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To identify the composition of iridoids from Hedyotis diffusa Willd and explore the mechanism on its anti-renal fibrosis effect based on network pharmacology, LC-Q/TOF-MS (liquid chromatograpy-quadrupole/time of flight mass spectrometry) was used to analyze the iridoid ingredients and the related targets of renal fibrosis were obtained by DisGeNET database and MalaCards database. The potential targets were screened by SYBYL-X7.3 software. We then imported the identified ingredients and potential target genes into Cytoscape3.7.1 to construct the compound-target network and the protein-protein interaction (PPI) network. Finally, the gene ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis of the selected core genes were made to explore the mechanism of iridoids against renal fibrosis. There were 10 active iridoid compounds and 111 corresponding targets including dimethylarginine dimethylaminohydrolase 1 (DDAH1), heparanase (HPSE), human kirsten rat sarcoma viral oncogene (KRAS), moesin (MSN), etc. in compound-target network. The GO functional enrichment analysis obtained 211 GO entries. Twenty related signal pathways including Toll-like receptor signaling pathway, transforming growth factor-beta (TGF-β) signaling pathway, renal cell carcinoma signaling pathway, and the Janus kinase/signal transducer and activator of tran-ions (Jak-STAT) signaling pathway were selected by KEGG enrichment analysis. We preliminarily investigated the mechanism of the iridoid compounds on renal fibrosis to provide guide information for the subsequent experimental research and clinical application.
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@#This research used inter simple sequence repeat(ISSR)markers to analyze the genetic diversity of Hedyotis diffusa Willd. from different origins. A total of 23 samples of Hedyotis diffusa Willd. in the Guangxi Zhuang Autonomous Region, Guangdong, Hunan, Zhejiang, Fujian, Jiangxi and Anhui, respectively were collected. 150 ISSR primers were used to amplify PCR and then POPGENE1. 32, NTSYS2. 10 software were used to analyze genetic diversity. 11 primers were screened, 115 polymorphic bands were amplified, the polymorphism ratio was 85. 22%, the number of alleles(Na)was 1. 852 2, the effective allele(Ne)was 1. 543 4, Neis gene diversity index(H)was 0. 316 5 and Shannon′s information index(I)was 0. 470 0. The results of cluster analysis show that the Hedyotis diffusa can be divided into three clades. The conclusion is that ISSR molecular markers can provide a insight for the identification of Hedyotis diffusa Willd. .
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Objective: To investigate the effects of Hedyotis diffusa Willd (HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms. Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition (MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein (RAP1GAP), RAS guanyl releasing protein 2 (RASGRP2), RAP guanine nucleotide exchange factor 3 (RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1) and G protein subunit alpha i1 (GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase (MAPK) pathway-related proteins [c-Jun N -terminal kinase (JNK), phospho-JNK (p-JNK), protein kinase B (PKB, Akt), phospho-Akt (p-Akt), extracellular signalregulated kinase (ERK) and phospho-ERK (p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells (P < 0.01), blocked cell cycle at S-phase (P < 0.01), and induced apoptosis (P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins (E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition (EMT)-related proteins (Vimentin and Snail1) (all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells (all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1 (all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells. Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.
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OBJECTIVE@#To investigate the therapeutic effects of Hedyotis diffusa Willd.on type Ⅱ collagen-induced rheumatoid arthritis in rats.@*METHODS@#According to the random number table, 60 SD rats were divided into the normal control group (=10, normal saline) and model group (=50).The collagen-induced arthritis model was established with the injection of type Ⅱ collagen into the back in rats other than the normal group and evaluated by arthritis score, then the model rats were randomly divided into model group (normal saline), tripterygium wilfordii polyglycoside (GTW) 6 mg/kg group (daily dose:0.4 mg/kg), HD 3, 6, 12 g/kg groups (daily dose:3, 6 and 12 g/kg, respectively), with 10 rats in each group. The rats were treated with corresponding agents by intragastric administration.The arthritis index and the pain threshold of all rats at different time points were observed and measured weekly.After treated by intragastric administration for 28 days, all rats were killed to measure the changes of serum cytokine levels including interleukin 1β (IL-lβ), tumor necrosis factor a (TNF-a), prostaglandin (PGE), receptor activator for nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG).@*RESULTS@#Compared with the control group, the arthritis index and the serum levels of IL-lβ, TNF-a, PGE, RANKL, OPG and RANKL/OPG of the model group were increased significantly (<0.05), the pain threshold of the model group was decreased significantly (<0.05); compared with the model group, the arthritis index and the serum levels of IL-lβ, TNF-a, PGE, RANKL, OPG and RANKL/OPG of the GTW group, HD low-dose, medium-dose, high dose groups were decreased significantly (<0.05), the pain threshold of the model group was increased significantly (<0.05).@*CONCLUSIONS@#Hedyotis diffusa Willd.can significantly reduce arthritis index and increase pain threshold, reduce the level of IL-lβ, TNF-a, PGE, RANKL, OPG, and RANKL/OPG, then can prevent CIA effectively.
