tetrahydroxystilbene-2-O-β-D-glucoside Research Articles

Tetrahydroxy stilbene glucoside rejuvenates aging hematopoietic stem cells with predilection for lymphoid differentiation via AMPK and Tet2

IntroductionAging of hematopoietic stem cells (HSCs) has emerged as an important challenge to human health. Recent advances have raised the prospect of rejuvenating aging HSCs via specific medical interventions, including pharmacological treatments. Nonetheless, efforts to develop such drugs are still in infancy until now. ObjectivesWe aimed to screen the prospective agents that can rejuvenate aging HSCs and explore the potential mechanisms. MethodsWe screened a set of natural anti-aging compounds through oral administration to sub-lethally irradiated mice, and identified 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) as a potent rejuvenating agent for aging HSCs. Then naturally aged mice were used for the follow-up assessment to determine the HSC rejuvenating potential of TSG. Finally, based on the transcriptome and DNA methylation analysis, we validated the role of the AMP-activated protein kinase (AMPK)-ten-eleven-translocation 2 (Tet2) axis (the AMPK-Tet2 axis) as the underlying mechanisms of TSG for ameliorating HSCs aging. ResultsTSG treatment not only significantly increased the absolute number of common lymphoid progenitors (CLPs) along with B lymphocytes, but also boosted the HSCs/CLPs repopulation potential of aging mice. Further elaborated mechanism research demonstrated that TSG supplementation restored the stemness of aging HSCs, as well as promoted an epigenetic reprograming that was associated with an improved regenerative capacity and an increased rate of lymphopoiesis. Such effects were diminished when the mice were co-treated with an AMPK inhibitor, or when it was performed in Tet2 knockout mice as well as senescent cells assay. ConclusionTSG is effective in rejuvenating aging HSCs by modulating the AMPK- Tet2 axis and thus represents a potential candidate for developing effective HSC rejuvenating therapies.

THSG alleviates cerebral ischemia/reperfusion injury via the GluN2B–CaMKII–ERK1/2 pathway

BackgroundThe potential therapeutic targeting of PINK1-PARK2-mediated mitophagy against cerebral ischemia/reperfusion (CI/R) injury involves the pathophysiological processes of neurovascular unit (NVU) and is closely associated with N-methyl-D-aspartate receptors (NMDARs) commonly expressed in NVU. 2,3,5,4′-Tetrahydroxy-stilbene-2-O-β-D-glucoside (THSG), a compound derived from the traditional Chinese medicine Polygonum multiflorum Thunb., has demonstrated notable neuroprotective properties against CI/R injury. However, it remains unclear whether THSG exerts its protective effects through GluN2B related PINK1/ PARK2 pathway. PurposeThis study aims to explore the pharmacological effects of THSG on alleviating CI/R injury via the GluN2B–CaMKII–ERK1/2 pathway. MethodsTHSG neuroprotection against CI/R injury was studied in transient middle cerebral artery occlusion/reversion (tMCAO/R) model rats and in oxygen and glucose deprivation/ reoxygenation (OGD/R) induced neurons. PINK1–PARK2-mediated mitophagy involvement in the protective effect of THSG was investigated in tMCAO/R rats and OGD/R-induced neurons via THSG and 3-methyladenine (3-MA) treatment. Furthermore, the beneficial role of GluN2B in reperfusion and its contribution to the THSG effect via CaMKII–ERK1/2 and PINK1–PARK2-mediated mitophagy was explored using the GluN2B-selective antagonist Ro 25–6981 both in vivo and in vitro. Finally, the interaction between THSG and GluN2B was evaluated using molecular docking. ResultsTHSG significantly reduced infarct volume, neurological deficits, penumbral neuron structure, and functional damage, upregulated the inhibitory apoptotic marker Bcl-2, and suppressed the increase of pro-apoptotic proteins including cleaved caspase-3 and Bax in tMCAO/R rats. THSG (1 μM) markedly improved the neuronal survival under OGD/R conditions. Furthermore, THSG promoted PINK1 and PARK2 expression and increased mitophagosome numbers and LC3-II–LC3-I ratio both in vivo and in vitro. The effects of THSG were considerably abrogated by the mitophagy inhibitor 3-MA in OGD/R-induced neurons. Inhibiting GluN2B profoundly decreased mitophagosome numbers and OGD/R-induced neuronal viability. Specifically, inhibiting GluN2B abolished the protection of THSG against CI/R injury and reversed the upregulation of PINK1–PARK2-mediated mitophagy by THSG. Inhibiting GluN2B eliminated THSG upregulation of ERK1/2 and CaMKII phosphorylation. The molecular docking analysis results demonstrated that THSG bound to GluN2B (binding energy: –5.2 ± 0.11 kcal/mol). ConclusionsThis study validates the premise that THSG alleviates CI/R injury by promoting GluN2B expression, activating CaMKII and ERK1/2, and subsequently enhancing PINK1–PARK2-mediated mitophagy. This work enlightens the potential of THSG as a promising candidate for novel therapeutic strategies for treating ischemic stroke.

