Organs-specific metabolomics and anticholinesterase activity suggests a trade-off between metabolites for therapeutic advantages of Trillium govanianum Wall. ex D. Don.

Trillium govanianum is traditionally used to treat innumerable alignments like sexual disorders, cancer, inflammation etc. Mainly rhizomes of T. govanianum have been explored for phytochemical profiling but comprehensive metabolomics of other parts has not been yet deeply investigated. Thus, current study was aimed for organs-specific (roots, rhizomes, rhizomatous buds, stems, leaves, and fruits) phytochemical profiling of T. govanianum via metabolomics approach. Targeted (steroidal saponins and free sugars) and non-targeted metabolomics were performed by UPLC-PDA/ELSD & UHPLC-Q-TOF-IMS. Among steroidal compounds, 20-hydroxyecdysone, pennogenin-3-O-ß-chacotrioside, dioscin were found predominantly in all samples while diosgenin was identified only in rhizomes. Further, four free sugars viz. 2-deoxyribose (116.24 ± 1.26 mg/g: leaves), fructose (454.76 ± 12.14 mg/g: rhizomes), glucose (243.21 ± 7.53 mg/g: fruits), and galactose (69.06 ± 2.14 mg/g: fruits) were found significant in respective parts of T. govanianum. Elemental analysis of targeted samples was determined by atomic absorption spectrophotometer. Heavy metals (Cd, Hg, Pd, As) were absent while micro- (Mn, Na, Zn, Cu) and macro- (Ca, Fe, Mg, K) elements were found in all samples. Furthermore, UHPLC-Q-TOF-IMS had identified 103 metabolites based on their mass fragmentation patterns and 839 were tentatively predicted using METLIN database. The multivariate statistical analysis showed organs specific clustering and variance of metabolites. Apart from this, extracts were evaluated for in vitro anticholinesterase activity, and found potentials inhibitors with IC50 values 2.02 ± 0.15 to 27.65 ± 0.89 mg/mL and 3.58 ± 0.12 to 16.81 ± 2.48 mg/mL of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme, respectively. Thus, comprehensive metabolomics and anti-cholinesterase activity of different parts of T. govanianum would lay the foundation for improving medicinal importance and health benefits of T. govanianum.

Biochemical, Toxicological, and in Silico Aspects of Trillium govanianum Wall. ex D.Don (Trilliaceae): A Rich Source of Natural Bioactive Compounds.

Trillium govanianum is a high-value medicinal herb, having multifunctional traditional and culinary uses. The present investigation was carried out to evaluate the phytochemical, biological and toxicological parameters of the T. govanianum Wall. ex D. Don (Family: Trilliaceae) roots collected from Azad Kashmir, Pakistan. Phytochemical profiling was achieved by determining total bioactive contents (total phenolic and flavonoid contents) and UHPLC-MS analysis. For biological evaluation, antioxidant activities (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelation assays) and enzyme inhibition activities (against AChE, BChE, glucosidase, amylase, and tyrosinase) were performed. Moreover, cytotoxicity was assessed against three human carcinoma cell lines (MDA-MB-231, CaSki, and DU-145). The tested extract was found to contain higher total phenolics (7.56 mg GAE/g dry extract) as compared to flavonoid contents (0.45 mg RE/g dry extract). Likewise, for the antioxidant activity, higher CUPRAC activity was noted with 39.84 mg TE/g dry extract values. In the case of enzyme assays, higher activity was pointed out against the cholinesterase, glucosidase and tyrosinase enzymes. The plant extract displayed significant cytotoxicity against the cell lines examined. Moreover, the in-silico studies highlighted the interaction between the important phytochemicals and tested enzymes. To conclude, the assessed biological activity and the existence of bioactive phytochemicals in the studied plant extract may pave the way for the development of novel pharmaceuticals.

Steroidal saponins from Trillium tschonoskii rhizome repress cancer stemness and proliferation of intrahepatic cholangiocarcinoma.

