Flavonoid Compounds from the Bark of Aglaia eximia (Meliaceae)

Three flavonoid compounds, kaempferol (1), kaempferol-3O-α-L-rhamnoside (2), and kaempferol-3O-β-D-glucosylα-L-rhamnoside (3), were isolated from the bark of Aglaia eximia (Meliaceae). The chemical structures of compounds 1–3 were identified with spectroscopic data, including UV, IR, NMR (H, C, DEPT 135°, HMQC, HMBC, H-HCOSY NMR), and MS, as well as a compared with previ ously reported spectra data. All compounds were evaluated for their cytotoxic effects against P-388 murine leukem ia cells. Compounds 1–3 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 1.22, 42.92, and >100 mg/mL, respectively.

Aglaia eximia is a higher plant and mainly distributed in the southern region of the island of Sumatera in Indonesia [16]. The plant's bark is used in Indonesian folk medicine to reduce fever, moisturize the lungs, treat contusions, coughs, and skin diseases [16][17]. During the course of our continuing search for bioactive compounds in Indonesian medicinal plants, we isolated and described three flavonoid derivates, kaempferol (1), kaempferol-3-O-α-L-rhamnoside (2), and kaempferol-3-O-β-D-glucosyl-α-L-rhamnoside (3), from the ethyl acetate extract of the bark of A. eximia (Meliaceae).

Materials and Methods
Equipments. UV spectra were measured by using a Shimadzu UV-160A ultraviolet-visible spectrometer, with MeOH. IR spectra were recorded with Perkin-Elmer 1760X FT-IR in KBr. 1 H, 13 C, DEPT 135°, HMQC, HMBC, and 1 H-1 H-COSY NMR spectra were obtained with a JEOL JNM A-500 spectrometer using TMS as the internal standard. The mass spectra were recorded with HR-ESI-TOFMS. Chromatographic separations were carried out on silica gel 60 (70-230 mesh and 230-400 mesh), 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 ethanol, followed by heating.

Plant material.
A. eximia (Meliaceae) bark was collected in the Bogor Botanical Garden, Bogor, West Java Province, Indonesia, in June 2011. The plant was identified by the staff of the Bogoriense Herbarium, Bogor, Indonesia, and a voucher specimen (No. Bo-1295315) was deposited at the herbarium.
Determination of cytotoxic activity. The P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3 × 10 4 cells/cm 3 . After 24 h of incubation for cell attachment and growth, various sample concentrations were added. The compounds added were first dissolved in DMSO at the required concentration. Then six concentrations were prepared using phosphate buffer solution (PBS, pH = 7.30-7.65). The control wells received only DMSO. The assay was terminated after a 48 h incubation period by adding the MTT reagent [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; also named as thiazole blue], and the incubation was continued for another 4 h, during which the MTT-stop solution containing sodium dodecyl sulfate (SDS) was added and another 24 h incubation period was conducted. Optical density was read by using a microplate reader at 550 nm. The IC 50 values were taken from the plotted graph of the percentage of live cells compared to control (%), which received only PBS and DMSO, versus the tested compound concentrations (mg/mL). The IC 50 value is the concentration required for 50% growth inhibition. All assays and analyses were each run in triplicate and averaged.

Results and Discussion
The bark of A. eximia was ground, and then n-hexane, EtOAc, and MeOH were extracted. The EtOAc extract showed flavonoid compounds. Therefore, the phytochemical analysis focused on the ethyl acetate extract. The EtOAc extract was subjected to column chromatography over silica gel with a gradient elution of nhexane-EtOAc-MeOH. The fractions were repeatedly subjected to column chromatography on silica gel (70-230 and 200-400 mesh) and preparative TLC on silica gel GF 254 to provide three flavonoid compounds 1-3 ( Figure 1).    Two meta-protons of ring A resonated at δ H 6.36 (d, 2.0), 6.16 (d, 2.0) for H-8 and H-6, respectively. Proton pairing was also confirmed with the 1 H-1 H-COSY spectrum. The 13 C-NMR (CD 3 OD 125 MHz) and DEPT 135° spectra showed the presence of six methines and nine quaternary carbons (14 sp 2 carbon) and a ketonic group, C-4, resonating at δ C 176.7. These functionalities accounted for eight of the total 11 degrees of unsaturation, and the remaining three degrees of unsaturation were consistent with the flavonoid structure.

Figure 1 The Structure of Compounds 1-3
A comparison of the NMR data of 1 with the data for kaempferol [19] revealed that the structures of the two compounds are very similar; compound 1 was identified as kaempferol.  [19] revealed that the structures of the two compounds are very similar; therefore, compound 2 was identified as kaempferol-3-O-α-L-rhamnoside.
The cytotoxicity effects of the three isolated compounds 1-3 against P-388 murine leukemia cells were investigated according to the method described in previous papers [18,21], and artonin E (IC 50 0.3 mg/mL) was used as a positive control [23].   1.22 ± 0.02 42,92 ± 0.12 >100 ± 0.01 The cytotoxicity activity of isolated compounds 1-3 are shown in Table 2. Activity of kaempferol (1) was influenced by the hydroxyl group in rings A, B, and C that have a role as hydrogen bond donors that can improve the reactivity of 1. The hydroxyl group in aromatic ring can support their reactivity and corrosiveness.
These properties can improve the denaturating effect on membrane protein-forming pores that may lead to cell death, whereas the hydroxyl in the sugar ring for 2 and 3, has a many chiral center can decrease of hydrogen bond donors [24] that affects their reactivity and corrosiveness. These results suggest that total sugar rings may lead to a decrease in the cytotoxic activity of the compound.
Three known flavonoid compounds (1-3) have been isolated from the bark of Aglaia eximia (Meliaceae) and was found in this plant for the first time. Compounds 1-3, were evaluated for their cytotoxic activity against P-388 murine leukimia cells, in vitro. As a results indicated that the presence of a sugar moeity in flavonoid skeleton can decrease cytotoxic activity.