Two novel prenylated kaempferol derivatives from fresh bud’s fur of Platanus acerifolia and their anti-proliferative activities

Abstract Two novel prenylated kaempferol derivatives (1, 2), together with seven known metabolites were isolated from ethanol extract of fresh Platanus acerifolia bud’s fur by multistep chromatographic processing. Structure of compounds 1 and 2 was confirmed by 1D, 2D NMR spectra and HR-ESI-MS. In addition, compound 1 was further analysed by X-ray crystallography. Anti-proliferative activities in vitro against human breast carcinoma (MCF-7) and human hepatocellular carcinoma (Hep-G2) cell lines for compound 1, 2 and 8 were evaluated. Compound 1 exhibited cytotoxic activity towards MCF-7 and Hep-G2 cell lines with the IC50 values 38.2 and 39.5 μM, respectively. Moreover, compound 2 showed weak cytotoxic activities against the two cell lines.


Introduction
Platanus acerifolia, belonging to the genus Platanus, is an interspecific hybrid between Platanus occidentalis and Platanus orientalis. Some plants of the genus have been used as folk medicines to heal diarrhoea, dysentery and to relieve toothache in Asia (Hartwell 1970;Nishanbaev et al. 2005). So far, the plant of Platanus species has been known as sources of flavonoids (Creuzet et al. 1988;Kaouadji & Ravanel 1990;Kaouadji et al. 1992), glycosides (Kaouadji 1990;Kaouadji et al. 1993) and organic acids (Mitrokotsa et al. 1993) from barks, leaves or buds. To discover chemical composition and potential use of the bud's fur, the ethanol extract of the fur has been further treated.
In this study, the cytotoxicity of compounds 1, 2 and 8 towards the two cell lines (MCF-7, Hep-G2) was evaluated with MTT assay (Table 1). Compound 1 showed effective activities against the two cell lines with IC 50 values of 38.2, 39.5 μM, respectively. Compound 2 showed weaker cytotoxic activities compared with compound 1, with IC 50 > 100. Compound 8 only showed potential cytotoxic activity against MCF-7 cell lines, with IC 50 values of 58.3 μM.

General experimental procedures
1 H and 13 C NMR spectra were measured on a Bruker Avance DRX-400 spectrometer. IR spectra were recorded on a NICoLET IR200 FT-IR spectrophotometer. Melting points were determined on a Kofler hot stage apparatus and are uncorrected. HR-ESI-MS was carried out on an Agilent Technologies 6224 ToF LC-MS apparatus. TLC was conducted on silica gel HSGF 254 plates (10-40 μm; Yantai Jiangyou Chemical, Inc.). Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical, Inc.), MCI Gel (CHP 20P, 75-150 μm, Mitsubishi Kasei Corporation). Cells were incubated in an HF-212uV Co 2 incubator and observed under an oLYMPuS CKX41 inverted microscope. Single-crystal X-ray diffraction data of compound 1 were collected using a Rigaku Saturn 924 diffractometer with Mo-Kα radiation (λ = 0.71073 Å).

Plant material
The fresh bud's fur of P. acerifolia was gathered from Nanjing in May, 2014. The species was identified by Prof Zhu-Nan Gong of College of Life Science, Nanjing Normal university, Nanjing, China. A voucher specimen (No. 14-05-25) was deposited in our laboratory.

Cytotoxicity activity experiments
The compounds 1, 2 and 8 were estimated in vitro against the cell lines Hep-G2, MCF-7 by MTT assay. The cells were plated in 96-well culture plates and incubated for 24 h at 37 °C in incubator with 5% Co 2 for 24 h. The compounds with five different concentrations (100.0, 50.0, 25.0, 10.0 and 5.0 μM) were added to each well for 24 h. Then, 20 μL solution (5 mg/mL MTT dissolved in PBS) was added to each well for 4 h cultivation. The oD values were then read on the microplate reader at 490 nm. IC 50 values were determined from the chart of cell viability (%) against compound concentration (μM). Matrine was used as positive control with 98% purity in HPLC (The control of pharmaceutical and biological products Chinese).

Conclusion
From the fur, nine compounds among which two novel prenylated kaempferol derivatives and seven known compounds were found. The cytotoxic activities of compound 1, 2 and 8 against MCF-7 and Hep-G2 cell lines were tested (Table 1). The two novel compounds showed potent cytotoxic activities against the two human cancer cell lines.

Supplementary material
The original spectra of NMR, HR-ESI-MS and IR for the new compounds and structures for compound 1-9 are listed on Supplementary material.