Neferine induces reactive oxygen species mediated intrinsic pathway of apoptosis in HepG2 cells

Evidence has accumulated concerning the medicinal application of Nelumbo nucifera in the treatment of various diseases. Neferine, an alkaloid from N. nucifera was found to exert cytotoxicity on liver cancer cells HepG2 in a dose-dependent manner. We evaluated its anticancer potential by studying its effect on mitochondrial membrane potential, intracellular calcium levels [Ca²⁺]_i, cell membrane integrity, apoptotic body formation and DNA fragmentation in cultured HepG2 cells. The reactive oxygen species level has been increased upon neferine treatment with concomitant decrease in reduced glutathione. Our data further indicate reduction of ΔψM and increased [Ca²⁺]_i during apoptosis induction by neferine with increased expression of apoptotic proteins such as Bax, Bad, cleaved forms of caspase 3, caspase 9 and PARP, with the downregulation of anti-apoptotic protein Bcl2 in HepG2 cells. Moreover, the expressions of tumour suppressor proteins p53 and PTEN were upregulated along with the downregulation of P-Akt. In addition, expression levels of TNF-α, p38 and ERK1/2 MAP kinases were increased upon neferine treatment. These results imply that mitochondrial-mediated ROS generation induced by neferine leads to caspase-dependent apoptosis in HepG2 cells.     

Immuno-enhancing effects of the herbal extract from Himalayan rhubarb Rheum emodi Wall. ex Meissn

In the present studies, the immuno-enhancing activity of Rheum emodi ethyl acetate rhizome extract has been carried out by conducting studies on the cell lines. The studies include effects on signal transduction parameters that could pave the way for understanding some of the treatment principles of the plant extracts. The data indicate that R. emodi may have an immuno-enhancing effect through the release of various cytokines. There is a dose dependent increase in the release of NO (nitric oxide) and cytokine TNF-α, IL-12 and a decrease in IL-10 by RAW 264.7 in macrophage cell lines in the presence of extract alone. However the cytokine level is concomitantly increased in the presence of stimulant as well as extract. The up-regulation of TNF-α, and IL-12 up to the level of 200 ng/ml and 530 ng/ml respectively switches the cell towards Th-1 immune response. TNF-α, IL-12 induces generation and proliferation of Th-1 cells which in turn produces NK and cytotoxic T-cells. On the other hand, the decreased levels of IL-10 switches off the Th-2 immune system and directs the cell towards Th-1 immune response only. Thus, indicating that R. emodi extract may have anti-tumour effects by regulating Th-1 and Th-2 immune response in vivo.     

Antioxidant and anti-inflammatory properties of Dichondra repens Forst. and its reference compounds

Dichondra repens (DR) is the main constituent in herbal beverages and consumed daily as a nutrition supplement for the liver in Taiwan. This study investigated the antioxidant and anti-inflammatory effects of D. repens ethanol extract (EDR) and its reference compounds ex vivo and in vivo. Fingerprint chromatograms (from HPLC) indicated that EDR contained vanillin, umbelliferone and scopoletin. EDR was evaluated for its antioxidant effects and LPS-induced NO production in RAW 264.7 cells. EDR decreased the LPS-induced NO production and expressions of iNOS and COX-2 in RAW 264.7 cells. In vivo anti-inflammatory activities of EDR were assessed in mouse paw oedema, induced by λ-carrageenan (Carr). We investigate the antioxidant mechanism of EDR via studies of the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the liver and the levels of malondialdehyde (MDA) and nitrite oxide (NO) in the oedematous paw. Serum NO and TNF-α were also measured. EDR exerts anti-inflammatory effects by suppressing TNF-α, NO, and might be related to the decrement of the level of MDA in the oedema paw via increasing the activities of CAT, SOD and GPx in the liver. The results show that EDR might be a natural antioxidant and anti-inflammatory agent.     

