Biotransformation-guided purification of a novel glycoside derived from the extracts of Chinese herb Baizhi.

Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (DgAS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with DgAS. Structural elucidation confirmed the compound as byakangelicin-7″-O-α-glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of DgAS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds.

A New Stilbene Glucoside from Biotransformation-Guided Purification of Chinese Herb Ha-Soo-Oh.

Ha-Soo-Oh is a traditional Chinese medicine prepared from the roots of Polygonum multiflorum Thunb. The herb extract has been widely used in Asian countries as a tonic agent and nutritional supplement for centuries. To identify new bioactive compounds in Chinese herbs, the biotransformation-guided purification (BGP) process was applied to Ha-Soo-Oh with Bacillus megaterium tyrosinase (BmTYR) as a biocatalyst. The result showed that a major biotransformed compound could be purified using the BGP process with preparative high-performance liquid chromatography (HPLC), and it was confirmed as a new compound, 2,3,5,3',4'-pentahydroxystilbene-2-O-ß-glucoside (PSG) following mass and nucleic magnetic resonance (NMR) spectral analyses. PSG was further confirmed as a biotransformation product from 2,3,5,4'-tetrahydroxystilbene-2-O-ß-glucoside (TSG) by BmTYR. The new PSG exhibited 4.7-fold higher 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging activity than that of TSG. The present study highlights the potential usage of BGP in herbs to discover new bioactive compounds in the future.

A new macrocyclic diterpenoid from Anisomeles indica.

A new macrocyclic diterpenoid, 4ß,5ß-dihydroxyovatodiolide (1), together with twenty-two known compounds (2-23) were isolated from the MeOH extract of the dried aerial parts of Anisomeles indica (L.) O. Kuntze (Labiatae). The structure of 1 was established on the basis of spectral evidence. Phenylethanoids, acteoside (5) and isoacteoside (6) showed significant inhibitory to IL-2 secretion of with respect to phorbol myristate acetate and anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T cells.

Tris(8-Hydroxyquinoline)iron induces apoptotic cell death via oxidative stress and by activating death receptor signaling pathway in human head and neck carcinoma cells.

BACKGROUND: 8-Hydroxyquinoline derivatives have highly sensitive fluorescent chemosensors for metal ions, which are associated with anti-oxidant, anti-tumor and anti-HIV-1 properties. Head and neck squamous cell carcinoma (HNSCC) is associated with a high rate of mortality and novel anti-HNSCC drugs must be developed. Therefore, effective chemotherapy agents are required to address this public health issue. HYPOTHESIS/PURPOSE: The aim of this study was to investigate the inhibitory effect of tris(8-hydroxyquinoline)iron (Feq3) on the HNSCC and the underlying mechanism. STUDY DESIGN/METHODS: A novel 8-hydroxyquinoline derivative, Feq3, was synthesized. The cell viabilities were analyzed using MTT reagent. Apoptosis and the cell cycle distributions were determined by flow cytometer. Reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, western blot, MitoSOX and CellROX stain assay were used to study the mechanism of Feq3. Feq3 combined with antioxidants NAC (N-acetylcysteine) and BSO (buthionine sulfoximine) measured the cell viability and intracellular ROS. RESULTS: Feq3 induced the death of HNSCC cells and caused them to exhibit the morphological features of apoptosis. Feq3 also induced apoptosis of SCC9 cells by cell cycle arrest during the G2/M phase and the induced arrest of SCC25 cells in the G0/G1 and G2/M phases, which was associated with decreased cyclin B1/cdc2 and cyclin D/cdk4 expressions. Feq3 increases reactive oxygen species (ROS) and reduces glutathione (GSH) levels, and responds to increased p53 and p21 expressions. Feq3 induced apoptosis by mitochondria-mediated Bax and cytochrome c up-expression and down-expression Bcl-2. Feq3 also up-regulated tBid, which interacts with the mitochondrial pathway and tumor necrosis factor-α (TNF-α)/TNF-Rs, FasL/Fas, and TNF-related apoptosis inducing ligand receptors (TRAIL-Rs)/TRAIL-dependent caspases apoptotic signaling pathway in HNSCC cells. However, Feq3 activates Fas but not FasL in SCC25 cells. Feq3 arrests the growth of HNSCC cells and is involved in the mitochondria- and death receptor (DR)-mediated caspases apoptotic pathway. CONCLUSION: This study is the first to suggest that apoptosis mediates the anti-HNSCC of Feq3. Feq3 has potential as a cancer therapeutic agent against HNSCC.

