Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosorbent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were successfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias%ranged from -12.2% to -5.2%,precision ranged from -12.4% to -1.4%,and the relative standard deviation(RSD)was less than 6.6% and 8.7%,respectively.The total error was less than 20.4%.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Tumor-targeting intravenous lipid emulsion of paclitaxel:Characteristics,stability,toxicity,and toxicokinetics

Lipid nanoemulsions are promising nanodrug delivery carriers that can improve the efficacy and safety of paclitaxel(PTX).However,no intravenous lipid emulsion of PTX has been approved for clinical treatment,and systemic safety profiles have not yet been reported.Here we outline the development of a PTXloaded tumor-targeting intravenous lipid emulsion(PTX Emul)and describe its characteristics,colloidal stability,and systemic safety profiles in terms of acute toxicity,long-term toxicity,and toxicokinetics.We also compare PTX Emul with conventional PTX injection.Results showed that PTX Emul exhibited an ideal average particle size(approximately 160 nm)with narrow size distribution and robust colloidal stability under different conditions.Hypersensitivity reaction and hemolysis tests revealed that PTX Emul did not induce hypersensitivity reactions and had no hemolytic potential.In addition,where the alleviated systemic toxicity of PTX Emul may be attributed to the altered toxicokinetic characteristics in beagle dogs,including the decreased AUC and increased plasma clearance and volume of distribution,PTX Emul alleviated acute and long-term toxicity as evidenced by the enhanced the median lethal dose and approximate lethal dose,moderate body weight change,decreased bone marrow suppression and organ toxicity compared with those under PTX injection at the same dose.A fundamental understanding of the systemic safety profiles,high tumor-targeting efficiency,and superior antitumor activity in vivo of PTX Emul can provide powerful evidence of its therapeutic potential as a future treatment for breast cancer.

Role of microRNAs in the process of metformin treating multiple diseases

Metformin as the first-line treatment for type 2 diabetes mellitus has been discovered to exert beneficial effects on many diseases for nearly ten years,but its specific mechanism is still unclear.As a new class of gene expression regulators with pleiotropic properties,microRNAs(miRNAs)participate in multiple physiological processes such as cell differentiation,proliferation,survival,and metabolism,which drive them to play a regulatory role in the occurrence,development and even treatment of various diseases.A substantial body of research has found the relationship between metformin and miRNAs,in which metformin can alter the expression profiles of miRNAs in multiple disease states and on the other hand the signal pathways involving miRNAs may contribute to the pharmacological actions of metformin.This review summarizes the effects of metformin on miRNAs and their relationship in different diseases(like tumor,metabolic diseases,etc.),which should be of a great help for our better understanding of the mechanism of metformin for treating multiple diseases.

High nitrogen stainless steel drug-eluting stent-Assessment of pharmacokinetics and preclinical safety in vivo

Pharmacokinetic analyses were performed using 20 pigs for 120-days implantation,while one sirolimus-eluting stent was implanted into one of their coronary artery.At different time points,the residual sirolimus on the stent,delivered locally(to artery wall),regionally(to adjacent and downstream muscle)and systemically(to plasma and visceral organs),was detected throughout 120 days.Preclinical safety evaluation was performed using 32 pigs for 180-days implantation to study the safety of metal platform material and the effectiveness of sirolimus eluting coating on the HNS stent.The neointima area,restenosis rate and inflammatory grade for HNS and control group stents were detected and analyzed.Approximately 80%sirolimus was eluted from the sirolimuseluting stents after 30-days implantation in vivo.Additionally,there was sustained sirolimus in the artery wall,cardiac muscle and heart throughout 120-days implantation,and sirolimus accumulated to the peak at 90-days implantation.It was inferred that the sirolimus eluting stent in this study was covered by neointima before 90-days implantation,indicating that the sirolimus eluting coating on the HNS stent was safe and effective.Very little sirolimus was distributed in visceral organs after 14-days implantation.HNS sirolimus-eluting stent exhibited lower restenosis rate and lower inflammatory grade than control group,which verified that the sirolimus-eluting coating design in this study was reasonable and practical.In addition,there were no significant difference in restenosis rate and inflammatory score between HNS bare-metal stent and drug-eluting stents,illustrating that HNS has good bio-compatibility and is suitable to use as coronary artery stent material.

