Secondary metabolites from Sarcosperma kontumense

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INTRODUCTION
The genus Sarcosperma (Sapotaceae) includes 11 species distributed in Asia, five of which are found in Viet Nam, namely S. affinis, S. angustifolium, S. kachinense, S. kontumense, and S. laurinum [1,2].The phytochemical and biological investigations of these plants are limited.Up to now, there has been only one publication on the chemical constituents of S. affinis, which reported the isolation and identification of some triterpenes, lignans, and polyphenols [3].However, the species Sarcosperma kontumense Gagnep.ex Aubrév.has not been studied yet.In our project for the biological activity screening of Vietnamese plants, the methanol extract of the leaves of this plant showed cytotoxic activity against KB cells with 32.31 % inhibition at 1 µg/mL.Therefore, a sample of this plant was collected in Lam Dong province for further study.As a result, in a recent paper, we reported on the isolation of two new compounds, sarcokontums A and B, from the stems and leaves of this plant [4].The isolation and structural elucidation of six ursane-type triterpenes (1 -6) and two flavonoids (7, 8) (Figure 1) from its leaves were described in this paper.

Plant materials
The leaves of Sarcosperma kontumense were collected from the Bidoup-Nui Ba National Park, Lam Dong province, Viet Nam, during May 2021 and identified by botanist Nguyen Quoc Dat at the Bidoup-Nui Ba National Park.The voucher specimen (VN-1436) has been deposited at the Institute of Marine Biochemistry (IMBC), VAST.

Extraction and isolation
The dried and ground leaves of S. kontumense (5.0 kg) were ultrasonically extracted with 70 % MeOH (15 L × 4) and the solvent was removed in vacuo to yield MeOH crude extract (SKM, 350 g).The SKM extract was suspended in H 2 O (1 L) and successively partitioned with n-hexane and ethyl acetate.The obtained extracts were concentrated under decreased pressure to yield the corresponding residues, SKH (140 g) and SKE (157 g) The residue SKE was chromatographed on a silica gel column eluting with the gradient solvent system of dichloromethane/methanol (20/1, 10/1, 2/1, 1/1, v/v) to give four fractions, SE1-SE4.
Compound 3 was obtained as a white powder.The NMR spectra of 3 showed similar signals to those of 2, except for the addition of a hydroxyl group at C-2 (Table 1) and the 23oxymethylene being replaced by a methyl group.The small coupling constants of H-3 (d, J = 1.2 Hz) indicated that H-3 was in β/equatorial orientation.The carbon chemical shifts of 3 were compared to those of euscaphic acid and found to match (Table 1).In addition, the ESI-MS spectrum of 3 gave the ion peak at m/z 511 [M + Na] + , corresponding to the molecular formula of C 30 H 48 O 5 .Therefore, 3 was identified as euscaphic acid (2α,3α,19α-trihydroxyurs-12-en-28oic acid) [5].
Table 2. 13 C-NMR spectral data of compounds 4, 5, 6 and the references.Compound 5 was isolated as a white powder.The 1 H and 13 C NMR data of 5 (Table 2) were similar to those of 4, except for the addition of a hydroxyl group at C-19.The signals of an urs-12-ene-28-oic acid were indicated, including one carbonyl, one oxymethine, one olefinic methine, and seven methyls.The large coupling constant of H-3 (J = 10.8)suggested its α/axial position.Furthermore, the singlet proton signals of CH 3 -29 and H-18, together with the downfield carbon chemical shift of C-19 at δ C 73.3, confirmed the group 19α-OH.The ESI-MS spectrum of 5 gave the ion peak at m/z 473 [M+H] + , corresponding to the molecular formula of C 30 H 48 O 4 .Accordingly, 5 was determined to be pomolic acid (3β,19α-dihydroxyurs-12-en-28oic acid) [5].
Compound 6 was isolated as a white powder.The 1 H and 13 C NMR spectra of 6 gave signals similar to those of 4, except for the addition of a hydroxyl group at C-2 and a trans-pcoumaroyloxy unit (Table 2).The signals of an urs-12-ene skeleton were revealed with one carbonyl; seven methines, including two oxymethines, one olefinic methine, and four methines; eight methylenes; seven methyls, including five singlet methyls and two doublet methyls; one oxygenated tertiary carbon; and six quarnatery carbons; The signals of the coumaroyloxy unit were also indicated at δ  Compounds 7 and 8 were isolated as yellow powders.Using TLC on a silica gel plate, they were identified by comparison with the authentic samples of (−)-catechin and (−)-epi-catechin, which were previously isolated from the flowers of Amesiodendron chinense (Sapindaceae) in our laboratory [11].Their 1 H, 13 C NMR spectra (Table 3) indicated signals of a flavanol skeleton, including five aromatic methines, five oxygenated aromatic carbons, two nonprotonated aromatic carbons of the A and B rings, and two oxymethines and one methylene of the C ring.The large proton coupling constant of H-2 at  H 4.59 (J = 7.2 Hz), together with the resonance of C-2 at  C 82.9, suggested the trans-2,3 stereochemistry of 7. Furthermore, the optical rotation of 7 with [α] D 25 = +17 (c 0.1, MeOH) confirmed 7 as (+)-catechin [11,12].On the contrary, the small proton coupling constant of H-2 at  H 4.81 (s), along with the resonance of C-2 at  C 79.9, suggested the cis-2,3 stereochemistry of 8.In addition, the optical rotation of 8 with [α] D 25 = −39 (c 0.1, MeOH) determined 8 as (−)-epi-catechin [11,12].

Table 3 .
The NMR spectral data for compounds 7, 8 and the references.