A phytochemical study was carried out on the extract of Trillium tschonoskii rhizomes, resulting in the isolation of thirty-six steroidal glycosides (1-36). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence, of which 1-25 were identified as new analogues. Herein, all the isolated analogues were screened for the cytotoxicity against intrahepatic cholangiocarcinoma (ICC) cell lines of HuCCT1 and RBE through tumor colony formation and CCK-8 survival analysis, and the results demonstrated that three compounds 9, 12, and 26 significantly repressed tumor colony and sphere formation in both cell lines, respectively. Furthermore, the three analogues possessed a remarkable inhibitory role of organoid formation established from hydrodynamic induced mouse primary intrahepatic cholangiocarcinoma. Moreover, the functional assays of flow cytometry analysis, cancer stemness related gene expression, and western blotting assays all indicated that compound 26 could significantly repress cancer stem markers. Taken together, these results demonstrate that steroidal glycosides derived from T. tschonoskii rhizomes could be potentially implicated in human ICC therapy.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. AIM OF THE STUDY: We aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. METHODS: Male Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. RESULTS: TSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3ß, and down-regulated phosphorylated ß-catenin and CRMP-2 expression. CONCLUSION: Taken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/ß-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

Steroidal saponins from Trillium govanianum as α-amylase, α-glucosidase, and dipeptidyl peptidase IV inhibitory agents.

OBJECTIVE: To provide the scientific basis for the utility of rhizome of Trillium govanianum as nutraceutical supplements in managing physiological glycemic levels. METHODS: The in vitro enzyme inhibitory activity of the extract, fractions, and the isolated steroidal saponins from the rhizome part of T. govanianum was carried out against α-amylase, α-glucosidase, and dipeptidyl peptidase IV. The molecular interactions, binding score, and pharmacokinetic parameters (absorption, distribution metabolism, and excretion) of steroidal saponins were analyzed by the Schrodinger molecular docking software. KEY FINDINGS: Current study explained that the extract, fractions, and isolated steroidal saponins from T. govanianum possess good α-amylase and α-glucosidase inhibitory activity while moderate dipeptidyl peptidase IV inhibitory activity. Moreover, in vitro results revealed that borassoside E (IC50 7.15 ± 1.78 µM), protodioscin (IC50 6.72 ± 0.04 µM), and diosgenin (IC50 12.75 ± 2.70 µM) are most effective in inhibiting the activity of α-amylase, α-glucosidase, and dipeptidyl peptidase IV, respectively. Current in silico and in vitro studies established an association between the steroidal saponins from T. govanianum and their molecular interactions with α-amylase, α-glucosidase, and dipeptidyl peptidase IV. CONCLUSION: The results of this investigation suggest that fractions and steroidal saponins from T. govanianum exhibit good antidiabetic activity which could be used as nutraceutical supplements for the management of systemic glucose level.

Chemical constituents of Trillium tschonoskii Maxim.

A new fatty acid-spirostan steroid glycoside ester, a new cholestane glycoside and a new stilbene trimer, along with three known steroidal saponins, were isolated from the 70% EtOH extract of the roots and rhizomes of Trillium tschonoskii Maxim. The structure of isolated compounds was elucidated by spectroscopic analysis. Compound 1-6 were assessed for their cytotoxicity against cancer cell lines (MCF-7, HCT-116, DU-145, SGC-7901, MCF-7/ADR, K562/ADR), and the result showed that compound 4 was highly toxic to six human tumor cell lines.

Furostanol Saponins from Trillium tschonoskii Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells.

Trillium tschonoskii is a medicinal plant known to biosynthesize steroidal saponins. A phytochemical investigation of the rhizomes of T. tschonoskii led to the isolation of nine new furostanol saponins (1-9) and 11 known analogues (10-20). Five of these new compounds were shown to have hydroxy groups at the C-5 and C-6 positions, while two possess a rare aglycone containing carbonyl groups at the C-16 and C-22 positions as well as a Δ17(20) double bond, and the others have conjugated double bonds in the E-ring or have different sugar chains at the C-3 position. All the isolates were tested for their effect on the expansion of human cord blood (CB) CD34+ hematopoietic stem and progenitor cells. It was found that CB CD34+ cells treated with compounds 6, 7, 9, 10, 14, 15, and 19 showed increased numbers of rigorously phenotype-defined hematopoietic stem cells. Notably, compounds 9, 10, 13, and 14 demonstrated an enhanced ability to increase the percentages and numbers of CB CD34+CD38- cells and multipotential progenitors. The present study is the first to report that furostanol saponins from T. tschonoskii rhizomes can promote hematopoietic stem/progenitor cell (HSPC) expansion.

