Bioactive chemical constituents from the resin of Aloe vera

2.1 Structure elucidation

The new C-glucosyl chromone viz., 7-methoxy-6′-O-coumaroylaloesin (1) along with the known dihydroisocoumarin feralolide (2) were isolated from the resin of A. vera (Fig. 1). The structure of the known dihydroisocoumarin viz., feralolide (2) was determined by comparing its NMR data to that reported in the literature [6].

Fig. 1:

Structures of compounds 1 and 2.

Compound 1 was isolated as a light-yellow amorphous solid and molecular formula was established to be C₂₉H₃₀O₁₁ based on the molecular ion peak in the ESI-MS spectrum and ¹³C NMR spectroscopic analysis. The IR bands at 3304, 1705 and 1640 cm⁻¹ illustrated the presence of hydroxyl, ester, and ketone functional groups as well as an aryl ring. Preliminary inspection of the ¹H NMR spectrum (see Experimental) showed the anomeric signal for one C-glucosyl group [δ_H=5.02 (d, J=10.2 Hz, 1 H, H-1′) ppm], which was further confirmed by the typical C-glucosyl signals in the ¹³C NMR spectrum (δ_C=80.2 (C-3′), 80.0 (C-5′), 75.1 (C-1′), 73.0 (C-2′), 72.1 (C-4′), 65.4 (C-6′) ppm [5], [7]). Additionally, the large coupling constant of (J=10.2 Hz) of the anomeric proton H-1′ suggests a β-configuration for the glucose moiety. Moreover, the NMR spectra of compound 1 illustrated the presence of a p-hydroxy cinnamoyl ester [δ_H=7.66 (d, J=15.6 Hz, 1 H, H-3″); δ_C=146.3; δ_H=6.45 (d, J=15.6 Hz, 1 H, H-2″); δ_C=114.8; δ_H=7.54 (d, J=8.2 Hz, 2 H, H-5″, H-9″); δ_C=131.0; δ_H=6.94 (d, J=8.2 Hz, 2 H, H-6″, H-8″); δ_C=115.4 ppm]. Clearly, the stereochemistry of the C-2″ and C-3″ bond is trans as is evident from the large coupling constant J=15.6 Hz. Moreover, attachment of p-hydroxy cinnamoyl ester was confirmed to be at C-6′ of the sugar unit from the clear HMBC correlation between H-6′ and C-1″. Finally, the OH-7″ group was placed due to the HMBC correlation between H-5″/H-9″ and C-7″as well as between H-6″/H-8″and C-7″.

The ¹H NMR spectrum of compound 1 furthermore displayed the presence of two aromatic singlets [δ_H=6.62 (s, 1H, H-6) and 6.10 (s, 1H, H-3) ppm] together with one aryl methyl group [δ_H=2.68 (s, 3H, 5-Me) ppm], whereas the ¹³C NMR spectrum showed a strong signal for a ketone of a chromone group at δ_H=181.1 ppm. Furthermore, NMR analysis of compound 1 showed the presence of an acetonyl group at the C-2 position, which is evident from the NMR peaks at δ_H=3.33 (br s, 2H, H-9), δ_C=47.1, δ_H=2.27 (s, 3H, H-11), and δ_C=29.8 ppm and a ketone carbonyl signal at δ_H=204.5 (C-10) ppm [5], [7]. The complete structural elucidation of compound 1 was accomplished by ¹H,¹H COSY, and HMBC experiments (Fig. 2) as well as by comparing the chemical shift values with 7-methoxy-6′-O-coumaroylaloesin (1) [7]. Furthermore, the key HMBC correlations between H-1′ and C-3′, C-5′, C-7 and C-8; H-6 with C-4a, C-5, C-7 and C-8; H-9 with C-2, C-3, C-10 and C-11; H-11 to C-9 and C10; Me-5 with C-4a, and C-6; OMe with C-7; H-3 with C-2, C-4, C-4a and C-9 confirmed the structure of compound 1 as 7-methoxy-6′-O-coumaroylaloesin.

Fig. 2:

Key COSY and HMBC correlations of 7-methoxy-6′-O-coumaroylaloesin (1).

Interestingly, various C-glucosyl chromones have been reported from A. vera and we have already reported two C-glucosyl chromones viz., aloeverasides A and B from the resin of said species [4]. In addition, Okamura et al. [8] also reported three C-glucosyl chromones from A. vera, which has a glucose moiety at the C-8 position. It is noteworthy that C-glucosyl chromones have also been detected in various other Aloe species viz., A. angelica, A. arenicola, A. comptonii, A. dabenorisana, A. distans, A. erinacea, A. melanacantha, A. meyeri, A. mitriformis, A. pearsonii, A. peglerae, and A. yavellana [9]. Moreover, organic acids (salicylic acid, malic acid) [10], [11], [12], [13], anthraquinones [12], [13], [14], and steroids [11], [15] have been found.

