Gallic acid ameliorates dextran sulfate sodium-induced ulcerative colitis in mice via inhibiting NLRP3 inflammasome.

Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease (IBD). The conventional drugs for UC may induce severe side effects. Herbal medicine is considered as a complementary and alternative choice for UC. Purpose: This study aims to estimate the effect of natural polyphenol gallic acid (GA) on the NLRP3 inflammasome with dextran sulfate sodium (DSS)-induced colitis in mice. Study design: The body weights and symptoms of BALB/c mice were recorded. Histological evaluation, ELISA, q-PCR, immunohistochemistry, and western blotting were carried out to observe the morphology, cytokine contents, mRNA expressions, and protein expressions, respectively. Lipopolysaccharide (LPS)-induced RAW264.7 macrophage was used to probe GA's effect on relative protein expression. Results: GA attenuated weight loss (p < 0.05), relieved symptoms, and ameliorated colonic morphological injury (p < 0.05) in mice with colitis induced by DSS. GA also lowered the contents of TNF-α, IL-1ß, IL-18, IL-33, and IFN-γ in the serum and colon of mice, which were elevated by DSS, downregulated protein, and mRNA expressions of the NLRP3 pathway in the colon tissue. Furthermore, GA downregulated the expressions of NLRP3 (p < 0.05), iNOS (p < 0.01), COX2 (p < 0.01), and P-p65 (p < 0.05), and suppressed NO release (p < 0.001) in LPS-induced RAW264.7 cells. Conclusion: GA ameliorated DSS-induced UC in mice via inhibiting the NLRP3 inflammasome. These findings furnish evidence for the anti-inflammatory effect of herbal medicines containing GA on UC.

Beware of the Potential Risks for Polygoni Multiflori Caulis-Induced Liver Injury.

Background: Reynoutria multiflora (Thunb.) Moldenke (PM) is a widely-used medicinal plant in China, whose root and stem are included in the Chinese Pharmacopoeia as Polygoni Multiflori Radix (RPM), Polygoni Multiflori Radix Preparata (PMP), and Polygoni Multiflori Caulis (PMC). The hepatotoxicity of RPM and PMP is concerned by the public, while the risk of PMC is ignored. Purpose: Here, we investigate the potential risks for PMC-induced liver injury from clinical, chemical, and animal features. Study design: First, we analyzed the 12-month usage of RPM, PMP, and PMC in Longhua Hospital. Second, we determined the contents of gallic acid, cis-2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside (cis-SG), trans-2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside (trans-SG), emodin-8-O-ß-D-glucoside (EG), physcion-8-O-ß-D-glucoside (PG), emodin, and physcion in the water extracts from 15 batches of RPM, PMP, and PMC. Third, we probed the hepatotoxic effect of RPM, PMP, and PMC in mice and explored the mechanism of cis-SG and trans-SG causing the liver injury at the dosages based on our results from the first and second parts. Results: PMC had nearly five times the amount of usage in both outpatient prescriptions and inpatient orders than RPM and PMP. Overall, 68% dosage of PMC was 30 g. The contents of cis-SG, trans-SG, and emodin in PMC water extracts were significantly lower than those in RPM and PMP water extracts. PMC induced milder idiosyncratic liver injury for its lower content of cis-SG and trans-SG than its root counterparts. Conclusion: The potential risks for PMC-induced liver injury should be fully aware of.

Fingerprinting and Determination of Hepatotoxic Constituents in Polygoni Multiflori Radix Praeparata of Different Producing Places by HPLC.

Polygoni Multiflori Radix Praeparata (PMRP) is used as Chinese herbal medicine with long history. However, reports about PMRP hepatotoxicity have increased recently, and producing area might be one reason. This article aims to figure out the relationship between producing area and hepatotoxic ingredients in PMRP. HPLC fingerprint for PMRP was established and the contents of gallic acid, trans-stilbene glycoside (TSG), emodin-8-O-ß-D-glucoside (EG), emodin and physcion were determined. Clustering heatmap was implemented by TCMNPAS software，and principal component analysis was implemented by SPSS and SIMCA-P software. Hepatotoxic constituents' contents of PMRP from separate producing area were different. PMRP from Guangxi had the highest content of gallic acid, TSG, EG, emodin and physcion, followed by Hubei, Guangdong, Guizhou, Yunnan. PMRP from Henan had the lowest contents of hepatotoxic components. Hepatotoxic components' contents of PMRP in southern were higher than central China. This study carried out a preliminary qualitative and quantitative investigation on the PMRP from different producing places, which provided a basis for safe medication of PMRP.

