The Populations of Two Differently Medicine-Used Plants of Hedyotis diffusa and Hedyotis corymbosa Shoot-Assembling Rich Bacterial and Fungal Communities with Varied Compositions but Conserved Structures.

The plant-colonized microbial communities have closely micro-ecological effects on host plant growth and health. There are many medicinal plants in the genus Hedyotis, but it is yet unclear about the shoot-assembled bacterial and fungal communities (SBFC) of Hedyotis plants. Hence, eight plant populations of Hedyotis diffusa (HD) and H. corymbosa (HC) were evaluated with 16S rRNA gene and ITS sequences, for comparing the types, abundance, or/and potential functions of SBFC at plant species- and population levels. In tested HD- and HC-SBFC, 682 fungal operational taxonomic units and 1,329 bacterial zero-radius operational taxonomic units were identified, with rich species compositions and varied alpha diversities. Notably, the SBFC compositions of HD and HC plant populations were exhibited with partly different types and abundances at phylum and genus levels but without significantly different beta diversities at plant species and population levels. Typically, the SBFC of HD and HC plant populations were presented with abundance-different biomarkers, such as Frankiaceae and Bryobacteraceae, and with similar micro-ecological functions of microbial metabolisms of lipids, terpenoids,and xenobiotics. Taken together, HD- and HC-SBFC possessed with varied rich compositions, conservative taxonomic structures, and similar metabolic functions, but with small-scale type and abundance differences at plant species- and population- levels.

Hedyotis diffusa Willd inhibits inflammation and oxidative stress to protect against chronic prostatitis via the NRF2/ARE signaling pathway.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a common and serious disease with unclear pathogenesis and recurrent symptoms. Hedyotis diffusa Willd (HDW) has been recognized for its potential in managing various chronic inflammatory diseases. This research aimed to interrogate the mechanism of HDW in treating CP/CPPS. Complete Freund Adjuvant (CFA) and LPS were utilized to establish the rat and cell models of CP/CPPS. Results showed that HDW decreased levels of inflammation-related factors in CP rat prostate tissue and LPS-elicited RWPE-1 cell injury model. Moreover, HDW administration impaired oxidative stress in the prostate and RWPE-1 cells. In addition, HDW treatment activated the NRF2/ARE signaling in rat prostate tissue and cell models. Interestingly, NRF2/ARE pathway inhibitor ML385 reversed the inhibition effects of cell apoptosis, inflammation, and oxidative stress triggered by HDW. In summary, HDW alleviated inflammation and oxidative stress by activating NRF2/ARE signaling in CP/CPPS rat model and human prostate epithelial cell injury model.

Six new iridoid glycosides from the whole plants of Hedyotis diffusa.

Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa.

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.

Hedyotis diffusa alleviate aflatoxin B1-induced liver injury in ducks by mediating Nrf2 signaling pathway.

Aflatoxin B1 (AFB1), the most harmful aflatoxins, is a frequent contamination in feed and food items, raising global concerns in animal production and human public health. Also, AFB1 induces oxidative stress, cytotoxicity, mutations, and DNA lesions through its metabolic transformation into aflatoxin B1-8,9-epoxide (AFBO) by cytochrome P450 (CYP450). Hedyotis diffusa (HD) is a traditional Chinese herbal medicine known for its multiple pharmacological activities, including antioxidant, anti-inflammatory, and immunomodulatory. Yet, the influence of HD on AFB1-induced liver injury in ducks is still unknown. Here, we investigated whether HD positively affects AFB1-induced liver injury in ducks. Results revealed that I) AFB1 caused significant changes in serum biochemical indices and decreased growth performance of ducks (such as ALT, AST, ALP, TP, ALB, final body weight, and body weight gain), whereas HD supplementation at 200 mg/kg mitigated these alterations. II) HD alleviated hepatic histopathological changes and liver index induced by AFB1 in ducks. III) HD significantly attenuated AFB1-induced oxidative stress, as measured by increased antioxidant enzyme activities such as SOD, GPx, and T-AOC and decreased MDA levels. Furthermore, HD reduced the level of AFB1-DNA adduct in duck liver. IV) HD significantly promoted the transcriptional expression of NF-E2-related nuclear factor 2 (Nrf2) and associated genes, including heme oxygenase 1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), glutamate-cysteine ligase catalytic (GCLC). In conclusion, these results demonstrated that HD could activate the Nrf2 pathway in ducks to reduce the hepatotoxicity driven by AFB1. This finding also provides theoretical and data support for a deeper understanding of the toxic mechanisms of AFB1 and its prevention.

