Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Qi-dan-dihuang decoction ameliorates renal fibrosis in diabetic rats via p38MAPK/AKT/mTOR signaling pathway.

CONTEXT: Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. OBJECTIVE: This study reveals the mechanism by which QDD ameliorates DKD. MATERIALS AND METHODS: The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. RESULTS: A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways. DISCUSSION AND CONCLUSION: QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD.

Mulberry (Morus alba L.) leaf water extract attenuates type 2 diabetes mellitus by regulating gut microbiota dysbiosis, lipopolysaccharide elevation and endocannabinoid system disorder.

ETHNOPHARMACOLOGICAL RELEVANCE: Mulberry (Morus alba L.) leaf is a well-known herbal medicine and has been used to treat diabetes in China for thousands of years. Our previous studies have proven mulberry leaf water extract (MLWE) could improve type 2 diabetes mellitus (T2D). However, it is still unclear whether MLWE could mitigate T2D by regulating gut microbiota dysbiosis and thereof improve intestinal permeability and metabolic dysfunction through modulation of lipopolysaccharide (LPS) and endocannabinoid system (eCBs). AIM OF STUDY: This study aims to explore the potential mechanism of MLWE on the regulation of metabolic function disorder of T2D mice from the aspects of gut microbiota, LPS and eCBs. MATERIALS AND METHODS: Gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. LPS, N-arachidonoylethanolamine (AEA) and 2-ararchidonylglycerol (2-AG) contents in blood were determined by kits or liquid phase chromatography coupled with triple quadrupole tandem mass spectrometry, respectively. The receptors, enzymes or tight junction protein related to eCBs or gut barrier were detected by RT-PCR or Western blot, respectively. RESULTS: MLWE reduced the serum levels of AEA, 2-AG and LPS, decreased the expressions of N-acylphophatidylethanolamine phospholipase D, diacylglycerol lipase-α and cyclooxygenase 2, and increased the expressions of fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), alpha/beta hydrolases domain 6/12 in the liver and ileum and occludin, monoacylglycerol lipase and cannabinoid receptor 1 in the ileum of T2D mice. Furthermore, MLWE could change the abundances of the genera including Acetatifactor, Anaerovorax, Bilophila, Colidextribacter, Dubosiella, Gastranaerophilales, Lachnospiraceae_NK4A136_group, Oscillibacter and Rikenella related to LPS, AEA and/or 2-AG. Moreover, obvious improvement of MLWE treatment on serum AEA level, ileum occludin expression, and liver FAAH and NAAA expression could be observed in germ-free-mimic T2D mice. CONCLUSION: MLWE could ameliorate intestinal permeability, inflammation, and glucose and lipid metabolism imbalance of T2D by regulating gut microbiota, LPS and eCBs.

Mulberry leaf water extract alleviates type 2 diabetes in mice via modulating gut microbiota-host co-metabolism of branched-chain amino acid.

Elevations in circling branched-chain amino acids (BCAAs) levels associated with insulin resistance and type 2 diabetes mellitus (T2DM). Morus alba L. water extracts (MLE) show hypoglycemic function, but the precise mechanism remains obscure. This study is designed to investigate the association of the antidiabetes effect of MLE with the BCAAs co-metabolism modulated by host and gut microbiota. Tissue-specific expressions of BCAA-catabolizing enzymes were detected by RT-PCR and western blot, respectively. The components of the intestinal microflora were analyzed by high-throughput 16S rRNA gene sequencing. The results showed that MLE administration improved blood glucose and insulin level, decreased inflammatory cytokines expression, and lowered serum and feces BCAAs levels. Furthermore, MLE reversed the abundance changes of the bacterial genera correlated with serum and feces BCAAs, such as Anaerovorax, Bilophila, Blautia, Colidextribacter, Dubosiella, Intestinimonas, Lachnoclostridium, Lachnospiraceae_NK4A136, Oscillibacter, and Roseburia. Functionality prediction indicated that MLE potentially inhibited bacterial BCAAs biosynthesis, and promoted the tissue-specific expression of BCAAs catabolic enzyme. More importantly, MLE had obvious impacts on BCAA catabolism in germ-free-mimic T2DM mice. Those results indicated that MLE improving T2DM-related biochemical abnormalities is associated with not only gut microbiota modification but also the tissue-specific expression of BCAAs catabolic enzyme.

