Antibacterial activity and synergistic antibiotic mechanism of trialdehyde phloroglucinol against methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a critical global concern. Identifying new anti-S. aureus agents or therapeutic strategies are urgently needed to treat S. aureus infection. The present study investigated the antibacterial activity of 16 phenolic compounds against MRSA, four of which exhibited antibacterial activity. Their antibacterial activities increased in a dose-dependent manner but showed different responses with the extension of treatment time. Trialdehyde phloroglucinol (TPG) and 2-nitrophloroglucinol (NPG) maintained stable antibacterial activity; however, that of dichlorophenol and myricetin decreased rapidly over 24 hr of treatment. Checkerboard and time-kill assays indicated that TPG and NPG exhibited strong synergistic antibacterial activities with penicillin or bacitracin. Microscopic observation and membrane integrity analysis showed that the combination of TPG and penicillin destroyed the MRSA cell membrane, resulting in the leakage of intracellular biomacromolecules, marked changes in surface zeta potential, and the collapse of membrane potential. Moreover, the combination significantly decreased penicillinase activity and penicillin-binding protein 2a mRNA expression, inhibiting MRSA growth. Taken together, these results demonstrated that the combination of the phloroglucinol derivative TPG and penicillin has significant synergistic anti-MRSA activity and can serve as a potential therapeutic strategy to treat MRSA infections.

Aesculetin exhibited anti-inflammatory activities through inhibiting NF-кB and MAPKs pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Aesculetin (6,7-dihydroxy-2H-1-benzopyran-2-one) has been reported to exhibit potent anti-inflammatory property both in vitro and in vivo. AIMS OF THIS STUDY: In this study, we evaluated the anti-inflammatory effect and investigated underlying molecular mechanisms of aesculetin in LPS-induced RAW264.7 macrophages and DSS-induced colitis. MATERIALS AND METHODS: In this study, the production of NO, TNF-α, and IL-6 were measured to identify the aesculetin with potent anti-inflammatory effect. Then, the underlying anti-inflammatory mechanisms were explored by western blotting in LPS-induced cells. Next, we verify the anti-inflammatory potential of aesculetin in DSS-induced colitis in vivo. The clinical symptoms of colitis, including weight loss, DAI, colon length and MPO activity, and the secretion of TNF-α and IL-6 were evaluated. Finally, Western blot analysis was applied to further investigate underlying mechanism in DSS-induced colitis model. RESULTS: Our studies showed that aesculetin exhibited anti-inflammatory potential by inhibiting NO, TNF-α, and IL-6 production and reducing iNOS and NLRP3 expression in LPS-induced RAW264.7 cells. Mechanically, we found that aesculetin significantly inhibited LPS-induced activation of NF-κB and MAPKs signaling pathways. In DSS-induced mouse model, the colitis-related symptoms were relieved by treatment with aesculetin. Besides, aesculetin also inhibited the secretion of TNF-α and IL-6, and the activation of NF-κB and MAPKs signaling pathways in DSS-induced colitis. CONCLUSIONS: The anti-inflammatory effect of aesculetin was connected with its inhibition on the activation of NF-κB and MAPKs signaling pathways both in vitro and in vivo. Therefore, aesculetin was expected to be developed as an anti-inflammatory drug.

Semisynthetic pleuromutilin antimicrobials with therapeutic potential against methicillin-resistant Staphylococcus aureus by targeting 50S ribosomal subunit.

A series of pleuromutilin analogs with a substituted 1,2,4-triazole were designed, synthesized and assessed for their in vitro and in vivo antibacterial activity. Initially, the MIC of the synthesized derivatives against five strains of Staphylococcus aureus (MRSA ATCC 43300, S. aureus ATCC 29213, clinical isolation of S. aureus AD3, S. aureus 144 and S. aureus SA17) were tested by the broth dilution method. Compounds 30a, 31b and 32a were the most active antibacterial agents in vitro against MRSA (MIC = 0.0625 µg/mL). The results of the time-kill curves showed that compounds 30a and 32a could reduce the amount of MRSA in vitro quickly (-7.70 log10 CFU/mL and -7.10 log10 CFU/mL reduction). In the experiment to further evaluate the in vivo antibacterial activity of compound 30a against MRSA, compound 30a (-1.71 log10 CFU/g) was effective in reducing MRSA load in thigh infected mice. Compound 30a (survival rate was 50%) displayed superior in vivo efficacy to that of tiamulin (survival rate was 30%) in the mouse systemic model. The results of further pharmacokinetic studies on compound 30a showed that the half-life (t1/2), clearance rate (Cl) and the area under the plasma concentration time curve (AUC0→∞) of compound 30a were 0.37 h, 5.43 L/h/kg and 1.84 µg h/mL, respectively. After affinity measurement by surface plasmon resonance (SPR), compound 30a exhibited high affinity with the 50S ribosome, with KD value of 1.95 × 10-6 M. Furthermore, the results of molecular docking studies revealed that compound 30a was successfully localized inside the binding pocket of 50S ribosomal subunit (ΔGb = -9.40 kcal/mol). Meanwhile, most of these compounds had no significant inhibitory effect on RAW 264.7 cells and 16HBE cells at the concentration of 8 µg/mL. The obtained outcomes showed that compound 30a could be utilized as an encouraging perspective in the development of a new therapeutic candidate for bacterial infection.