The dual-inhibitory effect of miR-338-5p on the multidrug resistance and cell growth of hepatocellular carcinoma

Chemotherapeutic treatments against hepatocellular carcinoma(HCC)are necessary for both inoperable patients to improve prospects for survival and surgery patients to improve the outcome after surgical resection.However,multidrug resistance(MDR)is a major obstacle to obtaining desirable results.Currently,increasing the chemotherapy sensitivity of tumor cells or discovering novel tumor inhibitors is an effective therapeutic strategy to solve this issue.In the present study,we uncovered the dual-inhibitory effect of miR-338-5p:on the one hand,it could downregulate ABCB1 expression and sensitize HCC cells to doxorubicin and vinblastine by directly targeting the 3′-untranslated region(3′-UTR)of ABCB1,while,on the other hand,it could suppress the proliferation of HCC cells by directly targeting the 3′-UTR of EGFR and reducing EGFR expression.Since EGFR regulates ABCB1 levels,the indirect action of miR-338-5p in ABCB1 modulation was revealed,in which miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway.These data indicate that the miR-338-5p/EGFR/ABCB1 regulatory loop plays a critical role in HCC,and a negative correlation between miR-338-5p and EGFR or ABCB1 was also detected in HCC clinical samples.In conclusion,these findings reveal a critical role for miR-338-5p in the regulation of MDR and proliferation of HCC,suggesting the potential therapeutic implications of miR-338-5p in HCC treatment.

Protective effect of mesenchymal stem cell-derived exosomal treatment of hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury

BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.

Scutellarein alleviates the dysfunction of inner blood-retinal-barrier initiated by hyperglycemia-stimulated microglia cells

AIM:To investigate the alleviation of scutellarein(SN)against inner blood-retinal-barrier(iBRB)dysfunction in microglia cells stimulated by hyperglycemia and to elucidate the engaged mechanism.METHODS:Microglia BV2 cells were stimulated by using 25 mmol/L D-glucose.The same concentration of mannitol(25 mmol/L)was applied as an isotonic contrast.Real-time PCR,Western-blot assay and immunofluorescence staining assay was performed.The dysfunction of iBRB in vitro was detected by using transendothelial electrical resistance(TEER)assay.Additionally,the leakage of fluorescein isothiocyanate(FITC)-conjugated dextran(70 kDa)was detected.RESULTS:SN abrogated microglia BV2 cells activation and reduced the phosphorylated activation of extracellular signal-regulated protein kinase(ERK)1/2.SN also decreased the transcriptional activation of nuclear factorκB(NFκB)and the elevated expression of tumor necrosis factorα(TNFα),interleukin(IL)-6 and IL-1βin BV2 cells treated with D-glucose(25 mmol/L).SN attenuated iBRB dysfunction in human retinal endothelial cells(HRECs)or choroid-retinal endothelial RF/6 A cells when those cells were treated with TNFα,IL-1βor IL-6,or co-cultured with microglia cells stimulated by D-glucose.Moreover,SN restored the decreased protein expression of tight junctions(TJs)in TNFα-treated HRECs and RF/6 A cells.CONCLUSION:SN not only alleviate iBRB dysfunction via directly inhibiting retinal endothelial injury caused by TNFα,IL-1βor IL-6,but also reduce the release of TNFα,IL-1βand IL-6 from microglia cells by abrogating hyperglycemia-mediated the activation of microglia cells.

Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2-hydroxybutyric acid

Liver injury caused by acetaminophen(AP)overdose is a leading public health problem.Although APinduced liver injury is well recognized as the formation of N-acetyl-p-benzoquinone(NAPQI),a toxic metabolite of AP,resulting in cell damage,emerging evidence indicates that AP-induced liver injury is also associated with gut microbiota.However,the gut microbiota-involved mechanism remains largely unknown.In our study,we found that vancomycin(Vac)pretreatment(100 mg/kg,twice a day for 4 days)attenuated AP-induced liver injury,altered the composition of gut microbiota,and changed serum metabolic profile.Moreover,we identified Vac pretreatment elevated cecum and serum 2-hydroxybutyric acid(2-HB),which ameliorated AP-induced cell damage and liver injury in mice by reducing AP bioavailability and elevating GSH levels.Our current results revealed the novel role of 2-HB in protecting AP-induced liver injury and add new evidence for gut microbiota in affecting AP toxicity.

