMR-ILI, screen the relevant hepatotoxic components and unveil its mechanism. METHODS: RT-qPCR was used to detect the effects of water extract of Polygoni Multiflori Radix (PMR) and its main components on the mRNA expression of CYP3A4 and UGT1A1 in human hepatic parenchyma cell line L02. High-performance liquid chromatography (HPLC) was employed to detect the content of major components in the PMR. And then, the stable CYP3A4 or UGT1A1 knockdown cells were generated using short hairpin RNAs (shRNA) in L02 and HepaRG cells. Hepatotoxic components were identified by cell viability assay when PMR and its four representative components, 2,3,5,4'-tetrahydroxy stilbene glycoside (TSG), emodin (EM), emodin-8-O-ß-D-glucoside (EG), and gallic acid (GA), acted on CYP3A4 or UGT1A1 knockdown cell lines. The PMR-ILI mechanism of oxidative stress injury and apoptosis in L02 and HepaRG cells were detected by flow cytometry. Finally, the network toxicology prediction analysis was employed to excavate the targets of its possible toxic components and the influence on the metabolic pathway. RESULTS: PMR and EM significantly inhibited the mRNA expression of CYP3A4 and UGT1A1 in L02 cells, while TSG and GA activated the mRNA expression of CYP3A4 and UGT1A1, and EG activated CYP3A4 expression while inhibited UGT1A1 expression. The contents of TSG, EG, EM and GA were 34.93 mg/g, 1.39 mg/g, 0.43 mg/g and 0.44 mg/g, respectively. The CYP3A4 or UGT1A1 knockdown cells were successfully constructed in both L02 and HepaRG cells. Low expression of CYP3A4 or UGT1A1 increased PMR cytotoxicity remarkably. Same as PMR, the toxicity of EM and GA increased in shCYP3A4 and shUGT1A1 cells, which suggested EM and GA may be the main components of hepatotoxicity in PMR. Besides, EM not only inhibited the expression of metabolic enzymes but also reduced the cytotoxicity threshold. EM and GA affected the level of ROS, mitochondrial membrane potential, Ca2+ concentration, and dose-dependent induced hepatocyte apoptosis in L02 and HepaRG cells. The network toxicology analysis showed that PMR-ILI was related to drug metabolism-cytochrome P450, glutathione metabolism, and steroid hormone biosynthesis. CONCLUSION: The inhibition of mRNA expression of CYP3A4 or UGT1A1 enhanced hepatotoxicity of PMR. EM and GA, especially EM, may be the main hepatotoxic components in PMR. The mechanism of PMR, EM and GA induced hepatotoxicity was proved to be related to elevated levels of ROS, mitochondrial membrane potential, Ca2+ concentration, and induction of apoptosis in liver cells.

Shikonin sensitizes A549 cells to TRAIL-induced apoptosis through the JNK, STAT3 and AKT pathways.

BACKGROUND: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, can selectively kill cancer cells with little or no cytotoxicity toward normal human cells and is regarded as a potential relatively safe antitumor drug. However, some cancer cells are resistant to TRAIL-induced apoptosis. Thus, reagents that potentiate TRAIL-induced cytotoxicity are needed. Herein, we investigated whether shikonin, a natural compound from the root of Lithospermum erythrorhizon, can sensitize TRAIL-resistant cells to TRAIL-induced cytotoxicity. RESULTS: The viability of A549 cells, which were resistant to TRAIL, was significantly decreased after treatment with TRAIL followed by shikonin. The underlying mechanisms by which shikonin sensitizes cells to TRAIL-induced cytotoxicity were also examined. Combined treatment with shikonin and TRAIL activated the caspase and JNK pathways, inhibited the STAT3 and AKT pathways, downregulated the expression of Mcl-1, Bcl-2, Bcl-xL, c-FLIP and XIAP and upregulated the expression of Bid. CONCLUSIONS: In conclusion, the results indicated that shikonin sensitized resistant cancer cells to TRAIL-induced cytotoxicity via the modulation of the JNK, STAT3 and AKT pathways, the downregulation of antiapoptotic proteins and the upregulation of proapoptotic proteins.

Hepatotoxicity in Rats Induced by Aqueous Extract of Polygoni Multiflori Radix, Root of Polygonum multiflorum Related to the Activity Inhibition of CYP1A2 or CYP2E1.