Cleome rutidosperma and Euphorbia thymifolia Suppress Inflammatory Response via Upregulation of Phase II Enzymes and Modulation of NF-κB and JNK Activation in LPS-Stimulated BV2 Microglia.

Cleome rutidosperma DC. and Euphorbia thymifolia L. are herbal medicines used in traditional Indian and Chinese medicine to treat various illnesses. Reports document that they have antioxidant and anti-inflammatory activities; nonetheless, the molecular mechanisms involved in their anti-inflammatory actions have not yet been elucidated. The anti-neuroinflammatory activities and underlying mechanisms of ethanol extracts of Cleome rutidosperma (CR) and Euphorbia thymifolia (ET) were studied using lipopolysaccharide (LPS)-stimulated microglial cell line BV2. The morphology changes and production of pro-inflammatory mediators were assayed. Gene expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1ß, and CC chemokine ligand (CCL)-2, as well as phase II enzymes such as heme oxygenase (HO)-1, the modifier subunit of glutamate cysteine ligase (GCLM) and NAD(P)H quinone dehydrogenase 1 (NQO1), were further investigated using reverse transcription quantitative-PCR (RT-Q-PCR) and Western blotting. The effects of CR and ET on mitogen activated protein kinases (MAPKs) and nuclear factor (NF)-κB signaling pathways were examined using Western blotting and specific inhibitors. CR and ET suppressed BV2 activation, down-regulated iNOS and COX-2 expression and inhibited nitric oxide (NO) overproduction without affecting cell viability. They reduced LPS-mediated tumor necrosis factor (TNF) and IL-6 production, attenuated IL-1ß and CCL2 expression, but upregulated HO-1, GCLM and NQO1 expression. They also inhibited p65 NF-κB phosphorylation and modulated Jun-N terminal kinase (JNK) activation in BV2 cells. SP600125, the JNK inhibitor, significantly augmented the anti-IL-6 activity of ET. NF-κB inhibitor, Bay 11-7082, enhanced the anti-IL-6 effects of both CR and ET. Znpp, a competitive inhibitor of HO-1, attenuated the anti-NO effects of CR and ET. Our results show that CR and ET exhibit anti-neuroinflammatory activities by inhibiting pro-inflammatory mediator expression and production, upregulating HO-1, GCLM and NQO1, blocking NF-κB and modulating JNK signaling pathways. They may offer therapeutic potential for suppressing overactivated microglia and alleviating neurodegeneration.

Anti-inflammatory effect and mechanism of the green fruit extract of Solanum integrifolium Poir.

The green fruit of Solanum integrifolium Poir. has been used traditionally as an anti-inflammatory and analgesic remedy in Taiwanese aboriginal medicine. The goal of this study is to evaluate the anti-inflammatory activity and mechanism of the green fruit extract of S. integrifolium. A bioactivity-guided fractionation procedure was developed to identify the active partition fraction. The methanol fraction (ME), with the highest phenolic content, exhibited the strongest inhibitory effect against LPS-mediated nitric oxide (NO) release and cytotoxicity in RAW264.7 macrophages. ME also significantly downregulated the expression of LPS-induced proinflammatory genes, such as iNOS, COX-2, IL-1ß, IL-6, CCL2/MCP-1, and CCL3/MIP1α. Moreover, ME significantly upregulated HO-1 expression and stimulated the activation of extracellular-signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with the HO-1 inhibitor zinc protoporphyrin and MEK/ERK inhibitor U0126 attenuated ME's inhibitory activity against LPS-induced NO production. Taken together, this is the first study to demonstrate the anti-inflammatory activity of green fruit extract of S. integrifolium and its activity may be mediated by the upregulation of HO-1 expression and activation of ERK1/2 pathway.

Antioxidant, anti-adipocyte differentiation, antitumor activity and anthelmintic activities against Anisakis simplex and Hymenolepis nana of yakuchinone A from Alpinia oxyphylla.

