Emodin-8-O-ß-D-glucopyranoside-induced hepatotoxicity and gender differences in zebrafish as revealed by integration of metabolomics and transcriptomics.

BACKGROUND: Emodin-8-O-ß-D-glucopyranoside (Em8G) is an active ingredient of traditional Chinese medicine Rhei Radix et Rhizoma and Polygonum multiflorum Thunb.. And it caused hepatotoxicity, while the underlying mechanism was not clear yet. PURPOSE: We aimed to explore the detrimental effects of Em8G on the zebrafish liver through the metabolome and transcriptome integrated analysis. STUDY DESIGN AND METHODS: In this study, zebrafish larvae were used in acute toxicity tests to reveal the hepatotoxicity of Em8G. Adult zebrafish were then used to evaluate the gender differences in hepatotoxicity induced by Em8G. Integration of transcriptomic and metabolomic analysis was used further to explore the molecular mechanisms underlying gender differences in hepatotoxicity. RESULTS: Our results showed that under non-lethal concentration exposure conditions, hepatotoxicity was observed in Em8G-treated zebrafish larvae, including changes in liver transmittance, liver area, hepatocyte apoptosis and hepatocyte vacuolation. Male adult zebrafish displayed a higher Em8G-induced hepatotoxicity than female zebrafish, as demonstrated by the higher mortality and histopathological alterations. The results of transcriptomics combined with metabolomics showed that Em8G mainly affected carbohydrate metabolism (such as TCA cycle) in male zebrafish and amino acid metabolism (such as arginine and proline metabolism) in females, suggesting that the difference of energy metabolism disorder may be the potential mechanism of male and female liver toxicity induced by Em8G. CONCLUSIONS: This study provided the direct evidence for the hepatotoxicity of Em8G to zebrafish models in vivo, and brought a new insight into the molecular mechanisms of Em8G hepatotoxicity, which can guide the rational application of this phytotoxin. In addition, our findings revealed gender differences in the hepatotoxicity of Em8G to zebrafish, which is related to energy metabolism and provided a methodological reference for evaluating hepatotoxic drugs with gender differences.

Safety evaluation of kaempferol glycosides-rich standardized roasted goji berry leaf extract.

Goji berry leaf (GL) has been used for medicinal foods for its pharmacological effects, including anti-oxidative and anti-obesity activities. Nevertheless, toxicological information on GL is limited for developing health functional ingredient. The aim of the research was to evaluate the single dose acute, 14-day repeated oral toxicity, and genotoxicity of standardized roasted GL extract (rGL) rich in kaempferol-3-O-sophoroside-7-O-glucoside. Tested rGL was found to be stable as kaempferol-3-O-sophoroside-7-O-glucoside, showing 0.7-2.1% of analytical standard variance. According to the single dose toxicity for 14 days, the lethal dose of rGL was determined to be ≥ 2000 mg/kg. Repeated doses of 0-1000 mg/kg of rGL per day for 14 days did not show any toxicity signs or gross pathological abnormalities. No genotoxic signs for the rGL treatment appeared via bacterial reverse mutation up to 5000 µg/plate. There was no significant increase in chromosomal aberration of rGL irrespective of metabolic activation by using CHO-K1 cells (p > 0.05). Regarding carcinogenic toxicity, chromosomal aberrations were not induced at 2000 mg of rGL/kg by using the in vivo bone marrow micronucleus test (p > 0.05). Results from the current study suggest that rGL could be used as a functional ingredient to provide various effects with safety assurance.

Reduction of emodin-8-O-ß-D-glucoside content participates in processing-based detoxification of polygoni multiflori radix.