A new application of Elasticnet regression based near-infrared spectroscopy model: Prediction and analysis of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-D-glucoside and moisture in Polygonum multiflorum

This study constructed a near-infrared spectroscopy (NIRS) quantitative analysis model of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) and Polygonum multiflorum's water content based on Elasticnet regression (ER). NIRS data, TSG and the moisture content data sets of 117 samples of P. multiflorum were obtained by using NIR scanning and high-performance liquid chromatography analysis (HPLC) methods. MSE was used to determine the hyperparameters α and λ of Elasticnet. The optimal α values of TSG and water content were 0.83 and 0.01, and the optimal λ values were 0.03 and 1.13, respectively. By preprocessing the original spectral data, the NIRS model was established based on the source data set using ER, compared with the model established by PLS and its optimization algorithm. The results show that the ER model has good performance compared with other methods, with R2c value was 0.9879, RMSEC value was 0.09, R2p value was 0.9784, RMSEP value was 0.13 and RPD value was 6.8. In addition, according to the properties of Elasticnet's variable selection, we speculate that the final coefficients of spectral features may indirectly reflect the spectral features of different components. The results showed better prediction accuracy and superior performance. This is a much more reliable method used to forecast the content of effective components in plants.

Chemical Profiling of Shen-Wu-Yi-Shen Tablets Using UPLC-Q-TOF-MS/MS and Its Quality Evaluation Based on UPLC-DAD Combined with Multivariate Statistical Analysis.

Shen-Wu-Yi-Shen tablets (SWYST) is a traditional Chinese medicine prescription used for treating chronic kidney disease (CKD). This study aims to characterize the constituents in SWYST and evaluate the quality based on the quantification of multiple bioactive components. SWYST samples were analyzed with ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and a data-processing strategy. As a result, 215 compounds in SWYST were unambiguously identified or tentatively characterized, including 14 potential new compounds. Meanwhile, strategies based on characteristic fragments for rapid identification were summarized, indicating that the qualitative method is accurate and feasible. Notably, the glucose esters of laccaic acid D-type anthraquinone were first found and their fragmentation patterns were described by comparing that of O-glycoside isomers. Besides, based on comparisons of the cleavage ways of mono-acyl glucose with different acyl groups or acylation sites, differences in fragmentation pathways between 1,2-di-O-acyl glucose and 1,6-di-O-acyl glucose were proposed for the first time and verified by reference substances. In addition, a validated UPLC-DAD was established for the determination of 11 major bioactive components related to treatment of CKD (albiflorin, paeoniflorin, 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG), 1-O-galloyl-2-O-cinnamoyl-β-d-glucose, emodin-8-O-β-d-glucoside, chrysophanol-O-β-d-glucoside, aloe-emodin, rhein, emodin, chrysophanol and physcion). Moreover, TSG and 1-O-galloyl-2-O-cinnamoyl-β-d-glucose were found as the quality markers related to the origins of SWYST based on multivariate statistical analysis. Conclusively, the findings in this work provide a feasible reference for further studies on quality research and mechanisms of action in treating CKD.

