The active fraction from the tuber of Bolboschoenus yagara inhibits melanoma B16 cells metastasis LPS-induced in vitro and in vivo.

This study was to identify anti-metastatic active fractions and compounds of Bolboschoenus yagara (B. yagara). The results indicated that 50 µg/mL ethyl acetate fraction (Et) can dramatically inhibit mouse melanoma B16 cells migration and invasion in vitro. In B16 cells pulmonary and hepatic metastasis assays, 50 µg/mL Et alleviated mouse lung and liver weights, the number of metastatic nodules and the levels of TNF-α and IL-6 in mouse serum and organs. Histological studies showed that Et fraction was able to prevent liver and lung metastasis. And the inhibition of 50 µg/mL Et fraction against hepatic metastasis was almost equivalent to that of 1 µM TAK242. In addition, fourteen compounds of Et were quantified by HPLC analysis, in which, isocoumarins, stilbenes and xanthones obviously abated LPS-modulated B16 cells migration and invasion.[Formula: see text].

Inhibition of p38 and ERK1/2 pathways by Sparstolonin B suppresses inflammation-induced melanoma metastasis.

BACKGROUND: Cancer related inflammation plays a fatal role in the metastatic process, which can foster tumor growth, angiogenesis and dissemination. Sparstolonin B (SsnB), derived from Chinese medicine of the tubers of Scirpus yagara, is a TLR2 and TLR4 antagonists. It has exhibited multiple activities of anti-inflammatory, anti-cancer, anti-obesity and anti-hepatitis. However, whether SsnB is involved in the regulation of inflammation-induced tumor metastasis is not well elucidated. PURPOSE: The aim of this study was to investigate the effectiveness of SsnB as a treatment of inflammation-induced tumor metastasis and identify the underlying mechanisms of its anti-tumor metastatic activity. METHOD: The anti-tumor metastatic activity in vitro was estimated by MTT, wound-healing assay, matrigel invasion analysis and extracellular matrix adhesion assay. Mice lung metastasis and hepatic metastasis experiments were performed to assess the activities in vivo. Lungs or livers were weighed and the number of metastatic nodules was determined after mice were sacrificed. The levels of pro-inflammatory cytokines in the serum, lungs and livers were detected by using enzyme-linked immunosorbent assay (ELISA). Micro-metastasis nodules in lungs or livers were analyzed by histological examination. Immunohistochemistry and western blot analysis were conducted to determine protein expression. RESULT: Herein, SsnB dose-dependently inhibited cell migration and invasion in mouse melanoma B16 cells with or without stimulation of lipopolysaccharide (LPS), Pam3csk4 or molecules from damaged tumor cells (DTC-Ms). The expression of matrix metalloproteinases (MMP)-2 was also significantly abated by SsnB in LPS-modulated B16 cells. And SsnB reduced LPS-activated B16 cells adhesion to extracellular matrix components collagen I and fibronectin in a dose-dependent manner. In vivo, SsnB obviously attenuated LPS-activated pulmonary metastasis in mice by reduction the number of metastatic nodules on the lung surfaces, lung weight and levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in serums and lungs. Moreover, in experimental hepatic metastasis model mice, SsnB remarkably repressed LPS-stimulated the number of metastatic nodules along with liver weight; and SsnB significantly suppressed LPS-activated increase levels of TNF-α and IL-6 in livers. Immunohistochemistry analysis indicated that SsnB inhibited the expression of TLR4 in livers. Furthermore, SsnB remarkably blocked p38 and ERK1/2 signaling pathway in LPS-induced B16 cells. P38 and ERK1/2 signaling silencing, using BIRB0796 (small molecular inhibitor of p38 MAPK) and PD184352 (inhibitor of MEK1/2 kinases that activate ERK1/2), significantly abated LPS-induced migration and invasion of B16 cells. CONCLUSION: The present study reports a novel use of SsnB in mitigating TLRs ligands-induced melanoma metastasis by inhibition of p38 and ERK1/2 pathway.

New isocoumarin and stilbenoid derivatives from the tubers of Sparganium stoloniferum (Buch.-Ham.).