Two new iridoid glycosides from Hedyotis diffusa.

Two new iridoid glycosides, named productasperulosidic acid butyl ester (1) and E-6-O-3-hydroxy-p-methoxycinnamoyl scandoside methyl ester (2), along with nine known ones (3-11), were isolated from Hedyotis diffusa Willd. The structures of them were elucidated by extensive 1D, 2D NMR and HR-ESI-MS spectral data. Compounds 1-11 showed no significant cytotoxic activity against HeLa cells.

Development and evaluation of Hedyotis corymbosa (L.) extract containing phytosomes: a preclinical approach for treatment of neuropathic pain in rodent model.

PURPOSE: The study was aimed to encapsulate Hedyotis corymbosa extract (HCE) into phytosomes to improve its therapeutic efficacy in neuropathic pain by enhancing the bioavailability of chief chemical constituent Hedycoryside -A (HCA). METHODS: For preparing phytosomes complexes (F1, F2, and F3), HCE and phospholipids were reacted in disparate ratio. F2 was chosen to assess its therapeutic efficacy in neuropathic pain induced by partial sciatic nerve ligation. Nociceptive threshold and oral bioavailability were also estimated for F2. RESULTS: Particle size, zeta potential and entrapment efficiency for F2 were analysed as 298.1 ± 1.1 nm, -3.92 ± 0.41 mV and 72.12 ± 0.72% respectively. F2 gave enhanced relative bioavailability (158.92%) of HCA along with a greater neuroprotective potential showing a significant antioxidant effect and augmentation (p < 0.05) in nociceptive threshold with the diminution in damage to nerves. CONCLUSION: F2 is an optimistic formulation for enhancing the HCE delivery for the effective treatment of neuropathic pain.

[Chemical constituents from whole herb of Hedyotis scandens].

This study aimed to investigate the chemical constituents in the water extract of the whole herb of Hedyotis scandens by silica gel, ODS, and MCI column chromatographies together with preparative high-performance liquid chromatography(HPLC). The structures of isolated constituents were identified by NMR, HR-ESI-MS, etc. Thirteen compounds were isolated and identified as methyl 4-benzoyloxy-3-methoxybenzeneacetate(1), 4-benzoyloxy-3-methoxybenzeneacetic acid(2), 3-(4-hydroxy-3-methoxyphenyl)-propanoic acid(3), salicylic acid(4), 3-hydroxy-4-methoxypyridine(5), syringic acid(6), hydroxycinnamic acid(7),(R)-6-methyl-4,6-bis(4-methylpent-3-enyl)cyclohexa-1,3-dienecarbaldehyde(8), 1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol(9), 1H-indole-3-carboxaldehyde(10), isoscopoletin(11), syringaresinol(12), and pinoresinol(13). Among them, compounds 1 and 2 were new phenolic acid compounds, compounds 3-5, 8-11, and 13 were isolated from this genus for the first time, and compounds 6, 7, and 12 were obtained from H. scandens for the first time. The activity test showed that compounds 1 and 10 had a certain inhibitory effect on Mycobacterium smegmatis, with MIC_(50) values of 58.5 and 33.3 µg·mL~(-1), respectively.

Phytochemical Composition and Anti-Aging Activity of Butanol Extract of Hedyotis diffusa in Caenorhabditis elegans.

Hedyotis diffusa Willd. (H. diffusa), a kind of traditional Chinese medicine, has been evaluated to potential display antioxidant and anti-aging effects in vitro experiments. In this work, we investigated the effects on lifespan and stress resistance of the butanol extract from H. diffusa (NHD) in vivo using a Caenorhabditis elegans (C. elegans) model. The phytochemicals of NHD were identified by UPLC-ESI-qTOF-MS/MS method. NHD-treated wild-type N2 worms showed an increase in survival time under both normal and stress conditions. Meanwhile, NHD promoted the healthspan of nematodes by stimulating growth and development, reducing the deposition of age pigment, increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase dismutase (GSH-Px), and decreasing the level of ROS without impairing fertility. Moreover, the upregulating of the expression of daf-16, gst-4, sod-3, hsp12.6 genes and the downregulating of the expression of daf-2 were involved in the NHD-mediated lifespan extension. Finally, the increasing of the expression of GST-4::GFP in CL2166 transgenic nematodes and the life-span-extending activity of NHD was completely abolished in daf-2 and daf-16 mutants further revealed that the potential roles for these genes in NHD-induced longevity in C. elegans. Collectively, our findings suggest that NHD may have an active effect in healthy aging and age-related diseases.

