B16 Melanoma 4A5 Research Articles

Structure-activity relationship study of 4,5-didehydroguadiscine, an aporphine alkaloid showing potent melanogenesis-inhibitory activity in B16 melanoma cells

Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure–activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b–12f) of 12a and five benzylisoquinoline analogs (13b–13f) lacking the C11a–C11b bond of 12b–12f were prepared. The inhibitory effects of 12b–12f and 13b–13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b–12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b–13f were significantly inferior to those of 12a and 12b–12f. These results suggest that the C11a–C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a–12e.

Chloroacridine derivatives as potential anticancer agents which may act as tricarboxylic acid cycle enzyme inhibitors

PurposeThis paper concerns the cytotoxicity of 9-chloro-1-nitroacridine (1a) and 9-chloro-4-methyl-1-nitroacridine (1b) against two biologically different melanoma forms: melanotic and amelanotic. Melanomas are tumors characterized by high heterogeneity and poor susceptibility to chemotherapies. Among new analogs synthesized by us, compound 1b exhibited the highest anticancer potency. Because of that, in this study, we analyzed the mechanism of action for 1a and its 4-methylated derivative, 1b, against a pair of biological melanoma forms, with regard to proliferation, cell death mechanism and energetic state. MethodsCytotoxicity was evaluated by XTT assay. Cell death was estimated by plasma membrane structure changes (phosphatidylserine externalization), caspase activation, and ROS presence. The energetic state of cells was estimated based on NAD and ATP levels, and the activity of tricarboxylic acid cycle enzymes (pyruvate dehydrogenase complex, aconitase, isocitrate dehydrogenase). ResultsThe chloroacridines affect biological forms of melanoma in different ways. Amelanotic (Ab) melanoma (with inhibited melanogenesis and higher malignancy) was particularly sensitive to the action of the chloroacridines. The Ab melanoma cells died through apoptosis and through death without caspase activation. Diminished activity of TAC enzymes was noticed among Ab melanoma cells together with ATP/NAD depletion, especially in the case of 1b. ConclusionOur data show that the biological forms of the tumors responded to 1a and its 4-methylated analog in different ways. 1a and 1b could be inducers of regulated melanoma cell death, especially the amelanotic form. Although the mechanism of the cell death is not fully understood, 1b may act by interfering with the TAC enzymes and blocking specific pathways leading to tumor growth. This could encourage further investigation of its anticancer activity, especially against the amelanotic form of melanoma.

Impedimetric melanoma invasion assay device using a simple paper membrane and stencil-printed electrode on PMMA substrate

The transwell assay is currently the most popular approach to studying cellular invasion due to its ease of use and readout, and the possibility for quantitative measurements. However, it only allows end-point measurements without the possibility for real-time tracking of the dynamics of cell movement during an invasion. Moreover, it requires cell labeling, and construction of customized devices is hampered by the commercial standard membrane inserts, only available in certain designs. Recently, paper has been used as a scaffold for three-dimensional (3D) cell cultures. Because of its microfibrous structure and easy handling, it could be a versatile alternative as a membrane insert in customized devices. Here, we develop a low-cost real-time invasion assay device using paper as an alternative membrane insert. The device was designed for two-electrode impedance measurements and fabricated using CNC micromilling. It also comprised a disposable low-cost stencil-printed working electrode on a poly(methyl methacrylate) substrate below the membrane and glassy carbon counter electrode above the membrane inserted in a specially designed lid. Thus, the impedance measurements during cell invasion addressed the entire membrane. We demonstrated the function of the device by monitoring the invasion of B16 melanoma 4A5 cells from a mouse using insulin growth factor-1 as the chemoattractant. The cell invasion on paper was visualized using scanning electron microscopy and confocal microscopy with Z-stack 3D imaging. Melanoma cell invasion could be observed within 7 h after the chemoattractant treatment, which was faster than the conventional assay and less likely to be influenced by cell proliferation.

Anticancer Properties of Amino Acid and Peptide Derivatives of Mycophenolic Acid.

