Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Latifolicinin A from a Fermented Soymilk Product and the Structure-Activity Relationship of Synthetic Analogues as Inhibitors of Breast Cancer Cell Growth.

The functional components in soymilk may vary depending upon the fermentation process. A fermented soymilk product (FSP) obtained by incubation with the microorganisms of intestinal microflora was found to reduce the risk of breast cancer. Guided by the inhibitory activities against breast cancer cells, two cytotoxic compounds, daidzein and (S)-latifolicinin A, were isolated from the FSP by repetitive extraction and chromatography. Latifolicinin A is the n-butyl ester of ß-(4-hydroxyphenyl)lactic acid (HPLA). A series of the ester and amide derivatives of (S)-HPLA and L-tyrosine were synthesized for evaluation of their cytotoxic activities. In comparison, (S)-HPLA derivatives exhibited equal or superior inhibitory activities to their L-tyrosine counterparts, and (S)-HPLA amides showed better cytotoxic activities than their corresponding esters. In particular, (S)-HPLA farnesyl amide was active to triple-negative MDA-MB-231 breast cancer cells (IC50 = 27 µM) and 10-fold less toxic to Detroit-551 normal cells.

Chemical constituents of Plectranthus amboinicus and the synthetic analogs possessing anti-inflammatory activity.

This study demonstrates that compounds 1-4 from an extract of Plectranthus amboinicus inhibit the binding of AP-1 to its consensus DNA sequence. Thymoquinone (5) was further identified as a nonpolar ingredient from the hexane extract of P. amboinicus to suppress the expression of lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α). We then synthesized 2-alkylidenyl-4-cyclopentene-1,3-diones as the designed biomimetics of thymoquinone, and found that compounds 8a, 8b and 8d were more potent TNF-α inhibitors.

The immunologically active oligosaccharides isolated from wheatgrass modulate monocytes via Toll-like receptor-2 signaling.

Wheatgrass is one of the most widely used health foods, but its functional components and mechanisms remain unexplored. Herein, wheatgrass-derived oligosaccharides (WG-PS3) were isolated and found to induce CD69 and Th1 cytokine expression in human peripheral blood mononuclear cells. In particular, WG-PS3 directly activated the purified monocytes by inducing the expression of CD69, CD80, CD86, IL-12, and TNF-α but affected NK and T cells only in the presence of monocytes. After further purification and structural analysis, maltoheptaose was identified from WG-PS3 as an immunomodulator. Maltoheptaose activated monocytes via Toll-like receptor 2 (TLR-2) signaling, as discovered by pretreatment of blocking antibodies against Toll-like receptors (TLRs) and also determined by click chemistry. This study is the first to reveal the immunostimulatory component of wheatgrass with well defined molecular structures and mechanisms.

HA-pseudotyped retroviral vectors for influenza antagonist screening.

Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.

Effects of rhubarb extract on radiation induced lung toxicity via decreasing transforming growth factor-beta-1 and interleukin-6 in lung cancer patients treated with radiotherapy.

BACKGROUND: Radiation induced lung toxicity (RILT) is the main adverse effect in the radiation therapy of lung cancer. However, the optimal management of RILT has not been defined. In this paper, we investigated the effects of rhubarb extract on RILT, pulmonary function (PF), transforming growth factor-beta-1 (TGF-beta1), and interleukin-6 (IL-6) in lung cancer patients treated with radiotherapy. PATIENTS AND METHODS: We conducted a randomized, double-blind, placebo-controlled trial. Eighty consecutive patients were randomly enrolled into two groups: trial group and control group. The trial group received three-dimensional conformal radiation therapy (3D-CRT) plus rhubarb (at a dose of 20 mg kg(-1) once a day) for 6 weeks. The control group received 3D-CRT plus a placebo containing starch for 6 weeks. Plasma TGF-beta1 and serum IL-6 were measured in all patients before, every 2 weeks during, and at 6 weeks after the completion of the treatment. RILT and PF were evaluated at 6 weeks and 6 months after the end of the treatment, respectively. The differences of TGF-beta1, IL-6, RILT, and PF between the two groups were analysed. RESULTS: The incidence of RILT in the trial group was significantly lower than that in the control group at 6 weeks and 6 months after treatment (32.4% versus 56.7% at week 6, and 27.0% versus 52.8% at month 6, both P<0.05). The plasma TGF-beta1 levels in the trial group were significantly lower than that in the control group during and after the treatment (P<0.05 or 0.01, respectively). The serum IL-6 levels in the trial group were significantly lower than that in the control group during the treatment (all P<0.01). The forced vital capacity (FVC), forced expiratory volume at 1s (FEV1) at 6 weeks and the diffusion capacity for carbon monoxide (DLCO) at 6 months in the trial group were significantly improved compared to the control group (P<0.05 or 0.01, respectively). CONCLUSIONS: The rhubarb extract significantly attenuated RILT and improved PF, probably by decreasing the level of TGF-beta1 and IL-6. These results may be of value for the prophylaxis of RILT, but the exact mechanisms underlying these prophylactic effects remain to be further explored.