Chinese quince (Chaenomeles sinensis) extract inhibits cell migration and cytokine release in HMC-1 cells

Chinese quince (Chaenomeles sinensis; CS) is known as a traditional oriental medicine for treating anaphylaxis and viral infection. Mast cells play an important role in a variety of inflammatory diseases. The inhibitory effects of CS extract on the inflammatory response of human mast cells were examined. CS extract inhibited HMC-1 cell migration in response to stem cell factor (SCF). Its mechanism is involved in the inhibition of a surface expression of c-kit binding to SCF. Tumor necrosis factor (TNF)-α expression is induced by phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CaI). CS extract inhibited the TNF-α expression by blocking the activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun-N-terminal kinase (JNK) in HMC-1 cells. CS extract suppressed the expression of interlukin (IL)-6, IL-8, and MCP-1 in human monocytic THP-1 cells, as well as the secretion of IL-6 in human keratinocytic HaCaT cells.     

Polyopes lancifolia extracts protect INS-1 pancreatic β cells against high glucose level induced apoptosis

Polyopes lancifolia extracts were investigated for protection of INS-1 pancreatic β cells against high glucose level-induced apoptosis using cell viability, lipid peroxidation generation, intracellular ROS, and NO assays. Expressions of the Bax and Bcl-2 proteins in INS-1 cells were evaluated using western blotting. Apoptosis induced by high glucose levels was determined based on analysis of FITC-annexin V/PI stained cells. A high glucose level-treatment induced INS-1 cells death. Cells treated with P. lancifolia extracts showed markedly reduced cytotoxicity in a dose-dependent manner, compared to the high glucose level-treated cells. Treatment with extracts dose-dependently decreased lipid peroxidation and intracellular ROS and NO levels, and increased cell viability in high glucose level-pretreated INS-1 cell, compared to the high glucose level-treated cells. Extract treatment significantly (p<0.05) reduced the Bax protein level and increased the anti-apoptotic Bcl-2 protein level, compared with the high glucose leveltreated cells. P. lancifolia extract treatment reduced the apoptosis that is induced by a high level glucose-treatment, based on annexin V/PI staining.     

Protective effect of antrosterol from Antrodia camphorata submerged whole broth against carbon tetrachloride-induced acute liver injury in mice

The hepatoprotective potential of antrosterol (ergostatrien-3β-ol, ST1) from Antrodia camphorata (AC) against carbon tetrachloride (CCl₄)-induced liver damage was evaluated in preventive models in mice. Pretreatment with ST1 markedly prevented the elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and liver lipid peroxides in CCl₄-treated mice. The activities of antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)] were significantly increased after treatment with CCl₄invivo. In addition, ST1 decreased the level of nitric oxide (NO) production and tumour necrosis factor-alpha (TNF-α) in CCl₄-treated mice. In this study, these results pointed out that ST1 can inhibit lipid peroxidation, enhance the activities of antioxidant enzymes, decreases the TNF-α level, nitric oxide production and inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expressions. Therefore, it was speculated that ST1 protects mice from liver damage through their anti-inflammation capacity.     

Mare's and cow's milk: promote similar metabolic effects and expression of innate markers in Caco-2 cells?

Mare's milk (MM) is very similar to human breast milk, as it consists of low concentrations of proteins and fat and high concentrations of lactose. Thus, MM can be considered as an alternative to cow's milk (CM) for human nutrition. In this study, proteolytic resistant peptides to gastrointestinal enzymes were measured by reverse phase-high-performance liquid chromatography (RP-HPLC-PDA-UV) to determine their relative uptake by intestinal epithelial (Caco-2) cells. Alterations in mitochondrial enzyme activities (MTT test), inner mitochondrial membrane potential (ΔΨm), cell cycle progression and the expression (mRNA) of inflammatory (IL-1β, TNF-α and NF-κB) markers were studied. This is the first study comparing the effects of proteolytic resistant peptides from the whey proteins of mare's milk to those originating from cow's milk on intestinal cells. Similar proteolytic resistant peptide patterns, but lower uptake rates, were observed for MM peptides (7–40%) compared to peptides from CM (40–60%), although both types of peptides, probably derived from α-lactalbumin, were still capable of impairing ΔΨm in intestinal cells (CM > MM).Both MM and CM increased the expression of TNF-α, whereas IL-1β levels were not affected. CM increased NF-κB mRNA levels. Overall, our data indicate that proteolytic resistant peptides from both MM and CM exert similar metabolic effects on mitochondrial metabolism (up to 30%) in intestinal Caco-2 cells. However, the low uptake rates of peptides from MM may be beneficial to the human diet, an alternative to CM. Further in vivo studies are needed to better understand the potential clinical relevance of the control and/or regulation of metabolic processes related to perturbations in the gut immune system associated with lymphoid tissue.     

