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.


Introduction
Labdanes, belonging to the bicyclic diterpenoid group, have been found as secondary metabolites in the tissues of fungi, insects, and marine organisms, and in essential oils, resins, and tissues of higher plants. Among plant materials, the gymnosperms, as well as the Asteraceae, Lamiaceae, and Zingiberaceae families, are the most important sources of labdane-type diterpenoids [1,2]. Labdanes have been reported to have a broad spectrum of biological activities, including antimicrobial, antiviral, cytotoxic, radical scavenging, anti-hypertensive, hepatoprotective, and anti-inflammatory activities [1,2]. The Zingiberaceae plant Alpinia galanga Swartz, which is known as Greater Galangal in English and Kulanjan in Hindi, is widely cultivated in China, India, and Southeast Asian countries such as Thailand, Indonesia, and the Philippines [3]. The fruit of this plant has been used for the treatment of stomachache, dyspepsia, emesis, diarrhea, asthma, osteoarthritis, and rheumatoid arthritis in several traditional medicine systems such as Ayurveda and Siddha [3][4][5][6][7][8][9][10]. In the course of our studies on the chemical constituents of A. galanga, we have isolated several phenylpropanoid, neolignan, and sesquineolignan constituents from 80% aqueous acetone extracts of the rhizome [11,12] and the fruit parts of A. galanga [13]. We have also reported that several phenylpropanoids showed gastroprotective [11], antiallergic [14], anti-inflammatory [12,15,16], and melanogenesis inhibitory activities [13]. Further separation of the constituents in the extract from the fruit part allowed us to isolate three new labdane-type diterpenes, galangalditerpenes A (1), B (2), and C (3), along with four known sesquiterpenes (4-7) and two known diterpenes (8 and 9). Here, we describe the isolation and structural elucidation of 1-3 as well as their inhibitory effects on theophylline-stimulated melanogenesis in mouse murine B16 melanoma 4A5 cells.

Structures of Galangalditerpenes A-C (1-3)
Galangalditerpene A (1) was obtained as a white powder with negative optical rotation ([α] 25 D −13.8 in CHCl3). The positive-ion EIMS spectrum of 1 showed a molecular ion peak at m/z 318 (M + ), and the molecular formula was determined as C20H30O3 by high-resolution MS. The UV spectrum exhibited maximum absorption at 230 nm, while the IR spectrum showed absorption bands at 1686 cm −1 ascribable to the conjugated aldehyde function. The 1 H-and 13 C-NMR spectra of 1 (CDCl3, Table 1), which were assigned with the aid of distortion-less enhancement by polarization transfer (DEPT), 1 H-1 H correlation spectroscopy (COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments ( Figure 2 and Figure S1), showed signals assignable to three methyls (

Effects on Theophylline-Stimulated Melanogenesis Inhibitory Activity
Melanin is a broad term for a group of natural pigments found in bacteria, fungi, plants, and animals. It is a heterogeneous, polyphenol-like biopolymer with a complex structure; the color varies from yellow to black through its development. The colors of mammalian skin and hair are determined by several factors, the most important being the degree and distribution of melanin pigmentation. The role of melanin is to protect the skin from ultra-violet (UV) damage by absorbing UV light and removing reactive oxygen species. However, excess production of melanin due to prolonged exposure to sunlight causes dermatological disorders such as melisma, freckles, post-inflammatory melanoderma, and solar lentigines. Melanin is secreted from melanocytes distributed in the basal layer of the dermis. Melanocytes are known to be stimulated by various factors, including UV radiation, α-melanocyte-stimulating hormone (α-MSH), or a phosphodiesterase inhibitor, theophylline [13,[24][25][26][27][28][29]. In our previous investigation of naturally occurring compounds possessing melanogenesis inhibitory activity, we reported that several alkaloids [24][25][26]28], phenylethanoid glycosides [26], phenylpropanoids [13], neolignans [13], methoxyflavones [27], and diterpenes [29] exhibited significant positive effects against theophylline-stimulated melanogenesis in B16 melanoma 4A5 cells. As a continuation of the above study, melanogenesis inhibitors from the fruit of A.  Table 2). The melanogenesis inhibitory activity was found to be more than 6-87-fold higher than that of arbutin (174 µM), a commercially available positive control [13,[24][25][26][27][28][29]. Table 2. Inhibitory effects of the isolates (1-9) from the fruit of A. galanga on theophylline-stimulated melanogenesis and viability in B16 4A5 cells. Each value represents the mean ± S.E.M. (n = 4); asterisks denote significant differences from the control group, * p < 0.05, ** p < 0.01; # cytotoxic effects were observed, and values in parentheses indicate cell viability (%) in MTT assay; commercial arubutin was purchased from Nakalai Tesque Inc., (Kyoto, Japan).

Effects on Mushroom Tyrosinase
Tyrosinase, a copper-containing enzyme widely distributed in microorganisms, animals, and plants, is a key enzyme in melanin biosynthesis and determines the color of skin and hair. It catalyzes the oxidation of both L-tyrosine and L-DOPA, following the oxidation of L-DOPA to dopaquinone and oxidative polymerization via several dopaquinone derivatives to yield melanin. Tyrosinase inhibitors are clinically used for the treatment of several dermatologic disorders associated with melanin hyperpigmentation. In addition, these inhibitors are commonly used as additives in cosmetics for skin whitening and/or depigmentation [13,24,[26][27][28][29]. As shown in Table S1, none of the isolates (1)(2)(3)(4)(5)(6)(7)(8)(9) showed inhibitory activity at effective concentrations when either L-tyrosine or L-DOPA was used as a substrate. This suggests that tyrosinase inhibition is minimally involved in the mechanisms of action of these melanogenesis inhibitors.

Plant Materials
The fruit of Alpinia galanga was collected in Nakhonsithammarat Province, Thailand, in September 2011. The plant material was identified by one of the authors (S.C.), and a voucher specimen (2011.09. Raj-01) of this plant is on file in our laboratory, as described in a previous report [13].

Conversion of Galanolactone (8) into Galangalditerpene B (2)
A solution of 8 (20.0mg, 0.06 mmol) in dry toluene (1.0 mL) at 0 • C was treated with BF 3 ·Et 2 O (100 µL, 0.08 mmol), and the entire mixture was stirred at 0 • C for 30 min. The reaction mixture was poured into cold aqueous saturated NaHCO 3 and extracted with EtOAc. The EtOAc extract was washed with brine, then dried over anhydrous Na 2 SO 4 and filtered. Removal of the solvent under reduced pressure gave a crude product, which was purified by HPLC (CH 3 CN-H 2 O (55:45, v/v)) to furnish 2 (4.5 mg, 23%).

Hydrogenation of Galangalditerpene C (3)
A solution of 3 (5.5 mg, 0.02 mmol) in dry THF (1.0 mL) was treated with H 2 in the presence of 5% Pd/C(en) (5.0 mg), and the entire mixture was stirred at room temperature for 6 h. The catalyst was removed by filtration, and the filtrate was evaporated under reduced pressure to give a crude product, which was purified by HPLC (MeOH-H 2 O (75:25, v/v)) to furnish 3a (5.0 mg, 90%).