Limonoids From Choerospondias axillaris

Abstract

Eight limonoids (1-8), including 1 new compound named as axillariol A (1), were isolated from the dichloromethane extract of Choerospondias axillaris branches and leaves using various chromatographic separations. Their structures were confirmed by detailed analysis of the 1-dimensional (1D), 2D nuclear magnetic resonance, and high-resolution electrospray ionization quadrupole time-of-flight mass spectra. In addition, toonaciliatins N (6) and toonaciliatins O (7) showed selective and significant cytotoxicity on the rhabdomyosarcoma human cancer cell line with half-maximal inhibitory concentration values of 9.12 and 7.79 µM, respectively.

Keywords

Anacardiaceae limonoid axillariol A cytotoxic activity

Limonoids are highly oxygenated triterpenes, which have lost the terminal 4 carbon atoms of the side chain, with the remaining side chain cyclized into a furan ring. Limonoids are widely found in leaves, bark, fruits, seeds, and kernels throughout the Meliaceae and Rutaceae.¹ This type of compound revealed various interesting biological effects such as antifeedant,^1,2 cytotoxic,^3,4 antibacterial, anti-human immunodeficiency virus, and human carboxylesterase 2 inhibitory⁵ activities.

As a part of our ongoing investigations on medicinal plants growing in Lamdong province, Vietnam,^6
-8 the methanol (MeOH) extract of Choerospondias axillaris branches and leaves wre found to exhibit cytotoxicity against hepatocellular carcinoma (Hep-G2) and rhabdomyosarcoma (RD) human cancer cell lines with half-maximal inhibitory concentration (IC₅₀) values of 8.02 and 10.08 µg/mL, respectively. In Vietnam, this plant is found in glades in semideciduous forests in Son La, Lao Cai, Bac Giang, Quang Ninh, Nghe An, Ha Tinh, Kon Tum, Dak Lak, and Lam Dong provinces. The stem bark and fruit of this plant have been used in folk medicine for their antibacterial, detox, and anti-inflammatory effects.⁹ Recent investigations of this plant reported the main chemical constituents as bridged-ring ketones,¹⁰ proanthocyanidins,¹¹ and flavonoids,¹² with some exhibiting cytotoxic,^10,12 antioxidant, antiangiogenic,¹¹ antihypoxia, and antibacterial¹² activities. In this article, we address the isolation and structure elucidation of 8 limonoids (1-8) from C. axillaris branches and leaves as well as their in vitro cytotoxic activity against 4 human cancer cell lines.

Results and Discussion

The dry MeOH extract of C. axillaris branches and leaves was suspended in water (H₂O) and partitioned in turn with n-hexane, dichloromethane (CH₂Cl₂), and ethyl acetate (EtOAc). Subsequently, 8 limonoids (1-8, Figure 1) were isolated from the CH₂Cl₂ extract by using various chromatographic procedures. Careful analysis of the 1-dimensional (1D), 2D nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) data, as well as comparison of them with the literature data, led to the elucidation of the known compounds as surenolactone (2),¹³ ouabanginone (3),¹⁴ toonaciliatins D (4),¹⁵ H (5),¹³ N (6),¹⁶ and O (7),¹⁶ and toonayunnanin I (8).¹⁷

Figure 1

The structures of compounds 1-8.