BACKGROUND: Alpinia oxyphylla is a common remedy in traditional Chinese medicine. Yakuchinone A is a major constituent of A. oxyphylla and exhibits anti-inflammatory, antitumor, antibacterial, and gastric protective activities. METHODS: Antioxidant and antitumor characteristics of yakuchinone A in skin cancer cells as well as novel mechanisms for the inhibition of adipocyte differentiation, cestocidal activities against Hymenolepis nana adults, and nematocidal activities against Anisakis simplex larvae are investigated. RESULTS: Yakuchinone A presents the ability of the removal of DPPH·and ABTS+ free radicals and inhibition of lipid peroxidation. Yakuchinone A suppresses intracellular lipid accumulation during adipocyte differentiation in 3 T3-L1 cells and the expressions of leptin and peroxisome proliferator-activated receptor γ (PPARγ). Yakuchinone A induces apoptosis and inhibits cell proliferation in skin cancer cells. The inhibition of cell growth by yakuchinone A is more significant for non-melanoma skin cancer (NMSC) cells than for melanoma (A375 and B16) and noncancerous (HaCaT and BNLCL2) cells. Treatment BCC cells with yakuchinone A shows down-regulation of Bcl-2, up-regulation of Bax, and an increase in cleavage poly (ADP-ribose) polymerase (PARP). This suggests that yakuchinone A induces BCC cells apoptosis through the Bcl-2-mediated signaling pathway. The anthelmintic activities of yakuchinone A for A. simplex are better than for H. nana. CONCLUSIONS: In this work, yakuchinone A exhibits antioxidative properties, anti-adipocyte differentiation, antitumor activity, and anthelmintic activities against A. simplex and H. nana.

Brazilein from Caesalpinia sappan L. Antioxidant Inhibits Adipocyte Differentiation and Induces Apoptosis through Caspase-3 Activity and Anthelmintic Activities against Hymenolepis nana and Anisakis simplex.

Brazilein, a natural, biologically active compound from Caesalpinia sappan L., has been shown to exhibit anti-inflammatory and antioxidant properties and to inhibit the growth of several cancer cells. This study verifies the antioxidant and antitumor characteristics of brazilein in skin cancer cells and is the first time to elucidate the inhibition mechanism of adipocyte differentiation, cestocidal activities against Hymenolepis nana, and reduction of spontaneous movement in Anisakis simplex. Brazilein exhibits an antioxidant capacity as well as the ability to scavenge DPPH(•) and ABTS(•+) free radicals and to inhibit lipid peroxidation. Brazilein inhibited intracellular lipid accumulation during adipocyte differentiation in 3T3-L1 cells and suppressed the induction of peroxisome proliferator-activated receptor γ (PPAR γ ), the master regulator of adipogenesis, suggesting that brazilein presents the antiobesity effects. The toxic effects of brazilein were evaluated in terms of cell viability, induction of apoptosis, and the activity of caspase-3 in BCC cells. The inhibition of the growth of skin cancer cells (A431, BCC, and SCC25) by brazilein is greater than that of human skin malignant melanoma (A375) cells, mouse leukemic monocyte macrophage (RAW 264.7 cells), and noncancerous cells (HaCaT and BNLCL2 cells). The anthelmintic activities of brazilein against Hymenolepis nana are better than those of Anisakis simplex.

trans-Caffeic acid stearyl ester from Paeonia suffruticosa inhibits melanin synthesis by cAMP-mediating down-regulation of α-melanocyte-stimulating hormone-stimulated melanogenesis signaling pathway in B16 cells.

trans-Caffeic acid stearyl ester (TCASE) from the root cortex of Paeonia suffruticosa ANDREWS is a traditional medicinal herb that has several beneficial properties. However, the inhibitory effect of TCASE on melanogenesis has not been explored. In the cell viability assay, TCASE did not show a cytotoxic effect at a dose of 65 µM for 48 h in B16, HaCaT and Hs68 cells. TCASE considerably inhibits melanin synthesis, and reduces intracellular cyclic adenosine monophosphate (cAMP) levels, tyrosinase activity and L-3-(3,4-dihydroxyphenyl)-alanine (DOPA) oxidase activity in a concentration-dependent manner in the presence of α-melanocyte-stimulating hormone (α-MSH) in B16 cells, and the inhibition efficiency of TCASE exceeds that of ascorbic acid and arbutin. TCASE reduces melanocortin-1 receptor (MC1R), microphthalmia transcription factor (MITF), tyrosinase, tyrosinase-related protein-2 (TRP-2) and TRP-1 mRNA and protein levels in B16 cells. Based on the findings, TCASE is posited to inhibit melanogenesis signaling while suppressing cAMP levels and, subsequently, MC1R, MITF, tyrosinase, TRP-2 and TRP-1 down-regulation, resulting in the suppression of tyrosinase activity, DOPA oxidase activity and melanin synthesis.

