Anti-tyrosinase and antimelanogenic effect of cinnamic acid derivatives from Prunus mahaleb L.: Phenolic composition, isolation, identification and inhibitory activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional use of Prunus species against skin diseases and especially for skin lightning cosmeceutical purposes is widespread in many cultures. Prunus mahaleb L. is a well known food plant and used in the baking industry for flavoring. The fruit kernels (endocarp) are used in India for hyperpigmentation. AIM OF THE STUDY: To investigate the chemical composition with the antimelanogenesis effect of P. mahaleb seed and kernel extracts and isolated compounds. MATERIALS AND METHODS: Isolation studies performed from the methanol extracts obtained from kernels and structures were determined using NMR and MS analysis. Antimelanogenesis effect was determined by mushroom tyrosinase assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. RESULTS: Five cinnamic acid derivatives were isolated and their structures (2-O-ß-glucopyranosyloxy-4-methoxy-hydrocinnamic acid (1), cis-melilotoside (2), dihydromelilotoside (3), trans-melilotoside (4), 2-O-ß-glucosyloxy-4-methoxy trans-cinnamic acid (5)) were elucidated using advanced spectroscopic methods. Mushroom tyrosinase enzyme inhibition of extracts, fractions and pure compounds obtained from P. mahaleb kernels were investigated and structure-activity relationship revealed. According to a detailed, comprehensive and validated LC-MS/MS technique analysis, vanilic acid (41.407 mg/g), protocatechuic acid (8.992 mg/g) and ferulic acid (4.962 mg/g) in the kernel ethylacetate fraction; quinic acid (14.183 mg/g), fumaric acid (8.349 mg/g) and aconitic acid (5.574 mg/g) were found as major phenolic compounds in the water fraction. The correlation of trace element copper content in extracts and fractions with mushroom enzyme activity was determined. By examining the enzyme kinetics of the compounds with effective cinnamic acid derivatives, inhibition types and enzyme binding constants Ki were calculated. Compounds 1,3 and 5 exhibited high noncompetitive tyrosinase inhibitory activity against L-tyrosine substrates, with IC50 values of 0.22, 0.31 and 0.37 mM respectively. In addition compounds 1, 3 and 5 showed dose-dependent inhibitory effects on intracellular tyrosinase and melanin levels in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. CONCLUSIONS: Potent tyrosinase inhibitory compounds and extracts of P. mahaleb kernels suggest that it could be a new, non-toxic and inexpensive resource for the cosmeceutical industry and in skin diseases associated with hyperpigmentation.

Identification of Maple Anthocyanin and its Antiproliferative Activity against LLC, T47D and C3H10T1/2 Cells.

BACKGROUND: The genus Acer contains around 200 species, with more than 400 garden varieties. There is considerable diversity in these species and garden varieties, and each can be characterized by morphology and chemical composition. The red appearance of Acer leaves is due to anthocyanin compounds, including cyanidin glycosides, delphinidin glycosides, and galloylated anthocyanins. Few studies have investigated the diversity of anthocyanin compounds in garden varieties, and no studies have examined the pharmacological effects of these compounds. OBJECTIVE: The purpose of this study was to identify the anthocyanins of Acer palmatum cv. 'Chishio', a garden variety of A. palmatum and evaluate their antiproliferative and antioxidant activities. METHODS: A methanol extract of fresh leaves was partitioned with ethyl acetate. The extract was purified by column chromatography and compounds were subsequently identified by 1H and 13C NMR and ESI-HRMS. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt (MTS) colorimetric assay. The antioxidant assay was evaluated by scavenging activity using the stable radical DPPH. RESULTS: The anthocyanins cyanidin-3-O-(6''-O-α-rhamnopyranosyl- ß-glucopyranoside), cyanidin-3-O- ß- glucopyranoside, cyanidin-3-O-[2''-O-(galloyl)-6''-O-(rhamnosyl)-ß-glucoside], and cyanidin-3-O-[2''-O-(galloyl)- ß-glucopyranoside] were isolated from A. palmatum cv. 'Chishio'. All four anthocyanin compounds showed antiproliferative activity against LLC and T47D cells, and galloylated anthocyanin showed antiproliferative activity against C3H10T1/2 cells. All four anthocyanins inhibited the activity of DPPH radicals in a dosedependent manner. CONCLUSION: Maple anthocyanins could be a new cancer therapeutic agent or prophylactic medicine.

