Three New Multiflorane-Type Triterpenes from Pumpkin (Cucurbita maxima) Seeds

Three new multiflorane-type triterpenes; 7α-methoxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (1), 7-oxomultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (2), and multiflora-7,9(11)-diene-3α,29-diol 3-p-hydroxybenzoate-29-benzoate (3), were isolated from seeds of Cucurbita maxima, along with three known compounds. Compound 3 and multiflora-7,9(11)-diene-3α-29-diol 3-benzoate (5) exhibited potent inhibitory effects on melanogenesis, with low cytotoxicities, and 2 exhibited single-digit micromolar cytotoxicity against HL-60 and P388 cells.


Introduction
Pumpkins, including Cucurbita moschata, C. pepo, and C. maxima are gourd squashes of the genus Cucurbita and the family Cucurbitaceae. Cucurbita moschata seeds have been used as an anthelmintic [1], and Cucurbita pepo seeds as an anthelmintic and a diuretic [2].
Cucurbita maxima (English name: squash, pumpkin, Japanese name: kabocha) is indigenous to the plateaus of central and south America, but is cultivated throughout the World. Its fruits, flowers, and seeds have been eaten as vegetables containing vitamins A, C, and E. Several triterpenes such as cucurbita-5,24-dienol [3] and  and -amyrin [4] are present in the seeds of Cucurbita maxima.
The known compounds 4 [7,8] and 5 [9] were identified by comparing MS and 1 H and 13 C-NMR data with published data, and 6 [7] by MS and 1 H NMR data . The six multiflorane triterpenes 1−6 from C. maxima were evaluated for inhibitory activities against -MSH-induced melanogenesis in B16 melanomas (Table 2). At a low concentration (10 M), 5 inhibited melanogenesis (76.9% of melanin content) with low cytotoxicity (99.5% of cell viability). 5 also inhibited melanogenesis (70.9% of melanin content) with low cytotoxicity (97.7% cell viability) at 30 M. At a high concentration (100 M), 3 and 5 exhibited inhibitory activities (51.8 and 67.4% of melanin content, respectively) with low cytotoxicity (95.1 and 99.6% of cell viability, respectively). The activity levels of compounds 5 at 10 and 30 M were comparable with or superior to those of the positive control, arbutin, which has been recognized as a useful depigmentation compound for skin whitening in the cosmetic industry [10]. It appears that two multiflorane-type triterpenes, 5 from C. maxima seeds, may be valuable as potential skin-whitening agents. The melanogenesis inhibitory activity of 2 (28.1% of melanin content at 100 M) is thought to be due to their cytotoxic action (69.0% of cell viability at 100 M). Table 2. Melanogenesis inhibitory activity and cytotoxicity in B16 mouse melanoma cells of multiflorane-type triterpenes isolated from Cucurbita maxima seeds a .  Six triterpenes and a reference compound, 5-fluorouracil (5-FU), were also evaluated for cytotoxic activities against human leukemia (HL-60) and murine leukemia (P388) cell lines by means of the MTT assay (Table 3). Compound 2 exhibited single-digit micromolar cytotoxicity with IC 50 values of 7.0 and 9.5 M against HL-60 and P388 cells, respectively. It was slightly less cytotoxic than 5-FU [IC 50 2.3 (HL-60); 1.9 (P388) M] ( Table 3).

Plant Material
The seeds of Cucurbita maxima, produced in Japan (Nara prefecture), were purchased from JA (Japan Agricultural Co-operation)-Takatsuki in 2011. A voucher specimen was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.

Cell Cultures
The cell lines HL-60 (human leukemia) and P388 (murine leukemia) were grown in RPMI 1640 medium, while B16 4A5 cells were grown in D-MEM. The medium was supplemented with 10% FBS and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin). The cells were incubated at 37 °C in a 5% CO 2 humidified incubator.

Determination of B16 4A5 Cells Proliferation
B16 4A5 cell proliferation was examined according to a method reported previously [11] with slight modifications. Briefly, B16 4A5 cells (obtained from the Riken Cell Bank, Tsukuba, Ibaraki, Japan) (3 × 10 4 cells in 500 L), preincubated for 24 h were treated for 48h with test samples dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100, 30, or 10 M, and MTT solution was added. After 3 h of incubation, 2-propanol containing 0.08 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 550 nm using a microplate reader.

Assay of Melanin Content
The assay of melanin content was performed as described previously [11] with small modifications. B16 4A5 cells (3 × 10 4 cells in 500 L) were pre-incubated as above in -MSH (100 nM)-containing medium. Test samples dissolved in DMSO were added to the medium and the cells were cultured for 48 h. The medium was removed and the cells were dissolved in 2 M NaOH containing 10% DMSO. The amount of melanin was determined spectrophotometrically by measuring absorbance at 450 nm using a microplate reader. The optical density of control cells was assumed to be 100%.

Cytotoxicity Assay against Cancer Cell Lines
The cytotoxicity assay against HL-60 and P388 cells was determined as described previously [12].