The objective of this study is to investigate the relationship between the hepatotoxicity induced by Polygoni Multiflori Radix (PMR, root of Polygonum multiflorum Thunb., He Shou Wu) and the activity of CYP1A2 or CYP2E1 in the rat liver. Levels of rat serum transaminases ALT and AST were not altered but the activity of CYP1A2 or CYP2E1 in the rat liver was significantly inhibited after oral administration of aqueous extract of PMR under the experimental dosage. However, levels of ALT and AST were significantly increased and the activity of CYP1A2 or CYP2E1 was significantly decreased after injection of specific inhibitor for CYP1A2 or CYP2E1 combined with oral administration of aqueous extract of PMR, especially under the repeated treatment over interval times. Liver histopathological observation showed that a moderate liver injury occurred in rats receiving PMR treatment with the activity of CYP1A2 or CYP2E1 inhibited, but there was no significant liver damage in rats receiving PMR treatment or CYP inhibitor alone. These suggested that low level activity of CYP1A2 or CYP2E1 from genetic polymorphism among people might be one of the important reasons for the hepatotoxicity induced by PMR in clinical practice.

Transformation of trollioside and isoquercetin by human intestinal flora in vitro.

The present study was designed to determine the intestinal bacterial metabolites of trollioside and isoquercetin and their antibacterial activities. A systematic in vitro biotransformation investigation on trollioside and isoquercetin, including metabolite identification, metabolic pathway deduction, and time course, was accomplished using a human intestinal bacterial model. The metabolites were analyzed and identified by HPLC and HPLC-MS. The antibacterial activities of trollioside, isoquercetin, and their metabolites were evaluated using the broth microdilution method with berberine as a positive control, and their potency was measured as minimal inhibitory concentration (MIC). Our results indicated that trollioside and isoquercetin were metabolized by human intestinal flora through O-deglycosylation, yielding aglycones proglobeflowery acid and quercetin, respectively The antibacterial activities of both metabolites were more potent than that of their parent compounds. In conclusion, trollioside and isoquercetin are totally and rapidly transformed by human intestinal bacteria in vitro and the transformation favors the improvement of the antibacterial activities of the parent compounds.

[Study on inhibitory effect of water extract of Polygonum multiflorum on CYP1A2 and CYP2E1 enzymatic activities and mRNA expressions in rat liver].

Rats were continuously given different doses of water extract of Polygonum multiflorum (1, 10 g x kg(-1)) for 7 days to prepare liver microsomes. Cocktail in vitro incubation approach and Real-time quantitative PCR technology were used to observe the effect of water extract of P. multiflorum on CYP450 enzymatic activities and mRNA expressions in rat liver. Compared with the blank control group, both 1, 10 g x kg(-1) water extract of P. multiflorum treated groups showed significant inhibitions in CYP2E1 enzymatic activities and mRNA expressions (enzymatic activities of CYP2E1, P < 0.01; mRNA expression of CYP2E1, P < 0.05 in 1 g x kg(-1) group, P < 0.01 in 10 g x kg(-1) group). They revealed a significant increase in the enzymatic activity of CYP3A1 (P < 0.01), but without significant change in mRNA expressions. The 10 g x kg(-1) group showed a significant inhibition in CYP1A2 enzymatic activities and mRNA expressions in rat livers (P < 0.01).

Chromatographic fingerprint analysis of the floral parts of Trollius chinensis.

This study provided a practical procedure, for the first time, to compare the component difference of the floral parts of Trollius chinensis and identify the characteristic peaks of each floral part using the high-performance liquid chromatographic fingerprint technique followed by similarity analysis. The results showed that the constituents of different floral parts exhibited lower similarity than those of the same part. It can be concluded that the procedure established herein is useful for analysis of variability in constituent distribution of herbal drugs, and the components are unevenly distributed in the floral parts of T. chinensis.

Distribution of Two bioactive compounds in flowers of Trollius chinensis.

