Two Flavonoid Compounds as Antiproliferative Activity Against SPC 1 Cancer Tongue Cells from the Leaves of Rasamala ( Altingia excelsa Nornha )

Two flavonoid compounds, kaempferol (1) and quercetin (2) have been isolated from the leaves of Rasamala (Altingia excelsa Nornha). The chemical structure of compounds 1 and 2 were identified by spectroscopic evidences including, UV, IR, 1D-NMR, 2D-NMR and MS as well as by comparing with previously reported spectral data.These compounds were isolated from this plant for the first time. Compounds 1 and 2 were evaluated for their antiproliferative activity against SP-C1 cancer tongue cells and showed IC50 values of 2.50 and 2.31 , respectively.


INTRODUCTION
Oral squamous cell cancer (OSCC) has high morbidity and mortality rates across the world because it is frequently found in advanced stages before therapy (Chen et al., 2008;Chen et al., 2013).The major environmental risk factors responsible for the development of cancer cell include betel nut chewing, cigarette smoking, alcohol consumption, and exposure to high-risk human papillomavirus.This cancer cell is a difficult disease to treat because of multidisciplinary and diverse treatment strategies and the varied natural behavior of the cancer.Local invasion and frequent regional lymph node metastases together with relative resistance to chemotherapeutic.The conventional strategies of cancer cell management still depend on surgery, radiotherapy, chemotherapy and targeted therapy (Shah and Gil 2009).The poor outcome of chemotherapy to oral squamous cell cancer contributes to the poor prognosis for this diseases (Scully and Bagan 2009).Therefore, novel, effective therapy for oral squamous cell cancer treatment is still needed.Due to this high incidence, the identification of novel compounds that inhibit cancer development has become a crucial objective for scientists.Hundreds of chemicals that have been and are being evaluated for their anti-cancer activities, natural products derived from medicinal plants rank among the most promising (Tan et al., 2011).During the course of our continuing search for novel anticancer compounds from Indonesian plants, the ethanol extract of the leaves of Altingia excelsa Nornha exhibited significant antiproliferative activity against SP-C1 cancer tongue cells.A. excelsa Nornha is known as Rasamala in Indonesia is higher plant.The plant is used in folklore and traditional medicine for the treatment of stomachache and coughs (Kanjijal et al., 2003;Pramono and Djam'an, 2002).Previous phytochemical study of this plant reported the presence of sesquiterpenoid from the leaves (Kanjijal et al., 2003), but antiproliferative compounds not yet reported.In this paper, we report the isolation and structure elucidation of two flavonoid compounds (1 and 2) as antiproliferative activity against SP-C1 cancer tongue cells.

General Experimental Procedure
UV spectra were measured by using a TECAN Infinite M200 pro, with MeOH.The IR spectra were recorded on a SHIMADZU IR Prestige-21 in KBr.The mass spectra were recorded with a Waters Xevo QTOF MS.NMR data were recorded on JEOL JNM A-500 spectrometer at 500 MHz for 1 H and 125 MHz for 13 C, chemical shifts are given on a  (ppm) scale with TMS as an internal standard.Column chromatography was conducted on silica gel 60.TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% H 2 SO 4 in EtOH, followed by heating and under UV light at wavelenght at 254 and 367 nm.

Plant Material
The leaves of A. excelsa Nornha were collected in Wayang Windu mountain, Pangalengan, West Java Province, Indonesia in August 2015.The plant was identified by the staff of the Laboratory of Taxonomy, Department of Biology, Universitas Padjadjaran and a voucher specimen (No. 256/HB/06/2015) was deposited at the herbarium.

Cell culture and treatment
The SP-C1 human tongue cancer cell line used in this study were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).For cell treatments, various concentrations of the sample were added to the cell culture medium.After 24 h, the cells were released from treatment, the medium was replaced, and cells were subsequently collected at the indicated times (Prayitno et al., 2013).

