Chemical Constituents of Cassia abbreviata and Their Anti-HIV-1 Activity

Abstract

Three new (1–3) and 25 known compounds were isolated from the crude extract of Cassia abbreviata. The chemical structures of new compounds were established by extensive spectroscopic analyses including 1D and 2D NMR and HRESIMS. Cassiabrevone (1) is the first heterodimer of guibourtinidol and planchol A. Compound 2 was a new chalcane, while 3 was a new naphthalene. Cassiabrevone (1), guibourtinidol-(4α→8)-epiafzelechin (4), taxifolin (8), oleanolic acid (17), piceatannol (22), and palmitic acid (28), exhibited potent anti-HIV-1 activity with IC₅₀ values of 11.89 µM, 15.39 µM, 49.04 µM, 7.95 µM, 3.58 µM, and 15.97 µM, respectively.

1. Introduction

Cassia abbreviata is a small-to-medium-sized branched tree of the Fabaceae. It is widely spread in the tropics, especially in southeast Africa, with a long history in traditional medicine for the treatment of numerous conditions [1], such as headaches, diarrhea, constipation, some skin diseases, malaria, syphilis, pneumonia, stomach troubles, uterine pains, and gonorrhea [2,3]. Pharmacological studies indicated that C. abbreviata showed a broad spectrum of biological activities, including CNS depression [4], hypoglycemia [5], anti-AIDS [6], hepatoprotection [7], antioxidant [8], antibacterial [9], etc. Although some fatty acid compositions were analyzed from its seed oil by gas chromatography (GC) [10], while several dimeric and trimeric flavonoids were proposed on the basis of the UPLC–MS spectroscopic data [11], the chemical component investigation on C. abbreviata was seldom reported. Up to now, only a new flavan [12] and two novel trimeric proanthocyanidins [9] were isolated.

Recently, we screened several crude extracts from different plants of Cassia species and found that C. abbreviata showed potent anti-HIV-1 activity. Therefore, a systematic phytochemical investigation was carried out, which led to the isolation of three new (1–3) and 25 known (4–28) compounds (Figure 1). Herein, we report the isolation, structure, and anti-HIV-1 activity of these compounds.

2. Results and Discussion

Compound 1 showed a molecular ion peak at m/z 535.1590 [M + H]⁺ in its positive HRESIMS (Figure S18), corresponding to the molecular formula of C₂₉H₂₆O₁₀. Its ¹H and ¹³C NMR spectroscopic data in DMSO-d₆ (Table 1) exhibited 29 carbon signals, consisting of one ABX [δ_H 6.12 (1H, d, J = 2.4 Hz, H-8); 6.15 (1H, dd, J = 8.4, 2.4 Hz, H-6); 6.40 (1H, d, J = 8.4 Hz, H-5); δ_C 102.0 (d, C-8), 108.3 (d, C-6), 118.6 (s, C-10), 129.5 (d, C-5), 155.3 (s, C-9), 155.8 (s, C-7)], one 1,4-disubsituted [δ_H 6.76 (2H, d, J = 8.5 Hz, H-3′,5′); 7.24 (2H, d, J = 8.6 Hz, H-2′,6′); δ_C 114.8 (d × 2, C-3′,5′), 129.3 (d × 2, C-2′,6′), 130.3 (s, C-1′), 157.1 (s, C-4′)] and one penta-substituted [δ_H 6.09 (1H, s, H-6″); δ_C 95.2 (d, C-6″), 97.4 (s, C-10″), 107.7 (s, C-8″), 151.7 (s, C-9″), 154.0 (s, C-5″), 155.4 (s, C-7″)] benzoic moieties, besides to one methyl [δ_H 0.97 (3H, s, Me-17″); δ_C 23.8 (q, C-17″)], two sp³ methylenes [δ_H 2.57 (dd, J = 17.7, 5.3 Hz, H-4″β), 2.67 (d, J = 17.7 Hz, H-4″α); 2.62 (dd, J = 19.0, 4.8 Hz, H-12″α), 2.98 (dd, J = 19.0, 11.6 Hz, H-12″β); δ_C 20.2 (t, C-4″), 31.1 (t, C-12″)], four oxygenated sp³ methines [δ_H 4.19 (d, J = 1.6 Hz, H-2″); 4.26 (br. t, J = 9.1 Hz, H-3); 4.39 (t, J = 2.5 Hz, H-3″); 4.48 (d, J = 9.5 Hz, H-2); δ_C 69.5 (d, C-3), 72.8 (d, C-3″), 78.7 (d, C-2″), 82.9 (d, C-2)], two sp³ methines [δ_H 2.36 (dd, J = 11.7, 4.6 Hz, H-11″); 4.40 (d, J = 9.1 Hz, H-4); δ_C 40.4 (d, C-4), 50.2 (d, C-11″)], one carbonyl (δ_C 174.5 s, C-13″), and one acetalic quaternary carbon (δ_C 115.6 s, C-15″).

