Exploration of 1-(indolin-1-yl)-2-(thiazol-2-yl)ethan-1-one derivatives as novel anti-HBV agent with potential TLR7-agonistic effect.

Hepatitis B virus (HBV) infection, as a serious global public health issue, is closely related to the immune dysfunction. Herein, thirty-seven 1-(indolin-1-yl)-2-(thiazol-4-yl)ethan-1-one derivatives were prepared as potential immunomodulatory anti-HBV agents. Anti-HBV activity evaluation confirmed compound 11a could significantly suppress the HBV DNA replication in both wild and resistant HBV stains, with IC50 values of 0.13 µM and 0.36 µM, respectively. Preliminary action mechanism studies showed that 11a had an inhibitory effect on cellular HBsAg secretion and could effectively activate TLR7, thereby inducing the secretion of TLR7-regulated cytokines IL-12, TNF-α and IFN-α in human PBMC cells. SPR analysis confirmed that 11a could bind to TLR7 protein with an affinity of 7.06 µM. MD simulation predicted that 11a could form tight interactions with residues in the binding pocket of TLR7. Physicochemical parameters perdition and pharmacokinetic analysis indicated that 11a displayed relatively favorable drug-like properties. Considering all the results, compound 11a might be a promising lead for developing novel immunomodulatory anti-HBV agents.

Design and evaluation of dibenzoazepine-tetrahydroisoquinoline hybrids as potential P-glycoprotein inhibitors against multidrug resistant K562/A02 cells.

Multidrug resistance (MDR) caused by P-glycoprotein (P-gp) is a main barrier to the success of cancer chemotherapies. In this study, fourteen novel dibenzoazepine-tetrahydroisoquinoline hybrids were prepared as potential P-gp inhibitors to surmount MDR caused by P-gp. Amongst them, 8a displayed the most potent inhibition effect on P-gp, thus effectively reversing P-gp-mediated drug resistance with a reversal fold (RF) value of 93.17 in K562/A02 cells. Excitingly, the EC50 value of 8a on MDR reversing effect was 48.74 nM, which was nearly two thousand-fold lower than its IC50 value (95.94 µM) for intrinsic cytotoxicity on K562/A02 cells. Further investigation showed that 8a exerted the MDR reversal effect through impairing P-gp function rather than affecting its expression. Molecular docking and CETSA results illustrated that 8a possessed a relatively high affinity for P-gp, thus effectively improving the stability of P-gp. Furthermore, 8a exhibited a much poorer inhibitory effect on CYP3A4 activity than CYP3A4 inhibitor ketoconazole, thus might not cause unfavorable drug-drug interactions. These data together suggested that 8a may be a promising lead to design P-gp inhibitors, and warranted further investigation on overcoming P-gp-mediated MDR.

Discovery of benzimidazole substituted 1, 2, 4-oxadiazole compounds as novel anti-HBV agents with TLR8-agonistic activities.

Hepatitis B virus (HBV) infection is a public health threat worldwide and characterized by a dysfunctional immune response. In the present work, a new series of benzimidazole substituted 1, 2, 4-oxadiazole compounds were designed as immunomodulatory anti-HBV agents. Data showed compound 11o displayed significant in vitro anti-HBV activities against wild-type and nucleos(t)ide analogues-resistant HBV with IC50 values of 0.53 and 0.44 µM, respectively. In contrast, nucleos(t)ide analogue lamivudine is only effective for wild-type HBV (IC50 < 0.1 µM) but not effective for resistant HBV (IC50 > 100 µM). Dual-luciferase reporter gene and ELISA assay revealed that 11o exhibited a dose-dependent effect on inducing TLR8-regulated NF-κB activity, and could promote the secretion of cytokines TNF-α and IL-12 in supernatant from human PBMC cells. Molecular docking studies found that 11o formed tight interactions with binding pocket residues located at the dimer interface of TLR8. Considering the potent in vitro anti-HBV activity, effective TLR8-agonistic potency, and relatively safe profile with a selectivity index (SI) value high above 37, compound 11o deserves further investigation as a potential immunomodulatory anti-HBV agent.