The Correlation between Traditional Chinese Medicine Constitution and Hyperuricemia and Gout: A Systematic Review and Meta-Analysis.

Objective: To investigate the correlation between the constitution of traditional Chinese medicine (TCM) and hyperuricemia (HUA) and gout. Method: Databases including China National Knowledge Infrastructure (CNKI), WanFang Data, China Science and Technology Journal Database (VIP), China Biology Medicine Disc (CBMdisc), PubMed, The Cochrane Library, Web of Science, and Excerpta Medical Database (Embase) were searched to collect observational studies about TCM constitution in HUA and gout from inception to November 21, 2021. The distribution of TCM constitution types in HUA and gout patients was presented by proportion, while the correlation was presented by odds ratio (OR) and 95% CI. Meta-analysis was performed using StataCorp Stata (STATA) version 16.0 software. Results: Twenty-one cross-sectional studies and 10 case-control studies involving 38028 samples were included, among which 27526 patients were diagnosed with HUA and 2048 patients with gout. Phlegm-dampness constitution (PDC), damp-heat constitution (DHC), and qi-deficiency constitution (QDC) are the most common types, accounting for 24% (20%-27%), 22% (16%-27%), and 15% (12%-18%), respectively, in HUA patients, while DHC, PDC, and blood stasis constitution (BSC) accounted for 28% (18%-39%), 23% (17%-29%), and 11% (8%-15%), respectively, in gout patients. PDC and DHC were the main constitution types in patients with HUA or gout in south China, east China, north China, southwest China, northwest China, and northeast China. There was no difference in the distribution of PDC and QDC in male or female patients with HUA, while males with DHC in HUA were more common than females. The proportion of PDC or DHC among HUA patients was 1.93 times and 2.14 times higher than that in the general population (OR and 95% CI: 1.93 (1.27, 2.93), 2.14 (1.47, 3.13)), while the proportions of PDC, DHC, and BSC were 3.59 times, 4.85 times, and 4.35 times higher than that of the general groups (OR and 95% CI: 3.59 (1.65, 7.80), 4.85 (1.62, 14.57), and 4.35(2.33, 8.11)). Conclusion: PDC, DHC, and QDC are the main constitution types of patients with HUA, while PDC and QDC may be the risk factors for HUA. DHC, PDC, and BSC are the main constitution types of patients with gout, and they may be the risk factors for gout. In clinical and scientific research, more attention should be paid to the relationship between the above-mentioned TCM constitution in HUA or gout. Nevertheless, because the quality of the included observational studies is low, more prospective cohort studies related to TCM constitution and HUA or gout can be carried out to verify the causality between TCM constitution and HUA or gout.

Jiawei Yanghe Decoction suppresses breast cancer by regulating immune responses via JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Yanghe Decoction (JWYHD) is a widely used traditional Chinese medicine prescription in the clinical setting for the treatment of autoimmune diseases. Many studies showed that JWYHD has anti-tumor activities in cell and animal models. However, the anti-breast cancer effects of JWYHD and the underlying mechanisms of action remain unknown. AIM OF STUDY: This study aimed to determine the anti-breast cancer effect and reveal the underlying mechanisms of action in vivo, in vitro and in silico. MATERIALS AND METHODS: Orthotopic xenograft breast cancer mouse model and inflammatory zebrafish model were used to observe the anti-tumor effect and immune cell regulation of JWYHD. Moreover, the anti-inflammatory effect of JWYHD were evaluated by the expression of RAW 264.7 cells. JWYHD active ingredients were obtained by UPLC-MS/MS and potential targets were screened by network pharmacology. The therapeutic targets and signaling pathways predicted by computer were assessed by Western blot, real-time PCR (RT-PCR), immunohistochemistry (IHC) staining, and Enzyme-linked immunosorbent assays (ELISA) to explore the therapeutic mechanism of JWYHD against breast cancer. At last, Colivelin and Stattic were used to explore the effect of JWYHD on JAK2/STAT3 pathway. RESULTS: JWYHD significantly decreased the tumor growth in a dose-dependent manner in the orthotopic xenograft breast cancer mouse model. Flow cytometry and IHC results indicated that JWYHD decreased the expressions of M2 macrophages and Treg while increasing M1 macrophages. Meanwhile, ELISA and Western blot results showed a decrease in IL-1ß, IL-6, TNFα, PTGS2 and VEGFα in tumor tissue of JWYHD groups. The results were also verified in LPS-induced RAW264.7 cells and zebrafish inflammatory models. TUNEL assay and IHC results showed that JWYHD significantly induced apoptosis. Seventy-two major compounds in JWYHD were identified by UPLC-MS/MS and Network pharmacology. It was found that the significant binding affinity of JWYHD to TNFα, PTGS2, EGFR, STAT3, VEGFα and their expressions were inhibited by JWYHD. IHC and Western blot analysis showed that JWYHD could decrease the expression of JAK2/STAT3 pathway. Furthermore, Colivelin could reverse the decrease effect of JWYHD in vitro. CONCLUSION: JWYHD exerts a significant anti-tumor effect mainly by inhibiting inflammation, activating immune responses and inducing apoptosis via the JAK2/STAT3 signaling pathway. Our findings provide strong pharmacological evidence for the clinical application of JWYHD in the management of breast cancer.