Design, synthesis and biological evaluation of pleuromutilin-Schiff base hybrids as potent anti-MRSA agents in vitro and in vivo.

A series of pleuromutilin derivatives with 1,2,4-triazole-3-substituted Schiff base structure were designed and synthesized under mild conditions. The in vitro antibacterial activities of the synthesized derivatives against 4 strains of Staphylococcus aureus (MRSA ATCC 43300, S.aureus ATCC 29213, S.aureus 144 and S.aureus AD3) and 1 strain of E. coli (ATCC 25922) were evaluated by the broth dilution method. Among these derivatives, compound 60 exhibited superior in vitro antibacterial effect against MRSA (MIC = 0.25 µg/mL) than tiamulin (MIC = 0.5 µg/mL), and compound 60 (-2.28 log10 CFU/mL) also displayed superior in vivo antibacterial efficacy than tiamulin (-1.40 log10 CFU/mL) in reducing MRSA load in the mouse thigh infection model. The time-kill study and the post-antibiotic effect study indicated that compound 60 showed a faster bactericidal kinetic and longer PAE time (exposure to 2 × MIC and 4 × MIC for 2 h, the PAE was 4.06 and 4.27 h) against MRSA compared with tiamulin (exposure to 2 × MIC and 4 × MIC for 2 h, the PAE was 1.72 and 2.14 h). Meanwhile, most of these compounds had no significant inhibitory effect on RAW 264.7 cells and HepG2 cells at the concentration of 4 µg/mL. Additionally, the development of resistance study showed that MRSA did not easily develop resistance against compound 60 compared with tiamulin after induction for 8 passages.

Efficiency comparison of apigenin-7-O-glucoside and trolox in antioxidative stress and anti-inflammatory properties.

OBJECTIVES: Chamomile has long been used as a medicinal plant due to its antioxidative and anti-inflammatory activity. Apigenin-7-O-glucoside (AG) is one of the major ethanol extract components from chamomile; however, the underlying mechanism remains unclear. METHODS: In this study, the antioxidant potential and the anti-inflammatory activities of AG were analysed and compared with those of trolox. We demonstrate the protective effects of AG on free radical-induced oxidative damage of DNA, proteins and erythrocytes. Flow cytometry assay was used to detect ROS production. Additionally, the expression of anti-oxidation-related and inflammation-related factors was detected by ELISA and Western blotting, respectively. KEY FINDINGS: AG and trolox showed different efficiency as antioxidant in different experimental systems. AG had similar effect as trolox to inhibit H2 O2 -induced ROS production in RAW264.7 cells, while exerted stronger inhibition against free radical-induced oxidative damage on erythrocytes than trolox. Interestingly, compared with trolox, AG also had stronger inhibitory effect on LPS-induced NF-κB/NLRP3/caspase-1 signalling in RAW246.7 cells. CONCLUSIONS: These results suggest the potential of AG as a pharmaceutical drug for anti-oxidation and anti-inflammation, and the combined usage of AG and trolox might promote its efficacy. Our findings will provide new insights into the development of new drugs with antioxidative and anti-inflammatory functions.

Contribution of Borneolum syntheticum to the Intervention Effect of Liuwei Dihuang Pill () on Experimental Retinal Degeneration.

OBJECTIVE: To observe the contribution of Borneolum syntheticum to the intervention effect of Liuwei Dihuang Pill (, LDP) on experimental retinal degeneration, and initially investigate the mechanism of Borneolum syntheticum as meridian-lead-in drug. METHODS: A total of 180 sodium iodateinduced retinital degeneration rats were randomly divided into three groups, including distilled water group, LDP group, and LDP+Borneolum syntheticum (LDP+BS) group. Twenty normal rats were fed regularly without any treatment as normal control. On day 7 and 14 after treatment, histopathological study and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test were performed to evaluate the retinopathy. Claudin-5 expression at blood-retina barrier (BRB) was detected by Western blot at different time points from 0.5 to 8 h after gavage. RESULTS: On day 7 and 14 after treatment, the retinal lesion grades were significantly different among the three groups (P<0.05). The grade in the LDP+BS group was significantly less than the LDP and distilled water groups (both P<0.05), no significant difference was observed between the LDP and distilled water groups (P>0.05). The apoptosis rates in the LDP+BS group was significantly less than the distilled water and LDP groups (both P<0.05), while there was no significant difference between LDP and distilled water groups (P>0.05). Expression of claudin-5 in LDP+BS group was significantly less than the other two groups at 0.5, 1 and 2 h after gavage (P<0.05). There was no apparent difference among the three groups at 4 and 8 h after gavage (P>0.05). CONCLUSION: Borneolum syntheticum could strengthen the effect of LDP on experimental retinal degeneration, indicated that Borneolum syntheticum might play the role of meridian-lead-in drug in the formula. The mechanism may be due to Borneolum syntheticum could promote the physiologically openness of bloodretina barrier through transiently affecting the expression of claudin-5.

Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated. RESULTS AND DISCUSSION: Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE2 but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6. CONCLUSION: These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.

Identification and determination of the saikosaponins in Radix bupleuri by accelerated solvent extraction combined with rapid-resolution LC-MS.

A method based on accelerated solvent extraction combined with rapid-resolution LC-MS for efficient extraction, rapid separation, online identification and accurate determination of the saikosaponins (SSs) in Radix bupleuri (RB) was developed. The RB samples were extracted by accelerated solvent extraction using 70% aqueous ethanol v/v as solvent, at a temperature of 120 degrees C and pressure of 100 bar, with 10 min of static extraction time and three extraction cycles. Rapid-resolution LC separation was performed by using a C(18) column at gradient elution of water (containing 0.5% formic acid) and acetonitrile, and the major constituents were well separated within 20 min. A TOF-MS and an IT-MS were used for online identification of the major constituents, and 27 SSs were identified or tentatively identified. Five major bioactive SSs (SSa, SSc, SSd, 6''-O-acetyl-SSa and 6''-O-acetyl-SSd) with obvious peak areas and good resolution were chosen as benchmark substances, and a triple quadrupole MS operating in multiple-reaction monitoring mode was used for their quantitative analysis. A total of 16 RB samples from different regions of China were analyzed. The results indicated that the method was rapid, efficient, accurate and suitable for use in the quality control of RB.

[Influence of Chinese drugs for cooling blood and dissolving stasis in different concentrations on morphology of choroidal neovascularization].

OBJECTIVE: To evaluate the influence of Chinese drugs for cooling blood and dissolving stasis (CBDS) in different concentrations on morphology of krypton laser induced choroidal neovascularization (CNV) in brown Norway (BN) rats. METHODS: Forty-eight rats received laser irradiation (659 nm) on fundus of one eye (power 360 mW, spot diameter 50 microm, time 0.05 s). They were divided into four groups equally: the control group (A) treated with normal saline, and the three CBDS groups treated respectively with high (B, 5.0 g/kg), median (C, 2.5 g/kg) and low (D, 1.25 g/kg) dosage of CBDS, twice every day via gastric perfusion for 21 successive days. Fundus fluorescein angiography (FFA) on 4 selected rats in each group was performed at the 7th, 14th and 21st day after photocoagulation, and histopathologic examination using light microscope with immuno-histochemical stain was conducted on them as well. RESULTS: FFA showed that CNV was firstly appeared on day 7 after photocoagulation, in Group A, it expanded gradually and reached the peak on day 21, but showed no significant expansion in the three CBDS groups. The fluorescein leakage in Group C (52343.13 +/- 12973.92 dots) and D (66252.78 +/- 20659.71 dots) was significantly less than that in Group A (91457.19 +/- 29309.11 dots) and B (95973.40 +/- 53950.43 dots) on day 21, all showing statistical significance (P<0.05). The variation of CNV in thickness showed that in Group A it increased gradually from day 7 and reached the peak on day 21 (55.3383 +/- 8.5036 microm); but in the CBDS groups, the peak was reached on day 14, then became thinned gradually, on day 21, it was 40.0913 +/- 13.3448 microm in Group B, 38.8473 +/- 7.9862 microm in Group C and 38.9372 +/- 5.1728 microm in Group D, all thinner than that in Group A significantly (P<0.05). CONCLUSION: CDBS can effectively suppress the krypton laser induced CNV proliferation and prevent the CNV leakage in BN rats.

Protective effect of icariin on DNA against radical-induced oxidative damage.

Icariin (2-(4'-methoxylphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(3'-methyl-2-butylenyl)-4-chromanone) is a flavonoid with a rhamnose as ligand. It is the major component in Herba epimedii, widely used for the treatment of atherosclerosis and neuropathy in Chinese traditional medicine, and its antioxidative property has attracted much scientific interest. The major objective of this work is to determine the antioxidative effect of icariin against oxidative DNA damage induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The oxidative damage of DNA was followed by measuring the formation of carbonyl compounds that can react with thiobarbituric acid (TBA) to form thiobarbituric acid reactive substance (TBARS). We found that icariin protects DNA against AAPH-induced oxidative damage in a concentration-dependent manner, although it does not affect the rate of AAPH-induced DNA damage. This result indicates that icariin is a concentration-dependent chemopreventor in protecting DNA against radical-induced damage.