Yudan Cao

Bio-Guided Isolation of the Cytotoxic Terpenoids from the Roots of Euphorbia kansui against Human Normal Cell Lines L-O2 and GES-1

The dried roots of Euphorbia kansui (kansui) have been used for centuries in China as a herbal medicine for edema, ascites, and asthma. The 95% ethanol extract showed a significant inhibition of cell proliferation against human normal cell lines L-O2 and GES-1. Bioassay-guided separation of the 95% ethanol extract from the roots of E. kansui led to the isolation of 12 diverse terpenoids whose structures were identified by 1H, 13C NMR spectroscopy and ESI-MS as kansuinine A (1), kansuinine B (2), kansuinine C (3), kansuiphorin C (4), 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol (5), 3-O-(2′E,4′Edecadienoyl)-20-O-acetylingenol (6), 3-O-(2′E,4′Z-decadienoyl)-20-deoxyingenol (7), 3-O-benzoyl-20-deoxyingenol (8), 5-O-benzoyl-20-deoxyingenol (9), kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5–11) exhibited a relatively lower IC50 value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb.

Anti-inflammatory effects of Huangqin tang extract in mice on ulcerative colitis

ETHNOPHARMACOLOGICAL RELEVANCE HuangqinTang (HQT) is a traditional Chinese formula which is composed of Scutellaria baicalensis Georgi, Paeonia lactiflora Pall, Glycyrrhiza uralensis Fisch, and Ziziphus jujube Mill. HQT has been used in China for a wide range of disorders, especially in gastrointestinal inflammation with symptoms of nausea, vomiting, diarrhea, abdominal cramps and so on. AIM OF THE STUDY To investigate the protective effects of HQT extract on 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) induced colitis in mice. MATERIALS AND METHODS Different doses of HQT extract (1, 2 and 4 g/kg/day) and salicylazosulfapyridine (SASP, 500 mg/kg/day) were administered by gavage for 7 days after the induction of colitis with TNBS. The effects were studied by macroscopic score, histological analysis, immunohistochemical study of Cyclo-oxygenase-2 protein expression, as well as by determination of inflammation markers such as myeloperoxidase (MPO) and mRNA expression levels of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6. RESULTS In TNBS induced group, mice body weight decreased gradually and did not recover at the end of the experiment, as compared with that of control group (p<0.01). Edema and redness were also discovered in the colons profoundly and scores representing inflammation were all high in this group (p<0.01). The level of colonic MPO activity and the tissue levels of TNF-α, IL-1β and IL-6 were markedly increased (p<0.01). The mice treated with HQT extract and SASP recovered significantly compared with the TNBS group (p<0.01). CONCLUSION Our results suggested that the efficacy of HQT extract, especially at the higher dose, was analogous to that of SASP, which implicated its potential application as a natural alternative medicine in colitis treatment.

Processing of Kansui Roots Stir-Baked with Vinegar Reduces Kansui-Induced Hepatocyte Cytotoxicity by Decreasing the Contents of Toxic Terpenoids and Regulating the Cell Apoptosis Pathway

Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased contents of five diterpene and one triterpene in kansui (GS-1) after stir-baking with vinegar (GS-2) was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm), decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS), ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb.

A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro

Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

Effects of Schizonepetin on Activity and mRNA Expression of Cytochrome P450 Enzymes in Rats

The aim of this study was to find out whether Schizonepetin influences the pharmacokinetics of the main substrates drugs of CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 in rats; the influence on the levels of CYP mRNA was also studied. Phenacetin, dapsone, chlorzoxazone, omeprazole and metoprolol were selected as probe substrates for CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 respectively. HPLC methods were employed for the determination of these substrates in plasma and the pharmacokinetic parameters were calculated. Real-time RT-PCR was used to determine the effects of Schizonepetin on the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 in the rat liver. After the rats were orally administrated with Schizonepetin once a day for seven consecutive days, there were significant differences in plasma concentration of phenacetin, dapsone, chlorzoxazone and metoprolol, but not omeprazole, as compared with pre-administration. In addition, Schizonepetin induced the expression of CYP3A1, CYP1A and CYP2E1 at dosages of 24 and 48 mg/kg. Our results indicated that Schizonepetin had significant induction effects on CYP3A1/2 and inhibition effects on CYP1A2, CYP2E1 or CYP2D6 as oriented from the pharmacokinetic profiles of the substrates. Moreover, in the mRNA expression levels, Schizonepetin could induce the mRNA expression of CYP3A1, CYP1A and CYP2E1. In conclusion, co-administration of some CYP substrates with Schizonepetin may lead to an undesirable herb-drug interaction.

