The Cytotoxic Activity of Dammarane-Type Triterpenoids Isolated from the Stem Bark of Aglaia cucullata (Meliaceae)

The Aglaia genus, a member of the Meliaceae family, is generally recognized to include a number of secondary metabolite compounds with diverse structures and biological activities, including triterpenoids. Among the members of this genus, Aglaia cucullata has been reported to have unique properties and thrives exclusively in mangrove ecosystems. This plant is also known to contain various metabolites, such as flavaglines, bisamides, and diterpenoids, but there are limited reports on the isolation of triterpenoid compounds from its stem bark. Therefore, this research attempted to isolate and elucidate seven triterpenoids belonging to dammarane-type (1–7) from the stem bark of Aglaia cucullata. The isolated compounds included 20S,24S-epoxy-3α,25-dihydroxy-dammarane (1), dammaradienone (2), 20S-hydroxy-dammar-24-en-3-on (3), eichlerianic acid (4), (20S,24RS)-23,24-epoxy-24-methoxy-25,26,27-tris-nor dammar-3-one (5), 3α-acetyl-cabraleahydroxy lactone (6), and 3α-acetyl-20S,24S-epoxy-3α,25-dihydroxydammarane (7). Employing spectroscopic techniques, the chemical structures of the triterpenoids were identified using FTIR, NMR, and HRESITOF-MS. The cytotoxic activity of compounds 1–7 was tested with the PrestoBlue cell viability reagent against MCF-7 breast cancer, B16-F10 melanoma, and CV-1 normal kidney fibroblast cell lines. The results displayed that compound 5 had the highest level of bioactivity compared to the others. Furthermore, the IC50 values obtained were more than 100 μM, indicating the low potential of natural dammarane-type triterpenoids as anticancer agents. These findings provided opportunities for further studies aiming to increase their cytotoxic activities through semi-synthetic methods.


Introduction
The Meliaceae family comprises flowering trees or shrubs that are commonly found in tropical and subtropical regions of Asia, Africa, and America [1]. Among the members of this family, the Aglaia genus has been reported to have the highest number of species, with more than 150 [2]. Furthermore, the Aglaia plant is primarily distributed in tropical and subtropical areas of Asia, Northern Australia, and the Pacific, with Indonesia being home to more than 65 species [2,3]. This plant has, over time, been implemented by Indonesians as traditional medication for wounds, fever, and skin disease [4]. Based on previous reports, the therapeutic effects exhibited can be attributed to their constituent secondary metabolites, such as diterpenoids [5,6], triterpenoids [7,8], sesquiterpenoids [9,10], limonoids [11,12], A total of 3.5 kg of dried crushed stembark of A. cucullata was macerated using EtOH and evaporated when the pressure was lower to produce 525 g of thick brown EtOH extract. The extract was dissolved in water and segmented based on differences in polarity, leading to the production of 64 g of n-hexane, 35 g of EtOAc, and 13 g of n-BuOH extracts. The nhexane sample was then subjected to the Liebermann-Burchard test, which showed intense positive results, indicating the presence of triterpenoid compounds. Therefore, the process of chemical separation and purification was carried out on n-hexane extract. Vacuum liquid chromatography (VLC) was carried out along with silica gel 60-column chromatography, and the reverse phase ODS yielded seven triterpenoids belonging to dammarane-type 1-7 ( Figure 1). All isolated compounds obtained in pure form were proofed by the TLC profile of each compound ( Figure S59). However, after a detailed analysis of NMR followed by a comparison with the literature, the purity of compounds 1-7 is reliable, and no mixture is observed. The structure identification of the isolated dammarane triterpenoids was discussed based on spectroscopic data.
