The combination of berberine and evodiamine ameliorates high-fat diet-induced non-alcoholic fatty liver disease associated with modulation of gut microbiota in rats

Nonalcoholic fatty liver disease (NAFLD) is considered to be a manifestation of hepatic metabolic syndrome. Some studies on the pathogenesis of NAFLD by targeting gut microbiota have attracted wide attention. Previous studies have demonstrated the positive effects of berberine and evodiamine on metabolic diseases and gut microbiota dysbiosis. However, it is not known whether the combination of berberine and evodiamine (BE) can prevent the development of high-fat diet (HFD)-induced NAFLD. Therefore, we aimed to explore the protective effects of BE on the development of HFD-induced NAFLD from the perspective of the gut microbiota. Gut microbiota profiles were established by high throughput sequencing of the bacterial 16S ribosomal RNA gene. The effects of BE on liver and intestinal tissue, intestinal barrier integrity, and hepatic inflammation were also investigated. The results showed that the abundance and diversity of gut microbiota were enriched by BE treatment, with an increase in beneficial bacteria, such as Lactobacillus, Ruminococcus, and Prevotella, and a decrease in pathogenic bacteria such as Fusobacterium and Lachnospira. In addition, BE effectively improved liver fat accumulation and tissue damage, inhibited the apoptosis of intestinal epithelial cells, increased the contents of intestinal tight junction proteins, and decreased the expression of pro-inflammatory factors. Consequently, BE treatment could be an effective and alternative strategy for alleviating NAFLD by modulating gut microbiota and safeguarding the intestinal barrier.

Study on Liver Injury of Different Components of Evodia Fructus in Vivo and in Vitro / 中国实验方剂学杂志

ObjectiveTo evaluate the in vivo and in vitro toxicity of Evodia Fructus water extraction and its index components， and provide a basis for basic research on the toxic substances of Evodia Fructus. MethodInstitute of Cancer Research（ICR） mice were divided into high， medium and low dose groups of water extraction of Evodia Fructus and a blank control group. The administration groups were respectively given 80，60，40 g·kg-1 water extraction of Evodia Fructus， the blank control group was given distilled water in equal volume， blood was taken 24 hours later to determine the serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）values， the liver was weighed and histopathological examination was performed. Evodia Fructus water extract， evodiamine， rutaecarpine and limonin were respectively acted on HepG2 cells for 24 h， and cell counting kit-8（CCK-8） method was used to investigate the cytotoxicity. The ICR Mice were divided into two groups， one group was given by oral gavage and the other group was given intraperitoneal injection. The two routes of administration were separately given 3 index components of Evodia Fructus， and the dosage was 200 mg·kg-1. Take blood 24 hours after administration to determine the activity of ALT and AST in serum， and take liver to calculate liver index. ResultCompared with the blank group， the high and medium dose groups of Evodia Fructus water extract were depressed 24 hours after administration， and the behavior of the low dose group was not significantly abnormal. The serum biochemical results showed that the activities of serum ALT and AST in the high and medium dose groups were significantly increased （P<0.01）， the activities of serum ALT and AST in the low dose group were significantly increased， and the histopathological results showed that the high and medium dose groups were significantly increased Punctate necrosis and vacuolar degeneration appeared in the liver of the medium dose group， and there was no obvious abnormality in the low dose group. Compared with the blank group， evodiamine and rutaecarpine had a certain inhibitory effect on the proliferation of HepG2 cells， but the inhibitory effect was not strong. Limonin had no significant inhibitory effect on the proliferation of HepG2 cells. Compared with the control group， the 3 index components of Evodia Fructus have no effect after oral administration. There was no significant difference in the activity of ALT and AST in serum of mice， and there was no significant difference in liver index. Intraperitoneal injection of evodiamine and rutaecarpine can cause the activity of serum ALT and AST to increase， and limonin can cause ALT activity was significantly increased （P<0.01）， and the liver index was significantly increased （P<0.05）. ConclusionEvodia Fructus water extract can cause acute liver injury in mice， Oral administration of evodiamine， rutaecarpine and limonin had no damage to the liver of mice. Intraperitoneal administration of evodiamine and rutaecarpine had no effect on liver injury in mice， and intraperitoneal administration of limonin could cause acute liver injury in mice.