Design and synthesis of novel quinazolinone derivatives as anti-HBV agents with TLR8 agonist effect.

Hepatitis B virus (HBV) infection is a worldwide threat to public health. In this work, a series of novel quinazolinone derivatives (5a-q) were synthesized and evaluated as novel anti-HBV agents. Among them, compound 5l exhibited potent inhibitory effect on HBV DNA replication in both wild type and drug resistant (lamivudine and entecavir) HBV strains with IC50 values of 0.15 and 0.10 µM, respectively. Notably, the selective index value of 5l was high above 66.67, indicating the favorable safety profile. Molecular docking study indicated that compound 5l well fitted into the binding pocket of TLR8 protein-protein interface. Dual-luciferase reporter gene assay further confirmed that compound 5l could dose-dependently activate TLR8, thus effectively inducing the activity of TLR8-dependent NF-κB. Collectively, compound 5l displayed potent anti-HBV activities and TLR8 agonist effect in vitro, and might be a potential immunomodulatory anti-HBV agent to warrant further investigation.

Synthesis and Evaluation of Novel Quinazolinone Derivatives as Potential Anti-HCC Agents.

Hepatocellular carcinoma (HCC), a common malignancy worldwide, has a high mortality rate and limited effective therapeutic options. In this work, a series of quinazolinone compounds (6a-t and 7a-i) were synthesized as potential anti-HCC agents. Among them, compound 7b more potently inhibited HepG2, HUH7 and SK-Hep-1 cells proliferation than classical anti-HCC drug sorafenib, indicating its potential anti-HCC effect. Interestingly, 7b could dose-dependently decrease Cyclin D1 and CDK2 levels, and increase p21 protein expression, thus inducing HepG2 cells cycle arrest at G0/G1 phase. In addition, 7b also displayed potent apoptosis-induced effect on HepG2 cells by interfering Bad, Bax, Bcl-2 and Bcl-xl proteins expression. Notably, 7b could efficiently block the activity of PI3K pathway by dose-dependently reducing the phosphorylation of PI3K (Y607) and AKT (S473). Moreover, predicted ADME properties indicated that 7b possessed a good pharmacokinetic profile. Collectively, compound 7b might be a promising lead to the development of novel therapeutic agents towards HCC.

MiR-122 Participates in Oxidative Stress and Apoptosis in STZ-Induced Pancreatic ß Cells by Regulating PI3K/AKT Signaling Pathway.

At present, there are few reports concerning the relationship between miR-122 and diabetes. In addition, the effect of miR-122 on streptozotocin- (STZ-) induced oxidative damage in INS-1 cells remains unclear. The present study aimed to investigate the role and modulatory mechanisms involving miR-122 in diabetes. STZ was used to induce INS-1 cell damage. Reverse transcription-quantitative PCR was used to investigate the expression of miR-122. A TUNEL cell apoptosis detection kit was used to detect apoptosis. Intracellular ROS levels were determined using dichlorofluorescein-diacetate. The activities of insulin secretion, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) were measured using ELISA kits. Western blotting was used to measure the expression levels of Bax, Bcl-2, PI3K, p-PI3K, caspase-3 and caspase-9, cleaved-caspase-3 and cleaved-caspase-9, AKT, and p-AKT. Then, LY294002 (LY, PI3K inhibitor) was used to treat INS-1 cells, and oxidative stress and apoptosis were measured. The results showed that STZ-induced inhibitory effects on insulin secretion were mitigated by miR-122 inhibitor, and the activities of SOD, CAT, and GSH-px were also increased. Furthermore, miR-122 inhibitor inhibited apoptosis and oxidative stress in STZ-induced INS-1 cells. Finally, the addition of LY increased insulin levels; reduced the activities of SOD, CAT, and GSH-px; and promoted apoptosis in STZ-induced INS-1 cells. In conclusion, interference with miR-122 can inhibit oxidative stress and apoptosis in STZ-induced INS-1 cells, involving a mechanism of action related to the PI3K/AKT pathway.