Characterization of the physiological parameters, effective components, and transcriptional profiles of Polygonum multiflorum Thunb. Under pH stress

Polygonum multiflorum Thunb. is a traditional Chinese medicine with extensive distribution and robust adaptability, but comprehensive research on its acid and alkali resistance is presently lacking. This study aimed to analyze the effects of 5 months of continuous pH stress on the physiological and photosynthetic parameters of P. multiflorum, and the content of effective components. Results revealed that pH stress significantly influenced the normal growth, physiological functions, and photosynthetic indicators of P. multiflorum. At soil pH 4.5, the tubers of P. multiflorum exhibited the highest levels of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-d-glucoside (THSG) and total anthraquinones at 5.41% and 0.38%, respectively. However, increased soil pH significantly reduced the content of THSG and total anthraquinones. Reference-free transcriptome analysis was further conducted on P. multiflorum treated at pH 4.5 and 9.5, generating a total of 47,305 unigenes with an N50 of 2118 bp, of which 31,058 (65.65%) were annotated. Additionally, 2472 differentially expressed genes (DEGs) were identified. Among them, 17 DEGs associated with the biosynthesis of THSG and anthraquinones were screened. A comprehensive analysis of differential gene expression and effective component content demonstrated a significant positive correlation between the content of effective components and the 14 DEGs’ expression but a negative correlation with soil pH. This study highlighted the influence of varying soil pH values on the effective component content of P. multiflorum. Specific acidic conditions proved beneficial for the synthesis and accumulation of THSG and total anthraquinones in P. multiflorum, thereby enhancing the quality of the medicinal material.

Protective effect on pancreatic β cells of α-glucosidase inhibitors screened from Rehmannia glutinosa

Rehmannia glutinosa (RG) has been traditionally used as Chinese folk medicine for thousands of years. Catalpol in RG has significant therapeutic effects on type 2 diabetes mellitus (T2DM) and its complications. However, very limited study on other chemicals in RG for the treatment of diabetes. In this study, the compounds with α-glucosidase inhibitory activity in RG were screened and isolated by microfractionation bioactivity evaluation method. For the first time, 2,3,5,4′ -tetrahydroxystilbene-2-O-β-D-glucoside (THSG) was found in RG, and the inhibitory type of its enzyme inhibitory activity was determined. In addition, INS-1 cells were treated with H2O2 to establish a model of oxidative damage to pancreatic β cells, and the protective effect of THSG on oxidative damage of INS-1 cells was evaluated by detecting cell viability, content of malondialdehyde (MDA), superoxide dismutase (SOD) and insulin secretion levels. The results showed that THSG had a non-competitive reversible inhibitory effect on α-glucosidase (IC50 = 0.186 ± 0.003 μmol/mL). In the oxidative damage model of INS-1 cells treated with 80 μM THSG, cell viability was increased from 52.13% to 76.26%, content of MDA was decreased by 146.21%, and activity of SOD was increased by 26.27%. Compared with the model group, the total amount of insulin increased by 59.37%. Without the influence of H2O2, the total amount of insulin increased by 22.02% compared with the normal group. These results suggested that THSG could regulate blood glucose by effectively inhibiting α-glucosidase, alleviating oxidative damage to pancreatic β cells, and promoting insulin secretion.

2,3,5,4'-Tetrahydroxystilbene (TG1), a Novel Compound Derived from 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG), Inhibits Colorectal Cancer Progression by Inducing Ferroptosis, Apoptosis, and Autophagy.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide and long-term survival is not guaranteed in metastatic disease despite current multidisciplinary therapies. A new compound 2,3,5,4'-Tetrahydroxystilbene (TG1), derived from THSG (2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside), has been developed, and its anticancer ability against CRC is verified in this study. HCT116, HT-29, and DLD-1 were treated with TG1 and the IC50 was measured using a sulforhodamine B assay. A Xenograft mouse model was used to monitor tumor growth. Apoptosis and autophagy, induced by TG1 in CRC cells, were examined. RNA-sequencing analysis of CRC cells treated with TG1 was performed to discover underlying pathways and mechanisms. The results demonstrated that treatment with TG1 inhibited CRC proliferation in vitro and in vivo and induced apoptotic cell death, which was confirmed by Annexin V-FITC/PI staining and Western blotting. Additionally, TG1 treatment increased the level of autophagy in cells. RNA-sequencing and GSEA analyses revealed that TG1 was associated with MYC and the induction of ferroptosis. Furthermore, the ferroptosis inhibitor Bardoxolone abrogated the cytotoxic effect of TG1 in CRC cells, indicating that ferroptosis played a crucial role in TG1-induced cytotoxicity. These findings suggest that TG1 might be a potential and potent compound for clinical use in the treatment of CRC by inhibiting proliferation and inducing ferroptosis through the MYC pathway.

