New dammarane-type triterpenoids from Aglaia elliptica (C.DC.) blume

Abstract Three new dammarane-type triterpenoids, namely elliptaglin A-C (1–3) were isolated from the stem bark of Aglaia elliptica (C.DC.) Blume along with three known derivatives, namely (20S)-hydroxydammar-24-en-3-on (4), cabralealactone (5), and E-25-hydroperoxydammar-23-en-20-ol-3-one (6). Subsequently, their chemical structures were determined using HR-ESI-MS, FTIR, 1D and 2D-NMR spectroscopic analysis as well as comparison with previous studies. The cytotoxicity activities of the isolated compounds against MCF-7 breast cancer and B16-F10 melanoma cell lines were then tested using PrestoBlue reagent. The analysis results showed that elliptaglin B (2) had the strongest activity against both cell lines with IC50 values of 60.98 and 51.83 µM, respectively. GRAPHICAL ABSTRACT


Introduction
Aglaia genus, also known as Mahagoni has more than 150 species of which only 65 are grown in Indonesia (Harneti and Supratman 2021).It is also a native plant in the tropical and subtropical regions of Asia, Australia, and the Pacific Islands.Consequently, it is widely distributed in India, Indonesia, Malaysia, Thailand, and Vietnam, while small populations are grown in the southern part of China as well as the northern part of Australia (Pannell 1992).As the largest genus in the Meliaceae family, Aglaia is known as an abundant source of secondary metabolites, such as sesquiterpenoids (Milawati et al. 2019), diterpenoids (Yodsaoue et al. 2012), triterpenoids (Harneti et al. 2014), protolimonoid (Farabi et al. 2017), limonoid (Sun et al. 2022), steroids (Farabi et al. 2018;Hutagaol et al. 2020), lignan (Sianturi et al. 2016), alkaloids (Sianturi et al. 2015), and flavaglines (An et al. 2016).Furthermore, several studies reported that it has diverse biological activities, such as cytotoxic (Farabi et al. 2017), insecticidal (Chaidir et al. 2001), antiviral (Joycharat et al. 2008), antifungal (Engelmeier et al. 2000), anti-inflammatory (Yodsaoue et al. 2012), and molluscicidal (Zhang et al. 2012).Aglaia elliptica is a rain forest plant that is mainly distributed in the northern part of Kalimantan Island, Indonesia.It is also used as an Indonesian folk medicine for the treatment of wounds, fever, coughs, and skin diseases (Cui et al. 1997;Muellner et al. 2005).Previous studies revealed that triterpenoids and steroids have been isolated from its stem bark, and they have cytotoxic activity against P388 murine leukemia cell lines (Hidayat et al. 2017;Farabi et al. 2018).To complete the chemical study on the plant as well as continue the investigation of the cytotoxicity of its natural product, three new dammarane-type triterpenoids (1-3), along with three known derivatives (4-6) were isolated and identified from its bark.Therefore, this study discusses the isolation and structural elucidation of six dammarane-type triterpenoids (1-6) (Figure 1) as well as their cytotoxic activity against MCF-7 breast cancer and B16-F10 melanoma cell lines.