Application of UHPLC-ESI-Q-TOF-MS to Identify Multiple Constituents in Processed Products of the Herbal Medicine Ligustri Lucidi Fructus

Ligustri Lucidi Fructus (LLF), the fruit of Ligustrum lucidum Ait. (Oleaceae), has been used as a common herbal medicine in clinical practice in China for nearly 2000 years. In most cases, LLF is prescribed in decoctions in the form of processed products rather than crude drugs. In this study, an ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) method was established for rapid separation and identification of multiple constituents in the 80% methanol extract of processed-LLF. A total of 50 compounds (one phenylethanoid, seven phenylethanoid glycosides, seven flavonoids, 25 iridoids, nine triterpenoids and one cyclohexanecarboxylic acid) were either unambiguously identified or tentatively characterized with the aid of authentic standards or published data. Luteolin-7-O-rutinoside, oleoside and secologanoside were detected in LLF for the first time. This study enriches the chemical profiling of processed-LLF and could provide valuable information for the quality control and further investigation of processed-LLF and crude LLF.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro

Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Toxicity of Pekinenin C from Euphorbia Pekinensis Radix on Rat Small Intestinal Crypt Epithelial Cell and Its Apoptotic Mechanism.

Pekinenin C is a casbane diterpenoid separated from the root of the traditional Chinese medicine, Euphorbia pekinensis Rupr., which is used as drug for the treatment of edema, ascites, and hydrothorax. Whereas pekinenin C exhibits severe cytotoxicity, the exact toxicity mechanism is unclear. In this study, the effects of pekinenin C on cell inhibition, cell cycle, and cell apoptosis were examined to explain its toxic mechanism. The proliferation of IEC-6 cells was accessed via MTT colorimetric assay after incubated with different concentrations of pekinenin C. Pekinenin C-treated IEC-6 cells labeled with RNase/PI and Annexin V/PI were analyzed by flow cytometric analyses for evaluation of cell cycle distribution and cell apoptosis, respectively. The apoptosis mechanism of pekinenin C on IEC-6 was investigated through assaying the activities of caspase-3, 8, 9 by enzyme-linked immunosorbent assay (ELISA), protein expression of Bax, Bcl-2, apoptosis-inducing factor (AIF), Apaf-1, Fas-associated death domain (FADD) and type 1-associated death domain (TRADD) by Western-blot, mRNA expression of Fas receptor (FasR), Fas ligand (FasL), tumor necrosis factor receptor (TNFR1) and NF-κB by RT-PCR. The results showed that pekinenin C has exhibited obvious IEC-6 cells toxicity and the IC50 value was 2.1 μg·mL−1. Typical apoptosis characteristics were observed under a transmission electron microscopy, and it was found that pekinenin C could cause G0/G1 phase arrest in IEC-6 cells in a dose-dependent manner and induce apoptosis of IEC-6 cells. Additionally, pekinenin C could increase the expressions of Bax, AIF, Apaf-1, FasR, FasL, TNFR1 and NF-κB, suppress the expression of Bcl-2, FADD and TRADD, then activate caspase-3, 8, 9 cascades, and at last result in apoptosis. These results demonstrated that pekinenin C effectively promoted cell apoptosis, and induced IEC-6 cells apoptosis through both the mitochondrial and death receptor pathways.