Melanogenesis inhibition by homoisoflavavone sappanone A from Caesalpinia sappan.

Homoisoflavanone, sappanone A, was isolated from Caesalpinia sappan and proven to dose-dependently inhibit both melanogenesis and cellular tyrosinase activity via repressing tyrosinase gene expression in mouse B16 melanoma cells. To our knowledge, sappanone A is the first homoisoflavanone to be discovered with melanogenesis inhibitory activity. Our results give a new impetus to the future search for other homoisoflavanone melanogenesis inhibitors.

Free radical scavenging activity of 4-(3,4-dihydroxybenzoyloxymethyl)phenyl-O-ß-D-glucopyranoside from Origanum vulgare and its protection against oxidative damage.

4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-ß-d-glucopyranoside (DBPG), a polyphenolic glycoside, isolated from Origanum vulgare has shown 1,1-diphenyl-2-picrylhydrazyl (DPPH(•))-scavenging capacity in previous work. This study demonstrated that DBPG exhibits antioxidant activity by a series of DPPH(•), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(•+)), and superoxide anion radical (O(2)(•-)) radical-scavenging assays. The inhibition of lipid peroxidation (LP) by DBPG exceeded that by l-ascorbic acid (AA) in a liposome model system. Adding DBPG to mouse liver and brain tissue inhibited the formation of thiobarbituric acid reactive substances (TBARS) to a greater extent than did trolox. In the oxygen stress test, BNLCL2 and HaCaT cells pretreated with DBPG showed increased activities of glutathione peroxidase (GPx), perhaps as a result of reduction of the production of reactive oxygen species (ROS). These findings proved that DBPG had antioxidant activities and a cytoprotective effect in hepatocytes and keratinocytes, suggesting that DBPG may be a useful food and cosmetic additive.

To accomplish preliminary Qigong simulation state by acupuncture.

OBJECTIVE: The purpose of the study was to find out how to enter the preliminary Qigong simulation state in a short period of time. METHODS: This is a non-randomized, human experiment with healthy participants. A multi-channel digital physiological data recorder was used to detect whether the participants had entered the Qigong state. Participants were assisted to enter the Qigong state (relaxation, tranquility and naturalness) by being given the sore (sour) feeling produced by acupuncturing Hegu (LI4), and suggestions (repeating words "relax" and "heat" from the hypogastrium). RESULTS: About 72.2% of the participants who had no Qigong experience were found entering the preliminary Qigong simulation state. Most of the physiological parameters measured after the participants entering the Qigong state showed significant changes compared with the baseline data. CONCLUSION: This study revealed that acupuncture-made sore feeling is able to induce the participants to quickly enter the preliminary Qigong simulation state; hence this can be seen as no longer a limited phenomenon, but can be commonly applied to everybody.

Extracts and constituents of Rubus chingii with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the fruits of Rubus chingii was studied in vitro. Ethanolic extract, ethyl acetate and n-butanol fractions from dried R. chingii fruits revealed strong DPPH free radical scavenging activity with IC(50) values of 17.9, 3.4 and 4.0 µg/mL, respectively. The ethyl acetate and n-butanol fractions were further purified by a combination of silica gel chromatography, Lobar RP-8 chromatography, and high-pressure liquid chromatography (HPLC). Nine compounds were isolated, where methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), vanillic acid (5), kaempferol (7), and tiliroside (9) showed stronger DPPH free radical scavenging activity than that of ascorbic acid (131.8 µM) with IC(50) values of 45.2, 34.9, 78.5, and 13.7 µM, respectively. In addition, rubusine (1) is a new compound discovered in the present study and methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), methyl dioxindole-3-acetate (3), and 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4) were isolated from the fruits for the first time.

