Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism

The obstruction of post-insulin receptor signaling is the main mechanism of insulin-resistant diabetes. Progestin and adipoQ receptor 3 (PAQR3), a key regulator of inflammation and metabolism, can negatively regulate the PI3K/AKT signaling pathway. Here, we report that gentiopicroside (GPS), the main bioactive secoiridoid glycoside of Gentiana manshurica Kitagawa, decreased lipid synthesis and increased glucose utilization in palmitic acid (PA) treated HepG2 cells. Additionally, GPS improved glycolipid metabolism in streptozotocin (STZ) treated high-fat diet (HFD)-induced diabetic mice. Our findings revealed that GPS promoted the activation of the PI3K/AKT axis by facilitating DNA-binding protein 2 (DDB2)-mediated PAQR3 ubiquitinated degradation. Moreover, results of surface plasmon resonance (SPR), microscale thermophoresis (MST) and thermal shift assay (TSA) indicated that GPS directly binds to PAQR3. Results of molecular docking and cellular thermal shift assay (CETSA) revealed that GPS directly bound to the amino acids of the PAQR3 NH2-terminus including Leu40, Asp42, Glu69, Tyr125 and Ser129, and spatially inhibited the interaction between PAQR3 and the PI3K catalytic subunit (P110α) to restore the PI3K/AKT signaling pathway. In summary, our study identified GPS, which inhibits PAQR3 expression and directly targets PAQR3 to restore insulin signaling pathway, as a potential drug candidate for the treatment of diabetes.

Synthesis, and anti-inflammatory activities of gentiopicroside derivatives / 中国天然药物

A series of 26 novel derivatives have been synthesized through structural modification of gentiopicroside, a lead COX-2 inhibitor. And their in vivo and in vitro anti-inflammatory activities have been investigated. The in vitro anti-inflammatory activities were evaluated against NO, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by LPS. Results showed that most compounds had good inhibitory activity. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Results demonstrated that several compounds were more active than the parent compound gentiopicroside. The inhibition rate of the most active compound P23 (57.26%) was higher than positive control drug celecoxib (46.05%) at dose 0.28 mmol·kg-1. Molecular docking suggested that these compounds might bind to COX-2 and iNOS. Some of them, e.g P7, P14, P16, P21, P23, and P24, had high docking scores in accordance with their potency of the anti-inflammatory activitiy, that downregulation of the inflammatory factors, NO, PGE2, and IL-6, was possibly associated with the suppression of iNOS and COX-2. Therefore, these gentiopicroside derivatives may represent a novel class of COX-2 and iNOS inhibitors.

Protective Effect of Gentiopicroside Against Liver Injury in Mice via TLR4/MyD88/NF-κB Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the mechanism of gentiopicroside （GPS） in preventing acute liver injury induced by carbon tetrachloride （CCl₄） in mice and its effect on the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） signaling pathway. Method:Sixty mice were randomly divided into a normal control group， a model group， a silymarin group （150 mg·kg^-1）， and high- （200 mg·kg^-1）， medium- （100 mg·kg^-1）， and low-dose （50 mg·kg^-1） GPS groups， with 10 in each group. The mice in the groups with drug intervention were administered correspondingly by gavage at 10 mL·kg^-1， and those in the normal control group and the model group receive an equal volume of distilled water， once per day. Ten days after administration， mice in the normal control group were subjected to the intraperitoneal injection of peanut oil （10 mL·kg^-1） and those in other groups were injected with peanut oil （10 mL·kg^-1） containing 0.12% CCl₄for the induction of acute liver injury model. After fasting for 16 hours， blood was collected from eyeballs and liver tissues were collected. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of liver tissues. The content or activities of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBIL）， alkaline phosphatase （ALP）， total superoxide dismutase （T-SOD）， and <italic>γ</italic>-glutamyl transpeptidase （<italic>γ</italic>-GT） in the serum， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） in liver tissues were determined by biochemistry techniques. The levels of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， and interleukin-6 （IL-6） in liver tissues were measured by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the protein expression of TLR4， MyD88， and NF-<italic>κ</italic>B in liver tissues. The expression of phosphorylated NF-<italic>κ</italic>B （p-NF-<italic>κ</italic>B） was detected by immunohistochemistry. Result:Compared with the normal control group， the model group showed increased levels of ALT， AST， ALP， TBIL， <italic>γ</italic>-GT， and MDA （<italic>P</italic><0.01）， and blunted activities of T-SOD and GSH-Px （<italic>P</italic><0.01）. Compared with the model group， the high- and medium-dose GPS groups exhibited declining levels of ALT， AST， ALP， TBIL， <italic>γ</italic>-GT， and MDA （<italic>P</italic><0.05， <italic>P</italic><0.01） and potentiated T-SOD and GSH-Px activities （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the normal control group， the model group displayed elevated levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and IL-6 in liver tissues （<italic>P</italic><0.01） and increased protein expression of TLR4， MyD88， and p-NF-<italic>κ</italic>B （<italic>P</italic><0.01）. Compared with the model group， the high- and medium-dose GPS groups showed decreased TNF-<italic>α</italic>， IL-1<italic>β</italic>， and IL-6 content in liver tissues （<italic>P</italic><0.05， <italic>P</italic><0.01） and dwindled TLR4， MyD88， and p-NF-<italic>κ</italic>B protein expression （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:GPS possesses a protective effect on mice with acute liver injury induced by CCl₄， and its mechanism of action may be related to the regulation of TLR4/MyD88/NF-<italic>κ</italic>B signaling pathway and inhibition of oxidative stress.