Cytotoxicity and underlying mechanism of evodiamine in HepG2 cells / 北京大学学报(医学版)

OBJECTIVE@#To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism.@*METHODS@#HepG2 cells were treated with EVO (0.04-25 μmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 μmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot.@*RESULTS@#The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 μmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 μmol/L, respectively. After exposure to EVO (0.2, 1 and 5 μmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated.@*CONCLUSION@#These results suggested that 0.2, 1 and 5 μmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

Research progress in pharmacology and toxicology of evodiamine / 中国中药杂志

Evodiamine, a bioactive indole alkaloid from Evodia rutaecarpa, E. rutaecarpa var. officinalis, or E. rutaecarpa var. bodinieri, has been extensively investigated due to its pharmacological activities in recent years. At present, evodiamine is proved to significantly suppress the proliferation of a variety of cancer cells and mediate cell processes such as cell cycle arrest and cell migration. In addition, evodiamine displays significant pharmacological activities against cardiovascular diseases(hyperlipidemia, etc.), and tinea manus and pedis. Recently, evodiamine has been found to have potential toxic effects, such as hepatotoxicity, nephrotoxicity, and cardiotoxicity. However, the pharmacological and toxicological mechanism of evodiamine is not clear, and its toxicity in vitro and in vivo has been rarely reported. Therefore, this study reviewed the pharmacological and toxicological articles of evodiamine in recent years, aiming at providing new ideas and references for future research.

Evodiamine inhibits high-fat diet-induced colitis-associated cancer in mice through regulating the gut microbiota / 中西医结合学报

OBJECTIVE@#High-fat diet is one of the main risk factors that disrupt the balance of gut microbiota, which eventually will induce colorectal cancer (CRC). Evodiamine (EVO) is a wildly used multifunctional traditional Chinese medicine extract. In this study, we investigated the role of gut microbiota in high-fat diet-propelled CRC and the potential of EVO for CRC chemoprevention.@*METHODS@#Gut microbiota, serum d-lactic acid and endotoxin from 38 patients with colon cancer and 18 healthy subjects were detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). In addition, body mass index, phospho-signal transducer and activator of transcription 3 (p-STAT3) expression in cancer tissues and paracancerous tissues were detected by immunohistochemistry. A mouse intestinal inflammatory tumor model was established by azomethane/sodium dextran sulfate, followed by treatment with EVO and 5-aminosalicylic acid (ASA). Gut microbiota and inflammatory factors were detected by quantitative polymerase chain reaction, while serum d-lactic acid and endotoxin were detected by ELISA. Furthermore, cell proliferation, cell apoptosis, and interleukin (IL)-6/STAT3/P65 pathway were evaluated by 5-ethynyl-2'-deoxyuridine, terminal-deoxynucleotidyl transferase-mediated nick-end labeling, and Western blot assays.@*RESULTS@#In patients with colon cancer, the numbers of Enterococcus faecalis and Escherichia coli were increased, while those of Bifidobacterium, Campylobacter and Lactobacillus were decreased. Serum endotoxin and d-lactic acid levels and p-STAT3 levels were significantly increased. In the mouse model, both EVO and ASA inhibited tumor formation, decreased the proliferation of tumor cells, and induced apoptosis of tumor cells. Compared with the control group, the numbers of E. faecalis and E. coli were decreased, while Bifidobacterium, Campylobacter and Lactobacillus numbers were increased. In the EVO group, serum endotoxin and d-lactic acid levels and inflammatory factors were significantly decreased. Further, the IL6/STAT3/P65 signaling pathway was inhibited in the EVO group.@*CONCLUSION@#EVO may inhibit the occurrence of colon cancer by regulating gut microbiota and inhibiting intestinal inflammation. The potential mechanism involves inhibition of the IL6/STAT3/P65 signaling pathway, revealing its potential therapeutic significance in clinical applications.