Hepatic Effect of 2,3,5,4'-tetrahydroxystilbene-2-O-β-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-β-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.

Molecular identification and functional characterization of two glycosyltransferases genes from Fallopia multiflora.

The traditional Chinese medicine plant Fallopia multiflora (Thunb.) Harald. contains various pharmacodynamically active glycosides, such as stilbene glycosides, anthraquinone (AQ) glycosides, and flavonoid glycosides. Glycosylation is an important reaction in plant metabolism that is generally completed by glycosyltransferase in the last step of the secondary metabolite biosynthesis pathway, and it can improve the beneficial properties of many natural products. In this study, based on the transcriptome data of F. multiflora, we cloned two Uridine-diphosphate-dependent glycosyltransferases (UGTs) from the cDNA of F. multiflora (FmUGT1 and FmUGT2). Their full-length sequences were 1602 and 1449 bp, encoding 533 and 482 amino acids, respectively. In vitro enzymatic reaction results showed that FmUGT1 and FmUGT2 were promiscuous and could catalyze the glycosylation of 12 compounds, including stilbenes, anthraquinones, flavonoids, phloretin, and curcumin, and we also obtained and structurally identified 13 glycosylated products from both of them. Further experiments on the in vivo function of FmUGT1 and FmUGT2 showed that 2, 3, 5, 4'- tetrahydroxy stilbene-2-O-β-d-glucoside (THSG) content in hairy roots was elevated significantly when FmUGT1 and FmUGT2 were overexpressed and decreased accordingly in the RNA interference (RNAi) groups. These results indicate that FmUGT1 and FmUGT2 were able to glycosylate a total of 12 structurally diverse types of acceptors and to generate O-glycosides. In addition, FmUGT1 and FmUGT2 efficiently catalyzed the biosynthesis of THSG, and promoted the production of AQs in transgenic hairy roots.

2,3,5,4-Tetrahydroxystilbene-2-O-Β-D-Glucoside Inhibiting Hepatocytes Injury Through Reducing the Expression of Tumor Necrosis Factor-a in LPSStimulated Kupffer Cells via Regulating PPAR-γ/NFκB

2,3,5,4'-Tetrahydroxystilbene-2-O-Β-D-Glucoside (TSG), a main bioactive component of polygonum multiflorum Thumb, exerts anti-oxidant, antitumor, anti-inflammatory effects. However, the protective effects of TSG on hepatocytes through anti-inflammatory effects have rarely been investigated. Mouse liver macrophages (Kupffer cells, KUP5 cells) were primed with various concentrations of TSG before Lipopolysaccharide (LPS) treatment, and AML12 hepatocytes were cultured with the supernatants of KUP5 cells. The results showed that TSG inhibits the expression of iNOS in KUP5 cells. Meanwhile, Tumor Necrosis Factor-a (TNF-a) and interleukin-1Β (IL-1Β) expression in KUP5 cells were reversed after TSG treatment. Additionally, the expression of Peroxisome Proliferators-Activated Receptors-γ (PPAR-γ) was up-regulated while the phosphorylation of p65 and IκB-a were decreased in KUP5 cells after TSG treatment. Furthermore, GW9662 (PPAR-γ antagonist) treatment could eliminate the anti-inflammatory effects of TSG. In AML12 hepatocytes, the level of AST, ALT, and apoptosis was decreased after being cultured with the supernatants of KUP5 cells pretreated with TSG. Moreover, TNF-a neutralization of KUP5 cells supernatants could decrease the level of AST, ALT, and AML12 hepatocyte apoptosis. These results indicate that TSG activates PPAR-γ, thereby decreasing nuclear factor kappa-B (NFκB) activation and reducing the release of TNF-a in macrophages to protect hepatocytes from injury. Our study may help further understand the protective effects of TSG on hepatocytes by suppressing liver inflammation.

