Engeletin attenuates the inflammatory response via inhibiting TLR4-NFκB signaling pathway in Crohn's disease-like colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra rhizome has a long history been used for clinical purposes in traditional Chinese medicinal for treating various inflammatory conditions. Engeletin1 (ENG) is one of the most abundant bioactive compounds found in Smilax glabra rhizome, with anti-inflammatory, antioxidant, and ulcer-preventing activities. AIM OF THE STUDY: The purpose of this study was to investigate the ability of ENG to alleviate inflammatory symptoms and improve epithelial barrier integrity utilize a 2,4,6-trinitrobenzene sulfonic acid2 (TNBS)-induced murine model in Crohn's disease3 (CD)-like colitis, and to characterize the underlying anti-inflammatory mechanisms of action. MATERIALS AND METHODS: A colitis model was established in BALB/c mice and treated with ENG for 7 days. RAW264.7 macrophages were pre-treated with ENG and lipopolysaccharide4 (LPS) stimulation. The mice's weight and colon length were assessed. qPCR and Western blotting were used to analyze gene expression and TLR4-NFκB pathway. Flow cytometry was used to analyze the polarization states of the macrophages. RESULTS: Treatment with ENG was sufficient to significantly alleviate symptoms of inflammation and colonic epithelial barrier integrity in treated mice. Significant inhibition of TNF-α, IL-1ß, and IL-6 expression was observed following ENG treatment in vivo and in vitro. ENG was also determined to be capable of inhibiting the expression of iNOS and CD86, inhibited M1 macrophage polarization in vitro, as well as the TLR4-NFκB signaling pathway. Molecular docking showed a highly stable binding between ENG and TLR4. CONCLUSION: ENG has been proven to alleviate inflammation and ameliorate the damage of epithelial barrier in CD-like colitis. ENG also suppressed the M1 macrophages polarization and the inhibited inflammatory cytokines. TLR4-NFκB signaling pathway, especially TLR4, may be the target of ENG. These data offer a new insight into the therapeutic mechanisms of ENG.

Intestinal lymphatic transport of Smilax china L. pectic polysaccharide via Peyer's patches and its uptake and transport mechanisms in mononuclear phagocytes.

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.

Effects of Smilax China L. extracts on Hyperuricemia chicken model via inhibiting xanthine oxidase activity.

Hyperuricemia (HUA) is a metabolic disorder caused by excessive production of uric acid (UA) or impaired uric acid metabolism. Smilax China L. has a wide range of pharmacological activities such as immunomodulatory, anti-inflammatory, and antioxidant. Its roots and rhizomes have been widely used for the treatment of HUA. However, its mechanisms for treating HUA and reducing renal impairment have not been fully elucidated. In the present study, we evaluated the effect of Smilax China L. extract (SC) on UA metabolism and further explored its mechanism of action by feeding a high-calcium and high-protein diet to chickens to induce a model of HUA in chickens. SC significantly reduced serum UA levels and improved renal function in hyperuricemic chickens. Meanwhile, SC was able to inhibit the activity of xanthine oxidase (XOD) in vivo and in vitro, reducing the production of uric acid. In addition, SC was able to increase the expression of Breast Cancer Resistance Protein (BCRP) in the kidney and ileum and increase uric acid excretion. Therefore, our results suggest that SC may be a candidate for anti-hyperuricemia.

Chemical profile, antioxidant potential and toxicity of Smilax brasiliensis Sprengel (Smilacaceae) stems.