BACKGROUND: The occurrence of severe liver injury by the herbal medicine Polygoni Multiflori Radix (PMR) has drawn significant attention. The fact that processing attenuates PMR-induced hepatotoxicity has been well accepted, but the mechanisms are still ambiguous. PURPOSE: This study aimed to illuminate the mechanism of processing-based attenuation of PMR hepatotoxicity. METHODS: The contents of emodin-8-O-ß-d-glucoside (EG) and emodin (EMD) in raw and processed PMR were quantified. The difference in toxicokinetic behaviors of EG and EMD was determined in vivo, and the disposition properties of EG were investigated in vitro and in vivo. RESULTS: Decreased EG content was found in processed (black bean) PMR. Processed PMR showed reduced adverse effects relative to raw PMR. In addition, less hepatic protein adduction derived from EMD was produced in mice after exposure to processed PMR than that in animals receiving raw PMR. Glucose transporters SGLT1 and GLUT2 participated in the absorption of EG, and effective hydrolysis of EG to EMD took place in the intestinal epithelial cells during the process of absorption. Cytosolic broad-specificity ß-glucosidase and lactase phlorizin hydrolase, as well as intestinal flora, participated in the hydrolysis of EG. The circulated EMD resulting from the deglycosylation of EG executed the hepatotoxic action. CONCLUSION: EG is a pre-toxin and can be metabolically activated to EMD participating in the hepatotoxic event. The reduction of EG content due to processing is a key mechanistic factor that initiates the detoxification of PMR.

[Toxicokinetics of emodin-8-O-ß-D-glucoside in rats in vivo].

This study aims to establish an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method for the determination of emodin-8-O-ß-D-glucoside(EG) and its metabolites in plasma, and to investigate the toxicokinetics(TK) behavior of them in rats. To be specific, the TK of EG and its metabolites from the first to the last administration in the repeated dose toxicity study was determined, and the kinetic parameters were calculated. The exposure of EG prototype and metabolites in rat plasma after oral administration of different doses of EG was evaluated. The result showed that the prototype of EG and its metabolites aloe-emodin-8-O-ß-D-glucoside, emodin, aloe-emodin, and hydroxyemodin could be detected in rats after oral administration of high-, medium-, and low-dose EG. The area under the curve(AUC) of the prototype and metabolites after the first and last administration was in positive correlation with the dose. The time to the maximum concentration(T_(max)) of EG and metabolites in the three administration groups was <6 h, and the longest in vivo residence time was 12 h. The T_(max) and in vivo residence time of EG were prolonged with the increase in the dose. The metabolites emodin, aloe-emodin, and hydroxyemodin all had two peaks. Both hydroxyemodin and aloe-emodin exhibited increased plasma exposure, slow metabolism, and accumulation in vivo. In addition, aloe-emodin-8-O-ß-D-glucoside and emodin disappeared with the increase in dose, suggesting the change of the metabolic pathway of EG in vivo in the case of high-dose administration. The mechanism of high-dose EG in vivo needs to be further explored. This study preliminarily elucidates the TK behavior of EG in rats, which is expected to support clinical drug use.

Stilbene glucoside: recent advances in pharmacology, bioinformatics investigation, toxicity and future opportunities.

Background: 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) from Polygonum multijiorum Thunb. (PMT), is a major bioactive component. This review is aimed to summarize the present development of TSG regarding pharmaceutics, pharmacology and toxicology, with a focus on the novel mechanism of drug-induced toxicity and provides insight for its potential developments and applications in the future on traditional Chinese medicine. Methods: Studies about TSG's activities and toxicity were searched and summarized. Targets and mechanisms were predicted and analyzed with network pharmacology methods. Affinities and binding modes of key targets with TSG were verified by AutoDock Vina software. Results: TSG plays an essential role among the chemical components of PMT because of multiple pharmacological activities, which suggests a potential application of TSG for a variety of diseases, like atherosclerosis, Alzheimer's disease, Parkinson's disease, cerebral I/R injury, diabetes, osteoporosis, colitis. However, mild liver toxicity of TSG is also pointed out. Conclusions: As a biologically active natural product in PMT, TSG has shown prospective pharmacological activities, particularly as an agent for cardiovascular protection and neuroprotection.

Investigation of toxicity attenuation mechanism of tetrahydroxy stilbene glucoside in Polygonum multiflorum Thunb. by Ganoderma lucidum.