Result and discussion
Elliptaglin A (1) was isolated as a colorless oil with a molecular formula of C 26 H 42 O 2, which was obtained from the HR-ESI-TOFMS spectrum (m/z 387.3254 ) and the NMR data, as shown in Table S1.Furthermore, the presence of functional groups was detected using the FTIR spectrum, such as hydroxyl, carbonyl, and olefinic groups at 3403 cm À1 , 1708 cm À1 , and 1647 cm À1 , respectively.S1.The FTIR spectrum of 2 showed high similarity with 1, which indicated that they relatively have the same functional groups.The 13 C NMR and DEPT spectra, as well as the HMQC experiment revealed that it contains 27 carbon resonances, which can be divided into six methyls, eight methylenes, eight methines including two olefinic at d C 162.6 and 130.1, one oxymethine at d C 76.3, and one aldehyde at d C 193.9 as well as five quaternary carbons of which one is oxygenated at d C 76.4.The 1 H NMR spectrum showed six tertiary methyls (d H 1.35, 0.92, 0.91, 0.87, 0.83, and 0.82), one aldehyde (d H 9.68, d, J ¼ 7.5 Hz, H-24), two olefinic (d H 6.84, d, J ¼ 15.5 Hz, H-22 and 6.32, dd, J ¼ 7.5, 15.5 Hz, H-23), and one oxymethine (d H 3.38, H-3).The coupling constant of aldehyde H-24 and olefinic H-23 at 7.5 Hz were identical, which indicated that they were in a neighboring position as an a,b-unsaturated aldehyde.The coupling constant of two olefinic protons, namely H-22 and H-23 at J ¼ 15.5 Hz were suitable for the E-configuration.The NMR data of 2 were also closely related to that of 1. Furthermore, the difference between the two compounds was that 1 contains 26 carbons, while 2 has 27.The HMBC correlations of five tertiary methyls revealed that 2 has a tetracyclic dammarane-type triterpenoid skeleton.The relationship of   Elliptaglin C (3) was obtained as a white amorphous powder, and its molecular formula was assigned to be C 30 H 50 O 2 based on the HR-ESI-TOFMS data (m/z 443.3883S29.Therefore, these findings showed that 3 is a new dammarane-type triterpenoid, 20S,25-epoxy-dammar-23-ene-3b-ol, which was named elliptaglin C. The structure of the known compounds 4-6 were identified using the spectroscopy methods as well as the comparison with previously reported data, namely (20S)hydroxydammar-24-en-3-one (4) (Heliawati et al. 2020), cabralealactone (5) (Retnowati et al. 2021), and E-25-hydroperoxydammar-23-en-20-ol-3-one (6) (Lee et al. 2001), which were isolated for the first time from Aglaia elliptica.
Compounds 1-6 were evaluated for their cytotoxic activities against MCF-7 breast cancer cell lines and B16F10 melanoma skin cancer cell lines.They were then compared to Cisplatin as a positive control, as shown in Table S2.The IC 50 of 2 indicated that it had the strongest activities against MCF-7 and B16F10 compared to the other isolated compounds.These results suggest that the presence of a,b-unsaturated aldehyde, as well as a hydroxyl group in the side chain of dammarane-type triterpenoid compounds, plays an important role in their cytotoxic activity.Another compound that increased their activity was the hydroperoxyl group.This finding was clearly evident in compound 6, which was the second most active isolated compound.

General experimental procedures
The mass spectra were measured using a Waters Xevo QTOFMS (Waters, Milford, MA, USA), while the IR spectra were measured on a One Perkin Elmer infrared-100 (Shelton, Connecticut, USA).Furthermore, the NMR data were recorded on a JEOL ECZ-500 spectrometer (Tokyo, Japan) at 500 MHz for 1 H and 125 MHz for 13 C using TMS as an internal standard.Chromatographic separations were then carried out on a silica gel G60 (Merck, Darmstadt, Germany) and octadecyl silane (Fuji Sylisia Chemical LTD., ChromatorexV R C 18 DM1020 M, 100-200 mesh).The TLC plates were precoated with silica gel GF 254 (Merck, 0.25 mm) and RP-18 F 254s plates (Merck), after which detection was performed by spraying with 10% H 2 SO 4 in ethanol, followed by heating.

Plant material
The stem bark of A. elliptica was obtained from the Bogor Botanical Garden, West Java, Indonesia, in June 2015.The plant was then identified and classified by the staff of Herbarium Bogoriense.Subsequently, a voucher specimen with No. Bo-1294562 was deposited at the herbarium.