Axillariol A (1) was isolated as a white powder. Its molecular formula was identified as C₂₆H₃₂O₈ by high-resolution (HR) ESI quadrupole time-of-flight (QTOF) MS having a quasi-molecular ion peak at m/z 495.2021 [M + Na]⁺ (Supplemental Figure S7). As in the case of 2-8, the ¹H and ¹³C NMR spectra of 1 (Figure 1) revealed a limonoid containing typical signals of 3 lactone carbonyl carbons [δ _C 166.5 (C-3), 171.2 (C-7), and 170.4 (C-23)], 3 oxygenated quaternary carbons [δ _C 85.2 (C-4), 84.1 (C-8), and 72.4 (C-14)], 1 oxymethine [δ _C 54.4 (C-15)/δ _H 3.72 (1H, s, H-5)], 1 dioxymethine [δ _C 98.6 (C-21)/δ _H 5.89 (1H, br s, H-21)], 2 double bonds [δ _C 160.4 (CH, C-1) and 120.8 (CH, C-2)/δ _H 6.67 (1H, d, J = 12.0 Hz, H-1) and 5.86 (1H, d, J = 12.0 Hz, H-2) and δ _C 168.8 (C, C-20) and 119.0 (CH, C-22)/δ _H 6.07 (1H, br s, H-22)], and 5 tertiary methyls [δ _C 22.3 (C-18), 14.8 (C-19), 31.7 (C-28), 26.7 (C-29), and 18.3 (C-30)/δ _H 1.01 (H-18), 1.34 (H-19), 1.39 (H-28), 1.41 (H-29), and 1.43 (H-30), each 3 H, s] (Supplemental Figures S1, S2). From the above observation, the ¹H and ¹³C NMR data of 1 (Table 1) were found to be similar to those of surenolactone (2),¹³ except for signals of the side chain. Detailed comparison of the ¹H and ¹³C NMR spectroscopic data of 1 with those of surenolactone (2),¹³ as well as analysis of the heteronuclear single quantum correlation, heteronuclear multiple bond correlation, and correlation spectroscopy (Supplmental Figures S3–S5) correlation, clearly confirmed the planar structure of the triterpene skeleton of 1 (Figure 2). The side chain of 1 contained signals for 1 trisubstituted double bond [δ _C 168.8 (C, C-20) and 119.0 (CH, C-22)/δ _H 6.07 (1H, d, J = 0.5 Hz, H-22)], 1 dioxygenated methine [δ _C 98.6 (C-21)/δ _H 5.89 (1H, br s, H-21)], and 1 lactone carbonyl carbon [δ _C 170.4 (C-23)], indicating a 21-hydroxy-20(22)-ene-21,23-γ-lactone moiety.^18,19 The low intensities of the ¹³C NMR signals for C-20, C-21, and C-22 of 1 relative to the other signals suggested that this compound might be a mixture of 2 epimers at C-21. The relative configuration for the triterpene skeleton of 1 was assigned by analogy with surenolactone (2),¹³ coexistence in C. axillaris, as well as by rotating frame Overhauser effect spectroscopy (ROESY) experiment (Supplemental Figure S6). The ROESY correlations of H-9 (δ _H2.33) with both H-5 (δ _H2.58) and H-18 (δ _H1.01) confirmed an α-orientation of H-5 and H-9, and the methyl group at C-13 (Figure 3). The similarity of the ¹H and ¹³C NMR spectra at C-14 and C-15 of 1 with those of surenolactone (2),¹³ as well as the biosynthetic background with the coexistence of these 2 compounds in C. axillaris, supported the same configuration of the epoxy group at C-14/C-15 for both compounds. In addition, H-17 (δ _H2.53) exhibited an ROE-correlation with H _β -12 (δ _H1.93), supporting a β-orientation of H-17, consistent with the computer-generated 3D model by PerkinElmer Chem3D Ultra version 18.2.0.48 software using MM2 force field calculations (Figure 3). Noteworthily, to the best of our knowledge, this is the first finding of limonoid constituents from C. axillaris to date.

Figure 2

Key correlation spectroscopy (▬) and heteronuclear multiple bond correlation () correlations of 1.

Figure 3

Key rotating frame Overhauser effect spectroscopy correlations and computer-generated 3-dimensional model using MM2 force field calculations of 1.

Table 1

¹H (500 MHz, DMSO-d ₆) and ¹³C NMR (125 MHz, DMSO-d ₆) Spectroscopic Data of 1.

C	δ _C	δ _H mult. (J in Hz)
1	160.4	6.67 d (12.0)
2	120.8	5.86 d (12.0)
3	166.5	−
4	85.2	-−
5	51.8	2.58 dd (2.0, 13.5)
6	37.3	2.52 dd (2.0, 13.5)/3.28 t (13.5)
7	171.2	−
8	84.1	−
9	52.4	2.33 t (5.5)
10	45.9	−
11	24.7	1.64 m
12	34.5	1.76 m/1.93 m
13	41.4	−
14	72.4	−
15	54.4	3.72 s
16	30.3	2.07 m
17	43.2	2.53 m
18	22.3	1.01 s
19	14.8	1.34 s
20	168.8	−
21	98.6	5.89 br s
22	119.0	6.07 br s
23	170.4	−
28	31.7	1.39 s
29	26.7	1.41 s
30	18.3	1.43 s

Abbreviation: DMSO, dimethyl sulfoxide.

All assignments were based on heteronuclear single quantum correlation, correlation spectroscopy, heteronuclear multiple bond correlation, and rotating frame Overhauser effect spectroscopy experiments.

