Optimization of alcohol precipitation process for Quhan Zhufeng Granules by overall desirability and central composite design / 国际中医中药杂志

Objective:To optimize the alcohol precipitation process of Quhan Zhufeng Granules.Methods:Taking the volume fraction of ethanol, the relative density of the concentrated solution and the standing time as the investigation factors, the Z value of the total evaluation of gentiopicrin content, oleanolic acid content and dry paste yield of gentiana macrophylla as the evaluation indexes, the star-point design-response surface method was used to optimize the alcohol precipitation process.Results:The optimal the alcohol precipitation process of Quhan Zhufeng Granules: concentration relative density 1.08 g/ml (90-95 ℃), alcohol precipitation at the end of volume fraction of 62% ethanol, standing for 16 h.Conclusion:The alcohol precipitation process using overall desirability and central composite design is stable and feasible, and has good predictability, which can provide experimental basis for further scale production.

Oleanolic acid alleviates steroid-induced osteonecrosis of the femoral head in rats by regulating Wnt/beta-catenin signaling pathway / 中国组织工程研究

BACKGROUND:Oleanolic acid can promote osteoblast proliferation and inhibit osteoclast proliferation,thereby improving steroid-induced osteonecrosis of the femoral head,but its specific mechanism of action is not yet fully understood. OBJECTIVE:To explore the mechanism by which oleanolic acid alleviates steroid-induced osteonecrosis of the femoral head in rats by regulating the Wnt/β-catenin signaling pathway. METHODS:Forty Sprague-Dawley rats were randomized into control group,model group,oleanolic acid group and oleanolic acid+sFRP1 group.An animal model of steroid-induced osteonecrosis of the femoral head was established by injecting prednisolone acetate in the latter three groups.Rats in the oleanolic acid group were gavaged with 10 mg/kg/d oleanolic acid and intramuscularly injected with the corresponding saline;rats in the oleanolic acid+sFRP1 group were gavaged with 10 mg/kg/d oleanolic acid and intramuscularly injected with 1 mg/kg/d Wnt inhibitor-sFRP1;and rats in the control and model groups were administered by gavage and intramuscularly injected with equal volumes of saline for 6 weeks.The levels of serum calcium,phosphorus,transforming growth factor-β1,and alkaline phosphatase were detected.Micro-CT was applied to detect femoral morphology.The morphology of femoral tissue was detected by hematoxylin-eosin staining.Cell apoptosis was detected by TUNEL.The levels of Bcl-2,Bax,β-catenin,and Wnt proteins were determined by western blot. RESULTS AND CONCLUSION:Compared with the control group,the trabeculae bone and femoral head of the model group were seriously injured,the serum levels of calcium,phosphorus,and transforming growth factor-β1 were significantly decreased,the levels of Bcl-2,Wnt,and β-catenin proteins in bone tissue were significantly reduced,and the serum alkaline phosphatase level,cell apoptosis rate,and Bax protein level were significantly increased(P<0.05).Compared with the model group,the degree of trabecular thinning in the oleanolic acid group was significantly improved,and the degree of femoral head damage was significantly reduced,serum alkaline phosphatase level,cell apoptosis rate,and Bax protein level were significantly reduced,serum levels of calcium,phosphorus,and transforming growth factor-β1,and levels of Bcl-2,Wnt,and β-catenin proteins in bone tissue were significantly increased(P<0.05).Compared with the oleanolic acid group,the oleanolic acid+sFRP1 group showed opposite changes in the above-mentioned indicators(P<0.05).To conclude,oleanolic acid can improve bone metabolism indicators and trabecular structure and attenuate femoral head necrosis in rats with steroid-induced osteonecrosis of the femoral head,which can be achieved by activating the Wnt/β-catenin signaling pathway.

Bioinformatics Analysis of Modified Lugen Formula in the Treatment of Influenza:Perspectives from the Virus-Host Interaction Network / 中药新药与临床药理

Objective To explore the therapeutic mechanism of Modified Lugen Formula(Phragmitis Rhizoma,Cicadae Periostracum,Batryticatus Bombyx,Lonicerae Japonicae Flos,Glycyrrhiza,Menthae Haplocalycis Herba,Notopterygii Rhizoma et Radix,Puerariae Lobatae Radix,Bupleuri Radix)in treating influenza from the virus-host interaction interface.Methods The phytocompounds were first collected from the HERB database,and then potential active compounds were screened out by Lipinski's rules of five.The targets of active compounds were further predicted through the SwissTargetPrediction platform.Differentially expressed genes(DEGs)were determined from the human H1N1 influenza dataset GSE90732 available in the Gene Expression Omnibus database(GEO).H1N1-Homo sapiens-related protein-protein interactions(PPIs)were gathered from the Pathogen-Host Interaction Search Tool(PHISTO).The above mentioned bioinformatic datasets were integrated.Then a PPI network and a Formula-virus-host interaction network were constructed using Cytoscape.Functional enrichment analyses were performed by using R software.Finally,molecular docking was carried out to evaluate the binding activities between the key compounds and targets.Results A total of 1 252 active compounds,1 415 targets,951 influenza-related DEGs,and 10 142 H1N1-Homo sapiens-related PPIs were obtained.There were 72 intersection targets between the Modified Lugen Formula and influenza.Functional enrichment analyses showed that these targets are closely related to host defense and programmed cell death.The network topological analysis showed that active compounds in the Modified Lugen Formula,such as oleanolic acid,γ-undecalactone,and longispinogenin,regulate viral proteins M2,NA,NS1,and HA and/or the host factors HSP90AA1,NRAS,and ITGB1,thus exert therapeutic effect.Molecular docking results confirmed that these compounds had a good binding ability with the targets.Conclusion Multiple active ingredients in Modified Lugen Formula directly target influenza virus proteins and/or host factors,thereby play an anti-influenza role in multiple dimensions,including inhibiting virus replication,regulating host defense and cell death.This study provides a theoretical basis for further experimental analysis of the action mechanism of the Modified Lugen Formula in treating influenza.