Trillin prevents proliferation and induces apoptosis through inhibiting STAT3 nuclear translocation in hepatoma carcinoma cells.

Trillin is a constituent of total Trillium Tschonoskii Maxim (TTM), which is extracted from TTM and displayed anti-tumor effect in many tumor cell lines. However, the anti-tumor mechanism of trillin is still unclear. This study demonstrated that trillin could dramatically inhibit hepatoma carcinoma cell proliferation, induce apoptosis and decrease migration and invasion through suppressing phosphorylated STAT3 translocated to nucleus. Trillin could down-regulate Bcl-2 and Survivin, up-regulate cleaved PRAP, leading to dramatically apoptosis; trillin could also down-regulate MMP1, MMP2, MucI and VEGF, which displayed an inhibition effect on hepatocellular tumor cells invasion and development. The results of this study indicated the potential utility of trillin as a STAT3 inhibitor for the treatment of cancers.

Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells.

A phytochemical study on the rhizomes of Trillium tschonoskii led to the isolation of fourteen new steroidal saponins, trillitschosides S1-S14 (1-14), along with ten known analogues (15-24). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence. All isolated compounds were screened for the cytotoxicity against HepG2 cells, and the results demonstrated that only the known compounds 21-24 exhibited the remarkable cytotoxic activity against HepG2 cells which is much better than the positive control of 5-FU.

Spirostanol and sesquiterpenoid glycosides from the rhizomes of Trillium tschonoskii.

Three new spirostanol glycosides, trilliumosides K-M (1-3), one new sesquiterpenoid glycoside, tritschsesuquiside A (4), along with three known analogues (5-7) were obtained from the rhizomes of Trillium tschonoskii. The structures of new glycosides were elucidated by spectroscopic analyses (HRMS and NMR) and chemical methods. Glycosides 5-7 displayed cytotoxicities against five human cancer cell lines with IC50 values ranging from 10.5 ±â€¯1.0 to 1.0 ±â€¯0.2 µM, with 7 being the most cytotoxic compound with IC50 values of 1.0 ±â€¯0.2, 2.2 ±â€¯1.2, and 3.4 ±â€¯0.4 µM against Huh7, CCRF-CEM, and HeLa cell lines, respectively. The flow cytometric results revealed that both 5 and 6 could induce apoptosis of HCT116 and Huh7 cells.

Novel steroidal saponin isolated from Trillium tschonoskii maxim. exhibits anti-oxidative effect via autophagy induction in cellular and Caenorhabditis elegans models.

BACKGROUND: Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration. OBJECTIVE: In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans. METHODS: The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H2O2-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H2DCFDA probe. RESULTS: The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H2O2-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect. CONCLUSION: Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo.

Molecular networking-based strategy for the discovery of polyacetylated 18-norspirostanol saponins from Trillium tschonoskii maxim.

Nine undescribed polyacetylated 18-norspirostanol saponins, trilliumosides A‒J, were obtained after a guidance based on a molecular networking strategy from the rhizomes of Trillium tschonoskii. Their structures were established by analysis of comprehensive spectroscopic data and chemical methods after their isolation in pure form. All isolated saponins were evaluated for their cytotoxicities against five selected human cancer cell lines (Huh7,A549,MCF-7,HepG2, and MOLT-4) and anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages. Trilliumoside D showed significant cytotoxicity against MOLT-4 cell lines with an IC50 value of 1.0 ±â€¯0.1 µM, whereas trilliumosides H and I displayed remarkable anti-inflammatory effects on NO production with inhibitory rates of 56.3 ±â€¯1.5 and 56.2 ±â€¯2.2% at the concentration of 1.0 µM, respectively.

[Inhibitory effect of Trillium saponins on invasion and migration of HuH-7 cells by promoting human ß defensin-2 expression].