Smilax brasiliensis Sprengel is a monocotyledon of the Smilacaceae family, native to the Brazilian Cerrado, popularly known as "salsaparrilha" or "japecanga". In this study, the ethanol extract (EE) and the hexane (HEXF), dichloromethane (DCMF), ethyl acetate (ACF), and hydroethanol (HEF) fractions of the stems were obtained. The chemical composition was determined, the contents of phenolic compounds and flavonoids were quantified, and the antioxidant potential and the cytotoxic effect on Artemia salina were evaluated. Fatty acid esters, hydrocarbons, and phytosterols were identified in the HEXF analyzed by gas chromatography - mass spectrometry (GC-MS). The EE and DCMF, ACF, and HEF were analyzed by liquid chromatography coupled to a diode array detector and mass spectrometer (LC-DAD-MS), and the identified constituents included glycosylated (rutin, 3-O-ß-galactopyranosyl quercetin, 3-O-ß-glucopyranosyl quercetin, O-deoxyhexosyl-hexosyl quercetin, O-deoxyhexosyl-hexosyl kaempferol, O-deoxyhexosyl-hexosyl O-methyl quercetin, and others), and non-glycosylated (quercetin) flavonoids, phenylpropanoids (3-O-E-caffeoyl quinic acid, 5-O-E-caffeoyl quinic acid, O-caffeoyl shikimic acid, and others), neolignan, steroidal saponin (dioscin), and N-feruloyltyramine. The EE, DCMF, and ACF showed high total contents of phenolic compounds (112.99, 175.71, and 524.02 µg of GAE/mg, respectively), and in the ACF and DCMF a great content of flavonoids was also quantified (50.08 and 31.49 µg of QE/mg, respectively). The EE, DCMF, ACF, and HEF exhibited great antioxidant potential by DPPH (IC50 1.71 - 32.83 µg/mL) and FRAP (IC50 0.63 - 6,71 µg/mL) assays. A maximum cytotoxic activity on A. salina of 60% was observed for the DCMF (LC50 = 856.17 µg/mL). This study contributes to the phytochemical study of S. brasiliensis since these compounds were identified for the first time in the stems of this species. The S. brasiliensis stems demonstrated to be a rich source of polyphenols compounds and exhibited high antioxidant potential without toxicity. Thus, extract and fractions obtained from the S. brasiliensis stems can be used in food supplements or as natural antioxidants in the food industry.

Studies on the phytochemical constituents of Smilax elegantissima Gagnep.

Nine compounds were isolated and elucidated from this species, among which, two new compounds (1, 2) and seven known compounds (3-9). Their structures were determined by means of extensively spectroscopic analysis including HR-ESI-MS, 1H NMR, 13C NMR, HSQC and HMBC. The bioactivities evaluation was referred to the cytotoxic assay on four human tumor cell lines of the ethanol extract, different fractions and 6 compounds. The results demonstrated that the dichloromethane fraction showed the strongest cytotoxicity, followed by the ethyl acetate fraction. Compounds 4 and 6 had significant effects on SMMC-7721 and Hela cells.

Steroidal Saponins from the Rhizomes of Smilax china and Their Inhibitory Effects on Lipopolysaccharide-Induced Proinflammatory Cytokines Expression.

Four new furostanol saponins (1:  - 4: ) and a new pregane-type saponin (5: ) along with six known steroidal saponins (6:  - 11: ) were isolated from the rhizomes of Smilax china. The structures of 1:  - 5: were elucidated by extensive analysis of NMR and HR-ESI-MS data in addition to enzymatic hydrolysis and other chemical methods. Compounds 1, 4: , and 11: showed inhibitory activity against the expression of proinflammatory mediators, inducible nitric oxide synthase, interleukin-1ß, interleukin-6, and tumor necrosis factor-α in lipopolysaccharide-induced RAW264.7 cells. Compound 1: , at a concentration of 20 µM, decreased the production of inducible nitric oxide synthase, interleukin-1ß, interleukin-6, and tumor necrosis factor-α by 36, 62, 72, and 67%, respectively, which is comparable to that of the positive control dexamethasone.

Smilax glabra Roxb.: A Review of Its Traditional Usages, Phytochemical Constituents, Pharmacological Properties, and Clinical Applications.