ETHNOPHARMACOLOGICAL RELEVANCE: The idiosyncratic hepatotoxicity of Polygonum multiflorum Thunb. (PM) has attracted great interest, and tetrahydroxy stilbene glucoside (TSG) was the main idiosyncratic hepatotoxicity constituent, but biological detoxification on idiosyncratic hepatotoxicity of PM was not well investigated. AIM OF THE STUDY: This study aimed to illustrate biological detoxification mechanism on PM-induced idiosyncratic hepatotoxicity by Ganoderma lucidum (G. lucidum). MATERIALS AND METHODS: G. lucidum was used for biological detoxification of tetrahydroxy stilbene glucoside (TSG)-induced idiosyncratic hepatotoxicity of PM. The TSG consumption and products formation were dynamically determined during transformation using high-performance liquid chromatography coupled with diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-MSn). The transformation invertases (ß-D-glucosidase and lignin peroxidase) were evaluated by using intracellular and extracellular distribution and activity assay. The key functions of lignin peroxidase (LiP) were studied by experiments of adding inhibitors and agonists. The entire TSG transformation process was confirmed in vitro simulated test. The cellular toxicity of TSG and the transformation products was detected by MTT. RESULTS: A suitable biotransformation system of TSG was established with G. lucidum, then p-hydroxybenzaldehyde and 2,3,5-trihydroxybenzaldehyde can be found as transformation products of TSG. The transformation mechanism involves two extracellular enzymes, ß-D-glucosidase and LiP. ß-D-glucosidase can remove glycosylation of TSG firstly and then LiP can break the double bond of remaining glycosides. The toxicity of TSG after biotransformation by G. lucidum was attenuated. CONCLUSIONS: This study would reveal a novel biological detoxification method for PM and explain degradation processes of TSG by enzymic methods.

Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding.

BACKGROUND: The outbreak of coronavirus (SARS-CoV-2) disease caused more than 100,000,000 people get infected and over 2,200,000 people being killed worldwide. However, the current developed vaccines or drugs may be not effective in preventing the pandemic of COVID-19 due to the mutations of coronavirus and the severe side effects of the newly developed vaccines. Chinese herbal medicines and their active components play important antiviral activities. Corilagin exhibited antiviral effect on human immunodeficiency virus (HIV), hepatitis C virus (HCV) and Epstein-Barr virus (EBV). However, whether it blocks the interaction between SARS-CoV-2 RBD and hACE2 has not been elucidated. PURPOSE: To characterize an active compound, corilagin derived from Phyllanthus urinaria as potential SARS-CoV-2 entry inhibitors for its possible preventive application in daily anti-virus hygienic products. METHODS: Computational docking coupled with bio-layer interferometry, BLI were adopted to screen more than 1800 natural compounds for the identification of SARS-CoV-2 spike-RBD inhibitors. Corilagin was confirmed to have a strong binding affinity with SARS-CoV-2-RBD or human ACE2 (hACE2) protein by the BLI, ELISA and immunocytochemistry (ICC) assay. Furthermore, the inhibitory effect of viral infection of corilagin was assessed by in vitro pseudovirus system. Finally, the toxicity of corilagin was examined by using MTT assay and maximal tolerated dose (MTD) studies in C57BL/6 mice. RESULTS: Corilagin preferentially binds to a pocket that contains residues Cys 336 to Phe 374 of spike-RBD with a relatively low binding energy of -9.4 kcal/mol. BLI assay further confirmed that corilagin exhibits a relatively strong binding affinity to SARS-CoV-2-RBD and hACE2 protein. In addition, corilagin dose-dependently blocks SARS-CoV-2-RBD binding and abolishes the infectious property of RBD-pseudotyped lentivirus in hACE2 overexpressing HEK293 cells, which mimicked the entry of SARS-CoV-2 virus in human host cells. Finally, in vivo studies revealed that up to 300 mg/kg/day of corilagin was safe in C57BL/6 mice. Our findings suggest that corilagin could be a safe and potential antiviral agent against the COVID-19 acting through the blockade of the fusion of SARS-CoV-2 spike-RBD to hACE2 receptors. CONCLUSION: Corilagin could be considered as a safe and environmental friendly anti-SARS-CoV-2 agent for its potential preventive application in daily anti-virus hygienic products.