A total of 3.5 kg of dried crushed stembark of A. cucullata was macerated using EtOH and evaporated when the pressure was lower to produce 525 g of thick brown EtOH extract. The extract was dissolved in water and segmented based on differences in polarity, leading to the production of 64 g of n-hexane, 35 g of EtOAc, and 13 g of n-BuOH extracts. The n-hexane sample was then subjected to the Liebermann-Burchard test, which showed intense positive results, indicating the presence of triterpenoid compounds. Therefore, the process of chemical separation and purification was carried out on n-hexane extract. Vacuum liquid chromatography (VLC) was carried out along with silica gel 60-column chromatography, and the reverse phase ODS yielded seven triterpenoids belonging to dammarane-type 1-7 ( Figure 1). All isolated compounds obtained in pure form were proofed by the TLC profile of each compound ( Figure S59). However, after a detailed analysis of NMR followed by a comparison with the literature, the purity of compounds 1-7 is reliable, and no mixture is observed. The structure identification of the isolated dammarane triterpenoids was discussed based on spectroscopic data. Compound 1, 20S,24S-epoxy-3α,25-dihydroxy-dammarane (1), was obtained as a white amorphous powder. The molecular composition of compound 1 according to HR-ESI-TOFMS was C30H52O3 (m/z 461.3993 [M+H] + calculated for C30H53O3 + , m/z 461.3995). Further analysis was carried out using NMR data, and the results were presented in Tables  1 and 2. Furthermore, the FTIR spectrum showed the appearance of hydroxyl (3457 cm −1 ), gem-dimethyl (1380 cm −1 ), and ether groups (1055 cm −1 ). The 13 C NMR (Table 1), along with the DEPT 135° and 1 H-NMR spectra (Table 2), demonstrated that the NMR data of 1 had high similarity with the 20S,24S-epoxy-3α,25-dihydroxy-dammarane isolated from A. elaeagnoidea [35]. A 2D NMR spectrum of this compound ( Figure 2) was also obtained to determine the exact structure of compound 1. According to the evaluation, the structure was identified as a dammarane-type triterpenoid, particularly 20S,24S-epoxy-3α,25-dihydroxy-dammarane, which was isolated for the first time from A. cucullata. Compound 1, 20S,24S-epoxy-3α,25-dihydroxy-dammarane (1), was obtained as a white amorphous powder. The molecular composition of compound 1 according to HR-ESI-TOFMS was C 30 Tables 1 and 2. Furthermore, the FTIR spectrum showed the appearance of hydroxyl (3457 cm −1 ), gem-dimethyl (1380 cm −1 ), and ether groups (1055 cm −1 ). The 13 C NMR (Table 1), along with the DEPT 135 • and 1 H-NMR spectra (Table 2), demonstrated that the NMR data of 1 had high similarity with the 20S,24S-epoxy-3α,25-dihydroxy-dammarane isolated from A. elaeagnoidea [35]. A 2D NMR spectrum of this compound ( Figure 2) was also obtained to determine the exact structure of compound 1. According to the evaluation, the structure was identified as a dammarane-type triterpenoid, particularly 20S,24S-epoxy-3α,25-dihydroxy-dammarane, which was isolated for the first time from A. cucullata.
Dammaradienon (2) was observed as a white amorphous, and its composition according to HR-ESI-TOFMS was identified as C 30 (Tables 1 and 2). The FTIR spectrum indicated the appearance of olefinic (3081 cm −1 for stretching of the C-H sp 2 group and 1641 cm −1 for the C=C group), gem-dimethyl (1375 cm −1 ) and carbonyl (1705 cm −1 ) groups. The 13 C NMR (Table 1), as well as the DEPT 135 • and 1 H-NMR spectra (Table 2), demonstrated that the NMR data of compound 2 were identical with the dammaradienone isolated from Chisocheton pendoliflorus [43]. Therefore, compound 2 was elucidated as a dammaranetype triterpenoid, namely dammaradienone, which was isolated from A. cucullata for the first time.  Tables 1 and 2. The FTIR spectrum showed the appearance of a hydroxyl (3448 cm −1 ), a gem-dimethyl (1378 cm −1 ), and a carbonyl (1704 cm −1 ) group. The 13 C NMR (Table 1), DEPT 135 • , and 1 H-NMR spectra ( Table 2) showed that the NMR data of compound 3 had high similarity with 20S-hydroxy-dammar-24-en-3-on isolated from A. elliptica [44]. Consequently, the structure of 3 was identified as a dammarane triterpenoid, namely 20S-hydroxy-dammar-24-en-3-on, which was isolated from A. cucullata for the first time.