Diprenylated flavonoids from licorice induce death of SW480 colorectal cancer cells by promoting autophagy: Activities of lupalbigenin and 6,8-diprenylgenistein.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a well-known herbal medicine, and we previously found that several licorice prenylated flavonoids could cause death of SW480 colorectal cancer cells by promoting autophagy. Given many kinds of prenylated flavonoids in licorice, the activities of other compounds deserve further investigation. In addition, the contribution of isoprenyl groups on the autophagy promotion activities has not been clarified. AIM OF THE STUDY: This study aimed to investigate whether lupalbigenin (LPB) and 6,8-diprenylgenistein (DPG), two licorice diprenylated flavonoids, could induce autophagic cell death of SW480 cells, and clarify the contribution of isoprenyl groups. MATERIALS AND METHODS: Cytotoxic activities of LPB and DPG were tested by using an MTT method, and apoptosis induction effects were evaluated by PI-Annexin V staining-based flow cytometry and protein levels of caspase-3 and PARP-1. Autophagy promotion effects of LPB and DPG were assessed by protein levels of LC3, p62, Akt and mTOR as well as number of autophagosomes in cells, and autophagy inhibitor chloroquine (CQ) was involved to identify the role of autophagy on LPB or DPG-caused death of SW480 cells. In addition, two groups of structurally similar diprenylated, mono-prenylated and free flavonoids were obtained from licorice, which were used to investigate the contribution of isoprenyl groups on their autophagy promotion activities. RESULTS: Both LPB and DPG significantly induced apoptosis of SW480 cells with strong cytotoxic activities, and meanwhile, they also promoted autophagy probably through the Akt/mTOR signaling pathway. Further studies indicated that LPB and DPG could induce autophagic cell death of SW480 cells. Moreover, isoprenyl groups contributed mainly to the cytotoxic and autophagy promotion activities of licorice prenylated flavonoids. CONCLUSION: This study reported for the first time that licorice diprenylated flavonoids LPB and DPG induced death of SW480 cells by promoting autophagy, which was mainly attributed to the isoprenyl groups. The results provided theoretical basis for researches on anti-colorectal cancer drugs and their structural modification.

Analysis of plasma free amino acids in diabetic rat and the intervention of Ginkgo biloba leaves extract using hydrophilic interaction liquid chromatography coupled with tandem mass-spectrometry.