Simultaneous determination of 13 bioactive components in Jiawei Zuojin Pill by HPLC-MS/MS / 中草药

Objective: To develop and validate an high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method for simultaneously qualitative and quantitative determination of 13 major bioactive components (ferulic acid, costunolide, baicalin, paeoniflorin, tetrahydropalmatine, rutecarpine, berberine, palmatine, evodiamine, naringin, hesperidin, saikosaponin a, and saikosaponin d) in Jiawei Zuojin Pills (JZP). Methods: The chromatographic separation was performed on a Thermo Syncronis C18 column (100 mm × 4.6 mm, 3.0 μm) with a gradient elution of methanol and 0.02 mol/L ammonium acetate in water at a flow rate of 0.35 mL/min, and the injection volume was 10 μL. The 13 major bioactive components were detected using an electrospray ionization source in ESI+ and ESI- ionization mode, quantified by multiple reaction monitor (MRM) scanning at the same time. Results: The linear ranges of ferulic acid, costunolide, baicalin, paeoniflorin, tetrahydropalmatine, rutecarpine, berberine, palmatine, evodiamine, naringin, hesperidin, saikosaponin a and saikosaponin d were 2-80 μg/mL (r = 0.999 2), 0.5-20.0 μg/mL (r = 0.999 1), 3.5-140.0 μg/mL (r = 0.999 8), 1-40 μg/mL (r = 0.999 2), 0.3-12.0 μg/mL (r = 0.999 1), 1-40 μg/mL (r = 0.999 2), 3-120 μg/mL (r = 0.999 7), 2.5-100.0 μg/mL (r = 0.999 5), 0.5-20.0 μg/mL (r = 0.999 3), 0.5-20.0 μg/mL (r = 0.999 1), 1-40 μg/mL (r = 0.999 1), 0.3-12.0 μg/mL (r = 0.999 2), 0.3-12.0 μg/mL (r = 0.999 2), and the average recoveries were 99.5% (RSD = 4.11%), 98.9% (RSD = 4.88%), 100.2% (RSD = 1.08%), 99.2% (RSD = 3.23%), 99.5% (RSD = 4.13%), 99.7% (RSD = 3.23%), 98.6% (RSD = 2.78%), 99.9% (RSD = 3.12%), 101.3% (RSD = 4.53%), 98.7% (RSD = 3.43%), 99.8% (RSD = 3.58%), 97.9% (RSD = 5.22%), and 101.3% (RSD = 5.13%), respectively. The contents of 12 batches of the 13 major bioactive components were 0.324-0.383, 0.051-0.072, 3.225-3.466, 0.154-0.198, 0.015-0.062, 0.144-0.199, 2.145-2.982, 0.441-0.953, 0.032-0.099, 0.062-0.089, 0.111-0.178, 0.012-0.065, 0.011-0.069 mg/g, respectively. Conclusion: The developed method is simple, specific, and sensitive, and it can be applied for the determination of 13 major bioactive components and the quality control of JZP.

Quality control of eight substances in Changwei San based on quantitative analysis of multi-components by single-marker / 国际药学研究杂志

Objective: To establish a quantitative analysis of multi-components by single marker(QAMS)method for the simultaneous determination of jaceosidin, eupatilin, limoni, evodiamine, rutaecarpine, cinnamyl alcohol, cinnamic acid and cinnamal-dehyde in Changwei San. Methods: The Waters Symmetry C18 column(250 mm×4.6 mm, 5 μm)was used for the separation, and the mobile phase was the acetonitrile(A)and 0.1% phosphoric acid(B)solution in a gradient elution at a flow rate of 1.0 ml/min. The detection wavelengths were set at 345 nm for jaceosidin and eupatilin, 215 nm for limoni, evodiamine and rutaecarpine, and 275 nm for cinnamyl alcohol, cinnamic acid and cinnamaldehyde. With evodiamine as an internal reference standard, the relative correction factors for the other 7 components were established and their contents were calculated with the relative correction factors to achieve the QAMS, and then the differences between the calculated values by QAMS and measured values by the external standard method(ESM) were compared to validate the accuracy and feasibility of the QAMS method. Results: Jaceosidin, eupatilin, limoni, evodiamine, rutaecarpine, cinnamyl alcohol, cinnamic acid and cinnamaldehyde showed good linear relationships within the ranges of 0.98-19.60, 2.67-53.40, 4.06-81.20, 1.98-39.60, 2.69-53.80, 0.56-11.20, 1.49-29.80, and 8.77-175.40 μg/ml(r≥0.9992), whose average recoveries(RSD) were 98.77%(0.96%), 99.38%(1.01%), 100.02%(0.83%), 97.80%(1.40%), 98.91%(1.18%), 96.99% (1.13%), 98.09%(1. 24%)and 99.10%(0.67%), respectively. No significant difference was observed between the calculated values by QAMS and the measured values by ESM. Conclusion: The established QAMS method is simple and accurate, which might be used to evaluate the quality of Changwei San.