Study on transdermal permeation of ethanol extracts from roots of Gentiana crassicaulis in vitro and in vivo / 中草药

Objective: To establish a determination method for kinds of chemical components of iridoids in the roots of Gentiana crassicaulis in transdermal absorption liquid, and research its transdermal permeability, so as to provide scientific basis for transdermal delivery system, clinical medication and reform of the traditional forms of G. crassicaulis. Methods: Based on the results of the previous investigation, in this paper, using the roots of G. crassicaulis as the research subject, a certain concentration solution of G. crassicaulis extract was prepared by the alcohol extraction method. Three kinds of common penetration enhancers, azone, borneol and propylene glycol were used. The effects of single penetration enhancer and dual compound penetration enhancers on the transdermal penetration of five kinds of chemical components of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside in vitro and the three kinds of chemical components of gentiopicroside, loganic acid and swertimarin in vivo were quantitatively studied by the method of HPLC to investigate the transdermal permeability of G. crassicaulis extract in mice skin model. Results: According to the experimental results, compared to the control group, penetration enhancers significantly increased the absorption of five chemical components of G. crassicaulis in vitro. Transdermal absorption rates (J) of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside were 12.306 0, 1.248 8, 4.187 5, 153.030 0 and 5.012 6 μg/(cm2∙h), respectively. The transdermal enhancer effects of A (5% azone), B (5% borneol), C (5% propylene glycol), A + B (2.5% azone and 2.5% borneol), A + C (2.5% azone and 2.5% propylene glycol), A + C (2.5% borneol and 2.5% propylene glycol) were 9.73, 2.57, 13.94, 15.92 and 8.08 times faster than the control group, respectively. Among these, the group of A + C had a marked osmotic enhancer effect in vitro. In comparison with the control group, the in vivo percutaneous penetration test indicated that the dual compound penetration enhancers of 2.5% azone and 2.5% propylene glycol had a marked effect for the permeability enhancement. Conclusion: This study showed azone and propylene glycol significantly promoted the percutaneous penetration effect of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside of G. crassicaulis, and this study laid a foundation for the quality control of percutaneous drug delivery preparation of G. crassicaulis.