Semen Cuscutae-Fructus Lycii improves spermatogenic dysfunction by repairing the blood-testis barrier in rats according to in silico and in vitro methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae and Fructus Lycii (SC-FL) is a commonly used herbal pair for male infertility treatment. Studies have found that the mechanism of SC-FL treatment may be related to repairing the blood-testis barrier (BTB). The application of network pharmacology can be used to explore the correlation between medicines and diseases and predict the potential pharmacological mechanisms of SC-FL. AIM OF THE STUDY: This study aimed to explore the specific effects and mechanisms of SC-FL in repairing the BTB and initially revealed the mechanism of Chinese medicine treating male infertility through network pharmacology and animal experiments. MATERIALS AND METHODS: We searched databases using the network pharmacology method and performed mass spectrometry analysis. We analyzed and predicted the active ingredients, targets and key pathways of SC-FL in male infertility treatment. Then, we designed animal experiments to verify the results. Thirty-six Sprague-Dawley rats were randomly divided into the normal control group (NC group), spermatogenic dysfunction group (SD group) and SC-FL treatment group (SCFL group). Glucosides of Tripterygium wilfordii Hook. F (GTW) (40 mg/kg/d) was administered for 4 weeks to generate a spermatogenic dysfunction model. The rats in the SCFL group were given the SC-FL suspension (6 g/kg/d) daily. After 4 weeks of treatment, we detected the sperm quality of each group of rats and observed the cell morphology. Western blotting and qRT-PCR were used to detect the expression of BTB-related proteins in testicular tissues. RESULTS: 213 chemical ingredients of SC and FL were retrieved from the TCMSP database, and 54 effective chemical ingredients were obtained. Mass spectrometry analysis showed the above results were credible. Then, we identified 44 potential targets for the treatment of male infertility, and we plotted a network diagram of the interaction network between the core targets and a diagram of herbal medicine-active ingredient-target-disease interactions. The target genes were enriched according to biological functions, and 22 biological processes, 49 cellular components, 1487 molecular functions, and 122 signaling pathways were obtained. The results of the animal experiments showed that the sperm concentration and motility of the SCFL group were significantly improved compared with those of the SD group. Compared with those in the SD group, the structure and morphology of the Sertoli cells and seminiferous tubules of rats in the SCFL group improved, and the number of spermatogenic cells increased significantly. Western blotting and qRT-PCR results showed that compared with that in the SD group, the expression of p38 MAPK decreased significantly, and the expression of c-Jun, Occludin, ZO-1 and connexin 43 increased significantly in the SCFL group. CONCLUSION: We predicted that the active ingredients of SC-FL can treat male infertility by interacting with the core targets JUN, IL6, MAPK1, TP53, MYC, CCND1, AR, EGF, FOS, and MAPK8, and the possible mechanism is related to the MAPK signaling pathway. SC-FL can regulate the MAPK pathway and affect the expression of Occludin, ZO-1 and connexin 43 to repair damaged BTB and improve spermatogenic dysfunction induced by GTW, which may be one of the possible mechanisms.

Pharmacokinetic studies unveiled the drug-drug interaction between trans-2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside and emodin that may contribute to the idiosyncratic hepatotoxicity of Polygoni Multiflori Radix.

Polygoni Multiflori Radix (PMR) has been a reputable tonifying traditional Chinese medicine for a long history. However, clinical side effects regarding its idiosyncratic hepatotoxicity are occasionally reported. The containing anthraquinones, particularly emodin, could cause liver injury in both in vitro and in vivo experiments. It is well-known that some compounds could influence other compounds' pharmacokinetic parameters significantly. In this work, the influence of trans-2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside (TSG) on the pharmacokinetic behavior of emodin in rats was evaluated by an ultra-high performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/MS-MS) approach. Pharmacokinetic parameters of emodin, PMR extract, and TSG-free PMR extract (prepared by a component "knock-out" strategy with TSG eliminated), in rats after one-day and seven-day administration were determined and compared. We found that, after seven-day administration of the whole PMR extract (rather than TSG-free extract), emodin in rats was accumulated. And accordingly, the exposure of emodin in rats pre-treated with single TSG for seven days could be significantly enhanced. The results indicate that TSG was able to accelerate the exposure and metabolism of emodin. The effect of TSG on the metabolic activities of cytochrome P450 enzymes was further assessed by an LC-MS cocktail method. The accelerated exposure and metabolism of emodin could result from the up-regulation activity of CYP450s, in particular CYP1A2 isozyme. The findings obtained in this work firstly unveiled DDI between TSG and emodin in the administration of PMR, thus may provide a basis for unveiling the underlying mechanism of PMR-induced liver injury.

