Melanogenesis Stimulation in Murine B16 Melanoma Cells by Umberiferae Plant Extracts and Their Coumarin Constituents

Abstract

Gray hair is caused by a genetic predisposition, aging, decrement of melanocytes by environmental stress, and decrement of the biosynthesis of melanin pigment or melanogenesis.1,2) Hair dye agents are used for the treatment of gray hair. However, there remain some problems with these agents, such as side effects due to the dyes. Thus, there is a need for anti-gray hair agents that exhibit satisfactory melanogenesis activity and gray hair prevention. For the development of gray hair prevention agents, we have carried out a screening program to find a potential stimulant of melanogenesis from natural resources by using cultured murine B16 melanoma cells with theophylline as a reference drug. In a preceding paper, 3) we reported that a methanolic extract from the leaves of Piper nigrum L. showed a significant stimulatory effect on melanogenesis in cultured murine B16 melanoma cells and that two lignans, (Ϫ)-cubebin and (Ϫ)-3,4-dimethoxy-3,4-desmethylenedioxycubebin, were isolated as its active constituents. As the next step in our herbal screening, we report here the melanogenesis stimulation effect of seven ethanolic extracts obtained from Umbelliferae -dokkatsu), and the fruits of Cinidium monnieri (L.) CUSSON (Jashoshi) and C. formosanum YABE (Taiwan-jashoshi). The melanogenesis stimulation effects of sixteen coumarins (1-16, Fig. 1) isolated from the Umberiferae crude drugs were also evaluated. MATERIALS AND METHODS MaterialsThe roots of Angelica biserrata (Todokkatsu), Notopterygium incisum (Kyokatsu), the fruits of Cinidium monnieri (Jashoshi) and Cinidium formosanum (Taiwan-jashoshi) were obtained from Osaka market. The roots of Angelica dahurica (Byakushi) and Heracleum candicans (Hakuryo-dokkatsu) were obtained from Chinese market (Yunnan Province). The roots of Heracleum lanatum (Gyubi-dokkatsu) were grown at the Osaka University of Pharmaceutical Sciences.Preparation of Ethanolic Extracts Seven crude drugs (each 10 g) were extracted with EtOH (100 ml) in a water bath at 70°C for 2 h and then filtrated. Each solution was concentrated under reduced pressure to give the corresponding extract: To-dokkatsu (1.00 g), Kyokatsu (1.45 g), Byakushi (0.70 g), Hakuryo-dokkatsu (1.00 g), Gyubidokkatsu (1.17 g), Jashoshi (0.40 g), and Taiwan-jashoshi (0.40 g).Drugs Fourteen coumarins (1-7, 9, 11-16, Fig. 1) were isolated from the Umbelliferae crude drugs.4-10) The isolation yields of each compound are depicted in Table 1. Synthetic melanin and 0.4 M HEPES buffer (pH 6.8) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Dulbecco's modified Eagle medium (D-MEM), Antibiotic-Antimycotic (a mixture of 10000 U/ml penicillin, 10000 mg/ml streptomycin sulfate, and 25 mg/ml amphotericine B), Dulbecco's phosphate buffered saline (Ca Isolation of Heraclenin (8) and Heraclenol (10) from Hakuryo-dokkatsu (H. candicans WALL.) Powdered Hakuryo-dokkatsu (500 g) was successively extracted with hexane (5 l) and EtOAc (5 l) under reflux for 2 h. Each extract was evaporated under reduced pressure to give a hexane ex-

Modifying skin pigmentation – approaches through intrinsic biochemistry and exogenous agents

“…The flavone quercetin, originally described as a tyrosinase inhibitor [121], turned out to be a strong inducer of melanogenesis in vitro [122]. Although some hydroxylated derivatives of coumarins were reported to inhibit melanogenesis [123], several coumarins of seven umberiferae plants stimulated melanogenesis in murine melanoma cells [124]. The number of putative hypo-or hyper-pigmenting agents is vast, but promising effects detected in vitro are often not confirmed in clinical tests.…”

Testosterone 5.ALPHA.-Reductase Inhibitory Active Constituents of Piper nigrum Leaf

“…3,4) Therefore, it was expected that agents which exhibit both melanogenesis stimulation activity and testosterone 5a-reductase inhibitory activity may be desirable ingredients of hair-care cosmetic products for prevention of gray hair and alopecia. During the course of our screening program using cultured murine B16 melanoma cells for the development of gray hair prevention agents from natural resources, we found that extracts of some Piper species (Piperaceae) showed a potent stimulation effect on melanogenesis without any effects on cell proliferation, and two lignans, (Ϫ)-cubebin (1) and (Ϫ)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2), were isolated as active constituents from the leaves of Piper nigrum L. [5][6][7] With the expectation that we might find new desirable agents from Piper species to prevent gray hair and alopecia as described above, the testosterone 5a-reductase inhibitory activity of 50% ethanolic extracts obtained from several different parts of six Piper species (P. nigrum L., P. methysticum FORST., P. betle L., P. kadsura (CHOISY) OHWI, P. longum L., and P. cubeba L.) was examined. This paper deals with the screening results of in vitro testosterone 5a-reductase inhibitory activity of Piper species, the activity-guided fractionation of P. nigrum leaf extract led to the isolation of active lignans, 1 and 2, and the in vivo anti-androgenic activity of the leaf extract.…”

Effect of Angelica gigas extract on melanogenesis in B16 melanoma cells

“…Among the Angelica species, coumarin derivatives extracted from Angelica dahurica have been reported to stimulate (29) or inhibit melanogenesis (30). In the case of Angelica sinensis, melanogenesis was stimulated (31).…”