Smilax glabra Roxb. (SGB) is a medicinal plant widely distributed in 17 countries worldwide. It is the primary raw material of the world-famous and best-selling functional food and beneficial tea. SGB was first recorded in Ben Cao Jing Ji Zhu of the Southern and Northern Dynasties (420-589 AD) and was reported for nutritional and medicinal properties for thousands of years. This review searched PubMed, Web of Science, and other databases for relevant literature on SGB species until April 2022. It aims to provide more integrated thinking, detailed awareness, and better knowledge of SGB. More than 200 chemical components have been discovered, including flavonoids, phenolic, phenolic acids, stilbenes, organic acids, phenylpropanoids, and others. Previous studies have demonstrated that SGB and its active ingredients show a wide range of pharmacological effects, including anti-infective, anti-cancer, anti-inflammatory, antioxidant, cardiovascular protection, etc. However, many studies on the biological activity of this plant were mainly based on crude extracts and active ingredients, and there is a lack of clinical studies and toxicity studies to support the development of drug design, development, and therapy. In summary, this review will provide specific and valuable suggestions and guidelines for further research and application of this plant in the medicinal field.

Scopolin obtained from Smilax china L. against hepatocellular carcinoma by inhibiting glycolysis: A network pharmacology and experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax china L. is a well-known traditional medicinal plant. In China, it is a common anti-cancer drug that has been inherited for thousands of years. Some in vitro and in vivo studies have confirmed its potential lipid-lowering, anti-inflammatory and anti-ovarian cancer effects. However, there is no research on the material basis and mechanism of the rhizome of Smilax china L. against hepatocellular carcinoma. AIM OF THE STUDY: To explore the material basis and mechanism of scopolin from Smilax china L. against hepatocellular carcinoma. METHODS: The potential targets and active components of Smilax china L. against hepatocellular carcinoma were screened by transcriptomics, network pharmacology and molecular docking. Microscale Thermophoresis (MST) detection was used to verify the affinity of small molecule compounds with potential proteins and protein-protein interaction. The Extract from HepG2 cells was used to measure the expression of glycolysis-related proteins, glucose consumption and lactate production. The expression of apoptosis-related factors and glycolysis-related proteins in vivo was detected by immunohistochemistry. RESULTS: The glycolysis-related proteins glucose-6-phosphate isomerase (GPI), glycerol-3-phosphate dehydrogenase, mitochondrial (GPD2) and phosphoglycerate kinase 2 (PGK2) screened by transcriptomics, network pharmacology showed strongly binding with scopolin by molecular docking. MST detection has also verified the affinity of scopolin with GPI and GPD2. It was the first time found that Heat shock protein HSP 90-alpha (Hsp90α) bound strongly to GPI and GPD2 in the worldwide, while scopolin was able to affect the interaction between Hsp90α and GPD2. In vitro and in vivo experiments further demonstrated that scopolin may play an anti-cancer role by affecting the stability of tumor-associated proteins. The results showed that scopolin obtained from Smilax china L. could regulate the expression of GPI, GPD2 and PGK2 and inhibit the interaction of protein-protein, reduce the energy metabolism of tumor tissue, thereby inhibit tumor growth. CONCLUSION: Scopolin obtained from Smilax china L. plays the role of anti-hepatocellular carcinoma by regulating the expression of glycolysis proteins GPI, GPD2 and PGK2. Scopolin could affect the interaction between Hsp90α and GPD2 may provide a novel potential treatment direction for hepatocellular carcinoma.