Although Mycophenolic Acid (MPA) is applied as prodrugs in clinic as an immunosuppressant, it also possesses anticancer activity. MPA acts as Inosine-5'-Monophosphate Dehydrogenase (IMPDH) inhibitor, where the carboxylic group at the end of the side chain interacts with Ser 276 of the enzyme via hydrogen bonds. Therefore, MPA derivatives with other polar groups indicated high inhibition too. On the other hand, potent anticancer agents like dacarbazine and cisplatin give numerous side-effects. Based on the literature data, MPA derivatives should be explored towards anticancer properties. Conversion of the carboxylic group of MPA to amide could maintain antiproliferative activity. Therefore, we decided to investigate several amino acid and peptide derivatives of MPA against chosen cancer cell lines in vitro. Amides of MPA hold threonine and arginine amino acid unit. These amino acid derivatives were tested as L and D enantiomers and both in free acid and methyl esters forms. Additionally, MPA was modified with tuftsin or retro-tuftsin as biologically active peptides, which could act as a drug carrier. Amino acid and peptide derivatives of MPA were investigated in vitro as potential anticancer agents on cell lines: Ab melanoma, A375 melanoma and SHSY5Y neuroblastoma. The activity of the tested compounds was compared to parent MPA and known chemotherapeutics: dacarbazine and cisplatin. Amino acid moiety and the sequence of amino acids in the peptide part influenced observed activity. The most active amino acid MPA analogues occurred to be D and L-threonine derivatives as methyl esters, probably due to better cell membrane penetration.

Anti-melanogenic activity of methanolic extract from leaves of Sorbaria sorbifolia var. stellipila Max. on \u03b1-MSH-stimulated B16 melanoma 4A5 cells

BackgroundMelanin is a pigment, which is widely distributed in organisms. In humans, melanin pigments determine eye, hair, and skin color and protect the skin from damage by ultraviolet radiation; however, immoderate distribution of melanin in the skin causes discoloration. In the present study, we screened methanolic extracts from leaves of 47 plant species, most of which are native to East Asia, for the inhibitory activity against melanogenesis.MethodsB16 melanoma 4A5 cells were used in all assays conducted in this study. Melanin content assay was performed using methanolic extracts from leaves of 47 plant species. Cytotoxicity of the extract from leaves of Sorbaria sorbifolia var. stellipila Max. (SME) was evaluated by WST-8 assay. Tyrosinase activity was determined using the lysate of α-MSH-stimulated B16 melanoma 4A5 cells and L-dopa as a colorimetric substrate. Melanogenic gene expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsSME showed the highest inhibitory activity among tested samples without cytotoxicity. SME exhibited the inhibition potency for the enzymatic activity of tyrosinase. In addition, qRT-PCR result displayed that SME downregulates the expression of melanogenic genes including tyrosinase, TRP-1, TRP-2, and the transcription factor MITF.ConclusionOverall results revealed that the extract from leaves of S. sorbifolia var. stellipila Max. has a potential to be used as a skin whitening agent.

Synthesis and Biological Evaluation of Acridine/Acridone Analogs as Potential Anticancer Agents.

The lack of efficacious therapy for advanced melanoma and neuroblastoma makes new approaches necessary. Therefore, many scientists seek new, more effective, more selective and less toxic anticancer drugs. We propose the synthesis of the new functionalized analogs of 1-nitroacridine/4- nitroacridone connected to tuftsin/retro-tuftsin derivatives as potential anticancer agents. Acridine and acridone analogues were prepared by Ullmann condensation and then cyclization reaction. As a result of nucleophilic substitution reaction 1-nitro-9-phenoxyacridine or 1- chloro-4-nitro-9(10H)-acridone with the corresponding peptides, the planned acridine derivatives (10a-c, 12, 17-a-d, 19) have been obtained. The cytotoxic activity of the newly obtained analogs were evaluated against melanotic (Ma) and amelanotic (Ab) melanoma cell lines and neuroblastoma SH-SY5Y by using the XTT method. Apoptosis and cell cycle were analyzed by flow cytometry. Among the investigated analogs compound 12 exhibited the highest potency comparable to dacarbazine action for amelanotic Ab melanoma cells. FLICA test (flurochrome-labeled inhibitors of caspases) showed that this analog significantly increased the content of cells with activated caspases (C+) among both neuroblastoma lines and only Ab melanoma line. Using phosphatidylserine (PS) externalization assay, 12 induced changes in the Ab melanoma plasma membrane structure as the externalization of phosphatidylserine (An+ cells). These changes in neuroblastoma cells were less pronounced. Analog 12 could be proposed as the new potential chemotherapeutic against amelanotic melanoma form especially.