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. AIMS OF THE STUDY: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. MATERIALS AND METHODS: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. RESULTS: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. CONCLUSION: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

Anti-Trypanosoma cruzi Activity in vitro of Phases and Isolated Compounds from Excoecaria lucida Leaves.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. This illness is found mainly in 21 Latin American countries and an estimated 8 million people are infected worldwide. The unsatisfactory chemotherapy provokes severe toxicity and resistant strains. Medicinal plants constitute a promising source of new drugs and remedies against all kinds of disorders, mainly infectious diseases arousing interest worldwide. OBJECTIVE: The aim of this study is the isolation, structural identification and evaluation of the trypanocidal activity of samples present in the Excoecaria lucida Sw. leaves. METHODS: Total extract (TE) of E. lucida Sw. leaves was obtained by ethanol extract therefore fractionated sequentially with hexane, ethyl acetate and n-butanol, to obtain three phases: Hex, EA and But, respectively. Ellagic acid (EL1) was purified from both EA and But phases, while EL2; a 1:1 stigmasterol-3-O-ß-D-glucopyranoside plus sitosterol-3-O-ß-D-glucopyranoside mixture was obtained from the Hex phase. Activity assays were performed using bloodstream and intracellular forms of T. cruzi and cytotoxicity assays using L929 fibroblasts. RESULTS: The EL1 and EL2 samples were more active against bloodstream trypomastigote forms with EC50 of 53.0±3.6 and 58.2±29.0 µg/mL, respectively; at 100 µg/mL. These samples also showed 70% of inhibition of L929 cells infection. Toxicity assays demonstrated that after 96 h of treatment only the fractions Hex and EA presented detectable cytotoxicity. CONCLUSION: Ellagic acid, stigmasterol-3-O-ß-D-glucopyranoside and sitosterol-3-O-ß-Dglucopyranoside are reported for the first time in E. lucida Sw. leaves as well as their biological activity studies supporting further investigations for Chagas disease treatment.

A research on the genotoxicity of stevia in human lymphocytes.

Stevia extracts are obtained from Stevia rebaudiana commonly used as natural sweeteners. It is ∼250-300 times sweeter than sucrose. Common use of stevia prompted us to investigate its genotoxicity in human peripheral blood lymphocytes. Stevia (active ingredient steviol glycoside) was dissolved in pure water. Dose selection was done using ADI (acceptable daily intake) value. Negative control (pure water), 1, 2, 4, 8 and 16 µg/ml concentrations which were equivalent to ADI/4, ADI/2, ADI, ADI × 2 and ADI × 4 of Stevia were added to whole-blood culture. Two repetitive experiments were conducted. Our results showed that there was no significant difference in the induction of chromosomal aberrations and micronuclei between the groups treated with the concentrations of Stevia and the negative control at 24 and 48 h treatment periods. The data showed that stevia (active ingredient steviol glycosides) has no genotoxic activity in both test systems. Our results clearly supports previous findings.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

Enhanced absorption and inhibited metabolism of emodin by 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside: Possible mechanisms for Polygoni Multiflori Radix-induced liver injury.