A comprehensive procedure was established, which combined a high-performance liquid chromatography (HPLC) assay for the simultaneous quantification of 2″-O-ß-L-galactopyranosylorientin (OGA) and veratric acid and a gravimetric analysis for the determination of the mass fraction of the floral parts (calyx, corolla, stalk, stamens and pistils and ovary) of Trollius chinensis, to investigate the distribution and identify the enriched floral part(s) of these two compounds in the flowers. The calculated mean distributions of OGA in calyx, corolla, stamens and pistils, stalk and ovary were 83.62, 7.76, 4.35, 2.92 and 1.35%, respectively, whereas those of veratric acid in the corresponding floral parts were 46.41, 9.01, 18.41, 4.11 and 22.06%, respectively, indicating the uneven and noncorresponding distribution of these two compounds. This study extends the application of the HPLC assay and favors the production of OGA and veratric acid from the flowers of T. chinensis in addition to the benefits of breeding, cultivation and utilization of these flowers.

Contribution evaluation of the floral parts to orientin and vitexin concentrations in the flowers of Trollius chinensis.

AIM: To provide a comprehensive procedure to evaluate the contribution of the floral parts to the yield of the major components from the flowers of Trollius chinensis, to underlay the selective breeding, cultivation, development, and utilization of the flowers. METHODS: Five floral parts from eleven batches of the flowers of T. chinensis were examined by HPLC analysis for the content of orientin and vitexin, and by gravimetric analysis for their respective mass fraction. The contribution of each floral part was calculated using mathematical methods based on the results of the content and mass fraction. Variance analysis was carried out by Kruskal-Wallis H test and PCA method. RESULTS: The calculated mean contributions of calyx, corolla, stamens and pistils, stalk, and ovary to the yield of both orientin and vitexin were 76.99% and 71.93%, 9.60% and 8.33%, 9.21% and 8.10%, 2.17% and 6.62%, and 2.03% and 5.02%, respectively. CONCLUSION: The floral parts contribute unequally to the yield of orientin and vitexin, and the calyx contributes the highest and makes a significant difference compared with any other part.

[Study on the hepatoxicity of polygoni multiflori radix and polygoni multiflori rodix praeparata in rats].

OBJECTIVE: To compare the hepatoxicity of Polygoni Multiflori Radix and Polygoni Multiflori Rodix Praeparata in rats. METHODS: Water extracts of Polygoni Multiflori Radix and Polygoni Multiflori Rodix Praeparata were prepared by concentional decocting method (named as RPWE and PPWE respectively). Both in 30 days and 60 days experiments, SD rats were randomly divided into 5 groups: normal control group, RPWE high dosage group (40 mg/kg x d) and PPWE low dosage group (20 mg/kg x d), PPWE high dosage group (40 mg/kg x d) and PPEW low dosage group (20 mg/kg x d). After daily administration,the general conditions of rats were observed. At the end of the experiments, the levels of ALT and AST in plasma were detected. The liver coefficient was calculated and HE staining was used to observe the changes of liver tissue sections. RESULTS: Both in 30 days and 60 days experiments, the treatment groups rats had hypokinesia and dry fur when compared with control groups, and in a dose-dependent manner. The liver coefficients, levels of ALT and AST of plasma weren't changed significantly when conpared with control groups. The liver had varying degrees of steatosis and inflammation cell infiltration in the treatment groups rats in 60 days experiment. CONCLUSION: Oral administration of Polygoni Multiflori Radix and Polygoni Multiflori Rodix Praeparata for 60 days have hepatoxic in rats.

[Effects of extract of Polygonum multiflorum on cell cycle arrest and apoptosis of human liver cell line L02].

OBJECTIVE: To analyze the chemical constituents of Polygonum multiflorum extract which may cause human liver cell damage and to explore the mechanism. METHODS: Raw and processed Polygonum multiflorum were extracted by 70% ethanol, then raw and processed Polygonum multiflorum water-eluted material (RW and PW), 50% ethanol-eluted material (R50 and P50) and 95% ethanol-eluted material (R95 and P95) were obtained by absorbing through AB-8 macroporous resin, followed by water, 50% ethanol and 95% ethanol elution in order. The water extracts of raw and processed Polygonum multiflorum (RWE or PWE) were obtained by boiling them in water as usual. Normal human liver L02 cells were treated by different concentrations of eluted Polygonum multiflorum materials for different time, and the cell growth inhibition of each group was determined by methylthiazolyldiphenyl-tetrazolium bromide method. The chemical constituents which had a significant cytotoxicity to L02 cells were analyzed by high-performance liquid chromatography (HPLC). Morphological changes of L02 cells were observed by Giemsa staining and cell cycle distribution was observed by flow cytometry. RESULTS: It was found that 95% ethanol-eluted extracts of raw and processed Polygonum multiflorum showed significant growth inhibition on normal human liver L02 cells, while the other components showed no significant inhibition on cell growth. HPLC analysis showed that the main component in 95% ethanol-eluted extract of raw and processed Polygonum multiflorum was emodin at content of (18.53+/-2.96)% and (10.28+/-1.34)% respectively. Cell cycle analysis showed that 95% ethanol-eluted material of Polygonum multiflorum and emodin had a similar significant effect of S phase arrest and all could induce L02 cell apoptosis. CONCLUSION: The main part of Polygonum multiflorum causing liver cell damage is the 95% ethanol-eluted extract, and emodin is one of the important chemical constituents leading to liver cell damage.