Diagnostic ion filtering targeted screening and isolation of anti-inflammatory iridoid glycosides from Hedyotis diffusa.

Efficient and targeted screening and isolation of bioactive compounds from complex natural products is still a challenging work. Herein, diagnostic ion filtering based high-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry was firstly developed to screen six main iridoid glycosides from Hedyotis diffusa. Then, online extraction-high-speed counter current chromatography was proposed for targeted enrichment and preparative isolation using ethyl acetate/n-butanol/water (4.5:0.5:5, v/v/v) as solvent system. After that, Sephadex LH-20 column chromatography using methanol as solvent system was selected for further purification of six iridoid glycosides with purities over 98%. They were finally identified as monotropein, desacetylasperuloside acid, asperuloside, 6-O-(Z)-p-coumaroyl scandoside methyl ester, 6-O-(Z)-feruloyl scandoside methyl ester, and 6-O-(E)-p-coumaroyl scandoside methyl ester. And their anti-inflammatory activities were evaluated and confirmed by lipopolysaccharide activated RAW 264.7 macrophages. Obviously, the results provide a scientific basis for the potential applications of H. diffusa, and the developed methodology is efficient and reliable for targeted screening and isolation of bioactive compounds from natural products.

The effectiveness of Hedyotis diffusa Willd extract in a mouse model of experimental autoimmune prostatitis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a frustrating and often debilitating disease. Current studies have shown that Traditional Chinese Medicine (TCM) can improve patients' quality of life and alleviate CP/CPPS symptoms. In this study, the efficacy of Hedyotis diffusa Willd aqueous extraction in experimental autoimmune prostatitis (EAP) mice models was revealed. The C57BL/6 mice were randomly assigned to three groups. Except for the control group, all other groups were subcutaneously injected with 0.2 ml emulsion of T2 peptide, on day 0 and day 14, for inducing EAP models. After the EAP modelling, oral saline was given to the model group, while the H. diffusa group was treated with aqueous extract of H. diffusa Willd. Micturition habits and withdrawal response frequencies were measured. Haematoxylin and eosin staining and immunohistochemistry were used to investigate inflammatory cell infiltration and TNF-α in the prostate tissue respectively. TNF-α levels in the serum were evaluated by ELISA. The H. diffusa Willd aqueous extraction considerably reduced the urine spots number and increased the pain threshold in H. diffusa group. H. diffusa group showed significantly reduced inflammatory lesion and inflammatory cell infiltration than the model group. The levels of TNF-α in H. diffusa group were considerably reduced.

N-butanol extract of Hedyotis diffusa protects transgenic Caenorhabditis elegans from Aß-induced toxicity.

Hedyotis diffusa Willd (Rubiaceae) is a widely used and resourceful traditional Chinese medicine that exerts protection against aging and age-related diseases. However, the underlying mechanisms of the protective effects remain largely unclear. Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which ß-amyloid (Aß)-induced toxicity has been suggested as a main cause. Herein, we use the transgenic Caenorhabditis elegans CL4176, CL2006, and CL2355 strains, which express human Aß1-42 peptide, to investigate the effects and the possible mechanisms of n-butanol extract of H.diffusa (HDB)-mediated protection against Aß toxicity in vivo. During the experiments, a method of quality control for HDB was established by HPLC. Additionally, we examined the effects of HBD on gene expression changes with qRT-PCR, aggregation of Aß plagues with thioflavin-S staining, and protein detection with GFP labeling. HDB improved lifespan, locomotion, and stress resistance. Further study showed that HDB decreased paralysis, the accumulation of ROS, and AChE activity. Moreover, HDB suppressed neuronal Aß-expression-induced defects in chemotaxis behavior and increased SOD activity. HDB also downregulated the Aß mRNA level and decreased the number of Aß deposits. Furthermore, HDB increased the expression levels of sod-3, daf-16, hsf-1, and hsp-16.2 gene and upregulated hsp-16.2::GFP and gst-4::GFP expression. Taken together, these results suggest that HDB may protect against Aß-induced toxicity in C. elegans via the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway.

