Chemical constituents from the leaves of Melia azedarach

Abstract Six compounds, benzyl 3-O-β-D-glucopyranosyl-7-hydroxybenzoate (1), spathulenol (2), 1,7,8-trihydroxy-2-naphtaldehyde (3), quercetin (4), astragalin (5) and 2-methoxy-4-(2-propenyl)phenyl β-D-glucoside (6), were isolated from the leaves of Melia azedarach L. The structure elucidation of compound 1 was discussed in detail based on its 2D-NMR data. Compound 1 showed weak cytotoxicity against the cell lines of T-24, NCI-H460, HepG2, SMMC-7721, CNE, MDA-MB-231 and B16F10 with the inhibition rates from 10.01% to 34.05% at the concentration of 80 μM.


Introduction
Melia azedarach L., a deciduous tree of family Meliaceae, is widely distributed in southern districts of the Yellow River in China (Editorial Committee of Flora of China 1997). The barks of M. azedarach have been used as an insect repellent for threadworm, and as a therapeutic medicine for tinea imbricata according to the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission 2015). The extracts of leaves and barks of this plant showed antidiabetic activity (Seifu et al. 2017), cytotoxic, antibacterial and antioxidant activities (Zahoor et al. 2015). The triterpenoids from this plant showed antifungal activity (Jabeen et al. 2011), limonoids showed antifeedant (Carpinella et al. 2002) and cytotoxic activity (Akihisa et al. 2013). The metabolites from the fungal endophyte isolated from this plant showed antifungal and antibacterial activities (Xiao et al. 2014). In our previous study, sterols and terpenoids were obtained from the barks of Melia azedarach (Tan et al. 2010), as part of our program to discover biologically active secondary metabolites from this plants, the leaves of this plant were investigated, which led to the isolation of six compounds (1-6). Their structures were identified as benzyl 3-O-b-D-glucopyranosyl-7-hydroxybenzoate (1)

Results and discussion
Compound 1 was obtained as yellowish amorphous powder. Its positive HR-ESI-MS showed the [M þ Na] þ peak at m/z 429.1163 (calcd for C 20 H 22 O 9 Na). The 1 H NMR and 13 C NMR spectra data were almost identical with those of benzyl 2-O-b-D-glucopyranosyl-2,6-dihydroxybenzoate (D'Abrosca et al. 2001), which possessed a benzene moiety containing three vicinal protons. The peak multiplicity and coupling constants in the 1 H NMR spectrum and the correlations between d H 7.28 (t, J ¼ 8.3, 1H) with d H 6.76 (dd, J ¼ 8.3, 0.7, 1H) and with 6.59 (dd, J ¼ 8.3, 0.7, 1H) in the COSY spectrum of 1 indicated that the possible ortho-tri-substituted patterns of benzoate moiety might be 2,3,4-or 2,3,7-as shown in supplementary material Figure S1. The key correlation in the HMBC spectrum between d H 4.95 (H-1 00 , d, J ¼ 7.5 Hz) with d C 158.3 indicated this quaternary carbon was attached to glucopyranosyl, and thus another quaternary carbon of d C 159.9 was to hydroxyl groups. The key correlation of 7.28 (1H, t, J ¼ 8.3 Hz) with d C 159.9 suggested that the hydroxyl group was substituted at C-7 in the possible structure of I or C-4 in II instead of C-3 in III as shown in supplementary material Figure S1. Although the structures of I and II were all explicable from the viewpoint of correlations in HMBC spectrum due to the groups were too closed to each other, structure II was unreasonable because of the steric hindrance of these groups. Thus, compound 1 was elucidated as benzyl 3-O-b-D-glucopyranosyl-7-hydroxybenzoate.
The cytotoxicity in vitro of compound 1 against T-24, NCI-H460, HepG2, SMMC-7721, CNE, MDA-MB-231 and B16F10 cell lines was evaluated by a modified MTT assay, and the inhibition rates (n ± s%) were 10.96 ± 0.47%, 19.82 ± 1.80%, 21.37 ± 1.09%, 34.05 ± 1.55%, 27.37 ± 0.45%, 10.01 ± 1.26%, and 27.45 ± 1.84%, respectively, at the concentration of 80 lM, which is its maximum solubility in DMSO. Due to the low inhibition rates against the tested cell lines, the IC 50 values haven't been calculated in this experiment. As far as we know, this is the first report on the cytotoxicity of compound 1 against the mentioned cell lines.

Conclusions
Compounds 1, 3, and 6 were isolated from Meliaceae for the first time, and the cytotoxicity in vitro against cell lines of T-24, NCI-H460, HepG2, SMMC-7721, CNE, MDA-MB-231 and B16F10 were tested and reported for the first time.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by the National Natural Science Foundation of China under Grant 81360477; Natural Science Foundation of Guangxi under Grant 2016GXNSFAA380268 and2016GXNSFAA380041;and Guangxi BaGui Scholars Fund under Grant [2017]143. This fund was issued by the Sciences and Technology Department of Guangxi Province, which was similar with "The Thousand Talents Plan" initiated by Chinese government. Another work (doi: 10.3390/ molecules23010121) completed by our colleagues was also supported by this fund.