Polygoni Multiflori Radix (PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. In our previous in vivo study, an interaction between stilbenes and anthraquinones has been discovered and a hypothesis is proposed that the interaction between stilbene glucoside-enriching fraction and emodin may contribute to the side effects of PMR. To further support our previous in vivo results in rats, the present in vitro study was designed to evaluate the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) on the cellular absorption and human liver microsome metabolism of emodin. The obtained results indicated that the absorption of emodin in Caco-2 cells was enhanced and the metabolism of emodin in human liver microsomes was inhibited after TSG treatment. The effects of the transport inhibitors on the cellular emodin accumulation were also examined. Western blot assay suggested that the depressed metabolism of emodin could be attributed to the down-regulation of UDP-glucuronosyltransferases (UGTs) 1A8, 1A10, and 2B7. These findings definitively demonstrated the existence of interaction between TSG and emodin, which provide a basis for a better understanding of the underlying mechanism for PMR-induced liver injury.

A nematicidal tannin from Punica granatum L. rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus).

The ethanol extract of Punica granatum L. rind was tested to show significant nematicidal activity against pine wood nematode. Three nematicidal compounds were obtained from the ethanol extract by bioassay-guided fractionation and identified as punicalagin 1, punicalin 2, and corilagin 3 by mass and nuclear magnetic resonance spectral data analysis. Punicalagin 1 was most active against PWN among the purified compounds with the LC50 value of 307.08µM in 72h. According to the enzyme assays in vitro, punicalagin 1 could inhibit the activity of acetylcholinesterase, amylase and cellulase from PWN with IC50 value of 0.60mM, 0.96mM and 1.24mM, respectively. The morphological structures of PWNs treated by punicalagin 1 were greatly changed. These physiological effects of punicalagin 1 on PWN may helpful to elucidate its nematicidal mechanism.

Plantamajoside from Plantago asiatica modulates human umbilical vein endothelial cell dysfunction by glyceraldehyde-induced AGEs via MAPK/NF-κB.

BACKGROUND: Plantago asiatica has been traditionally used for traditional medicine around East Asia. Plantamajoside (PM), which is isolated from this plant, is known for biological properties including anti-inflammation and antioxidant activity. To demonstrate the biological activity of PM against endothelial dysfunction induced by advanced glycation end-products (AGEs), a cellular inflammatory mechanism system was evaluated in human umbilical vein endothelial cells (HUVECs). METHODS: We obtained PM through previous research in our laboratory. We formed the AGEs from bovine serum albumin with glyceraldehyde in the dark for seven days. To confirm the modulation of the inflammatory mechanism in endothelial dysfunction, we quantified the various pro-inflammatory cytokines and endothelial dysfunction-related proteins in the HUVECs with Western blotting and with real-time and quantitative real-time polymerase chain reactions. RESULTS: Co-treatment with PM and AGEs significantly suppressed inflammatory cytokines and adhesion molecule expression. Moreover, the PM treatment for down-regulated inflammatory signals and blocked monocyte adhesion on the HUVECs. CONCLUSIONS: Theses results demonstrated that PM, as a potential natural compound, protects AGE-induced endothelial cells against inflammatory cellular dysfunction.

Cycloartane triterpenoid and its glucoside isolated from Cassia occidentalis.

In the present study, one new cycloartane triterpenoid, named cycloccidentalic acid C (1) and its glucoside, cycloccidentaliside VI (2) were isolated from the whole plant of Cassia occidentalis. Their structures were elucidated by a combinational analyses of 1D and 2D NMR data and HRMS. Compound 2 showed modest anti-HIV-1 activity with EC50 value of 1.44 µmol·L-1 and TI (Therapeutic Index) value of 15.59.

Acute and repeated doses (28 days) oral toxicity study of Vicenin-1, a flavonoid glycoside isolated from fenugreek seeds in laboratory mice.

