Chemistry
For the first time, six known compounds (1–6) were identified from the methanolic extract of C. canescens (CCE) utilizing chromatographic methods and analyses of spectral data namely 1H NMR, MS, IR, and 13C NMR (see supplemental file) and correlated with those reported in the literature (Fig. 1).
Compound 1 [(1R,9S,Z)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene] was obtained as a yellowish solid with Rf value of 0.8 in hexane and ethyl acetate (4:1). Mol. formula: C15H24. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 0.986 (s, 6H), 1.083–1.127 (m, 1H), 1.334–1.378 (m, 1H), 1.685–1.687 (m, 4H), 1.955–1.993 (m, 3H), 1.993–2.157 (m, 4H), 2.510–2.520 (d, 1H, J = 4 Hz), 2.922 (s, 1H), 4.605–4.609 (d, 1H, J = 0.8 Hz), 4.855–4.857 (d, 1H, J = 0.8 Hz), 5.162–5.189 (m, 1H); 13C NMR (400 MHz, CDCl3): 22.21 (C-12), 27.42 (C-5), 29.29 (C-14/15), 30.86 (C-1), 36.82 (C-9/10), 40.13 (C-4), 42.29 (C-11), 51.61 (C-7), 54.52 (C-6), 114.13 (C-13), 126.83 (C-2), 137.31 (C-3), 154.90 (C-8). ESI-MS (positive mode) m/z: 205 [M + H+], calcd. m/z for C15H24: 204.19 [M]. FT-IR (in KBR): 757.67, 830.97, 1037.70, 1088.51, 1129.48, 1160.81, 1239.96, 1342.80, 1420.75, 1455.71, 1503.45, 1581.58, 1619.37, 1657.04, 2864.85, 2926.91, 2958.57 cm−1.
Compound 2 [(1S,5R,9R)-10,10-dimethyl-2,6-dimethylenebicyclo[7.2.0]undecan-5-ol] was obtained as a pale yellow solid with Rf value of 0.4 in chloroform and ethyl acetate (3:7). Mol. formula: C15H24O. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 0.944 (s, 6H), 1.044–1.082 (m, 1H), 1.295–1.320 (m, 1H), 1.320–1.333 (m, 1H), 1.532–1.543 (m, 2H), 1.613–1.635 (m, 1H), 1.783–1.882 (m, 1H), 1.902–1.926 (m, 1H), 2.013–2.038 (m, 2H), 2.116–2.141 (m, 2H), 2.444–2.504 (m, 1H), 2.992 (s, 1H, -OH), 4.563–4.567 (dd, 2H, J = 0.8 Hz), 4.813–4.817 (dd, 2H, J = 0.8 Hz); 13C NMR (400 MHz, CDCl3): 22.80 (C-4), 24.05 (C-14/15), 28.98 (C-3), 29.56 (C-8), 30.03 (C-9), 31.59 (C-10), 37.06 (C-11), 46.37 (C-6), 49.29 (C-5), 72.64 (C-1), 107.12 (C-12), 108.74 (C-13), 147.5 (C-2), 147.87 (C-7). ESI-MS (positive mode) m/z: 221 [M + H+], calcd. m/z for C15H24O: 220.18 [M]. FT-IR (in KBR): 758.70, 1462.79, 28.55.53, 2921.95 cm−1.
Compound 3 [(1R,4R,10S)-4,12,12-trimethyl-9-methylene-5-oxatricyclo[8.2.0.04,6]dodecane] was obtained as a white powder with Rf value of 0.6 in hexane and ethyl acetate (1:1). Mol. formula: C15H24O. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 0.960 (s, 6H), 1.160 (s, 3H), 1.239–1.279 (m, 4H), 1.389–1.630 (m, 2H), 1.647–1.691 (m, 2H), 1.898–1.942 (m, 1H), 2.054–2.175 (m, 2H), 2.190–2.205 (m, 1H), 2.461–2.519 (m, 1H), 4.829–4.831 (dd, 2H, J = 0.8 Hz); 13C NMR (400 MHz, CDCl3): 22.87 (C-12), 27.28 (C-4), 32.01 (C-9), 32.22 (C-14/15), 38.07 (C-8), 39.75 (C-10), 40.40 (C-3), 45.22 (C-11), 54.54 (C-6), 59.44 (C-5), 65.15 (C-2), 67.48 (C-1), 116.90 (C-13), 156.03 (C-7). ESI-MS (positive mode) m/z: 221 [M + H+], calcd. m/z for C15H24O: 220.18 [M]. FT-IR (in KBR): 757.43, 1164.22, 1243.88, 1280.23, 1371.49, 1462.62, 1625.60, 2855.31, 2920.71 cm−1.
