New Triterpenoids from Lansium domesticum Corr. cv kokossan and Their Cytotoxic Activity

Lansium domesticum Corr. is a member of the Meliaceae family that is widely spread in tropical and subtropical region of Asia and America. Traditionally, the fruit of this plant has been consumed because of its sweet taste. However, the fruit peels and the seeds of this plant have been rarely utilized. The previous chemical investigation of this plant showed the presence of secondary metabolites with many biological activities, including cytotoxic triterpenoid. Triterpenoids is a class of secondary metabolites which contain thirty carbon atoms in the main skeleton. The high modification of this type of compound, including the ring opening, highly oxygenated carbons, and the degradation of its carbon chain to give the nor-triterpenoid structure, is responsible for its cytotoxic activity. In this paper, we isolated and elucidated the chemical structure of two new onoceranoid triterpenes, kokosanolides E (1) and F (2), from the fruit peels of L. domesticum Corr., along with a new tetranortriterpenoid, kokosanolide G (3), from the seeds of L. domesticum Corr. The structural determination of compounds 1–3 was undertaken through FTIR spectroscopic analysis, 1D and 2D NMR, mass spectrometry, as well as through a comparison of the chemical shifts of the partial structures of compounds 1–3 with the literature data. The cytotoxic properties of compounds 1–3 were tested against MCF-7 breast cancer cells using the MTT assay. Moderate activity was shown by compounds 1 and 3, with IC50 values of 45.90 and 18.41 μg/mL, respectively, while compound 2 showed no activity (IC50 168.20 μg/mL). For the onoceranoid-type triterpene, the high symmetrical structure of compound 1 is presumably the reason for its better cytotoxic activity compared with that of compound 2. Compound 3 showed moderate activity, mainly because of the presence of the furan ring, which, based on the literature, gives better cytotoxic activity in a tetranortriterpenoid-type structure. The findings of three new triterpenoid compounds from L. domesticum indicate the significant value of this plant as a source of new compounds.


Introduction
Lansium domesticum Corr., as one of plant species from the Meliaceae family, is a source of triterpenoid compounds with various biological activities. This plant is widely distributed in Southeast Asia [1,2], Suriname, Puerto Rico, and Australia [3]. The latest taxonomic system of Meliaceae does not assign a specific rank for L. domesticum [4]. However, there are three varieties of L. domesticum that are widely known in Java, Indonesia, namely, duku, bidjitan or langsat, and kokossan. Duku has small, ellipsoidal, glabrous, pale yellow fruits without latex from trees with glabrous leaves and small flowers. Meanwhile, bidjitan or langsat has larger, ellipsoidal, glabrescent, pale yellow fruits with a little latex from trees with larger flowers and leaves, which are ±pilose abaxially. Kokossan has smaller, globose, orange-yellow fruits with latex and a tough pericarp from trees with the largest flowers and the most pubescent leaves. Hasskarl in Mabberley et al. (1995) [5] proposed the vernacular names as subdivisions of Lansium domesticum in Java, and these could be taken as cultivar names. Therefore, L. domesticum cv. kokossan or L. domesticum 'kokossan' were used to refer to the varieties of kokossan.
Almost all parts of L. domesticum plants have long been widely used for traditional therapeutic purposes. Boiled water from the bark of L. domesticum Corr., which is mixed with the bark of Pterocarpus indica Wild, is commonly used as a medicine for dysentry. The green seeds are very bitter, and when crushed with water, they can be used as an anthelmintic and as antipyretics. The stems can be used to treat malaria. The resin is thought to be non-toxic and useful for stopping diarrhea. The leaf extract of L. domesticum can be used as eye drops to prevent inflammation. The fruit skin of the plants can be used as a mosquito repellent through the smoke of the burning dry fruit skin. The fruit flesh and tree trunks are used by traditional communities for poisoning arrows [6].
Molecules 2023, 28, 2144 2 of 10 latex from trees with larger flowers and leaves, which are + pilose abaxially. Kokossan has smaller, globose, orange-yellow fruits with latex and a tough pericarp from trees with the largest flowers and the most pubescent leaves. Hasskarl in Mabberley et al. (1995) [5] proposed the vernacular names as subdivisions of Lansium domesticum in Java, and these could be taken as cultivar names. Therefore, L. domesticum cv. kokossan or L. domesticum 'kokossan' were used to refer to the varieties of kokossan. Almost all parts of L. domesticum plants have long been widely used for traditional therapeutic purposes. Boiled water from the bark of L. domesticum Corr., which is mixed with the bark of Pterocarpus indica Wild, is commonly used as a medicine for dysentry. The green seeds are very bitter, and when crushed with water, they can be used as an anthelmintic and as antipyretics. The stems can be used to treat malaria. The resin is thought to be non-toxic and useful for stopping diarrhea. The leaf extract of L. domesticum can be used as eye drops to prevent inflammation. The fruit skin of the plants can be used as a mosquito repellent through the smoke of the burning dry fruit skin. The fruit flesh and tree trunks are used by traditional communities for poisoning arrows [6].