Objective To investigate the effect and mechanism of Trillium saponins on the invasion and migration of HuH-7 cells regulated by human ß-defensin 2 (HBD-2). Methods HuH-7 cells were treated with 0.5, 1.0, 2.0, 4.0 mg/L Trillium saponins. Cell proliferation was detected by MTT assay. TranswellTM chamber was used to measure cell invasion and migration. The levels of matrix metalloproteinase-2 (MMP2), HBD-2 and matrix metalloproteinase-9 (MMP9) were detected by Western blot analysis. HBD-2 small interfering RNA (siRNA) was transfected into HuH-7 cells. The interference effect of Trillium saponins treatment was verified by real-time quantitative PCR and Western blot analysis, and TranswellTM assay was used to detect cell invasion and migration. Results Except that 0.5 mg/L Trillium saponins had no effect on the proliferation of HuH-7 cells, the proliferation of HuH-7 cells was inhibited by all other doses of Trillium saponins. (0.5, 1.0) mg/L of Trillium saponins down-regulated the levels of MMP2 and MMP9 proteins, increased the level of HBD-2 protein and inhibited cell invasion, migration. HBD-2 siRNA transfection significantly reduced the level of HBD-2 in HuH-7 cells. Down-regulation of HBD-2 increased the invasive, migratory ability and increased the levels of MMP2 and MMP9 proteins in HuH-7 cells treated with Trillium saponins. Conclusion Trillium saponins can inhibit the invasion and migration of HuH-7 cells by promoting the expression of HBD-2.

Effects of total saponins from Trillium tschonoskii rhizome on grey and white matter injury evaluated by quantitative multiparametric MRI in a rat model of ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Trillium tschonoskii rhizome (TTR), a medicinal herb, has been traditionally used to treat traumatic brain injury and headache in China. Although the potential neuroprotective efficacy of TTR has gained increasing interest, the pharmacological mechanism remains unclear. Steroid saponins are the main bioactive components of the herb. AIM OF THE STUDY: To investigate the protective and repair-promoting effects of the total saponins from TTR (TSTT) on grey and white matter damages in a rat model of middle cerebral artery occlusion (MCAO) using magnetic resonance imaging (MRI) assay. MATERIALS AND METHODS: Ischemic stroke was induced by MCAO. TSTT and Ginaton (positive control) were administered orally to rats 6h after stroke and daily thereafter. After 15 days of treatment, the survival rate of each group was calculated. We then conducted neurological deficit scores and beam walking test to access the neurological function after ischemic stroke. Subsequently, T2-weighted imaging (T2WI) and T2 relaxometry mapping were performed to measure infarct volume and grey and white matter integrity, respectively. Moreover, diffusion tensor imaging (DTI) was carried out to evaluate the grey and white matter microstructural damage. Additionally, arterial spin labelling (ASL) - cerebral blood flow (CBF) and magnetic resonance angiography (MRA) images provided dynamic information about vascular hemodynamic dysfunction after ischemic stroke. Finally, haematoxylin and eosin (HE) staining was carried out to evaluate the stroke-induced pathological changes in the brain. RESULTS: The survival rate and neurological behavioural outcomes (Bederson scores and beam walking tests) were markedly ameliorated by TSTT (65mg/kg) treatment within 15 days after ischemic stroke. Moreover, T2WI and T2 relaxometry mapping showed that TSTT (65mg/kg) significantly reduced infarct volume and attenuated grey and white matter injury, respectively, which was confirmed by histopathological evaluation of brain tissue. The results obtained from DTI showed that TSTT (65mg/kg) not only significantly alleviated axonal damage and demyelination, but also promoted axonal remodelling and re-myelination. In addition, TSTT treatment also enhanced vascular signal density and increased CBF in rats after MCAO. CONCLUSION: Our results suggested the potential protective and repair-promoting effects of TSTT on grey and white matter from damage induced by ischemia. This study provides a modern pharmacological basis for the application of TSTT in managing ischemic stroke.

Bioactive Steroids and Saponins of the Genus Trillium.

The species of the genus Trillium (Melanthiaceae alt. Trilliaceae) include perennial herbs with characteristic rhizomes mainly distributed in Asia and North America. Steroids and saponins are the main classes of phytochemicals present in these plants. This review summarizes and discusses the current knowledge on their chemistry, as well as the in vitro and in vivo studies carried out on the extracts, fractions and isolated pure compounds from the different species belonging to this genus, focusing on core biological properties, i.e., cytotoxic, antifungal and anti-inflammatory activities.

[Determination of three saponins in rhizoma and fibrous root of Trillium tschonoskii and Trillium kamtschaticum].