Vicenin-1 (fenugreek glycoside) has been proven to possess potent anti-inflammatory and anti-oxidant activity. The objective of the present investigation was to determine in-vivo acute and subacute (28-days repeated dose) oral toxicity of Vicenin-1 isolated from fenugreek seed. Vicenin-1 (93%) was isolated from a hydroalcoholic extract of fenugreek seed and characterized using HPLC, TLC, 1H NMR and 13C NMR. Acute oral toxicity (AOT) and subacute toxicity studies of Vicenin-1 were carried out according to OECD 425 (up-and-down procedure) and OCED 407 guidelines in Swiss albino mice. In AOT, Vicenin-1 showed 10% mortality when administered at a dose of 5000 mg/kg. However, when vicenin-1 was administered for at doses of 37.5, 75, or 150 mg/kg 28-days it did not show any mortality at the administered doses. Vicenin-1 (75 mg/kg) did not show observational, behavioral, biochemical or histopathological toxic effects. There were minor alterations in body weight, hematology, and histopathology of mice administered with Vicenin-1 (150 mg/kg), but these changes were within normal laboratory ranges. The highest concentration of Venicin-1 was found in liver (3.46%) followed by lung (0.65%). In conclusion, Vicenin-1 showed median lethal dose (LD50) of 4837.5 mg/kg with no-observed-adverse-effect levels (NOAEL) at 75 mg/kg and lowest adverse effect levels (LOAEL) at 150 mg/kg for both sexes of mice during AOT and sub-acute toxicity study, respectively.

Synergic actions of polyphenols and cyanogens of peanut seed coat (Arachis hypogaea) on cytological, biochemical and functional changes in thyroid.

In animals, long-term feeding with peanut (Arachis hypogaea) seed coats causes hypertrophy and hyperplasia of the thyroid gland. However, to date there have been no detailed studies. Here, we explored the thyroidal effects of dietary peanut seed coats (PSC) in rats. The PSC has high levels of pro-goitrogenic substances including phenolic and other cyanogenic constituents. The PSC was mixed with a standard diet and fed to rats for 30 and 60 days, respectively. Animals fed with the PSC-supplemented diet showed a significant increase in urinary excretion of thiocyanate and iodine, thyroid enlargement, and hypertrophy and/or hyperplasia of thyroid follicles. In addition, there was inhibition of thyroid peroxidase (TPO) activity, 5'-deiodinase-I (DIO1) activity, and (Na+-K+)-ATPase activity in the experimental groups of rats as compared to controls. Furthermore, the PSC fed animals exhibited decreased serum circulating total T4 and T3 levels, severe in the group treated for longer duration. These data indicate that PSC could be a novel disruptor of thyroid function, due to synergistic actions of phenolic as well as cyanogenic constituents.

Nonclinical safety of the sodium-glucose cotransporter 2 inhibitor empagliflozin.

Empagliflozin, a selective inhibitor of the renal tubular sodium-glucose cotransporter 2, was developed for treatment of type 2 diabetes mellitus. Nonclinical safety of empagliflozin was studied in a battery of tests to support global market authorization. Safety pharmacology studies indicated no effect of empagliflozin on measures of respiratory or central nervous system function in rats or cardiovascular safety in telemeterized dogs. In CD-1 mouse, Wistar Han rat, or beagle dogs up to 13, 26, or 52 weeks of treatment, respectively, empagliflozin exhibited a toxicity profile consistent with secondary supratherapeutic pharmacology related to glucose loss and included decreased body weight and body fat, increased food consumption, diarrhea, dehydration, decreased serum glucose and increases in other serum parameters reflective of increased protein catabolism, gluconeogenesis, and electrolyte imbalances, and urinary changes such as polyuria and glucosuria. Microscopic changes were consistently observed in kidney and included tubular nephropathy and interstitial nephritis (dog), renal mineralization (rat) and tubular epithelial cell karyomegaly, single cell necrosis, cystic hyperplasia, and hypertrophy (mouse). Empagliflozin was not genotoxic. Empagliflozin was not carcinogenic in female mice or female rats. Renal adenoma and carcinoma were induced in male mice only at exposures 45 times the maximum clinical dose. These tumors were associated with a spectrum of nonneoplastic changes suggestive of a nongenotoxic, cytotoxic, and cellular proliferation-driven mechanism. In male rats, testicular interstitial cell tumors and hemangiomas of the mesenteric lymph node were observed; both tumors are common in rats and are unlikely to be relevant to humans. These studies demonstrate the nonclinical safety of empagliflozin.