A new isocoumarin derivative 8,5'-dihydroxy-6'-methoxy-4-phenyl-5,2'-oxidoisocoumarin (1) and a new stilbenoid derivative methyl 5-hydroxy-2-(2-hydroxyphenyl)benzofuran-4-carboxylate (2) together with nine known compounds (3-11) were isolated from the tubers of Sparganium stoloniferum Buch.-Ham.. Another stilbenoid derivative (3) and a xanthone (4) were identified as new natural products and compounds 5-10 were obtained for the first time from the genus Sparganium. All their structures were elucidated by comprehensive spectroscopic analysis and comparison with available literature information.

Interleukin-23 Secreted by Activated Macrophages Drives γδT Cell Production of Interleukin-17 to Aggravate Secondary Injury After Intracerebral Hemorrhage.

BACKGROUND: Neuroinflammation plays a key role in intracerebral hemorrhage (ICH)-induced secondary brain injury, but the specific roles of peripheral inflammatory cells such as macrophages and lymphocytes remain unknown. The purpose of this study was to explore the roles of macrophages, T lymphocytes, and the cytokines they secrete as potential targets for treating secondary brain injury after ICH. METHODS AND RESULTS: Our results showed that peripheral macrophages and T lymphocytes successively infiltrated the brain, with macrophage counts peaking 1 day after ICH and T-lymphocyte counts peaking after 4 days. These peaks in cellular infiltration corresponded to increases in interleukin (IL)-23 and IL-17 expression, respectively. We found that hemoglobin from the hematoma activated IL-23 secretion by infiltrating macrophages by inducing the formation of toll-like receptor (TLR) 2/4 heterodimer. This increased IL-23 expression stimulated γδT-cell production of IL-17, which increased brain edema and neurologic deficits in the model mice as a proinflammatory factor. Finally, we found that sparstolonin B (SsnB) could ameliorate brain edema and neurologic deficits in ICH model mice via inhibition of TLR2/TLR4 heterodimer formation, and notably, SsnB interacted with myeloid differentiation factor 88 Arg196. CONCLUSIONS: Together, our results reveal the importance of the IL-23/IL-17 inflammatory axis in secondary brain injury after ICH and thus provide a new therapeutic target for ICH treatment.

Chemical constituents from the tubers of Scirpus yagara and their anti-inflammatory activities.

A new natural compound, dehydrophyllodulcin (1) was isolated from the tubers of Scirpus yagara, together with 11 known compounds. Among them, compounds 2, 5-8, and 10-12 were isolated from this plant for the first time. (1)H NMR, (13)C NMR, and 2D NMR data of compound 1 are first reported in this article, though it was synthesized in 1996. The structures of all compounds were determined by comprehensive analyses of their spectroscopic data and compared with literature information. Moreover, the anti-inflammatory effects of compounds 1, 3, 4, 6, and 9 against inflammatory cytokines production in Lipopolysaccharide - or Pam3csk4-stimulated macrophage RAW264.7 cells were evaluated by Enzyme-linked immunosorbent assay. And these compounds significantly inhibited the tumor necrosis factor (TNF)-α, interleukin (IL)-6 productions in RAW264.7 cells, with IC50 values less than 20 µM.

Determination of sparstolonin B by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry: application to pharmacokinetic study of sparstolonin B in rat plasma.

Sparstolonin B (SsnB), a spontaneous isocoumarin compound isolated from the tuber of Scirpus yagara Ohwi. (Cyperaceae), possesses potent anti-inflammatory and antitumor activity. In the present study, a rapid and simple UHPLC/MS/MS method for determination of SsnB in rat plasma was developed and validated. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate containing rhein as an internal standard and separated on a C18 column at 35 °C, with a gradient mobile phase consisting of acetonitrile and water containing 0.2% (v/v) formic acid within 2.1 min. MS/MS detection was accomplished in multiple reaction monitoring mode with negative electrospray ionization. The precursor-product ion transitions were m/z 266.9 [M-H](-) → m/z 211.0 for SsnB and m/z 283.2 [M-H](-) → m/z 239.0 for IS. The intra- and inter-day precision (RSD) was <8.98% and the accuracy (RE) ranged from -7.40 to 4.50%. The extraction recoveries ranged from 96.28 to 97.30%. The pharmacokinetic parameters were calculated using Win Nonlin53 software. The absolute bioavailability of SsnB was estimated to be 6.98%. The proposed method was successfully applied to a pharmacokinetic study of SsnB in rats after intravenous administration with a dose of 0.5 mg/kg and oral administration at a dose of 5 mg/kg.