Macrophage senescence in health and diseases

Macrophage senescence,manifested by the special form of durable cell cycle arrest and chronic low-grade inflammation like senescence-associated secretory phenotype,has long been consid-ered harmful.Persistent senescence of macrophages may lead to maladaptation,immune dysfunction,and finally the development of age-related diseases,infections,autoimmune diseases,and malignancies.However,it is a ubiquitous,multi-factorial,and dynamic complex phenomenon that also plays roles in remodeled processes,including wound repair and embryogenesis.In this review,we summarize some general molecular changes and several specific biomarkers during macrophage senescence,which may bring new sight to recognize senescent macrophages in different conditions.Also,we take an in-depth look at the functional changes in senescent macrophages,including metabolism,autophagy,polarization,phagocytosis,antigen presentation,and infiltration or recruitment.Furthermore,some degenerations and diseases associated with senescent macrophages as well as the mechanisms or relevant genetic regulations of senescent macrophages are integrated,not only emphasizing the possibility of regulating macrophage senescence to benefit age-associated diseases but also has an implication on the finding of potential tar-gets ordrugs clinically.

Targeting PI3K/AKT signaling for treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis(IPF) is a chronic progressive fibrotic interstitial pneumonia with unknown causes. The incidence rate increases year by year and the prognosis is poor without cure.Recently, phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(PKB/AKT) signaling pathway can be considered as a master regulator for IPF. The contribution of the PI3 K/AKT in fibrotic processes is increasingly prominent, with PI3 K/AKT inhibitors currently under clinical evaluation in IPF. Therefore,PI3 K/AKT represents a critical signaling node during fibrogenesis with potential implications for the development of novel anti-fibrotic strategies. This review epitomizes the progress that is being made in understanding the complex interpretation of the cause of IPF, and demonstrates that PI3 K/AKT can directly participate to the greatest extent in the formation of IPF or cooperate with other pathways to promote the development of fibrosis. We further summarize promising PI3 K/AKT inhibitors with IPF treatment benefits, including inhibitors in clinical trials and pre-clinical studies and natural products, and discuss how these inhibitors mitigate fibrotic progression to explore possible potential agents, which will help to develop effective treatment strategies for IPF in the near future.

Ferulic acid prevents liver injury and increases the anti-tumor effect of diosbulbin B in vivo

The present study is designed to investigate the protection by ferulic acid against the hepatotoxicity induced by diosbulbin B and its possible mechanism, and further observe whether ferulic acid augments diosbulbin B- induced anti-tumor activity. The results show that ferulic acid decreases diosbulbin B-increased serum alanine transaminase/aspartate transaminase （ALT/AST） levels. Ferulic acid also decreases lipid peroxide （LPO） levels which are elevated in diosbulbin B-treated mice. Histological evaluation of the liver demonstrates hydropic degeneration in diosbulbin B-treated mice, while ferulic acid reverses this injury. Moreover, the activities of copper- and zinc-containing superoxide dismutase （CuZn-SOD） and catalase （CAT） are decreased in the livers of diosbulbin B-treated mice, while ferulic acid reverses these decreases. Further results demonstrate that the mRNA expressions of CuZn-SOD and CAT in diosbulbin B-treated mouse liver are significantly decreased, while ferulic acid prevents this decrease. In addition, ferulic acid also augments diosbulbin B-induced tumor growth inhibition compared with diosbulbin B alone. Taken together, the present study shows that ferulic acid prevents diosbulbin B-induced liver injury via ameliorating diosbulbin B-induced liver oxidative stress injury and augments diosbulbin B-induced anti-tumor activity.

Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals

Chlorogenic acid（CGA）, a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen（AP）-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase（ALT/AST） in vivo. The effect of CGA on cytochrome P450（CYP） enzymatic（CYP2E1, CYP1A2, and CYP3A4） activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde（MDA）, reactive oxygen species（ROS）, and glutathione（GSH） levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased m RNA expression of peroxiredoxin（Prx） 1, 2, 3, 5, 6, epoxide hydrolase（Ephx） 2, and polymerase（RNA） II（DNA directed） polypeptide K（Polr2k）, and nuclear factor erythroid-2-related factor 2（Nrf2）. In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2 k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury.

