Isolation of cytotoxic triterpenes from the mangrove plant , Scyphiphora hydrophyllacea C . F . Gaertn ( Rubiaceae )

Purpose: To isolate active cytotoxic compounds from the hexane and chloroform extracts of the leaves of the mangrove plant, Scyphiphora hydrophyllacea C.F. Gaertn (Rubiaceae), grown in Sri Lanka. Methods: Dried pulverized leaves of S. hydrophyllacea were extracted with hexane and chloroform. Vacuum liquid chromatography (VLC), column chromatography (size exclusion chromatography, Sephadex LH-20) and reversed phase preparative recycling high performance liquid chromatography (HPLC) techniques were used to isolate three compounds (compounds 1, 2 and 3). The structures of the isolated compounds were established with the aid of H, C and two-dimensional nuclear magnetic resonance (2D-NMR) and electron ionization-mass spectrometry (EI-MS) techniques. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects of the compounds on oestrogen receptor positive breast (MCF-7) and non-small cell lung (NCIH-292) cancer cells. Results: The isolated compounds were identified as oleanolic acid (1), ursolic acid (2) and eichlerianic acid (3). Ursolic acid and eichlerianic acid showed strong cytotoxic effects {IC50ursolic acid: 8.47 μg/mL (24 h, MCF-7), 7.78 μg/mL (24 h, NCI-H292) and eichlerianic acid: 8.86 μg/mL (24 h, MCF-7), 10.15 μg/mL (24 h, NCI-H292)} in MCF-7 and NCI-H292 cancer cells at 24, 48 and 72 h post-incubation periods. Conclusion: Hexane and chloroform extracts of the leaves of S. hydrophyllacea yielded three compounds namely oleanolic acid, eichlerianic acid and ursolic acid. Ursolic acid and eichlerianic acid have been isolated for the first time from the leaves of S. hydrophyllacea grown in Sri Lanka and demonstrate in-vitro cytotoxic effects in oestrogen receptor positive (MCF-7) and non-small lung cancer (NCI-H-292) cells.


INTRODUCTION
Cancer is considered a major cause of death in the world [1].Lung and breast cancers are the most commonly diagnosed cancer among men and women respectively.Despite the availability of modern cancer treatment options, there is no permanent cure for cancer [2,3].Radiotherapy, chemotherapy, hormone therapy and surgery are the main available treatment options for cancer [4,5].Chemotherapy and radiotherapy will be the ultimate treatment option for metastatic cancer when surgery causes some functional defects in the organs and systems of the patient.However, chemotherapy and radiotherapy cause severe side effects [6].Therefore identification of alternative treatment options for cancer with fewer side effects is of paramount importance.Phytochemicals have gained much attention in the development of cancer drugs and several plant derived natural compounds have been developed as anti-cancer drugs [7].
Mangroves are considered as one of the main productive coastal eco-systems.Mangroves have widely been used in traditional medicine to treat cancers and a number of natural compounds with different bio-activities have been reported from several mangrove plants [8].S. hydrophyllacea (Family: Rubiaceae) is one of the common mangrove plants found mainly in South Asia including Sri Lanka [8,9].Several flavonoids, terpenoids and iridoids have been isolated from S. hydrophyllacea by previous researchers [9].Cytotoxic and apoptotic properties of leaf and bark extracts of S. hydrophyllacea and isolation of hopenone-I from the hexane leaf extract of S. hydrophyllacea have reported in our previous studies [10,11].The present study was aimed at to isolate cytotoxic compound/s in the active hexane and chloroform extracts of S. hydrophyllacea leaves and to evaluate their cytotoxic effects in nonsmall cell lung (NCI-H292) and breast cancer (MCF-7) cells.

EXPERIMENTAL General
Cell lines and cell culture reagentsused in this studuy were purchased from the ATCC (American Type Culture Collection , Manassas, USA) and all the chemicals in the study were purchased from the Sigma-Aldrich (St Louis, MO, USA) unless otherwise stated.Bruker AV-500 and AV-600 NMR instruments (Fällanden, Switzerland) were used to record NMR spectra.Synergy™ HT Multi-Mode micro plate reader (Bio-Tek, Winooski, VT) was used to measure absorbance.All the solvents used in this study were of analytical grade.

Collection of plant material
Leaves of S. hydrophyllacea were collected from the same plant used for our previous studies [10,11].Leaves of the plant were collected from the National Aquatic Resources Research and Development Agency (NARA) Negombo, Sri Lanka and the plant material was identified by Mr. W.A. Sumanadasa, Research Officer at NARA.Voucher specimen (no.S-11) of the plant was deposited at the herbarium of IBMBB, University of Colombo, Sri Lanka.