Protective effects of the active fraction from the tuber of Scirpus yagara in mouse endotoxin shock model.

ETHNOPHARMACOLOGICAL RELEVANCE: Scirpus yagara Ohwi is a perennial, aquatic plant, whose dry tubers have long been used as Traditional Chinese Medicine (TCM) "Sanleng" for the treatment of postpartum abdominal pain, hyperemesis gravidarum, amenorrhea, dyspepsia and several inflammatory related diseases. Although it is known to have anti-inflammatory activities, its mechanism of action on lipopolysaccharide (LPS)-induced inflammation has not yet been identified in detail.This study was designed to investigate the anti-inflammatory activity of the active fraction (AF) from the tuber of Scirpusyagara both in vitro and in vivo. MATERIALS AND METHODS: RAW264.7 macrophage was incubated for 16h with 1µg/ml of LPS in absence or presence of AF (0, 10, 50 and 100µg/ml) and the secretions of tumor necrosis-alpha (TNF-α) and interleukin-6 (IL-6) in the medium were determined by using an enzyme-linked immunosorbent assay (ELISA). In the in vivo study, mice were orally administrated with AF (50 and 300mg/kg) for three days consecutively. 1h after the last AF administration, the mice were intraperitoneally injected with LPS (15mg/kg), and the life span of LPS-challenged mice were determined. Furthermore, the levels of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were measured using ELISA kit, and histological change in lungs was examined by light microscopy. Additionally, the components of AF were analyzed by high performance liquid chromatography (HPLC) using a C18 column. RESULTS: AF significantly decreased TNF-α and IL-6 production induced by LPS in RAW264.7 macrophage. In LPS-induced mouse endotoxin shock model, AF pre-treatment significantly improved the survival rate of mice. And LPS-induced increases of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were markedly suppressed by AF. Moreover, the histopathological examination indicated that AF could significantly attenuate lung tissues injury in endotoxemic mice. In addition, eight compounds (protocatechuic acid, vanillic acid, p-coumaric acid, ferulic acid, methyl-3,6-dihydroxy-2-[2-(2-hydroxyphenyl)-ethynyl] benzoate, sciryagarol I, sparstolonin B, SanLeng diphenyllactone) of AF were quantified by HPLC analysis. CONCLUSIONS: These results suggested that AF protected mice against LPS-induced lethality by inhibiting the production of multiple cytokines and organ dysfunction. Thus AF may prove beneficial in the prevention and treatment of endotoxin shock.

Toll-like receptor 2/4 heterodimer mediates inflammatory injury in intracerebral hemorrhage.

OBJECTIVE: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. However, the upstream events that initiate inflammatory responses following ICH remain elusive. Our previous studies suggested that Toll-like receptor 4 (TLR4) may be the upstream signal that triggers inflammatory injury in ICH. In addition, recent clinical findings indicated that both TLR2 and TLR4 may participate in ICH-induced brain injury. However, it is unclear how TLR2 functions in ICH-induced inflammatory injury and how TLR2 interacts with TLR4. METHODS: The role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury was investigated in both in vivo and in vitro models of ICH. RESULTS: TLR2 mediated ICH-induced inflammatory injury, which forms a heterodimer with TLR4 in both in vivo and in vitro models of ICH. Hemoglobin (Hb), but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. MyD88 (myeloid differentiation primary response gene 88), but not TRIF (Toll/IR-1 domain-containing adaptor protein inducing interferon-beta), was required for ICH-induced TLR2/TLR4 heterodimerization. Mutation of MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. INTERPRETATION: Our results suggest that a novel TLR2/TLR4 heterodimer induced by Hb initiates inflammatory injury in ICH. Interfering with the assembly of the TLR2/TLR4 heterodimer may be a novel target for developing effective treatment of ICH.

Bioactive cis-stilbenoids from the tubers of Scirpus yagara.

