Quantification of the Ability of Natural Products to Prevent Herpes Virus Infection.

Background: Herpes simplex virus (HSV) is usually dormant and becomes apparent when body conditions decline. We investigated the anti-HSV activity of various natural and synthetic compounds for future clinical application. Methods: Mock- and HSV-infected Vero cells were treated for three days with various concentrations of samples. For short exposure, 100-fold concentrated virus were preincubated for 3 min with samples, diluted to normal multiplicity of infection (MOI), before the addition to the cells. Anti-HSV activity was evaluated by the chemotherapy index. Results: Alkaline extracts of the leaves of Sasa sp. (SE) and pine cone (PCE) showed higher anti-HSV activity than 20 Japanese traditional herb medicines (Kampo formulas), four popular polyphenols, and 119 chromone-related compounds. Exposure of HSV to SE or PCE for 3 min almost completely eliminated the infectivity of HSV, whereas much longer exposure time was required for Kakkonto, the most active Kampo formulae. Anti-HSV activity of PCE and Kakkonto could be detected only when they were dissolved by alkaline solution (pH 8.0), but not by neutral buffer (pH 7.4). Anti-HSV activity of SE and povidone iodine was stable if they were diluted with neutral buffer. Conclusions: The present study suggests the applicability of SE and PCE for treatment of oral HSV and possibly other viruses.

Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics.

Yucca schidigera is mainly distributed in southwestern US and the northern desert of Mexico. Its extract is widely used as a food additive for its antimicrobial activity. However, this antimicrobial activity is subject to significant variability across production lots. Yucca extracts are natural products and their composition is affected by their cultivation area and weather. Manufacturer deal with natural products such as food additives pay particularly close attention to quality control. In the present study, NMR metabolomics methods were used to screen the antimicrobial activity of yucca extracts. Yucca extracts were subjected to principal component analysis (PCA) and categorized on a score plot of their 1H NMR spectral data according to their antimicrobial activity. Furthermore, hierarchical cluster analysis (HCA) was also used to classify yucca extracts based on their antimicrobial activity. Classification using PCA and HCA was dependent upon saponin content, particularly that of schidigera-saponin A1 and D1, which was further confirmed by HPLC analysis of the yucca extracts. We demonstrated that NMR-based metabolomics is a potentially useful tool to use in combination with conventional quality control methods for yucca extracts used as food additives. We envisage this method as tool for initially screening the extracts prior to carrying out the officially recommended quality control tests.

Antiviral and Antitumor Activity of Licorice Root Extracts.

BACKGROUND: In the search for anti-viral and antitumor substances from natural resources, antiviral and antitumor activities of licorice root extract and purified ingredients were investigated. MATERIALS AND METHODS: Viability of cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antiviral activity was quantified by the selectivity index, defined as the ratio of the 50% cytotoxic concentration (CC50) to the 50% effective concentration against human immunodeficiency virus (HIV) or herpes simplex virus (HSV)-infected cells (EC50). The tumor specificity was calculated by the ratio of CC50 against human normal oral cells to that against human oral squamous cell carcinoma cell lines. Licorice flavonoids and lower molecular polyphenols were subjected to quantitative structure-activity relationship analysis. RESULTS: Alkaline extract of licorice root had higher anti-HIV activity than did water extracts, confirming our previous reports. On the other hand, water extract, especially the flavonoid-rich fraction, had higher anti-HSV activity than did the alkaline extract. The flavonoid-rich fraction was more cytotoxic against human oral squamous cell carcinoma cell lines compared to normal oral cells, suggesting their tumor-specific cytotoxicity. CONCLUSION: The present study suggests that water and alkaline extracts of licorice root exert different mechanisms of actions against these two viruses. Physicochemical properties, rather than the category of compounds, may be important in determining their anti-HSV activity.

Efficient utilization of licorice root by alkaline extraction.