A review of structural modification and biological activities of oleanolic acid / 中国天然药物

Oleanolic acid (OA), a pentacyclic triterpenoid, exhibits a broad spectrum of biological activities, including antitumor, antiviral, antibacterial, anti-inflammatory, hepatoprotective, hypoglycemic, and hypolipidemic effects. Since its initial isolation and identification, numerous studies have reported on the structural modifications and pharmacological activities of OA and its derivatives. Despite this, there has been a dearth of comprehensive reviews in the past two decades, leading to challenges in subsequent research on OA. Based on the main biological activities of OA, this paper comprehensively summarized the modification strategies and structure-activity relationships (SARs) of OA and its derivatives to provide valuable reference for future investigations into OA.

Protective effect and mechanism of oleanolic acid on kidney in rats with type 2 diabetes mellitus / 中国糖尿病杂志

Objective To investigate the protective effect and mechanism of oleanolic acid(OA)on kidneys in rats with type 2 diabetes mellitus(T2DM).Methods A total of 35 Sprague-Dawley(SD)rats were enrolled in this study.25 SD rats were randomly selected to establish T2DM model,after modeling,20 rats remained and divided into T2DM group(n=6),low-dose oleanolic acid group(LOA,n=6)and high-dose oleanolic acid group(HOA,n=8).And ten rats were selected as normal control group(NC,n=10).The biochemical indicators,24 h urine volume and 24 h urinary microalbumin(UAlb)were compared among the four groups.Renal lipid deposition was evaluated by Oil red O staining.The protein expressions of Adenosine 5'-monophosphate-activated protein kinase(AMPK),p-AMPK and peroxisome proliferator-activated receptor γ coactivator-1 α(PGC-1 α)were detected by Western blot.Results Compared with the NC group,the levels of 24 h urine volume,fasting blood glucose(FPG),serum total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),serum creatinine(Scr),serum uric acid(SUA)and 24 hUAlb were increased(P<0.05),while the body weight,high density lipoprotein cholesterol(HDL-C),p-AMPK and PGC-1α were decrease in the T2DM group(P<0.05).Compared with the T2DM group,the expressions of HDL-C,p-AMPK and PGC-1α were increased(P<0.05),while the levels of 24 h urine volume,FPG,TG,TC and LDL-C,Scr,SUA and 24 hUAlb were decreased in the LOA and HOA groups(P<0.05).Compared with LOA group,the expressions of HDL-C,p-AMPK and PGC-1α were increased(P<0.05),while the levels of 24 h urine volume,FPG,TG,TC,LDL-C,Scr,SUA and 24 h UAlb were decreased in the HOA group(P<0.05).Compared with the NC group,a large number of red-stained lipid droplets were deposited in the renal tubular epithelial cells in the T2DM group.Compared with the T2DM group,the lipid droplet deposition was reduced in the LOA and HOA groups,and the improvement was more significant in the HOA group.Conclusion OA can alleviate renal injury in T2DM rats,which may be linked to activation of AMPK/PGC-1α pathway.

Effect of Oleanolic Acid on Abnormal Water Metabolism of Mice with Water-dampness Retention Caused by Spleen Deficiency / 中国实验方剂学杂志

