Isolation, structural elucidation, and immunoregulation properties of an arabinofuranan from the rinds of Garcinia mangostana.

In our search for bioactive polysaccharides as immunomodulatory agents, an arabinofuranan (GMP90-1) was purified and characterized from the rinds of Garcinia mangostana L. GMP90-1 (absolute molecular weight: 5.30 × 103 g/mol) was found to be composed of arabinose, galactose, and rhamnose. The backbone of GMP90-1 was determined as (1→5)-linked α-l-Araf, (1→2,3,5)-linked α-l-Araf, (1→3,5)-linked α-l-Araf, (1→6)-linked ß-d-Galp, and (1→2)-linked α-l-Rhap. Conformational analysis revealed GMP90-1 to exist as a rigid rod structure in sodium chloride solution. To explore its potential as immunomodulatory agents, an in vitro cell screening was performed and GMP90-1 was found to significantly enhance the phagocytic uptake of neutral red and improve the secreted level of nitric oxide (NO), interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α) of macrophages. Furthermore, the cellular immunomodulatory activities were confirmed by the in vivo zebrafish experiment, which suggested that GMP90-1 with immunomodulatory effects could be considered as a potential immunomodulatory for immune diseases.

Evaluation of phyto-medicinal efficacy of thymoquinone against Arsenic induced mitochondrial dysfunction and cytotoxicity in SH-SY5Y cells.

BACKGROUND: It is evaluated that a few million individuals worldwide are experiencing Arsenic (As) harmfulness coming about because of anthropogenic discharges. There is likewise proof to propose that As can affect the peripheral, as well as, the central nervous system (CNS). On the contrary, thymoquinone (TQ), a biologically active ingredient of Nigella sativa has exhibited numerous neuro-pharmacological traits since ancient times. HYPOTHESIS/PURPOSE: In the present study, the neuroprotective efficacy of TQ was explored by primarily studying its antioxidant and anti-apoptotic potential against Arsenic trioxide (As2O3) induced toxicity in SH-SY5Y human neuroblastoma cell lines. STUDY DESIGN: For experimentation, cells were seeded in 96 well tissue culture plates and kept undisturbed for 24 h to attain proper adhesion. After 75-80% confluence, cells were pretreated with 10 µM and 20 µM thymoquinone (TQ) for 1 h After adding 2 µM As, cells were set aside for incubation for 24 h without changing the medium. METHODS: The mitigatory effects of TQ with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. RESULTS: Pretreatment of SH-SY5Y cells with TQ (10 and 20 µM) for an hour and subsequent exposure to 2 µM As2O3 protected the SH-SY5Y cells against the neuro-damaging effects of the latter. Also, the SH-SY5Y cells were better preserved with increased viability, repaired DNA, less free radical generation and balanced transmembrane potential than those exposed to As2O3 alone. TQ pretreatment also inhibited As2O3-induced exacerbation in protein levels of BAX and PARP-1 and restored the loss of Bcl2 levels. CONCLUSION: The findings of this study suggest that TQ may prevent neurotoxicity and As2O3-induced apoptosis and cytotoxicity. It is, therefore, worth studying further for its potential to reduce the risks of arsenic-related neurological implications.

Ameliorative effect of Homalium zeylanicum against carbon tetrachloride-induced oxidative stress and liver injury in rats.

Objective is to evaluate the ameliorative effects of Homalium zeylanicum in carbon tetrachloride-induced oxidative stress and liver injury in rats. To establish the nature of antioxidant principles in the bioactive ethyl acetate fractions of bark (HZEB) and leaf (HZEL); oxygen radical absorbance capacity (ORAC) and cell-based antioxidant protection in erythrocytes (CAP-e) assays were performed. From acute toxicity study, HZEB and HZEL at 200 and 300 mg/kg b.w., were relatively safe at their effective doses. The degree of protection was measured by using biochemical parameters such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin (TB) and total protein (TP) contents. Hepatic markers e.g. thiobarbituric acid reactive species (TBARS), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were evaluated along with histopathological observations of liver tissues. Both fractions showed significant improvement in restoring SGOT, SGPT, ALP, TB and TP level. TBARS, SOD, CAT and GSH levels were significantly altered towards normal values. Both fractions at 300 mg/kg showed remarkable improvement in liver markers as compared to silymarin. Histopathological examinations showed reduction in hepatic necrosis and appeared normal hepatocellular architecture in HZEB and HZEL treated groups. In CAP-e assay, IC50 of HZEB (54.66 mg/mL) was higher than HZEL (60.88 mg/mL) and in ORAC assay, AUC of HZEB and HZEL were 33.46, 21.29 respectively and results were comparable with trolox. GC-MS and LC-MS analysis identified a total no. of 44 compounds. Few compounds were identified as bioactive compounds e.g. catechol (7.23%), tetraacetyl-d-xylonic nitrile (3%), oleic acid (0.49%), 2,6-bis(1,1-dimethylethyl)-phenol (3.71%), 3,4,5-trimethoxy-phenol (0.31%), and conifer alcohol (7.41%). The presence of antioxidant principles in both fractions were responsible for hepatoprotective activities, however, the presence of catechol (7.23%) in the bark part imparted better activities in protecting liver than leaf of H. zeylanicum.