Results and Discussions
After the separation of fruit peels and seeds from the fruit of L. domesticum, the drying in room temperature was carried out, followed by grinding, to give 1.7 kg powder of fruit peels and 3.8 kg powder of seeds. Two different extraction techniques were applied for the fruit peels and seeds due to the amount of material used.
The powder of fruit peels was extracted exhaustively with n-hexane, EtOAc, and MeOH. The n-hexane extract was chosen for the next separations and purifications due to the presence of the triterpenoid compound, which was identified qualitatively by the Liebermann-Burchard test. After the series of column chromatography (normal and reversed phase), compounds 1 and 2 were obtained.
The powder of seeds was extracted exhaustively with methanol. After the solvent removal under reduced pressure, the methanol extract was dissolved in water and then partitioned by n-hexane, EtOAc, and n-BuOH. The EtOAc extract was chosen for the next separations and purifications due to the presence of the triterpenoid compound, which was identified qualitatively by the Liebermann-Burchard test. After the series of column chromatography (normal and reversed phase), compound 3 was obtained.
The structure elucidation of the new triterpenoid compounds 1-3 was discussed based on the spectroscopic data and a comparison with the literature, along with its cytotoxic assay against MCF-7 breast cancer cell lines.

Structure Elucidation of the Isolated Compounds
Compound 1 was discovered as an amorphous and colorless powder. According to the NMR data and HRTOFMS data results that indicated a molecular ion peak at m/z 443.3881 [M+H] + (calculated for C 30 H 51 O 2 at m/z 443.3889), the molecular formula of 1 was determined to be C 30 H 50 O 2 , with six degrees of unsaturation. Fifteen carbon signals, including four methyls, four methylenes, four methines (involving one olefinic and one oxygenated carbon), and three quaternary carbons (including one olefinic carbon), were identified through 13 C NMR (DEPT) data (Table 1) and HSQC spectra. The IR spectrum suggested the existence of hydroxyl (3448 cm −1 ), gem-dimethyl (1385 and 1458 cm −1 ), and ether (1022 cm −1 ) functionalities. The above data revealed that 1 is a symmetric onoceranoid-type triterpenoid with a total of 30 carbon signals [7]. The two olefinic groups accounted for two degrees of unsaturation, leaving four degrees of unsaturation for the four-rings core of 1. In the 1 [7]. The two hydroxyl methines positioned at C-3 and C-21 were deduced by the HMBC correlations between Me-23/Me-24 to C-3 and Me-29/Me-30 to C-21. Furthermore, the correlations of Me-26 to C-7, C-8, and Me-27 to C-14, C-15 verified the formation of two double-bond pairs at C-7/C-8 and C-14/C-15. A careful analysis of the 1D and 2D NMR showed that 1 was similar to 3β-hydroxyonocera-8(26),14-dien-21-one [10], with the main differences being the double-bond movement at C-7/C-8 and C-14/C-15 in the B ring and the ketonic replacement at C-21 by a hydroxyl moiety. In addition, the hydroxyl group on C-3 (C-21) was determined to be an α-orientation by the J coupling value compared to the 3β-hydroxyl in its analog [10], referencing the reflection form of the stereocenter's orientation in the symmetrical structure of the onoceranoid-type. Thus, compound 1 was elucidated as a new onoceranoid triterpene derivative, 3,21-dihydroxy-onocera-7,14-diene, and trivially named as kokosanolide E (1).    (Tables S4-S6), respectively. Compounds 1 and 3 exhibited moderate activity against MCF-7, while compound 2 showed no activity. The significant difference in cytotoxic activity between the onoceranoid triterpenes 1 and 2 was most probably due to the symmetrical structure of 1 compared to the structure of 2. Additionally, the presence of a carbonyl group at 2 was expected to decrease the cytotoxic activity. Compound 3 showed the highest cytotoxic activity among all isolated compounds, probably due to the presence of a furan ring and a highly oxygenated structure.   (Table 1) showed the presence of a total of thirty carbon signals, which were assigned as eight methyls, nine methylenes, six methines (involving one olefinic and one oxygenated carbon), and seven quaternary carbons (including one olefinic and two oxygenated carbons), together with the aid of DEPT and HSQC spectra. The above data suggested that 2 is an onoceranoid-type triterpenoid [7]. The presence of one double-bond pair and one carbonyl ketone accounted for two degrees of unsaturation, leaving four degrees of unsaturation for the four-rings core of 2. In the 1 H NMR spectrum, eight tertiary methyls at δ H (ppm) 1 [7]. The presence of a ketonic group at C-3 and a hydroxyl methine group at C-21 was verified by the correlations between Me-23/Me-24 to C-3 and Me-29/Me-30 to C-21. Furthermore, the correlations of Me-27 to C-14 and Me-26 to C-7, C-8 revealed the attachment of a hydroxyl in the quaternary carbon at C-14 and the double-bond form at C-7/C-8. A careful analysis of the 1D and 2D NMR indicated that compound 2 was closely related to kokosanolide B [14], with the main difference being the hydroxyl moiety at C-21. The relative configuration of each stereocenter carbon in 2 was determined by an NOESY experiment. The NOESY spectrum showed correlations between H-21/H-17/H-3 and H-28/H-27. According to the biosynthesis of the onoceranoid-type triterpene, the orientation of H-17 is -oriented, which indicates that the H-21 proton has a orientation. Based on this result, it can be concluded that the hydroxyl group at C-21 is α-oriented, while the hydroxyl group at C-14 is -oriented. Thus, compound 2 was elucidated as a new onoceranoid-type triterpene, 14,21α-dihydroxy-onocera-7-en-3-one, and trivially named as kokosanolide F (2).

