A Novel 3α-p-Nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate and Two New Triterpenoids from the Seeds of Zucchini (Cucurbita pepo L)

Three novel multiflorane-type triterpenoids, 3α-p-nitrobenzoylmultiflora-7:9(11)-diene-29-benzoate (1), 3α-acetoxymultiflora-7:9(11)-diene-29-benzoate (2), and 3α-acetoxymultiflora-5(6):7:9(11)-triene-29-benzoate (3), along with two known related compounds 4 and 5 were isolated from the seeds of zucchini (Cucurbita pepo L). Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HREIMS. Triterpenoids possessing a nitro group were not isolated previously.


Introduction
The species Cucurbita pepo is a cultivated plant of the genus Cucurbita that includes varieties of squash, gourd, and pumpkin.Cucurbita pepo L (zucchini, also known as field pumpkin or summer squash) (Cucurbitaceae) are widely cultivated in America, Europe, and Asia.The zucchini is a hybrid of the cucumber, and has been a commercially important crop in many countries since the 1950-1960s.It is a highly nutritional low caloric food that requires relatively little effort to prepare.It is full of nutrients like vitamin A, vitamin C, potassium, folate and fiber-all of which support a healthy OPEN ACCESS metabolism.Zucchini, grows well in warm climates.This readily available vegetable can also be an important part of weight loss efforts.

General Procedures
Melting points were determined on a Yanagimoto micro-melting point apparatus and are uncorrected.Optical rotations were measured using a JASCO DIP-1000 digital polarimeter.IR spectra were recorded using a Perkin-Elmer 1720X FTIR spectrophotometer. 1 H-and 13 C-NMR spectra were obtained on a Varian INOVA 500 spectrometer with standard pulse sequences, operating at 500 and 125 MHz, respectively.CDCl 3 was used as the solvent and TMS, as the internal standard.EIMS were recorded on a Hitachi 4000H double-focusing mass spectrometer (70 eV).Column chromatography was carried out over silica gel (70-230 mesh, Merck, Darmstadt, Germany) and MPLC was carried out with silica gel (230-400 mesh, Merck, Darmstadt, Germany).HPLC was run on a JASCO PU-1586 instrument equipped with a differential refractometer (RI 1531).Fractions obtained from column chromatography were monitored by TLC (silica gel 60 F 254 , Merck).

Cytotoxicity Assay
The cytotoxicity assay was determined previously [18].Briefly, the HL-60 and P388 cell lines (each 1 × 10 4 cells in 100 μL) were treated with test compounds for 72 h, and MTT solution was added to the wells.The grown cells were labeled with 5 mg/mL MTT in phosphate-buffered saline (PBS), and the absorbance of formazan dissolved with 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was measured at 550 nm using a microplate reader (Model 450, BioRad, Richmond, CA).

3 a
Melanin content (%) and cell viability (%) were determined based on the absorbances at 450 nm, and 550 nm, respectively, by comparison with those for DMSO (100%).Each value represents the mean ± S.D. of three determinations.Concentration of DMSO in the sample solution was 2 μL/mL; b Reference compound.
a Assignments were based on 1 H-1 H COSY, HMQC, HMBC and NOESY spectroscopic data.
a HL-60 and P388 cell lines (each 1 × 10 4 cells in 100 μL) were treated with test compounds for 72 h, and

Table 3 .
Melanogenesis inhibitory activities and cytotoxicities in B16 mouse melanoma cell line of multiflorane-type triterpenes isolated from Cucurbita pepo a .