Inhibition of melanin production by anthracenone dimer glycosides isolated from Cassia auriculata seeds

The methanol extract of Cassia auriculata seeds was found to inhibit melanogenesis in B16 melanoma 4A5 cells under conditions of theophylline stimulation. Two new phlegmacin-type anthracenone dimer glycosides, auriculataosides A and B, were isolated from the active methanol fraction, and their inhibitory effects were observed in the concentration range of 0.03 to 0.3 μM. Inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression was observed, suggesting that the inhibition of these factors is part of the mechanism of action underlying melanogenesis inhibition.

Anti-proliferative effect of auriculataoside A on B16 melanoma 4A5 cells by suppression of Cdc42-Rac1-RhoA signaling protein levels.

Auriculataoside A, an anthracenone dimer glycoside isolated from Cassia auriculata seed, shows anti-proliferative effects on cell line B16 melanoma 4A5 cells with an IC50 value of 0.82μM. However, it shows no such effect on normal human dermal fibroblast (HDF) cells. To evaluate the mode of action underlying the anti-proliferative effect of auriculataoside A on cells, we examined changes in whole protein expression after treatment with auriculataoside A and found that the expression Cdc42, RhoA, and Rac1, which are Rho family GTPases, was reduced. Auriculataoside A also arrested the cell cycle at G1 phase. These results suggest that the suppression of the above proteins induced G1 arrest. In addition, auriculataoside A also suppressed the expression of β-catenin and c-Myc proteins. This action of auriculataoside A could be one of the mechanisms underlying its selective anti-proliferative effect on B16 melanoma cells.

Melanization of Bomirski hamster amelanotic melanoma cells (Ab line) depends on the type of culture medium.

The effect of melanogenesis intensity on melanoma biology remains an open question, and the biological differences between melanotic and amelanotic melanoma cells have not yet been satisfactorily documented. As a result, the melanization of melanoma cells in in vitro cultures is not considered among experimental procedures. The aim of this study was to investigate the effect of the medium used to culture Bomirski amelanotic Ab melanoma cells on the melanogenesis process. Amelanotic melanoma cells (Ab) were cultured in two media recommended for in vitro melanoma cell cultures, RPMI and DMEM. The melanization was evaluated by determining the melanin and tyrosinase presence in the cells using spectrophotometrical and western blot methods, respectively. Changes in Ab melanoma cells' ultrastructure were determined using electron microscopy (EM). The medium with higher level of tyrosine (DMEM) induced significant melanization of amelanotic melanoma cells (Ab) after only 24 h, while the RPMI medium, with a lower level of tyrosine, weakly affected melanin production. Melanization of Ab cells was paralleled by an increase in the amount of tyrosinase protein. Induced melanization was easily observed on EM-micrographs in the form of newly formed melanosomes containing melanin pigment. Melanosomes at stages from one (I) to four (IV) were observed. Culture medium has an important effect on the in vitro biology of amelanotic melanoma cells, since it can affect the rate of cellular melanization. The appropriate medium should be carefully selected, taking into account the known biology of the melanoma cells being used.

Novel therapeutic compound acridine\u2013retrotuftsin action on biological forms of melanoma and neuroblastoma

