Phenolic composition, antioxidant, cytotoxic activities and cardioprotective effect of hydroalcoholic extract from aerial-parts of Hypericum attenuatum Fisch. ex Choisy.

This study investigated phenolic metabolites, antioxidant, cytotoxic and cardioprotective effects of the hydroalcoholic extract from the aerial parts of Hypericum attenuatum Fisch. ex Choisy. The total phenolic and flavonoid contents of the extract were 132.40 ± 2.06 mg GAE/g and 101.46 ± 1.47 mg QE/g respectively. The extract exhibited antioxidant activities with an EC50 value against DPPH radical of 0.099 ± 0.03 mg/mL and a FRAP value of 1.22 ± 0.086 mmol/L Fe2+. The extract could protect H9c2 cardiomyoblasts from the injury of H2O2, while it restored the H9c2 cell viability to 82.69 ± 2.33% at 100 µg/mL. The extract possessed cytotoxicity on MGC803, C666-1 and SW620 cells with IC50 values of 69.77 ± 2.43 µg/mL, 74.97 ± 1.08 µg/mL and 58.91 ± 1.81 µg/mL, respectively. Moreover, it could promote apoptosis of the tested cancer cells. This research provided useful information for the utilization of H. attenuatum as herbal medicine.

α-Glucosidase Inhibition Action of Major Flavonoids Identified from Hypericum Attenuatum Choisy and Their Synergistic Effects.

Hypericum attenuatum Choisy is a traditional Chinese herbal plant with multiple therapeutic effects. In this study, bioactivity-guided fractionation of Hypericum attenuatum Choisy extracts afforded three major flavonoids (including astragalin, guaijaverin and quercetin), which possessed α-Glucosidase inhibitory activity with IC50 values of 33.90±0.68 µM, 17.23±0.75 µM and 31.90±0.34 µM, respectively. Circular dichroism analysis revealed that all the three compounds could interact with α-glucosidase by inducing conformational changes of the enzyme. Molecular docking results indicated that they could bind to the active site in α-glucosidase, and the binding force was driven mainly by hydrogen bond. Additionally, isobolographic analysis of the interactions between two compounds showed that all the combinations presented a synergistic α-glucosidase inhibitory effect at lower concentrations, and the combination between quercetin and guaijaverin or astragalin exhibited the best synergistic effect. This research might provide a theoretical basis for the application of Hypericum attenuatum Choisy in treating hyperglycemia.

LHH1, a novel antimicrobial peptide with anti-cancer cell activity identified from Lactobacillus casei HZ1.

Antimicrobial peptides have been attracting increasing attention for their multiple beneficial effects. In present study, a novel AMP with a molecular weight of 1875.5 Da, was identified from the genome of Lactobacillus casei HZ1. The peptide, which was named as LHH1 was comprised of 16 amino acid residues, and its α-helix content was 95.34% when dissolved in 30 mM SDS. LHH1 exhibited a broad range of antimicrobial activities against Gram-positive bacteria and fungus. It could effectively inhibit Staphylococcus aureus with a minimum inhibitory concentration of 3.5 µM and showed a low hemolytic activity. The scanning electron microscope, confocal laser scanning microscope and flow cytometry results showed that LHH1 exerted its antibacterial activity by damaging the cell membrane of Staphylococcus aureus. Meanwhile, LHH1 also exhibited anti-cancer cell activities against several cancer cells via breaking the cell membrane of MGC803, HCT116 and C666-1 cancer cells.

EtOAc extract of H. attenuatum Choisy inhibits inflammation by suppressing the NF-κB and MAPK pathways and modulating the gut microbiota.