Anti-inflammatory Effect of Gentiopicroside in Adjuvant-induced Arthritis Rats / 中国实验方剂学杂志

Objective::To study the therapeutic and inflammatory effects of gentiopicroside(GPS) on adjuvant-induced arthritis (AA) rats. Method::The 36 male SD rats were randomly divided into six groups, namely the model group, GPS groups (low, medium and high dose), and the methotrexate (MTX) group, with six rats in each group. AA rats were induced through intradermal injection with 0.1 mL complete Freund's adjuvant (CFA) into the right hind paw, except the normal group. After modeling, rats in each group were treated with drugs for 7 days, once a day. The doses were 30, 60, 120 mg·kg-1 in the GPS groups, and 0.2 mg·kg-1 in the MTX group. The normal group and the model group were intragastrically treated with the same volume of normal saline. During the experiment, the paw thickness and paw volume of rats were recorded everyday by the digital vernier calipers and the toe volume measuring instrument. On the seventh day, X-ray and histopathological examination of the ankle joints were performed by the small animal living imaging instrument and hematoxylin eosin stain. Blood samples were collected from the abdominal aorta at the end of the experiment to determine the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in serum by enzyme-linked immunosorbent assay(ELISA) kits. The mRNA levels of IL-6 and TNF-α in synovial tissues were determined by Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR). Result::Compared with the normal group, the results of each index in the model group were significantly different (P<0.01). Compared with the model group, the results of paw volume and paw thickness decreased significantly (P<0.01), TNF-α level decreased significantly (P<0.01), and IL-6 and TNF-α mRNA levels decreased significantly (P<0.05, P<0.01) in drug treated groups. The results of X-ray and histopathological examinations indicated that GPS had a protective effect on the ankle joints of AA rats. Conclusion::GPS has the therapeutic effect on AA rats by inhibiting levels of proinflammatory cytokines in serum and relevant mRNA levels in synovial tissues.

Effects of Gentiopicroside on the Apoptosis of Human Pancreatic Cancer Cells PANC- 1 and IL- 6/JAK2/STAT3 Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of gentiopicroside on the apo ptosis o f human pancreatic cancer cells PANC- 1，and to explore its mechanism from the perspective of IL- 6/JAK2/STAT3 signaling pathway. METHODS ：Using PANC- 1 cells as model ， the proliferation inhibition rate of cells was tested by MTT assay after treated with 0（negative contro ），2，4，8，16，32，64，128 mg/L gentiopicroside for 72 h and IC 50 were calculated. The cells were divided into negative control group ，gemcitabine group （positive control，4 mg/L）and gentiopicroside low-concentration ，medium-concentration and high-concentration groups （15，30，60 mg/L）. After cultured for 1，3，5，7 d，Trypan blue exclusion staining was used to count the survival cell ，and the growth of cells was investigated. After cultured for 72 h，colony formation assay was used to observe colony formation rate of cells ；the apoptotic rate of cells was detected by Hoechst 33258 staining；the mRNA and protein expressions of IL- 6，JAK2，STAT3 in cells were detected by RT-PCR and Western blotting assay. RESULTS ：4-28 mg/L gentiopicroside could inhibit the proliferation of cells （P＜0.05 or P＜ 0.01），in concentration dependent trend ；IC50 was 9.54 mg/L. Compared with negative control group ，survival cell count （cultured from 3，5，7 d），mRNA and protein expressions of IL- 6，JAK2 and STAT 3 in cells were decreased significantly in gemcitabine group ， gentiopicroside medium-concentration and high-concentration groups （P＜0.05 or P＜0.01），while the apoptotic rate was increased significantly （P＜0.01）. The colony formation rate of cellswere decreased significantly in gemcitabine group and gentiopicroside high-concentration group （P＜0.01）. mail：hb.gz@163.com Compared with gemcitabine group ，there was no statistical significance in above indexes of gentiopicroside high- 6716008。 concentration group （P＞0.05）. CONC LUSIONS：30，60 mg/L gentiopicroside could inhibit the proliferation and induce apoptosis of PANC- 1 cells，and 60 mg/L gentiopicroside is similar to gemcitabine in the effects. Its mechanism may be related to inhibiting the activation of IL- 6/JAK2/STAT3 signaling pathway.