Amino acids (AAs) are important metabolites that are related with diabetes. However, their roles in the initiation and development of diabetes mellitus (DM), especially in the treatment of Ginkgo biloba leaves extract (GBE) have not been fully explored. Thus, we investigated the roles that AAs played in the progression and GBE supplementation of DM rat induced by streptozotocin. The rats were randomly divided into a normal control group treated with drug-free solution, a normal control group treated with GBE, a DM group treated with drug-free solution, and DM group treated with GBE; and maintained on this protocol for 9 weeks. Rat plasma was collected from the sixth week to the ninth week and then analyzed with the optimized hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method. A total of 17 AAs with differential levels were monitored to indicate dysfunction of AAs metabolism to confirm the occurrence and development of DM. Treatment with GBE partially reversed the changes seen in seven AAs including leucine, isoleucine, tyrosine, glutamic acid, asparagines, lysine and alanine in DM rats, indicating that GBE could prevent the occurrence and development of DM by acting on AAs metabolism. The improvement of those AAs metabolism disorders may play a considerable role in the treatment of GBE on the occurrence and development of DM. Those findings potentially promote the understanding of the pathogenic progression of DM and reveal the therapeutic mechanism of GBE against DM.

Morus alba L. water extract changes gut microbiota and fecal metabolome in mice induced by high-fat and high-sucrose diet plus low-dose streptozotocin.

Gut microbiota plays a key role in the pathophysiology of type 2 diabetes mellitus (T2D). Mulberry leaf has a hypoglycemic effect, but the potential mechanism is not fully understood. This study aimed to explore the influences and potential mechanisms of mulberry leaf water extract (MLWE) intervention on mice with T2D induced through a high-fat and high-sucrose diet combined with streptozotocin by the combination of fecal metabolomics and gut microbiota analysis. Results showed that MLWE could decrease fasting blood glucose and body weight while ameliorating lipid profiles, insulin resistance, liver inflammation, and the accumulation of lipid droplets in T2D mice. MLWE could reverse the abundances of the phyla Actinobacteria and Bacteroidetes and the ratio of Firmicutes/Bacteroidetes, and increase the abundances of the phyla Cyanobacteria and Epsilonbacteraeota in the feces of T2D mice. The abundances of genera Alloprevotella, Parabacteroides, Muribaculaceae, and Romboutsia in the feces of T2D mice could be reversed, while Oscillatoriales_cyanobacterium and Gastranaerophilales could be reinforced by MLWE supplementation. The levels of nine metabolites in the feces of T2D mice were improved, among which glycine, Phe-Pro, urocanic acid, phylloquinone, and lactate were correlated with Romboutsia and Gastranaerophilales. Taken together, we conclude that MLWE can effectively alleviate T2D by mediating the host-microbial metabolic axis.

Jia Wei Xiao Yao San ameliorates chronic stress-induced depression-like behaviors in mice by regulating the gut microbiome and brain metabolome in relation to purine metabolism.

BACKGROUND: The pathogenesis of depression remains largely unknown. Accumulating evidence demonstrates the existence of a complex relationship between gut microbiome composition and brain functions. Jia Wei Xiao Yao San (JWXYS) is considered a potential antidepressant. However, the pharmacological mechanisms of JWXYS have not yet been clarified. PURPOSE: This study aimed to explore the effects of JWXYS on chronic stress-induced depression-like behaviors in mice. METHODS: A chronic restraint stress mouse model of depression was established. JWXYS was administered, and the responses of these mice to treatment were evaluated through several behavioral tests. The activity of astrocytes and microglia was detected by specific fluorescent labels. Inflammatory cytokines were quantified in intestinal and cerebral tissues. An integrated approach with full-length 16S rRNA sequencing and different types of untargeted metabolomics was conducted to investigate the relationship between the gut microbiome at the species level, metabolic brain functions, and JWXYS. RESULTS: We found that behavioral symptoms were associated with the relative abundance of Lactobacillus animalis. After JWXYS treatment, the relative abundance of Ileibacterium valens with enzymes potentially involved in purine metabolism was also described. The activation of astrocytes and microglia was negatively correlated with the relative abundance of L. animalis. Combined with network pharmacological analysis, several targets predicted based on JWXYS treatment focused on purine metabolism, which was also enriched from cerebral metabolites regulated by JWXYS. CONCLUSION: Our study suggests that L. animalis is involved in depression-like behaviors in mice. JWXYS increases the abundance of I. valens with potential enzymes in relation to cerebral purine metabolism, which is positively correlated with the activation of astrocytes in the amygdala.

Amelioration of lipid accumulations and metabolism disorders in differentiation and development of 3T3-L1 adipocytes through mulberry leaf water extract.