In the heteronuclear multiple bond connectivity (HMBC) spectrum, a diagnostic ketal-γ-lactone moiety could easily be deduced according to correlations of H-2″ to C-3″/C-15″, H-3″ to C-10″, H-11″ to C-3″/C-13″, H₂-12″ to C-13″/C-15″, and H₃-17″ to C-11″/C-15″, which by further cross peaks of H₂-4″ to C-5″/C-9″/C-10″ constructed the fragment of planchol A (6) [13]. Moreover, HMBC correlations of H-2 to C-1′/C-2′/C-9, H-3 to C-1′/C-2/C-4/C-10, and H-4 to C-5/C-9/C-10 and the large coupling constant of H-2/H-3 (³J_H2-H3 = 9.5 Hz) could be used to establish another fragment of guibourtinidol [14]. These two fragments could be connected via C-4 and C-8″ by the key HMBC correlations of H-4 to C-7″/C-9″ (Figure 2). According to the large coupling constant between H-3 and H-4 (³J_H3-H4 = 9.1 Hz), the stereochemistry of C-4 was assumed to be α-orientation [15]. On the basis of the above evidence and from the perspective of the biosynthetic pathway, the structure of 1 was then determined to be guibourtinidol (4α→8) planchol A, and named cassiabrevone.

Although dimers or trimers of flavonoids were commonly found in nature, cassiabrevone is the first example of a heterodimer formed by flavanol and planchol A. It might be biosynthesized from a natural chalconol by dehydration, oxidation, and final endo-attacking via neighboring group participation induced Friedel–Crafts reaction (Figure 3).

Compound 2 was assigned the molecular formula C₁₅H₁₆O₆ from its positive HRESIMS at m/z 293.0947 [M + H]⁺. The ¹H and ¹³C NMR spectroscopic data of 2 were very similar to those of epifiliferol [16,17], except that an ABX aromatic ring instead of an AX benzoic moiety was found in 2. The assumption was confirmed by the HMBC correlation of H₂-4 (δ_H 2.75, 1H, dd, J = 16.2, 3.0 Hz, H-4a; 3.14,1H, dd, J = 16.2, 4.0 Hz, H-4b) to C-5 (δ_C 156.7 s)/C-9 (δ_C 131.7 d)/C-10 (δ_C 111.9 s). The small coupling constant between H-2 and H-3 (³J_H2/H3 = 4.0 Hz) further confirmed the erythro-configuration of 2. Accordingly, the structure of 2 was assigned as 9-dehydroxyepifiliferol. Interestingly, it might be originated by the same biosynthetic precursor as 1, via a nucleophilic displacement reaction, followed by the oxidation reaction.

The molecular formula of compound 3 was assigned to be C₂₄H₃₀O₁₃ according to its sodium adduct ion peak at m/z 557.1792 [M + H]⁺, suggesting ten degrees of unsaturation. The ¹H and ¹³C NMR spectra (Table 1) of 3 were almost the same as those of cassiaglycoside II (17) [18], except that the β-d-glucopyranosyl moiety at the C-6 position was shifted to the C-2′ position. This was evidenced by the downfield shift from 74.9 to 79.8 of the C-2′ position. Further confirmation could be observed by the HMBC correlations of H-1″ (δ_H 5.23, 1H, d, J = 7.9 Hz) to C-2′ (δ_C 79.8 d) and H-1′ (δ_H 1H, 4.75, d, J = 7.7 Hz) to C-8 (δ_C 156.5 s). By detailed analysis of its HSQC, COSY, and HMBC NMR spectroscopic data, compound 3 was then elucidated as 6-deglucopyranosyl-2′-glucopyransoyl cassiaglycoside II, and named cassiaglycoside V.

