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

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.


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)(2)(3) and 25 known (4-28) compounds ( Figure 1). Herein, we report the isolation, structure, and anti-HIV-1 activity of these compounds.
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 1 H and 13 C 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″ (δH 5.23, 1H, d, J = 7.9 Hz) to C-2′ (δC 79.8 d) and H-1′ (δH 1H, 4.75, d, J = 7.7 Hz) to C-8 (δC 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], 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 endoattacking via neighboring group participation induced Friedel-Crafts reaction (Figure 3).
The molecular formula of compound 3 was assigned to be C 24 H 30 O 13 according to its sodium adduct ion peak at m/z 557.1792 [M + H] + , suggesting ten degrees of unsaturation. The 1 H and 13 C 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.

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 (δH 2.50/δC 39.5) and CD3OD (δH 3.0/δC 49.0) as references. Column chromatography (CC) was performed on silica gel, Sephadex LH-20, and ODS (octadecyl silane).

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-d 6 (δ H 2.50/δ C 39.5) and CD 3 OD (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).

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.

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 CHCl 3 , 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 CHCl 3 -MeOH (0→100%) to obtain eight fractions (Fr.1-Fr.8).

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 OD 540 and OD 690 using a POLARstar Omega Plate Reader (BMG Labtech, Ortenberg, Germany). Data were normalized to cells without treatment. Values of OD 540 −OD 690 were calculated to determine IC 50 values in Prism. The entry inhibitors, enfuvirtide, and AMD3100 (Sigma Aldrich, Liège, Belgium), were used as positive controls.