Tradescantia pallida extract inhibits biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is capable of biofilm formation. In this study, we investigated the effects of aqueous Tradescantia pallida extract on Pseudomonas aeruginosa growth and biofilm formation. Aqueous Tradescantia pallida extracts significantly inhibited both bacterial growth and biofilm formation. However, methanolic Tradescantia pallida extracts inhibited neither. Aqueous Tradescantia pallida extracts were deactivated by heating but were not deactivated by light exposure. The ingredients retained the inhibitory effect on the bacterial growth and biofilm formation after ultrafiltration of aqueous Tradescantia pallida extract. Furthermore, polyphenol-rich Tradescantia pallida extracts inhibited bacterial growth, thus, polyphenols are possible to be an active ingredient. We observed the biofilm by scanning electron microscopy, and quantitative and qualitative differences in the biofilm and cells morphology. Interestingly, the biofilm treated aqueous Tradescantia pallida extracts remained premature. We postulated that premature biofilm formation was due to the inhibition of swarming motility. Indeed, aqueous Tradescantia pallida extracts inhibited swarming motility. These results demonstrate that Peudomonas aeruginosa growth and biofilm formation are inhibited by aqueous Tradescantia pallida extracts.

Sasa veitchii extract protects against carbon tetrachloride-induced hepatic fibrosis in mice.

BACKGROUND: The current study aimed to investigate the hepatoprotective effects of Sasa veitchii extract (SE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. METHODS: Male C57BL/6J mice were intraperitoneally injected with CCl4 dissolved in olive oil (1 g/kg) twice per week for 8 weeks. SE (0.1 mL) was administered orally once per day throughout the study, and body weight was measured weekly. Seventy-two hours after the final CCl4 injection, mice were euthanized and plasma samples were collected. The liver and kidneys were collected and weighed. RESULTS: CCl4 administration increased liver weight, decreased body weight, elevated plasma alanine aminotransferase, and aspartate aminotransferase and increased liver oxidative stress (malondialdehyde and glutathione). These increases were attenuated by SE treatment. Overexpression of tumor necrosis factor-α was also reversed following SE treatment. Furthermore, CCl4-induced increases in α-smooth muscle actin, a marker for hepatic fibrosis, were attenuated in mice treated with SE. Moreover, SE inhibited CCl4-induced nuclear translocation of hepatic nuclear factor kappa B (NF-κB) p65 and phosphorylation of mitogen-activated protein kinase (MAPK). CONCLUSION: These results suggested that SE prevented CCl4-induced hepatic fibrosis by inhibiting the MAPK and NF-κB signaling pathways.

Potentiating effect of acetaminophen and carbon tetrachloride-induced hepatotoxicity is mediated by activation of receptor interaction protein in mice.

When multiple drugs or chemicals are used in combination, it is important to understand the risk of their interactions and predict potential additive effects. The aim of the current study was to investigate the molecular mechanism(s) accounting for the additive/synergistic effect of combination treatment with acetaminophen (APAP) and carbon tetrachloride (CCl4). Mice were intraperitoneally administered vehicle or 100 mg/kg (5 mL/kg) APAP and 30 min after vehicle or 15 mg/kg (5 mL/kg) CCl4. Sixteen hours after treatment, mice from each group were sacrificed and the livers were removed. CCl4 administration caused slight glycogen depletion; this effect was more pronounced following co-administration of APAP and CCl4. ATP and NADPH levels showed the same trend as glycogen levels. The levels of receptor interacting protein 1 and 3 increased following combination treatment with APAP and CCl4. In contrast, levels of the glutamate cysteine ligase catalytic subunit and glutamate cysteine ligase modifier subunits were not significantly affected by combination treatment. APAP and CCl4 co-administration potentiated the phosphorylation of c-Jun N-terminal kinase and p38 kinases, although phosphorylated activation of extracellular signal-regulated kinase was not changed. Our results suggest that APAP and CCl4 co-administration potentiates hepatotoxicity in an additive/synergistic manner via receptor interacting protein activation.

Non-toxic Level of Acetaminophen Potentiates Carbon Tetrachloride-Induced Hepatotoxicity in Mice.