Smilax glabra Roxb. flavonoids protect against pathological cardiac hypertrophy by inhibiting the Raf/MEK/ERK pathway: In vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra Roxb., the dry rhizome of Sarsaparilla, which is also known as Tu fuling (TFL) in China, is a well-known traditional CHINESE medicine that is widely used for detoxication, relieving dampness and as a diuretic. We have previously shown that the extracted TFL flavonoids (designated TFLF) possess anti-cardiac hypertrophy effects in vitro. However, the anti-cardiac hypertrophy effects of TFLF in vivo and the underlying mechanisms remain to be elucidated. AIM OF THE STUDY: To reveal the underlying therapeutic mechanism of TFLF on cardiac hypertrophy by using transverse aortic constriction (TAC) model and cellular assays in vitro. MATERIAL & METHODS: Cardiac hypertrophy was replicated by TAC surgery in rats or by isoprenaline treatment of rat H9C2 myocardial cells in vitro. Cardiac structure and function were evaluated by echocardiographic and hemodynamic examinations in vivo and histological analysis of tissues ex vivo. Biochemical kits and quantitative PCR were used to analyze markers of cardiac hypertrophy. Expression and phosphorylation of key proteins in the Raf/MEK/ERK pathway were quantified by Western blotting. We further confirmed our findings in H9C2 rat cardiomyocytes treated with isoprenaline and the ERK inhibitor in vitro. RESULTS: TFLF attenuated cardiac hypertrophy and fibrosis and improved cardiac dysfunction in TAC rats. TFLF treatment induced a strong reduction in serum NT-proBNP levels. Cardiac hypertrophy marker gene (ANP, BNP and ß-MHC) expression and the phosphorylation levels of c-Raf and ERK1/2 were decreased by TFLF treatment. TFLF also protected H9C2 cells from isoprenaline-induced hypertrophy in vitro via a similar molecular mechanism as that observed in the rat heart. Moreover, pretreatment with TRLF and the ERK inhibitor further inhibited the mRNA overexpression of hypertrophic genes in vitro. CONCLUSIONS: TFLFs may protect against pathological cardiac hypertrophy via negative regulation of the Raf/MEK/ERK pathway. Thus, TFLFs are implicated as a potential pharmacological agent for treating cardiac hypertrophy in clinical practice.

Therapeutic potential of Smilax fluminensis ethanolic extract: antitumoral activity in murine melanoma cells.

The aim of the study was to investigate the in vitro and in vivo antitumor activity of leaves ethanol extract from Smilax fluminensis on murine melanoma. The extract was performed by ethylic alcohol and submitted to classical chemical analysis. Cytotoxicity test were performed on neoplastic cells, where antitumor activity was expressed in GI50 (concentration that inhibits 50% of cell growth) and the determination of selectivity index using a normal cell line. In addition, BALB/c mice models were used to evaluate the in vivo antitumor activity of extract in two different concentrations against B16-F10 melanoma cells. The tumor inhibition ratio was determined and the histopathological analyses of nodules and liver were compared. The chemical analysis indicated a major presence of phenolic compounds and flavonoids. Cytotoxicity test results that S. fluminensis extract was active in B16-F10 line (GI50: 4.37 µg/mL), being the extract considered a promising antineoplastic agent. In the experimental model, the inhibition percentage of tumoral growth was between 78.77 and 83.49%. Histopathology analysis of nodules showed necrotic cells reduction, adipocytes presence, melanin deposition, vascularization, and inflammatory process in a concentration-dependent manner. On the liver, the animals treated with the extract on both concentrations showed normal hepatic organization, normal hepatocytes, and absence of inflammatory focus. The results indicate that S. fluminensis extract demonstrated both in vitro and in vivo antitumor activity, reducing the tumoral growth in B16-F10 and could therefore be a promising antineoplastic agent.

Pectic polysaccharide from Smilax china L. ameliorated ulcerative colitis by inhibiting the galectin-3/NLRP3 inflammasome pathway.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the colon and rectum. Although galectin-3 (Gal-3) has been reported to play a proinflammatory role in UC, it is unknown whether pectic polysaccharide, a Gal-3 inhibitor in tumor metastasis, can alleviate UC by inhibiting Gal-3. The aim of this study was to investigate the anti-inflammatory effects and underlying mechanisms of SCLP, a pectic polysaccharide purified from Smilax china L. in our previous work, on dextran sulfate sodium-induced UC in BALB/c mice. The results showed that SCLP could significantly improve symptoms, alleviate histopathological damage and reduce the secretion of inflammatory mediators in mice with UC. Analysis of the anti-colitis mechanisms indicated that SCLP could inhibit the Gal-3/NLRP3 inflammasome/IL-1ß pathway by suppressing the expression of Gal-3 and the interaction of Gal-3 and NLRP3. Our results suggested that SCLP could be a promising candidate for prevention and treatment of UC.