ObjectiveTo explore the effect of oleanolic acid （OA） on water metabolism in mice with water-dampness retention caused by spleen deficiency and the mechanism. MethodThe 60 SPF Kunming （KM） mice were randomized into blank group （n=10） and modeling group （n=50）. Through long-term living in damp place and irregular diet， water-dampness retention caused by spleen deficiency was induced in modeling mice. Then the model mice were randomly classified into model group， natural recovery group， and low-dose， medium-dose， and high-dose OA groups. The mice in the blank group， model group， and natural recovery group were given （ig） 10 mL·kg-1·d-1 normal saline， and mice in the low-dose， medium-dose， and high-dose OA groups received 50， 100， 200 mg·kg-1·d-1 OA， respectively. The intervention lasted 7 days. Before and after modeling and administration， the general conditions of the mice were observed and body weight of mice was measured. The water content in feces and tissues was detected with the oven-drying method， and water load index and organ coefficient were measured with the weighing method. Enzyme-linked immunosorbent assay （ELISA） was employed to detect the urinary D-xylose excretion， serum gastrin （GAS）， total protein （TP）， albumin （ALB）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， interleukin-6 （IL-6）， antidiuretic hormone （AVP）， aquaporin 1 （AQP1） in renal medulla， and liver Na+-K+-ATPase. At the same time， OA was docked with ALB， IL-6， AQP1， and Na+-K+-ATPase. ResultCompared with the blank group， the model group showed withered hair， emaciation， laziness， bradykinesia， slow weight growth， infrequent spontaneous activities， high water content in feces and tissues， low weight loss after water loading， high coefficient of each organ （P<0.05， P<0.01）. Moreover， the model group had less urinary D-xylose excretion， lower serum levels of GAS， TP， ALB， and HDL-C， higher levels of TC， LDL-C， AVP， and IL-6， lower expression of Na+-K+-ATPase in the liver， and higher expression of AQP1 in renal medulla than the blank group （P<0.05， P<0.01）. The three OA groups demonstrated better general conditions， faster weight gain， more frequent spontaneous activities， lower water content in feces and tissues， larger weight loss after water loading， and lower coefficient of each organ than the model group （P<0.05， P<0.01）. Moreover， compared with the model group， the three OA groups had high D-xylose excretion， high serum levels of GAS， TP， ALB， and HDL-C， low serum levels of TC， LDL-C， AVP， and IL-6， high expression of Na+-K+-ATPase in liver， and low expression of AQP1 in renal medulla （P<0.05， P<0.01）. The recovery in each OA group was better than that in natural recovery group. Molecular docking results also confirmed that OA had high binding affinity with ALB， IL-6， AQP1， and Na+-K+-ATPase. ConclusionOA can alleviate the abnormal water metabolism in mice with water-dampness retention caused by spleen deficiency， which lays a basis for its potential clinical application.

Evaluación de una nueva formulación de compuestos naturales sobre la modificación de los componentes del síndrome metabólico en adultos con sobrepeso

El sobrepeso y la obesidad, condiciones cada vez más frecuentes en Chile y el Mundo, se definen como una enfermedad crónica caracterizada por la acumulación excesiva de grasa corporal que puede ser perjudicial para la salud. El exceso de grasa corporal es el principal factor patogénico para el desarrollo de resistencia a la insulina y síndrome metabólico (SM), este último caracterizado por la agrupación de una serie de anormalidades metabólicas que determinan un mayor riesgo de padecer enfermedades metabólicas y mortalidad cardiovascular. El objetivo de este estudio piloto transversal y observacional fue evaluar una nueva formulación de compuestos naturales, Delphinol®, resveratrol y ácido oleanólico, frente a parámetros asociados al SM en una población joven con exceso de peso, al ingerir durante 22 semanas la nueva formulación. La evaluación fue realizada en base al estado clínico y antropométrico de 20 sujetos voluntarios (ambos sexos, 18 a 30 años e IMC 26 a 42), mediante valoración médica y exámenes de laboratorio clínico para cada componente del SM (obesidad abdominal, triglicéridos, HDL, presión arterial y glicemia en ayunas). De esta manera, se observó que con la ingesta diaria de esta formulación existe una reducción significativa de los niveles de glicemia, índice HOMA-IR, triglicéridos y aumento de HDL-colesterol. Estos resultados preliminares se traducirían en un beneficio sobre el estado de salud de los individuos con sobrepeso y obesidad, lo cual impactaría en reducir los índices de SM y de esta manera enlentecer la progresión hacia enfermedades crónicas como diabetes y dislipidemia, y contribuir también a disminuir el riesgo cardiometabólico asociado.

Overweight and obesity, increasingly frequent conditions in Chile and the world, are defined as chronic diseases characterized by the excessive accumulation of body fat, that can be detrimental to health. Excess body fat is the main pathogenic factor for the development of insulin resistance and metabolic syndrome, the latter characterized by a cluster of metabolic abnormalities that increase risk of metabolic diseases and cardiovascular mortality. The objective of this preliminary cross-sectional, observational study was to evaluate a new formulation of natural compounds, Delphinol®, resveratrol and oleanolic acid, against metabolic syndrome components in a young population with excess weight, through the intake during 22 weeks of the new formulation. The evaluation was carried out based on the clinical and anthropometric status of 20 volunteer subjects (both gender, 18 to 30 years and BMI 26 to 42), medical evaluation and laboratory tests using each metabolic syndrome component (abdominal obesity, triglycerides, HDL, blood pressure y fasting glicaemia). It was observed that with the daily intake of this formulation there was a significant reduction in blood glucose levels, HOMA-IR index, triglycerides and an increase in HDL-cholesterol. These preliminary results would translate into a benefit in the health status of overweight and obese individuals, which would impact on reducing rates of metabolic syndrome. Thus, slowing down the progression of chronic diseases such as diabetes and dyslipidemia, and also contributing to decreasing the associated cardiometabolic risk.