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Animals , Rats , Arthritis, Experimental , Arthritis, Rheumatoid , Collagen Type II , Hedyotis , RANK Ligand , Rats, Sprague-DawleyABSTRACT
Objective: Based on the tumor-bearing rat model, using rutin, quercitrin, and isoquercitrin from Hedyotis diffusa as the research object, the pharmacokinetics of those three flavonoid glycosides in the pathological state was studied. Methods: Establishing a method for the comparison of the pharmacokinetics of flavonoid extracts in normal rats and tumor-bearing rats, which was analyzed by HPLC-MS/MS in subcutaneous tumor models of SD rat made with tumour cell of Walker-256. Results: Compared with the pharmacokinetics parameters of flavonoid glycosides in normal rats, the Cmax and AUC0-∞ of rutin, quercetin, and isoquercitrin in the tumor-bearing rats were significantly decreased, t1/2z was prolonged, and the metabolic time of three components was prolonged to 24 h, which revealed the effect of pathological condition on the pharmacokinetic characteristics of flavonoid glycosides. Conclusion: The method established in this study is simple, fast, sensitive, and suitable for the pharmacokinetic study of flavonoid glycosides in rats in vivo. The pharmacokinetic characteristics of flavonoids in normal and tumor-bearing rats are different.
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Hedyotis diffusa and H. corymbosa belong to the genus Hedyotis Linn. in family Rubiaceae. They shared similar heat-clearing and toxin-resolving effects and pharmacological activities, such as anti-cancer activity, and H. diffusa is frequently adulterated or substituted by H. corymbosa in clinical application. But whether they can completely replace with each other or whether the therapeutic effect could be changed after the substitution, is still a problem needs to be resolved. Therefore, the two herbs were compared in terms of the textual research, medical history, mixed use, commercial medicine status, macro-characters, micro-characteristic, chemical compositions and pharmacologic activities, and found that they shared similar macro-characters and micro-characteristic but were obviously different in chemical composition and pharmacologic activities, and now they could be identified quickly and accurately by molecular identification method. Therefore, we suggest that they should be used as two different medicinal materials at this stage.
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Objective: To investigate the intestinal absorption characteristics of five kinds of flavonoids from Hedyotis diffusa extract in rats. Methods: The in situ rat single-pass intestinal perfusion model was used. The contents of rutin, isoquercitrin, quercetin, kaempferol, and quercetin in perfusates were determined by HPLC-DAD, and the rat intestinal absorption parameters of flavonoids were calculated. Results: In perfusion model, the effective permeability coefficients (Peff) of all components were low. The absorption rate constant (Ka) and Peff had no significant difference when the concentration of total flavonoids from H. diffusa was 0.5-4.0 g/L. In the 2.0 g/L concentration, Ka of rutin, isoquercitrin, quercetin, kaempferol, and quercetin were 0.0113, 0.0154, 0.0102, 0.0305, and 0.0275 min-1, respectively; The Ka sequences of rutin and isoquercitrin in different intestinal segments were ileum > duodenum > jejunum ≈ colon and jejunum > duodenum > ileum ≈ colon. Conclusion: The absorption of the five flavonoids from H. diffusae is a first-order process with the passive diffusion mechanism. The absorption rates of each flavonoid are significantly different. The absorption rate of flavonoid glycosides is lower than that of aglycones. The flavonoids from H. diffusa could be absorbed in all the intestinal segments. The best parts of intestine to absorb rutin and isoquercitrin are ileum and jejunum.
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OBJECTIVE: To develop an HPLC method for simultaneous determination of multiple-components in Hedyotis diffusa Willd. METHODS: The HPLC analysis was carried out on a C18 column (4.6 mm×250 mm, 5 μm) by gradient elution with acetoni-trile-water[both containing 0.1‰ (V/V) acetic acid] as mobile phase at a flow rate of 0.8 mL·min-1, the column temperature at 35°C, and the detection wavelength was set at 238 nm. External standard method and quantitative analysis of multi-components by single marker (QAMS) method were adopted for simultaneous determination of six components in Hedyotis diffusa Willd, respectively. RESULTS: The linear ranges for asperulosidic acid, quercetin-3-O-[2-O-(6-O-E-feruloyl)-β -D-glucopyranosyl]-β-D-glucopyrano-side, kaempferol-3-O-[2-O-(6-O-E-feruloyl)-β-Z) -gfucopyranosyl]-β-D-galactopyranoside, (E)-6-O-p-coumaroyl scandoside methyl ester, (E)-6-O-feruloyl scandoside methyl ester, (Z)-6-O-p-coumaroyl scandoside methyl ester were 2.34-93.50, 2.61-104.33, 0.67-26.69, 3.42-136.84, 0.65-26.07, and 1.10-44.17 μg·mL-1 (r<0.9993), respectively. The RSD values of precision, reproducibility, and sample stability were not more than 2.2%. The average recoveries of the six components were 99.8%-101.1% with RSDs not more than 1.2%. The P values of external standard method and QAMS by paired t-test were greater than 0.05. CONCLUSION: There is no significant difference in the content analysis results of the two methods, which can both used for simultaneous determination of the four iridoids and two flavonoids in Hedyotis diffusa Willd.