Smilax glabra Roxb. Inhibits Collagen Induced Adhesion and Migration of PC3 and LNCaP Prostate Cancer Cells through the Inhibition of Beta 1 Integrin Expression.

Smilax glabra Roxb. (SGR) has been used as a traditional medicine for brucellosis and syphilis. In this study, we investigated whether nontoxicological levels of water extract of SGR (WESGR) are effective for suppressing steps in the progression of prostate cancer, such as collagen-mediated migration and adhesion and identified the target molecule responsible for such effects. We found that nontoxicological levels of WESGR did not attenuate PC3 and LNCaP cell adhesion to serum but did significantly do so with collagen. In addition, using the Boyden chamber assay, we found that nontoxicological levels of WESGR did not inhibit the migration of PC3 and LNCaP cells to a serum-coated area but did significantly attenuate migration to a collagen-coated area. Interestingly, the expression of α2ß1 integrin, a known receptor of collagen, was not affected by ectopic administration of WESGR. However, WESGR significantly attenuated the expression of ß1 integrin, but not α2 integrin when PC3 and LNCaP cells were placed on a collagen-coated plate, resulting in attenuation of focal adherent kinase phosphorylation. Finally, 5-O-caffeoylquinic acid was determined as a functional single component which is responsible for antiprostate cancer effects of WESGR. Taken together, our results suggest a novel molecular mechanism for WESGR-mediated antiprostate cancer effects at particular steps such as with migration and adhesion to collagen, and it could provide the possibility of therapeutic use of WESGR against prostate cancer progression.

Smilax guianensis Vitman Extract Prevents LPS-Induced Inflammation by Inhibiting the NF-κB Pathway in RAW 264.7 Cells.

Nutraceutical treatments can reduce inflammation and prevent the development of inflammatory diseases. In this study, the anti-inflammatory effects of Smilax guianensis Vitman extract (SGE) were examined. SGE suppressed lipopolysaccharide (LPS)-mediated nitrite production in RAW 264.7 cells. SGE also prevented the LPS-induced expression of inducible nitric oxide synthase (iNOS) but not cyclooxygenase (COX)-2. Western blot analysis showed that SGE attenuated LPS-induced phosphorylation of IκB kinase (IKK), inhibitor of kappa B (IκB), and p65. Additionally, SGE inhibited LPS-induced IκB degradation in RAW 264.7 cells. Western blot analysis of the cytosolic and nuclear fractions, as well as immunofluorescence assay results, revealed that SGE suppressed LPS-induced p65 nuclear translocation in RAW 264.7 cells. Moreover, SGE reduced LPS-induced interleukin (IL)- 1ß, IL-6, and tumor necrosis factor-α (TNF-α) mRNA expression and IL-1ß and IL-6 protein expression in RAW 264.7 cells. Collectively, these results indicate that SGE suppresses the NF-κB signaling pathway and thereby inhibits the production of NO, IL-1ß, and IL-6.

Transcriptome profiling reveals multiple pathways responsible for the beneficial metabolic effects of Smilax glabra flavonoids in mouse 3T3-L1 adipocytes.

