LIMONOIDS FROM FRUITS OF MELIA DUBIA CAV. IN VIET NAM

Although the genus Melia of the Meliaceae family contains about five poorly defined species in the old-world tropics, almost every part of the plants of this genus are used as traditional herbal medicines, such as being anthelmintics, for the treatment of leprosy, eczema, asthma, malaria, fevers, and venereal diseases, as well as cholelithiasis, acariasis, and pain. The genus Melia is well-known as a rich and valuable source of bioactive limonoids. The genus Melia has 3 species (M. azedarach, M. dubia, and M. toosendan) in Viet Nam. Melia dubia Cav. has been shown to be a source of interesting tetranortriterpenoids. In addition, it is well known for its traditional medicinal properties and other biological activities such as insect or larval growth inhibition and as an antifeedant. Therefore, Melia dubia has been used to anti-insect and larval in agriculture in recent days. A phytochemical study from fruits of Melia dubia Cav. collected in Quy Hop, Nghe An province led to the isolation of three secondary metabolites, including 21α-O-methylmelianodiol (1), 21β-O-methylmelianodiol (2) and (21S,23R,24R)21,23-epoxy-24-hydroxy-21β-methoxytirucalla-7,25-dien-3-one (3). The chemical structures of these compounds were determined based on 1D-, 2DNMR, UV, IR, and MS analytical results and compared with reported data. All three compounds (1–3) were isolated from this plant for the first time.


INTRODUCTION
Although the genus Melia of the Meliaceae family contains about five poorly defined species in the old world tropics, almost every part of the plants of this genus are used as traditional herbal medicines, such as being anthelmintics, for the treatment of leprosy, eczema, asthma, malaria, fevers, and venereal diseases, as well as cholelithiasis, acariasis, and pain [1,2]. The genus Melia is well-known as a rich and valuable source of bioactive limonoids [3]. The genus Melia has 3 species (M. azedarach, M. dubia, and M. toosendan) in Viet Nam [4,5]. Therein, M. dubia Cav. is listed as a synonym of Melia azedarach L., it is not clear they are the same species or different. Therefore, M. dubia and M. azedarach were analyzed by DNA barcoding. The results based on Bayesian analysis of the concatenated dataset strongly support the view that M. dubia be considered a distinct species; not a synonym of M. azedarach [6].
Melia dubia is well known as a high tree in the Ghat forests of India and is generally termed the giant neem [7,8]. The tree enjoys a broad geographical distribution (e.g., in Viet Nam, Indonesia and Philippines) and is well known for its traditional medicinal properties and other biological activities such as insect or larval growth inhibition and as an antifeedant [8]. M. dubia has been shown to be a source of interesting tetranortriterpenoids [9,10]. However, to our knowledge, there are no documents on phytochemical research of fruits of M. dubia in Viet Nam. In this study, three triterpenoids such as 21α-O-methylmelianodiol (1), 21β-Omethylmelianodiol (2) and (21S,23R,24R)-21,23-epoxy-24-hydroxy-21β-methoxytirucalla-7,25dien-3-one (3) have been isolated from fruits of M. dubia.

General
Melting points were measured using Yanagimoto MP-S3 apparatus without corrections. Optical rotations were determined using a JASCO DIP-370 polarimeter. The UV spectra were obtained on an Agilent UV-3210 spectrophotometer and IR spectra were recorded on a Bruker FTIR-8501 spectrophotometer. 1 H-and 13 C-NMR, DEPT, COSY, NOESY, HSQC, and HMBC spectra were recorded on the Bruker AV-III 500 NMR spectrometer, with tetramethylsilane (TMS) as the internal standard and chemical shifts were reported in δ values (ppm). The electrospray ionization mass spectra (ESI-MS) were measured using an Agilent 1200 LC-MSD Trap spectrometer. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70-230 mesh and 230 -400 mesh, E. Merck). The preparative HPLC was conducted on an Agilent 218 Purification System. Thin-layer chromatography (TLC) was conducted on precoated Kieselgel 60 F 254 plates (Merck) and the compounds were visualized by spraying with 10 % (v/v) H 2 SO 4 followed by heating at 110 °C for 10 min.