Effects of Qing Chang Suppository Powder and its Ingredients on IL-17 Signal Pathway in HT-29 Cells and DSS-induced Mice.

BACKGROUND: Qingchang Suppository, a formula used for more than 30 years in Longhua Hospital, has shown satisfactory clinical effects on Ulcerative Colitis (UC). However, its therapeutic mechanism has not been fully elucidated. PURPOSE: The study aims to investigate the effects of Qingchang Suppository powder (QCSP) and its ingredients by regulating the IL-17A signaling pathway which plays an important role in the development of UC. METHODS: HPLC was used to analyze the main ingredients (Gallic acid, Indigo, Indirubin) in QCSP. HT-29 cells were induced by rhIL-17A and TNF-α, and IL-17A related protein expressions were determined by western blot. BALB/C mice were induced by 4% Dextran Sodium sulfate (DSS). The effects of QCSP and its ingredients were evaluated by measuring weight loss, disease activity index (DAI), colon length, histological analysis. Western blot was used for analysis of IL-17A and MAPK related proteins p-ERK, p-JNK, p-P38. Quantitative reverse transcription polymerase chain reaction (q-PCR) was used to detect the expression of IL-17A, HSP90 and ACT1 in colon tissue. Cytokines such as IL-17A, IL-1ß, IFN-γ and TNF-α were determinated by enzyme-linked immunosorbent assay (ELISA). RESULTS: QCSP had good therapeutic effect on DSS-induced colitis in mice. QCSP significantly relieved weight loss, restored colon length, repaired colon lesions, reduced histological scores and DAI, decreased TNF-α, IL-1ß, IL-17 and IFN-γ contents, significantly suppressed the gene expressions of IL-17A, ACT1 and HSP90, and up-regulated the expressions of tight junction proteins like ZO-1 and Occludin. IL-17A pathway related proteins such as IL-17A, IL-17RA, HSP90, MAPKs, P-iκbα and iNOS were significantly increased in vitro and in vivo. CONCLUSIONS: This paper reveals that QCSP inhibited the IL-17A signaling pathway in HT-29 cells and DSS induced mice, presenting a new mechanism of QCS on treating UC.

Bufothionine induces autophagy in H22 hepatoma-bearing mice by inhibiting JAK2/STAT3 pathway, a possible anti-cancer mechanism of cinobufacini.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini is extracted from the skins and parotid venom glands of the toad for treating symptoms like swelling and pain in ancient times. Nowadays, cinobifucini injection has also achieved satisfactory therapeutic effects on hepatocellular carcinoma (HCC) in China. AIM OF THE STUDY: Our previous work found that bufothionine, an alkaloid abundant in cinobufacini injection, induced mitochondria-mediated apoptosis. In this work, the underlying effects of bufothionine on autophagy in HCC and its possible dependent pathway were investigated. METHODS: CCK-8 and Hoechst staining assays were performed to verify effects of drugs on proliferation and apoptosis of SMMC7721 cell. H22-tumor-bearing mice model was established by inoculating ascites fluid. HE staining was used to observe pathological changes in liver and tumor tissues. ELISA and Western blot experiments were conducted to investigate IL-6/JAK2/STAT3 signaling pathway. The effects of drugs on expressions of autophagic relative proteins were investigated by Western blot in vitro and in vivo. RESULTS: In vitro, CCK-8 and Hoechst staining assays showed that bufothionine inhibited SMMC7721 cell proliferation and promoted apoptosis at 100 µM. In vivo, bufothionine relieved symptoms of H22-tumor-bearing mice and exerted anti-inflammation activity. ELISA and Western blot demonstrated that bufothionine significantly reduced serum IL-6 concentration, suppressed p-Stat3tyr705, p-Stat3ser727 and Jak2 expressions in tumor tissues and upregulated Atg5, Atg7 and LC3Ⅱ expressions in SMMC7721 cell and H22 tumor. CONCLUSION: This is the first report showing that bufothionine might induce autophagy in HCC by inhibiting JAK2/STAT3 pathway, presenting a possible anti-cancer mechanism of bufothionine in cinobufacini injection.

Effects and possible mechanism of Ruyiping formula application to breast cancer based on network prediction.