To compare the differences of main components between in rhizoma and fibrous root of Trillium tschonoskii and T. kamtschaticum, a simple, accurate and reliable high performance liquid chromatography coupled with the charged aerosol detector (HPLC-CAD) method was developed and then successfully applied for simultaneous quantitative analysis of three compounds, including polyphyllin Ⅶ (T1),pennogenin 3-O-α-L-rhamnopyranosyl-(1→2) [α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside (T2),polyphyllin Ⅵ (T3), in 16 batches of rhizome and 14 batches of fibrous root. The analytes were well separated from other constituents on TSK gel ODS (4.6 mm×250 mm, 5 µm) with acetonitrile-water (43∶57) at a flow rate of 1.0 mL•min⁻¹. The injection volume was 20 µL. The nitrogen inlet pressure for the CAD system was 35 psi and the nebulizer chamber temperature was 35 ℃.The method was validated for linearity (r>0.999 0), intra and inter-day precision (0.29%-3.0%), repeatability (0.45%-1.4%), stability (1.9%-2.6%), recovery (100.1%-100.2%, 1.2%-1.8%), limits of detection (0.002 g•L⁻¹), and limits of quantification (0.005 g•L⁻¹).The obtained datasets were processed by principal component analysis (PCA) and it showed that there was almost no difference in rhizoma of T. tschonoskii and T. kamtschaticum from different areas of China. However, the 3 major compounds existed in rhizoma were different from those in fibrous root of T. tschonoskii and T. kamtschaticum.

A new steroidal saponin, furotrilliumoside from Trillium tschonoskii inhibits lipopolysaccharide-induced inflammation in Raw264.7 cells by targeting PI3K/Akt, MARK and Nrf2/HO-1 pathways.

A new steroidal saponin, furotrilliumoside (FT) was isolated from the roots and rhizomes of Trillium tschonoskii Maxim. Its structure was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. FT showed superior activity of inhibiting NO production of RAW264.7 cells induced by lipopolysaccharide (LPS) in the preliminary biological screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of FT were investigated in LPS-induced RAW264.7 cells. The results showed that FT could reduce LPS-induced expression of inducible nitric oxide synthase (iNOS) and then resulted in the decrement of NO production. More meaningful, FT could down-regulate the expression of cyclooxygenase-2 (COX-2) and decrease the expressions of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß), in both gene and protein levels. In mechanism study, FT blocked the LPS-induced upregulation of phosphorylated phosphoinositide-3-kinase and Akt (PI3K/Akt). Furthermore, FT inhibited the translocation of nuclear factor-kappa B (NF-κB) through the prevention of inhibitory factor kappa B alpha (IκBα) phosphorylation and degradation and also suppressed the mitogen-activated protein kinases (MAPK) signaling pathway in LPS-stimulated RAW264.7 macrophages. In addition, FT upregulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). Taken together, FT might act as a natural agent to treat some inflammatory diseases by targeting PI3K/Akt, MARK and Nrf2/HO-1 pathways.

Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP­2/9 production via the p38 MAPK signaling pathway.

Metastasis is the primary cause of mortality in osteosarcoma. Targeting metastasis is a major strategy in osteosarcoma treatment. As a traditional Chinese medicine, Trillium tschonoskii Maxim has been widely used in the therapy of various diseases, including cancer. However, currently there is no evidence regarding the anti­metastasic effect of Paris saponin VII (PS VII), which is extracted from Trillium tschonoskii Maxim, on osteosarcoma cells and its underling mechanisms. The present study aimed to examine the effect of PS VII on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Migration and invasion of osteosarcoma cells was then detected using scratch wound healing assays and Transwell assays, respectively. Additionally, the expression of matrix metalloproteinase (MMP)­2 and ­9 was determined at the mRNA and protein level following treatment with PS VII. Mitogen­activated protein kinase (MAPK) expression was also detected by western blot analysis. Finally, an inhibitor of p38 MAPK was used to verify the effect of PS VII on the expression of MMP­2 and ­9, as well as the migration and invasion osteosarcoma cells. This demonstrated that the proliferation, migration and invasion of the osteosarcoma cells were suppressed following treatment with PS VII. PS VII downregulated the expression of MMP­2 and ­9 in a dose­ and time­dependent manner. PS VII also exerted its ability to downregulate the phosphorylation of p38 MAPKs. Furthermore, by using a p38 inhibitor, SB203580, the role of PS VII in MMP­2 and ­9 expression and osteosarcoma cell invasion was revealed. Taken together, these results demonstrated that PS VII suppresses the migration and invasion of osteosarcoma cells via the p38 MAPK signaling pathway.

