New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes.

Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata) Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9) and 3,4-dihydroxy-5-methoxybiphenyl-2'-O-ß-d-glucopyranoside (13), as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver-Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

Neurocytoprotective Effects of Aliphatic Hydroxamates from Lovastatin, a Secondary Metabolite from Monascus-Fermented Red Mold Rice, in 6-Hydroxydopamine (6-OHDA)-Treated Nerve Growth Factor (NGF)-Differentiated PC12 Cells.

Lovastatin, a secondary metabolite isolated from Monascus-fermented red rice mold, has neuroprotective activity and permeates the blood-brain barrier. The aim of this study was to enhance the activity of lovastatin for potential use as a treatment for neuronal degeneration in Parkinson's disease. Six lovastatin-derived compounds were semisynthesized and screened for neurocytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in human neuroblastoma PC12 cells. Four compounds, designated as 3a, 3d, 3e, and 3f, significantly enhanced cell viability. In particular, compound 3f showed excellent neurocytoprotective activity (97.0 ± 2.7%). Annexin V-FITC and propidium iodide double staining and 4',6-diamidino-2-phenylindole staining indicated that compound 3f reduced 6-OHDA-induced apoptosis in PC12 cells. Compound 3f also reduced caspase-3, -8, and -9 activities, and intracellular calcium concentrations elevated by 6-OHDA in a concentration-dependent manner, without inhibiting reactive oxygen species generation. JC-1 staining indicated that compound 3f also stabilized mitochondrial membrane potential. Thus, compound 3f may be used as a neurocytoprotective agent. Future studies should investigate its potential application as a treatment for Parkinson's disease.

Stimulation of osteogenic activity in human osteoblast cells by edible Uraria crinita.

Uraria crinita is an edible herb used as a natural food for childhood skeletal dysplasia. Ethyl acetate, n-butanol, and aqueous fractions of a 95% ethanol crude extract of U. crinita were obtained and the active ingredients isolated and purified using a bioguided method. In this manner, we isolated and identified a new active flavone glycoside, apigenin 6-C-ß-d-apiofuranosyl(1→2)-α-d-xylopyranoside (3) and 10 known components with stimulatory activity on human osteoblast cells. The new compound 3 at 100 µM significantly increased alkaline phosphatase activity (114.10 ± 4.41%), mineralization (150.10 ± 0.80%), as well as osteopontin (1.39 ± 0.01-fold), bone morphogenetic protein-2 (BMP-2, 1.30 ± 0.04-fold), and runt-related transcription factor 2 (Runx2, 1.43 ± 0.10-fold) mRNA expression through the activation of the BMP-2/Runx2 pathway. Two other components, dalbergioidin (1) and byzantionoside B (9), displayed similar effects. These results show that U. crinita and its active compounds may have the potential to stimulate bone formation and regeneration.

Chemical and pharmacological investigation of micropropagated Hygrophila pogonocalyx produced from leaf explants.

BACKGROUND: An optimized method for indirect shoot organogenesis from the leaf explants of Hygrophila pogonocalyx, a rare and endemic species in Taiwan, was developed to supply enough quantity of plant materials for the first chemical and pharmacological investigation. RESULTS: Incubation of the young leaves on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (0.5 mg/l) and indole-3-acetic acid (0.1 mg/l) resulted in the best multiplication rate for organogenesis. The average number of adventitious buds per leaf was 22.8 ± 1.9 after 8-week culture. The adventitious buds rooted and developed into plantlets when cultured simply on MS medium. Using this protocol, up to 37,600 plants were produced from a single leaf explant in one year. From the ethanol extract of the leaves of this micropropagated plant, 13 compounds were isolated and identified, including two flavones (1, 11), four flavonols (9, 10, 12, and 13), three phenylethanoid glycosides (6-8), two alkylated glycosides (2-3), and two steroids (4-5). Of these, acteoside (7) exhibited anti-tyrosinase activity in human epidermal melanocytes and luteolin 7-O-ß-D-glucopyranoside (11) exhibited the greatest neurocytoprotective activity. CONCLUSIONS: The method, indirect shoot organogenesis from leaf explants of H. pogonocalyx, could be developed to supply enough quantity of plant materials for the chemical and pharmacological investigation. In the present study, the isolated active compounds may develop for whitening agents or treating neurodegenerative diseases in the future.