Design and semisynthesis of oleanolic acid derivatives as VEGF inhibitors: Inhibition of VEGF-induced proliferation, angiogenesis, and VEGFR2 activation in HUVECs / 中国天然药物

Angiogenesis inhibitors targeting the VEGF signaling pathway are developed into drugs for the treatment of vaious diseases, such as cancer, rheumatoid arthritis, and age-related macular degeneration. Recent studies have revealed that oleanolic acid (OA), a natural pentacyclic triterpenoid, inhibited the VEGF/VEGFR2 signaling pathway and angiogenesis in HUVECs, which may represent an attractive VEGF inhibitor. In this paper, rational structural modification towards OA was performed in order to improve its inhibitory effects aganist VEGF and anti-angiogenesis potential. As a result, a series of novel OA derivatives, possessing α,β-unsaturated ketone system in ring A and amide functional group at C-28, were prepared and evaluated for cytotoxicity and their ability to inhibit VEGF-induced abnormal proliferation of HUVECs. The results showed that two promising derivatives, OA-1 and OA-16, exhibited no in vitro cytotoxicity against HUVECs but showed more potent inhibitory activity against VEGF-induced proliferation and angiogenesis in HUVECs, compared with OA. The results of Western blot indicated that OA-1 and OA-16 inhibited VEGF-induced VEGFR2 activation. Furthermore, small interfering RNA experiments were performed to confirm that both compounds inhibited VEGF-induced angiogenesis via VEGFR2. Thus, the present study resulted in the discovery of new promising OA-inspired VEGF inhibitors, which can serve as potential lead compounds for the treatment of angiogenesis-related diseases.

Protective effect of oleanolic acid on liver injury induced by acute exposure to mercury chloride and its possible mechanism / 环境与职业医学

Background Acute exposure to mercury chloride (HgCl2) can cause liver damage. Whether oleanolic acid (OA) as a hepatoprotective drug can protect against liver injury induced by acute exposure to HgCl2 and related mechanism of action remain unclear. Objective To investigate the protective effect and possible mechanism of OA on liver injury in mice caused by acute exposure to HgCl2. Methods Forty SPF C57BL/6 male mice were randomly divided into four groups with 10 mice in each group according to body weight. The four groups were named control group, OA group (300 mg·kg−1), HgCl2 group (5 mg·kg−1), and OA + HgCl2 group (300 mg·kg−1 OA + 5mg·kg−1 Hgcl2). Soybean oil and OA solution were administered intragastric once a day for two consecutive days. HgCl2 solution was injected intraperitoneally 2 h after the second intragastric administration. Mice were sacrificed after 48 h, and their serum and liver were collected. Liver coefficient was calculated. The changes of liver structure and iron deposition were observed by hematoxylin-eosin (HE) staining and Prussian blue staining. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total superoxide dismutase (T-SOD), reduced glutathione (GSH), malondialdehyde (MDA), and tissue iron content were measured with commercial kits. Western blotting was used to detect nuclear factor erythroid-2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 4 (Gpx4), transferrin receptor 1 (TFR1,) and solute carrier family 7 member 11 (SLC7A11). Results The AST and ALT levels of the HgCl2 group were (76.447±9.695) U·g−1 and (98.563±24.673)U·g−1, respectively, which were higher than those of the control group (P<0.05). After the OA pretreatment, the liver coefficient and the above indexes were decreased to (4.769±0.237)%, (57.086±10.087) U·g−1, and (87.294±27.181)U·g−1, respectively. The liver coefficient and AST level of the OA + HgCl2 group were significantly different from those of the HgCl2 group (P<0.05). After acute exposure to HgCl2, the hepatocytes of mice were disordered, accompanied by inflammatory infiltration, positive blue particles appeared in Prussian blue staining of liver tissue, and the above changes in liver tissue were alleviated after the OA pretreatment. The iron content in the HgCl2 group was (3.646±0.238) μmol·g−1, which was higher than that in the control group, (2.948±0.308) μmol·g−1. After the OA pretreatment, the iron content decreased to (3.429±0.415) μmol·g−1. Compared with the control group, acute exposure to HgCl2 resulted in decreased levels of GSH and T-SOD, decreased protein expression levels of Nrf2, HO-1, SLC7A11, and Gpx4, increased level of MDA, and increased protein expression level of TFR1 (P<0.05). After the OA pretreatment, all indicators were improved including increased GSH level, decreased MDA level, increased Nrf2, HO-1, and SLC7A11 protein expression levels, and decreased TFR1 protein expression level; compared with the HgCl2 group, the differences were statistically significant (P<0.05). Conclusion Acute HgCl2 exposure could induce liver injury in mice, and its mechanism may involve iron overload and ferroptosis. OA may alleviate the liver injury caused by acute HgCl2 exposure by affecting iron overload and the ferroptosis-related protein expression.