[Effect of "Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma" in treatment of lung adenocarcinoma based on network pharmacology].

To explore the mechanism of Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma(HDH-SGR) in treating lung adenocarcinoma based on big data bioinformatics combined with network pharmacology analysis and molecular docking technology. The chemical components and potential therapeutic targets of HDH-SGR were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Lung adenocarcinoma-related genes were obtained from The Cancer Genome Atlas(TCGA), Therapeutic Target Database(TTD), Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB), Online Mendelian Inheritance in Man(OMIM), DrugBank, and GeneCards. "Drug component-target" network was constructed using Cytoscape to screen out key compounds. STRING was used to build protein-protein interaction(PPI) network and core targets were screened out by Cytoscape-CytoNCA topology analysis. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses of target genes were performed by R-clusterProfiler. Finally, key compounds were docked to core target genes using AutoDock. The results showed that 22 active compounds and 499 potential therapeutic targets were obtained from HDH-SGR. A total of 14 332 lung adenocarcinoma-related targets were screened out through six data platforms, including 182 common targets. Fifteen core targets were screened out from the PPI network. GO and KEGG analyses revealed significant enrichment of relevant target genes in various biological processes, cellular functions(e.g., response to lipopolysaccharide, nuclear receptor activity, and ligand-activated transcription factor activity) and close relationship between target genes and non-small cell lung cancer signaling pathways. Based on the results of molecular docking validation, diosgenin, quercetin, naringenin, taxifolin, 2-methoxy-3-methyl-9,10-anthraquinone, stigmasterol, and ß-sitosterol were able to bind tightly to the core targets. HDH-SGR can intervene in lung adenocarcinoma through multiple targets and signaling pathways, such as non-small cell lung cancer signaling pathways. The binding of active components in Chinese medicine to key targets is presumedly one of the mechanisms that produce therapeutic effects.

Protective Antioxidant Effects of Amentoflavone and Total Flavonoids from Hedyotis diffusa on H2 O2 -Induced HL-O2 Cells through ASK1/p38 MAPK Pathway.

In this study, total flavonoids and total triterpenoid acid were extracted with ethyl acetate from Hedyotis diffusa Willd, and hepatoprotective activities of them and five compounds from total flavonoids against H2 O2 induced hepatocyte damage on HL-02 cells were determined. In particular, amentoflavone and total flavonoids had influence on the leakage of ALT, AST, LDH, the activities of SOD and the content of MDA. They effectively reduced the loss of MMP, the release of Cyt C, and then inhibited activation of caspase-3/caspase-9 cascade in hepatotoxic cells. The contents of ROS were significantly reduced to inhibit p38 in amentoflavone and flavonoids groups which decreased ASK1 and p-p38 levels through increasing thioredoxin Trx1 and reductase TrxR1. These results suggesting that the antioxidant protection of amentoflavone and flavonoids might be reducing ROS to inhibit the H2 O2 -induced upstream of pathway via increasing levels of Trx1 and TrxR1, which were pivotal in blocking the down streaming effectors of ASK1/p38 MAPK pathway and alleviating hepatotoxicity.

Bioassay-Guided Isolation of Anti-Alzheimer Active Components from the Aerial Parts of Hedyotis diffusa and Simultaneous Analysis for Marker Compounds.

Previous studies have reported that Hedyotis diffusa Willdenow extract shows various biological activities on cerebropathia, such as neuroprotection and short-term memory enhancement. However, there has been a lack of studies on the inhibitory activity on neurodegenerative diseases such as Alzheimer's disease (AD) through enzyme assays of H. diffusa. Therefore, H. diffusa extract and fractions were evaluated for their inhibitory effects through assays of enzymes related to AD, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and on the formation of advanced glycation end-product (AGE). In this study, ten bioactive compounds, including nine iridoid glycosides 1-9 and one flavonol glycoside 10, were isolated from the ethyl acetate and n-butanol fractions of H. diffusa using a bioassay-guided approach. Compound 10 was the strongest inhibitor of cholinesterase, BACE1, and the formation of AGEs of all isolated compounds, while compound 5 had the lowest inhibitory activity. Compounds 3, 6, and 9 exhibited better inhibitory activity than other compounds on AChE, and two pairs of diastereomeric iridoid glycoside structures (compounds 4, 8, and 6, 7) showed higher inhibitory activity than others on BChE. In the BACE1 inhibitory assay, compounds 1-3 were good inhibitors, and compound 10 showed higher inhibitory activity than quercetin, the positive control. Moreover, compounds 1 and 3 were stronger inhibitors of the formation of AGE than aminoguanidine (AMG), the positive control. In conclusion, this study is significant since it demonstrated that the potential inhibitory activity of H. diffusa on enzymes related to AD and showed the potential use for further study as a natural medicine for AD treatment on the basis of the bioactive components isolated from H. diffusa.