Ruyiping (RYP), a Chinese herbal formula, can remove toxin and clear nodular, showing ability of preventing postoperative recurrence of breast cancer. In this study, network was performed to predict possible targets, genes and pathways associated with RYP and breast cancer. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to quantitatively study RYP formula and its single herbs. MTT methods, Luciferase reporter systems, zebrafish model and western blotting were respectively adopted to verify network prediction. Results showed that the quality of RYP could be controlled and icariin could be selected as mark ingredient; RYP expressed anti-breast tumor effects, which could be associated with inhibiting expression of Transforming Growth Factor ß (TGFß), promoting cells apoptosis and anti-angiogenesis. Parts of these results were consistent with network predictions in some degree, but not all. Network can help us narrow areas, focus on crucial factors, save money as well as time, but the results predicted by network should be confirmed by further experiments.

Compatibility Study of Danggui Buxue Tang on Chemical Ingredients, Angiogenesis and Endothelial Function.

Danggui Buxue Tang (DBT) is a classic Chinese herbal formula which consists of Astragali mongholici Radix and Angelica sinensis Radix (ASR). For chemical ingredients, HPLC were performed. Results showed compared with single herbs, DBT decoction could promote the dissolution of ingredients such as ferulic acid and calycosin. Furthermore, when ratio of AMR to ASR was 5 to 1, synthetic score was the best. For angiogenesis, normal and injured zebrafish model were applied. Results showed DBT and its ingredients had angiogenesis effects on Sub Intestinal vessels (SIVs) of normal zebrafish. Meanwhile, DBT and its single herbs could also recover Inter-Segmental Vessels (ISVs) injured by VRI. Angiogenesis effects of DBT on ISVs were better than single herbs. AMR extract, Total Saponins of AMR, Polysaccharide of ASR, ferulic acid, calycosin and calycosin-7-glucoside could be effective ingredients for angiogenisis. For endothelium functions, Lysoph-Osphatidyl choline was used to damage rat endothelial function of thoracic aorta. The results showed DBT and its single herbs could improve endothelial dysfunctions in dose-dependence. Both ferulic acid and calycosin-7-glucoside could also improve endothelium dysfunction in dose dependence. Therefore, compatibility of DBT was reasonable. Compared with single herbs, DBT could promote dissolution of effective ingredients, enhance angiogenesis and relieve endothelial dysfunction.

Chemosynthesis pathway and bioactivities comparison of saponins in radix and flower of Panax notoginseng (Burk.) F.H. Chen.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (Burk.) F.H. Chen is a well known medicinal plant. Its radix is used in the history while its flower is recently used for health care. In this study we compared chemical ingredients and bioactivities in cell culture for radix and flower of Panax notoginseng (Burk.) F.H. Chen. MATERIALS AND METHODS: The liquid chromatography-mass spectrometry system was applied to determine the contents of saponins in flower and radix of Panax notoginseng (Burk.) F.H. Chen. Transcription specific luciferase reporter assay and qPCR method for selected RNA were carried out to assess the impacts of flower and radix extract on the transcription signal pathways. RESULTS: The results of chemical analysis showed that the contents of saponins in flower and radix are very different: the contents of Rg1, Rb1, Re, R1, Rg3-20R, Rh1 and Rf in radix are abundant; in contrast, the contents of Rb3, Fc, Ft1, Rb2 and Rh2-20s in flowers are plentiful. There are substantial variations of those saponin contents from one batch vs another. Based on relative content of saponins, the chemosynthesis pathway of ingredients in radix and flower are proposed: for radix, both PPT (Protopanaxatriol) and PPD (Protopanaxadiol) type triterpenoids are involved, the main pathway is PPT→Rb1→Rg1→R1 or PPD→Rh2 20s→Rg3(20s)→Rd→Rb1; for flowers, only PPD is main passage with PPD→Rh2 (20s)→Rg3(20s)→Rd→Rb2→Fc. The results of signal transcription assays demonstrated that herb water extract of radix and flower had no significant influences on most of transcription activities. However, total saponins of radix and flower which have highly content of saponins were able to inhibit the inflammatory related transcriptional activities and their related mRNA expression of IFNα, TNFα, il-6 and TGFß as well as induce anti-oxygen NrF2 activities. In summary, although chemical ingredients and chemosynthesis pathway of flower and radix for Panax notoginseng (Burk.) F.H. Chen were different, these differences might not result in their differences of pharmacological activities.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing "congealed" blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. MATERIAL AND METHODS: In this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. RESULTS: Our results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25-50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. CONCLUSIONS: The pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Optimization of high pressure machine decocting process for Dachengqi Tang using HPLC fingerprints combined with the Box-Behnken experimental design.