Extraction and isolation
A. elliptica stem bark was ground to dried powder of which 2.3 kg was extracted exhaustively with methanol (12 L Â 5) at room temperature.Subsequently, a total of 321.5 g extract was obtained after the solvent was evaporated under reduced pressure.It was then partitioned with n-hexane, ethyl acetate, and n-butanol to produce 22.6 g, 31.4 g, and 34.5 g of the extracts, respectively.
22.6 g of the n-hexane extract was fractionated by vacuum liquid chromatography (r: 5 cm; h: 12 cm) on silica gel (300 g of G60 silica gel) using a gradient elution of nhexane-ethyl acetate (10:0-0:10, stepwise 10%; v: 500 mL) continued by ethyl acetatemethanol (10:0-0:10, stepwise 10%; v: 500 mL) to give five fractions (A-E).Subsequently, 6 g of C was separated with silica gel column chromatography (70-230 mesh, 120 g) using a gradient elution of n-hexane-ethyl acetate (10:0-1:1 stepwise 5%; r: 4 cm; h: 15 cm; v: 400 mL) to give five fractions (C1-C5).A total of 2.1 g C3 was then separated with silica gel column chromatography (70-230 mesh, 63 g) using a gradient elution of n-hexane-ethyl acetate (10:0-7:3 stepwise 2.5%; r: 2 cm; h: 15 cm; v: 240 mL) to give eight fractions (C3a-C3h).516 mg of fraction C3c was separated with silica gel column chromatography (230-400 mesh, 21 g) using n-hexane-ethyl acetate in a ratio of 23:2 to give three fractions (C3c1-C3c3).105 mg of fraction C3c1 was then separated with silica gel column chromatography (230-400 mesh, 4.2 g) using n-hexanemethylene chloride-ethyl acetate in a ratio of 7:3:4 to give four fractions (C3c1a-C3c1d).Furthermore, 44 mg of C3c1b was separated with silica gel column chromatography with silica gel column chromatography (230-400 mesh, 1.8 g) using n-hexane-methylene chloride-ethyl acetate in a ratio of 7:3:1 to produce 11.3 mg of compound 4. A total of 134 mg C3c3 was then separated with silica gel column chromatography with silica gel column chromatography (230-400 mesh, 5.7 g) using n-hexane-ethyl acetate in a ratio of 17:3 to give six fractions (C3c3a-C3c3f).21 mg of C3c3c was then separated with ODS column chromatography (1.3 g of ODS silica gel) using methanol-water in a ratio of 10:1 to produce 4 mg of 1. 270 mg of C3d was separated with silica gel column chromatography (230-400 mesh, 11 g) using n-hexane-ethyl acetate (18:2-17:3 stepwise 1%; r: 1 cm; h: 15 cm; v: 10 mL) to give three fractions (C3d1-C3d3).73 mg of C3d2 was separated with ODS column chromatography (3.7 g of ODS silica gel) using acetonitrile-water in a ratio of 17:3 to produce 10 mg of compound 5. Subsequently, a total of 232 mg C3e was separated with silica gel column chromatography (230-400 mesh, 9.3 g) using methylene chloride-ethyl acetate in a ratio of 9:1 to give six fractions, namely C3e1-C3e6.23 mg of C3e3 was then separated with ODS column chromatography (1.2 g of ODS silica gel) using methanol-water in a ratio of 17:3 to produce 2 mg of compound 2. 50 mg C3e4 was separated with the same technique (2.5 g of ODS silica gel) using methanol-water in a ratio of 17:3 to The 13 C NMR spectrum along with the HMQC experiment successfully identified 26 resonated carbons.They were classified into six methyls, eight methylenes, seven methines including one olefinic at d C 126.8, one oxymethine at d C 76.3, and one aldehyde at d C 191.6 as well as five quaternary carbons including one olefinic at d C 167.8.These functionalities accounted for two out of the total six degrees of unsaturation, while the remaining four were caused by the tetracyclic dammarane-type triterpenoid skeleton.The 1 H NMR spectrum showed six tertiary methyls (d H 2.11, 0.96, 0.93, 0.90, 0.84, and 0.82), one aldehyde (d H 9.98, d, J ¼ 8.0 Hz), one olefinic (d H 5.86, d, J ¼ 8.0 Hz), and one oxymethine (d H 3.38).The identical coupling constant of the olefinic and aldehyde protons indicated that the two functional groups are related to each other by forming an a,b-unsaturated aldehyde.Furthermore, the comparison of the NMR data of 1 with notoginsenoside ST8 isolated from Panax notoginseng (Gu et al. 2015) showed some similarities, specifically in the side chain part, in terms of losing four carbons to give an a,b-unsaturated aldehyde.The structure of 1 was deduced by HMBC and 1 H-1 H COSY experiments, as shown in Figure S9 and S10.The correlations observed between the five tertiary methyl protons and their neighboring carbons revealed that they have a tetracyclic dammarane-type triterpenoid core.The relationship of the downfield methyl signal at Me-21 (d H 2.11) with C-17 (d C 51.3), C-20 (d C 167.8), and C-22 (d C 126.8) indicated the presence of a double bond at C-20/C-22.The occurrence of the 1 H-1 H COSY cross peak at H-22/H-23 also showed that the compound is a degraded dammarane-type triterpenoid, which lost four carbons to
H 1.35) with C-17 (d C 50.4), C-20 (d C 76.4), and C-22 (d C 162.6) indicated the presence of hydroxyl group at C-20.The study of NMR spectra characteristic of numbers of isolated 20-hydroxy dammarane-type triterpenoid by Ruan et al. (2016) revealed that the d C of C-21 can be used for determination of absolute configuration at C-20.The d C of compound 2 was 27.3 (C-21), thus suitable for the 20S-configuration (20 R if d C was 21.3) (Ruan et al. 2016).The occurrence of 1 H-1 H COSY cross peak of H-22/H-23/H-24 suggests that a,b-unsaturated aldehyde was formed at C-24, as shown in Figure S19.Consequently, the structure of 2 was established as a new dammarane-type triterpenoid, (E)-3b,20S-dihydroxy-25,26,27-trinordammar-22-ene-24enal, which was named elliptaglin B.