BACKGROUND: Hypericum attenuatum Choisy, a traditional Chinese herb, has been shown to be effective in the treatment of diseases associated with inflammation and has been used to treat rheumatic arthritis in China for centuries. However, the underlying mechanism of its anti-inflammatory effect is poorly understood. HYPOTHESIS/PURPOSE: In this study, we aimed to investigate the anti-inflammatory mechanisms of EtOAc fractions of H. attenuatum Choisy (Ha-EtOAc) on lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation and hypothesized that Ha-EtOAc could attenuate inflammation in the colon. STUDY DESIGN: LPS was utilized to induce RAW264.7 cells inflammation. The anti-inflammatory effect of Ha-EtOAc in RAW264.7 cells was evaluated by measuring the inhibition ratio of nitric oxide (NO) production. Murine ulcerative colitis (UC) was induced by treatment with 2.5% dextran sulfate sodium (DSS). The basic indexes of the mice, including body weight, food intake and hematochezia, were recorded during mice experiments. METHODS: The expression levels of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1ß, were measured by quantitative real-time PCR and western blot. Additionally, the influences of Ha-EtOAc on the NF-κB and MAPK signaling pathways were determined by western blot and immunofluorescence assays. In addition, the impact of Ha-EtOAc on gut microbiota of mice with UC was detected by 16S rDNA sequencing. RESULTS: Ha-EtOAc inhibited the LPS-induced production of NO and decreased the release of TNF-α, IL-6 and IL-1ß in RAW264.7 cells in a dose-dependent manner. In addition, pretreatment with Ha-EtOAc could suppress the nuclear translocation of p65 and the phosphorylation of Erk1/2, p38 and JNK. Ha-EtOAc treatment ameliorated murine UC, as reflected by a reduced body weight loss, improved colon shortening, alleviated mucosal damage and decreased releases of pro-inflammatory cytokines. Furthermore, Ha-EtOAc could modulate the composition of microbial communities. CONCLUSION: Our results demonstrated that Ha-EtOAc exhibited anti-inflammatory effects mainly by suppressing the NF-κB and MAPK pathways, and Ha-EtOAc treatment may be a potent therapy for the treatment of ulcerative colitis.

Induction of apoptosis in human cervical carcinoma HeLa cells by active compounds from Hypericum ascyron L.

Hypericum ascyron L. (Great St. Johnswort), which belongs to the Hypericaceae family, has been used for the treatment of hematemesis, metrorrhagia, rheumatism, swelling, stomach ache, abscesses, dysentery and irregular menstruation for >2,000 years in China. The aim of the present study was to clarify the anticancer activity compounds from H. ascyron L. and the underlying molecular mechanism. Anticancer activity of H. ascyron L. extract was evaluated using an MTT assay. To confirm the anticancer mechanism of activity compounds, Hoechst 33258, Annexin V-fluorescein isothiocyanate/propidium iodide, 2',7'-dichlorodihydrofluorescein diacetate, rhodamine 123 staining and caspase-3 activity analysis were performed. The results demonstrated that the anti-proliferative action of the mixture of kaempferol 3-O-ß-(2″-acetyl) galactopyranoside (K) and quercetin (Q) (molar ratio, 1:1) was significantly increased compared with either of these two compounds separately, and the active fraction of the H. ascyron L. extract |(HALE). HALE, indicating that the anti-proliferative function of H. ascyron L. may be a synergic effect of K and Q. Furthermore, the inhibitory effect of KQ on the growth of HeLa cells was mediated by the induction of apoptosis. To the best of our knowledge, the present study is the first to identify that KQ exhibits significant anti-proliferation activity on HeLa cells via the apoptotic pathway, and is also the first to evaluate the anticancer potential of H. ascyron L. The results of the present study may provide a rational base for the use of H. ascyron L. in the clinic, and shed light on the development of novel anticancer drugs.

Preparation of antioxidative corn protein hydrolysates, purification and evaluation of three novel corn antioxidant peptides.