Antioxidant and antimelanogenic behaviors of Paeonia suffruticosa.

Antioxidant properties of eight Paeonia suffruticosa (Ps) extracts (Ps-1 to Ps-8) were evaluated. The respective half maximally effective concentration (EC(50)) values of Ps-1 ~ 8 were 10.0, 9.8, 63.6, >100, 3.8, 85.1, 6.9, and 0.7 µg/ml for 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) radical scavenging efficiency and 22.9, 11.4, 53.1, >100, 7.5, 97.6, 43.7, 4.2 µg/ml for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS·(+)) radical scavenging capacity. The Ps-8 exhibited high free radical scavenging capacity, ion-chelating ability, reducing power, and inhibition of lipid peroxidation, which may have been attributable to its abundant phenolic and flavonoid content. In Hs68 and B16 cells treated with 100 µg/ml Ps-1, Ps-3, Ps-4 and Ps-6, expressions of toxic activities were lower than those in cells treated with arbutin and ascorbic acid. The antimelanogenesis properties were also tested in B16 cells. Extract Ps-1, and particularly extract Ps-6, considerably inhibited cellular tyrosinase and 3,4-dihydroxyphenylalanine (DOPA) oxidase activity and also reduced melanin content in B16 cells by down-expression of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins-1 (TRP-1). The results suggest that P. suffruticosa extracts have antioxidant and antimelanogenesis activities with potential applications in cosmetic materials or food additives.

Murine tyrosinase inhibitors from Cynanchum bungei and evaluation of in vitro and in vivo depigmenting activity.

Two natural acetophenone derivatives, 2,5-dihydroxyacetophenone (2,5-DHAP) and 2,6-DHAP, were purified from Cynanchum bungei and identified as murine tyrosinase inhibitors. Investigation into 2,5-DHAP showed it to be an uncompetitive inhibitor of murine tyrosinase (K(I) 0.28 mm). 2,5-DHAP strongly inhibited both melanogenesis and cellular tyrosinase activity in vitro in 3-isobutyl-1-methylxanthin-stimulated B16 mouse melanoma cells or in vivo in zebrafish and mouse models, but showed no cytotoxicity at the concentrations used. In B16 cells, 2,5-DHAP inhibition was dose-dependent and was fourfold greater than that of arbutin. 2,5-DHAP had no effect on the expression of tyrosinase protein or mRNA, as confirmed by Western blotting and quantitative real-time reverse transcription polymerase chain reaction, respectively. A 2% gel preparation of 2,5-DHAP applied to the skin of mice significantly increased the average skin-whitening index (L value), indicating its potential use as a treatment for skin hyperpigmentation in humans.

Inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells.

The inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells was investigated. The results showed that the P. acidula extract (PAE) inhibited melanogenesis in 3-isobutyl-1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner, with an IC50 value of 33.5 µg/ml. In addition, PAE also inhibited cellular tyrosinase activity. Moreover, western blot and real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses respectively confirmed that PAE down-regulated levels of tyrosinase protein and its mRNA in IBMX-stimulated B16 cells. These results demonstrated that PAE inhibits melanogenesis of B16 cells by reducing tyrosinase gene expression. From the present study, PAE is proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation.

Inhibition of melanogenesis and oxidation by protocatechuic acid from Origanum vulgare (oregano).

Antioxidant and antimelanogenesis activities of protocatechuic acid (1) from Origanum vulgare (oregano) were investigated. The antioxidative capacity of 1 was confirmed from its free-radical-scavenging activities, inhibition of lipid peroxidation, and suppression of reactive oxygen species in H(2)O(2)-induced BNLCL2 cells. The inhibition by 1 of tyrosinase and DOPA oxidase activity and melanin production was possibly related to the down-regulation of melanocortin-1 receptor, microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related proteins-2, and tyrosinase-related proteins-1 expression in α-melanocyte-stimulating hormone-induced B16 cells. After a gel containing 1 was applied to mice, the values of L* slightly increased, and a* and erythema-melanin levels of skin were reduced by comparing the values of untreated control groups, indicating 1 can reduce melanin production. These results suggest that 1 may act as an effective quencher of oxidative attackers with antimelanogenesis properties.