Simultaneous determination of multiple constituents of Qi-Lin pill by UPLC-MS/MS: Applications to pharmacokinetics and testicular tissue distribution in rats

Qi-Lin pill (QLP) is an effective traditional Chinese medicine prescription (TCMP) that has been used for the treatment of the oligoasthenozoospermia in China. Recently, some articles described the pharmacological effects of QLP and multiple ingredients in QLP contribute to its effects. However, the pharmacokinetic and target tissue distribution data of QLP are still unknown. In the present study, according to the Bioanalytical Method Validation Guidance of FDA, a sensitive and selective UPLC-MS/MS method was developed and validated for simultaneous determination of multiple constituents in rat plasma and testicular tissue, including morusimic acid A, codonopyrridium B, magnoflorine, emodin, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), ecliptasaponin A, paeoniflorin, albiflorin, gallic acid, danshensu, salvianolic acid A, catechin, isosinensetin, nobiletin, formononetin, calycosin, icariside II, icariin and epimedin C. For 19 analytes, the LLOQs reached 0.01–4 ng/mL. And all calibration curves showed favorable linearity (r ≥ 0.9903) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.92 %, and the accuracies (as relative error) were in the range of − 6.44 % to 6.22 %. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The method was successfully applied for the pharmacokinetics and testis distribution of multiple chemical constituents in QLP after a single oral dose. As a result, high exposure of danshensu, gallic acid, paeoniflorin and albiflorin were observed in rat plasma and testicular tissue. Among the flavonoids, isosinensetin and nobiletin had high exposure in testicular tissue. Moreover, alleviation of progesterone reduction was evaluated in H2O2-induced R2C leydig cells, and danshensu, gallic acid, paeoniflorin, albiflorin and nobiletin showed potent activity. Therefore, these five components were considered to be the effective components of QLP due to their relatively high exposure in vivo and biological activity. This finding also provided relevant information on action mechanism of QLP in the treatment of oligoasthenozoospermia.

2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside ameliorates bleomycin-induced pulmonary fibrosis via regulating pro-fibrotic signaling pathways.

2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) is the main active ingredient extracted from Polygonum multiflorum Thunb. (PMT), which has been reported to possess extensive pharmacological properties. Nevertheless, the exact role of THSG in pulmonary fibrosis has not been demonstrated yet. The main purpose of this study was to investigate the protective effect of THSG against bleomycin (BLM)-induced lung fibrosis in a murine model, and explore the underlying mechanisms of THSG in transforming growth factor-beta 1 (TGF-β1)-induced fibrogenesis using MRC-5 human lung fibroblast cells. We found that THSG significantly attenuated lung injury by reducing fibrosis and extracellular matrix deposition. THSG treatment significantly downregulated the expression levels of TGF-β1, fibronectin, α-SMA, CTGF, and TGFBR2, however, upregulated the expression levels of antioxidants (SOD-1 and catalase) and LC3B in the lungs of BLM-treated mice. THSG treatment decreased the expression levels of fibronectin, α-SMA, and CTGF in TGF-β1-stimulated MRC-5 cells. Conversely, THSG increased the expression levels of SOD-1 and catalase. Furthermore, treatment of THSG profoundly reduced the TGF-β1-induced generation of reactive oxygen species (ROS). In addition, THSG restored TGF-β1-induced impaired autophagy, accompany by increasing the protein levels of LC3B-II and Beclin 1. Mechanism study indicated that THSG significantly reduced TGF-β1-induced increase of TGFBR2 expression and phosphorylation of Smad2/3, Akt, mTOR, and ERK1/2 in MRC-5 cells. These findings suggest that THSG may be considered as an anti-fibrotic drug for the treatment of pulmonary fibrosis.

2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside improves female ovarian aging.

