Antioxidant Constituents from the Bark of Aglaia eximia (Meliaceae)

The genus Aglaia is a a rich source of different compounds with int eresting biological activities. A part of our continuing search for novel biologically active com pounds from Indonesia Aglaia plants, the ethyl acetate extract of bark of Aglaia eximia showed significant antioxidant activity. Four antioxidant compounds, kaempferol ( 1), kaempferol-3O-α-L-rhamnoside (2), kaempferol-3O-β-D-glucoside (3) and kaempferol-3O-β-D-glucosyl-(1→4)-αL-rhamnoside (4) were isolated from the bark of Aglaia eximia (Meliaceae). The chemical structures of compounds 1-4 were identified on the basis of spectroscopic datas including UV, IR, NMR and MS along with by c omparison with those spectra datas previously reported. All compounds showed DPPH radical-scavenging activi ty with IC50 values of 1.18, 6.34, 8.17, 10.63 μg/mL, respectively.

The different parts of the genus Aglaia have been reported to contain biologically active classes of flavonoid compound [11]. It was suggested, for the same genus, that there are possibilities to generate the derivate compounds based on biosynthesis pathways of plants [12]. The flavonoids are a class of widely distributed phytochemicals, and scavenging of free radicals seems to play a considerable part in the antioxidant activity [13].
To the best of our knowledge, antioxidant activity of compounds or extracts from some members of Aglaia have been described previously [11,12], but no infor-mation is available on the antioxidant activity of kaempferol and their glycosides from the species of A. eximia. In the further screening for antioxidant activity against DPPH radical-scavenging on polar fraction from A. eximia, we found that the ethyl acetate extract of the bark of A. eximia showed a DPPH radical-scavenging activity with an IC 50 values of 20 µg/mL. We herein report the isolation and structure elucidation of kaempferol and their glycosides from the bark of A. eximia together with antioxidant activity against DPPH radical-scavenging.

Materials and Methods
General. Melting points were measured on an electrothermal melting point apparatus and are uncorrected. Optical rotations on an ATAGO AP-300 automatic polarimeter. UV spectra were measured by using Shimazu UV-160A UV-Vis spectrophotometer. The IR spectra were measured on a Perkin-Elmer spectrum-100 FT-IR in KBr. Mass spectra with a Water Qtof HR-MS XEV otm mass spectrometer; the-NMR spectra were measured with a JEOL JNM A-500 spectrometer using TMS as an internal standard. Chromatographic separations were carried out on silica gel 60 (70-230 mesh and 230-400 mesh, Merck). Preparative TLC glass and TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and detection was achieved by spraying with 5% AlCl 3 and 10% H 2 SO 4 in ethanol, followed by heating on a hotplate at 100 o C for 2-5 minutes.
Plant material. The bark of A. eximia was collected in Bogor Botanical Garden, Bogor in June of 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.

Results and Discussion
The phytochemical test for the EtOAc extract showed the presence of flavonoids. By using DPPH radicalscavenging assay to guide separations, the EtOAc fraction was separated by column chromatography over silica gel by gradient elution. The fractions were repeatedly subjected to normal-phase column chromatography and preparative TLC on silica gel GF 254 and yielded four antioxidant flavonoid compounds 1-4 ( Figure 1).  Table 1. Comparison of the NMR data of 1 with kaempferol [16] showed high similarity, consequently compound 1 was identified as kaempferol.  [19]. In comparison the NMR data of 3 with the literature data [19,20], and compound 3 was identified as kaempferol-3-O-α-Dglucoside. Hz was assigned for axial-equatorial position which occurred to be the βand α-configuration, respectively [18]. A comparison the carbon signals of 4 with the glucosyl moiety [19], the glucose C-2′′, C-1′′ experienced downfield and upfield as well as the C-5′′; C-6′′, shift due to the αand β-effects of rhamnosylation. In comparison, the NMR data of 4 with the literature data [17,18,20], compound 4 was identified as kaempferol-3-O-β-D-glucosyl-(1→4)-α-L-rhamnoside.
Compounds 1-4 antioxidant activity were evaluated against DPPH radical-scavenging. The antioxidant activiy of compounds 1-4 are shown in Table 2. Compounds 1-4 showed weaker activity compared with standard compound, ascorbic acid. Among those isolated compounds, compound 1, showed strongest activity with IC 50 value of 1.18 ± 0.02 µg/mL. Previous structure-activity studies of flavonoids have pointed to the importance of the number and location of OH groups and could be more important for the antiradical efficacy. The effectivity of radical scavenging activity of 1, is proposed by 4-hydroxy in the B ring as electron donating and being a radical target, together with 3-OH moiety in the C ring is also beneficial as antioxidant activity. A conjugated double bond between C2-C3 with 4-keto group further enhances the radical-scavenging capacity through electron delocalization from the B ring; the presence of hydroxyl group on C-3 and C-5 in combination with an α-β-unsaturated-4-carbonyl can improve the radical scavenging activity of 1 [19], whereas sugar moiety of compounds 2-4 were showed the steric effect. That could reduce the free radical scavenging activity as well as weaken the electronic distribution of flavonoid molecules. Dihedral angles of compounds 2 >3 >4 caused by the sugar unit in the Cring, lead the conformation to reverse and make molecule Based on frontier molecular orbital theory, the HOMO of flavonoid is mainly distributed in the B-ring, while the LUMO is distributed in the C-ring. The electrondonating capability of a molecule can be determined by the values of HOMO corresponds with a strong capability for donating electrons [19,20]. It was suggested the higher of DPPH radical-scavenging was more focused on B-ring and the conjugate part than the C ring. It show us, that there is no significance to the IC 50 vaules of compound 1-4 ( Table 2).