Is diabetes mellitus a risk factor for low bone density: a systematic review and meta-analysis.

BACKGROUND: This systematic review aimed to investigate whether diabetes mellitus is a risk factor for low bone density, as this might be important and necessary for doctors specialized in treating patients with low bone density. METHODS: PubMed, Embase, CINAHL, and SciELO were searched for cohort, case-control, and cross-sectional studies that investigated the effects of diabetes mellitus on bone mineral density till January 2020. Data screening and extraction are done independently, whereas the methodological quality of the studies was assessed according to the Newcastle-Ottawa Scale (NOS). RESULTS: A total of 14 studies that met the eligibility criteria including 24,340 participants were enrolled. The overall quality of the studies had a scale of over 6 points. The overall odds ratio (OR) regarding the risk of diabetes mellitus in low bone density patients was 1.20 [95% confidence interval (CI)0.80-1.79, P = 0.30], and type 2 diabetes mellitus (T2DM) (OR = 0.69 [0.11, 4.55], P = 0.70). Subgroup analysis revealed that whether females or males, developed or developing countries, T2DM, studies after 2015, and quality over 7 points (all P values > 0.05) showed no significant differences with the risk of low bone density, except type 1 diabetes mellitus (T1DM) (OR = 3.83 [1.64, 8.96], P = 0.002), and studies before 2015 (OR = 1.76 [1.06, 2.92], P = 0.03), and quality below 7 points (OR = 2.27 [1.50, 3.43], P = 0.0001). Funnel plot showed no significant asymmetry. CONCLUSIONS: These findings revealed no relationship between T2DM and low bone density, and also, the evidence between T1DM and low bone density is inadequate, requiring further analysis of well-designed cohort studies.

Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells.

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential antileukemia agents against drug resistant K562/A02 cell overexpressing P-gp. Among them, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could significantly inhibit K562/A02 cells proliferation with an IC50 value of 0.96 µM. Interestingly, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity (CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression, reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide might be a promising lead to discover novel antileukemia agents against P-gp overexpressing leukemic cells.

Synthesis and evaluation of new phenyl acrylamide derivatives as potent non-nucleoside anti-HBV agents.

As a continuation of our previous work, a series of new phenyl acrylamide derivatives (4Aa-g, 4Ba-t, 5 and 6a-c) were designed and synthesized as non-nucleoside anti-HBV agents. Among them, compound 4Bs could potently inhibit HBV DNA replication in wild-type and lamivudine (3TC)/entecavir resistant HBV mutant strains with IC50 values of 0.19 and 0.18 µM, respectively. Notably, the selective index value of 4Bs was above 526, indicating the favorable safety profile. Interestingly, unlike nucleoside analogue 3TC, 4Bs could significantly inhibit 3.5 kb pgRNA expression. Molecular docking study revealed that 4Bs could fit well into the dimer-dimer interface of HBV core protein by hydrophobic, π-π and H-bond interactions. Considering the potent anti-HBV activity, low toxicity and diverse anti-HBV mechanism from that of nucleoside anti-HBV agent 3TC, compound 4Bs might be a promising lead to develop novel non-nucleoside anti-HBV therapeutic agents, and warranted further investigation.