Smilax glabra Roxb. (SG) is a well-known traditional Chinese medicine that has been extensively used as both food and folk medicine in many countries. Although many beneficial health effects of SG and its primary components have been reported, their action on adipocyte function remains unknown. In the present study, we investigated the effects of the total flavonoids from Smilax glabra Roxb. (SGF) on lipid accumulation in mouse 3T3-L1 adipocytes and further elucidated its potential mechanism using RNA-Seq transcriptome technique. Our results showed that SGF exposure significantly decreased the lipid droplet size and the levels of cellular free fatty acids, while triglyceride accumulation was not affected by SGF. Transcriptome analysis revealed that SGF induced the expression of genes involved in triglyceride storage, fatty acid ß-oxidation and mitochondrial biogenesis. Furthermore, we also observed an increased cellular ATP level and mitochondrial mass after SGF exposure, indicating that SGF enhanced mitochondrial function. The other relevant transcriptional changes appeared to be involved in AMPK/PGC-1α signaling, inflammatory response, as well as PI3K/AKT and calcium signaling pathways, which might contribute to the beneficial metabolic effects of SGF on adipocyte function. The results of Western blotting confirmed that SGF could increase the phosphorylation of AMPK while decrease the phosphorylation of AKT in adipocytes. Altogether, our results provided novel information about the molecular mechanism responsible for the effects of SGF on fat storage in adipocytes and highlights the potential metabolic benefits of SGF on human obesity and its related chronic diseases.

Smilax China-derived yellow-fluorescent carbon dots for temperature sensing, Cu2+ detection and cell imaging.

Here, we report an environmentally friendly fabrication strategy of bright yellow fluorescent carbon dots (y-CDs) and construct a rapid and accurate multifunctional sensing platform for the effective detection of temperature and Cu2+. The y-CDs were favorably obtained through a one-step hydrothermal treatment of natural Smilax China for the first time and exhibit long-wavelength emission at 542 nm under an excitation wavelength of 470 nm. Additionally, the obtained y-CDs exhibit superior biocompatibility and distinguished stability under different conditions, and display a respectable fluorescence quantum yield of up to 22.37%. Appealingly, the as-prepared y-CDs were implemented as temperature probes within ranges of 25 °C-40 °C and 45 °C-80 °C. Significantly, based on the static quenching effect, the as-prepared y-CDs were developed as an effective platform for fluorescence sensing of Cu2+, with linear ranges of 0.5 µM-10 µM, 75 µM-225 µM and 250 µM-350 µM, achieving a detection limit of 28 nM. Furthermore, confocal fluorescence imaging of PC12 cells was achieved successfully, which indicated that the as-synthesized y-CDs could visualize Cu2+ fluctuations in living cells.

Smilax china leaf extracts suppress pro-inflammatory adhesion response in human umbilical vein endothelial cells and proliferation of HeLa cells.

Objective: The objective of this study was to investigate the anti-inflammatory and anticancer effects of the leaves of Smilax china.Methodology: The aqueous extract was examined for its anti-inflammatory effects on tumour necrosis factor (TNF)-α-induced inflammation in HUVECs whereas the aqueous (water), ethyl acetate (EA), butanol (B) and methylene chloride (MC) extracts were examined for their anticancer effect on HeLa cells.Results: The aqueous extract suppressed the (TNF)-α-induced expression of ICAM-1, VCAM-1 and TNF-R1 and attenuated the expression of MCP-1, MMP-9, NF-kB and IFN-γ. The MC extract suppressed the proliferation of HeLa cells at all doses employed (50, 150, and 300 µg/ml). The EA extract demonstrated appreciable anti-proliferative effect whereas the BuOH extract demonstrated mild anti-proliferative activity. The aqueous extract did not show any significant anti-proliferative effect. None of the extracts were toxic to the normal cells (HUVECs).Conclusion: Smilax china leaf extracts possess significant anti-inflammatory and anticancer effects.

Endothelium-dependent vasodilatory effect of Smilax china Linn. water extract via PI3K/Akt signaling.