By comparison of the NMR and MS data with those published in the literature, 25 known compounds were determined to be guibourtinidol-(4α→8)-epiafzelechin (4) [15], guibourtinidol-(4α→8)-epicatechin (5) [15], planchol A (6) [13], (+)-afzelechin (7) [19], taxifolin (8) [20], dihydrokaempferol (9) [20,21], naringenin (10) [22], rhusopolyphenol E (11) [23], cascaroside D (12) [24], 1″-deoxyaloin B-1-O-β-d-glucopyranoside (13) [24], 10-hydroxycascaroside C (14) [24], cassialoin (15) [24], chrysophanol (16) [25], oleanolic acid (17) [26], erythrodiol (18) [26], lupeol (19) [27], β-sitosterone (20) [28], β-sitosterol (21) [29], piceatannol (22) [30], markhamioside F (23) [31], vanillic acid (24) [32], cassiaglycoside II (25) [18], (7S, 8S)-syringoylglycerol (26) [33], β-d-glucopyranosyl (1→2)-β-d-glucopyranoside (27) [34], and palmitic aicd (28) [35].

The crude extract of Cassia abbreviata and all isolated compounds were assessed for their anti-HIV activity in MT4 cells infected by the reference strain HIV-1 IIIB (Figure 3). Cassiabrevone (1), guibourtinidol-(4α→8)-epiafzelechin (4), taxifolin (8), oleanolic acid (17), piceatannol (22), and palmitic acid (28) inhibited HIV-1 infection at noncytotoxic concentration and showed IC₅₀ values ranging from 3.58 to 49.04 µM (Table 2). Enfuvirtide and Plerixafor are entry inhibitors for positive controls.

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded on Bruker 500 MHz spectrometer using TMS as an internal standard. The HRESIMS spectra were measured on a Waters Xevo G2 Q-TOF mass spectrometer. Optical rotations were measured with an Anton Paar MCP100 polarimeter. Chemical shifts were recorded in δ values using solvent signals DMSO-d₆ (δ_H 2.50/δ_C 39.5) and CD₃OD (δ_H 3.0/δ_C 49.0) as references. Column chromatography (CC) was performed on silica gel, Sephadex LH-20, and ODS (octadecyl silane).

3.2. Plant Material

The mature shrubs of the plant (3 kg) were collected in Makueni County, Kenya, and the identity of Cassia abbreviata was confirmed by DNA barcoding.

3.3. Extraction and Isolation

Barks and roots of Cassia abbreviata were pulverized and extracted with 95% EtOH four times at room temperature. The extracts were combined and concentrated to provide a crude extract (CE, ca 420 g). Then, it was suspended in deionized water and partitioned successively with CHCl₃, EtOAc, and n-BuOH, to provide an EtOAc-soluble extract (306.7 g) and an n-BuOH-soluble extract (103.5 g). The EtOAc extract was subjected to CC over silica gel eluting with a gradient CHCl₃-MeOH (0→100%) to obtain eight fractions (Fr.1–Fr.8). Fr.2 was further separated by ODS and Sephadex LH-20 chromatography, and finally obtained 16 (58.1 mg) and 19 (16.8 mg) by preparative thin-layer chromatography (prep. TLC). Compounds 20 (32.7 mg) and 21 (68.5 mg) were purified from Fr.3 by repeated ODS CC, Sephadex LH-20 chromatography, and prep. TLC. Fr.5 was subjected to ODS CC, Sephadex LH-20 chromatography, and prep. TLC to give 9 (9.3 mg), 10 (11.5 mg), 11 (5.1 mg), 17 (0.8 mg), 18 (3.5 mg), 24 (11.5 mg), and 28 (6.9 mg). Purification of Fr.6 by ODS and Sephadex LH-20 chromatography, followed by prep. TLC led to the isolation of 1 (2.4 mg), 2 (3.4 mg), 4 (7.6 mg), 5 (6.5 mg), 6 (17.8 mg), 7 (40.9 mg), 8 (17.1 mg), 15 (140.3 mg), 22 (75.9 mg), and 26 (11.4 mg). The n-BuOH-soluble part was separated via ODS CC and Sephadex LH-20 chromatography, respectively. Final purification by prep. TLC obtained 3 (14.8 mg), 12 (20.0 mg), 13 (24.0 mg), 14 (22.0 mg), 23 (11.0 mg), 25 (21.0 mg), and 27 (28.7 mg).