Cytotoxicity of the Roots of Trillium govanianum Against Breast (MCF7), Liver (HepG2), Lung (A549) and Urinary Bladder (EJ138) Carcinoma Cells.

Trillium govanianum Wall. (Melanthiaceae alt. Trilliaceae), commonly known as 'nag chhatri' or 'teen patra', is a native species of the Himalayas. It is used in various traditional medicines containing both steroids and sex hormones. In folk medicine, the rhizomes of T. govanianum are used to treat boils, dysentery, inflammation, menstrual and sexual disorders, as an antiseptic and in wound healing. With the only exception of the recent report on the isolation of a new steroidal saponin, govanoside A, together with three known steroidal compounds with antifungal property from this plant, there has been no systematic pharmacological and phytochemical work performed on T. govanianum. This paper reports, for the first time, on the cytotoxicity of the methanol extract of the roots of T. govanianum and its solid-phase extraction (SPE) fractions against four human carcinoma cell lines: breast (MCF7), liver (HEPG2), lung (A549) and urinary bladder (EJ138), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide cytotoxicity assay and liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry analysis of the SPE fractions. The methanol extract and all SPE fractions exhibited considerable levels of cytotoxicity against all cell lines, with the IC50 values ranging between 5 and 16 µg/mL. Like other Trillium species, presence of saponins and sapogenins in the SPE fractions was evident in the liquid chromatography mass spectrometry data. Copyright © 2016 John Wiley & Sons, Ltd.

[The protective effects and mechanisms of Trillium tschonoskii Maxim on rats' liver damage induced by lipopolysaccharide].

OBJECTIVE: To study the protective effects of Trillium tschonoskii maxim (TTM) on rats' oxidative stress and hepatotoxicity in-duced by lipopolysaccharide (LPS). METHODS: Sixty SD rats were randomly divided into TTM high, medium and low dose groups, model group, Dexamethasone (DEX) control group and blank control group with ten rats in each group. The TTM high, medium and low dose groups were treated with 8, 4, 2 g/(kg·d) TTM by intragastric administration and model group, DEX control group and blank control group were treated with the same amount of distilled water respectively. The TTM high, medium and low dose groups, model group, DEX control group were injected intraperitoneal with 1 mg/kg LPS and the DEX control group was injected intraperitoneal with 5 mg/kg DEX, the blank control group was injected with same amount of normal saline every five days. The indexes of rats' thymus and spleen were measured in 30 days. The activities of serum nitric oxide synthase (NOS), superoxide dismutase (SOD) and the contents of nitric oxide (NO), glutathione (GSH), glu-cosinolates barbituric acid reaction product(TBARS), white cells interleukin-6(IL-6), IL-10 and tumor necrosis factor-α(TNF-α), the activi-ties of liver SOD, GSH-Px and the contents of GSH and TBARS were measured. RESULTS: TTM high dose group was significantly different in body weight in 19~30 days(P<0.05); The index of thymus in TTM high, medium and low dose groups and the index of spleen in TTM high dose group were decreased significantly compared with those of the model group. The activity of serum NOS and the contents of TBARS and NO in TTM high, medium and low dose groups were decreased significantly(P<0.05). The activity of serum SOD in TTM high dose group and the contents of GSH in TTM medium and low dose groups were increased significantly(P<0.05). The contents of serum IL-6 and TNF-αin TTM high, medium dose groups were decreased significantly and the contents of serum IL-10 were increased significantly(P<0.05). The con-tents of liver TBARS in TTM high, medium dose groups were decreased significantly. The activity of liver SOD in TTM high, medium and low dose groups, the activity of GSH-Px in TTM high, medium dose groups and the contents of GSH in TTM high dose group were increased signifi-cantly(P<0.05). CONCLUSIONS: TTM has a certain effect to delay the rats' atrophy of thymus and spleen generated by LPS. It can effectively reduce the activity of NOS in serum, reduce the formation of NO, improve the activity of SOD, GSH-Px and the contents of GSH, reduce lipid peroxidation, decrease the excessive secretion of IL-6, TNF-α, increase the contents of IL-10, which can resist inflammation and protect the liver.