Discovery of thiosemicarbazone-containing compounds with potent anti-proliferation activity against drug-resistant K562/A02 cells.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle to successful chemotherapy for leukemia. In this study, a series of thiosemicarbazone-containing compounds (4a-b, 7a-q) were synthesized. Biological evaluation showed that the most active compound 7e displayed potent anti-leukemia activity against P-gp overexpressing drug-resistant K562/A02 cells, with an IC50 value of 0.44 µM. Notably, compound 7e exhibited a selective killing effect on K562/A02 cells by dose-dependently increasing the intracellular levels of reactive oxygen species (ROS), thus exerting a potential collateral sensitivity (CS)-promoting effect in vitro. Moreover, compound 7e could inhibit HDAC1 and HDAC6, and induce the apoptosis of K562/A02 cells by increasing the expression of Bax, decreasing Bcl-2 protein level, and promoting the cleavage of caspase-3 and PARP, respectively. Overall, 7e may be a potential anti-cancer agent against drug-resistant myelogenous leukemia.

Discovery of novel quinazolinone derivatives as potential anti-HBV and anti-HCC agents.

As a continuation of earlier works, a series of novel quinazolinone derivatives (5a-s) were synthesized and evaluated for their in vitro anti-HBV and anti-hepatocellular carcinoma cell (HCC) activities. Among them, compounds 5j and 5k exhibited most potent inhibitory effect on HBV DNA replication in both drug sensitive and resistant (lamivudine and entecavir) HBV strains. Interestingly, besides the anti-HBV effect, compound 5k could significantly inhibit the proliferation of HepG2, HUH7 and SK- cells, with IC50 values of 5.44, 6.42 and 6.75 µM, respectively, indicating its potential anti-HCC activity. Notably, the in vitro anti-HCC activity of 5k were more potent than that of positive control 5-fluorouracil and sorafenib. Further studies revealed that compound 5k could induce HepG2 cells apoptosis by dose-dependently upregulating Bad and Bax expression and decreasing Bcl-2 and Bcl-xl protein level. Considering the potent anti-HBV and anti-HCC effect, compound 5k might be a promising lead to develop novel therapeutic agents towards HBV infection and HBV-induced HCC.

Synthesis and assessment of phenylacrylamide derivatives as potential anti-oxidant and anti-inflammatory agents.

Oxidative stress and inflammation are major causes of numerous life-threatening human diseases. In the present study, we synthesized a series of phenylacrylamide derivatives as novel anti-oxidant and anti-inflammatory agents. Biological evaluation showed that compound 6a could more potently protect HBZY-1 mesangial cells from H2O2-caused oxidative stress than positive controls resveratrol and sulforaphane by dose- and time-dependently impairing the ROS accumulation. Preliminary anti-oxidant mechanism studies indicated that compound 6a could activate Nrf2 and increase the protein and mRNA expression of downstream anti-oxidant enzymes, ie. NQO-1, HO-1, GCLM and GCLC. Notably, 6a could inhibit the production of NO and the activity of NF-κB in LPS-stimulated HBZY-1 mesangial cells, indicating its potential anti-inflammatory activity. Interestingly, both effects could be significantly attenuated by Nrf2 inhibitor TRG, HO-1 inhibitor ZnPP or GCL inhibitor BSO at non-toxic concentrations, confirming that the anti-oxidant and anti-inflammatory activity of 6a is related to the activation of Nrf2 signaling pathway. These results, together with the relatively safety profile, indicated that compound 6a could be a promising lead to develop novel anti-oxidant and anti-inflammatory agents, thus preventing diseases induced by oxidative stress and inflammation.

Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors.

Hepatitis B virus (HBV) infection is a worldwide public health issue. Search for novel non-nucleoside anti-HBV agents is of great importance. In the present study, a series of quinazolinones derivatives (4a-t and 5a-f) were synthesized and evaluated as novel anti-HBV agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with IC50 values of 1.54 µM and 0.71 µM, respectively. Interestingly, the selective index values of 5f was higher than that of lead compound K284-1405, suggesting 5f possessed relatively safety profile than K284-1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC50 values of 1.90 and 0.84 µM, confirming their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein dominated by hydrophobic interactions. Notably, their binding modes were different from the lead compound K284-1405, which may be attributed to the additional substituent groups in the quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further research as potential non-nucleoside anti-HBV candidates.