[Mechanism of "Scutellaria barbata-Hedyotis diffusa" against breast cancer based on network pharmacology].

To explore the mechanism of Scutellaria barbata-Hedyotis diffusa against breast cancer based on network pharmacological methods. The active components and corresponding target proteins of S. barbata and H. diffusa were screened by using Traditional Chinese Medicine Systems Pharmacology Database(TCMSP), and the targets of breast cancer were screened through the Genecards and OMIM databases. Venn online software was used to obtain the common targets of drugs and the disease, and then the "compound-target-disease" network diagram was constructed by using Cytoscape 3.7.2 software. The STRING database was used to draw the protein-protein interaction(PPI) network, and the David database was used to perform GO function and KEGG pathway enrichment analysis on effective targets. The results showed that 29 S. barbata compounds, 7 H. diffusa compounds, and 109 effective targets were screened. The 59 common targets for drugs and disease were obtained through Venn diagrams. The PPI network showed that MYC, CCND1, EGFR, ESR1, CASP3, VEGFA, etc. might be the key targets for "S. barbata-H. diffusa" in the treatment of breast cancer. In the GO function analysis, 88 entries were found, involving the regulation of transcription factor activity, receptor activity, cytokine activity, enzyme activity, and biosynthetic processes. In KEGG pathway analysis, 37 entries related to cancer, cell cycle, apoptosis, angioge-nesis and other pathways were found, including p53 signaling pathway, ErbB signaling pathway, nod like receptor signaling pathway, MAPK signaling pathway, VEGF signaling pathway, Wnt signaling pathway and mTOR signaling pathway and other classic signaling pathways. This study preliminarily revealed the key targets, biological processes and signal pathway of "S. barbata-H. diffusa" against breast cancer. It was found that its function was in a multi-target and multi-channel manner, which laid a foundation for future molecular biological experiments.

Metabolic effects of Hedyotis diffusa on rats bearing Walker 256 tumor revealed by NMR-based metabolomics.

Hedyotis diffusa, a traditional Chinese herbal medicine, is widely used for oncotherapy and shows a positive effect in the clinical treatment. But its mechanism of anticancer activities is complicated and unclear. This study was undertaken to assess the therapeutic effects and reveal detailed mechanisms of H. diffusa for oncotherapy. A Walker 256 tumor-bearing rat model was established, and metabolomic profiles of plasma and urine were obtained from 1 H NMR technique. Multivariate statistical analysis methods were used to characterize the discriminating metabolites between control (C), Walker 256 tumor-bearing rats model (M), and H. diffusa treatment (H) groups. Finally, 13 and 10 metabolomic biomarkers in urine and plasma samples were further identified as characteristic metabolites in M group, whereas H group showed a partial metabolic balance recovered, such as ornithine, N-acetyl-l-aspartate, l-aspartate, and creatinine in urine samples, and acetate, lactate, choline, l-glutamine, and 3-hydroxybutyrate in plasma samples. On the basis of the methods above, we hypothesized H. diffusa treatment reduced the injury caused by Walker 256 tumor and maintained a metabolic balance. Our study demonstrated that this method provided new insights into metabolic alterations in tumor-bearing biosystems and researching on the effects of H. diffusa on the endogenous metabolism in tumor-bearing rats.

Scandoside Exerts Anti-Inflammatory Effect Via Suppressing NF-κB and MAPK Signaling Pathways in LPS-Induced RAW 264.7 Macrophages.