Research progress on anti-inflammatory activity of three kinds of iridoid glycosides in Chinese materia medica / 中草药

Iridoid glycosides are widely distributed in Chinese materia medica (CMM) with various biological activities such as anti-inflammation. They are also used as quality control constituents in some Chinese medicines. Iridoid glycosides are usually divided into nine-carbon skeleton iridoid glycoside type, ten-carbon skeleton iridoid glycoside type, and secoiridoid glycoside type. In this paper, 15 representative iridoid glycosides from three types which are received extensive attention (including geniposide, catalpol, gentiopicroside, etc) have been selected. Their anti-inflammatory effects and possible related mechanisms are summarized to find out the acting feature of different types. Through comparing the structures and function characteristics, it was concluded that the anti-inflammatory effects of iridoid glycosides were mostly related to NF-κB pathway and MAPK pathway. They have obvious inhibitory effects on TNF-α, IL-6, and IL-1β inflammatory factors, some of which could play a role by reducing the expression of iNOS and COX-2 in NLRP3, Nrf2/HO-1, PI3K, and other pathways. From the structure-activity relationship, the double bond on the cyclopentane, the C-11 substituent and the bond formation after ring opening in iridoid glycosides all have important effects on its anti-inflammatory activities.

Simultaneous determination of eight components before and after processing of Gentiana crassicaulis by UPLC-Q-Orbitrap HRMS / 中草药

Objective: To develop and validate a ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) Methods: for simultaneously qualitative and quantitative determination of eight chemical components (gentiopicroside, loganic acid, swertiamarin, sweroside, luteolin, isovitexin, apigenin, and kaempferol) in raw and processed products of Gentiana crassicaulis. Methods Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column was used for gradient elution with methanol-0.1% formic acid in mobile phase. The volume flow was 0.3 mL/min and the column temperature was 35 ℃. The mass spectrometer was detected using negative ion detection mode. Results: After frying and wine frying, the content of iridoid in G. crassicaulis was significantly increased. Statistically, the differences between gentiopicroside and loganic acid after frying were significant. The content of flavonoids changed little after frying and wine frying. The total content changes of the two components were as follows: the content of iridoids: frying > wine frying > raw products; flavonoids content: raw products > wine frying > frying. Conclusion: Based on UPLC-Q-Orbitrap HRMS, the quantitative analysis method of eight components of raw and processed products of G. crassicaulis was established, which provided a basis for optimizing the processing technology of G. crassicaulis and improving the clinical curative effect of G. crassicaulis.

Correlation Between Effective Components Content and Color Values of Gentianae Radix et Rhizoma Based on Color Difference Principle / 中国实验方剂学杂志

Objective:To measure the color values of Gentianae Radix et Rhizoma and the contents of gentiopicroside,the appearance index value of the representative color was correlated with the intrinsic index value representing the quality,in order to explore the correlation between the contents of active ingredients and the color values,and provide basis for the quality evaluation of Gentianae Radix et Rhizoma. Method:The color difference values of Gentianae Radix et Rhizoma powder was measured by colorimeter.The content determination method of gentiopicroside in the 2015 edition of China Pharmacopoeia was adopted. The content of gentiopicroside was determined by HPLC,and the correlation and regression analysis was carried out by SPSS 21.0 software. Result:There was a significantly positive correlation between the contents of gentiopicroside and L* (representing colorshade)and E* ab(representing total color difference)(Pa* (representing color red-green direction) and b* (representing color yellow-blue direction) had a significantly negative correlation (PConclusion:The color value of Gentianae Radix et Rhizoma has a certain correlation with the content of gentiopicroside,and yellowish Gentianae Radix et Rhizoma contain more active ingredients, with a better quality.The contents of gentiopicroside active ingredients in Gentianae Radix et Rhizoma can be quickly predicted by determining the color difference values,which can provide a new idea for quality evaluation of this herb.

Anti-inflammatory activities of gentiopicroside against iNOS and COX-2 targets / 中草药·英文版