Preparation of evodiamine-glycyrrhizic acid micelles with glycyrrhizic acid as carrier and their anti-hepatic fibrosis activity / 中国中药杂志

This study aimed to prepare evodiamine-glycyrrhizic acid(EVO-GL) micelles to enhance the anti-hepatic fibrosis activity of evodiamine. Firstly, EVO-GL micelles were prepared with use of thin film dispersion method. With particle size, encapsulation efficiency, loading capacity of micelles and the solubility of evodiamine as the indexes, the effect of different factors on micelles was observed to screen the optimal preparation methods and process. Then the pharmaceutical properties and the therapeutic effects of EVO-GL micelles prepared by optimal process were evaluated on CCl_4-induced hepatic fibrosis. The results showed that the micelles prepared by the thin film dispersion method had an even size, with an average particle size of(130.80±12.40)nm, Zeta potential of(-41.61±3.12) mV, encapsulation efficiency of 91.23%±1.22%, drug loading of 8.42%±0.71%, high storage stability at 4 ℃ in 3 months, and slow in vitro release. Experimental results in the treatment of CCl_4-induced hepatic fibrosis in rats showed that EVO-GL micelles had a synergistic anti-hepatic fibrosis effect, which significantly reduced the liver function index of hepatic fibrosis rats. In conclusion, the EVO-GL micelles prepared with glycyrrhizic acid as a carrier would have a potential application prospect for the treatment of hepatic fibrosis.

Pharmacokinetics and in situ intestinal absorption of evodiamine lipidic nanoparticle / 中国药科大学学报

@#The aim of this study was to investigate the in vivo pharmacokinetic behavior characteristics and in situ intestinal absorption characteristics of the evodiamine lipidic nanoparticle in rats. Evodiamine lipidic nanoparticle was prepared by the solvent evaporation methods. The particle size and zeta potential of evodiamine lipidic nanoparticle were measured by dynamic light scattering analysis. Male SD rats were divided into two groups randomly. Each group was given single dose of evodiamine and evodiamine lipidic nanoparticle by gavage at evodiamine dose of 250 mg/kg,respectively. The blood samples were collected at scheduled time points. The content of evodiamine in plasma samples was determined by high performance liquid chromatography (HPLC) method. The main pharmacokinetic parameters of evodiamine and evodiamine lipidic nanoparticle were calculated using DAS 2.1.1 software. Moreover,the single-pass intestinal perfusion model was also established in rats to investigate the in situ intestinal absorption characteristics of evodiamine lipidic nanoparticle. The mean particle size and mean zeta potential of evodiamine lipidic nanoparticle were 180.10 nm and -17.90 mV,respectively. The area under the curve of evodiamine and evodiamine lipidic nanoparticle were (862.60±14.03) and (4084.31±17.21) μg/L·h,respectively,and the peak concentration were (163.40±13.27) and (616.90±21.04) μg/L,respectively. Moreover,the absorption of evodiamine lipidic nanoparticle was significantly higher than that of evodiamine in each segment of intestinal tract in rats (P<0.05). The absorption of evodiamine lipidic nanoparticle in colon was better than those of evodiamine lipidic nanoparticle in stomach,duodenum,jejunum and ileum. The absorption rate constant of evodiamine lipidic nanoparticle in stomach,duodenum,jejunum,ileum and colon were (45.10±6.08)×10-5,(48.20±1.21)×10-5,(22.10±3.18)×10-5,(59.10±1.21)×10-5 and (90.00±3.85)×10-5 s-1,respectively,and the effective permeability coefficient in duodenum,jejunum,ileum and colon was (44.10±0.51)×10-5,(17.21±0.77)×10-5,(35.36±0.31)×10-5 and (40.33±0.34)×10-5 cm/s,respectively.All in all, evodiamine lipidic nanoparticle remarkably improved the in situ intestinal absorption of evodiamine in different segments of the intestinal tract in rats and its oral bioavailability in rats.