Immunostimulatory effect of β-cryptoxanthin in vitro and in vivo

Beta-cryptoxanthin (β-Crp), a mono-hydroxylated β-carotene, is a major dietary provitamin A xanthophyll. The immunostimulatory effect of β-Crp was examined using human hybridoma HB4C5 cells and mouse primary lymphocytes in vitro and using mice in vivo. IgM production by the HB4C5 cells and both IgA and IgG productions by mouse primary lymphocytes from mesenteric lymph node and spleen were accelerated in vitro by the treatment of β-Crp in dose-dependent manners. Afterwards 6-week-old female BALB/c mice were administered with a low or high dose of β-Crp for 14 days, mesenteric lymph node and spleen were excised and the immunoglobulin production levels were evaluated using lymphocytes from mesenteric lymph node and spleen in vivo. Results exhibited that all of IgA, IgG, and IgM productions by lymphocytes from mesenteric lymph node were increased. Especially, the IgG production increased with statistically significant differences in both of the low and high dose groups against the control group. The immunostimulatory effect on splenocytes was also observed although not as clear as on lymphocytes from mesenteric lymph node. Overall results suggest that β-Crp may stimulate the humoral immunity in mammals. Hence, β-Crp might have a potentially significant impact on human health and on prevention of diseases.     

Anti-inflammation of epicatechin mediated by TMEM35A and TMPO in bovine mammary epithelial cell line cells and mouse mammary gland

Epicatechin (EC) has significant antiinflammation, antioxidation, and anticancer activities. It also provides a new alternative treatment for mastitis, which can result in great economic losses in the dairy industry if left untreated. The purpose of this study was to investigate the anti-inflammatory effects of EC on mastitis and the underlying mechanism using in vivo and in vitro systems. The use of ELISA and immunohistochemistry assays showed that EC treatment at 1.5, 7.5, 15, and 30 mg/mL decreased protein expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase; inflammatory cytokines, which were composed of IL-1β, TNF-α, and IL-6 in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cell line (MAC-T); and mouse mammary gland, together with reduced filtration of T lymphocytes in the mouse mammary gland. Furthermore, EC treatment reduced LPS-induced phosphorylation levels of p65 and inhibitor of NF-κB, and blocked nuclear translocation of p65 as revealed by western blot and immunofluorescence test in MAC-T cells and the mouse mammary gland. Epicatechin also attenuated LPS-induced phosphorylation levels of mitogen-activated protein kinase members (i.e., p38, c-Jun N-terminal kinase 1/2 and extracellular regulated protein kinases 1/2). Using RNA-seq and tandem mass tag analyses, upregulation of TMEM35A and TMPO proteins was disclosed in MAC-T cells cotreated with LPS and EC. Although clustered regularly interspaced short palindromic repeats/Cas9-based knockdown of TMEM35A and TMPO attenuated abundance of phosphorylated (p)-p65, p-p38, TNF-α, and iNOS, overexpression of TMEM35A reversed EC-mediated effects in TMPO knockdown cells. Moreover, interaction between TMEM35A and TMPO was detected using the co-immunoprecipitation method. In conclusion, our data demonstrated that EC inhibited LPS-induced inflammatory response in MAC-T cells and the mouse mammary gland. Importantly, TMEM35A mediated the transmembrane transport of EC, and the interaction between TMEM35A and TMPO inhibited MAPK and NF-κB pathways.     

Increasing espresso coffee brew antioxidant capacity using phenolic extract recovered from hazelnut skin waste

The simultaneous consumption of different classes of phytochemical antioxidants in the diet can result in more beneficial effects than when consumed alone. In the present study, the in vitro and in vivo antioxidative effects of espresso coffee brew (EC) (rich in chlorogenic acids) with added crude hazelnut skin phenolic extract (HSPE) from hazelnut skin waste (rich in flavonoids) were studied. Both post-brewing and pre-brewing phenolic-enriched espresso coffees (PE-ECs) were analysed for total phenols and screened for their in vitro antiradical ability. Moreover, the in vivo biological effect on the antioxidant potential of plasma in rats was evaluated. The PE-ECs showed increased both in vitro and in vivo antiradical activity proportional to the added HSPE. The in vivo experiments suggested that HSPE was much more antioxidant active than the phenolic fraction naturally contained in EC. Moreover, evidence of possible synergic effects of EC and HSPE phenolics was observed in vivo.     