2.2 Biological activities

The effect of compound 1 on cell viability was tested on the breast cancer cell line, MDA-MB-231. After treating the cells with varying concentrations (25, 50, 75 and 100 μM) of compound 1 for 24 h, an MTT assay was performed and cell viability was measured. It was observed that although it was able to induce a more than 30% decline in cell survival at a concentration of 25 μM and higher, the inhibition of cell growth was not concentration dependent. No significant difference in the degree of cell growth inhibition at increasing concentrations of compound 1 was seen (Fig. 3). Similarly, the cytotoxic effects of compound 2 on MDA-MB-231 (breast cancer) cells was also evaluated using different concentrations for a treatment duration of 24 h. The results of an MTT assay showed that for the breast cancer cell line, compound 2 was significantly able to induce a reduction in cell viability in a concentration-dependent manner (Fig. 4). To corroborate the findings, compound 2 was further tested on the SKOV-3 cell line (ovarian cancer) and was found to cause a concentration-dependent decrease in cell proliferation in ovarian cancer cells (Fig. 4). However, it was noted that the IC₅₀ value of compound 2 for MDA-MB-231 breast cancer cells was slightly lower (51.1 μM) than that for the SKOV-3 ovarian cancer cells (66.05 μM). This indicates that compound 2 is relatively more cytotoxic towards breast cancer cells. Therefore, from Figs. 3 and 4, it may be concluded that compound 2 is much more active in inhibiting the growth of cancer cells compared with compound 1 because it is able to reduce the cancer cell viability up to 86% at the high concentration of 100 μM, as against the 33% cell growth inhibition at the same concentration in the case of compound 1.

Fig. 3:

MDA-MB-231 cells were incubated with the indicated concentrations of compound 1 for 24 h. The effect on cell proliferation was evaluated by performing an MTT assay as described in ‘Biological activities’. All results are expressed as a percentage of control±SD.

Fig. 4:

Cells from MDA-MB-231 and SKOV-3 cancer cell lines were incubated with the indicated concentrations of compound 2 for 24 h. The effect on cell proliferation was evaluated by performing an MTT assay as described in ‘Biological activities’. All results are expressed as a percentage of control±SD.

Compounds 1 and 2 were additionally evaluated against urease, α-glucosidase, and DPPH radical scavenging potential. It was noted that only compound 2 exhibited a good inhibition (50±1.5%) against urease enzyme whereas against the α-glucosidase enzyme and DPPH radical scavenging assay, compound 2 showed weak inhibition with 30±1.5% and 40±1.0%, respectively. Compound 1 was, however, not active in the abovementioned assays.

3.1 General

¹H and ¹³C NMR spectra were recorded on a Bruker NMR spectrometer operating at 600 MHz (150 MHz for ¹³C). The chemical shift values are reported in ppm δ units and the coupling constants J are given in Hz. IR spectra were recorded on a Bruker, ATR-Tensor 37 spectrophotometer. Optical rotations were measured on a KRUSS P P3000 polarimeter (A. Kruss Optronic, Germany). ESI-MS was recorded on Waters Quattro Premier XE Mass Spectrometer (Waters, Milford, MA, USA). For TLC, precoated aluminum sheets (silica gel 60F-254, E. Merck) were used. Visualizations of the TLC plates were achieved under UV light at 254 and 366 nm by spraying with the reagent ceric sulfate.

3.2 Sample collection and identification

The resin of A. vera was purchased from a local market in Nizwa, Sultanate of Oman, and authenticated by Mr. Saif Al-Hatmi (plant taxonomist) at the Oman Botanical Garden, Muscat. A voucher specimen (no. AFS-08/2016) was deposited with the Oman Botanical Garden, Muscat.

3.3 Extraction and isolation

The air-dried and finely powdered resin (1042 g) of A. vera was extracted with methanol (5 L) at room temperature (three times×15 days). Evaporation of the methanol in vacuo at 45°C yielded a crude MeOH extract (987.0 g), which after being suspended in water was successively fractionated into n-hexane (0.9 g), dichloromethane (CH₂Cl₂, 8.5 g), ethyl acetate (EtOAc, 73.2 g), butanol (n-BuOH, 602.0 g), and aqueous fractions (289.0 g) on the basis of an increasing polarity of organic solvents. TLC of the CH₂Cl₂ and EtOAc fractions exhibited sufficient similarity to be combined (81.7 g). The combined material was applied to a silica gel chromatographic column (70–230 mesh; Merck), and sequentially eluted using n-hexane-EtOAc, EtOAc-MeOH, and pure MeOH with 10% increments of the polarity of the eluent to afford 28 fractions (Fr. 1–28). Fraction 16 (Fr. 16), eluted with the 60% EtOAc-n-hexane eluent, was further subjected to repeated column chromatography to afford two new compounds; 1 (8 mg, MeOH-EtOAc (1:9)) and 2 (50 mg, EtOAc-n-hexane (6:4)).

3.4 Anticancer activity