Compound 4 [(3S,3aS,6R,8aS)-3,7,7-trimethyl-8-methyleneoctahydro-1H-3a,6-methanoazulene] was obtained as a grayish solid with Rf value of 0.4 in hexane and ethyl acetate (1:1). Mol. formula: C15H24. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 0.893–0.918 (m, 1H), 1.053–1.066 (d, 3H, J = 5.2 Hz), 1.144–1.169 (m, 1H), 1.292–1.298 (m, 1H), 1.386 (s, 6H), 1.433–1.438 (m, 2H), 1.486–1.515 (m, 1H), 1.543–1.574 (m, 2H), 1.568–1.574 (m, 1H), 1.660–1.687 (m, 2H), 2.071–2.106 (t, 2H, J = 6.8, 7.2 Hz), 4.975–4.980 (dd, 2H, J = 0.8 Hz); 13C NMR (400 MHz, CDCl3): 12.17 (C-15), 21.42 (C-9), 23.62 (C-13/14), 23.77 (C-4), 28.97 (C-10), 34.26 (C-5), 35.55 (C-2), 38.14 (C-8/11), 43.31 (C-7), 43.50 (C-3), 52.69 (C-6), 101.50 (C-12), 156.57 (C-1). ESI-MS (positive mode) m/z: 205 [M + H+], calcd. m/z for C15H24: 204.19 [M]. FT-IR (in KBR): 758.59, 1466.21, 2853.30, 2919.84 cm−1.
Compound 5 [(1R,2S,6S,7S,8S)-8-isopropyl-1,3-dimethyltricyclo[4.4.0.02,7]dec-3-ene] was obtained as a greenish semi-solid with Rf value of 0.6 in chloroform and ethyl acetate (3:7). Mol. formula: C15H24. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 1.044 (s, 3H), 1.057 (s, 3H), 1.061 (s, 3H), 1.215–1.318 (m, 1H), 1.326–1.331 (m, 1H), 1.409–1.434 (m, 2H), 1.488–1.512 (m, 1H), 1.606–1.755 (m, 2H), 1.811–1.871 (m, 2H), 1.912 (s, 1H), 1.913 (s, 3H), 2.050–2.093 (m, 1H), 5.529 (t, 1H, J = 0.8 Hz); 13C NMR (400 MHz, CDCl3): 22.26 (C-5/13/14), 25.62 (C-15), 26.07 (C-11), 30.44 (C-12), 33.80 (C-9), 38.10 (C-6), 39.41 (C-7), 44.84 (C-8), 46.31 (C-4), 47.30 (C-3), 49.31 (C-2), 120.87 (C-10), 143.44 (C-1). ESI-MS (positive mode) m/z: 205 [M + H+], calcd. m/z for C15H24: 204.19 [M]. FT-IR (in KBR): 757.07, 831.97, 1036.44, 1089.60, 1130.24, 1160.95, 1258.86, 1344.88, 1419.78, 1455.32, 1505.34, 1580.19, 1621.23, 1653.08, 2875.04, 2962.67 cm−1.
Compound 6 [(3aR,6R,7S,8aS)-6,8,8-trimethyl-3-methyleneoctahydro-1H-3a,7-methanoazulene] was obtained as a pale brownish solid with Rf value of 0.4 in chloroform and ethyl acetate (1:1). Mol. formula: C15H24. Purity: above 95% pure (based on TLC and NMR observations). 1H NMR (400 MHz, CDCl3): 0.880–0.893 (s, 3H, J = 5.2 Hz), 0.937 (s, 6H), 1.061–1.069 (m, 2H), 1.111–1.166 (m, 1H), 1.175–1.180 (m, 2H), 1.185–1.189 (m, 1H), 1.362–1.368 (m, 1H), 1.387–1.429 (m, 4H), 2.054–2.092 (d, 2H, J = 15.2 Hz), 4.587–4.595 (t, 1H, J = 0.8, 2.4 Hz); 13C NMR (400 MHz, CDCl3): 22.03 (C-13), 26.08 (C-14/15), 29.07 (C-11), 30.59 (C-3), 32.88 (C-4), 35.35 (C-10), 35.44 (C-5), 37.96 (C-8), 46.59 (C-1), 52.74 (C-6), 56.18 (C-2), 60.45 (C-7), 100.53 (C-12), 157.13 (C-9). ESI-MS (positive mode) m/z: 205 [M + H+], calcd. m/z for C15H24: 204.19 [M]. FT-IR (in KBR): 699.83, 805.16, 849.70, 1163.13, 1205.87, 1268.76, 1321.28, 1447.21, 1499.94, 1625.06, 2857.43, 2921.21, 3356.81 cm−1.