Reduced fertility associated with normal aging may reflect the over-maturity of oocytes. It is increasingly important to reduce aging-induced infertility since recent trends show people marrying at later ages. 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, has been reported to have anti-inflammatory and anti-aging properties. To evaluate whether THSG can reduce aging-related ovarian damage in a female mouse model of aging, THSG was administered by gavage at a dose of 10 mg/kg twice weekly, starting at 4 weeks of age in a group of young mice. In addition, the effect of THSG in a group of aged mice was also studied in mice starting at 24 weeks of age. The number of oocytes in the THSG-fed group was higher than in the untreated control group. Although the percentage of secondary polar bodies (PB2) decreased during aging in the THSG-fed group, it decreased much more slowly than in the age-matched control group. THSG administration increased the quality of ovaries in young mice becoming aged. Western blotting analyses also indicated that CYP19, PR-B, and ER-β expressions were significantly increased in 36-week-old mice. THSG also increased oocyte numbers in aged mice compared to mice without THSG fed. Studies of qPCR and immunohistochemistry (IHC) analyses of ovaries in the aged mice groups were conducted. THSG increased gene expression of anti-Müllerian hormone (AMH), a biomarker of oocyte number, and protein accumulation in 40-week-old mice. THSG increased the expression of pgc1α and atp6, mitochondrial biogenesis-related genes, and their protein expression. THSG also attenuated the fading rate of CYP11a and CYP19 associated with sex hormone synthesis. And THSG maintains a high level of ER-β expression, thereby enhancing the sensitivity of estrogen. Our findings indicated that THSG increased or extended gene expression involved in ovarian maintenance and rejuvenation in young and aged mice. On the other hand, THSG treatments significantly maintained oocyte quantity and quality in both groups of young and aged mice compared to each age-matched control group. In conclusion, THSG can delay aging-related menopause, and the antioxidant properties of THSG may make it suitable for preventing aging-induced infertility.

A Systematic Strategy for the Characterization of 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside Metabolites In Vivo by Ultrahigh Performance Liquid Chromatography Coupled with a Q Exactive-Orbitrap Mass System.

2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (THSG), a polyphenol stilbene compound, is the main active constituent in Polygonum multiflorum. In this study, a comprehensive analytical strategy was developed for the characterization of THSG metabolites in vivo (rat plasma, bile, urine, heart, liver, spleen, lung, kidney, and stomach) utilizing ultrahigh performance liquid chromatography coupled with Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q Exactive-Orbitrap MS) based on multiple data-processing techniques. As a result, a total of 75 metabolites were characterized in bio-samples, and calculated Clog P values were further employed to assign the chemical structures of some isomers. Glucoside hydrolysis, hydrogenation, hydroxylation, glucuronide conjugation, and sulfate conjugation would be the major metabolic pathways of THSG. It appeared that most metabolites would generally undergo phase I reactions followed by phase II reactions. These results provided valuable information for in-depth understanding of the safety and efficacy of THSG and showed a valuable methodology for metabolic characterization.

2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside ameliorates NAFLD via attenuating hepatic steatosis through inhibiting mitochondrial dysfunction dependent on SIRT5

Non-alcoholic fatty liver disease (NAFLD) is becoming more and more common in clinic in the world, and the study on its mechanism and treatment strategy has already been a research hotspot. Natural chemical compound 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG) is isolated from Polygonum multiflorum Thunb. that has already been reported to have the lipid-lowering activity. The purpose of this research was to observe the improvement of TSG on methionine and choline deficient (MCD) diet-induced NAFLD in mice and to further elucidate its engaged mechanism. NAFLD was induced in mice fed by MCD diet for 6 weeks. The accumulation of lipids in hepatocytes was induced by 0.5mM non-esterified fatty acid (NEFA). Biochemical parameters in serum or livers from mice were tested. Protein and mRNA expression and stability were measured. Mitochondrial dysfunction was analyzed both in vivo and in vitro. The Label-free quantitative proteomic analysis was used to find potential involved key molecules. TSG attenuated hepatic parenchymal cells injury, liver inflammatory responses and hepatic fibrosis, and markedly ameliorated liver steatosis in mice from MCD group. In vitro results indicated that TSG reduced the accumulation of cellular lipids in hepatocytes induced by NEFA. TSG reduced reactive oxygen species (ROS) formation and attenuated mitochondrial dysfunction both in vivo and in vitro. The label-free quantitative proteomic analysis predicted the crucial participation of NAD-dependent protein deacylase sirtuin-5 (SIRT5). Next experimental results further evidenced that TSG enhanced SIRT5 expression in mitochondria both in vitro and in vivo. The TSG-supplied inhibition on ROS formation and mitochondrial dysfunction in hepatocytes was disappeared after the application of SIRT5 siRNA. TSG increased the expression and enzymatic activity of carnitine palmitoyltransferase 1A (CPT1A), but this enhance was diminished in hepatocytes transfected with SIRT5 siRNA. Additionally, the TSG-provided inhibition on cellular lipids accumulation was also disappeared in hepatocytes transfected with SIRT5 siRNA. Further results demonstrated that TSG increased SIRT5 expression by regulating its mRNA stability through enhancing the binding of SIRT5 mRNA with serine/arginine-rich splicing factor 2 (SRSF2), which is an RNA-binding protein (RBP). TSG attenuated liver steatosis and inhibited NAFLD progression through preventing oxidative stress injury and improving mitochondrial dysfunction, and SIRT5 played a key role in this process.

TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2.

Objectives To investigate the effect of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its molecular mechanism. Methods After TSG treatment of rBMSCs, alkaline phosphatase (ALP) activity was compared between the drug treatment group and the control group. The effects of TSG on alkaline phosphatase positive cloning and mineralized nodule formation were also detected. Total mRNA and protein were extracted, and the effects of TSG on the expression levels of osteopontin (OPN), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), Osterix, and Col1a1 were detected by real-time fluorescence quantitative PCR. Western blotting was used to detect the inhibitory effect of TSG on KDM5A. BMSCs were transfected with small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and pcDNA3.1 KMD5A. Results TSG significantly increased the activity of ALP, the number of alkaline phosphatase clones, and calcified nodule formation. The OPN, OCN, Runx2, and Osterix expression levels were significantly increased among the osteoblasts after TSG treatment. A mechanistic study showed that the effect of TSG is realized by inhibiting KDM5A. Conclusions KDM5A signaling may be involved in the regulation of osteogenic differentiation of rBMSCs. TSG can promote osteogenic differentiation and maturation of rBMSCs at 0.1–50 μmol/L. The mechanism of action was realized by inhibiting the expression of KDM5A.

2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside alleviates lipopolysaccharide-induced acute lung injury in rats

To observe the protective effect of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside (TSG) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and explore the underlying mechanism. Thirty-six SD rats were randomized equally into 4 groups: the normal control group, ALI model group, and low- and high-dose TSG groups (treated with 50 and 100 mg/kg TSG via intragastric administration, respectively). In all but the normal control group, the rats were subjected to tail vein injection of LPS to induced ALI. The rats were euthanized at 6 h after the injection for pathological examination of the lungs. The wet/dry weight ratio (W/D) of the lungs were calculated, and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the lung tissues and serum levels of TNF-α and IL-6 were determined. Western blotting was performed to detect the levels of NF-κB p65 in the lungs. Compared with those in LPS group, the TSGtreated rats showed significantly milder lung pathologies (P < 0.001) and had lower serum TNF-α and IL-6 levels (P < 0.001) and W/D of the lung tissues (P < 0.001), higher SOD activity (P < 0.001) and lower MDA content in the lungs (P < 0.001), and significantly lower expression of NF-κB p65 in the lungs (P < 0.001). None of these indices showed significant differences between the lowand high-dose TSG treatment groups (P>0.05). TSG can ameliorate LPS-induced ALI in rats possibly by suppressing the NF-κB pathway to improve the antioxidant capacity and decrease the release of inflammatory factors.

The Protective Effect of Tetrahydroxystilbene Glucoside on High Glucose-Induced Injury in Human Umbilical Vein Endothelial Cells through the PI3K/Akt/eNOS Pathway and Regulation of Bcl-2/Bax

Endothelial dysfunction plays a central role in the patho­genesis of diabetic vascular complications. 2,3,5,4′-tetra­hydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from the roots of Polygonum multiflorum Thunb, has been shown to have strong antioxidant and antiapoptotic activities. In the present study, we investigated the protective effect of TSG on apoptosis induced by high glucose in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms. Our data demonstrated that TSG significantly reversed the high glucose-induced decrease in cell viability, suppressed high glucose-induced generation of intracellular reactive oxygen species (ROS), the activity of caspase-3, and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, we found that TSG not only increased the expression of Bcl-2, while decreasing Bax expression, but also activated phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) with subsequent nitric oxide production and ultimately reduced high glucose-induced apoptosis. However, the antiapoptotic effects of TSG were abrogated by pretreatment of the cells with PI3K inhibitor (LY294002) or eNOS inhibitor NG-L-nitro-arginine methyl ester, respectively. These results suggest that TSG inhibits high glucose-induced apoptosis in HUVECs through inhibition of ROS production, activation of the PI3K/Akt/eNOS pathway, and upregulation of the Bcl-2/Bax ratio, and thus may demonstrate significant potential for preventing diabetic cardiovascular complications.