Wound healing and anti-inflammatory activities induced by a Plantago australis hydroethanolic extract standardized in verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a popular plant found to be widely spread in Latin America. In folk medicine, the seeds and leaves are used mainly for anti-inflammatory, wound healing, among others. The verbascoside, a phenolic glycoside, is an active chemical component described in this species of plant, which has antioxidant, anti-inflammatory and healing effects. PURPOSE: The aim of the present study was to evaluate whether P. australis hydroethanolic extract (PAHE) standardized in verbascoside could promote wound healing associated with anti-inflammatory action within both in vitro and in vivo models. METHODS: For the wound healing activity, we used a Scratch Test, an assay capable of evaluating the migratory ability of keratinocyte cells (HaCat) in vitro and thereby confirming the activity in rats. For the anti-inflammatory activity, the inflammation was induced with LPS in microglial murine cells (N9). Inflammatory mediators (IL-6, IL-10, IL-12p70, INFγ, MCP-1 and TNFα) were measured and the activity of superoxide dismutase (SOD), catalase (CAT), and mitochondrial membrane potential were evaluated. In addition, using paw edema induced by carrageenan in rats, the anti-inflammatory activity in vivo was analyzed. RESULTS: The PAHE and verbascoside, induced a significant increase in migration of keratinocytes, at all concentrations tested when compared to the negative control. The wound healing activity in vivo showed that the PAHE accelerated the process. The treatments with PAHE and verbascoside induce increases in the antioxidants enzymes, suggesting a possible activation of these enzymes. However, this did not result in an increase in the expression of inflammatory mediators in microglial cells. In LPS activated cells the verbascoside displayed a significant reduction of TNFα, IL-6, IL-12p70, MCP-1 and INFγ, while the PAHE only displayed statistically significant reduction in TNFα. Interestingly, both the compounds could reduce the oxidative parameters in N9 cells activated by LPS. Additionally, pretreatment with PAHE inhibited the paw edema in rats. CONCLUSION: The results suggest that PAHE has wound healing activity, improving cells migration and, as well as was able to reverse the oxidation effect in LPS-activated N9 cells. The wound-healing and anti-inflammatory activities of PAHE were confirmed in vivo. In addition, the presence of verbascoside can be related to PAHE effects, since this compound was capable of increase keratinocytes migration and inhibiting inflammation mediators.

Nutraceutical potential of hemp (Cannabis sativa L.) seeds and sprouts.

In this study the antioxidant effect of Cannabis sativa L. seeds and sprouts (3 and 5 days of germination) was evaluated. Total polyphenols, flavonoids and flavonols content, when expressed on dry weight basis, were highest in sprouts; ORAC and DPPH (in vitro assays), CAA-RBC (cellular antioxidant activity in red blood cells) and hemolysis test (ex vivo assays) evidenced a good antioxidant activity higher in sprouts than in seeds. Untargeted analysis by high resolution mass spectrometry in negative ion mode allowed the identification of main polyphenols (caffeoyltyramine, cannabisin A, B, C) in seeds and of ω-6 (linoleic acid) in sprouts. Antimutagenic effect of seeds and sprouts extracts evidenced a significant decrease of mutagenesis induced by hydrogen peroxide in Saccharomyces cerevisiae D7 strain. In conclusion our results show that C. sativa seeds and sprouts exert beneficial effects on yeast and human cells and should be further investigated as a potential functional food.

In vitro screening of neuroprotective activity of Indian medicinal plant Withania somnifera.

Canine cognitive dysfunction (CCD) is an age-dependent neurodegenerative condition characterised by changes in decline in learning and memory patterns. The neurodegenerative features of CCD in ageing dogs and cats are similar to human ageing and Alzheimer's disease (AD). Discovering neuroprotective disease-modifying therapies against CCD and AD is a major challenge. Strong evidence supports the role of amyloid ß peptide deposition and oxidative stress in the pathophysiology of CCD and AD. In both the human and canine brain, oxidative damage progressively increases with age. Dietary antioxidants from natural sources hold a great promise in halting the progression of CCD and AD. Withania somnifera (WS), an Ayurvedic tonic medicine, also known as 'Indian ginseng' or ashwagandha has a long history of use in memory-enhancing therapy but there is a dearth of studies on its neuroprotective effects. The objective of this study was to investigate whether WS extract can protect against Aß peptide- and acrolein-induced toxicity. We demonstrated that treatment with WS extract significantly protected the human neuroblastoma cell line SK-N-SH against Aß peptide and acrolein in various cell survival assays. Furthermore, treatment with WS extract significantly reduced the generation of reactive oxygen species in SK-N-SH cells. Finally, our results showed that WS extract is also a potent inhibitor of acetylcholinesterase activity. Thus, our initial findings indicate that WS extract may act as an antioxidant and cholinergic modulator and may have beneficial effects in CCD and AD therapy.