Eichlerianic acid (4) was observed as a white amorphous, and its composition according to HR-ESI-TOFMS and NMR data was C 30 Tables 1 and 2). The FTIR spectrum showed the appearance of hydroxyl (3421 cm −1 ), gem-dimethyl (1376 cm −1 ), carbonyl (1704 cm −1 ), and ether (1078 cm −1 ) groups. The 13 C NMR (Table 1), DEPT 135 • , and 1 H-NMR spectra ( Table 2) presented that the NMR data of compound 4 were identical with eichlerianic acid isolated from A. foveolata [45]. Two-dimensional NMR spectra of this compound ( Figure 2) were also obtained to determine its exact structure. Therefore, the structure was identified as a dammarane-type triterpenoid, namely eichlerianic acid, which was isolated from A. cucullata for the first time.  (Tables 1 and 2). The FTIR spectrum indicated the appearance of olefinic (3081 cm −1 for stretching of the C-H sp 2 group and 1641 cm −1 for the C=C group), gem-dimethyl (1375 cm −1 ) and carbonyl (1705 cm −1 ) groups. The 13 C NMR (Table 1), as well as the DEPT 135° and 1 H-NMR spectra (Table 2), demonstrated that the NMR data of compound 2 were identical with the dammaradienone isolated from Chisocheton pendoliflorus [43]. Therefore, compound 2 was elucidated as a dammarane-type triterpenoid, namely dammaradienone, which was isolated from A. cucullata for the first time.
Compound 3, 20S-hydroxy-dammar-24-en-3-on, was obtained as a white amorphous, and its molecular composition according to HR-ESI-TOFMS and NMR data was identified as C30H50O2 (m/z 443.3817 [M+H] + calculated for C30H51O2 + , m/z 443.3811), as presented in Tables 1 and 2. The FTIR spectrum showed the appearance of a hydroxyl (3448 cm −1 ), a gem-dimethyl (1378 cm −1 ), and a carbonyl (1704 cm −1 ) group. The 13 C NMR (Table Compound 5, (20S,24RS)-23,24-epoxy-24-methoxy-25,26,27-tris-nor dammar-3-one was observed as a white amorphous, and its composition according to HR-ESI-TOFMS and NMR data was C 28 1 and 3). The FTIR spectrum presented the appearance of gem-dimethyl (1377 cm −1 ), carbonyl (1703 cm −1 ), and ether (1083 cm −1 ) groups. The 13 C NMR (Table 1) and DEPT 135 • spectra gave the resonance of 28 carbon signals, which were classified into seven methyls (including one methoxy at δ C 54.5), ten methylenes, five methines (consisting of one acetal at δ C 104.7), and six quaternary carbons (including one oxygenated quaternary carbon at δ C 88.1 and one ketone at δ C 218.4). The functionalities of one of the six levels of unsaturation and the leftover five levels of unsaturation were suitable with a tetracyclic dammarane-like triterpenoid core with an additional epoxy ring on the side  (Table 3) showed seven tertiary methyls (δ H 0.87, 0.92, 0.99, 1.03, 1.07, 1.12, and 3.30; each 3H) and one acetal proton at δ H 4.91 (1H, br.s). The position of each functional group was determined by HMBC and 1 H-1 H COSY spectra. Furthermore, ketone was formed in C-3, as shown by the HMBC correlation of H-28 and H-29 to C-3, C-4, and C-5. HMBC correlations of H-1 to C-24 were used to determine the exact position of the methoxy group at C-24. The 1 H-1 H COSY cross-peaks between H-22/H-23/H-24 provided information about the location of the acetal group at C-24, as well as the formation of an epoxide ring at C-20/C-24. An NMR data ratio of compound 5 with (20S,24RS)-23,24epoxy-24-methoxy-25,26,27-tris-nordammar-3-one obtained from the oxidation product of dipterocarpol through chemical synthesis [46,47] showed that the two compounds were identical. Furthermore, this was the first report on the isolation and detailed assignment of NMR data of this compound from a natural source, which was often previously obtained from synthetic products. According to these findings, the structure of 5 was identified as a dammarane triterpenoid, namely (20S,24RS)-23,24-epoxy-24-methoxy-25,26,27-tris-nor dammar-3-one.  (Table 3) demonstrated that the NMR data of compound 6 had high simi-larity with cabraleahydroxy lactone isolated from A. elaeagnoidea [35]. The sole distinction was the addition of an acetyl group at compound 6, which was identified based on the addition of two carbon atoms. Therefore, its structure was identified as a dammarane-type triterpenoid, namely 3α-acetyl-cabraleahydroxy lactone, which was isolated for the first time from A. cucullata.