Evodiamine induces apoptosis of human hepatoma cell line BEL-7402 by Hippo-YAP pathway / 中草药

Objective: To investigate the effects and the underlying mechanisms of evodiamine (EVO) on apoptosis of hepatocellular carcinoma (HCC) BEL-7402 cells. Methods: Cell counting kit-8 (CCK-8) assay was used to detect the effect of EVO on proliferation activity of BEL-7402 cells. Hoechst 33258 staining was used for observing morphological changes of apoptosis. The cell apoptosis and cycle distribution were analyzed by flow cytometry. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay were used to detect the expression levels of key genes from Hippo-YAP pathway in HL-7702 and BEL-7402 cells, including mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor 1 (LATS1), and Yes-associated protein (YAP), then to examine the effect of EVO on the expression levels of these genes in BEL-7402 cells. The effect of EVO on the expression of YAP in hepatocellular carcinoma BEL-7402 cells was observed by immunofluorescence assay. Results: The proliferation of BEL-7402 cells were significantly inhibited by EVO in a dose- and time-dependent manners. Hoechst 33258 staining showed that EVO induced BEL-7402 cell typical apoptotic morphology, such as nuclear chromatin concentration and edge accumulation. Besides, flow cytometry tests showed that BEL-7402 cell apotosis were increased and cell cycle arrested in G2/M phase after being treated with EVO (P < 0.01). qRT-PCR and Western blotting showed that the expression level of MST2 and LATS1 were lower in BEL-7402 cell (P < 0.05), while the transcription and protein levels of YAP were significantly higher (P < 0.05). EVO could activate Hippo signal pathway, upregulate the expression of MST2 and LATS1 and then inhibit the expression of YAP in BEL-7402 cell (P < 0.05). Immunofluorescence assay also validated that EVO would significantly inhibit the overexpression of YAP in BEL-7402 cell (P < 0.01). Conclusion: EVO can induce the apoptosis of BEL-7402 cells, which may be through activating Hippo signal pathway and then down-regulate the expression of YAP.

Pharmacokinetic study on asparaginase-evodiamine loaded core-shell lipidic nanoparticles / 中国药理学通报

To investigate the pharmacokinetic behavior of core-shell lipidic nanoparticles (AELNs) loaded with asparaginase and evodiamine in rats, and compare the bioavailability of AELNs with free asparaginase (ASNase)/evodiamine (EVO). Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups and injected with AELNs, free ASNase and free EVO, respectively. After injection, blood samples were taken from the eyelids of rats at different time. The concentration of EVO and ASNase in plasma samples was determined by HPLC and Nessler reagent colorimetry method, respectively. To analyze the differences in pharmacokinetic behavior be-tween AELNs and free ASNase or EVO, their pharmacokinetic parameters were calculated using DAS software. Results and MRTom4thy of ASNase in AELNs were 1. 83 and 1. 94 times higher than those of free ASNase, respectively. The AUC(0-48h) of EVO in AELNs was 29. 94 times that of free EVO. Conclusions Compared with free ASNase and EVO, AELNs have a higher bioavailability in rats.

Evodiamine Reduces Caffeine-Induced Sleep Disturbances and Excitation in Mice

Worldwide, caffeine is among the most commonly used stimulatory substances. Unfortunately, significant caffeine consumption is associated with several adverse effects, ranging from sleep disturbances (including insomnia) to cardiovascular problems. This study investigates whether treatment with the Evodia rutaecarpa aqueous extract (ERAE) from berries and its major molecular component, evodiamine, can reduce the adverse caffeine-induced sleep-related and excitation effects. We combined measurements from the pentobarbital-induced sleep test, the open field test, and the locomotor activity test in mice that had been dosed with caffeine. We found that ERAE and evodiamine administration reduced the degree of caffeine-induced sleep disruption during the sleep test. Additionally, we found that evodiamine significantly inhibits caffeine-induced excitation during the open field test, as well as decreasing hyperlocomotion in the locomotor activity test. Additional in vitro experiments showed that caffeine administration decreased the expression of γ-aminobutyric acid (GABA)(A) receptor subunits in the mouse hypothalamus. However, evodiamine treatment significantly reversed this expression reduction. Taken together, our results demonstrate that ERAE and its major compound, evodiamine, provide an excellent candidate for the treatment or prevention of caffeine-induced sleep disturbances and excitatory states, and that the mechanism of these beneficial effects acts, at least in part, through the GABA(A)-ergic system.