BACKGROUND: Obesity is a worldwide problem that resulted from the excessive fat accumulation in adipose tissue, leading to the impairment of individual health. Mulberry leaf is an important traditional Chinese medicine and has been used to alleviate obesity for a long term. However, its underlying molecular mechanisms have not been fully elucidated yet. PURPOSE: In this study, we aimed to investigate the inhibition effects of mulberry leaf water extract (MLWE) on lipid accumulation during the process of differentiation of 3T3-L1 preadipocytes and development of mature adipocytes through the combination of molecular biology assays and metabolomic analysis. METHODS: The quality consistency and main chemical ingredients of MLWE were analyzed by high performance liquid chromatography and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), respectively. Oil red O staining was used to mirror lipid accumulation. Lipogenesis-, lipolysis- and inflammation-related genes were evaluated by real-time PCR and western blot, respectively. Untargeted metabolomics were performed by LC-MS/MS. RESULTS: Prepared method and quality of MLWE were stable and reliable. A total of 34 compounds were identified and 14 of them were undoubtedly confirmed. MLWE supplementation could dose-dependently inhibit the aggregation of lipid droplets, and the expressions of sterol regulatory element-binding protein (SREBP)-1c, peroxisome proliferator-activated receptor (PPAR) γ, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), tumor necrosis factor (TNF)-α and interleukin (IL)-6, and increase the expressions of adenosine monophosphate-activated protein kinase (AMPK), hormone-sensitive lipase (HSL) and IL-10 in the differentiation of preadipocytes. Furthermore, MLWE treatment could dose-dependently decrease the level of triglycerides and the expressions of ACC, FAS, TNF-α, and IL-6, and up-regulate the level of glycerol and the expressions of PPARα, adiponectin (ADPN), adiponectin receptor (AdipoR) 1, AdipoR2, AMPK, HSL, and IL-10 in the development of mature adipocytes. Untargeted metabolomics showed that a total of 5 and 18 differential metabolites were reversed by MLWE intervention in the differentiation of preadipocytes and the development of mature adipocytes, respectively, which involved in the biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism. CONCLUSION: Taken together, this study firstly verified that MLWE could effectively alleviate lipid accumulation and inflammation by regulating ADPN/AMPK-mediated signaling pathways and relevant metabolic disturbances including biosynthesis of unsaturated fatty acids, arachidonic acid metabolism and glycerophospholipids metabolism.

Prenylated phenolic compounds from licorice (Glycyrrhiza uralensis) and their anti-inflammatory activity against osteoarthritis.

Osteoarthritis is a significant driver of disability in the elderly with increasing prevalence, and inflammation plays a vital role on its etiology. Licorice is commonly used as a traditional Chinese medicine or food additive, and its prenylated phenolic compounds were recently reported to be able to inhibit osteoarthritis with anti-inflammatory activity. In order to explore more anti-osteoarthritic prenylated phenolic compounds from licorice, we isolated ten compounds (1-10), with three new ones (1-3), from the ethyl acetate extract of Glycyrrhiza uralensis. Compound 2 (glycyuralin R) was a racemic 3-phenoxy-chromanone, and we achieved its chiral separation for the first time. Compounds 1, 2, 7 and 8 showed significant NO inhibitory ability in IL-1ß-stimulated mouse primary chondrocytes, and we further confirmed the anti-inflammatory activity of 1 (glycyuralin Q) by evaluating its effect on osteoarthritis-related iNOS, COX-2, TNF-α, IL-6, MMP3, MMP13 and NF-κB based on various experimental methods. These results clarified the potential of several prenylated phenolic compounds, especially 1 in licorice, as the lead compounds for osteoarthritis.

Isoangustone A induces autophagic cell death in colorectal cancer cells by activating AMPK signaling.