Compounds 1-7 were classified as dammarane-type triterpenoids. Although those were known compounds, compounds 1-7 were isolated from A. cucullata for the first time. This indicated that the species, along with other members of the Aglaia genus, could be used as a source of triterpenoid compounds. Dammarane-type triterpenoids were known for their biological activity, including cytotoxicity. Therefore, this study was consistent with previous reports where this plant was identified as a source of bioactive compounds [38,40,42].

Cytotoxic Activity Compounds 1-7
A normal cell (CV-1 kidney fibroblast cells) and two cancer cell lines (MCF-7 breast cancer and B16-F10 melanoma cells) were used to test the cytotoxic activities of compounds 1-7. Furthermore, cisplatin (positive control) was used during the assessment, as presented in Table 4. In this experiment, two wavelengths (570 and 600 nm) were used because the measured cells were live cells so, to obtain the value/number of live cells, measurements were carried out at two wavelengths, namely before the reaction (blue) and after the reaction (pink) to produce a corrected absorbance value, which was interpreted as the number live cells after treatment [48]. The results arising from the experiments, along with IC 50 calculation graphs and cell morphology at each sample concentration, are presented in Figures S38-S58. In MCF-7 cells, compounds 2 and 5 showed cytotoxicity with an IC 50 close to cisplatin due to its chemical structure. In compound 3, the presence of the olefinic group at C-20 increased the cytotoxic activity compared with compound 4 with the hydroxyl group at C-20. A similar observation also occurred in compound 2, where the additional olefinic group at C-20/C-21 suggested an increase in cytotoxic effect. Based on previous studies, the degradation of three carbon atoms to form an acetal group in the side chain, which was attached to a five-membered ring in compound 5, was believed to increase cytotoxicity against cells [31].
Compounds 1, 3, and 5 showed similar cytotoxicity against B16-F10 melanoma cells compared to the positive control cisplatin. Compared to MCF-7, the appearance of the hydroxyl group at C-20 in compound 3 increased the activity against B16-F10 compared to compound 2 with the olefinic group at C-20. The results also demonstrated that compound 5 had better cytotoxicity against both cancer cells compared to the others. Furthermore, a significant difference was the existence of an acetal group in the side chain, suggesting that this group contributed to its bioactivity.
Among all isolated compounds, 4 showed the weakest cytotoxicity to both cancer cells, suggesting that its constituent ring A served as a negative factor. The compounds obtained in this study were also tested against CV-1 normal cell lines to determine their safety toward normal cells. Furthermore, 3, 5, and 7 exhibited high IC 50 of >300 µg/mL (>300 µM), indicating that they were relatively safe. Based on morphological observation, there was no cell death at all concentration levels.
The cytotoxicity assessment revealed that 5 was the most active compound in terms of cytotoxicity as opposed to MCF-7 and B16-F10, as well as the safest of the CV-1 normal cell lines. Furthermore, the IC 50 range obtained for the natural dammarane-type triterpenoids was more than 100 µM, indicating that they were still far from being considered potential compounds for cytotoxic activity. A literature review by Cao et al. [49] stated that several herbal plants with high potential in treating various diseases, including cancer, contained dammarane-type triterpenoids. According to our results, dammarane-type triterpenoids alone give low cytotoxicity, suggesting that these compounds showed the synergistic effect with other active components in plant extracts to give a more significant effect. The semisynthesis of dammarane-type triterpenoids also offered a pathway to obtain more active compounds through extensive modifications of their structure.