Plant material
The fruits of Melia dubia Cav. were collected in Quy Hop, Nghe An province, Viet Nam, in July 2018 and identified by Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST. A voucher specimen (TDT-20180718) was deposited at the Herbarium of the School of Chemistry, Biology and Environment, Vinh University, Viet Nam.

RESULTS AND DISCUSSION
Compound 1 was obtained as a white amorphous powder. A molecular formula of 1 (C 31 H 50 O 5 ) was assigned on the basis of its ESI-MS, 13 C-NMR and DEPT spectral data. The 1 H-NMR, 13 C-NMR and DEPT spectra of 1 (Table 1)  Besides, based on the 13 C-NMR chemical shifts, it was apparent that a saturated carbonyl ketone at δ C 219.2/C-3 and an oxygenated tertiary carbon at δ C 78.7/C-25 were present in the molecule of 1. All of the above-mentioned NMR observations suggested that compound 1 is a triterpene possessing one methoxy group. The locations of seven methyl groups were assigned at C-4, C-10, C-13, C-14 and C-25 on the basis of the HMBC correlations seen in Fig.  1. Also, the presence of a tetrahydrofuran ring in the side chain was assigned based on the observed correlations in its 2D NMR ( 1 H-1 H COSY, HMQC and HMBC) spectra.  The side chain possessing tetrahydrofuran ring in compound 1 was found to be similar to that of holstinone A [11], the 21-methoxy group analogue of melianodiol [12]. Furthermore, the NOESY correlations between OMe-21 and H-23 (δ H 3.37/4.19), OMe-21/H-23 (δ H 3.37/4.79) also showed the relative configuration of the methoxy group at C-21 in the α-orientation with respect to the tetrahydrofuran ring. Based on ESI-MS, 1 H-, 13 C-NMR, DEPT, 1 H-1 H COSY, NOESY, HSQC and HMBC spectral data and comparison with the reported spectral data of 21α-O-methylmelianodiol [13], compound 1 had been identified as 21α-O-methylmelianodiol.
Compound 2 was isolated as a white powder. The ESI-MS of 2 determined the molecular formula C 31 H 50 O 5 , which is the same as that of 1. Both the 1 H-and 13 C-NMR spectroscopic data of compound 2 (Table 1) were comparable to those of 1, suggesting it was also a triterpene possessing a methoxy group. The gross structure of compound 2 was assigned in the same way as that of compound 1 based on the observed correlations in its 2D NMR ( 1 H-1 H COSY, HSQC and HMBC) spectra. The same correlations as that of 1 were observed in the HMBC spectrum of 2 ( Fig. 1). However, the signals for C-17 and C-21 were relatively upfield at δ C 48.0 and 106.0, while the signal for C-23 was downfield at δ C 79.6, suggesting a γ-gauche effect of the oxygenated substituent on C-21β [14]. Additionally, in contrast to compound 1, the NOESY correlation between OMe-21 and H-23 (δ H 3.37/4.19) was not observed in its NOESY experiment. Therefore, the relative configuration of the methoxy group at C-21 in compound 2 was assigned as a 21β. Based on the above spectral evidence and comparison of these data with those in the literatures [13], the structure of 2 was elucidated as 21β-O-methylmelianodiol.
Compound 3 was attributed to molecular formula C 31 H 48 O 4 by ESI-MS giving m/z 507.3 ([M+Na] + ). The optical rotation of 3 is the same with those reported of compound in the literatures [15]. The 1 H-NMR and 13 C-NMR signals of 3 typical for the tirucallane triterpenoids showed six methyl groups at δ C/H 22. 6