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Objective To study the antitumor activities and immunity regulation of Hedyotis diffusa Willd injection. Methods Inhibitory effects of Hedyotis diffusa Willd injection on cell proliferation was detected with MTT method. Then the inhibitive rate was calculated. The immunomdulatory effects of Hedyotis diffusa Willd injection were investigated. Results Hedyotis diffusa Willd injection inhibited the proliferation of A549, SGC-7901, HEP-G2, Helacells in the dose of 100～20 ?g/mL, and enhance the immunity regulation on S180 tumor transplanted in mice. Conclusion Hedyotis diffusa Willd injection has the antitumor activities and can enhance the immunity.
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Objective To study the lab-scale simulated method of three-effect dynamic countercurrent extraction from Hedyotis diffusa by multi-index optimization and SPSS software.Methods Polyphenol content,flavonoids content,scavenging activity of samples on DPPH radicals,and extracting rate were considered as the optimization indexes.Orthogonal design method was used to optimize the four main factors of alcohol concentration,ratio of solid to solution,extracting temperature,and extracting times.Results The optimum condition is 70 min extraction time,solid to solution ratio of 1∶14,50% alcohol,extracting temperature 85 ℃.Conclusion The condition of large-scale industrialized production might be explored by studying the lab-scale simulated method of dynamic countercurrent extraction from H.diffusa.
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Objective The absorption and distribution of nitrogen(N),phosphorus(P),and potassium(K) of Hedyotis diffusa was studied in field tests.Methods Through regular sampling,dry matter accumulation was studied,the contents of N,P,and K were analyzed by conventional methods.Results The uptake amount of N,P,and K by H.diffusa was lower at seedling stage,but increased rapidly at branching,blossoming,and fruiting stages,and the uptake amount of N and P decreased and K was negatively absorbed at mature stage.The uptake amount of N by H.diffusa was the highest,following was K2O,while P2O5 was the lowest.In the growing season of H.diffusa,the ratio of N,P2O5,and K2O was 1.00:0.14:0.54.N Mainly distributed in leaf at seedling,branching,and blossoming stages,while P2O5 and K2O mainly distributed in stem.The mineral nutrition mostly distributed in stem,flowers,and fruits at fruiting and mature stages,N and P2O5 mostly distributed in flowers and fruits,but K2O mainly accumulated in stem.Conclusion A little fertilizer should be applied at seedling stage,oppositly the use amount of fertilizer should be increased at branching,blossoming,and fruiting stages,particularly P and K at fruiting stage.
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AIM:To establish an HPLC method for determining the contents of caffeic acid,quercetin and campherenol in Hedyotis diffusa Willd. METHODS:The samples were separated on an Alltima C 18 (250 mm? 4.6 mm,5 ?m) column with the mobile phase of MeOH(A)-0.5% glacial acetic acid solution;gradient elution(0～15 min,30%～60% A;15～30 min,60%～60% A).Flow rate was 1.0 mL/min. The detection wavelength was set at 350 nm.Column temperature was at 30 ℃. RESULTS:The contents of caffeic acid,quercetin and campherenol were 14.218～23.695 ?g/g,9.919～25.564 ?g/g and 6.229～18.160 ?g/g in Hedyotis diffusa Willd from different sources. The linear range of caffeic acid was 0.005 0～0.200 0 ?g(r=0.999 9),the average recovery was 102.35%,RSD was 2.31%(n=6);The linear range of quercetin was 0.006 2～0.244 0 ?g(r=0.999 9),the average recovery was 101.84%,RSD was 1.79%(n=6);The linear range of campherenol was 0.007 8～ 0.310 6 ?g(r=0.999 9),the average recovery was 99.04%,RSD was 2.90%(n=6). CONCLUSION:The method for quantifying of caffeic acid,quercetin and campherenol in Hedyotis diffusa Willd is accurate and reliable.