The iridoids of Hedyotis diffusa Willd play an important role in the anti-inflammatory process, but the specific iridoid with anti-inflammatory effect and its mechanism has not be thoroughly studied. An iridoid compound named scandoside (SCA) was isolated from H. diffusa and its anti-inflammatory effect was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Its anti-inflammatory mechanism was confirmed by in intro experiments and molecular docking analyses. As results, SCA significantly decreased the productions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibited the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 messenger RNA (mRNA) expression in LPS-induced RAW 264.7 macrophages. SCA treatment suppressed the phosphorylation of inhibitor of nuclear transcription factor kappa-B alpaha (IκB-α), p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The docking data suggested that SCA had great binding abilities to COX-2, iNOS and IκB. Taken together, the results indicated that the anti-inflammatory effect of SCA is due to inhibition of pro-inflammatory cytokines and mediators via suppressing the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which provided useful information for its application and development.

Discovery of the Potential Biomarkers for Discrimination between Hedyotis diffusa and Hedyotis corymbosa by UPLC-QTOF/MS Metabolome Analysis.

Hedyotis diffuse Willd. (HD) and Hedyotis corymbosa (L.) Lam. (HC), two closely related species of the same genus, are both used for health benefits and disease prevention in China. HC is also indiscriminately sold as HD in the wholesale chain and food markets. This confusion has led to a growing concern about their identification and quality evaluation. In order to further understand the molecular diversification between them, we focus on the screening of chemical components and the analysis of non-targeted metabolites. In this study, UPLC-QTOF-MSE, UNIFI platform and multivariate statistical analyses were used to profile them. Firstly, a total of 113 compounds, including 80 shared chemical constituents of the two plants, were identified from HC and HD by using the UNIFI platform. Secondly, the differences between two herbs were highlighted with the comparative analysis. As a result, a total of 33 robust biomarkers enabling the differentiation were discovered by using multivariate statistical analyses. For HC, there were 18 potential biomarkers (either the contents were much greater than in HD or being detected only in HC) including three iridoids, eight flavonoids, two tannins, two ketones, one alcohol and two monoterpenes. For HD, there were15 potential biomarkers (either the contents were much greater than in HC or being detected only in HD) including two iridoids, eight flavonoids, one tannin, one ketone, and three anthraquinones. With a comprehensive consideration of the contents or the MS responses of the chemical composition, Hedycoryside A and B, detected only in HC, could be used for rapid identification of HC. The compounds 1,3-dihydroxy-2-methylanthraquinone and 2-hydroxy-3-methylanthraquinone, detected only in HD, could be used for rapid identification of that plant. The systematic comparison of similarities and differences between two confusing Chinese herbs will provide reliable characterization profiles to clarify the pharmacological fundamental substances. HC should not be used as the substitute of HD.

[Regulatory effect and relevant mechanisms of fraction from heat-clearing and detoxifying herb couplet on macrophage M1/M2 phenotypes].

To explore the regulatory effect and relevant mechanisms of the fraction of Hedyotis diffusa and Scutellaria barbata herb couple(YDW11) on polarization of macrophage between M1/M2 phenotypes.RAW264.7 cells were induced with LPS/IFN-γ or IL-4/IL-13 to establish M1 or M2 macrophage cell model. MTT assay was used to measure the cell cytotoxicity of YDW11. Griess reaction was used to detect the changes of nitrite accumulation in the cell supernatant. Trans-well assay was used to measure the migration capability. QRT-PCR was used to assay mRNA expressions of iNOS, IL-1ß, Arg-1 and MR. Western blot was used to detect the effect of YDW11 on iNOS and Arg-1 protein expressions. Taqman MicroRNA RT-PCR was used to detect the effect of YDW11 on miR155 expression under M1 and M2 phenotype conditions. In addition, MS-UPLC assay was carried out to identify the constituents in YDW11. The results showed that the ethyl acetate of H. diffusa and S. barbata extracted in 1:1 ratio with water (YDW11) showed the activity in suppressing the nitrite content in M1 macrophages without cytotoxicity. YDW11 also inhibited the migration of breast cancer cells with the help of M2 macrophages by blocking their polarization towards M2. YDW11 decreased iNOS, IL-1ß, Arg-1and MR mRNA expressions and iNOS and Arg-1 protein expressions. YDW11 down-regulated miR155 expression in M1 phenotype, and up-regulated miR155 expression in M2 phenotype. Based on MS-UPLC,four compounds were identified in YDW11, including 4'-hydroxyacetophenone, scutellarin, luteolin and apigenin. YDW11 inhibited M1/M2 phenotypes of macrophages by regulating the expression of miR155.