Table S1 .
(Zhang et al. 2010)C 30 H 51 O 2 þ ) as well as the NMR spectral data, as shown in Furthermore, the results revealed that the compound requires six degrees of unsaturation.The FTIR spectrum showed the presence of hydroxyl group at 3356 cm À1 and olefinic group at 1645 cm À1 .The 13 C NMR spectrum indicated that it contains 30 carbon resonances, which were classified using DEPT and HMQC spectra as eight methyls, nine methylenes, seven methines including two olefinic at d C 123.6 and 140.6, one oxymethine at d C 76.4, as well as six quaternary carbons of which two are oxygenated at d C 75.7 and 72.1.The 1 H NMR spectrum showed the presence of eight tertiary methyls at d H 1.33, 1.31, 1.15, 0.94, 0.92, 0.85, 0.84, and 0.82.It also revealed that the compound contains two sp 2 methines at d H 5.62 and 5.39, as well as one oxymethine at d H 3.38.A comparison of the NMR data of 3 with aglaiabbreviatin D isolated from A. abbreviata(Zhang et al. 2010)showed high similarity.Furthermore, the main difference between the compounds was the presence of a hydroxyl group at C-3 in 3 instead of the carbonyl group.The dammarane-type triterpenoid skeleton was confirmed in the HMBC spectrum of five tertiary methyls, as well as protons correlations in 1 H-1 H COSY spectrum, as shown in FigureS1.The correlation of Me-21 (d H 1.15) with C-17 (d C 50.6), C-20 (d C 75.7), and C-22 (d C 36.7) as well as Me-26,27 (d H 1.31, 1.33) with C-24 (d C 140.6) and C-25 (d C 72.1) confirmed the formation of ether bridge between C-20 and C-25.The olefinic group is located at C-23/C-24 based on the correlation of H-22 at d H 2.21 with C-23 at d C 123.6 and the 1 H-1 H COSY cross peak of H-22/H-23/H-24.The relative stereochemistry was determined unambiguously in the NOESY spectrum, as shown in Figure