Antioxidant activity
Initially, CCE was exposed to an initial test against DPPH [7] and superoxide [8] assays, and its IC50 values were found to be 60.0 and 62.5 μg/mL, respectively, whereas standard (ascorbic acid) value was 27.8 and 32.1 μg/mL, respectively. Based on the preliminary antioxidant analysis of CCE, we subjected its metabolites (1–6) for antioxidant activity. Among all the tested compounds, only compounds 2 and 3 showed moderate inhibition of DPPH and superoxide free radicals. The concentration of 2 needed for 50% inhibition of DPPH and superoxide free radicals was found to be 169.0 and 180.0 μg/mL, respectively, while 3 with 285.0 and 230.0 μg/mL, respectively (Fig. 2).
Anti-inflammatory activity
The anti-inflammatory effect of metabolites (1–6) and CCE were evaluated based on their inhibitory activities against COX-1 and 2 [9], 5-LOX [10] and XO [11] enzymes, initially at 100 μg/mL concentration. During initially screening, CCE showed better inhibitor profile against COX-1, COX-2, 5-LOX, and XO enzymes with 72.50 ± 3.44, 80.80 ± 4.30, 70.67 ± 4.57, and 75.90 ± 4.25 %enzyme inhibition, respectively. Besides, among the metabolites, only compound 2 at 100 μg/mL concentration displayed significant inhibitory profile on particularly COX-1 and 2 enzymes with 58.17 ± 3.67 and 56.50 ± 4.50 %enzyme inhibition, respectively. Hence, the active samples, i.e., compound 2 and CCE, are further examined at 25, 50, 75, and 100 μg/mL concentrations with standard drugs (indomethacin, diclofenac, and allopurinol) at 2.5, 5.0, 7.5, and 10.0 μg/mL concentrations. From the obtained results, IC50 values calculated by plotting concentration against percentage enzyme inhibition.
The 50% COX-1 enzyme inhibitory concentration required for compound 2 and CCE was found to be 87.0 and 45.0 μg/mL, respectively, whereas indomethacin with 4.2 μg/mL (Fig. 3a). Similarly, the IC50 values of compound 2 and CCE on COX-2 were found to be 88.0 and 63.1 μg/mL, respectively, while indomethacin with 4.2 μg/mL (Fig. 3b). The concentration needed for 50% inhibition of 5-LOX and XO enzymes of CCE was determined to be 42.5 and 56.0 μg/mL, respectively, whereas standard drugs (diclofenac and allopurinol) with 2.7 and 2.6 μg/mL, (Fig. 3c, d).
Anticancer activity
Firstly, all the isolated metabolites (1–6) and CCE tested against MCF-7 (breast), DLD-1 (colon), HeLa (cervical), A549 (lung), and normal human mammary epithelial (NHME) cell lines at 100 μg/mL concentration. From the primary screening of SRB assay [12], it noticed that CCE showed a prominent degree of specificity against the tested series of cancer cell lines. At 100 μg/mL concentration, CCE potently inhibited the growth of DLD-1 and A549 with 81.71 ± 8.53 and 75.26 ± 8.55 %cell death, respectively, than standard drug doxorubicin (10 μg/mL) with 76.17 ± 7.71 and 69.54 ± 5.10 %cell death. Besides, metabolite 2 showed a significant degree of specificity against all the tested panel of cancer cell lines, while compound 3 against only DLD-1 and A549 and compound 1 against only A549.
Secondly, the samples showed above 50% of cell death, examined for further analysis at 25, 50, 75, and 100 μg/mL concentrations with the standard drug (doxorubicin) at 2.5, 5.0, 7.5, and 10.0 μg/mL concentrations. IC50 values calculated by plotting concentration against percentage cell growth inhibition. The IC50 values of 2 and CCE on MCF-7 were found to be 82.0 and 61.5 μg/mL, respectively, whereas doxorubicin with 3.2 μg/mL (Fig. 4a). The concentration of 2, 3, and CCE needed for 50% cell death of DLD-1 was found to be 52.5, 72.5, and 32.5 μg/mL, respectively, while doxorubicin with 4.2 μg/mL (Fig. 4b). From the results of SRB assay on HeLa, the IC50 values of 2 and CCE were found to be 96.0 and 78.0 μg/mL, respectively, whereas doxorubicin with 3.8 μg/mL (Fig. 4c). Similarly, the concentration needed for 50% cell death of A549 of 1, 2, 3, and CCE determined to be 99.5, 88.0, 76.0, and 56.5 μg/mL, respectively, while doxorubicin with 3.8 μg/mL (Fig. 4d). Besides, all the isolated compounds (1–6) and CCE showed a very mild degree of specificity against NHME which indicates that the samples are non-toxic to normal human cells.