Effect of bengkoang (Pachyrhizus erosus) fiber extract on murine macrophage-like J774.1 cells and mouse peritoneal macrophages

Bengkoang (Pachyrhizus erosus (L) Urban) is an edible root vegetable containing fairly large amounts of carbohydrates and crude fibers. The purpose of this study is to evaluate the effects of the bengkoang fiber extract (BFE) on macrophages in vitro and in vivo. BEF was prepared by heat-extraction from the bengkoang fiber in distilled water at 121 °C for 20 min. BFE stimulated the phagocytotic activity of J774.1 cells. In addition, BFE significantly facilitated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by J774.1 cells and mouse peritoneal macrophages. BFE also facilitated the gene expression levels of inducible nitric oxide synthase and cyclooxygenase-2. The phagocytotic activity of mouse peritoneal macrophages was significantly enhanced by oral administration of BFE to BALB/c mice for 7 days. Ex vivo analysis revealed that production and gene expression of TNF-α and IL-6 in peritoneal macrophages from the BFE-administered mice were significantly enhanced. These results demonstrated that BFE activates macrophages in vitro and in vivo.     

Chemical properties and in vivo gastric protective effects of bamboo salt

Bamboo salt chemical properties and in vivo gastric protective effects were evaluated. Scanning electron microscopy revealed the diameter of 9× bamboo salt was approximately 5 μm, the smallest of all samples. The Ca, Mg, Fe, Mn, P, S, and K levels of bamboo salts were higher than for common salts. X-ray diffraction and X-ray photoelectron spectroscopy analyses showed bamboo salts had a complex structure composed of NaCl, KCl, and MgCl₂. Bamboo salt structure complexity increased with increased baking times. Purified, solar, and 1× bamboo salt inhibited gastric injury by 19.3%, 28.0%, and 77.8% in Sprague-Dawley (SD) rats, respectively. The 9× bamboo salt showed a high rate of gastric injury inhibition (98.7%). The effect of omeprazole was 99.3%. Bamboo salts reduced gastric secretion and increased gastric juice pH by increased mineral and phytochemical contents, and increased antioxidant activities. Bamboo salt can be used as a functional food to protect against gastric injury.     

Cytoprotective effects of pu-erh tea on hepatotoxicity in vitro and in vivo induced by tert-butyl-hydroperoxide

The in vitro and in vivo protective effects of water extract of pu-erh tea (WEPT) on tert-butyl-hydroperoxide (t-BHP)-induced oxidative damage in hepatocytes of HepG2 cells and in rat livers were investigated. After treatment with 200 μg/ml of samples, the survival rate of HepG2 cells induced by t-BHP increased. WEPT concentration-dependently inhibited reactive oxygen species (ROS) generation in HepG2 cells in response to the oxidative challenge induced by t-BHP. Administration of WEPT (0.2, 0.5 and 1.0 g/kg of body weigh) to rats for 56 consecutive days before a single dose of t-BHP (0.5 mmol/kg, i.p.) exhibited a significant (p < 0.01) protective effect by lowering serum levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), as well as reducing the formation of malondialdehyde. Taken together, these results demonstrate that WEPT is able to protect against hepatic damage in vitro and in vivo, suggesting that the drinking of pu-erh tea may protect liver tissue from oxidative damage.     

Antioxidant,antimutagenic, and In vivo buccal mucosa cancer preventive effects of Fructus Malvae

Fructus Malvae is functional food known for antioxidant, anti-mutagenic, and in vivo anticancer effects. Fructus Malvae extracts demonstrated high antioxidant activities in DPPH and hydroxyl radical scavenging activity assays. In an Ames mutagenicity test, Fructus Malvae exhibited antimutagenicty in association with MNNG (Nmethyl-N’-nitro-N-nitrosoguanidine) in Salmonella Typhimurium TA100 cells. U14 squamous carcinoma cells were injected into the buccal mucosa of KM (Kunming) mice. The wound at the injection site was smeared with a Fructus Malvae solution, which was also administered to mice by gavage. Tumor volumes were reduced and tissue section analysis of buccal mucosa cancer and cervical lymph node tissues showed anti-cancer effects in Fructus Malvae treated groups. Fructus Malvae possesses good antioxidant and antimutagenic activities and exerts a preventive effect against buccal mucosa cancer in vivo.     