In vitro release behavior and in situ absorption of evodiamine loaded microemulsion / 中国药理学通报

Aim To study the characteristics of in vitro release and in situ absorption of evodiamine load-ed microemulsion (EDM).Methods EDM was pre-pared, its release in pH 1.2 HCl solution and pH 6.8 phosphate buffer solution were studied by dialysis , and the cumulative release rates were calculated .The sin-gle-pass intestinal perfusion was used to study the ab-sorption of EDM in duodenum , jejunum , ileum and co-lon, the absorption in stomach was also studied , and the absorption rate constant ( Ka ) and effective perme-ability (Pef ) of the drug were calculated.The concen-tration of ED was measured by HPLC .Results The cumulative release rate of EDM and ED in pH 1.2 HCl solution was ( 64.76 ±0.73 )％, ( 13.98 ±0.49 )％, respectively , and that of EDM was 4.63 times of ED . In pH 6.8 phosphate buffer solution the cumulative re-lease rate was ( 91.72 ± 0.51 )％, ( 18.34 ± 0.20)％, respectively, which was 5.01 times of ED. The Ka of EDM was more than 3 times of ED, and Pef was more than 2 times of ED .Conclusion Microe-mulsion can improve in vitro release and in situ absorp-tion of ED.

Pharmacokinetics and bioequivalence of evodiamine butyryl derivative-loaded lipid nanoparticles in rats / 中草药

Objective To study the pharmacokinetic characteristics and bioequivalence of evodiamine butyryl derivative (EAB) and evodiamine butyryl derivative-loaded lipid nanoparticles (EABLN) in rats. Methods EABLN were prepared by a film ultrasound method, 16 male SD rats were randomly divided into two groups, and their blood were extracted from eye socket after they were intragastric administrated EAB or EABLN (100 mg/kg) for a given time period. Plasma concentration of EAB was determined by HPLC, then pharmacokinetic data, bioequivalence between EAB and EABLN were analyzed by DAS software. Results The main pharmacokinetic parameters of EABLN were listed, AUC(0-72 h), Cmax, and tmax were (11 535.39 ± 261.08) μg∙h/L, (886.73 ± 15.40) μg/L, and (2.00 ± 0.17) h, respectively. Relative bioavailability of EABLN to EAB was 143%, the bioequivalence of AUC(0-72 h) and Cmax in EAB and EABLN were eligible, but bioequivalence of tmax was not eligible. Conclusion EABLN improved the pharmacokinetics of EAB in rats, meanwhile, oral bioavailability of EAB was significantly increased, and there was not bioequivalence between EABLN and EAB.

Simultaneous determination of nine bioactive components in Sishen Pills by HPLC-ESI-MS/MS / 中草药

Objective To develop and validate an high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method for simultaneously qualitative and quantitative determination of nine major bioactive components (deoxyschizandrin, γ-schizandrin, schizandrin, schizandrol B, schisantherin A, psoralen, isopsoralen, evodiamine, and rutaecarpine) in Sishen Pills. Methods The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C18 column (100 mm × 4.6mm, 3.5 μm) with a gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.4 mL/min, and the injection volume was 20 μL. The nine major bioactive components were detected using an electrospray ionization source in positive ionization mode (ESI+) and quantified by multiple reaction monitor (MRM) scanning at the same time. Results The linear ranges of deoxyschizandrin, γ-schizandrin, schizandrin, schizandrol B, schisantherin A, psoralen, isopsoralen, evodiamine, and rutaecarpine were 8.50-850.00 ng/mL (r = 0.999 7), 1.32-132.00 ng/mL (r = 0.997 4), 9.60-960.00 ng/mL (r = 0.999 8), 12.00-1 200.00 ng/mL (r = 0.999 3), 11.50-1 150.00 ng/mL (r = 0.997 9), 21.70-2 170.00 ng/mL (r = 0.999 7), 23.80-2 380.00 ng/mL (r = 0.999 6), 10.70-1 070.00 ng/mL (r = 0.999 5), 8.54-854.00 ng/mL (r = 0.998 0), and the average recoveries were 98.3% (RSD = 2.21%), 100.3% (RSD = 1.78%), 99.2% (RSD = 2.19%), 100.4% (RSD = 2.23%), 99.1% (RSD = 2.18%), 97.7% (RSD = 3.03%), 99.0% (RSD = 2.51%), 98.9% (RSD = 2.72%), and 100.3% (RSD = 2.10%), respectively. The contents of eight batches of the nine major bioactive components were 67.6-425.6, 0-131.5, 2.1-258.0, 0-71.2, 23.2-678.8, 806.4-1310.8, 718.5-1293.7, 11.5-123.2, and 10.9-62.4 μg/g, respectively. Conclusion The developed method is simple, specific, and sensitive, and it can be applied for the determination of nine major bioactive components and the quality control of Sishen Pills collected from different production batches.