Objective: To isolate high-purity gentiopicroside from the Chinese herbal Gentiana officinalis and investigate its anti-inflammatory activity against iNOS and COX-2 targets. Methods: The purity and structures of gentiopicroside were determined by HPLC, IR, NMR, and MS. The anti-inflammatory effects of gentiopicroside were investigated by in vivo, in vitro, and molecular experiments. Results: In vitro experiment results showed that gentiopicroside inhibited nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-6 (IL-6) production in mouse macrophages RAW 264.7 stimulated by lipopolysaccharide. In vivo experiment found that xylene-induced mouse ear swelling was inhibited by gentiopicroside with an inhibition rate of 34.17%. Molecular docking of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) with gentiopicroside showed that hydrogen bonds (H-bonds) were formed between the sugar fragments in gentiopicroside structure with Tyr355, Ser353, Leu352, Ser530, Arg120, and His90 of COX-2, and Glu377, Asp382, Tyr373, Tyr347, Gln263, Asn370, and Gly371 of iNOS. Thus, gentiopicroside had a lower docking score and displayed satisfactory anti-inflammatory activities. Conclusion: These results suggested that the mechanism of anti-inflammatory activity of gentiopicroside was associated with the downregulation of inflammatory cytokines, such as NO, PGE2, and IL-6, and the suppression of iNOS and COX-2. Therefore, gentiopicroside is a potential and selective iNOS and COX-2 inhibitor.

Effects of different drying methods on iridoids and flavonoids in Gentiana crassicaulis based on UPLC-ESI-HRMSn / 中草药

Objective The effects of far infrared, vacuum, freezing, hot air, dry in the shade and “sweating” drying methods on the quality of Gentiana crassicaulis were evaluated through chemical component content determination. Methods A UPLC-ESI-HRMSn method was used to determine quality score of four iridoids (swertiamarin, gentiopicroside, loganic acid, and sweroside) and four flavonoids (luteolin, isovitexin, apigenin, and kaempferol) of G. crassicaulis. In order to determine the best drying method, the results were combined with analysis of variance, cluster analysis and TOPSIS analysis, which could comprehensively evaluate the G. crassicaulis obtained with different drying methods. Results The contents of swertiamarin in samples dried in different methods were similar. The contents of gentiopicroside, loganic acid, sweroside, luteolin, isovitexin, apigenin, and kaempferol in samples dried in different methods were obviously different. Among them, the four iridoids were best preserved in the freezing drying samples, and the four flavonoids were best preserved in the “sweating” drying samples. All compounds significantly degraded in the sample dried in the shade. Cluster analysis and TOPSIS analysis showed that the samples of “sweating” and freezing drying methods had a similar and high quality, follow by the samples of hot air and far infrared ray. The samples dried in the shade had the worst quality. Conclusion It was noteworthy that the effects of different drying methods on the quality of G. crassicaulis. This study showed that the “sweating” drying method as official method listed in Chinese Pharmacopoeia was verified to be scientific. However, the traditional “sweating” method needed a long time for drying. In order to enhance the drying efficiency, further research should focus on the combination of “sweating” method and modern drying techniques, such as hot air, freeze, vacuum drying methods, which could shorten the drying time on the basis of the stable quality of G. crasicaulis herbs.

Effect of gentiopicroside on AMPKα signaling pathway of rats with non-alcoholic fatty liver disease / 中草药

Objective To investigate the therapeutic effect of gentiopicroside has a on rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat and high-sucrose diet, and explore its mechanism. Methods After 10 d adaptive feeding, 60 male SD rats were randomly divided into the normal group, model group, gentiopicroside low, medium, high dose treatment groups, and positive drug polyeno phosphoryl choline (PPC) intervention group. Except for the normal group, the rats in other groups were received a high fat and glucose diet for 12 weeks to establish NAFLD model; After model successfully established, gentiopicroside low, medium, and high dosetreatment groups were given 50, 100, and 200 mg/(kg•d) gentiopicroside, PPC group was ig given 23 mg/(kg•d) PPC, and 500 μL/(kg•d) saline was given to the normal and model groups. After treated for eight weeks, the rats were sacrificed, and the serum was collected from rats to detect the liver function, blood lipid, serum oxidation, antioxidant capacity, and inflammatory factors. HE staining was used to observe pathological changes of liver. In addition, western blotting and qRT-PCR were used to detect the expression of AMPKα and p-AMPKα. Results HE staining showed that the size of liver cells in the normal group was uniform and the nuclei were evenly distributed, there were obvious vacuoles and a certain inflammatory reaction in the model group. Compared with the model group, gentiopicroside treatment group and PPC group (especially the gentiopicroside middle and high dose group) had a significant improvement, but there were still some differences compared with the normal group; Compared with the normal group, the AST, ALT, HDL-C, LDL-C, MDA, IL-1, and IL-6 in the model group were significantly increased (P 0.05); The results of Western blotting and qRT-PCR showed that compared with the normal group, the expression of p-AMPKα protein and AMPKα mRNA in the model group was significantly decreased (P<0.05). After gentiopicroside and PPC administration, they were significantly increased (P<0.05), and gentiopicroside groups showed a significant dose-dependent manner, and the middle dose and high dose of gentiopicroside groups were better than the PPC group (P<0.05), while the expression of AMPKα protein has no significant difference in each group (P<0.05). Conclusion The NAFLD rats showed a obvious hepatic fat infiltration and dyslipidemia, the liver function index and inflammatory factors levels were elevated, and the anti-oxidant capacity was decreased. Gentiopicroside significantly improved above symptoms, which may be associated with the increased expression of p-AMPKα in liver tissue of NAFLD rats by gentiopicroside.