Comparative Studies on Multi-Component Pharmacokinetics of Polygonum multiflorum Thunb Extract After Oral Administration in Different Rat Models.

The clinical use of Polygonum multiflorum Thunb (PM) has been restricted or banned in many countries, due to its hepatotoxic adverse effects. Its toxicity research has become a hot topic. So far, the pharmacokinetic studies of PM, focusing on prototype compounds such as 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), emodin, and physcion, have been considered the main basis of pharmacodynamic material or of toxic effect. However, pharmacokinetic studies of its phase II metabolites have not yet been reported, mainly because the quantifications of such metabolites are difficult to do without the reference substance. In addition, pharmacokinetic studies on different pathological models treated with PM have also not been reported. On the other hand, toxic effects of PM have been reported in patients diagnosed with different liver pathologies. In the present work, a simultaneous quantitation method for eight prototypes components of PM and their five phase II metabolites has been performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and used for the pharmacokinetic study of PM in two different liver pathological models in rats (normal, alpha-naphthylisothiocyanate (ANIT), and carbon tetrachloride (CCl4)). The results showed that the main blood-entering components of PM are TSG, emodin, physcion, emodin-8-O-β⁃D⁃glucoside (E-Glu), physcion-8-O-β⁃D⁃glucoside (P-Glu), aloe-emodin, gallic acid, resveratrol and catechin, among which TSG, emodin, and catechin were primary metabolized in phase II, while resveratrol was converted to all phase II metabolites, and the others were metabolized as drug prototypes. Meanwhile, their pharmacokinetic parameters in the different models also exhibited significant differences. For instance, the AUC (0-∞) values of the TSG prototype and its phase II metabolites were higher in the ANIT group, followed by CCl4 group and the normal group, while the AUC (0-∞) values of the emodin prototype and its phase II metabolites were higher in the CCl4 group. To further illustrate the reasons for the pharmacokinetic differences, bilirubin metabolizing enzymes and transporters in the liver were measured, and the correlations with the AUC of the main compounds were analyzed. TSG and aloe-emodin have significant negative correlations with UGT1A1, BSEP, OATP1A4, OCT1, NTCP, MRP2 and MDR1 (p < 0.01). These data suggest that when the expression of metabolic enzymes and transporters in the liver is inhibited, the exposure levels of some components of PM might be promoted in vivo.

Comparative study on the stability/intestinal absorption kinetics of 2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside derived from Polygoni Multiflori Radix and its herb pairs

The 2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside (THSG) is a main active ingredient of Polygoni Multiflori Radix (PMR). In this study, THSG was found to be a differential chemical composition between PMR and its compatible herb pairs through chemometrics techniques. The effects of the herb pairs Codonopsis Radix-PMR, Astragali Radix-PMR, and Ophiopogonis Radix (OR)-PMR on the stability and absorption behavior of THSG were investigated by simulating the digestive environment in vitro and using an everted gut sac model. The results indicated that the compatible pairs were able to significantly increase the stability of THSG in the gastrointestinal tract and delay the absorption of THSG in the small intestine. OR had the most obvious influence on THSG as the t0.9 values of THSG increased by 14.74- and 6.68-fold in simulated gastric juice and small intestinal fluid, and the apparent permeability coefficient of THSG were 2.07- and 1.97-fold lower for OR-PMR in the ileum and duodenum, respectively. Thus, the compatibility could potentially be used to change the biopharmaceutical properties of THSG. Moreover, the present study established a research method for describing the compatibilities of traditional Chinese medicine based on stability-intestinal absorption, which may provide important reference values for the compatibilities of traditional Chinese medicine.