Effects of oleanolic acid on early brain injury after subarachnoid hemorrhage through SIRT1 / 国际生物医学工程杂志

Objective:To investigate the effect of oleanolic acid (OA) on Sirtuin1 (SIRT1)-mediated high-mobility group box 1(HMGB1) deacetylation in the early brain injury after subarachnoid hemorrhage (SAH).Methods:A total of 176 male Sprague-Dawley rats were randomly divided into Sham operation (Sham group) ( n=48), SAH group ( n=48), OA group ( n=48) and Sirtinol group ( n=32). Rats in the SAH group, OA group and the Sirtinol group all adopted internal carotid artery puncture to construct SAH model, while rats in the sham group did not adopt puncture. One hour after modeling, the rats in the OA group were given intraperitoneal injection of OA (20 mg/kg), and the rats in the Sirtinol group were given intracerebroventricular injection of Sirtinol (2 mmol/L, 30 μL/kg). The rats in the sham group and SAH group were injected with equal volumes of sodium chloride injection. The SAH score and neurological score were performed 24 h after SAH, and the water content in the brain tissue and Evans blue exudation rate were measured. The expressions of HMGB1, SIRT1 and acetylated HMGB1 proteins in the brain tissue of rats were detected by Western Blot. The expression of HMGB1 mRNA in the brain of the rats was detected by quantitative real-time PCR. The distribution of HMGB1 protein in the brain of the rats was observed by immunofluorescence staining. TUNEL staining was used to observe the neuronal apoptosis in the brain tissue of the rats. Results:Compared with the SAH group, the SAH score of the OA group was significantly reduced ( P<0.001), the Garcia score was increased ( P<0.01), and the brain water content and Evans blue exudation rate were both reduced (all P<0.01). Compared with the OA group, the SAH score of the Sirtinol group was increased ( P<0.01), the Garcia score was significantly decreased ( P<0.001), and the brain water content and Evans blue exudation rate were both increased (all P<0.01). The results of Western Blot and real-time fluorescent quantitative PCR showed that, compared with the SAH group, the protein level ( P<0.01) and mRNA level ( P<0.05) of HMGB1 in the OA group were decreased, the expression of SIRT1 protein was significantly increased ( P<0.001), and the expression of acetylated HMGB1 protein was decreased ( P<0.01). Immunofluorescence staining showed that OA inhibited the migration of HMGB1 protein from the nucleus to the cytoplasm. TUNEL staining showed that OA could effectively reduce the number of TUNEL-positive cells. Compared with the OA group, Sirtinol significantly increased the number of TUNEL-positive cells. Conclusions:OA can reduce the release of HMGB1 through the SIRT1/HMGB1 pathway, thereby protecting the early brain injury after SAH.

Research advances in phytochemistry, pharmacology and toxicology of oleanolic acid / 中国药理学与毒理学杂志

Oleanolic acid (OA) is a pentacyclic triterpenoid chemical component that exists in natural plants with a molecular formula of C30H48O3 and a molecular weight at 456.71 g·mol-1. OA is widespread in traditional Chinese herbal medicine (Ligustri Lucidi Fructus, Achyranthis Bidentate Radix, Red Sage) and berries (blueberries, grapes). In recent years, because of the extensive pharmacological effects of OA, its advantages in disease treatment have become increasingly prominent and gradually attracted the attention of pharmaceutical researchers. OA has effective therapeutic effects on a series of chronic diseases such as inflammation, cancer, diabetes, and cardiovascular diseases through mul?tiple signaling pathways and various targets. Especially in cancers, such as colorectal cancer, liver cancer, gastric cancer, lung cancer, breast cancer and other malignancies, OA presents substantial efficacy. However, its poor aqueous solubility, needy bioavailability, and unsatisfactory pharmacological activity excessively restrict its clinical application. More impor?tantly, the improper utilization of OA can cause adverse reactions, toxic effects and even damage to organs in some spe?cific situations. With the discovery of various pharmacological effects, the complex action mechanisms of OA, the contin?uous progress in structural modification of OA, as well as the synthesis of OA derivatives, its application is expand?ing gradually. Among numerous studies, there is a clear indication that OA and its derivatives, if fully developed, may provide an alternative and cheaper treatment for a variety of chronic diseases. However, the specific molecular mecha?nisms of OA and its derivatives as an alternative therapy and supplementary therapy for cancer, diabetes, cardiovascular disease and other chronic diseases remain to be clarified. Therefore, it is necessary to further study the pharmacokinet?ics, pharmacological activity, specific targets and related mechanisms of OA to lay a solid foundation for drug devel?opment and the application of OA in clinical settings.

RP-HPLC analysis of methanol extract of Viscum articulatum

Background: Viscum articulatum Burm. (Family: Loranthaceae) is commonly known as mistletoe. Inayurveda, the plant parts are used in “Kapha”, “Vata”, diseases of the blood, ulcer, and epilepsy. The plantparts are also used in urinary tract infection and wound infection. The plant contains five triterpenoidssuch as a-amyrin, lupeol, betulin, betulinic acid and oleanolic acid, exhibiting several pharmacologicalactivities including antimicrobial, anti-HIV, antitumor, antiviral activity.Objective: To ensure the content of uniformity of oleanolic acid, a RP-HPLC method has been developedfor estimation of oleanolic acid in V. articulatum aerial part.Material and methods: The RP-HPLC method was carried out in reverse phase C18 column, using methanol and water as mobile phase in the ratio of 95:5 (v/v), at the flow rate of 1 mL/min. The pH of aqueousphase was adjusted 3.2 with 1% (v/v) glacial acetic acid. The lmax was set at 210 nm.Results: The retention time of oleanolic acid was found at 21.5 ± 0.05 min. The linearity of the response wasfound to be 10e800 mg/mL. The coefficient of determinants of oleanolic acid was found to be (r2) 0.995 andequation Y ¼ 19462X þ 16,172. The LOD and LOQ were found to be for oleanolic acid (1.96% w/w) 0.197 ± 0.63and 0.623± 0.87 mg/mL, respectively. The developed method was accurate, specific, precise and reproducible.Conclusion: This RP-HPLC may be useful for quantitative estimation of the chemical constituents presentin the plant extract as well as the quality assessment of the herbal product.