Berberine and S allyl cysteine mediated amelioration of DEN+CCl4 induced hepatocarcinoma.

BACKGROUND: Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN+CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated. METHODS: ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1-pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry. RESULTS: CytochromeP4502E1 mediated bioactivation of DEN+CCl4 induced Akt dependent pMdm2-HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2-HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure. CONCLUSION AND GENERAL SIGNIFICANCE: SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN+CCl4 induced hepatocarcinoma.

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro.

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100-400 µg/mL), arrest cell cycle in the G0/G1 phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.

Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

BACKGROUND & AIMS: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.

Calyxin Y induces hydrogen peroxide-dependent autophagy and apoptosis via JNK activation in human non-small cell lung cancer NCI-H460 cells.

Calyxin Y has been recently isolated from Alpinia katsumadai which has a folk use as an anti-tumor medicine. Calyxin Y induced caspase-dependent cell death in NCI-H460 cells, and concomitantly, provoked cytoprotective autophagy with the upregulation of critical Atg proteins. The cleavage of Atg proteins by caspases acted as a switch between autophagy and apoptosis induced by calyxin Y. Intracellular hydrogen peroxide (H2O2) production was triggered upon exposure to calyxin Y via the induction of autophagy and apoptosis. We provided evidence that activated JNK was upstream effectors controlling both autophagy and apoptosis in response to elevated H2O2. Therefore, our findings demonstrate that calyxin Y serves multiple roles as a promising chemotherapeutic agent that induces H2O2-dependent autophagy and apoptosis via JNK activation.

Anti-inflammatory activity of edible brown alga Eisenia bicyclis and its constituents fucosterol and phlorotannins in LPS-stimulated RAW264.7 macrophages.

Although individual phlorotannins contained in the edible brown algae have been reported to possess strong anti-inflammatory activity, the responsible components of Eisenia bicyclis have yet to be fully studied. Thus, we evaluated their anti-inflammatory activity via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) and tert-butylhydroperoxide (t-BHP)-induced reactive oxygen species (ROS), along with suppression against expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), in RAW 264.7 cells. The anti-inflammatory activity potential of the methanolic extract and its fractions of E. bicyclis was in the order of dichloromethane>methanol>ethyl acetate>n-butanol. The strong anti-inflammatory dichloromethane fraction was further purified to yield fucosterol. From the ethyl acetate fraction, six known phlorotannins were isolated: phloroglucinol, eckol, dieckol, 7-phloroeckol, phlorofucofuroeckol A and dioxinodehydroeckol. We found that these compounds, at non-toxic concentrations, dose-dependently inhibited LPS-induced NO production. Fucosterol also inhibited t-BHP-induced ROS generation and suppressed the expression of iNOS and COX-2. These results indicate that E. bicyclis and its constituents exhibited anti-inflammatory activity which might attribute to inhibition of NO and ROS generation and suppression of the NF-κB pathway and can therefore be considered as a useful therapeutic and preventive approach to various inflammatory and oxidative stress-related diseases.

Antioxidant effects of diallyl trisulfide on high glucose-induced apoptosis are mediated by the PI3K/Akt-dependent activation of Nrf2 in cardiomyocytes.

BACKGROUND: Hyperglycemia-induced reactive oxygen species (ROS) generation contributes to development of diabetic cardiomyopathy. Nuclear factor E2-related factor 2 (Nrf2), a redox-sensing transcription factor, induces the antioxidant enzyme expressions. Diallyl trisulfide (DATS) is the most powerful antioxidant among the sulfur-containing compounds in garlic oil. We investigated whether DATS inhibits hyperglycemia-induced ROS production via Nrf2-mediated activation of antioxidant enzymes in cardiac cells exposed to high glucose (HG). METHODS AND RESULTS: Treatment of H9c2 cells with HG resulted in an increase in intracellular ROS level and caspase-3 activity, which were markedly reduced by the administration of DATS (10 µM). DATS treatment significantly increased Nrf2 protein stability and nuclear translocation, upregulated downstream gene HO-1, and suppressed its repressor Keap1. However, apoptosis was not inhibited by DATS in cells transfected with Nrf2-specific siRNA. Inhibition of PI3K/Akt signaling by LY294002 (PI3K inhibitor) or PI3K-specific siRNA not only decreased the level of DATS-induced Nrf2-mediated HO-1 expression, but also diminished the protective effects of DATS. Similar results were also observed in high glucose-exposed neonatal primary cardiomyocytes and streptozotocin-treated diabetic rats fed DATS at a dose of 40 mg/kg BW. CONCLUSIONS: Our findings indicate that DATS protects against hyperglycemia-induced ROS-mediated apoptosis by upregulating the PI3K/Akt/Nrf2 pathway, which further activates Nrf2-regulated antioxidant enzymes in cardiomyocytes exposed to HG.