Since the MeOH extract of C. axillaris was found to exhibit cytotoxicity to Hep-G2 (hepatocellular carcinoma) and RD (rhabdomyosarcoma) cancer cell lines, isolated compounds 1-8 were subsequently evaluated using the sulforhodamine B (SRB) method for their cytotoxic effects against the human cancer cell lines Hep-G2, Lu (lung adenocarcinoma), RD, and RD (cervix carcinoma).^20,21 Only compounds 6 and 7 showed selective and significant cytotoxicity on the RD cells with corresponding IC₅₀ values of 9.12 and 7.79 µM, relative to the positive control ellipticine (IC₅₀ = 0.73 µM). The other compounds did not show significant cytotoxic activity with IC₅₀ >10 µM (Table 2).

Table 2

Cytotoxic Activity of Compounds 1-8 on 4 Human Cancer Cell Lines

Coumpounds	IC₅₀ values (µM)
Coumpounds	Hep-G2	Lu	RD	RD
1	(−)	(−)	(−)	(−)
2	(−)	(−)	(−)	(−)
3	(−)	(−)	(−)	(−)
4	(−)	(−)	(−)	(−)
5	(−)	(−)	(−)	(-)
6	(−)	(−)	9.12	(−)
7	(−)	(−)	7.79	(−)
8	(−)	(−)	(−)	(−)
Ellipticine^a	1.18	1.26	0.73	0.89

Abbreviation: IC₅₀, half-maximal inhibitory concentration.

^aEllipticine was used as positive control; (−): IC₅₀ >10 µM; results are the average means of triplicate experiments.

Experimental

General

Optical rotations were determined on a JASCO P-2000 polarimeter (Tokyo, Japan), HR-MS on a MicroQ-TOF III mass spectrometer (Bruker Daltonics, Germany), and ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra on an AVANCE III HD 500 FT-NMR spectrometer (Bruker, Germany), with trimethylsilane used as an internal standard. Medium pressure liquid chromatography (MPLC) was carried out on a Biotage-Isolera One system (SE-751 03 Uppsala, Sweden), and column chromatography (CC) on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck, Darmstadt, Germany), reversed-phase silica gel (ODS-A, 12 nm S-150 mm, YMC Co., Ltd.), and Sephadex LH-20 (Sigma) resins. Thin-layer chromatography (TLC) used precoated silica gel 60 F₂₅₄ (1.05554.0001, Merck) and RP-18 F_254S plates (1.15685.0001, Merck), and compounds were visualized by spraying with aqueous 10% sulfuric acid, followed by heating for 3-5 minutes.

Plant Material

Choerospondias axillaris (Roxb.) Burtt et Hill (Anacardiaceae) branches and leaves were collected in Lamdong province, Vietnam, in June 2018. The voucher specimen (No: TN3/201) was identified by Dr Nong Van Duy and deposited at the Tay Nguyen Institute of Scientific Research, VAST, Vietnam.

Extraction and Isolation

Air-dried powder of C. axillaris branches and leaves (3 kg) was extracted with MeOH (5 L) under ultrasonic condition (3 times, 1 hour each). The resulting solutions were filtered, combined, and concentrated in vacuum to give the MeOH residue (M, 250 g). This was suspended in H₂O (2 L) and partitioned in turn with n-hexane (H; 3 × 2 L), CH₂Cl₂ (D; 3 × 2 L), and EtOAc (E; 3 × 2 L) to obtain the corresponding n-hexane (40.0 g), CH₂Cl₂ (14.5 g), and EtOAc (15.5 g) extracts, and H₂O residue. Extract D (14.5 g) was separated into 4 fractions, D1-D4, by using silica gel MPLC with step-wise elution using n-hexane-acetone (10:1, 5:1, 3:1, and 1:1, v/v). Fraction D2 (3.0 g) was further separated by silica gel CC with n-hexane-CH₂Cl₂-EtOAc (1:2:0.2, v/v) to obtain 6 subfractions, D2A-D2F. Purification of subfraction D2B (740 mg) on ODS-A CC with acetone-H₂O (2:1, v/v) furnished compound 2 (11.0 mg). Subfraction D2D (400 mg) was further purified by ODS-A CC with MeOH-H₂O (2:1, v/v), followed by silica gel CC with n-hexane-CH₂Cl₂-acetone (4:10:1, v/v) to give compounds 5 (4.5 mg) and 8 (10.1 mg). Compound 4 (11.5 mg) was purified from subfraction D2E (300 mg) after subjecting it to ODS-A CC with acetone-H₂O (2:1, v/v), followed by Sephadex LH-20 CC with MeOH-H₂O (2:1, v/v). Subfraction D2F (300 mg) was treated in the same manner as for subfraction D2D to obtain compound 3 (3.5 mg). Fraction D4 (7 g) was separated by ODS-A CC with acetone-H₂O (1:1, v/v) to obtain 3 subfractions, D4A-D4C. Subfraction D4A (2.1 g) was further separated into 4 smaller fractions, D4A1-D4A4, using silica gel CC with CH₂Cl₂-EtOAc-MeOH (5:1:0.5, v/v). Compound 1 (1.1 mg) was obtained from fraction D4A1 (70 mg) after subjecting it to Sephadex LH-20 CC with MeOH-H₂O (1.5:1, v/v). Subfraction D4B (1.0 g) was treated in the same manner as subfraction D4A to give 4 smaller fractions, D4B1-D4B4. Finally, compounds 6 (4.5 mg) and 7 (3.0 mg) were purified from fraction D4B4 (100 mg) after subjecting it to silica gel CC with CH₂Cl₂-EtOAc-MeOH (10:1:0.25, v/v), followed by ODS-A CC with acetone-H₂O (1:1, v/v).