Treatment with rhubarb improves brachial artery endothelial function in patients with atherosclerosis: a randomized, double-blind, placebo-controlled clinical trial.

Rhubarb has been used to decrease plasma cholesterol levels and reduce vascular endothelial cellular damage in recent years. However, it is not known whether reported lipid-lowering effects are associated with the improvement of endothelial function. This work aimed to elucidate the therapeutic effects of rhubarb on serum lipids and brachial artery endothelial function, as well as to investigate the relationship between them. One hundred and three patients with atherosclerosis were randomly divided into two groups: patients in the control and the trial group received a placebo and rhubarb, respectively, in addition to the 6 month baseline therapy. Serum lipids and brachial artery endothelial functions were measured in all patients before and after treatment. A total of 83 patients completed the 6-month follow-up protocol. Serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the trial group decreased significantly and LDL-C was significantly lower than that in the control group. Flow-mediated dilation (FMD) in the trial group was significantly higher after treatment in comparison to the baseline and to the control group. Improvement in FMD correlated with the decreased magnitude of TC and LDL-C levels. The results obtained appeared to confirm that rhubarb significantly improves endothelial function mainly due to lipid-lowering effects in patients with atherosclerosis.

Effects of Quyu Xiaoban capsules on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris.

OBJECTIVES: To investigate the effects of the Traditional Chinese Medicine (TCM) Quyu Xiaoban capsules (QYXB) on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris (UAP) and phlegm and blood stasis syndrome. DESIGN: Ninety (90) UAP patients were randomly divided into two groups: the control group received a loading dose of 300 mg aspirin and a maintenance dose of 100 mg of aspirin plus baseline therapy for 4 weeks, and the trial group received the same doses of aspirin and baseline therapy plus QYXB for 4 weeks. The severity of anginal attacks, alterations of TCM symptoms and signs, and electrocardiographic (ECG) changes were observed in all patients before and after treatment. Plasma platelet aggregation (PAG) rate and P-selectin level were measured in all patients at baseline and at the end of the fourth week. RESULTS: After treatment for 4 weeks, both group of patients showed improvement in the severity of angina pectoris and TCM symptoms and signs, and there was a significant difference of the total effective rate in clinical improvement between the two groups, whereas no difference of the total effective rate in ECG improvement between the two groups was found. Compared with the baseline level, PAG rate in both groups decreased significantly at the end of the fourth week (63.74 +/- 11.18% versus 55.69 +/- 10.40 % in the control group, and 63.83 +/- 12.70% versus 50.04 +/- 8.91% in the trial group). Similar changes of P-selectin levels were observed in both groups (9.40 +/- 1.25 ng/mL versus 8.90 +/- 1.34 ng/mL in the control group and 9.56 +/- 1.16 ng/mL versus 7.80 +/- 0.98 ng/mL in the trial group). However, both PAG rate and P-selectin level decreased to a greater extent in the trial group than in the control group after treatment, and the difference between treatment was significant (both p<0.05). Nevertheless, these biochemical changes were too small to explain fully the beneficial clinical outcomes achieved by QYXB capsules. CONCLUSIONS: On the background of baseline and aspirin therapy, QYXB capsules significantly attenuated anginal attacks and improved TCM symptoms and signs in patients with UAP, and the exact mechanisms underlying these therapeutic effects remain to be explored.

Effects of Quyu xiaoban capsules on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris.

OBJECTIVES: To investigate the effects of the Traditional Chinese Medicine (TCM) Quyu Xiaoban capsules (QYXB) on clinical outcomes and platelet activation and aggregation in patients with unstable angina pectoris (UAP) and phlegm and blood stasis syndrome. DESIGN: Ninety patients with UAP were randomly divided into two groups: a control group that received a loading dose of 300 mg aspirin and a maintenance dose of 100 mg of aspirin plus baseline therapy for 4 weeks, and a trial group that received the same doses of aspirin and baseline therapy plus QYXB for 4 weeks. The severity of anginal attacks, alterations of TCM symptoms and signs, and electrocardiographic (ECG) changes were assessed in all patients before and after treatment. The plasma platelet aggregation (PAG) rate and P-selectin level were measured in all patients at baseline and at the end of the fourth week of treatment. RESULTS: After treatment for 4 weeks, both group of patients showed improvement in the severity of angina pectoris and TCM symptoms and signs, but there was a significant difference in the two groups' rates of clinical improvement, whereas the rate of ECG improvement of the two groups showed no difference. As compared with the baseline value, the PAG rate in both groups decreased significantly at the end of the fourth week (63.74 +/- 11.18% vs. 55.69 +/- 10.40% in the control group, and 63.83 +/- 12.70% vs. 50.04 +/- 8.91% in the trial group). Similar changes in P-selectin levels were observed in the two groups (9.40 +/- 1.25 ng/mL vs. 8.90 +/- 1.34 ng/mL in the control group, and 9.56 +/- 1.16 ng/mL vs. 7.80 +/- 0.98 ng/mL in the trial group). However, both the PAG rate and P-selectin level decreased to a greater extent in the trial group than in the control group after treatment, and the difference between the two groups was significant (both p < 0.05). Nevertheless, these biochemical changes were too small to fully explain the beneficial clinical outcomes achieved with QYXB capsules. CONCLUSIONS: In comparison with both the respective baseline values and with aspirin therapy, QYXB capsules significantly attenuated anginal attacks and improved TCM symptoms and signs in patients with UAP. The exact mechanisms underlying these therapeutic effects remain to be explored.