A wide range of medications are routinely used to maintain and improve human health. Hence, it is essential that we understand and predict adverse effects caused by the combined use of multiple medications. In the present study, we investigated whether the combination of carbon tetrachloride (CCl4) and acetaminophen (APAP) had a detrimental effect on the liver. Mice injected with APAP (100 mg/kg) showed no significant changes in hepatic injury markers (alanine aminotransferase and aspartate aminotransferase), histopathological findings, pro-inflammatory cytokine levels, or hepatic oxidative stress. In contrast, a single injection of CCl4 (15 mg/kg) led to a significant increase in hepatic injury, in addition to an increase in pro-inflammatory cytokine levels and oxidative stress. Co-administration of APAP and CCl4 resulted in exacerbation of these hepatic injuries. Our results suggest that a non-toxic dose of APAP has the potential to increase CCl4-induced liver damage and oxidative stress.

Retinoic acid receptor agonist activity of naturally occurring diterpenes.

Recent accumulating evidence indicates that all-trans retinoic acid (ATRA) may be useful for preventing or treating inflammation, allergy, and autoimmune diseases, despite its severe side effects. In this study, screening of 99 crude drugs for retinoic acid receptor (RAR) ligands by luciferase reporter assay demonstrated that the methanol extract of Aralia cordata Rhizoma most effectively activates the transcriptional activity of RARα. Pimaradienoic acid (ent-pimara-8(14),15-dien-19-oic acid) was subsequently isolated as the constituent capable of activating RAR. Pimaric acid and abietic acid, which have similar structures to pimaradienoic acid, were also found to be novel RAR agonists, although abietic acid only slightly activated peroxisome proliferator-activated receptor gamma. These three natural RAR agonists with diterpene structures, while structurally different from ATRA, were able to increase the mRNA levels of the constitutive androstane receptor in HepG2 cells, induce F9 cell differentiation followed by Cyp26a1 mRNA expression, and differentiate HL-60 cells via RAR activation in a different manner from ATRA. These results demonstrate that some diterpenes exist as naturally occurring RAR agonists and that the differences in chemical structure between ATRA and these diterpenes may induce distinct gene activation and a specific cellular response.

Iridoid content and biological activities of Veronica cuneifolia subsp. cuneifolia and V. cymbalaria.

CONTEXT: The genus Veronica L. (Plantaginaceae) is represented by 79 species, 26 of which are endemic in Turkey. Some Veronica species are used for the treatment of different inflammatory diseases and cancer in traditional medicine. In addition, chemotaxonomy of the genus is important for the reclassification of the family Plantaginaceae after different phylogenetic studies. OBJECTIVE: Veronica cuneifolia subsp. cuneifolia D. Don and V. cymbalaria Bodard were studied from the view point of iridoid glucosides which are known as chemotaxonomical markers for this genus. Radical scavenging and cytotoxic activities of the extracts were also determined in this study. MATERIAL AND METHODS: Major compounds, isolated from iridoid fractions of V. cuneifolia subsp. cuneifolia were used as the standard compounds for HPLC after determination of their structures, and investigated for their presence in iridoid fractions of V. cymbalaria. Additionally, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and SO radical scavenging and cytotoxic activities against three cancer and a noncancerous cell lines of both extract were also tested using the MTT method. RESULTS: While aucubin, catalpol, verproside, amphicoside, verminoside, and veronicoside were obtained from V. cuneifolia subsp. cuneifolia, two more iridoid glucosides, 6-O-veratroylcatalposide and 6-O-isovanilloylcatalpol, were isolated from V. cymbalaria. Comparing both species, V. cuneifolia subsp. cuneifolia showed stronger radical scavenging and cytotoxic activities than V. cymbalaria. DISCUSSION: Our results demonstrated that the iridoid contents of both species were very close to each other confirming to the chemotaxonomic studies on Veronica species and their different bioactivity range make the plants interesting from the view point of natural drug discovery research.

Antinociceptive, free radical-scavenging, and cytotoxic activities of Acanthus hirsutus Boiss.