Phytochemicals, especially flavonoids, have been widely investigated for their diversified pharmacological activities including anticancer activities. Previously we identified isoangustone A from licorice-derived compounds as a potent inducer of cell death. In the present study, the exact mechanism by which isoangustone A induced cell death was further investigated, with autophagy as an indispensible part of this process. Isoangustone A treatment activated autophagic signaling and induced a complete autophagic flux in colorectal cancer cells. Knockdown of ATG5 or pre-treatment with autophagy inhibitors significantly reversed isoangustone A-induced apoptotic signaling and loss of cell viability, suggesting autophagy plays an important role in isoangustone A-induced cell death. Isoangustone A inhibited Akt/mTOR signaling, and overexpressing of a constitutively activated Akt mildly suppressed isoangustone A-induced cell death. More importantly, isoangustone A inhibited cellular ATP level and activated AMPK, and pre-treatment with AMPK inhibitor or overexpression of dominant negative AMPKα2 significantly reversed isoangustone A-induced autophagy and cell death. Further study shows isoangustone A dose-dependently inhibited mitochondrial respiration, which could be responsible for isoangustone A-induced activation of AMPK. Finally, isoangustone A at a dosage of 10 mg/kg potently activated AMPK and autophagic signaling in and inhibited the growth of SW480 human colorectal xenograft in vivo. Taken together, induction of autophagy through activation of AMPK is an important mechanism by which isoangustone A inhibits tumor growth, and isoangustone A deserves further investigation as a promising anti-cancer agent.

Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes.

BACKGROUND: Protection of pancreatic islet cells against dysfunction or death by regulating autophagy is considered to be an effective method for treatment of type 2 diabetes mellitus (T2DM). Morus alba leaves (mulberry leaves), a popular herbal medicine, have been used for prevention of T2DM since ancient times. PURPOSE: This study aimed to clarify whether Morus alba leaves ethanol extract (MLE) could protect islet cells in vivo and in vitro by regulating autophagy in T2DM, and explore the possible mechanism of action. METHODS: The main chemical constituents in MLE were analyzed by HPLC. The T2DM rat model was induced via high-fat diet combined with peritoneal injection of low-dose streptozotocin, and MLE was administered by oral gavage. Fasting blood glucose (FBG) and plasma insulin were measured, and homeostatic model assessment of ß cell function (HOMA-ß) and insulin resistance (HOMA-IR) were determined. The histomorphology of pancreas islets was evaluated by haematoxylin and eosin staining. In palmitic acid (PA)-stressed INS-1 rat insulinoma cells, cell viability was assayed by an MTT method. Expression of the autophagy-related proteins LC3 I/II, p62, p-AMPK and p-mTOR in islet tissues and INS-1 cells was evaluated by western blotting or immunohistochemistry analysis. RESULTS: The four main chemical constituents in MLE were identified as chlorogenic acid, rutin, isoquercitrin and quercitrin. MLE ameliorated hyperglycemia, insulin resistance and dyslipidemia of T2DM rats with prominent therapeutic effect. Further study indicated that MLE observably improved islet function, alleviated islet injury of T2DM rats, and inhibited PA-induced INS-1 cell death. On the other hand, MLE significantly induced autophagy in islet cells both in vivo and in vitro, and autophagy inhibitors abolished its therapeutic effect on T2DM rats and protective effect on islet cells. Apart from this, MLE markedly activated the AMPK/mTOR pathway in INS-1 cells, and the AMPK inhibitor prevented the autophagy induction ability of MLE. CONCLUSION: Together, MLE could protect islet cells against dysfunction and death by inducing AMPK/mTOR-mediated autophagy in T2DM, and these findings provide a new perspective for understanding the treatment mechanism of Morus alba leaves against T2DM.

Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored. AIM OF STUDY: To clarify the metabolism of liquiritigenin and liquiritin in mice. MATERIALS AND METHODS: In this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching. RESULTS: A total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation. CONCLUSION: This study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice.

Segmented scan modes and polarity-based LC-MS for pharmacokinetic interaction study between Fufang Danshen Dripping Pill and Clopidogrel Bisulfate Tablet.