Cytotoxic Activity of Isolated Compounds
Compound 3 was originally discovered as a colorless oil. According to the NMR data (Table 1), together with its HR-TOFMS analysis, compound 3 showed an [M+H] + ion peak at m/z 501.2125 (calcd. 501.2131 for C 27 H 33 O 9 ), which was consistent with the formula C 27 H 32 O 9 , requiring 12 degrees of unsaturation. The UV spectrum suggested the presence an α, -unsaturated ketone by an absorption maximum at 282 nm. The IR spectrum showed the existence of a hydroxyl at 3453 cm −1 , a carbonyl ketone at 1707 cm −1 , an ester at 1670 cm −1 , a gem-dimethyl at 1367 and 1379 cm −1 , and an ether at 1278 cm −1 . In the 1 H-NMR spectrum, three tertiary methyls at δ H 0.91, 0.95, and 1.07 and one secondary methyl at δ H 1.27 (d, J = 6.4 Hz), as well as the downfield tertiary methyl group, resonating at δ H 3.80, indicated the presence of a methoxy group that was identified. The signals corresponding to a tetranortriterpenoid bearing an α-substituted furan ring at δ H 6.41, 7.40, and 7.44 were obviously observed. Moreover, the signal for an olefinic of an α,β-unsaturated ketone at δ H 5.97 (1H, m), another olefinic signal at δ H 6.39 (1H, t, J = 1.0 Hz), and two protons corresponding to the oxymethine group at δ H 4.32 (1H, d, J = 1.0 Hz) and 5.02 (1H, s) were also found. The 13 C-NMR (DEPT) data revealed a total of twenty-seven carbon signals, including characteristics of a furan moiety (δ C 143.1 (d), 141.1 (d), 120.0 (s), and 110.2 (d)), two ketonic groups (δ C 211.5 and 208.1), two ester groups (δ C 174.1 and 166.3), two oxymethine carbons (δ C 73.8 and 80.8), one quaternary oxygenated carbon (δ C 80.4), a double-bond at an unsaturated ketone (δ C 159.7 and 110.4), and an additional double-bond (δ C 141.5 and 126.7). According to the 1D NMR data, the functionality of 3 accounted for eight degrees of unsaturation, leaving four degrees of unsaturation suitable for a tetracyclic tetranortriterpenoid core with a furan ring. The main skeleton of the tetranortriterpenoid compound was further proven by the 1 H-1 H COSY cross-peaks of H-24/H-25, H-9/H-11/H-12, and H-6/H-5/H-10. The study of the kokosanolide-type tetranortriterpenoid skeleton in the genus of Lansium revealed that the NMR data of 3 were related to the kokosanolide A isolated from the same species [14]. The difference featuring the ether ring opening (C-1/C-9) in kokosanolide A, which resulted in the formation of a double-bond at C-8/C-9 of 3, was indicated by the HMBC correlation of H-22 to C-8 and C-9, as well as the 1 H-1 H COSY of H-9/H-11. This one ring opening also led to the formation of an additional carbonyl group at C-1, which was observed through a correlation of H-22 and H-19 to C-1. Other structural characteristics of 3 are similar to the characteristics of kokosanolide A, including the formation of a lactone ring at C-16/C-17, which was shown by the correlation of H-15 to C-16, and the presence of a methyl ester at C-27, which arose from the correlation of H-27 to C-7. Finally, the furan moiety at C-17 was evidenced by the correlation of H-26 to C-17. The relative stereochemistry of 3 was mainly determined by the similarity of the 1 H and 13 C NMR chemical shifts, the J coupling constants, as well as the NOESY spectrum of kokosanolide A. From the NOESY spectrum, a cross-peak arising at H-6/H-5/H-10 indicated that those three protons were in the same face. Therefore, the structure of 3 was elucidated as the new tetranortriterpenoid and trivially named as kokosanolide G (3).  (Tables S4-S6), respectively. Compounds 1 and 3 exhibited moderate activity against MCF-7, while compound 2 showed no activity. The significant difference in cytotoxic activity between the onoceranoid triterpenes 1 and 2 was most probably due to the symmetrical structure of 1 compared to the structure of 2. Additionally, the presence of a carbonyl group at 2 was expected to decrease the cytotoxic activity. Compound 3 showed the highest cytotoxic activity among all isolated compounds, probably due to the presence of a furan ring and a highly oxygenated structure.