The immuno-regulatory effects of Schisandra chinensis and its constituents on human monocytic leukemia cells.

Many diseases occur when the immune system is weakened. Intracellular signals activate immuno-responsive cells to produce cytokines that modulate the immune response. Schisandra chinensis has been used traditionally to treat general fatigue, neurasthenia, and spontaneous sweating. In the present study, the effect of constituents of S. chinensis on cytokine release by human monocytic leukemia cells (THP-1) was tested using microparticle-based flow cytometric analysis. Two major lignans, schizandrin (Sch) and gomisin A (Gom A), were identified and shown to induce interleukin (IL)-8, macrophage inflammatory protein-1ß (MIP-1ß), and granulocyte-macrophage-colony stimulating factor (GM-CSF) release by THP-1 cells. By reverse transcription polymerase chain reaction (RT-PCR) or quantitative real-time PCR, there was a dose-dependent increase of IL-8, MIP-1ß and GM-CSF mRNA levels. Thus, Sch and Gom A from S. chinensis enhance cytokine release by THP-1 cells and this effect occurs through mRNA upregulation. Upregulation of MIP-1ß and GM-CSF in particular may have clinical applications. Therefore, S. chinensis may be therapeutically beneficial by promoting humoral and cell-mediated immune responses.

Neurocytoprotective effects of the bioactive constituents of Pueraria thomsonii in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 cells.

Chronic neurodegenerative disorders are having an increasing impact on public health as human longevity increases. Parkinson's disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine-producing brain cells. Pueraria thomsonii Benth. (Fabaceae) is an herbal medicine that has traditionally been used as an antipyretic agent. In the present study, the active constituents, daidzein and genistein, were isolated from P. thomsonii. Both compounds exhibited neurocytoprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in nerve growth factor (NGF)-differentiated PC12 cells. Neither daidzein nor genistein affected 6-OHDA-induced cellular reactive oxygen species (ROS) generation according to flow cytometric analysis. Rather, they inhibited caspase-8 and partially inhibited caspase-3 activation, providing a protective mechanism against 6-OHDA-induced cytotoxicity in NGF-differentiated PC12 cells. The present results imply that daidzein and genistein may be useful in the development of future strategies for the treatment of PD.

Active constituents from Sophora japonica exhibiting cellular tyrosinase inhibition in human epidermal melanocytes.

AIM OF THE STUDY: There is greater consumer awareness of plant-based skin-care products. Sophora japonica L. (Fabaceae) has been used traditionally as a hemostatic agent and also has skin-care and whitening benefits. The effect of the isolated active compounds of Sophora japonica L. (Fabaceae) that inhibits tyrosinase activity in human epidermal melanocytes (HEMn) was examined. MATERIALS AND METHODS: We used the mushroom tyrosinase inhibitory assay to isolate active constituents from the extracts. The structures of these constituents were characterized by physical and spectroscopic analyses. Cellular tyrosinase kinetics were analyzed and showed by Lineweaver-Burk plot. RESULTS: A new compound, N-feruloyl-N'-cis-feruloyl-putrescine (8), together with four flavonoids and three putrescine derivatives were obtained after assay-guided isolation of S. japonica. In HEMn, compound 8 was minimally cytotoxic (cell viability >90% at 100 microM) and the IC(50) value for suppression of cellular tyrosinase activity was estimated as 85.0 microM. Zymography analysis demonstrated the compound's concentration-dependent effects and the kinetic analysis indicated the compound's mixed-inhibitory action. CONCLUSIONS: We concluded that the new compound 8 is the most potent component of S. japonica yet discovered. Its pigment inhibition activity may be exploitable cosmetically.

The effects of antibiotics combined with natural polyphenols against clinical methicillin-resistant Staphylococcus aureus (MRSA).