Identification on triterpenoid saponins of Clematis species by UPLC-LTQ-Orbitrap combined with molecular network / 中草药

Objective: To construct molecular network and analyze rapidly the saponins of six species of Clematis plants. Methods: The mass spectral data, acquired with UHPLC-LTQ Orbitrap MS, were uploaded to the GNPS analysis platform to build molecular network, visualized by Cytoscape software. On the other hand, the triterpenoid saponins from six plants were identified on the basis of the fragmentation regularity of the standard and the reported literature. Results: Twenty-five triterpenoid saponins, including 16 hederagenin saponins and nine oleanolic acid-type saponins, were determined from six kinds of plants. The distribution of the triterpenoid saponins in the six kinds of plants were profiled by the pie chart of each node in molecular network. Twenty compounds were found in at least two species of Clematis. Clematichinenoside A and oleanolic acid 3-O-ribopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside were the common constituents in five species. Six saponins were distributed in in single species of Clematis. Conclusion: Compared with traditional phytochemical methods, the molecular network technology of UPLC-LTQ-Orbitrap MS can quickly and visually distinguish different triterpenoid saponins in six kinds of plants. There are similarities and differentiates among the triterpenoid saponins in the six kinds of plants, which provides the basis for the substitution of medicinal materials.

Research on molecular mechanism of Qiwei Tongbi Oral Liquid in treatment of rheumatoid arthritis via network pharmacology / 中草药

Objective: To investigate the major active components and potential molecular mechanism of Qiwei Tongbi Oral Liquid in treatment of rheumatoid arthritis. Methods: After protein targets related with rheumatoid arthritis (RA) were collected by mining literature, DrugBank, TTD and OMIM database, the potentional protein targets based on molecular docking were projected into KEGG databases to illustrate the molecular mechanism of Qiwei Tongbi Oral Liquid. Then RAW264.7 cells induced by LPS were employed to test the predict results. Results: Data analysis showed that 107 potential components acted on 116 RA related targets and 237 pathways. Herb-compound-target-pathway network analysis showed that triterpenoid, flavonoids, sterols and alkaloids isolated from Qiwei Tongbi Oral Liquid were the main active ingredients. These compounds could interact with a group of targets and pathways that might participate in anti-inflammatory, analgesic, immune regulation, proliferation, apoptosis, migration and invasion of synovial fibroblasts, osteoclast differentiation, migration and bone resorption. Some compounds against anti-inflammatory activity were verified by in vitro. Conclusion: The active components of Qiwei Tongbi Oral Liquid could regulate multiple biological pathways by acting with multiple target proteins, playing a role in reducing inflammation and swelling of joints, preventing and reducing the destruction of joint bones, and promoting the repair of damaged joint bones. The research results not only reveal its molecular mechanism and pharmacodynamic components, but also provide a theoretical basis for the subsequent experimental research of Qiwei Tongbi Oral Liquid.

Anewoleanolicacid-typesaponinfromrootsofPanaxginseng / 中草药

Objective To systematically investigate the chemical constituents of the roots of Panaxginseng. Methods Mild cold-soaked extraction by 70% aqueous ethanol, successive solvent extraction by ethyl acetate and n-butanol, column chromatography by D101 macroporous absorption resin and reversed-phase silica gel, and semi-preparative HPLC, were used for compounds isolation and purification, while high-resolution mass spectrometry, 1D and 2D NMR data were analyzed for compounds identification. Results A new oleanolic acid tetraglycoside (1) and 19 known ginsenosides (2-20) were isolated and identified. Compound 1 was identified as oleanolic acid 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, named ginsenoside Ro1 (1). The 19 known ginsenosides were notoginsenoside FP1 (2), ginsenoside Re3 (3), notoginsenoside Rt (4), 20-O-glucosyl ginsenoside Rf (5), ginsenoside Re2 (6), ginsenoside Rg2 (7), ginsenoside Ra2 (8), ginsenoside Rb1 (9), ginsenoside Rc (10), ginsenoside Ra1 (11), malonylginsenoside Rb1 (12), malonylfloralginsenoside Rd5 (13), malonylginsenoside Rc (14), ginsenoside Ro (15), ginsenoside Rd (16), ginsenoside F2 (17), 20(R)-ginsenoside Rh2 (18), ginsenoside F3 (19) and ginsenoside F1 (20). Conclusion Compound1is a new compound. Compound 2, notoginsenoside FP1, is isolated from this plant for the first time.