Antioxidant capacity of Typha angustifolia extracts and two active flavonoids

Abstract Context: The pollen of Typha angustifolia L. (Typhaceae) has been used as a traditional Chinese medicine for improving the microcirculation and promoting wound healing. Flavonoids are the main constituent in the plant, but little is known about the antioxidant activity of the principal constituent of the pollen in detail. Objectives: To assess the antioxidant activities of ethanol and water extracts and two constituents of the pollen. Materials and methods: Plant material (1 g) was extracted by 95% ethanol and water (10 mL × 2, 1 h each), respectively. The extracted activities (0.8–2.6 mg/mL) were measured by DPPH and the reducing activity of ferric chloride (1.7–2.6 mg/mL). Typhaneoside and isorhamnetin-3-O-neohesperidoside (I3ON) (2.8–70 μmol/L) were investigated on the relationship between NO, MDA and SOD in HUVECs treated with 100 μg/mL of LPS for 24 h. Results: Nine compounds were identified by UPLC-MS. Ethanol extract showed IC50 values in DPPH (39.51 ± 0.72) and Fe3+ reducing activity (82.76 ± 13.38), higher than the water extract (50.85 ± 0.74) and (106.33 ± 6.35), respectively. Typhaneoside and I3ON promoted cell proliferation at the respective concentration range of 2.8 to 70 μmol/L (p < 0.01). This two compounds decreased MDA (1.91 ± 0.10, 1.80 ± 0.34, p < 0.05) and NO levels (14.64 ± 0.08, 13.10 ± 0.88, p < 0.01), respectively, and increased SOD level (22.94 ± 2.48, 23.57 ± 2.38, p < 0.01) at the concentration of 70 μmol/L compared with LPS group. Conclusions: The constituents from Typha angustifolia could be a novel therapeutic strategy for LPS-induced inflammation.

Anti-thrombotic and pro-angiogenic effects of Rubia cordifolia extract in zebrafish

ETHNOPHARMACOLOGICAL RELEVANCE Rubia cordifolia is a common traditional Chinese medicine that promotes blood circulation and eliminates blood stasis, and has been used to cure diseases related to blood stasis syndrome (BSS) clinically for many years. It has been previously demonstrated that anti-thrombosis and pro-angiogenesis can improve BSS. However, the anti-thrombotic and pro-angiogenic activities of Rubia cordifolia have not been well investigated. AIM OF STUDY To determine the potential anti-thrombotic and pro-angiogenic activities of Rubia cordifolia and to elucidate the underlying mechanisms. In addition, the major chemical constituents of Rubia cordifolia extract (QC) were qualitatively analysed by UPLC-Q-TOF/MS to explore the association between pharmacological activity and chemical constituents. MATERIAL AND METHODS The QC samples were composed of a 95% ethanol extract and an aqueous extract following extraction using 95% ethanol. UPLC-Q-TOF/MS was used to analyse the major chemical constituents of QC. For the anti-thrombotic experiment of QC, a phenylhydrazine (PHZ)-induced AB strain zebrafish thrombosis model was used. The zebrafish larvae were stained using O-dianisidine, and the heart and caudal vein of the zebrafish were observed and imaged with a fluorescence microscope. The staining intensity of erythrocytes in the heart (SI) of each group and the morphology of thrombus in the caudal vein were used to assess the anti-thrombotic effect of QC. For the pro-angiogenic assay of QC, the intersegmental blood vessel (ISV) insufficiency model of Tg(fli-1: EGFP)y1 transgenic zebrafish (Flik zebrafish), which was induced by the VEGF receptor tyrosine kinase inhibitor II (VRI), was used. The morphology of the intact ISVs and defective ISVs was observed to evaluate the pro-angiogenic activity of QC. The mechanism involved in promoting angiogenesis was studied with real-time PCR. RESULTS A total of 12 components in QC were identified based on standard compounds and references, including nine anthraquinones and three naphthoquinones. After treatment with QC, the PHZ-induced thrombosis in AB strain zebrafish larvae decreased to a certain degree, which we believe was related to its dosages, and the therapeutic effect within the 50-200 µg/mL QC treatment groups was especially prominent (P < 0.01, P < 0.001) compared to that in the PHZ model group. Similarly, QC also recovered the loss of the ISVs, which was induced by VRI in Flik zebrafish larvae, which have a certain dose-effect relationship. The pro-angiogenic activity of QC was also conspicuous (P < 0.01, P < 0.001) compared to that of the VRI model group. The following real-time PCR assay proved that QC significantly restored the VRI-induced downregulation of vWF, VEGF-A, kdrl, and flt-1 in Flik zebrafish (P < 0.05, P < 0.01, P < 0.001). CONCLUSIONS A total of 12 compounds from QC were analysed by UPLC-Q-TOF/MS. The data of the pharmacological experiments demonstrated that QC presented anti-thrombotic and pro-angiogenic activities in zebrafish, and the principal active components were likely anthraquinones and naphthoquinones. Thus, the current study provided a theoretical basis for the clinical use of Rubia cordifolia as a traditional Chinese medicine in promoting blood circulation and eliminating stasis.