Novel therapies are needed to address the public health problem posed by methicillin-resistant STAPHYLOCOCCUS AUREUS (MRSA). In this study, we determined the effects of combinations of antibiotics and plant polyphenols against 20 clinical isolates of MRSA. The IN VITRO activities of 10 antibiotics and 15 natural polyphenols against the isolates were evaluated by determining minimum inhibitory concentrations (MICs). All isolates were susceptible to vancomycin and resistant to rifampicin, while susceptibilities to ciprofloxacin varied. Among the 15 natural polyphenols, kaempferol (3,4',5,7-tetrahydroxyflavone) and quercetin (3,3',4',5,7-pentahydroxyflavone) showed the lowest MICs. In checkerboard assays, combinations of rifampicin and either kaempferol or quercetin acted synergistically or partially synergistically against the clinical MRSA isolates. Rifampicin combined with kaempferol or quercetin exhibited good beta-lactamase inhibitory effects (57.8 % and 75.8 %, respectively) against a representative isolate according to nitrocefin analysis. The study results and ready availability and low toxicity of plant polyphenols warrant further investigations on the therapeutic potential of combination therapies for MRSA infections.

Phenolic constituents of Malus doumeri var. formosana in the field of skin care.

Plant phenolic compounds isolated from a 70% aqueous acetone extract of the leaves of Malus doumeri A. CHEV. var. formosana (KAWAK. & KOIDZ.) S. S. YING, a type of Taiwanese indigenous plant, were evaluated for potential application in the field of skin care. A phytochemical investigation of the active fractions resulted in the isolation of seven compounds of which the structures were identified by spectroscopic characterization. In the present study, the isolated phenolic compounds were evaluated for their free radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radicals, anti-elastase, and for their anti-matrix metalloproteinase-1 (MMP-1) activity in human skin fibroblast cells. Of these compounds, 3-hydroxyphloridzin (2), 3-hydroxyphloretin (6), and quercetin (7) exhibited the strongest DPPH and superoxide radical-scavenging activities. The IC50 values of these compounds were 9.2, 7.7, and 15.4 microM, respectively, for the DPPH radical, and 25.0, 19.6, and 42.6 microM, respectively, for the superoxide radical. 3-Hydroxyphloridzin (2) and 3-hydroxyphloretin (6) also showed xanthine oxidase inhibitory activity, with IC(50) values of 52.1 and 22.4 muM, respectively. In the test for elastase inhibitory activity, phloretin (5) and 3-hydroxyphloretin (6) were the most potent compounds. Phloretin (5), 3-hydroxyphloretin (6), and quercetin (7) showed better inhibition of MMP-1 production in fibroblast cells. To the best of our knowledge, this is the first time that the active phenolic compounds from M. doumeri var. formosana have been isolated, reported, and described. The above results suggest that the extract of M. doumeri var. formosana containing phenolic compounds could be suitable naturally occurring active constituents for use in anti-aging or cosmetic products.

Cosmetic applications of selected traditional Chinese herbal medicines.

Because tyrosinase catalyzes melanin synthesis, tyrosinase inhibitors are important in cosmetic skin-whitening. Oxidative stress contributes to skin aging and can adversely affect skin health, which means antioxidants active in skin cells may support skin health. We examined 25 traditional Chinese herbal medicines that might be useful for skin-whitening and skin health. Extracts (100microg/mL) were tested for cytotoxicity on human epidermal melanocytes (HEMn); 12 exhibited low cytotoxicity. Their effects on tyrosinase and melanin inhibitory activities and free radical scavenging activities were further assessed. Phenolic contents were evaluated using Folin-Ciocalteu reagent. Four herbs, Pharbitis nil, Sophora japonica, Spatholobus suberectus, and Morus alba, exhibited potent inhibitory effects on tyrosinase (IC(50) values 24.9, 95.6, 83.9, and 78.3microg/mL, respectively). Melanin inhibition was not dose-dependent. Sophora japonica (IC(50): 14.46microg/mL, 1,1-diphenyl-2-picrylhydrazyl (DPPH); 1.95microg/mL, hydroxyl radical) and Spatholobus suberectus (IC(50): 10.51microg/mL, DPPH; 4.36microg/mL, hydroxyl radical) showed good antioxidative activities and high phenolic contents (255 and 189mg of gallic acid/g extract, respectively). Among active anti-tyrosinase extracts, Sophora japonica and Spatholobus suberectus were especially potent in HEMn cells in terms of free radical scavenging effects and high phenolic contents, making them the strongest candidates for cosmetic application found in the current study.