A review by Ruan et al. [50] explained that in addition to cytotoxic activity, dammarane triterpenoids also provided a broad spectrum of activity. Glycosylated dammarane triterpenoids showed potential anti-inflammation activity, whereas aglycon dammarane triterpenoids with double bonds in its side chain from Panax ginseng displayed immunomodulatory activity. Sapogenin-type dammarane triterpenoids performed remarkable antineoplastic activity. It was shown that glycosylated and additional double bonds in the side chain significantly increased biological activity. These phenomena give insightful thought that our compounds can be used after small functional group modifications to give desired activity.

General Experimental Procedures
High-resolution mass spectra (HRESI-TOFMS) were acquired using a Waters Xevo Q-TOF direct probe/MS system utilizing ESI+ mode with a microchannel plate MCP detector (Milford, MA, USA), and optical rotations were determined using an ATAGO AP-300 automated polarimeter (Saitama, Japan). Additionally, the One Perkin Elmer infrared-100 (Shelton, CT, USA) was used to measure the IR spectra. On a JEOL ECZ-500 spectrometer (Tokyo, Japan), the NMR data were collected for 1 H (500 MHz) and 13  TLC plates with GF254 (Merck, 0.25 mm) was followed by detection, which was carried out by spraying 10% H 2 SO4 in ethanol and then heating.

Plant Material
The stem bark of A. cucullata was derived from the Manggar River in Balikpapan, East Kalimantan, Indonesia. The sample was assessed by the Herbarium Wanariset staff, Balikapapan, in December 2020, and the specimen was deposited at the herbarium (collection No. FF7.20).

Determination of Cytotoxic Activity
The PrestoBlue assay was utilized to conduct the cytotoxic bioassay. Additionally, Presto Blue reagent from Thermo Fisher Scientific, Uppsala, Sweden, was used to measure cell viability, which allowed for rapid evaluation of various resazurin-based cells. The rate of proliferation of the samples was then determined quantitatively through live-cell reduction capabilities. According to the findings, healthy cells kept a reduced habitat in their cytoplasm. By reducing resorufin (purple) with absorbance or fluorescence outputs, resazurin reduction (blue) acted as a viability indicator. The conversion correlated with the number of cells that were metabolically active. MCF-7, B16-F10, and CV-1 cell lines were cultured until they reached 70% confluence, and then they were removed, counted with a hemocytometer, and diluted with complete culture RPMI media. A total of 170,000 cells per well in 96-well plates with the samples were used. After an overnight growth period, the samples were treated with compounds 1-7 at escalating concentrations (3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, and 5000 µg/mL) in PBS, with 2% DMSO as the co-solvent. Additionally, cisplatin served as the positive control, and all samples were incubated for 24 h at 37 • C with 5% CO 2 . Following incubation, the medium was immediately changed to 10 µL of the PrestoBlue reagent in 90 µL of the RPMI medium. As resorufin began to form, the color of the plates changed from blue to purple after being incubated for another one to two hours. Using a microplate reader to measure absorbance at 570 nm and 600 nm, the concentration required to inhibit growth by 50% was calculated as the IC 50 value. The concentration was calculated using a plot of cytotoxicity against sample concentrations, and the result revealed 50% cytotoxicity (IC 50 ). The results of each test and analysis were averaged after being run twice.

Conclusions
In this study, seven dammarane-type triterpenoids (1-7) were successively isolated for the first time from the stembark of A. cucullata using various extraction and chromatography methods. Furthermore, their chemical structures were unambiguously determined using various spectroscopic methods. All isolated compounds were tested utilizing PrestoBlue reagent toward MCF-7 breast cancer, B16-F10 melanoma, and CV-1 normal kidney fibroblast cell lines. Based on the results, compound 5 was found to be the most active compared to the others.