Anti-rheumatoid arthritic effect of volatile components in notopterygium incisum in rats via anti-inflammatory and anti-angiogenic activities

Notopterygium incisum(QH) has been used for the treatment of rheumatoid arthritis(RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis(AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner(2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils(5, 15, and 45 μg·mL^(-1)) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.

Yes-associated protein(YAP)and transcriptional coactivator with a PDZ-binding motif(TAZ):a nexus between hypoxia and cancer

Hypoxia is a common feature of solid tumors.As transcription factors,hypoxia-inducible factors(HIFs)are the master regulators of the hypoxic microenvironment;their target genes function in tumorigenesis and tumor development.Intriguingly,both yes-associated protein(YAP)and its paralog transcriptional coactivator with a PDZ-binding motif(TAZ)play fundamental roles in the malignant progression of hypoxic tumors.As downstream effectors of the mammalian Hippo pathway,YAP and/or TAZ(YAP/TAZ)are phosphorylated and sequestered in the cytoplasm by the large tumor suppressor kinase 1/2(LATS1/2)-MOB kinase activator 1(MOB 1)complex,which restricts the transcriptional activity of YAP/TAZ.However,dephosphorylated YAP/TAZ have the ability to translocate to the nucleus where they induce transcription of target genes,most of which are closely related to cancer.Herein we review the tumor-related signaling crosstalk between YAP/TAZ and hypoxia,describe current agents and therapeutic strategies targeting the hypoxia-YAP/TAZ axis,and highlight questions that might have a potential impact in the future.

Opportunities and challenges of incretin-based hypoglycemic agents treating type 2 diabetes mellitus from the perspective of physiological disposition

The treatment of patients with diabetes mellitus, which is characterized by defective insulin secretion and/or the inability of tissues to respond to insulin, has been studied for decades. Many studies have focused on the use of incretin-based hypoglycemic agents in treating type 2 diabetes mellitus(T2DM). These drugs are classified as GLP-1 receptor agonists, which mimic the function of GLP-1,and DPP-4 inhibitors, which avoid GLP-1 degradation. Many incretin-based hypoglycemic agents have been approved and are widely used, and their physiological disposition and structural characteristics are crucial in the discovery of more effective drugs and provide guidance for clinical treatment of T2DM.Here, we summarize the functional mechanisms and other information of the drugs that are currently approved or under research for T2DM treatment. In addition, their physiological disposition, including metabolism, excretion, and potential drug drug interactions, is thoroughly reviewed. We also discuss similarities and differences in metabolism and excretion between GLP-1 receptor agonists and DPP-4 inhibitors. This review may facilitate clinical decision making based on patients' physical conditions and the avoidance of drug drug interactions. Moreover, the identification and development of novel drugs with appropriate physiological dispositions might be inspired.

Discovery of nitazoxanide-based derivatives as autophagy activators for the treatment of Alzheimer’s disease

Drug repurposing is an efficient strategy for new drug discovery.Our latest study found that nitazoxanide(NTZ),an approved anti-parasite drug,was an autophagy activator and could alleviate the symptom of Alzheimer’s disease(AD).In order to further improve the efficacy and discover new chemical entities,a series of NTZ-based derivatives were designed,synthesized,and evaluated as autophagy activator against AD.All compounds were screened by the inhibition of phosphorylation of p70S6K,which was the direct substrate of mammalian target of rapamycin(mTOR)and its phosphorylation level could reflect the mTOR-dependent autophagy level.Among these analogs,compound 22 exhibited excellent potency in promotingβ-amyloid(Aβ)clearance,inhibiting tau phosphorylation,as well as stimulating autophagy both in vitro and in vivo.What’s more,22 could effectively improve the memory and cognitive impairments in APP/PS1 transgenic AD model mice.These results demonstrated that 22 was a potential candidate for the treatment of AD.

Deubiquitination complex platform:A plausible mechanism for regulating the substrate specificity of deubiquitinating enzymes

Deubiquitinating enzymes(DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin-proteasome degradation and are critical for regulating protein expression levels in vivo.Therefore,dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling pathways.To data,extensive studies on the ubiquitin chain specificity of DUBs have been reported,but substrate protein recognition is still not clearly understood.As a breakthrough,the scaffolding role may be significant to substrate protein selectivity.From this perspective,we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate selectivity.Furthermore,we proposed a deubiquitination complex platform(DCP) as a potentially generic mechanism for DUB substrate recognition based on known examples,which might fill the gaps in the understanding of DUB substrate specificity.

Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2.Here we report that nelfinavir,an FDA approved drug for the treatment of HIV infection,is effective against SARS-CoV-2 and COVID-19.Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2(IC50=8.26μM),while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93μM(EC50).In comparison with vehicle-treated animals,rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals.At necropsy,nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude.A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center,which were randomized(1:1)to nelfinavir and control groups,showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days(9.0 vs.14.5 days,P=0.055)and the duration of fever time by 3.8 days(2.8 vs.6.6 days,P=0.014)in mild/moderate COVID-19 patients.The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients,together with its well-established good safety profile in almost all ages and during pregnancy,indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

Comparative metabolomics analysis of unique yellow hawthorn(Crataegus pinnatifida)and red-skinned cultivars reveals a different polyphenol biosynthesis flux and antioxidative and antidiabetic potential

Objectives:This paper aims to investigate the polyphenol metabolite variation and bioactivities of different-colored hawthorn berries(Crataegus pinnatifida).Materials and Methods:Comparative metabolomics analysis between peel and flesh of yellow hawthorn'Jinruyi'(JRY)and red-skinned ones was carried out by UPLC-MS/MS.Antioxidant activities andα-glucosidase inhibition capacity were also tested among different colored hawthorn samples.Results:A total of 453 polyphenols was characterized,among which phenolic acids and flavonoids were abundant,and were closely relevant to the antioxidant capacity of hawthorn fruits.Polyphenol profile showed accession-specific accumulation in peel or flesh of different colored hawthorn.The unique yellow hawthorn was found to have few anthocyanins but showed enhanced flux to synthesize flavones and flavonols,especially flavone C-glycosides and acylated flavonol glycosides.The specific acylation decoration included acetylation,p-coumaroylation,and malonylation.In addition,yellow hawthorn showed excellentα-glucosidase inhibitory effect,which might be associated with the high concentra­tion of 8 polyphenols including 5 phenolic acids,2 flavone C-glycosides,and an acylated flavonol glycoside,namely,quercetin-7-O-(6ʹʹ-malonyl)glucoside.Such acylated flavonol showed the strongest correlation with the inhibition effect of hawthorn fruits onα-glucosidase,and was pre­dicted to have the lowest binding energy with the enzyme according to molecular docking analysis,indicating its great potential as a strongα-glucosidase inhibitor and an important antidiabetic ingredient in yellow hawthorn.Conclusions:The acylated flavonol glycosides and C-glycosyl flavones might be chemotaxonomic markers differentiating varieties and bioactivities of yellow hawthorn from the traditional red-skinned ones.These findings complement the existing knowledge on the metabolite composition and nutritional properties of hawthorn fruits.

Toxicity comparison of different active fractions extracted from radix Sophorae tonkinensis in zebrafish

Radix Sophorae tonkinensis(RST) is a widely used herb in Traditional Chinese Medicine(TCM) for treating infectious and inflammatory diseases. However, the toxicity data for RST are limited. The aim of this work is to assess and compare the toxicity of the whole RST extract and its five active fractions using the zebrafish model. Five active fractions of RST were prepared using five different types of solvents, which included dealkalized water, ethanol, n-butyl ethanol, dichloromethane, and diethyl ether. The chemical profiles of the active fractions were determined by high-performance liquid chromatography(HPLC), and the toxicity observed in the zebrafish model was confirmed using mouse models. In the zebrafish model, cardiovascular toxicity was observed for the fraction extracted using diethyl ether, and hepatotoxicity was observed for the whole RST extract and the fractions extracted using water and ethanol, whereas both cardiovascular and hepatic toxicities were observed for the fractions extracted using n-butyl ethanol and dichloromethane. The hepatotoxicity of the fractions extracted using n-butyl ethanol and dichloromethane was also observed in mice. Our findings provide the toxicity data for RST and its five active fractions through modeling in a zebrafish, and indicate that the different fractions may each have a different toxicity, which is helpful for the optimal use of RST in clinical practice.