蜂胶乙醇提取物对小鼠主动脉内皮细胞损伤的保护作用

目的:探究蜂胶乙醇提取物（ethanol extracts of propolis,EEP）对脂多糖（lipopolysaccharide,LPS）诱导的小鼠主动脉内皮细胞（mouse aortic endothelial cell,MAEC）炎症因子损伤的保护作用。方法:将细胞分为对照组,LPS模型组,蜂胶低（2.5 μg/mL）、中（5 μg/mL）、高（10 μg/mL）剂量组。采用CCK-8检测MAEC的细胞增殖率,ELISA酶联免疫吸附实验测定MAEC炎症细胞中TNF-α、IL-6的含量,Western Blot法测定MAEC炎症细胞中ICAM-1、VCAM-1、MCP-1的表达水平。结果:与对照组相比,LPS组MAEC的细胞增殖率极其显著降低（P<0.001）,ICAM-1、VCAM-1、MCP-1、TNF-α以及IL-6的水平极其显著升高（P<0.001）。经不同浓度EEP处理后,MAEC的细胞增殖率显著上升（P<0.05或P<0.01）,TNF-α、IL-6的含量以及ICAM-1、VCAM-1、MCP-1表达水平降低,各蜂胶组与LPS组相比均有显著性差异（P<0.01或P<0.001）。结论:EEP能够抑制LPS诱导的MAEC中炎症因子的表达,对血管内皮细胞具有保护作用。     

Antioxidant activities of malt extract from barley (Hordeum vulgare L.) toward various oxidative stress in vitro and in vivo

The antioxidant activities of malt extract from barley were evaluated by various methods in vitro and in vivo. Scavenging effects on the hydroxyl and superoxide radicals, and protection against reactive oxygen species induced lipid, protein and DNA damage were evaluated. The d-galactose induced mouse aging model was used to evaluate ability of malt extract to behave as an antioxidant in vivo. The extract exhibited high antioxidant activities both in vitro and in vivo, evidenced by its ability to scavenge hydroxyl- and superoxide-radicals, high reducing power, and protection against biological macromolecular oxidative damage. Furthermore, malt extract prevented the decrease of antioxidant enzyme activities, decreased liver and brain malondialodehyde levels and carbonyl content, and improved total antioxidant capability in d-galactose-treated mice. In conclusion, these results demonstrate potential antioxidant activities and antiaging effect of malt, providing scientific support for the empirical use of malt as an antioxidant for diseases caused by reactive oxygen species.     

Nonclinical safety evaluation of boric acid and a novel borate-buffered contact lens multi-purpose solution,Biotrue™ multi-purpose solution

Multipurpose solutions (MPS) often contain low concentrations of boric acid as a buffering agent. Limited published literature has suggested that boric acid and borate-buffered MPS may alter the corneal epithelium; an effect attributed to cytotoxicity induced by boric acid. However, this claim has not been substantiated. We investigated the effect of treating cells with relevant concentrations of boric acid using two cytotoxicity assays, and also assessed the impact of boric acid on corneal epithelial barrier function by measuring TEER and immunostaining for tight junction protein ZO-1 in human corneal epithelial cells. Boric acid was also assessed in an in vivo ocular model when administered for 28 days. Additionally, we evaluated Biotrue multi-purpose solution, a novel borate-buffered MPS, alone and with contact lenses for ocular compatibility in vitro and in vivo. Boric acid passed both cytotoxicity assays and did not alter ZO-1 distribution or corneal TEER. Furthermore, boric acid was well-tolerated on-eye following repeated administration in a rabbit model. Finally, Biotrue multi-purpose solution demonstrated good ocular biocompatibility both in vitro and in vivo. This MPS was not cytotoxic and was compatible with the eye when administered alone and when evaluated with contact lenses. We demonstrate that boric acid and a borate-buffered MPS is compatible with the ocular environment. Our findings provide evidence that ocular effects reported for some borate-buffered MPS may be incorrectly attributed to boric acid and are more likely a function of the unique combination of ingredients in the MPS formulation tested.