Synthesis and biological evaluation of bifendate derivatives bearing acrylamide moiety as novel antioxidant agents.

Oxidative stress plays a significant role in the pathogenesis of various human diseases. In this study, a series of bifendate derivatives bearing acrylamide moiety were synthesized and evaluated as anti-oxidant agents. Biological evaluation indicated that compounds 6a and 6e displayed more potent cytoprotective effect against H2O2-induced HBZY-1 mesangial cells death than lead compound bifendate and positive control resveratrol and sulforaphane. Preliminary anti-oxidant mechanism studies showed that compound 6e could diminish the ROS accumulation by dose- and time-dependently activating Nrf2 and increasing the expression of downstream detoxification enzymes NQO-1, HO-1, GCLM and GCLC at protein and mRNA levels, thus displaying potent anti-oxidant activity. Interestingly, the Nrf2 activating effect of 6e is achieved, at least partly, in Michael acceptor and Keap1-dependent manners. These results, together with the low intrinsic cytotoxicity, suggested that compound 6e might be a promising lead for the development of novel anti-oxidant agents to prevent diseases induced by oxidative stress.

Assessment of a bifendate derivative bearing a 6,7-dihydro-dibenzo[c,e]azepine scaffold as a potential anti-metastatic agent.

Multidrug resistance (MDR) and metastasis are major causes of mortality in patients with cancer. We recently reported a bifendate derivative bearing a dibenzo[c,e]azepine scaffold (4i) as a P-gp and BCRP-medicated MDR reversal agent. As a continuation of the previous research, its ability to inhibit cancer metastasis was investigated in MDA-MB-231 cells in the present work. Wound-healing and chamber migration assays showed that 4i could significantly attenuate the migration of MDA-MB-231 cells. Additionally, 4i obviously suppressed the invasive activity of MDA-MB-231 cells, thus displaying potential anti-metastasis activity. Preliminary mechanism studies indicated that the anti-metastasis activity of 4i was associated with the inhibitory effect on the activity and expression of MMP-2 and MMP-9. These results, together with the previous findings, suggest that compound 4i could be a promising lead for the development of novel anti-cancer agents with anti-MDR and metastatic activities.

Application of a simple desolvation method to increase the formation yield, physical stability and hydrophobic drug encapsulation capacity of chitosan-based nanoparticles.

As one of the classical nano drug carriers, chitosan-based nanoparticles (CsNps) are getting increasing attentions for their non-toxicity, biocompatibility and biodegradability. The ionotropic gelation is the most favorable method for CsNps preparation, while it's beset by some issues, i.e. low particle formation yield, poor physical stability and low hydrophobic drug loading capacity. To solve these problems, a simple desolvation method was attempted in this study for CsNps preparation. Using this method, an increased formation yield of CsNps (about 100%) could be obtained, and after diluting the particles by various buffers (PBS 7.0, 7.4 and 8.0, DMEM medium and 1640 medium), an improved stability in diameter could be observed. The drug loading results showed paclitaxel loading efficiency of 9.8% could be obtained when the ratio of paclitaxel to Cs was set at 0.2, higher than the ionotropic gelation method (0.43%). The particles formation mechanisms were studied by the molecular dynamics simulation, and the results showed the Lennard-Jones interaction of Cs-Cs, which could be adjusted by the pH value of system, played a great role in formation of particles with the desolvation method. This study provided an alternative preparation technology for CsNps with high process yield, physical stability and hydrophobic drug encapsulation capacity.

Design, synthesis and evaluation of novel phenyl propionamide derivatives as non-nucleoside hepatitis B virus inhibitors.