Cassiabrevone (1): pale yellow amorphous powder; ${\left[\alpha \right]}_D^{20}$ −12.7 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 535.1590 [M + H]⁺ (calcd for C₂₉H₂₇O₁₀, 535.1599).

9-Dehydroxyepifiliferol (2): white amorphous powder; ${\left[\alpha \right]}_D^{20}$ −12.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 293.1009 [M + H]⁺ (calcd for C₁₅H₁₇O₆, 293.1020).

Cassiaglycoside V (3): pale yellow amorphous powder; ${\left[\alpha \right]}_D^{20}$ −90.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 557.1851 [M + H]⁺ (calcd for C₂₅H₃₃O₁₄, 557.1865).

3.4. Anti-HIV-1 Infection Bioassay

MT4 cells were obtained through the NIH AIDS Reagent Program and cultured in RPMI 1640 (Lonza, Wijchen, the Netherlands) supplemented with 10% heat-inactivated fetal bovine serum (Lonza, the Netherlands) and 2mM l-glutamine (Invitrogen, Gosselies, Belgium). MT-4 cells were incubated with the crude extract of Cassia abbreviatta or the tested compounds alone to assess cytotoxicity, or HIV-1 IIIB alone or a mixture of the tested extract and compounds and HIV-1 IIIB viruses to assess protection against HIV-1 infection. After five days, protection from viral infection and the cytotoxicity were evaluated in parallel using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, Liège, Belgium) by measuring OD₅₄₀ and OD₆₉₀ using a POLARstar Omega Plate Reader (BMG Labtech, Ortenberg, Germany). Data were normalized to cells without treatment. Values of OD₅₄₀−OD₆₉₀ were calculated to determine IC₅₀ values in Prism. The entry inhibitors, enfuvirtide, and AMD3100 (Sigma Aldrich, Liège, Belgium), were used as positive controls.

4. Conclusions

From Cassia abbreviata, three new compounds, cassiabrevone, 9-dehydroxyfiliferol, and cassiaglycoside V, were isolated along with 25 known ones. Noteworthily, cassiabrevone is the first heterodimer by flavanol guibourtinidol and tetracyclic phenolic planchol A. Moreover, six compounds showed inhibition against HIV-1 infection with IC₅₀ values ranging from 3 to 50 µM.

Author Contributions

X.Y. isolated all compounds. X.Y., Z.H., and N.W. analyzed the chemical data and wrote the manuscript. Y.Z. performed the bioactive experiments and the barcoding analysis. M.M. provided the plant material and performed the in silico ligand-based studies. J.-C.S. designed the study with A.S. and C.S.-D. C.S.-D. analyzed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the “Fonds National de la Recherche” of Luxembourg [PHD AFR grant 1189522] and the Luxembourg Institute of Health (MESR grant 20150415).

Data Availability Statement

Not available.

Acknowledgments

Authors thank Jean-Yves Servais and Morgane Lemaire for their excellent technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Compounds 1–28 from Cassia abbreviata.

Figure 2. The key HMBC correlations of 1–3.

Figure 3. Protective effects of the crude extract (CE) along with compounds 1, 4, 8, 17, 22, and 28 of Cassia abbreviata against HIV-1 infection (n = 3).

Table 1

¹H (500 Hz) and ¹³C (125 Hz) NMR spectroscopic data of 1–3 (δ in ppm, J in Hz within parentheses).