PurposeAs a continuation of our search for anticancer agents, we have synthesized a new acridine-retrotuftsin analog HClx9-[Arg(NO2)-Pro-Lys-Thr-OCH3]-1-nitroacridine (named ART) and have evaluated its activity against melanoma and neuroblastoma lines. Both tumors develop from cells (melanocytes, neurons) of neuroectodermal origin, and both are tumors with high heterogeneity and unsatisfactory susceptibility to chemotherapies. Thus, we analyzed the action of ART on pairs of biological forms of melanoma (amelanotic and melanotic) and neuroblastoma (dopaminergic and cholinergic) with regard to proliferation, mechanism of cell death, and effect on the activity of tricarboxylic acid cycle (TAC) enzymes.MethodsThe cytotoxicity of ART was evaluated by XTT and trypan blue tests. Cell death was estimated by plasma membrane structure changes (phosphatidylserine and calreticulin externalization), caspase activation, presence of ROS (reactive oxygen species), activity of tricarboxylic acid cycle enzymes (pyruvate dehydrogenase complex, aconitase, and isocitrate dehydrogenase), NAD level, and ATP level.ResultsART influences the biological forms of melanoma and neuroblastoma in different ways. Amelanotic (Ab) melanoma (with the inhibited melanogenesis, higher malignancy) and SHSY5Y neuroblastoma (with cholinergic DC cells) were especially sensitive to ART action. The Ab melanoma cells died through apoptosis, while, with SH-SY5Y-DC neuroblastoma, the number of cells decreased but not as a result of apoptosis. With Ab melanoma and SH-SY5Y-DC cells, a diminished activity of TAC enzymes was noticed, along with ATP/NAD depletion.ConclusionOur data show that the biological forms of certain tumors responded in different ways to the action of ART. As a combination of retrotuftsin and acridine, the compound can be an inducer of apoptotic cell death of melanoma, especially the amelanotic form. Although the mechanism of the interrelationships between energy metabolism and cell death is not fully understood, interference of ART with TAC enzymes could encourage the further investigation of its anticancer action.

Structural Requirements of Alkylglyceryl-l-Ascorbic Acid Derivatives for Melanogenesis Inhibitory Activity.

l-Ascorbic acid has multifunctional benefits on skin aesthetics, including inhibition of melanin production, and is widely used in cosmetics. It, however, has low stability and poor skin penetration. We hypothesize that alkylglyceryl-l-ascorbic acid derivatives, highly stable vitamin C–alkylglycerol conjugates, would have similar anti-melanogenic activity with better stability and penetration. We test 28 alkylglyceryl-l-ascorbic acid derivatives (1–28) on theophylline-stimulated B16 melanoma 4A5 cells to determine if they inhibit melanogenesis and establish any structure–function relationships. Although not the most potent inhibitors, 3-O-(2,3-dihydroxypropyl)-2-O-hexyl-l-ascorbic acid (6, IC50 = 81.4 µM) and 2-O-(2,3-dihydroxypropyl)-3-O-hexyl-l-ascorbic acid (20, IC50 = 117 µM) are deemed the best candidate derivatives based on their inhibitory activities and low toxicities. These derivatives are also found to be more stable than l-ascorbic acid and to have favorable characteristics for skin penetration. The following structural requirements for inhibitory activity of alkylglyceryl-l-ascorbic acid derivatives are also determined: (i) alkylation of glyceryl-l-ascorbic acid is essential for inhibitory activity; (ii) the 3-O-alkyl-derivatives (2–14) exhibit stronger inhibitory activity than the corresponding 2-O-alkyl-derivatives (16–28); and (iii) derivatives with longer alkyl chains have stronger inhibitory activities. Mechanistically, our studies suggest that l-ascorbic acid derivatives exert their effects by suppressing the mRNA expression of tyrosinase and tyrosine-related protein-1.

Labdane-Type Diterpenes, Galangalditerpenes A-C, with Melanogenesis Inhibitory Activity from the Fruit of Alpinia galanga.

In our continuing study of biologically active natural products from the fruit of Alpinia galanga (Zingiberaceae), we newly isolated three new labdane-type diterpenes, termed galangalditerpenes A–C (1–3), along with four known sesquiterpenes (4–7) and two diterpenes (8 and 9). The stereostructures of 1–3 were elucidated on the basis of their spectroscopic properties. The melanogenesis inhibitory activities in theophylline-stimulated murine B16 melanoma 4A5 cells of these isolates, including the new diterpenes (1–3, IC50 = 4.4, 8.6, and 4.6 μM, respectively), were found to be more than 6–87-fold higher than that of arbutin (174 μM), a commercially available positive control.

Melanogenesis Inhibitory Activity of Diterpenoid and Triterpenoid Constituents from the Aerial Part of Isodon trichocarpus

A methanol extract from the aerial part of Isodon trichocarpus (Labiatae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50 = 1.6 μg/mL). From the extract, nine diterpenoids (1–9) and four triterpenoids (10–13) were isolated. Among the isolates, enmein (1, IC50 = 0.22 μM), isodocarpin (2, 0.19 μM), nodosin (4, 0.46 μM), and oridonin (6, 0.90 μM) showed an inhibitory effect without notable cytotoxicity at the effective concentrations. These diterpenoids (1, 2, 4, and 6) inhibited the expression of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA, which could be the mechanism of melanogenesis inhibitory activity.