As an ongoing search for potent non-nucleoside anti-HBV agents with novel structures, we described a series of phenyl propionamide derivatives (3a-b, 4a-e, 7a-g, 8a-h and 9a-b) by pharmacophore fusion strategy in the present work. All the compounds exhibited an anti-HBV activity to some extent. Among them, compounds 8d and 9b displayed most potent anti-HBV activity with IC50 values on HBV DNA replication of 0.46 and 0.14 µM, respectively. And the selective index values of 8d and 9b were more than 217.39 and 153.14, suggesting that 8d and 9b exhibited favorable safety profiles. Interestingly, 8d and 9b possessed significantly antiviral activities against lamivudine and entecavir resistant HBV mutants with IC50 values of 0.77 and 0.32 µM. Notably, preliminary anti-HBV action mechanism studies showed that 8d could inhibit intracellular HBV pgRNA and RT activity of the HBV polymerase. Molecular docking studies suggested that compound 8d could fit into the dimer-dimer interface of HBV core protein by hydrophobic interaction. In addition, in silico prediction of physicochemical properties showed that 8d conformed well to the Lipinski's rule of five, suggesting its potential for use as a drug like molecule. Taken together, 8d possessed significantly anti-HBV activity, low toxicity, diverse anti-HBV mechanism and favorable physicochemical properties, and warranted further investigation as a promising non-nucleoside anti-HBV candidate.

Synthesis and Biological Evaluation of Matijin-Su Derivatives as Potential Antihepatitis B Virus and Anticancer Agents.

A series of Matijin-Su (MTS, (2S)-2-{[(2S)-2-benzamido-3-phenylpropanoyl]amino}-3-phenylpropyl acetate) derivatives were synthesized and evaluated for their anti-HBV and cytotoxic activities in vitro. Six compounds (4g, 4j, 5c, 5g, 5h and 5i) showed significant inhibition against HBV DNA replication with the IC50 values in range of 2.18 - 8.55 µm, which were much lower than that of positive control lamivudine (IC50 82.42 µm). In particular, compounds 5h (IC50 2.18 µm; SI 151.59) and 5j (IC50 5.65 µm; SI 51.16) displayed relatively low cytotoxicities, resulting in high SI values. Notably, besides the anti-HBV DNA replication activity, compound 4j also exhibited more potent in vitro cytotoxic activity than 5-fluorouracil in two hepatocellular carcinoma cell (HCC) lines (QGY-7701 and SMMC-7721), indicating that 4j may be a promising lead for the exploration of drugs with dual therapeutic effects on HBV infection and HBV-induced HCC.

LC-MS/MS method for the simultaneous quantification of 11 compounds of Ginkgo biloba extract in lysates of mesangial cell cultured by high glucose.

The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC-MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.

Screening for Potential Bioactive Components in Ginkgo biloba Extract by the Rat Renal Tubular Epithelial Cell Extraction and LC-MS/MS.

Rat renal tubular epithelial cell (RTEC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN). The compounds in GBE binding with cell membrane or entering into cell are still unknown, which may be potential bioactive components. In this paper, a powerful method for screening and analyzing the potential bioactive components from GBE was developed using cell extraction coupled with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 8 prototype compounds and 5 metabolites were obtained, among which 6 prototype compounds and 1 metabolite were identified or tentatively characterized as rutin, bilobalide, ginkgolide B, ginkgolide C, genkwanin, apigenin and diosmetin by comparing their retention times and MS spectra with those of authentic standards or literature data. The 6 prototype compounds were further quantitatively analyzed using electrospray ionization in negative mode multiple reaction monitoring (MRM). The results showed that high glucose changed the Tmax, MRT(0-t), Cmax and AUC(0-t) of all observed compounds and decreased the t1/2 of genkwanin and apigenin, significantly. The overall findings indicate that 8 prototype compounds may be the potential bioactive components of GBE with preventive effect against DN and the method of RTEC extraction coupled with LC-MS/MS technology screening method we developed is a feasible, rapid, and useful tool for screening and analyzing potential bioactive components.