Mechanism of sovereign medicines in Sanren Decoction on COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To investigate the mechanism of sovereign medicines in Sanren Decoction on coronavirus disease 2019 (COVID-19) through network pharmacology and molecular docking methods. Methods: The main active ingredients of sovereign medicines in Sanren Decoction (Armeniacae Semen Amarum, Amomun kravanh Pierre ex Gagnep, and Coicis Semen) were obtained and screened from by TCMSP and TCMID V2.0, combined with related research. Using UniPort database to query the target proteins corresponding to the active ingredients, then a component-target network was constructed by Cytoscape 3.7.2. PPI network was constructed through the STRING website, and cytoHubba was used to analysis the key subnetworks. CTD database was used to analyze GO and KEGG enrichment of the active ingredient target proteins of Sanren Decoction. Using the active ingredients of sovereign medicines in Sanren Decoction and related chemical drugs such as lopinavir as ligands, molecular docking with the SARS-CoV-2 3CL hydrolase was performed through the CB-Dock website. Results: Sovereign medicines in Sanren Decoction had 39 active ingredients, corresponding to 168 target proteins. The GO enrichment analysis obtained 25 biological processes (BP) items, 14 related items of cell composition (CC), and two molecular function (MF) item, respectively. KEGG enrichment screened 36 signaling pathways such as innate immune system, cytokine signaling in immune system, signaling by interleukins. The molecular docking results suggested that the active ingredients of mairin, ziziphin_qt, and oleanolic acid of sovereign medicines in Sanren Decoction had good binding energy with SARS-CoV-2 3CL hydrolase, and the Vina score of them were similar to those of lopinavir (the 3CLpro inhibitor) and remdesivir (RNA-dependent RNA polymerase inhibitor). Conclusion: Sovereign medicines in Sanren Decoction may participate in inflammation-related signaling pathways by regulating inflammatory factors, regulating multiple physiological processes of the disease with multi-components, multi-targets, and multi-pathways. It plays a certain intervention role in the treatment of COVID-19 and its active ingredients have potential resistance to SARS-CoV-2.

Study on antitumor activity of triterpenoid saponins from Tujia ethnomedicine Kouziqi / 中草药

Objective: To study the active triterpenoid saponins of Tujia ethnomedicine Kouziqi (Panax japonicus var. major). The antitumor activity was screened and the relationship between the structure and activity of the compounds was discussed. Methods: The ethanol extract of Kouziqi was isolated by Silica gel, ODS and MCI column chromatograph and purified by preparative HPLC. The structures were elucidated on the basis of spectroscopic analysis and compared with literatures. Using MTT assay to detect the cytotoxicity of 14 compounds in BGC-823, HCT-116, Hela, HepG-2 cells. Results: A total of 14 known compounds were isolated from Tujia ethnomedicine Kouziqi and determined as chikusetsusaponin IVa methyl ester (1), chikusetsusaponin IVa butyl ester (2), chikusetsusaponin IV (3), chikusetsusaponin IVa (4), 28-desglucosylchikusetsusaponin IVa (5), oleanolic acid-3-O-β-D-(6'- methylester)-glucuronopyranoside (6), (24R)-majonoside R1 (7), (24R)-pseudoginsinoside F11 (8), (20S)-notoginsinoside-R2 (9), (20S)-ginsenoside Rg2 (10), ginsenoside Rg1 (11), ginsenoside Re (12), ginsenoside Rd (13) and chikusetsusaponin-V methyl ester (14). Among the 14 compounds, compounds 5 and 6 showed dose-dependent cytotoxicity to BGC-823, HCT-116, Hela and HepG-2 cells. Compound 5 had cytotoxicity in BGC-823 and HCT-116 cells with IC50 values of 9.94 and 14.17 μmol/L, respectively. Compound 6 had the best cytotoxicity in HepG-2 cells with IC50 value of 12.70 μmol/L. Conclusion: Compound 6 is isolated from Kouziqi for the first time and its spectral data were reported. The antineoplastic activity of Tujia ethnomedicine Kouziqi is based on the oleanolic acid-type triterpenoid saponins and related to the substituents of C-28, but the mechanism still needs to be deeply studied.

Chemical compositions and anticancer activity of Mollugo pentaphylla / 中草药

Objective: To investigate the chemical compositions and anticancer activity of Mollugo pentaphylla. Methods: These compounds were isolated by various technologies, such as silica gel, Sephadex LH-20 and high performance liquid chromatography. Their structures were elucidated on the basis of physicochemical properties and spectroscopic data. The cytotoxic activities of compounds 1-5 against five cancer cell lines were tested by MTT. Results: Six compounds were isolated from this plant and identified as mollugoside E (1), 3-O-[α-L-rhamnopyranosy1 (1→2)-α-L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl (1→6)-β-D- glucopyranosyl]oleanolic acid (2), raddeanoside R8 (3), raddeanin A (4), mollugogenol A (5), and oleanolic acid (6). The cytotoxic activities results showed that compounds 1-5 had certain inhibitory effects on human prostate cancer DU145 cell lines, cervical cancer Hela cell lines and early-juvenile leukemia HL 60 cell lines, especially on HL 60 cells with IC50 value of 10.21, 38.43, 40.28, 20.59, and 83.16 μmol/L, respectively. Conclusion: Compound 1 is a new triterpenoid saponin and compounds 2-4 are isolated first time from M. pentaphylla. The cytotoxic activities results showed that compounds 1-5 had certain inhibitory effects on DU145, Hela and HL 60 cell lines.