Triterpenoid saponins from Polaskia chichipe Backbg. and their inhibitory or promotional effects on the melanogenesis of B16 melanoma cells.

Five new oleanane-type saponins 1-5 together with a known saponin 6 and a steroidal glycoside 7 were isolated from Polaskia chichipe Backbg., and their structures were determined from their 1D and 2D NMR and HRFABMS spectral data. The six isolated saponins 1-6 were tested for their effects on the melanogenesis of B16 melanoma 4A5 cells. Compound 1 exerted an inhibitory effect at 100μM whereas compound 3 promoted melanogenesis at the same concentration, even though these two compounds contain the same aglycon structure. The dose-dependent activities of compounds 1 and 3 on melanin synthesis were investigated.

Melanogenesis inhibitory activity of a 7-O-9′-linked neolignan from Alpinia galanga fruit

An aqueous acetone extract from the fruit of Alpinia galanga (Zingiberaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50=7.3μg/mL). Through bioassay-guided separation of the extract, a new 7-O-9′-linked neolignan, named galanganol D diacetate (1), was isolated along with 16 known compounds including 14 phenylpropanoids (2–15). The structure of 1, including its absolute stereochemistry in the C-7 position, was elucidated by means of extensive NMR analysis and total synthesis. Among the isolates, 1 (IC50=2.5μM), 1′S-1′-acetoxychavicol acetate (2, 5.0μM), and 1′S-1′-acetoxyeugenol acetate (3, 5.6μM) exhibited a relatively potent inhibitory effect without notable cytotoxicity at effective concentrations. The following structural requirements were suggested to enhance the inhibitory activity of phenylpropanoids on melanogenesis: (i) compounds with 4-acetoxy group exhibit higher activity than those with 4-hydroxy group; (ii) 3-methoxy group dose not affect the activity; (iii) acetylation of the 1′-hydroxy moiety enhances the activity; and (iv) phenylpropanoid dimers with the 7-O-9′-linked neolignan skeleton exhibited higher activity than those with the corresponding monomer. Their respective enantiomers [1′ (IC50=1.9μM) and 2′ (4.5μM)] and racemic mixtures [(±)-1 (2.2μM) and (±)-2 (4.4μM)] were found to exhibit melanogenesis inhibitory activities equivalent to those of the naturally occurring optical active compounds (1 and 2). Furthermore, the active compounds 1–3 inhibited tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA expressions, which could be the mechanism of melanogenesis inhibitory activity.

Simultaneous quantitative analysis of 12 methoxyflavones with melanogenesis inhibitory activity from the rhizomes of Kaempferia parviflora.

A methanol extract from the rhizomes of Kaempferia parviflora Wall. ex Baker (Zingiberaceae) has shown inhibitory effects against melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells (IC50 = 9.6 μg/mL). Among 25 flavonoids and three acetophenones isolated previously (1-28), several constituents including 5-hydroxy-7,3',4'-trimethoxyflavone (6, IC50 = 8.8 μM), 5,7,3',4'-tetramethoxyflavone (7, 8.6 μM), 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (12, 2.9 μM), and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (13, 3.5 μM) showed inhibitory effects without notable cytotoxicity at the effective concentrations. Compounds 6, 7, 12, and 13 inhibited the expression of tyrosinase, tyrosine-related protein (TRP)-1, and TRP-2 mRNA, which could be the mechanism of their melanogenesis inhibitory activity. In addition, a quantitative analytical method for 12 methoxyflavones (1, 2, 4-11, 13, and 14) in the extract was developed using HPLC. The optimal condition for separation and detection of these constituents were achieved on an ODS column (3 μm particle size, 2.1 mm i.d. × 100 mm) with MeOH-0.1 % aqueous acetic acid solvent systems as the mobile phase, and the detection and quantitation limits of the method were estimated to be 0.08-0.66 ng and 0.22-2.00ng, respectively. The relative standard deviation values of intra- and interday precision were lower than 0.95 and 1.08 %, respectively, overall mean recoveries of all flavonoids were 97.9-102.9 %, and the correlation coefficients of all the calibration curves showed good linearity within the test ranges. For validation of the protocol, extracts of three kinds of the plant's rhizomes collected from different regions in Thailand (Leoi, Phetchabun, and Chiang Mai provinces) were evaluated. The results indicated that the assay was reproducible, precise, and could be readily utilized for the quality evaluation of the plant materials.