Extraction and isolation of compounds
Air-dried pulverized leaves of S. hydrophyllacea (1.5 kg) were extracted with hexane (3×1.75L) and chloroform (3 × 1.75 L) by sonication at room temperature.Resulting chloroform extract was concentrated (32 g) and subjected to vacuum liquid chromatography (VLC) with a gradient solvent system composed of hexane: ethyl acetate (100:0 to 0:100) to yield 10 fractions.Fraction 9 (7.6 g), which was found to be cytotoxic, was seperated in a Sephadex LH-20 column (120 × 3.5 cm) with 50 % (v/v) dichloromethane and methanol to yield 17 fractions.The collected fractions were combined according to TLC profiles to make 4 fractions which were then subjected for cytotoxic evaluations.Most active fraction (fraction 3, 30 mg) was then subjected to preparative recycling HPLC in a normal-phase column with isocratic elution [5% (v/v) MeOH in CHCl 3 ; flow rate: 5 mL/min].Peaks were detected using refractive index detector (RI detector) at a sensitivity of 20nm/RIU.Two recycled base line separated peaks were collected to afford compounds 1 (7 mg) and 2 (12 mg).

Structure elucidation
Structures of the isolated compounds were established through of UV and IR spectroscopy,

Cell culture
Oestrogen receptor positive breast cancer (MCF-7) and non-small cell lung (NCI-H292) cancer cells were plated in 96-well cell culture plates in Dulbecco's Modified Eagle's Medium (DMEM) at 37 °C in 95 % air and 5 % CO 2 atmosphere with 95 % humidity.

Cytotoxic assay
MTT assay was carried out as previously described methods [12,13].Cancer cells plated in 96 well plates were treated with different concentrations (6.25, 12.5, 25, 50 and 100 µg/mL) of compounds and incubated for 24, 48 and 72 h.After incubation, MTT reagent (20 μL of 1 mg/mLstock solution) was added to each well and plates were incubated for 4 h at 37 °C.Following incubation, content was removed from each well and 100 μL of isopropanol/HCl mixture was added.Absorbance of treated and untreated wells were measured at 570 nm in a micro plate reader.Paclitaxel was used as the positive control.

Statistical analysis
Data are shown as mean ± SD (n = 3).Statistical differences as well as half-maximal inhibitory concentration (IC 50 ) values of the three compounds in two different cancer cells were analyzed using GraphPad Prism 5 (San Diego, California, USA) software package.P < 0.05 was taken as statistically significant.

Structure of isolated compounds
Structure of compound 1 and 2 were confirmed as oleanolic acid and ursolic acid by comparing NMR and mass spectroscopic data of these two compounds with reported data (Table 1 and 2 Comparative NMR chemical shift values of compound 3 are given in Table 3. Structure of compound 3 was further confirmed from 2D-NMR spectroscopic techniques (HSQC, COSY, HMBC and NOESY).Structures of the isolated compounds are shown in Figure 1.

Cytotoxic effects of isolated compounds
Cytotoxic effects of isolated compounds are shown in the Table 4.All three compounds were cytotoxic to cell lines tested.Compared to compound 1 (oleanolic acid), compound 2 (ursolic acid) and compound 3 (eichlerianic acid) were highly cytotoxic to both cancer cell lines tested.However compound 3 appeared to exert a higher effect in breast cancer cells than in lung cancer cells.

Morphological observations
Changes in cell structure such as membrane blebbing and apoptotic body formation changers in cell shape and decrease in cell number were evident in lung and breast cancer cells after treatment with the three compounds at 24, 48 and 72 h incubations (Figure 2 and Figure 3).

DISCUSSION
Scyphiphora hydrophyllacea is a mangrove plant of the family Rubiaceae which is widely distributed in Asia and Western Pacific [9].Presence of flavonoids, terpinoids and iridoids in the mangrove plant S. Hydrophyllacea has already been reported [9].Structures of three compounds isolated from the hexane and chloroform extracts of leaves of S. Hydrophyllacea were elucidated by means of 1 H-and 13 C-NMR data and the compounds were identified as oleanolic acid (compound 1), ursolic acid (compound 2) and eichlerianic acid (compound 3).Although isolation and structure elucidation of ursolic acid and oleanolic acid from several other mangrove varieties has been reported [16,17], this is the first report on the isolation eichlerianic acid from S. Hydrophyllacea leaves.Cytotoxicity results from the cytotoxic assays demonstrated that, among the three compounds ursolic acid and eichlerianic acid exerted strong cytotoxic effects on non-small lung cancer cells (NCI-H292) and oestrogen receptor positive (MCF-7) breast cancer cells at 24, 48 and 72 h post incubation periods.Morphological changes (changes in cell shape, volume and decrease in cell number) observed in ursolic acid and eichlerianic acid-treated cancer cells also confirmed cancer cell growth inhibitory effects of these two compounds.Oleanolic acid showed less cytotoxic effects in both the cancer cell lines tested.
Several researchers have also evaluated the cytotoxic effects of ursolic acid and eichlerianic acid [18-21] isolated from other plant species in several cancer cell lines including MCF-7 breast cancer cells [22,23] and those results are some