Study on the Effects of Oleanolic Acid on the Proliferation ，Invasion and Metastasis of Human Ovarian Cancer SKOV3 Cells and Its Mechanism / 中国药房

OBJECTIVE：To investigate the effects and m echanism of oleanolic acid on inhibiting the proliferation ，invasion and metastasis of human ovarian cancer SKOV 3 cells. METHODS ：CCK-8 assay was used to detect the effects of different concentrations of oleanolic acid （10，20，40，60，80，100 μmol/L）on the proliferation of ovarian cancer SKOV 3 cells at 12，24， 36 and 48 h. The effects of low-dose and high-dose of oleanolic acid （20，40 μmol/L）on the metastasis and invasion ability of SKOV3 cells for 24 h were observed in Transwell assay. Western blotting assay was used to detect the effects of low-dose and high-dose of oleanolic acid on the protein expression of NF-κB p65，PRL-3，TNF-α，IL-6 and E-cadherin in SKOV 3 cells. Through LPS induction and NF-κB p65 plasmid transfection ，Western blotting and RT-qPCR assay were used to investigate the effects of low-dose and high-dose oleanolic acid on the expression of NF-κB/PRL-3 pathway related proteins and their mRNA. RESULTS ： With the increase of the concentration and action time of oleanolic acid ，the proliferation capacity of ovarian cancer SKOV 3 cells was decreased ，the surval rates of administration groups were significantly lower than that of the control group （P＜0.05 or P＜ 0.01）. Low-dose and high-dose of oleanolic acid could significantly reduce the number of migrating and invading cells （P＜0.05 or P＜0.01）. The protein relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 in SKOV 3 cells were significantly decreased ， while the protein relative expression of E-cadherin was significantly increased （P＜0.05 or P＜0.01）. After LPS induction ，protein and mRNA relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 were increased significantly in LPS model group ，while protein and mRNA relative expression of E-cadherin were significantly decreased （P＜0.05 or P＜0.01）. The protein and mRNA relative expression of NF-κ B p65，PRL-3，TNF-α and IL-6 were significantly decreased ，and protein and mRNA relative expression of E-cadherin were significantly increased in low-dose and high-dose of oleanolic acid group （P＜0.05 or P＜0.01）. In SKOV3 cells with over-expressed NF-κB p65，low-dose and high-dose of oleanolic acid c ould significantly down-regulat the proteinexpression of NF-κ B p65，PRL-3，TNF-α and IL-6，while upregult the protein relative expression of E-cadherin （P＜0.05 E-mail：122821905@qq.com or P＜0.01）. CONCLUSIONS ： Oleanolic acid can inhibit SKOV3 cells proliferation，invasion and metastasis by regulating NF-κB/PRL-3 signaling pathway.

Optimization of the Purification Technology of Total Triterpenoids of Ligustrum lucidum by Box-Behnken Re- sponse Surface Methodology / 中国药房

OBJECTIVE：To i mprove the transfer rate and purity of oleanolic acid and ursolic acid in total triterpenoids from Ligustrum lucidum ，so as to optimize the purification technology. METHODS ：Oleanolic acid and ursolic acid were used as representative components of total triterpenoids ，and their contents were determined by HPLC. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of methanol- 0.02％ ammonium acetate solution （80∶20，V/V）at the flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm，and column temperature was 30 ℃. The sample size was 20 μ L. In single factor tests，using transfer rate of oleanolic acid and ursolic acid as index ，the effects of water precipitation temperature and time ，the amount of redissolved ethanol on the purification technology was investigated ；using transfer rate and purity of two components as indexes ，the effects of the amount of activated carbon and volume fraction of crystallization ethanol were investigated. Based on it ，using the amount of redissolved ethanol and activated carbon ，volume fraction of crystallization ethanol as factors ，Box-Behnken response surface methodology was used to optimize the purification technology ，and validation tests were performed. RESULTS ：The optimal purification technology was adding 4-fold（mL/g，the same below ）water in L. lucidum concentrated solution ，placing for 2 hours at 0 ℃（water precipitation ）；adding 1-fold ethanol to dissolve （redissolution）； adding 4％ activated carbon （edulcoration）；finally adding water to adjust the volume fraction of ethanol to 80％，placing at 4 ℃ for 12 hours（crystallization），centrifuging and drying. The results of 3 times of validation tests showed that the transfer rates of oleanolic acid and ursolic acid in total triterpenoids prepared by optimized technology were 61.11％ and 65.78％，the purities of them were 53.44％ and 19.79％，and RSDs were both lower than 3％. CONCLUSIONS ：The optimized purification technology has high extraction efficiency and simple operation ，which can be used for industrial production of purification of total triterpenoids from L. lucidum and the development of corresponding preparations.