[Anti-tumor effect of ethanol extracts from Thymus quinquecostatus Celak on human leukemia cell line].

OBJECTIVE: To screen the anti-tumor fraction of ethanol extracts from Thymus quinquecostatus Celak and investigate its anti-tumor effect on human leukemia cell line. METHODS: Ethyl acetate, n-butanol and acetone fractions were separated from the ethanol extracts of wild Thymus quinquecostatus Celak. Growth inhibiting effects of these extracts on human leukemia cell lines K562 and HL-60 were determined by live cell counting and cell growth curve analysis. The possible anti-tumor mechanism was studied by morphological analysis with norcantharidin as a positive control. RESULTS: Ethyl acetate fraction could significantly inhibit the proliferations of K562 and HL-60 cells, and the inhibiting effect depended on the concentration of ethyl acetate fraction. Ethyl acetate fraction could induce apoptosis of K562 and HL-60 cells. The n-butanol and acetone fractions had no significant inhibiting effect on K562 and HL-60 cells. CONCLUSION: Ethyl acetate fraction is the major anti-tumor fraction in ethanol extracts from Thymus quinquecostatus Celak.

Cholesterol depletion inhibits the degradation of amyloid beta-peptide in rat pheochromocytoma (PC12) cells.

Amyloid beta-peptide (Abeta) is a very important pathogenic factor for Alzheimer's disease (AD). It is a constitutive product of cellular metabolism and occurs in normal biological fluids, such as CSF and plasma. As the steady state levels of all peptides in vivo are a direct consequence of the balance between their anabolism and catabolism, peptide accumulation can arise not only from increased production but also from decreased breakdown. Although increasing evidence indicates that cholesterol is a risk factor for AD development, the exact mechanisms of cholesterol on Abeta metabolism or deposition have remained unclear. Here, we focus our attention on the effect of cholesterol depletion on the degradation of Abeta1-40. Cholesterol depletion of cultured rat pheochromocytoma (PC12) cells was achieved by methyl-beta-cyclodextrin (MbetaCD) extraction. It was found that cholesterol depletion by MbetaCD extraction can suppress Abeta degradation in PC12 cells.

[Original studies on anti-tumor and immunological effect of extracts from Thymus quinquecostatus Celak in mice].

OBJECTIVE: To study the anti-tumor and immunological effect of extracts from Thymus quinquecostatus Celak on mice transplanted S180 tumor cells. METHODS: Different doses of volatile oil and alcohol extracted substances from Thymus quinquecostatus Celak were given to mice bearing S180 tumor for 9 days. Tumor inhibition rates and coefficients of spleen and thymus were determined. RESULTS: Tumor inhibition rates of the groups with alcohol extracts (40 g crude drug.kg(-1).d(-1) and 20 g crude drug.kg(-1).d(-1)) were 51.5% (P<0.01) and 36.4% (P<0.05) respectively, and those of the groups with volatile oil (40 g crude drug.kg(-1).d(-1) and 20 g crude drug.kg(-1).d(-1))were both 39.4% (P<0.05). CONCLUSION: The extracts from Thymus quinquecostatus Celak have anti-tumor activities. The coefficient of spleen in group with alcohol extracts (40 g crude drug.kg(-1).d(-1))was close to normal value, and its coefficient of thymus was between that of the negative control group and the group with cyclophosphamide (0.02 g.kg(-1).d(-1)). The anti-tumor activity of the alcohol extracts was significantly higher than that of the control group and the tumor inhibition rate was depending on drug concentration. Depending on index of immunity,the extracts from Thymus quinquecostatus Celak may have some influences on immunity.