In vivo antinociceptive and in vitro radical scavenging and cytotoxic activities of Acanthus hirsutus Boiss. aqueous extract were investigated to give a new insight into plant usage in traditional medicine. The extract showed significant antinociceptive activity in acetic acid-induced writhing test in mice after oral application and did not change the hind-leg retraction period in the hot-plate test for any dose applied. In addition, the extract showed radical-scavenging activity against 2,2-diphenyl-1-picryl-hydrazyl, nitric oxide, and superoxide radicals similar to those of standard compounds 3-t-butyl-4-hydroxyanisole, ascorbic acid (vitamin C), and quercetin. The gallic acid equivalent total phenolic content of the plant was found to be 65.4 mg/g dry extract. Cytotoxic activity of the aqueous extract was tested against 3 different cancer cell lines-Hep-2 (human larynx epidermoid carcinoma), RD (human rhabdomyosarcoma), and L20B (transgenic murine L cells)-and 1 noncancerous cell line (VERO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Through the phytochemical studies, the following compounds were isolated: 3 lignan glucosides [(-)-syringaresinol-di-O-ß-glucopyranoside, (-)-medioresinol-di-O-ß-D-glucopyranoside, (-)-pinoresinol-4'-O-ß-glucopyranoside], 2 benzoxazinoids [2-hydroxy-1,4-benzoxazin-3(4H)-one, (2R)-2-O-ß-glucopyranosyl-1,4-benzoxazin-3(4H)-one], 4 phenylethanoid glycosides (acteoside, leucosceptoside A, martynoside, hattushoside), and 2 phenylpropanoid glucosides (sinapyl aldehyde-4-O-ß-glucopyranoside, sinapyl alcohol-4-O-ß-glucopyranoside). Cytotoxic and radical-scavenging activities of the isolated compounds were also determined. 2-hydroxy-1,4-benzoxazin-3(4H)-one and acteoside were the most active compounds in both experiments.

The herbal medicine compound falcarindiol from Notopterygii Rhizoma suppresses dendritic cell maturation.

Dendritic cells (DCs) are important for regulating the immune response. We report an herbal medicine compound called falcarindiol that affects DC function. Ethanol extracts of 99 crude drugs that are the main components of 210 traditional Japanese medicines (Kampo medicine) approved by the Ministry of Health, Labor and Welfare in Japan were prepared and screened using the murine epidermal-derived Langerhans cell line XS106. Notopterygii Rhizoma strongly suppressed major histocompatibility complex (MHC) class II expression in XS106 cells. Activity-guided fractionation led to the isolation and identification of falcarindiol as a principal active compound in Notopterygii Rhizoma. Falcarindiol (1-5 microM) dose-dependently suppressed MHC II expression in XS106 cells. Fresh-isolated bone marrow-derived DCs were examined for the production of MHC II, CD80, CD86, interleukin (IL)-12p70, and IL-10. Treatment of bone marrow-derived DCs with 5 muM falcarindiol significantly inhibited lipopolysaccharide-induced phenotype activation and cytokine secretion and inhibited MHC II expression by CD40 ligation, but not phorbol 12-myristate 13-acetate + ionomycin or IL-12. Falcarindiol inhibited DC maturation by blocking the canonical pathway of nuclear factor-kappaB and phosphorylated p38. Topical application of 0.002 and 0.01% falcarindiol before sensitization dose-dependently suppressed delayed-type hypersensitivity to ovalbumin (p < 0.01). Falcarindiol induces immunosuppressive effects in vitro and in vivo and might be a novel therapy for autoimmune or allergic diseases.

Purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin by high-speed countercurrent chromatography.

Curcuminoids are substances of great interest because of their important pharmacological activities, particularly anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activities. In this study, we report the first procedure and effect of processing for the high, efficient, and useful purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric powder. Purification involves high-speed countercurrent chromatographic (HSCCC) separation of these curcuminoids using a simple two-phase solvent system composed of n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v). The HSCCC-fractionated effluent peaks indicated that the peak resolutions were 1.7 between curcumin and demethoxycurcumin and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded turmeric powder. These purified substances were analyzed by liquid chromatography-tandem mass spectrometry with scan and daughter scan negative modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (>98% purity). Moreover, the antioxidant effect of curcuminoids was measured by a 1,1-diphenyl-2-picrylhydrazil assay. The order of antioxidant activity was purified curcumin > purified demethoxycurcumin > purified bisdemethoxycurcumin > turmeric powder. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin can be used for various evaluations of their pharmacological activities.

Phellifuropyranone A: a new furopyranone compound isolated from fruit bodies of wild Phellinus linteus.

A new furopyranone, phellifuropyranone A, was isolated from fruit bodies of wild Phellinus linteus as well as phelligridin G, and their chemical structures were determined by various spectroscopic methods including measurement of NMR spectra. Phellifuropyranone A together with meshimakobnol A and meshimakobnol B showed antiproliferative activity against mouse melanoma cells and human lung cancer cells in vitro.

