Pentacyclic Triterpenes from the Ethyl Acetate Fraction of the Bark of Platanus acerifolia Wild and Antitumor Activities In Vitro

Three pentacyclic triterpenes, named betulinic acid (1), 11α-hydroxy-β-amyrin (2) 3β-acetoxy-20 (29)-lupen28-aldehyde (3) were isolated from the ethyl acetate fraction of the bark of Platanus acerifolia Willd. The molecular structure of 1 and were established on the basis of various spectroscopic analyses. The molecular structure of (3) was determined by single-crystal X-ray diffraction. Compound (2) and (3) were obtained from the title plant for the first time. Cytotoxicity of the isolated compounds against three human cancer cell lines, HepG-2, MCF-7 and HL-60 were also determined with the cell counting kit-8 (CCK-8) assay. The target compounds showed the high cytotoxicity, with IC50 values in the range 2.2-9.1 μM. These results indicated that pentacyclic triterpenes from the bark of Platanus acerifolia Willd could be explored as potential cancer prevention agents. *Corresponding author: Chuan-Jin Wang, Department of Pharmaceutical Engineering, Institute of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, 210094, China, Tel: +862584315514; E-mail: wangchuanjin@njust.edu.cn Received February 11, 2016; Accepted March 27, 2016; Published March 31, 2016 Citation: Wang HZ, Wang CJ, Li W (2016) Pentacyclic Triterpenes from the Ethyl Acetate Fraction of the Bark of Platanus acerifolia Wild and Antitumor Activities In Vitro. Mod Chem appl 4: 178. doi:10.4172/2329-6798.1000178 Copyright: © 2016 Wang HZ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Platanus acerifolia Willd, one of the famous street and garden trees, is a very large, wide spreading, and long-lived hardwood species native to Eurasia [1]. The bark of Platanus acerifolia Willd has been used as the traditional Chinese medicine in the treatment of dysentery, diarrhea, toothache and tumor [2]. In order to find some bioactive compounds, the chemical constituents of Platanus acerifolia Willd bark were investigated and three compounds, named betulinic acid (1), 11α-hydroxy-β-amyrin (2) and 3β-acetoxy-20 (29)-lupen-28aldehyde (3) were isolated. The structures of the three compounds were identified by their physicochemical properties and spectral analysis. In addition, the isolated compounds were also evaluated for cytotoxic efficacy against HepG-2, MCF-7 and HL-60 cell lines in vitro.

General experimental procedures
Melting points were determined on RD-2 micromelting point apparatus and are uncorrected. The 1 HNMR (500 MHz) and 13 C NMR (500 MHz) spectra were recorded on a Bruker AvanceⅢ-500 spectrometer and tetramethylsilane (TMS) was used as an internal standard. Silica gel (200-300 mesh for Column Chromatography (CC) and GF 254 for TLC) was obtained from Qingdao Marine Chemical Company (Qingdao, China). Sephadex LH-20 was obtained from Amersham Biosciences (Uppsala, Sweden). Single-crystal structure of compound 2 was measured on an Enraf-Nonius CAD4 diffractometer etc.

Plant material
The bark of Platanus acerifolia Willd was collected in Nanjing County, Jiangsu Province, China, in January 2010.

Compounds identification
Compound (1): Compound (1) was readily identified as betulinic acid by the analysis of their NMR spectra and by the comparison with the data reported in literature [3]. 1

Antitumoral cytotoxic assay
HepG-2, MCF-7 and HL-60 cell lines were provided by the Nanjing University of Traditional Chinese Medicine Immunization Center. All cell lines were cultured in multi-well plates at 37°C in a humidified atmosphere of 5% CO 2 with RPMI-1640 medium containing 10% fetal bovine serum with 50U/mL penicillin and 50 µg/mL streptomycin.
In vitro anti-cell proliferative effects of compound (1) and (2) 11α-hydroxy-β-amyrin were determined on HepG-2, MCF-7 and HL-60 cell lines using the CCK-8 assay. Briefly, cells were counted, transferred into 96 well microtiter plates, and incubated for 24 h prior to the addition of test compounds. Compounds were dissolved in DMSO and diluted in sterile media, as necessary, to obtain the appropriate concentration. Exponentially growing cells of HepG-2, MCF-7 and HL-60 were made into single cell suspensions with 0.25% trypsin, at a cell concentration of 1 × 10 5 /mL. 90 µL cells (9 × 10 3 ) were seeded into each well of a 96-well plate. HepG-2, MCF-7 and HL-60 cells were incubated for 24 h before they were treated with compound (1) and (2) which were in a medium containing 0.1% DMSO, which showed no inhibitory effect on cell growth. This experiment was performed using 6 different final drug concentrations (3.125, 6.25, 12.5, 25, 50, 100 μM). To each well was added 10μL of the appropriate drug. Control cells were treated with an equal volume of serum-free RPMI 1640 containing 0.1% DMSO. After cells had been cultured for 24 h, 10 µL CCK-8 was added to each well. One hour later, the cell concentrations were recorded with an automated microplate reader at 450 nm. Each sample was assayed in triplicate, and each assay was repeated twice. Results are expressed as the concentration yielding 50% inhibition (IC 50 ).
The inhibition rate (%) = ( ) Data were expressed as mean ± SD. One-way analysis of variances and Fisher's least significant difference was performed using SAS 8.13. Differences were significant at P<0.05.

Results and Discussion
Compound (1) was readily identified as betulinic acid by the analysis of their NMR spectra and by the comparison with the data reported in literature (Figure 1) [3]. 1 H-NMR and 13 C-NMR spectra showed the typical pattern of pentacyclic triterpene. Especially, the 1 H-NMR spectrum of compound was characteristic of the presence of a vinyl protons at δ5.24 (1H, d) and 4.21 (1H, dd). Two C singlets at δ147.1 and 120.7 indicated the presence of C-C double bond. On the basis of the above evidences, compound (2) was suggested to 11α-hydroxy-βamyrin. The NMR data of compound was in good agreement with the previous data of 11α-hydroxy-β-amyrin ( Figure 1) [6]. Compound (3), was obtained as colorless crystals. The NMR data of compound (3) was in good agreement with the previous data of 3β-acetoxy-20 (29)-lupen-28-aldehyde [7]. Further single-crystal X-ray diffraction analysis confirmed the molecular structure of compound (3) (Figure 1) [4].
Cell Counting Kit-8 (CCK-8) is a reagent box used to detect cell proliferation, cell survival and cell toxicity based on a water soluble tetrazolium salt, WST-8{2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-benzene disulfonate)-2Htetrazolium monosodium salt}. CCK-8 is an alternative to MTT assay. During the process of metabolism of living cells, in the presence of 1-methoxy PMS, the WST-8 in cells produces soluble orange formazon. The formazan generated is proportional to the number of living cells. Compared with MTT, CCK-8 has significant advantages. The formazan generated by MTT is not water-soluble, and requires specific solvents to dissolve it, such as dimethyl sulfoxide. However, formazan generated by CCK-8 solution is water-soluble, and thus organic solvents need not be used in the experiment. On the other hand, CCK-8 solution is fairly stable, not toxic to cells, and can be used directly [8].