Antioxidative characteristics and inhibition of alpha-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare.

The antioxidant activities of vanillin and vanillic acid isolated from Origanum vulgare are investigated. These compounds may serve as agents for antimelanogenesis. Vanillic acid is a stronger antioxidant than vanillin, in terms of free radical scavenging activity, reducing power and inhibition of lipid peroxidation. The inhibition of cellular reactive oxygen species (ROS) in H(2)O(2)-treated BNLCL2 cells by vanillic acid exceeds that of ascorbic acid (AA) or trolox. In B16F0 cells stimulated with alpha-melanocyte-stimulating hormone (alpha-MSH), vanillic acid reduced cellular tyrosinase activity, DOPA oxidase and melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins 2 (TRP-2) and TRP-1. Vanillin did not express inhibition of tyrosinase activity. These results supported that vanillic acid is a significantly stronger antioxidant than vanillin and exhibited stronger antimelanogenesis performance because of the structural presence of the carboxyl group.

5,6,3',4'-Tetrahydroxy-7-methoxyflavone as a novel potential proteasome inhibitor.

Proteasome inhibition is one of the new pharmaceutical approaches to study antitumor activity. Although the active components are not yet identified for either treating or preventing cancer, the low toxicity plant Anisomeles ovata R. Br. has been used in traditional herbal medicine for more than 1000 years. In this study, the methanol extract from Anisomeles ovata showed potent inhibition of proteasome activity. Twenty compounds, two macrocycylic diterpenoids, six aromatics, seven flavonoids, and five phenylethanoids were isolated from Fang Feng Cao the dried aerial parts of A. ovata. Their structures have been established on the basis of spectral evidence. Using a proteolysis assay for inhibition of 26S proteasome from pig red blood cells, we found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 90.5 %, 85.4 % and 73.1 % the chymotrypsin-like, caspase-like and trypsin-like activities of 26S proteasome with IC (50) values of 14.0, 5.4 and 24.1 microM, respectively, when Suc-LLVY-AMC, Z-LLE-AMC and Ac-RLR-AMC were used as substrates. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone had a higher inhibitory at 15 minutes. A combination of 5,6,3',4'-tetrahydroxy-7-methoxyflavone and 5,6,4'-trihydroxy-7,3'-dimethoxyflavone increased the inhibition ability on 26S enzymatic activity. This combination appears to be a potentially attractive chemotherapy approach. We have found that 5,6,3',4'-tetrahydroxy-7-methoxyflavone has the highest inhibitory effects on 26S proteasome activities when compared to the other 11 flavonoids. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S proteasome activity.

Inhibition of melanogensis by a novel origanoside from Origanum vulgare.

BACKGROUND: Natural and synthetic substances are becoming increasingly utilized as tyrosinase inhibitors of depigmentation and developed cosmetics industry. However, few have been employed as skin-whitening agents, primarily because of numerous safety concerns. OBJECTIVE: A novel compound was found, and then its safe concentrations and inhibition effect of hyperpigmentation by the regulation of the tyrosinase family of proteins were examined. METHODS: A novel phenolic glucoside, origanoside (1), was isolated from Origanum vulgare. The structure of the origanoside (1) was established on the basis of spectral evidence and the safe concentrations were determined by MTT assay. Skin-whitening capacity in skin fibroblast Hs68 and melanoma B16 cells and in vivo animal test for origanoside (1) were investigated. RESULTS: Origanoside (1) is non-toxic in concentrations of 0-100 microg/ml in both cells. The ability of origanoside (1) to inhibit cellular tyrosinase and DOPA oxidase in B16 cells was investigated. Origanoside (1) significantly reduced expressions of microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related proteins 2 (TRP-2) in vitro and in vivo, suggesting that origanoside (1) is responsible for the antimelanogenic effect. Smearing origanoside (1)-gel samples on 12 mice for 10 days increased L*, reduced a* and erythema-melanin (E/M), and b* was almost unchanged compared with those of samples and untreated groups, indicating that the skin lightened. CONCLUSION: Experimental data demonstrate that origanoside (1) causes depigmentation and may be useful for novel food additives and skin-whitening cosmetics.