Corn gluten meal is a major co-product of corn wet milling. Corn gluten meal was hydrolyzed with Alcalase, Flavourzyme, Alcalase+Flavourzyme and Flavourzyme+Alcalase. At the substrate concentration of 10%, corn protein hydrolysate catalyzed by Alcalase had a degree of hydrolysis of 17.83%, which was higher than that by Flavourzyme (3.65%). The hydrolysate catalyzed by Alcalase+Flavourzyme exhibited better antioxidant activities and was further purified. Three novel antioxidant peptides were purified by a series of chromatographic techniques. Sequences of the three peptides were identified as Cys-Ser-Gln-Ala-Pro-Leu-Ala, Tyr-Pro-Lys-Leu-Ala-Pro-Asn-Glu and Tyr-Pro-Gln-Leu-Leu-Pro-Asn-Glu, respectively. Among the three peptides, Cys-Ser-Gln-Ala-Pro-Leu-Ala exhibited good reducing power and excellent scavenging capacities for DPPH radical and superoxide anion radical, with IC50 values of 0.116 and 0.39mg/ml, respectively. The results from our study indicate antioxidant potency of corn protein hydrolysates and peptides separated from corn gluten meal and can provide basic understanding for the application of corn protein hydrolysates as natural antioxidants.

Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

Functional polymorphism in the 5'-UTR of CR2 is associated with susceptibility to nasopharyngeal carcinoma.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a squamous cell cancer endemic in Southern China and Southeast Asia. It has been shown that inflammatory and immune responses during EBV infection contribute to the development of NPC. The complement receptor 2 (CR2) gene plays central roles during inflammatory and immune responses and, therefore, is a good candidate susceptibility gene for NPC. We performed PCR-based sequencing to identify multiple single-nucleotide polymorphisms (SNPs) within the exon regions of the CR2 gene in a Cantonese population. Two SNPs were screened in 528 NPC patients and 408 normal individuals to perform a case-control study matched according to age, gender and residence. Furthermore, we cloned the entire 5'-UTR and entire CR2 promoter into a luciferase report system and compared the luciferase activities between the different allelic constructs. A SNP in the 5'-UTR of CR2 (24 T/C, rs3813946) showed a significant association (P<0.01) with NPC in the Cantonese population studied. The subjects were categorized into 2 age groups: group 1, age ≤45 years and group 2, age >45 years. In group 1, the allelic frequencies of 24 T/C in the patients were significantly different from those of the controls (P=0.0034). The odds ratio (OR=1.81) also indicated a higher risk of NPC in individuals who carried the minor allele C. All constructs exerted allelic differences on luciferase activities, but only the susceptible allele +24C construct showed increased activity. Our findings implicate CR2 as a susceptibility gene for NPC and suggest that enhanced CR2 expression may be involved in the oncogenesis and development of NPC.

[Differential expression of toll-like receptors in chronic suppurative otitis media and cholesteatoma].

OBJECTIVE: To investigate the differential expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and their potential role in the pathogenesis of chronic suppurative otitis media and cholesteatoma. METHODS: Normal canal skin of 30 patients with tympanosclerosis were enrolled as control, 30 cases with chronic suppurative otitis media and 30 patients with cholesteatoma were studied. Real-time PCR, Western blot and Immunohistochemistry were preformed to detect the expression of TLR2/TLR4 in normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma, and the differential expression were analyzed. RESULTS: (1) the mRNA and protein expression of TLR2 and TLR4 were detected in all normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma. (2) Both mRNA and protein level of TLR2/TLR4 in mucosa of chronic suppurative otitis media and cholesteatoma were higher than those in normal canal skin, but lower in cholesteatoma epithelium, there was no significant difference in mucosa of the two otitis media groups. (3) The mRNA and protein expression of TLR2/TLR4 in granulation tissue of chronic suppurative otitis media and cholesteatoma were significant increased when compared with normal canal skin, and TLR2 expression level was higher in granulation tissue of cholesteatoma than in chronic suppurative otitis media. (4) TLR2/TLR4 positive cells mainly infiltrated in granulations, significantly more than in normal skin, while fewer in the epithelium of cholesteatoma. CONCLUSIONS: Differential expression of TLR2 and TLR4 in mucosa suggests middle ear is a TLR2/TLR4 participated functional modulation of the innate immune system and also suggests that they may play a different role in the pathophysiology of chronic otitis media and cholesteatoma.