Objective: The objective of this study was to investigate the pharmacological effect of Smilax china Linn. water extract (SCLWE) on vascular relaxation and its underlying biochemical mechanisms.Methodology: Isolated rat aortic rings were pre-constricted with phenylephrine (PE). This was followed by the cumulative addition of SCLWE. The effect of endothelial nitric oxide and PI3K/Akt on the SCLWE-induced vasodilation was investigated by the pretreatment of endothelium-intact aortic strips with or without NG-nitro-L-arginine methyl ester (L-NAME) or wortmanin before constriction with PE.Results: Treatment of PE (1 µM)-pre-contracted aortic strips with SCLWE induced endothelium-dependent relaxation, which was attenuated by L-NAME and wortmanin. Further studies using HUVECs indicated that nitrite production, eNOS and PI3K/PKB (Akt) phosphorylations were increased after exposure to SCLWE but was attenuated by pretreatment with wortmanin.Conclusion: These results suggest that SCLWE induces vasodilation by augmenting NO production in endothelial cells via PI3K/Akt-dependent eNOS phosphorylation.

In vitro anti-inflammatory properties of Smilax campestris aqueous extract in human macrophages, and characterization of its flavonoid profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. MATERIALS AND METHODS: The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. RESULTS: In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. CONCLUSION: These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.

Purification, structural elucidation and anti-inflammatory activity in vitro of polysaccharides from Smilax china L.

Smilax china L. is a traditional Chinese medicine mainly used for the treatment of pelvic inflammation. Polysaccharide might be one of the anti-inflammatory components of Smilax china L. Based on this hypothesis, this work aimed at extraction, purification and structural elucidation of Smilax china L. polysaccharides and their preliminary anti-inflammatory effects were also studied. Two polysaccharides named SCLP1 (Smilax china L. polysaccharide 1, 42.1kDa) and SCLP3-2 (Smilax china L. polysaccharide 3-2, 16.8kDa) were for the first time purified from Smilax china L. The structures of SCLP1 and SCLP3-2 were elucidated by chemical and spectral analysis. The results revealed that SCLP1 was a neutral polysaccharide composed of glucose and mannose (54.5:1.0). Its backbone was 1,4­linked α­Glcp interspersed with 1,2­linked α­Glcp and Manp; the branches were 1,6­linked α-Glcp and terminated with α­Glcp. SCLP3-2 was composed of galacturonic acid, arabinose, galactose and rhamnose (23.3:2.1:1.7:1.0) and was a pectin-type polysaccharide with an α­1,4­linked homogalacturonan backbone which was partially methyl-esterified and slightly acetylated. The side chains consisted of α­Rhap, ß­Galp and α­Araf. SCLP1 and SCLP3-2 could inhibit the production of NO, IL-6 and TNF-α in LPS-stimulated RAW264.7 cells via NF-κB and MAPKs (ERK1/2, JNK) pathways, indicating that they possessed a potential anti-inflammatory activity.

Cell cycle, energy metabolism and DNA repair pathways in cancer cells are suppressed by Compound Kushen Injection.

BACKGROUND: In this report we examine candidate pathways perturbed by Compound Kushen Injection (CKI), a Traditional Chinese Medicine (TCM) that we have previously shown to alter the gene expression patterns of multiple pathways and induce apoptosis in cancer cells. METHODS: We have measured protein levels in Hep G2 and MDA-MB-231 cells for genes in the cell cycle pathway, DNA repair pathway and DNA double strand breaks (DSBs) previously shown to have altered expression by CKI. We have also examined energy metabolism by measuring [ADP]/[ATP] ratio (cell energy charge), lactate production and glucose consumption. Our results demonstrate that CKI can suppress protein levels for cell cycle regulatory proteins and DNA repair while increasing the level of DSBs. We also show that energy metabolism is reduced based on reduced glucose consumption and reduced cellular energy charge. RESULTS: Our results validate these pathways as important targets for CKI. We also examined the effect of the major alkaloid component of CKI, oxymatrine and determined that it had no effect on DSBs, a small effect on the cell cycle and increased the cell energy charge. CONCLUSIONS: Our results indicate that CKI likely acts through the effect of multiple compounds on multiple targets where the observed phenotype is the integration of these effects and synergistic interactions.