In Vitro Antioxidant and Anti-Propionibacterium acnes Activities of Cold Water, Hot Water, and Methanol Extracts, and Their Respective Ethyl Acetate Fractions, from Sanguisorba officinalis L. Roots.

Identification of medicinal plants and naturally derived compounds as new natural antioxidant and antibacterial sources for topical acne treatment has long been important. To determine anti-Propionibacterium acnes activity and in vitro antioxidant activities, Sanguisorba officinalis L. root (SOR) was extracted with cold water (CWE), hot water (HWE), and methanol (ME), and each extract was fractionated successively with hexane, ethyl acetate (EA), and butanol to determine whether the activities could be attributed to the total phenolic, flavonoid, terpenoid, and condensed tannin contents. Pearson's correlation coefficients were analyzed between the respective variables. The SOR CWE, HWE, ME, and their respective EA fractions showed anti-P. acnes activity based on the paper disc diffusion method on agar plates, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC). The MIC against P. acnes had a moderate (+) correlation with the total phenolic content, but not with the other measures. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity (SC) had a strong (⁻) correlation with the total phenolic content and a moderate (⁻) correlation with the total flavonoid content. The total antioxidant capacity had a strong (+) correlation with the condensed tannin content. Linoleic acid peroxidation inhibition had a strong (⁻) correlation with the total phenolic content. To elucidate the major active phytochemicals in the CWE-EA, HWE-EA, and ME-EA fractions, high performance liquid chromatography-ultraviolet (HPLC-UV) and ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) were performed. The HPLC-UV analysis showed the presence of nine compounds in common (arjunic acid and/or euscaphic acid, gallic acid, kaempferol, caffeic acid, ferulic acid, tannic acid, and coumarin, quercetin). The UHPLC-QTOF-MS analysis showed the presence of nine compounds in common (gallic acid; caffeic acid; umbelliferone; arjunic acid, euscaphic acid, and/or tormentic acid; pomolic acid; rosamultic acid; and benzoic acid). When standards of the identified phytochemicals were tested against the same bacterium, quercetin, coumarin, and euscaphic acid showed antibacterial activity against P. acnes.

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs).

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1ß and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Generation of a Novel Staphylococcus aureus Ghost Vaccine and Examination of Its Immunogenicity against Virulent Challenge in Rats.

Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of infections in humans and animals. Bacterial ghosts are nonliving, empty cell envelopes and are well represented as novel vaccine candidates. In this study, we examined the immunogenicity and protective efficacy of S. aureus ghosts (SAGs) against a virulent challenge in rats. Nonliving SAGs were generated by using the MIC of sodium hydroxide. The formation of a transmembrane lysis tunnel structure in SAGs was visualized by scanning electron microscopy. To investigate these SAGs as a vaccine candidate, rats were divided into four groups, A (nonimmunized control), B (orally immunized), C (subcutaneously immunized), and D (intravenously immunized). The IgG antibody responses were significantly stronger in the SAG-immunized groups than in the nonimmunized control group (P < 0.05). Moreover, a significant increase in the populations of CD4(+) and CD8(+) T cells was observed in all three immunized groups (P < 0.05). We also found that serum bactericidal antibodies were significantly elicited in the SAG-immunized groups (P < 0.05). Most importantly, the bacterial loads in the immunized groups were significantly lower than those in the nonimmunized control group (P < 0.01). These results suggest that immunization with SAGs induces immune responses and provides protection against a virulent S. aureus challenge.

PCR-RFLP-based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea.

A polymerase chain reaction (PCR) using two sets of primers designed from published Tomato yellow leaf curl virus (TYLCV) genomes was developed to distinguish from the TYLCV-IL groups. The specificity of the two sets of primers was proven by testing against control TYLCV genomes and the symptomatic leaves of 34 different tomato cultivars naturally infected with TYLCV in greenhouses. One set for TYLCV-IL strain-specific primers (TYLCV-UNI-F and TYLCV-UNI-R) amplified full-length genome fragments from all the 34 tomato cultivars. Another set for TYLCV-IL group-II strain-specific primers (TYLCV-GPII-F and TYLCV-GPII-R) amplified target DNA fragments from only 9 tomato cultivars. Digestion by BglII and EcoRV of the PCR amplicons produced restriction fragment length polymorphism pattern that distinguished the TYLCV-IL group-I with two fragments from the TYLCV-IL group-II with no digested fragment. PCR coupled with BglII and EcoRV digestion confirmed that the 9 tomato cultivars were infected with the TYLCV-IL group-II and the remained 25 tomato cultivars were infected with the TYLCV-IL group-I.