Genetic polymorphisms of TLR3 are associated with Nasopharyngeal carcinoma risk in Cantonese population.

BACKGROUND: Nasopharyngeal carcinoma is endemic in Southern China, displays a strong relationship with genetic susceptibility and associates with Epstein-Barr virus infection. Toll-like receptor 3 (TLR3) plays an important role in the antivirus response. Therefore, we examined the association between TLR3 gene polymorphisms and NPC susceptibility. METHODS: We performed a case-control study of 434 NPC cases and 512 healthy controls matched on age, sex and residence. Both cases and controls are of Cantonese origin from Southern China. Genetic variants in TLR3 were determined by polymerase chain reaction (PCR)-based DNA direct sequencing and four SNPs were genotyped in all samples. RESULTS: Our results showed that allele C for SNP 829A/C increased NPC risk significantly ((p = 0.0068, OR = 1.49, 95%CI:1.10-2.00). When adjusted for age, gender and VCA-IgA antibody titers, the NPC risk was reduced significantly among individuals who carried the haplotype "ATCT" compared to those who carried the most common haplotype "ACCT" (p = 0.0054, OR = 0.028; 95% CI (0.002-0.341). CONCLUSION: The TLR3 polymorphisms may be relevant to NPC susceptibility in the Cantonese population, although the reduction in NPC risk is modest and the biological mechanism of the observed association merits further investigation.

Energy transfer among chlorophylls in trimeric light-harvesting complex II of Bryopsis corticulans.

A study on energy transfer among chlorophylls (Chls) in the trimeric unit of the major light-harvesting complex II (LHC II) from Bryopsis corriculan, was carried out using time-correlated single photon counting. In the chlorophyll Q region of LHC II, six molecules characterized as Chlb628, Chlb646, Chlb652(654,657), Chla664(666), Chla674(677,680) and Chla682(683) were discriminated according to their absorption spectrum and fluorescence emission spectrum. Then, excited by pulsed light of 628 nm, fluorescence kinetics spectra in the chlorophyll Q region were measured. In accordance with the principles of fluorescence kinetics, these kinetics data were analyzed with a multi-exponential model. Time constants on energy transfer were obtained. An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97 picoseconds (ps), which shows that, before transferring energy to another Chl, the excited Chl might convert energy to vibrations of a lower state with different multiplicity (intersystem crossing). Energy transfer at the level of approximately 10 ps was also obtained, which was interpreted as the excited Chls may go through internal conversion before transferring energy to another Chl. Although with a higher standard deviation, time constants at the femtosecond level can not be entirely excluded, which can be attributed to the ultrafast process of direct energy transfer. Owing to the arrangement and direction of the dipole moment of Chls in LHC II, the probability of these processes is different. The fluorescence lifetimes of Chlb652(654,657), Chla664(666), Chla674(677,680) and Chla682(683) were determined to be 1.44 ns, 1.43 ns, 636 ps and 713 ps, respectively. The percentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in the trimeric unit of LHC II. These results are important for a better understanding of the relationship between the structure and function of LHC II.

Steady state fluorescence spectroscopy of the photosystem II core complex.

Spectroscopic properties within the core complex of photosystem II were investigated by studying the influence of the wavelength of excitation on the fluorescence emission spectrum. At two temperatures, when the core complex of PSII isolated from spinach was excited at six different excitation wavelengths ranging from 436 nm to 520 nm, there is no difference in the maxima of the emission spectra of the core complex, and when the core complex was excited at 480, 489, 495 and 507 nm respectively, fluorescence intensities of maxima decrease with increasing of the absorbance of the beta-carotene molecules at the four excitation wavelengths. The extent of change of the shoulder of the spectra beyond 700 nm depends on the kind of pigment molecule excited. The excitation wavelength can influence the way of energy transfer in the core complex of photosystem II. By Gaussian deconvolution analysis, at least seven groups of chlorophyll a molecules were discovered. They are Chl a(660), Chl a(670), Chl a(680), Chl a(682), Chl a(684), Chl a(687) and Chl a(690).