Evodiamine activates autophagy and inhibits the proliferation in colon cancer cell / 中草药

Objective To investigate the effect of evodiamine (Evo) on the autophagy and proliferation of colon cancer HCT-116 cells and the underlying mechanism. Methods The effect of Evo on proliferation of HCT-116 cells was detected by CCK-8 method. After being processed with Evo (3 and 6 μmol/L) for 48 h, the number of autophagic vesicles were detected by MDC method. The amount of ROS in HCT-116 cells was measured by DHE assay, and the protein related with autophagy and AMPK/mTOR pathway was detected by Western blotting. After the treatment of Evo (6 μmol/L) combined with autophagy inhibitor 3-MA (3-methyladenine) or apoptosis inhibitor Z-DEVD-FMK respectively for 48 h, Western blotting was used to detect the expression of autophagy and apoptosis-related protein in HCT-116 cells. Results Compared with the control group, Evo inhibited the proliferation of HCT-116 cells in a dose-dependent manner; After treated with Evo (3 and 6 μmol/L) for 48 h, the amount of intracellular ROS and autophagic vesicles were increased, the protein expression levels of LC3, p-AMPK, and mTOR were increased while the expression of p62 was increased. After being treated with Evo and autophagy inhibitor, the protein expression of LC3 was decreased while activated Caspase-3 was increased; Combination of Evo and apoptosis inhibitor increased the expression of LC3 and inhibited the expression of activated Caspase-3. Conclusion Evo can activate autophagy of HCT-116 cells through AMPK/mTOR pathway and inhibit the proliferation, and the effect of autophagy and apoptosis on cells are complementary.

Recent advances in the antitumor effects of evodiamine / 上海交通大学学报（医学版）

Evodiamine is the natural component extracted from Euodiae Fructus. Recently, growing evidence has proved that evodiamine has great effects on suppressing cell viability and proliferation, arresting cell cycle, inducing apoptosis, promoting autophagy, inhibiting the formation of microvascular angiogenesis as well as affecting epigenetic modification in cancer. Recent studies have continuously revealed related signal pathways involved in evodiamine such as PI3K-Akt and JAK-STAT pathways, as well as the impact of evodiamine on survivin, vascular endothelial growth factor and miRNAs. With the development and synthesis of evodiamine derivatives and related herbal formulations, the understanding of antitumor activity of evodiamine is gradually deepening. The important clinical significance and market value of evodiamine can be prospected.

Effect of evodiamine on the proliferation and radiosensitivity of endometrial carcinoma cells / 中华放射医学与防护杂志

Objective To investigate the effect of evodiamine on the proliferation and sensitivity of endometrial cancer cells to irradiation.Methods After administration of evodiamine,cell proliferations of human endometrial carcinoma cell lines of Ishikawa,HEC-1A,AN3CA were detected by MTT and the half maximal inhibitory concentration (IC50) of drug was calculated.The cell lines most sensitive to drug were screened for further experiment and administered with evodiamine (IC50) and 8 Gy irradiation.Then,cell apoptosis was detected by flow cytometry,the levels of Cleaved Caspase-3,p38 and p-p38 were measured by Western blot,and the level of intracellular ROS was detected by a ROS kit.Cell clone survival was also detected to evaluate cell radiosensitivity.Results 1,2,4,6 and 8 μmol/L evodiamine could inhibit the proliferation of the cell line of Ishikawa,HEC-1A,and AN3CA with IC50 of(8.32 ± 0.95),(3.98 ± 0.84) and (4.78 ± 0.64) μmol/L,respectively.Compared with radiation alone,after radiation in combination with 4 μmol/L evodiamine,the apoptosis rate of HEC-1A cells was increased from (45.54 ±4.25)％ to (65.87 ±2.93)％ (t =11.010,P ＜0.05) and cell viability decreased from (41.84±4.18)％ to (33.27 ± 3.52)％ (t =7.484,P ＜0.05),and the levels of ROS,Cleaved Caspase-3 and p-p38 were also enhanced.In addition,the sensitivity ratio of evodiamine for HEC-1A cells was calculated to be 1.628.Conclusions Evodiamine could inhibit the proliferation,promote apoptosis and enhance the radiosensitivity of endometrial carcinoma cells,in which the intracellular ROS and p38 signaling pathway may be involved.