Using Dachengqi Tang (DCQT) as a model, high performance liquid chromatography (HPLC) fingerprints were applied to optimize machine extracting process with the Box-Behnken experimental design. HPLC fingerprints were carried out to investigate the chemical ingredients of DCQT; synthetic weighing method based on analytic hierarchy process (AHP) and criteria importance through intercriteria correlation (CRITIC) was performed to calculate synthetic scores of fingerprints; using the mark ingredients contents and synthetic scores as indicators, the Box-Behnken design was carried out to optimize the process parameters of machine decocting process under high pressure for DCQT. Results of optimal process showed that the herb materials were soaked for 45 min and extracted with 9 folds volume of water in the decocting machine under the temperature of 140 °C till the pressure arrived at 0.25 MPa; then hot decoction was excreted to soak Dahuang and Mangxiao for 5 min. Finally, obtained solutions were mixed, filtrated and packed. It concluded that HPLC fingerprints combined with the Box-Behnken experimental design could be used to optimize extracting process of traditional Chinese medicine (TCM).

Study on spectrum-effect relationship of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and their formula.

Rhizoma Rhei, cortex Magnoliae Officinalis and fructus Aurantii Immaturus compose dachengqi tang (DCQT), a classical formula of traditional Chinese medicine (TCM) that is used for acute intestinal obstruction and has been proven to be effective and economic. However, the ingredients of TCM are complicated, and it is unclear which ingredients are the most important for its effects. In this paper, the relationship between the spectra and effects is discussed to provide a powerful method and some insights into the quality control of the herbs and their formula. High-performance liquid chromatographic (HPLC) fingerprint analysis was performed to investigate the chemical structures in different batches of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and DCQT. Hierarchical clustering analysis was employed to evaluate the similarities between fingerprints. Animal model of small intestinal propulsion was established to study the purgative functions of the herbs and DCQT. The relationship between the chemical ingredients and the effects was explored by regression analysis. HPLC fingerprint analysis results demonstrated variations between ingredients in different batches of rhizoma Rhei, cortex Magnoliae Officinalis, fructus Aurantii Immaturus and DCQT. The origin, collection time and preparation process may have contributed to these differences. Small intestinal propulsion results showed that, compared with the control group, the positive and therapeutic groups including single herbs and formula were significantly effective (P < 0.05). Spectrum-effect relationship results indicated that seven peak ingredients, hesperidin, aloe-emodin, honokiol, rhein, magnolol, emodin and sennoside A, were inducted in the regression equation, among which, the influence of sennoside A was the largest and most positively associated with the effects. The data analysis results indicated that many ingredients contributed to the purgative effects, among which, sennoside A might be the most important effective component; therefore, sennoside A should be determined for quality control. Furthermore, the spectrum-effect relationship is simple, operative and suitable for the quality evaluation of TCM.

Bufothionine, a possible effective component in cinobufocini injection for hepatocellular carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini has been traditionally used in China for the treatment of tumor since hundreds years ago. For recent years, its modern preparation,cinobifucini injection has also obtained satisfactory therapeutic functions for cancer. MATERIALS AND METHODS: High performance liquid chromatography (HPLC) analysis was applied to determine the content of cinobufagin, resibufogenin and bufothionine in cinobufacin extract liquid and injection; MTT assay and flow cytometric analysis were also respectively used to study the effect of cinobufacini extract liquid, injection and three chemical structures on cells and cell cycles. RESULTS: HPLC results demonstrated that in cinobufacini extract liquid three ingredients (cinobufagin, resibufogenin and bufothionine) were all monitored while in cinofacini injection only bufothinone was detected; MTT assays showed bufothionine could obviously inhibit the proliferation of human hepatocellular carcinoma cell lines such as SMMC-7721 and BEL-7402 in a dose- and time-dependent manner as well as cinobufagin and resibufogenin; further flow cytometric analysis indicated obvious increases in G2/M phase and decrease in G0/G1 phase when SMMC-7721 cell line exposure to bufothionine (480 µg/ml). CONCLUSIONS: These results suggested bufothionine could be involved in treatment of human cancer for cinobufacini injection and the mechanism might be relative to induce G2/M phase cell cycle arrest.