Fufang Danshen Dripping Pill (FDDP) and Clopidogrel Bisulfate Tablet (CBT) are usually combined for treatment of coronary artery diseases in clinical. To investigate the pharmacokinetic interaction between FDDP and CBT after oral administration of FDDP, CBT and their combination in rats, a novel LC-MS method with segmented scan modes (multiple reaction monitoring and selected ion monitoring) and polarity (positive and negative ionization) was developed. Clopidogrel and the main active ingredients of FDDP, with different chemical and ionization properties, were simultaneously quantified in plasma in a single run. The method was validated in terms of specificity, linearity, precision, accuracy, recovery, matrix effect and stability. As a result, co-administration of FDDP and CBT significantly altered the pharmacokinetic parameters of danshensu, ginsenoside Rb1, dihydrotanshinone I, tanshinone I and tanshinone IIA of FDDP, as well as clopidogrel. Mechanism studies suggested that induction of liver cytochrome P450 isozymes CYP2C11 and CYP3A1 by co-administration, as well as inhibition of carboxyl esterase 1, was partly responsible for FDDP-CBT pharmacokinetic interactions. The developed LC-MS method could be used to simultaneously quantify different types of in vivo analytes in a single run, and the results could be used for clinical medication guidance of FDDP and CBT.

P450 enzymes-based metabolic interactions between monarch drugs and the other constituent herbs: A strategy to explore compatibility mechanism of Sangju-Yin.

BACKGROUND: Herbal compatibility of compound formulas can enhance therapeutic effects or reduce side effects of the monarch drugs, but majority of compatibility mechanisms are still unknown. Sangju-Yin, a well-known Chinese compound formula, is currently used to treat common cold in clinical. PURPOSE: In this study, we proposed a strategy to explore the compatibility mechanism of Sangju-Yin by investigating P450 enzymes-based metabolic interactions between monarch drugs and the other constituent herbs. METHODS: Under the guidance of traditional Chinese medicine theory, the constituent herbs of Sangju-Yin were divided into four groups, including monarch drugs, monarch drugs with addition of minister drugs, monarch drugs with addition of minister and adjuvant drugs, as well as the whole recipe, namely monarch drugs with addition of minister, adjuvant and conductant drugs. Their effects on rats in vivo P450 (CYP1A2, CYP2A3, CYP2C6, CYP2C11 and CYP3A1) activities after oral administration were evaluated using probe drug assay based on LC-MS/MS. Moreover, effects of the four groups of herbs on mRNA expression of P450 enzymes after oral administration, as well as in vitro P450 activities after co-incubation, were investigated to explore the underlying mechanisms. RESULTS: Comparing with monarch drugs, addition of different constituent herbs significantly enhanced CYP1A2 and CYP2C6 activities, and inhibited CYP2A3 and CYP3A1 activities, indicating their possible influences on plasma concentrations of active constituents in the monarch drugs. Mechanism study suggested that these herbs affected P450 activities by transcriptional regulation and/or direct interaction with the enzymes. CONCLUSION: This study clarified the compatibility mechanism of Sangju-Yin from the aspect of P450 enzymes-based metabolic interactions, which would benefit better understanding of the therapeutic basis of Sangju-Yin.

Interaction of clopidogrel and fufang danshen dripping pills assay in coronary heart disease based on non-target metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Clopidogrel is the recommended treatment by current clinical practice guidelines to prevent adverse cardiovascular events in patients with coronary heart disease (CHD), but this treatment regimen still fails and 5-40% patients display inadequate antiplatelet responses. Fufang Danshen Dripping Pill (FDDP), a Chinese patient drug, was used as the combination with clopidogrel to improve the therapeutic effect. However, the mechanism of the interaction between clopidogrel and FDDP has not been elucidated. MATERIALS AND METHODS: We have used non-targeted metabolism method based on GC-MS and LC-MS for the investigation of drug interactions between clopidogrel and FDDP. 63 patients were divided into four groups with different dosage regimen and the serum samples were collected for the analysis. RESULTS: We have found 5 and 55 differential metabolites between health volunteer group and CHD patients group, respectively. The contents of these differential metabolites had diverse changes in clopidogrel group, FDDP group, and drug combination group, indicating that the clopidogrel and FDDP combination can adjust the glycometabolism, lipid metabolism, and phospholipid metabolism, sequentially made the content of downstream related metabolites towards to the health volunteer group. CONCLUSION: This work has explained the mechanism of the interaction between clopidogrel and FDDP from the point of view of metabolic product change, and revealed the potential metabolic pathways it affects, which provided the new ideas for clinical medication.