Optimization of preparation of Haoqin-Huaban granules / 国际中医中药杂志

Objective@#To optimize the preparation process for Haoqin-Huaban granules so as to provide experimental basis for the development and utilization of the compound.@*Methods@#Taking the extraction rate of gentiopicroside from Gentiana macrophylla Pall as the observation index, L9(34)orthogonal test was used to investigate the amount of water, extraction time, and extraction frequency for optimizing the water extracting technology of the Haoqin-Huaban granules. Then the dry paste was used for the raw material, the ratio of the diluent (dextrin) and the concentration and dosage of the binder (ethanol) were investigated by single factor, and the particles were prepared.@*Results@#The optimum water extraction process was A2B2C3, which indicated that the prescribed medicinal materials with 8 times water were extracted 1 h for 3 times. The extract was concentrated into paste, vacuum drying, pulverizing, mixing according to the ratio of 5:1, with ethanol, made of soft material, pelletizing, drying at 60 ℃ for granulation, in order to make Haoqin-Huaban granules.@*Conclusions@#The preparation process is reasonable and feasible, which provides experimental basis for the industrial production of Haoqin-Huaban granules.

Prescription Optimization and Evaluation of Quality of Gentiopicroside Nanoemulsion / 中国中医药信息杂志

Objective To optimize prescription of gentiopicroside nanoemulsion; To evaluate its quality and security. Methods According to the basic properties of gentiopicroside, single-factor test and pseudo-ternary phase diagram were adopted to optimize prescription of gentiopicroside nanoemulsion. The physicochemical property was investigated, and the content was evaluated by HPLC. Results Optimal prescription was obtained as gentiopicroside/ethyl acetate/ EL-35/ absolute ethyl alcohol/ ultrapure water (1.77:7.66:5.94:11.91:72.71); the particle size of nanoemulsion was 23.08 nm; the content of gentiopicroside was determined by HPLC; the specificity of the method was good with a linear range of 0.004 28–0.214 mg/mL; stability of prepared gentiopicroside nanoemulsion was good in the state of different temperatures, illumination intensity and humidity conditions, without significant changes. Conclusion The optimized preparation technology of gentiopicroside nanoemulsion is simple, repeatable and stable, which can provide references for improving the bioavailability of gentiopicroside.

Gentiopicroside,originated from Gentiana macrophylla Pall,possesses anti-arthritic efficacy in adjuvant-induced arthritis rats / 中国药理学与毒理学杂志

OBJECTIVE This work aimed to investigate the anti-rheumatoid arthritic effect of gentio-picroside from Gentiana macrophylla Pall using an animal model of adjuvant induced arthritis. METH-ODS Adjuvant arthritis was induced in fifty SD male rats,which were randomly divided into five groups (n=10):control(0.5% CMC-Na)group,AIA(rats with CFA)group,dexamethasone(1 mg·kg-1)group, gentiopicroside(50 mg·kg-1)group,and gentiopicroside(100 mg·kg-1)group.Rats were administered intragastrically with drugs or CMC-Na once a day for a period of 2 weeks.Paw swelling,arthritic index, histological changes were assessed to evaluate the anti-arthritic effect.Weight growth,spleen and thymus indexes were also investigated in.RESULTS Gentiopicroside at dose of 100 mg·kg-1significantly inhibited the secondary paw swelling(P<0.05)and arthritis index(P<0.05),decreased synovial inflammatory infil-tration, synovial hyperplasia and bone erosion. Furthermore, gentiopicroside showed no immunosup-pressive adverse effects in body weight, index of spleen and thyums compared with dexamethasone administration (P<0.05, P<0.01). CONCLUSION Gentiopicroside possessed anti-arthritic efficacy in AIA rats without immunosuppressive effects.