Phenylethanoid and phenylpropanoid glycosides with melanogenesis inhibitory activity from the flowers of Narcissus tazetta var. chinensis

A methanol extract of the flowers of Narcissus tazetta var. chinensis Roem. (Amaryllidaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the extract, four new phenylethanoid glycosides, tazettosides A–D (1–4), and a new phenylpropanoid glycoside, tazettoside E (5), were isolated along with 23 known compounds (6–28). Of the isolates, 1 (IC50 = 22.0 μM) and 4 (82.5 μM), 3-methoxy-8,9-methylenedioxy-3,4-dihydrophenanthridine (13, IC50 = 28.5 μM), 5,6-dihydrobicolorine (14, 23.7 μM), tazettine (16, 60.8 μM), benzyl β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (18, 27.8 μM), 2-(3,4-dimethoxyphenyl)ethyl β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (21, 74.6 μM), 3-phenylpropyl β-D-glucopyranoside (22, 59.0 μM), and cinnamyl β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (24, 88.0 μM) showed inhibitory effects without notable cytotoxicity at the effective concentrations.

Inhibitors of melanogenesis in B16 melanoma 4A5 cells from flower buds of Lawsonia inermis (Henna).

The methanolic extract of Lawsonia inermis L. (henna) showed significant inhibitory activity toward melanogenesis in B16 melanoma 4A5 cells. Among the constituents isolated from the methanolic extract, luteolin, quercetin, and (±)-eriodictyol showed stronger inhibitory activity than the reference compound, arbutin. Several structure-activity relationships of the flavonoids were suggested, and OGlc<H=OH at the 3-position, OGlc<OH at the 4'-position, and the double bond between the 2- and 3-positions were important. The active constituents suppressed tyrosinase, tyrosinase related protein (TRP)-1, and TRP-2 mRNA expression. The suppression was considered as one of the mechanisms of action. Furthermore, the methanolic extract and several constituents, including luteoloside and spiraeoside, showed anti-plasmin activity, which is considered to play a key role in UV-stimulated melanogenesis in human skin.

Dimeric pyrrolidinoindoline-type alkaloids with melanogenesis inhibitory activity in flower buds of Chimonanthus praecox

A methanol extract of the flower buds of Chimonanthus praecox (L.) Link (Calycanthaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the extract, five dimeric pyrrolidinoindoline alkaloids and four sesquiterpenes were isolated, together with 16 known compounds. Among them, (-)-chimonanthine (1, IC50=0.93μM), (-)-folicanthine (2, 1.4μM), and (-)-calycanthidine (3, 1.8μM) showed potent inhibitory effects without notable cytotoxicity at the effective concentrations. The most potent alkaloid (1) inhibited both tyrosinase and tyrosine-related protein-1 mRNA expressions, to which the melanogenesis inhibitory activity would be ascribable.

Lignan Dicarboxylates and Terpenoids from the Flower Buds of Cananga odorata and Their Inhibitory Effects on Melanogenesis

The methanolic extract from the flower buds of Cananga odorata showed an inhibitory effect on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extract, two new lignan dicarboxylates, canangalignans I and II, three new terpenoids, canangaterpenes I, II, and III, and eight known compounds were isolated. The structures of these compounds were elucidated on the basis of chemical/physicochemical evidence. Several mono- and sesquiterpene analogues significantly inhibited melanogenesis. In particular, canangaterpene I and (3R,3aR,8aS)-3-isopropyl-8a-methyl-8-oxo-1,2,3,3a,6,7,8,8a-octahydroazulene-5-carbaldehyde exhibited a potent inhibitory effect on melanogenesis [inhibition (%): 34.7±4.2 (p<0.01), 45.5±5.7 (p<0.01) at 1 μM, respectively] without inducing cytotoxicity. Moreover, the biological effect of these compounds was much stronger than that of the reference compound, arbutin. Thus, these isolated terpenoid derivatives may be promising therapeutic agents for the treatment of several skin disorders.