General Experimental Procedures
High-resolution electrospray ionization (HRESIMS) was acquired on a waters Xevo QTOFMS (Waters, Milford, MA, USA). IR spectra were performed on a One Perkin Elmer infrared-100. NMR spectra were obtained on a JEOL ECZ-500 spectrometer at 500 MHz for 1 H NMR and at 125 MHz for 13 C NMR, with tetramethylsilane (TMS) as the internal reference. For the column chromatography, silica gel G60 (Merck, Darmstadt, Germany) and C18 silica gel (Merck, Darmstadt, Germany) were used. TLC was performed on precoated GF 254 (Merck, 0.25 mm) silica gel plates, and TLC spots were detected using 10% sulfuric acid in ethanol and then heated.

Plant Material
The seeds and fruit peels of L. domesticum were collected in April 2018 from Cililin, West Java, Indonesia (6 • 57 2 S, 107 • 27 25 E, 667 msl). A plant specimen (10188), identified by the staff of the Laboratory of Plant Taxonomy, was deposited in the Department of Biology, Universitas Padjadjaran, Indonesia.
The dried seeds of this species (3.8 kg) were extracted exhaustively with MeOH (5 × 5 L) at room temperature. The evaporation of the organic layer gave a concentrated MeOH extract (143.5 g). The crude MeOH extract was partitioned between H 2 O and n-hexane, EtOAc, and n-BuOH, successively. The EtOAc soluble fraction (10 g) was fractionated by VLC on silica gel using a stepwise gradient of nhexane-EtOAc 5% to yield eight fractions (I-P). Fraction L (1.38 g) was chromatographed by a silica gel column (nhexane-CH 2 Cl 2 -EtOAc, 4:3:3) to give six fractions (L1-L6). The separation of subfraction L4 (324 mg) by a silica gel column with a stepwise gradient elution of n-hexane-EtOAc 5% gave three subfractions (L4.1-L4.3). Subfraction L4.3 (106 mg) was then purified by a silica gel column (n-hexane-CH 2 Cl 2 -EtOAc, 4.5:4:1.5) to yield 3 (9.8 mg). Fraction L was chosen based on TLC profiles because there was a new spot, which indicated the presence of a new compound.

Cytotoxic Bioassay
All isolated compounds were tested for their cytotoxicity against human breast cancer cells (MCF-7) using the MTT (methyl thiazoldiphenyl-tetrazoliumbromide) method. The cells were cultured in Roswell Park Memorial Institute (RPMI) Medium (DMEM), 10% (v/v) Fetal Bovine Serum (FBS), and an antibiotic of 1% (v/v) (100U) penicillin-streptomycin mixture solution. The incubation was carried out at 37 • C for 24 h. The media were replaced by a mixture of fresh media with the addition of the isolated compounds at various concentrations (7.81; 15.63; 31.25; 62.50; 125.00; 250.00; 500.00; 1000.00 µg/mL). After 24 h, the mixture of 200 µL of DMSO and the formed formazan crystal was used to replace the media from each well. The absorbance was measured at 450 nm, and the IC 50 can be calculated through linier regression with Microsoft Excel software.

Conclusions
Three new triterpenoids, kokosanolides E-G (1-3), were isolated from L. domesticum Corr. Compounds 1-2 were isolated from the fruit peels part, whereas compound 3 was iso-lated from the seeds part. Extensive spectroscopic methods were used for the determination of the chemical structure of 1-3. Compounds 1 and 2 belong to an onoceranoid triterpene and compound 3 has a tetranortriterpenoid structure. All of the isolated compounds were tested for cytotoxic activity against the MCF-7 breast cancer cell line using MTT methods, which showed that compounds 1 and 3 have moderate activity, whereas compound 2 has no activity against MCF-7 cell lines. The highly symmetrical structure of 1 and the highly oxygenated nature and the presence of a furan ring in 3 are suspected to play important roles in cytotoxic activity.