No.	1 a		1 b		2 b		3 b
	δ C	δ H	δ C	δ H	δ C	δ H	δ C	δ H
1							153.7 C
2	82.9 CH	4.48 (d, 9.5)	84.3 CH	4.56 (d, 9.6)	80.1 CH	4.90 overlap	123.3 C
3	69.5 CH	4.26 (dd, 9.5, 9.1)	72.3 CH	4.40 (dd, 9.6, 9.1)	67.8 CH	4.19 (br. t, 4.0)	135.1 C
4	40.4 CH	4.40 (d, 9.1)	41.8 CH	4.58 (d, 9.1)	34.1 CH2	3.14 (dd, 16.2, 4.2)	119.6 CH	6.86 s
						2.75 (dd, 16.2, 3.0)
5	129.5 CH	6.40 (d, 8.4)	130.2 CH	6.60 (d, 8.6)	156.7 C		104.8 CH	6.64 (d, 2.0)
6	108.3 CH	6.15 (dd, 8.4, 2.4)	109.9 CH	6.24 (dd, 8.6, 2.4)	104.0 CH	(d, 2.4)	157.9 C
7	155.8 C		157.0 C		157.8 C		102.7 d	6.74 (d, 2.0)
8	102.0 CH	6.12 (d, 2.4)	103.3 CH	6.24 (d, 2.4)	109.6 CH	(dd, 8.2, 2.4)	156.5 C
9	155.3 C		156.9 C		131.7 CH	6.90 (d, 8.2)	109.3 C
10	118.6 C		120.6 C		111.9 C		139.5 C
1′	130.3 C		131.7 C		132.2 C		104.8 CH	4.75 (d, 7.7)
2′	129.3 CH	7.24 (d, 8.6)	130.3 CH	7.31 (d, 8.5)	115.4 CH	6.99 (d, 1.7)	79.8 CH	3.89 m
3′	114.8 CH	6.76 (d, 8.5)	116.0 CH	6.81 (d, 8.5)	145.9 C		78.0 CH	3.34 m
4′	157.1 C		158.5 C		146.0 C		78.2 CH	3.76 m
5′	114.8 CH	6.76 (d, 8.5)	116.0 CH	6.81 (d, 8.5)	116.0 CH	6.78 (d, 8.2)	71.0 CH	3.50 m
6′	129.3 CH	7.24 (d, 8.6)	130.3 CH	7.31 (d, 8.5)	119.4 CH	6.82 (dd, 8.2, 1.7)	66.9 CH2	3.23 m; 3.92 m
1″							100.4 CH	5.23 (d, 7.9)
2″	78.7 CH	4.19 (d, 1.6)	81.0 CH	4.22 (d, 2.4)			78.4 CH	3.54 m
3″	72.8 CH	4.39 (dd, 5.3, 1.6)	75.0 CH	4.45 (ddd, 5.1, 2.4, 1.5)			78.0 CH	3.34 m
4″	20.2 CH2	2.67 (d, 17.7)	21.3 CH2	2.89(d, 17.9)			75.4 CH	3.25 m
		2.57 (dd, 17.7, 5.3)		2.64 (dd, 17.9, 5.1)
5″	154.0 C		155.6 C				71.0 CH	3.50 m
6″	95.2 CH	6.09 s	96.3 CH	6.09 s			62.3 CH2	3.74 m; 3.93 m
7″	155.4 C		157.1 C
8″	107.7 C		109.4 C
9″	151.7 C		153.0 C
10″	97.4 C		99.6 C
11″(11)	50.2 CH	2.36 (dd, 11.7, 4.7)	52.1 CH	2.39 (dd, 11.6, 4.5)			208.3 C
12″(12)	31.1 CH2	2.98 (dd, 19.0, 11.7)	32.8 CH2	2.93 (dd, 19.2, 11.6)			32.7 CH3	2.58 s
		2.62 (dd, 19.0, 4.7)		2.60 (dd, 19.2, 4.5)
13″(13)	174.8 C		177.2 C				20.2 CH3	2.23 s
15″	115.9 C		118.4 C
17″	23.8 CH3	0.97 s	24.4 CH3	1.10 s

^a Recorded in DMSO-d₆. ^b Recorded in CD₃OD.

Table 2

The IC₅₀ values of compounds 1, 4, 8, 17, 22, and 28 harboring an anti-HIV-1 activity.

Compounds	IC50 (µM)
	HIV-1 Infection (µM)	Cytotoxicity
CE a	9.98 ± 3.88 (µg/mL)	>1000 (μg/mL)
Cassiabrevone (1)	11.89 ± 2.14	>333
Guibourtinidol-(4α→8)-epiafzelechin (4)	15.39 ± 9.09	>333
Taxifolin (8)	49.04 ± 5.02	>333
Oleanolic acid (17)	7.95 ± 2.57	>333
Piceatannol (22)	3.58 ± 0.27	>333
Palmitic acid (28)	15.97 ± 3.04	>333
Enfuvirtide (T20) b	0.0096 ± 0.001	>1
Plerixafor (AMD3100) b	0.075 ± 0.009	>1

^a CE: crude extract of Cassia abbreviate. ^b Enfuvirtide and Plerixafor: positive controls. Each experiment was conducted three times and data were expressed as means ± SD.

Supplementary Materials

The following are available online, Figures S1–S17: The 1D and 2D NMR spectra of 1–3.

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