Two new cis-stilbenoids, sciryagarol I (1) and II (2) were isolated from the EtOAc extract of the tubers of Scirpus yagara, together with four known compounds. The structures of all compounds were determined by comprehensive analyses of their spectroscopic data and comparison with literature information. The compounds 3, 4 and 6 were isolated for the first time from this genus. Some compounds were tested for their cytotoxicity against human tumor cell lines and antimicrobial activity. Compounds 1-4 showed significant cytotoxicity against the Hela cell lines with IC(50) values ranging from 7.21 to 61.21µM. 1 and 2 exhibited some antimicrobial activity against Staphylococcus aureus and Candida albicans with uniform MICs of 79.3µl/ml for 2, and 152µl/ml for 1, respectively.

[Study on quality evaluation of Sparganii rhizoma by biopotency determination method].

OBJECTIVE: To establish a method for determining anticoagulation potency of Sparganii Rhizoma, and evaluate the effect of Sparganii Rhizoma herbs from different producing areas on promoting blood circulation and removing blood stasis; and study the material basis of Sparganii Rhizoma through the correlation analysis on its anticoagulation potency, ferulic acid and total flavonoid content. METHOD: The anticoagulation time of Sparganii Rhizoma from different producing areas with activeated partial thromboplastin time for their active extracts. Their biopotency was calculated by using the method of "parallel lines of dose effect" (3, 3). The degree of correlation between their anticoagulation potency and chemical constituents were calculated by using Pearson correlational analysis method. RESULT: Sparganii Rhizoma and is control drugs had a good linear relationship between dose and effect (Y = 172.76X - 193.39, R2 = 0.9955). The method had better accuracy (RSD 4.7%), repeatability (RSD 2.3%) and intermediate precision (RSD 5.4%), finding that the biopotency of Sparganii Rhizoma from different producing areas ranged between 52.33-238.58 U x g(-1), and all of them passed the test on reliability. The results of correlation analysis showed no remarkable relationship between the anticoagulation potency of Sparganii Rhizoma and the contents of the two chemical constituents. CONCLUSION: This biopotency determination method established in the experiment can be used as one of approaches for qulaity evaluation on Sparganii Rhizoma.

A new cytotoxic casbane diterpene from Euphorbia pekinensis.

A new cytotoxic casbane diterpene, named pekinenal, was isolated from the roots of Euphorbia pekinensis. Its structure was elucidated as 5alpha-hydroxy-1betaH,2alphaH-casba-3Z,7E,11E-triene-18-al by a combination of 1D- and 2D-NMR techniques and confirmed by X-ray crystallography. Pekinenal showed cytotoxic activity against all four human cancer cell lines tested.

A new elemanolide sesquiterpene lactone from Elephantopus scaber.

A new elemanolide sesquiterpene lactone, named elescaberin (1), together with two known compounds, namely, isodeoxyelephantopin (2) and deoxyelephantopin (3), was isolated from the whole plant of Elephantopus scaber. The structure of 1 was elucidated on the basis of spectroscopic analysis. All three compounds exhibited significant inhibitory activities against human SMMC-7721 liver cancer cells in vitro (IC(50) 8.18-14.08 micromol/l).

[Structure modification on sesquiterpene lactones from Elephantopus scaber].

In order to look for lower toxic and strongly active compounds, the structure of sesquiterpene lactones from Elephantopus scaber was modified. Deoxyelephantopin and scabertopin were isolated from the whole plant of Elephantopus scaber and hydrogenated and epoxidated. Five sesquiterpenoide derivatives were prepared and their structures were identified by IR, NMR and MS spectra analysis. Four sesquiterpenoides are new compounds. Part of the compounds show definite antitumor activity.

[Studies on the constituents from the herb of Vernonia patula].

OBJECTIVE: To isolate and elucidate the chemical constituents of the whole plant of Vernonia patula, and to provide samples for biological activity screening. METHOD: The chromatography on silica gel was used and the structures were determined by IR, NMR and MS spectra analysis and physicochemical property comparion. RESULT: Four triterpenoids were isolated and identified as bauerenyl acetate(I), friedelin(II), epifriedelanol(III), 20(30)-taraxastene-3 beta, 21 alpha-diol(IV). CONCLUSION: Compounds I and IV were isolated from genus Vernonia for the first time and compounds II and III were isolated from the whole plant of V. patula for the first time. The four compounds show no inhibitory effect on the growing of PC-12 cells.