Sesquiterpene lactone suppresses vascular smooth muscle cell proliferation and migration via inhibition of cell cycle progression.

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration are involved in restenosis following percutaneous transluminal angioplasty (PTCA) as well as in the development and progression of atherosclerosis. We investigated the mechanisms underlying the inhibitory effect of the sesquiterpene 3-oxo-5alphaH,8betaH-eudesma-1,4(15),7(11)-trien-8,12-olide (1) on rat VSMC proliferation and migration. VSMCs were isolated from rat aorta, and then the effect of 1 on cell proliferation and migration was examined using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and chemotaxis assays, respectively. Compound 1 had a potent inhibitory effect on fetal calf serum-induced VSMC proliferation. This effect correlated with reduced expression of cyclin D(1). In addition, 1 also inhibited platelet derived growth factor (PDGF)-induced migration of VSMCs. These results indicate that 1 is a promising candidate for additional biological evaluation to further define its potential as an inhibitory modulator of VSMC responses that contribute to restenosis following PTCA and to the development and progression of atherosclerosis.

Chemopreventive action of xanthone derivatives on photosensitized DNA damage.

Photosensitized DNA damage participates in solar-UV carcinogenesis, photogenotoxicity and phototoxicity. A chemoprevention of photosensitized DNA damage is one of the most important methods for the above phototoxic effects. In this study, the chemopreventive action of xanthone (XAN) derivatives (bellidifolin [BEL], gentiacaulein [GEN], norswertianin [NOR] and swerchirin [SWE]) on DNA damage photosensitized by riboflavin was demonstrated using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. GEN and NOR effectively inhibited the formation of piperidine-labile products at consecutive G residues by photoexcited riboflavin, whereas BEL and SWE did not show significant inhibition of DNA damage. The four XAN derivatives decrease the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidative product of G, by photoexcited riboflavin. The preventive action for the 8-oxodGuo formation of these XAN derivatives increased in the following order: GEN>NOR>>BEL>SWE. A fluorescence spectroscopic study and ab initio molecular orbital calculations suggested that the prevention of DNA photodamage is because of the quenching of the triplet excited state of riboflavin by XAN derivatives through electron transfer. This chemoprevention is based on neither antioxidation nor a physical sunscreen effect; rather, it is based on the quenching of a photosensitizer. In conclusion, XAN derivatives, especially GEN, may act as novel chemopreventive agents by the quenching mechanism of an excited photosensitizer.

Antioxidants from rape (Brassica campestris vir. Japonica Hara) oil cake.

A simple but novel compound, S-1-methoxy-1-(3,5-dimethoxy-4-hydroxyphenyl)ethane, was isolated as a moderately antioxidative compound from rape (Brassica campestris L. subsp. napus) oil cake together with 5 known compounds. Three of these compounds, indolacetonitrile, 4-hydroxyindolacetonitrile, and 4-hydroxyphenylacetonitrile, showed strong antioxidative activity evaluated by the ferric thiocyanate method.

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon.

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.

Multiple mechanisms involved in the inhibition of proinflammatory cytokine production from human monocytes by N-(p-coumaroyl)serotonin and its derivatives.

We have reported that N-(p-coumaroyl)serotonin(CS) isolated from safflower oil cake (Carthamus tinctorius L.) inhibits the production of proinflammatory cytokines by endotoxin (LPS)- stimulated human monocytes. In this study, the effects of CS and its three derivatives, N-(trans-cinnamoyl)serotonin (Cin.S), N-(trans-cinnamoyl)tryptamine (Cin.T), and N-(p-coumaroyl)tryptamine (CT) on the production of proinflammatory cytokines were compared. Cin.S possessed radical scavenging activity at a comparable level to CS, while CT and Cin.T exhibited lower activity, suggesting that hydroxyl group in serotonin is essential for the antioxidative activity. CS and CT strongly inhibited the production of proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, and TNF-alpha) from LPS-stimulated human monocytes. However, Cin.S inhibited the production of only IL-1alpha and IL-1beta, and Cin.T inhibited none of these cytokines production. CS and CT markedly inhibited the protein synthesis in monocytes, the inhibitory effect of Cin.S was moderate, and that of Cin.T was quite weak. These results indicate that CS and its derivatives inhibit the production of proinflammatory cytokines through multiple mechanisms.