Activity of Cytotoxic Flavanoids against a P-388 Murine Leukemia Cell Line from the Stem Bark of Aglaia elliptica (Meliaceae)

Two mixtures of flavanoid compounds (1 and 2), a mixture of catechin (1a) and epicatechin (1b), and a mixture of gallocatechin (2a) and epigallocatechin (2b), were isolated from the active fraction of the stem bark of Aglaia elliptica methanol extract. The chemical structure of the compounds was identified with spectroscopic data, including UV, IR, NMR (H, C, DEPT 135°, HMQC, HMBC, H-H COSY), and MS, and additionally compared with previously reported spectral data. All compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compound 2 showed cytotoxicity against the P-388 murine leukemia cell, with an IC50 value of 7.79 μg/mL, but compound 1 was found not to be active (more than 100 μg/mL).

A. elliptica is a tree, mainly distributed in the northern part of Sulawesi in Indonesia [15]. Previous phytochemical study of this plant reported the presence of diamide and cycloartane-type triterpenoid from the leaves [17] and novel cytotoxic lH-cyclopenta[b]benzo furan from the fruits [16]. In the present paper, we elucidate the isolation and structure of the mixture of flavonoid compounds, catechin (1a), epicatechin (1b), gallocatechin (2a), and epigallocatechin (2b), together with their cytotoxic activity against murine leukemia cells.

Materials and Methods
Equipment. UV spectra were measured using a Shimadzu UV-160A ultraviolet-visible spectrometer, with MeOH (Kyoto, Japan). The IR spectra were recorded on a Perkin-Elmer 1760X FT-IR in KBr (Waltham, MA, USA). The mass spectra were recorded with a Synapt G2 mass spectrometer instrument (Waters, Milford, MA, USA). NMR data were recorded on a JEOL ECZ-600 spectrometer at 600 MHz for 1 H and 150 MHz for 13 C (Tokyo, Japan), using TMS as an internal standard. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan). 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.
Plant material. The stem bark of A. elliptica was collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia, in June 2015. The plant was identified by the staff of the Bogoriense Herbarium, Bogor, and a voucher specimen (No. Bo-1294562) was deposited at the Herbarium.
Plant extraction. Dried ground stem bark (2.3 kg) of A. elliptica was extracted with methanol (12 L) at room temperature for 3 days. After removal of the solvent under vacuum, the viscous concentrate of MeOH extract (321.5 g) was first suspended in H 2 O and then partitioned successively with n-hexane, EtOAc, and nbutanol. Evaporation resulted in the crude extracts of nhexane (22.6 g), EtOAc (31.4 g), and n-butanol (34.5 g), respectively. The n-hexane, EtOAc, and n-butanol extracts exhibited cytotoxic activity against P-388 murine leukemia cells, with IC 50 values of 67.72, 32.69, and >100 µg/mL, respectively. The EtOAc soluble fraction (20 g) was fractionated by column chromatography on silica gel using a gradient n-hexane and EtOAc to give fractions A-E, combined according to the TLC results. Fraction D (1.73 g) was subjected to column chromatography over silica gel, using a gradient mixture of CHCl 3 :Me 2 CO (10:0-1:1) as eluting solvents to afford six subfractions (D1-D6). Subfraction D4 (460 mg) was chromatographed on a column of silica gel, eluted with CHCl 3 -MeOH (10:0-4:1), to give five subfractions (D4A-D4E). Subfraction D4D was chromatographed on preparative TLC, eluted with CHCl 3 -MeOH (8.5:1.5), to give compound 1 (47.5 mg). Subfraction D5 (600 mg) was chromatographed on a column of silica gel, eluted with CHCl 3 -MeOH (10:0-7:3), to give five subfractions (D5A-D5E). Subfraction D5D was chromatographed on a column of silica gel, eluted with CHCl 3 -MeOH (10:0-1:1), to give compound 2 (127 mg). [8,11,20]. The P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3 x 10 4 cells cm -3 . After 24 h of incubation for cell attachment and growth, varying concentrations of samples were added. The compounds added were first dissolved in DMSO at the required concentration. Six subsequent desirable concentrations were prepared using phosphoric buffer solution (PBS, pH=7.30-7.65). Control wells received only DMSO. The assay was terminated after a 48 h incubation period by adding MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, also named thiazol blue), and the incubation was continued for another 4 h, in which the MTT-stop solution containing sodium dodecyl sulphate (SDS) was added, and another 24 h incubation was conducted. Optical density was read using a micro plate reader at 550 nm. IC 50 values were taken from the plotted graph of the percentage of live cells compared to control (%) receiving only PBS and DMSO, versus the tested concentration of compounds (µM). The IC 50 value is the concentration required for 50% growth inhibition. Each assay and analysis was run in triplicate and averaged.