RESULT AND DISCUSSION
The methanol extract from the dried leaves of A. excelsa Nornha were concentrated and extracted successively with n-hexane, ethyl acetate and n-butanol.The ethyl acetate extracts showed strongest antiproliferative activity against SP-C1 cancer tongue cells.By using antiproliferative activity assay to guide separations, the ethyl acetate fraction was separated by combination of column chromatography on silica gel G60 and preparative TLC on silica gel GF 254 to afford two flavonoid compounds 1 and 2.
Kaempferol  Compound 1 was obtained as yellow amorphous powder.The molecular formula was established to be C 15 H 10 O 6 from its LC-MS spectral data (m/z 286) and NMR spectra, thus requiring eleven degrees of unsaturations.The UV spectrum of 1 showed  max at 272 and 364 nm and exhibited a bathochromic shift by added NaOH and AlCl 3 suggested the presence of flavonoid structure with 4 and 5-hydroxyl groups.The IR spectrum of 1 showed the absorption band correspond to hydroxyl (3420 cm -1 ), carbonyl (1690 cm -1 ) and double bond (1605 cm -1 ) groups.The 1 H-NMR spectrum of 1 showed the presence of meta-coupled of aromatic protons at  H 6.28 (1H, d, J=1.95 Hz) and 6.52 (1H, d, J=1.95 Hz) corresponds to H-6 and H-8, respectively.The 1 H-NMR spectrum of 1 also showed the presence of two doublet signals at  H 7.15 (2H, d, J=6.80 Hz, H-2 and H-6) and 7.01 (2H, d, J=6.80 Hz, H-3 and H-5) corresponds to four aromatic protons in ring B, characteristics for the 1,4disubstituted flavone.A total fifteen carbon signals were observed in the 13 C-NMR spectrum.These were assigned by DEPT experiments to fourteen sp 2 carbons and a carbonyl signal at  C 176.6.The degree of unsaturation was accounted for eight out of the total eleven double bond equivalents.The remaining three degree of unsaturation were consistent to flavonol structure (Kim et al., 2016;Aisyah et al., 2017).A comparison of the NMR data of 1 with those of kaempferol (Castenada et al., 2016;Aisyah et al., 2017), revealed that the structures of the two compounds are very similar, therefore, compound 1 was identified as kaempferol, which shown in this plant for the first time.
Compound 2 was obtained as a yellow amorphous powder.The LC-MS of 1 gave a ion peak at m/z 300, compatible with the molecular formula C 15 H 10 O 7 .Its UV absorptions in MeOH were consistent with the presence of a 3, 5, 7, 3, 4pentahydroxyflavone structure (Kim et al., 2016;Aisyah et al., 2017).The 1 H-and 13 C-NMR spectra of 1 exhibited resonances due to aromatic systems.The 13 C-NMR signals of 1 were assigned with the help of a DEPT experiment.In the 1 H-NMR spectrum of 1, the aromatic region exhibited an ABX system at  H 7.73 (1H, d, J=2.0 Hz, H-2), 7.62 (1H, dd, J=2.0, 7.5 Hz, H-6), and 6.87 (1H, d, J=8.0 Hz, H-5) due to a 3, 4 disubstitution of ring B and a typical meta-coupled pattern for H-6 and H-8 protons ( C 6.17 and 6.37, d, J=2.5 Hz).The 13 C-NMR spectrum of 1 showed the presence of 15 aromatic carbon signals.Based on the NMR data and comparison of the data given in the literature previously, the structure of compound 2 was identified as quercetin (Huang et al., 2013), which shown in this plant for the first time.
The effect of kaempferol and quercetine on the viability of SP-C1 cells was evaluated according to the methodology described in previous papers (Prayitno et al., 2013).The treatment of cancer SP-C1 cell lines with kaempferol and quercetine resulted in a dosedependent inhibition of cell growth, as demonstrated by the MTT assay.Twenty-four hours of treatment with kaempferol and quercetine inhibited the proliferation of SP-C1cells with an IC 50 value of 2.50 and 2.31 , indicating that both compounds are potential for further application in cancer treatment.
Quercetin showed stronger antiproliferative activity against SP-C1 cancer tongue cells, suggested the presence of additional hydroxyl group in flavonoid structure can increase antiproliferative activity.