Gentiopicroside ameliorates LKB1/AMPK-dependent alcoholic hepatosteatosis via P2x7R-NLRP3 inflammasome / 中国药理学与毒理学杂志

OBJECTIVE Regulating P2x7R- NLRP3 inflammasome activation might be a potentialtherapeutic strategy to treat alcoholic hepatosteatosis. We investigated whether this process would be modulated by gentiopicroside (GPS), which is attributed to the bitterness of gentian root extract. METHODSAn in vivo model was established by intragastrically treating mice with ethanol, and an in vitro modelwas created by treating HepG2 cells with ethanol or treating RAW 264.7 macrophages and murinebone marrow- derived macrophages (BMDMs) with lipopolysaccharides (LPS) plus adenosine triphos-phate (ATP). RESULTS In alcoholic hepatosteatotic mice model, GPS decreased serum aminotrans-ferase and triglyceride accumulation. GPS regulated sterol regulatory element-binding protein-1 (Srebp1),peroxisome proliferators- actived receptors α (PPARα) and acetyl CoA carboxylase (ACC) expressionvia elevating liver kinase B1 (LKB1)/AMP-activated Kinase (AMPK). Suppression of nucleotide-bindingoligomerization domain-like receptor protein 3 (NLRP3), caspase-1 and expression by GPS resulted inthe inhibition of interleukin-1β (IL-1β) production. In ethanol-exposed HepG2 cells, GPS reduced lipo-genesis and promoted lipid oxidation via P2x7R- NLRP3 inflammasome activation. P2x7R silencingenhanced AMPK activity, and reduced Srebp1 expression in ethanol-treated hepatocytes. GPS down-regulated P2x7R-mediated inflammatory response to extracellular ATP in LPS-primed RAW 264.7 macro-phages and BMDMs. Additionally, P2x7R deficiency attenuated IL- 1β cleavage in RAW 264.7 macro-phages, and GPS further suppressed IL-1β cleavage. CONCLUSION Activation of LKB1/AMPK signalingby GPS might be mediated by P2x7R-NLRP3 inflammasome, suggesting a therapeutic utility of P2x7Rblockade in alcoholic hepatosteatosis treatment.

Determination of the raw material feeding (Gentiana scabra, Katsumade Galangal Seed and dried tangerine peel) in compound gentian tincture by HPLC-MS/MS / 国际中医中药杂志

Objective To establish a HPLC-MS/MS method for comprehensive monitor and control of the raw material feeding (Gentiana scabra,Katsumade Galangal Seed and dried tangerine peel),and determination of Gentiopicroside,Alpinetin,Cardamonin and Hesperidin in Compound gentian tincture.Methods The separation was performed on an Inertsil ODS-3 (4.6 mm× 150 mm,5 μm) analytical column with the mobile phase consisting of acetonitrile-0.1％ formic acid solution by gradient elution program,and the column temperature was 40 ℃.Active ingredients were separated by HPLC.The Electrospray Ionization Mass (ESI) source was applied and operated in the negative ion mode,and reactions ion monitoring mode (MRM) for quantitative analysis were selected.Results Through the analysis of the samples with mixed extract the same characteristic peak in MS was found to determine the proprietary Chinese medicine according to the prescription feeding process.The calibration curve of Gentiopicroside,Alpinetin,Cardamonin and Hesperidin were linear:103.26-619.56 μg (r=0.999 0),109.50-657.00 μg (r=0.999 5),105.50-633.00 μg (r=0.996 9),105.02-630.12 μg (r=0.999 5).The precision was Gentiopicroside 0.81％,Alpinetin 0.48％,Cardamonin 0.61％ and Hesperidin 1.55％ respectively.The average recovery rate were Gentiopicroside 95.08％,Alpinetin 93.28％,Cardamonin 94.78％ and Hesperidin 95.04％ respectively.Conclusions The method was proved to be simple,accurate,reliable,high sensitivity and can be used for determination and control of the raw material feeding (Gentiana scabra,Katsumade Galangal Seed and dried tangerine peel).