Results and Discussion
The stem bark of A. elliptica was ground and successively extracted with MeOH and partitioned with n-hexane, ethyl acetate, and n-butanol. All the extracts were evaluated for cytotoxic activity against P-388 murine leukemia cells. The ethyl acetate extracts exhibited the strongest cytotoxic activity against P-388 murine leukemia cells, with IC 50 values of 32.69 µg/mL. Subsequent phytochemical analysis was therefore focused on the EtOAc extract of A. elliptica. The EtOAc extract was chromatographed over a column packed with silica gel 60 by gradient elution. The fractions were repeatedly subjected to normal-phase column chromatography to afford flavonoid compounds 1 and 2 ( Figure 1). (1)    Compounds 1a and 1b were isolated as a mixture, which was obtained as a yellow powder. On the basis of their 1 H NMR and 13 C NMR spectra, the ratio of compounds 1a and 1b was deduced as 3:5 in the mixture. Most of the signals were well-resolved for both compounds. The HR-TOFMS spectrum showed [M+H] + m/z 291.0878 (calcd. m/z 290.0790), which corresponded to the molecular formula of C 15 H 14 O 6 and thus required nine degrees of unsaturation, originating from six C sp 2 , and the remaining tricyclic flavanoids. UV spectra in MeOH showed the presence of a flavan-3-ol skeleton [18]. The IR spectra showed absorption peaks at 3330, 1572, 1146, 1051, and 827 cm -1 , suggesting the presence of hydroxyl groups, C=C olefin rings, symmetric and asymmetric C-O-C, and a substituted benzene ring, respectively. The 1 H-NMR of the 1a (CD 3 OD 600 MHz) spectrum showed the presence of five olefinic methine groups, resonating at δ  (Figure 2). The flavan-3-ol skeleton in ring C was evidenced by 1 H-1 H COSY cross peak H-2/H-3/H-4 also from the HMBC correlation from H-2 to C-9 and C-1', H-3 to C-2 and C-4, and H-4 to C-5, C-9, and C-10. The 13 C NMR of 1a (CD 3 OD 150 MHz) and DEPT 135⁰ spectra showed the presence of five olefinic methines and seven quaternary olefinic carbons (12 sp 2 carbons), two oxymethines, and one methylene. These functionalities accounted for six of the total nine degrees of unsaturation, and the remaining three degrees of unsaturation were consistent with the flavan-3-ol structure. The 1 H NMR and 13 C NMR chemical shifts of 1a and 1b were similar, the main difference being the conformation of C-3. In 1a, the coupling constant between H-2/H-3 ( 3 J) was 7.8 Hz, indicating that the  13

C, in CD 3 OD) for Compounds 1 and 2
conformations of C-2 and C-3 were axial-axial, respectively. Otherwise, in 1b, 3 J of H-2/H-3 was 1.6 Hz, indicating that the conformations of C-2 and C-3 were axial-equatorial, respectively. A comparison of NMR data of 1a and 1b with data of catechin and epicatechin isolated from green tea (Camellia sinensis) [19] revealed that the structure of the compounds was very similar; therefore, compound 1 was identified as a mixture of compound catechin (1a) and epicatechin (1b), respectively. These compounds were isolated from A. elliptica for the first time.
Compounds 2a and 2b were isolated as a mixture, which was obtained as a dark brown powder. On the basis of their 1 H NMR and 13 C NMR spectra, the ratio of compounds 2a and 2b in the mixture was deduced as 2:3. Most of the signals were well-resolved for both compounds. The HR-TOFMS spectrum showed [M+H] + m/z 307.0825 (calcd. m/z 306.0740), which corresponded to the molecular formula of C 15 H 14 O 7 and thus required nine degrees of unsaturation, originating from six C sp 2 , and the remaining tricyclic flavanoids. UV spectra in MeOH showed the presence of a flavan-3-ol skeleton [18]. The IR spectra showed absorption peaks at 3325, 1577, 1144, 1062, and 829 cm -1 , suggesting the presence of hydroxyl groups, C=C olefin rings, symmetric and asymmetric C-O-C, and a substi  , H-4b). The 13 C NMR of the 2a (CD 3 OD 150 MHz) and DEPT 135⁰ spectra showed the presence of six`olefinic methines, eight quaternary olefinic carbons (14 sp 2 carbons) and one methylene. The NMR chemical shift showed that compound 2a is similar to 1a, as catechin derivatives. The main difference was the presence of an additional hydroxy group at C-5' instead of an olefinic methine in 1a, observed in the HMBC correlation of H-1' and H-6' to C-3', C-4', C-5', C-1'. The NMR chemical shift of 2a and 2b was similar, the main difference being the conformation of C-3. In 2a, the coupling constant between H-2/H-3 ( 3 J) was 7.4 Hz, indicating that conformations of C-2 and C-3 were axial-axial, respectively. Otherwise, in 2b, 3 J of H-2/H-3 was 1.7 Hz, indicating that conformations of C-2 and C-3 were axial-equatorial, respectively. A comparison of NMR data of 2a and 2b with data of gallocatechin and epigallocatechin from green tea (Camellia sinensis) [19] revealed that the structure of the compounds was very similar; therefore, compound 2 was identified as a mixture of gallocatechin (2a) and epigallocatechin (2b), respectively. These compounds were isolated from A. elliptica for the first time.
The cytotoxicity effects of the two isolated compounds 1 and 2 against P-388 murine leukemia cells were December 2017 Vol. 21  No. 4 investigated according to the method described in previous papers [8,11], and an artonin E (IC 50 0.3 µg/mL) was used as a positive control [20].
The cytotoxic activity of isolated compound 2 is not active, having an IC 50 value of more than 100 µg/mL, whereas compound 2 is active, having an IC 50 value of 7.79 µg/mL. The activity of 1 was stronger than that of 2, indicating that an additional hydroxy group at C-5' in the aromatic ring can support their reactivity and corrosiveness. This hydroxy group at C-5' in 2 can also act as a hydrogen bond donor that increases the affinity of the compound in the active site of the enzyme or in the biological receptor [21].

Conclusions
Two mixtures of known flavanoids, here labeled compound 1 and compound 2, were isolated from the stem bark of Aglaia elliptica. Compounds 1 and 2 were evaluated for their cytotoxic activity against P-388 murine leukemia cells, in vitro. It was found that compound 2 is active, indicating that the presence of a hydroxyl group in the aromatic ring can increase cytotoxic activity.