Axillariol A (1)

White amorphous powder.

[α]_D ²⁵ + 35 (c 0.05, MeOH);

¹H (dimethyl sulfoxide [DMSO-d ₆], 500 MHz) and ¹³C NMR (DMSO-d ₆, 125 MHz) data are given in Table 1.

HR ESI QTOF MS m/z 495.2021 [M + Na]⁺ (calcd. for C₂₆H₃₂NaO₈ ⁺, 495.1995).

Cytotoxic Assays

Cytotoxic evaluations were performed by SRB colorimetric assay^20,21 employing 4 human cancer cell lines: Hep-G2, Lu, RD, and RD. The assay was adapted for the quantitative measurement of cell growth and viability. Briefly, cancer cells were seeded at 5 × 10³ cells per well in 96-well microtiter plates and incubated at 37 °C (48 hours, 5% carbon dioxide). Cultures were fixed with trichloroacetic acid and stained for 30 minutes with 0.4% (w/v) SRB. The protein-bound dye was extracted with 10 mM Tris base for determination of optical density (OD) at a single wavelength of 564 nm in a 96-well plate reader. The percentages of cell survival of test samples were calculated using the following formula: % cell survival = 100% × [OD_(sample) – OD_{(day 0)}]/[OD_(DMSO) – OD_{(day 0)}]. IC₅₀ values of test samples were calculated by interpolation by linear regression analysis.

Conclusions

From C. axillaris branches and leaves, 8 limonoids, including 1 new compound named axillariol A (1), were isolated and their structures elucidated. Among the isolated compounds, toonaciliatins N (6) and O (7), showed selective and significant cytotoxicity to the RD human cancer cell line. With this significant cytotoxicity, compounds 6 and 7 may be potential candidates for further investigations on the molecular mechanism of action on anticancer targets. Furthermore, consideration of the structures of 1-8 suggested that the formation of a γ-lactone ring at C-4/C-7 might play an important role in the cytotoxicity of these compounds.

Supplemental Material

Figure S1 - Supplemental material for Limonoids From Choerospondias axillaris

Supplemental material, Figure S1, for Limonoids From Choerospondias axillaris by Nguyen Huu Toan Phan, Nguyen Thi Dieu Thuan, Nguyen Thi Thu Hien, Pham Van Huyen, Tran Thi Hong Hanh, Le Thi Vien, Tran Hong Quang, Nguyen Xuan Cuong, Nguyen Hoai Nam, Phan Van Kiem and Chau Van Minh in Natural Product Communications

Footnotes

Acknowledgments

The authors are grateful to Dr Tran Thi Nhu Hang, Institute of Natural Products Chemistry, VAST for the cytotoxic evaluation, and the Institute of Chemistry, VAST for measurement of the NMR spectra.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by Vietnam national project of the Tay Nguyen Program (TN18/C09).

ORCID iDs

Nguyen Thi Dieu Thuan

Nguyen Xuan Cuong

Phan Van Kiem

Supplemental Material

Supplemental material for this article is available online.

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