Spectroscopic data
Compound 1. IR (cm-1) 3324, 1698, 1608. 1H NMR (400 MHz, CD3OD). δH: 7.14 (2H, s, H-2′″/6′″), 7.09 (2H, s, H-2′/6′), 7.01 (2H, s, H-2″″/6″″ ), 6.98 (2H, s, H-2″″′/6′″″), 6.94 (2H, s, H-2′′/6′′), 6.26 (1H, d, J=8.0 Hz, H-1 ), 5.90(1H, m, H-4), 5.65 (1H, m, H-5), 5.61 (1H, m, H-2), 4.42 (1H, m, H-3), 4.39 (2H, m, H-6). 13C NMR (100 MHz, CD3OD). δc : 93.8 (CH, C-1), 74.1 (CH, C-3), 72.2 (CH, C-4), 70.8 (CH, C-2), 68.4 (CH, C-5), 62.2 (CH2, C-6). Galloyl i: 119.7 (CH, C-1′), 110.3 (CH, C-2′/6′), 140.0 (CH, C-4′), 146.2 (CH, C-3′/5′), 166.2 (C=O, C-7′), Galloyl ii: 120.2 (CH, C-1″), 110.3 (CH, C-2″/6″), 140.1 (CH, C-4″)146.4 (CH, C-3″/5″), 166.9 (C=O, C-7″); Galloyl iii; 121.1 (CH, C-1″′), 110.7 (CH, C-2″′/6″′), 146.5 (CH, C-3″′/5″′), 140.8 (CH, C-4″′), 167.9 (C=O, C-7″′); Galloyl iv; 120.2 (CH, C-1″′′), 110.4 (CH, C-2″′′/6″′′), 146.4 (CH, C-3″′′/5″′′), 140.3 (CH, C-4″′′), 167.0 (C=O, C-7″′′). Galloyl v: 120.2 (CH, C-1″′′′), 110.4 (CH, C-2″′′′/6″′′′), 146.4 (CH, C-3″′′′/5″′′′), 140.3 (CH, C-4″′′′), 167.0 (C=O, C-7″′′′). TOF HRMS m/z 963.1135 [M+Na]+ (calculated 963.1080).
Structural elucidation of the isolated compound
Compound 1 was isolated as a brown amorphous powder. The compound gave a strong blue-black colour on the thin layer chromatography when sprayed with ferric chloride, indicating the presence of phenolic moiety. IR spectrum displayed absorption at 3324, 1698, 1608, 1536 cm-1. Positive Time-of-Flight High Resolution Mass Spectrometry (TOF HRMS) gave a signal at m/z 963.1135 [M+Na]+ (calculated 963.1080) for a molecular mass C41H32O26. The proton NMR spectrum displayed five singlets at δH 7.14, 7.09, 7.01, 6.98, and 6.94, characteristic of multiply substituted gallotannins. The appearance of a doublet at 6.26 ppm with a large coupling constant (J= 8.4 Hz) indicates the presence of a β-anomeric proton. The sugar protons were assigned based on the COSY and HMBC spectra. In the COSY spectrum, these pairs of correlating protons were observed between the signals at δH 6.26 / 5.61; 5.61 /6.26, 4.42, 5.90 /5.65, while in the HMBC spectrum long range correlations were observed between the protons at δH 6.26 and δc 72.9, 164.7; δH 4.42 and δc 69.5, 166; δH 5.90 and δc 62.1, 73.0, 92.6 and 166. Each galloyl group was placed based on the correlation between the sugar protons as well as the aryl singlet protons with the galloyl carbonyl carbons. Compound 1 was identified as β-penta-O-galloyl glucose (PGG) (molecular weight: 940.68) (Figure 1) by comparison of the spectroscopic data with literature values [17].
Cytotoxicity of the isolated compound
Compound 1 showed concentration-dependent toxicity to HeLa, MRC5 and MRC5-SV2 cells following 24 h exposure to varying concentrations up to 100 μg/ml (Figure 2). The IC50 obtained for Compound 1 was 71.45 μg/ml (76 μM), 52.24 μg/ml (56 μM) and 84.33 μg/ml (90 μM) in HeLa, MRC5-SV2 and MRC5 cells respectively; the selectivity index for Compound 1, which is the ratio of its IC50 in MRC5 and MRC5-SV2 cells, was calculated as 1.61. Doxorubicin, used as a standard anti-cancer drug, also demonstrated concentration-dependent toxicity to HeLa, MRC5 and MRC5-SV2 cells following 24 h exposure to it (Figure 2), with IC50 of 4.65, 31.70 and 40.64 μM in HeLa, MRC5-SV2 and MRC5 cells respectively. The selectivity index for doxorubicin was 1.28.
The concentration-dependent toxicity of Compound 1 and that of the positive control doxorubicin were correlated with morphological damage that worsened as the concentration of each compound increased. As shown in Figure 3 for the HeLa, MRC5-SV2 and MRC5 cells, a higher concentration of Compound 1 or doxorubicin caused loss of cells and rounding up of several or nearly all remaining cells, while control cells not exposed to either compound were confluent and their connections were intact.
While the MRC5-SV2 cell was more sensitive to Compound 1 than the HeLa cell, it was nearly seven times less sensitive to doxorubicin than the HeLa cell. Compound 1 also showed better selectivity for cancer cells (as against normal cells) than doxorubicin. We thus decided to explore in a cancer cell aspects of the time course of its induction of cytotoxicity by comparing its effects with those of doxorubicin, following treatment with each compound at the same range of concentrations as was tested before but for much shorter durations of 3 h and 6 h. This was assessed using the HeLa cell that was less sensitive to Compound 1 than doxorubicin.
As shown in Figure 4, following 3 h and 6 h exposure of HeLa to Compound 1 and doxorubicin, Compound 1 demonstrated concentration- and time-dependent toxicity, which was significant at 50 and 100 μg/ml, while doxorubicin showed no toxicity at the two time points. This observation establishes a key difference in the cytotoxicity time-course profiles of Compound 1 and doxorubicin. While Compound 1 rapidly induced cytotoxicity, initiated from as early as 3 h following exposure of cells to it and progressively increasing up to 24 h, doxorubicin’s toxicity revealed a much-slower time-course, with significant, concentration-dependent toxicity only observed after 24 h exposure to it. For drug discovery and development purposes, this property of Compound 1 could make it uniquely promising, as a shorter time of exposure to an anti-cancer agent could ensure less damage to normal cells and less side effects.
Induction of reactive oxygen species (ROS) as a potential mechanism by which compound elicits cytotoxicity
As we established Compound 1 as rapidly and selectively cytotoxic to cancer cells, we assessed whether the generation of ROS was a mechanism by which it induced its toxicity in cancer cells, at least, in part. Figure 5 reveals that, at 3 h post-treatment in HeLa cells, Compound 1 up to 25 μg/ml did not induce significant ROS but at 100 μg/ml caused a significant increase in ROS levels. This almost 3-fold increase in ROS could be correlated with an almost 50% decrease in viability at the 3 h time point (cf. Figure 4, top panel, left hand side - for 3 h).