Effect of Evodiamine on Hypoxia Induced Cardiomyocytes Injury / 中国药师

Objective:To investigate the protective effect of evodiamine on cardiomyocytes hypoxia injury. Methods:H9c2 myo-cardial cells were exposed to hypoxia for 24 h to induce myocardial cell injury model. The cells were pretreated with different concentra-tions of evodiamine for 12 h. The cardiomyoctes viability was detected by CCK-8 assay. The transcription of inflammatory cytokines was detected by RT-PCR. The cardiomyocyte apoptosis was detected with Tunel staining. The alteration of signal pathway was detected by immunoblotting. Results:Four concentrations of evodiamine did not affect the activity of cardiomyocytes in the basal condition (P>0.05). After 24-hour hypoxia,the viability of cardiomyocytes decreased significantly when compared with that in the control group with significant difference (P<0.05) among 1,5 and 10 μmol·L-1evodia in a dose-dependent manner. The transcription of pro-inflam-matory cytokines(TNF-α,IL-1 and IL-6) significantly increased and the cells apoptosis increased. Evodiamine pretreatment increased the cell viability after hypoxia injury, reduced the transcription of inflammatory cytokines and reduced the number of apoptotic cells when compared with that in the control group with significant differences(P<0.05) between 1 μmol·L-1and 10 μmol·L-1evodia. The results of western blot showed that evodiamine activated AMPKα and AKT, inhibited the activity of NF- kappa B, and compared with the control group,there were significant differences(P<0.05) between 1 μmol·L-1and 10 μmol·L-1of evodia. Conclusion:Evodiamine can protect cardiomyocytes from hypoxia injury and may become a new anti-myocardial ischemia drug.

Evodiamine activates cellular apoptosis through suppressing PI3K/AKT and activating MAPK in glioma / 中国药理学与毒理学杂志

OBJECTIVE Glioblastoma multiforme (GBM) is the most malignant primary tumor of the central nervous system and is associated with a very poor prognosis. No further improvements in outcomes have been reported since radiotherapy-temozolomide therapy was introduced.Therefore,de-veloping new agents to treat GBM is important. This study aimed to evaluate the anti-tumor effect of evodiamine (Evo) on GBM cells, and to determine the underlying mechanisms involved. METHODS U251,LN229,HEB and PC12 cells were treated with various concentrations of evodiamine for 24 and 48 hours,cell viability was measured by MTT assay.The U251 and LN229 cells were treated with evo-diamine(0-10 μmol·L-1)for 24 h,and then stained with Hoechst 33258.An Annexin V-FITC Apoptosis Detection Kit was used to detect apoptosis in the cells.Reactive oxygen species(ROS)production was detected using dichlorofluorescein diacetate (DCFH-DA) staining. The changes in mitochondrial mem-brane potential (MMP) were assessed by JC-1 after cells were treated with evodiamine. The expres-sion levels of p-PI3K,PI3K,p-Akt,Akt,Bax,Bcl-2,p-p38,p38,p-JNK,JNK,p-ERK,ERK,Cytochrome c, Caspase-3, cleaved Caspase-3, PRAP, and cleaved PARP were measured by Western blot analy-ses. RESULTS According to MTT assay results, Evo significantly inhibited the cell proliferation in a time- and dose-dependent manner. Fluorescence microscopy and flow cytometry analyses revealed that Evo induced cell apoptosis in a concentration-dependent manner.Moreover,Evo induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) disruption. Finally, Evo induced apoptosis in cancer cells by suppressing PI3K/AKT signaling and inducing MAPK phos-phorylation(p38 and JNK,but not ERK)to regulate apoptotic proteins(Bax,Bcl-2,Cytochrome c,Cas-pase-3, and PARP). CONCLUSION In summary, Evo inhibits cell proliferation by inducing cellular apoptosis via suppressing PI3K/AKT and activating MAPK in GBM;these results indicate that Evo may be regarded as a new approach for GBM treatment.