Compared to studies of water extracts of plants, those utilising alkaline extracts are limited. Both water and alkaline extracts from licorice root were compared regarding their biological activities. Licorice root was successively extracted first with water or alkaline solution (pH 9 or 12), and the alkaline (pH 12.0) extract was further separated into 50% ethanol-soluble and -insoluble fractions. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone using human recombinant CYP3A4. Radical intensity of superoxide and hydroxyl radicals was determined by electron spin resonance spectroscopy. Alkaline extraction yielded slightly higher amounts of dried materials compared to water extraction. Alkaline extract showed higher anti-HIV and antibacterial activities, and similar magnitudes of CYP3A4 inhibitory and superoxide and hydroxyl radical-scavenging activities, compared to water extract. When alkaline extract was fractionated by 50% ethanol, anti-HIV activity was recovered from the insoluble fraction representing approximately 3% of the alkaline extract, whereas antibacterial activity was concentrated in the soluble fraction rich in glycyrrhizid acid, flavanones and chalcones. All extracts and sub-fractions led to bimodal hormetic dose-response (maximum hormetic response=238%) on the bacterial growth. The present study demonstrated the superiority of alkaline extraction over water extraction for preparing anti-HIV and antibacterial agents at higher yield from licorice root.

Evaluation of cytotoxiciy and tumor-specificity of licorice flavonoids based on chemical structure.

BACKGROUND: The mechanism of cytotoxicity induction by flavonoids has been studied by many investigators, but their tumor specificity is not clear. To address this point, 10 licorice flavonoids were subjected to quantitative structure-activity relationship (QASR) analysis with cytotoxicity assay with four human oral carcinoma and three normal cell lines. MATERIALS AND METHODS: Cytotoxicity was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. Physico-chemical, structural, and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method. RESULTS: Licurazid and isoliquiritigenin had the highest cytotoxicity against tumor cells, and liquiritin, isoliquiritin and licurazid had the highest tumor specificity, suggesting an antitumor potential for licurazid. Chalcones had slightly higher cytotoxicity and tumor specificity than flavanones. The number of sugar units in the molecule was somewhat negatively-correlated with cytotoxicity, but not with tumor specificity. Parameters that reflect the three-dimensional structure, molecular volume and number of phenolic OH groups were significantly correlated with cytotoxicity, but not with tumor specificity. On the other hand, solvation energy was significantly correlated with tumor specificity, but not with cytotoxicity. CONCLUSION: These physicochemical descriptors may be useful to estimate cytotoxicity or tumor specificity of structurally-related compounds to these licorice flavonoids.

Anti-UV activity of lignin-carbohydrate complex and related compounds.

BACKGROUND: We recently reported that an alkaline extract of the leaves of Sasa senanensis Rehder (SE) and Lentinus edodes mycelia extract (LEM), exhibiting lignin-carbohydrate complex (LCC)-like activity, protected cells from UV-induced injury (referred to as anti-UV activity). We investigated whether LCC is the major active components responsible for anti-UV activity. MATERIALS AND METHODS: Human oral squamous cell carcinoma HSC-2 cells were exposed to short UV irradiation in phosphate-buffered saline, containing different concentrations of LCC. After culturing for 48 h in fresh culture medium, the viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. From the dose-response curve, the 50% cytotoxic concentration (CC(50)) and the concentration that increased the viability of the UV-irradiated cells to 50% of the control value (EC(50)) were determined. The selectivity index (SI) was determined by the following equation: SI=CC(50)/EC(50). RESULTS: LCCs (Fr. VI) of pine cones and seed shell, and sulfated LCC exhibited relatively high anti-UV activity (SI=7.1-38), compared with that of SE and LEM. LCCs with lower lignin content (Fr. VII) exhibited anti-UV activity, approximately one half that of Fr. VI. However, polysaccharides (laminarin, pullulan, dextran) introduced with dimethylaminoethyl- or sulfate groups with different substitution ratios were totally inactive (SI<1). The introduction of a sulfate group to LCC did not enhance the anti-UV activity of LCC. Sodium ascorbate and vanillin were the most active (SI=65), whereas gallic acid (SI=5), epigallocatechin gallate (SI=2.6), ar-trumeron (SI<1), and turmeric extract (SI<1) were much less active. CONCLUSION: The prominent anti-UV activity of SE and LEM seems to be generated by LCCs present in the extract.