Antioxidant and heme oxygenase-1 (HO-1)-induced effects of selected Taiwanese plants.

Recent studies have shown biological effects of heme oxygenase-1 (HO-1) induction and antioxidation in cardiovascular disorders. The ethanol extracts of leaves of 12 selected indigenous Taiwanese plants were investigated for their antioxidant activities, evaluated using assays of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide radicals scavenging and reducing power activities as well as the induction of heme oxygenase-1 (HO-1). Acer albopurpurascens, Cinnamomum kanehirai, Diospyros discolor, Excoecaria kawakamii, Koelreuteria henryi, and Syzygium formosanum showed better DPPH-scavenging activities than the other plants. IC(50) values ranged from 1.7 to 8.7 microg/mL. Excepting Millettia pulchra var. microphylla and Pittosporum moluccanum, the extracts displayed hydroxyl-scavenging activities (IC(50) of 0.16-0.67 microg/mL). A. albopurpurascens, D. discolor, K. henryi, and S. formosanum also showed good superoxide anion radical scavenging activities and IC(50) values ranged from 12.9 to 28.5 microg/mL. D. discolor, K. henryi, and S. formosanum showed potent reducing power and M. pulchra var. microphylla and S. formosanum exhibited potent HO-1 induced activity. These active plant extracts also contained abundant phenolic constituents. The present results provide candidates to isolate the active constituents and develop natural antioxidants.

Antimicrobial activity of antibiotics in combination with natural flavonoids against clinical extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae.

Extended-spectrum beta-lactamases (ESBLs) are plasmid-mediated class A enzymes commonly found in the family Enterobacteriaceae, mainly in Klebsiella pneumoniae. Flavonoids have also been reported to possess antimicrobial activity. In this study, the in vitro activities of 18 antibiotics and 12 flavonoids against 20 ESBL-producing K. pneumoniae isolates were evaluated. All of these isolates were susceptible to imipenem and cefmetazole, but were resistant to ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefoperazone, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, piperacillin and ticarcillin. Susceptibilities to amikacin, amoxicillin/clavulanate, cefoxitin, ciprofloxacin and gentamicin were variable. Myricetin, a flavonol, inhibited ESBL-producing K. pneumoniae isolates at a high minimum inhibitory concentration (MIC) (MIC(90) value 256 mg/mL), but exhibited significant synergic activity against ESBL-producing K. pneumoniae in separate combination with amoxicillin/clavulanate, ampicillin/sulbactam and cefoxitin. Because of the low-toxic nature of flavonoids, the combination of antibiotics and flavonoids is a potential new strategy for developing therapies for infections caused by ESBL-producing bacteria in the future.

Monoamine oxidase B (MAO-B) inhibition by active principles from Uncaria rhynchophylla.

Attenuation of monoamine oxidase B (MAO-B) activity may provide protection against oxidative neurodegeneration. For this reason, inhibition of MAO-B activity is used as part of the treatment of Parkinson's and Alzheimer's patients. The hook of Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) is a traditional Chinese herbal drug that is generally used to treat convulsive disorders. In this study, the fractionation and purification of Uncaria rhynchophylla extracts using a bioguided assay isolated two known compounds, (+)-catechin and (-)-epicatechin. The compounds inhibited MAO-B, as measured by an assay of rat brain MAO-B separated by electrophoresis on a 7.5% native polyacrylamide gel. The IC(50) values of (+)-catechin and (-)-epicatechin were 88.6 and 58.9 microM, respectively, and inhibition occurred in a dose-dependent manner, as measured by the fluorescence method. The Lineweaver-Burk plot revealed K(i) values for (+)-catechin and (-)-epicatechin of 74 and 21 microM, respectively. This suggests that these two compounds, isolated here for the first time from Uncaria rhynchophylla, might be able to protect against neurodegeneration in vitro, and, therefore, the molecular mechanism deserves further study. This finding may also increase interest in the health benefits of Uncaria rhynchophylla.