Rapid identification and quantitative analysis of chemical constituents of Gentiana veitchiorum by UHPLC-PDA-QTOF-MS

ABSTRACT Gentiana veitchiorum Hemsl., Gentianaceae, a traditional Tibetan medicine, was used for the treatment of liver jaundice with damp-heat pathogen, as well as for headache and chronic pharyngitis. A rapid ultra-performance liquid chromatography, photodiode array detector, quadrupole time-of-flight mass spectrometry method was developed for the fast and accurate identification and quantification of the chemical constituents of G. veitchiorum. In fact, eighteen compounds were detected and identified on the basis of their mass spectra, fragment characteristics and comparison with published data. Especially, the MS fragmentation pathways of iridoid glycosides and flavone C-glycosides were illustrated. Five compounds among them were quantified by UHPLC-PDA, including swertiamarin, gentiopicroside, sweroside, isoorientin, and isovitexin. The proposed method was then validated based on the analyses of linearity, accuracy, precision, and recovery. The overall recoveries for the five analytes ranged from 96.54% to 100.81%, with RSD from 1.05% to 1.82%. In addition, ten batches of G. veitchiorum from different areas were also analyzed. The developed method was rapid and reliable for both identification and quantification of the chemical constituents of G. veitchiorum, especially for simultaneous qualitative and quantitative analysis of iridoid glycosides and flavone C-glycosides.

Optimization of Extraction Technology of Yigu Granule by Orthogonal Test / 中国药房

OBJECTIVE:To optimize the extraction technology of Yigu granule. METHODS:L9(34)orthogonal test was used, using comprehensive scores of transfer rate of icariin,gentiopicroside,loganic acid and yield rate as evaluation indexes,alcohol volume fraction,the amount of solvent,extraction times and extraction time as investigation factors,extraction technology of Yigu granule was optimized,and the verification test was conducted. RESULTS:The optimized extraction technology was 10-fold 60%ethanol reflux extraction for twice,1 h once. Results of verification test showed,average transfer rates of icariin,gentiopicroside and loganic acid were 81.28%(RSD=1.54%,n=3),48.71%(RSD=2.37%,n=3)and 59.82%(RSD=2.52%,n=3);aver-age yield rate was 31.48%(RSD=1.97%,n=3). CONCLUSIONS:The optimized extraction technology for Yigu granule is sta-ble and feasible with good reproducibility,which can provide basis for the follow-up study of production process.

Simultaneous Determination of Gentiopicroside,Gardenia and Baicalin in Xiegan Ashen Pills by HPLC / 中国药师

Objective: To establish an HPLC method for the determination of gentiopicroside,gardenia and baicalin in Xiegan Anshen pills.Methods: The analysis was carried out on a Phenomenex Luna C18(2) column(250 mm×60 mm, 5 μm).The mobile phase consisted of methanol-0.2% phosphoric acid solution with gradient elution and the flow rate was 1.0 ml·min-1.The column temperature was 30℃, the detection wavelength was 254nm, and the sample size was 10 μl.Results: The linear range of gentiopicroside was 10.55-168.80 μg·ml-1 (r=0.999 9),and the average recovery was 98.68%(RSD=1.2%, n=6).The linear range of gardenia was 29.85-477.60 μg·ml-1 (r=0.999 8), and the average recovery was 98.76%(RSD=1.1%, n=6).The linear range of baicalin was 43.05-688.8 μg·ml-1 (r=0.999 7), and the average recovery was 98.36%(RSD=1.4%, n=6).Conclusion: The method is simple and accurate, and can determine the contents of the three ingredients simultaneously, which can be used for the quality control of Xiegan Anshen pills.