Identification of histone demethylases in Saccharomyces cerevisiae.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

Effects of Quyu Xiaoban Capsule on vascular endothelial function in patients with atherosclerosis.

OBJECTIVE: To evaluate the therapeutic effects of Quyu Xiaoban Capsule (QYXBC) on endothelial dependent vascular relaxation (EDVR) function in patients with atherosclerosis (AS) with ultrasonic technique. METHODS: Tested were the endothelial function and blood lipids of 42 patients with AS in the treated group and 30 healthy volunteers in the control group. And re-examination of these parameters was carried out on the AS patients after they had been treated with QYXBC for 10 months. RESULTS: Before treatment, the reactive hyperemia induced changes in artery diameter in the treated group was significantly lower than that in the control group (P < 0.01), while insignificant difference was found between the two groups in response to nitroglycerin. In the treated group after treatment, with D%-R improved significantly (P < 0.01), total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) decreased by 16.3%, 5.6%, 10.2% respectively and high-density lipoprotein cholesterol (HDL-C) increased by 7.5%. EDVR was correlated negatively with the serum TC, LDL-C concentrations and the baseline brachial diameter (D(0)) (r = -0.41, -0.66, -0.59, respectively, all P < 0.01), but correlated positively with HDL-C (r = 0.62, P < 0.05). The ameliorative extent of EDVR was correlated positively to the decreased magnitude of TC and LDL-C concentrations (r = 0.67, 0.59, both P < 0.01). CONCLUSION: QYXBC can lower the level of blood lipids and improve significantly EDVR function.

[Clinical study of acoustic densitometry technique in detecting atherosclerotic plaque].

OBJECTIVE: To investigate the effect of Quyu Xiaoban Capsule (QYXB) on the regressive treatment of atherosclerosis (AS) with acoustic densitometry (AD) technique. METHODS: Eighty patients with AS were randomly divided into two groups, trial group was treated with QYXB and conventional medicine, and control group was treated with conventional medicine alone. Normal arterial wall and different types of atherosclerotic plaques were detected with AD technique before treatment and 10 months later. RESULTS: The corrected averages in intimal echo intensity (AIIc%) were elevated in both groups but without significant difference, AIIc% of fatty plaques were increased in both groups and the value after treatment was significantly higher than that of pre-treatment in the trial group (68.12 +/- 5.54 vs 61.43 +/- 5.37, P < 0.05). The increment rate of AIIc% in trial group was significantly higher than that in control group (10.9 +/- 5.1% vs 2.5 +/- 5.5%, P < 0.05). CONCLUSION: QYXB can stabilize the atherosclerotic plaque by increasing its acoustic density. Acoustic densitometry technique can differentiate the different histological plaques and monitor the histological changes of plaques during treatment.

[Clinical study on effect of quyu xiaoban capsule on regression of atherosclerosis].

OBJECTIVE: To study the effect of Quyu Xiaoban capsule (QXC) on regression and stabilization of atherosclerotic plaque with high-frequency ultrasound technique. METHODS: Eighty patients with atherosclerosis (AS) were randomly divided into the treated group and the control group, level of blood lipids was measured, and intima-media thickness (IMT) and corrected value of image average echo intensity (AIIc%) were determined by ultrasound technique at the beginning of experiment and after being treated for six months. RESULTS: The levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) were significantly decreased in the treated group after treatment (68.12 +/- 5.54 vs 61.43 +/- 5.37, P<0.05). The AIIc% of fatty atherosclerotic plaque was significantly increased after treatment (68.12 +/- 5.54 vs 61.43 +/- 5.37), and the change rate of AIIc% in the treated group was significantly different to that in the control group (10.9 +/- 5.1% vs 2.5 +/- 5.5%, P < 0.05). CONCLUSION: QXC can significantly lower the blood lipids level, delay the progress and enhance the stability of atherosclerotic plaque.

Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses.

Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.