93
94
Figure 4.1. Dose-response curve for viability using MTT assay in Caco-2, HepG2, and Hek-293 cells exposed to test samples for 24 h.
Compound 1: 3-(4-methoxybenzyl)-5,7-dimethoxychroman-4-one, Compound 2: 3-(4-methoxybenzyl)-6- hydroxy-5,7-dimethoxychroman-4-one, Compound 3 3-(4-hydroxybenzylidene)-5,7-dihydroxychroan-4-one.
DCM – dichloromethane, EtOAc – ethyl acetate and MeOH – methanol.
A reduction in cell viability for Caco-2 (61%), HepG2 (95%), and Hek-293 (65%) were observed after treatment with the DCM extract (50.12 µg mL-1) (Figure 4.1). At a higher concentration (1000 µg mL-1), a further reduction in cell viability was observed; Caco-2 (9%), HepG2 (14%), and Hek-293 (6%). Cell viability reduced from 100% (control) to 47% (Caco- 2), 82% (HepG2), and 19% (Hek-293) after treatment with the ethyl acetate (EtOAc) extract (50.12 µg mL-1). The cytotoxicity profile for the EtOAc extract was U-shaped, similar to the DCM extract (Figure 4.1). Cytotoxicity was most prominent with the methanol (MeOH) extract against Caco-2 and HepG2 cells, with IC50 concentrations of 7.79 µg mL-1 and 9.29 µg mL-1, respectively. Additionally, a reduction in cell viability from 100% (control) to 4% (Caco-2), 7% (HepG2), and 12% (Hek-293) was noted at a concentration of 50. 12 µg mL-1.
95 Mitochondrial membrane potential (MMP, ΔΨm)
Caco-2, HepG2, and Hek-293 cells were treated with IC80 and IC50 concentrations of the three homoisoflavonoids and three crude extracts. The results are expressed as fold change (Figure 4.2). The MMP did not significantly decrease in Caco-2 (1.01-fold) and the increase in HepG2 (1.09-fold), and Hek-293 (1.07-fold) was also not significant after treatment with an IC80 dose ofcompound 1. At the IC50 concentration of compound 1, 1.01 and 1.05- fold increase in MMP was observed in Caco-2 and HepG2 cell lines respectively, while a1.02-fold decrease was seen in Hek-293. Treatment with an IC80 dose of compound 2 resulted in a 1.09, 1.52 (p =0.023) and 1.03-fold increase in Caco-2, HepG2, and Hek-293. In addition, the IC50 dose showed a 1.40 (p =0.0436) and 2.19 (p =0.0008) fold increase in Caco-2 and HepG2 cells, with a 1.03- fold decrease in Hek-293 cells. The IC80 dose of compound 3 showed a 1.05-fold increase in Hek-293 but a 1.45 (p =0.0061) and 1.02-fold decrease in Caco-2 and HepG2 cells.
Furthermore, the IC50 dose showed a 1.15, a 1.01, and 1.14-fold decrease in Caco-2, HepG2, and Hek-293 cells. Among the three pure compounds, compound 2 showed a significant increase in the MMP of HepG2 cell lines at both IC80 and IC50 concentration, while compound 3 significantly decrease MMP in Caco-2 cell at IC80 concentration.
The DCM extract at an IC80 dose showed a significant decrease in Caco-2 (1.82-fold, p
=0.0337), no significant decrease in Hek-293 (1.02-fold), but a significant increase (p
=0.0173) was observed in HepG2 (1.44-fold). At the IC50 dose, a decrease was observed in Caco-2 (1.11-fold), a significant increase (p =0.0285) in HepG2 (1.52-fold) and 1.05-fold increase in Hek-293. Exposure to the EtOAc extract at an IC80 dose showed a decrease in MMP in Caco-2 (1.30-fold), a significant increase (p =0.0056) in HepG2 (1.20-fold) and a 1.01-fold increase in Hek-293 cell lines. The IC50 dose showed a significant decrease (p =0.0377) in Caco-2 (1.32-fold) a significant increase (p =0.0013) in HepG2 (1.40-fold) and a 1.04-fold increase in Hek-293. Treatment with the methanol extract at the IC80 dose showed a significant
96
decrease (p =0.0135) in Caco-2 (1.64-fold), 1.03-fold decrease in HepG2, and a significant increase (p =0.0369) in Hek-293 (1.10-fold) and at the IC50 dose, an increase in MMP was observed in Caco-2 (1.02-fold) and Hek-293 (1.02-fold), but a significant decrease (p =0.0306) in the HepG2 (1.04-fold).
Figure 4.2 Effect of IC80 and IC50 concentrations of tested samples on the mitochondrial membrane potential (MMP) of Caco-2, HepG2 and Hek-293 cell lines.
Compound 1: 3-(4-methoxybenzyl)-5,7-dimethoxychroman-4-one, Compound 2: 3-(4-methoxybenzyl)-6- hydroxy-5,7-dimethoxychroman-4-one, Compound 3 3-(4-hydroxybenzylidene)-5,7-dihydroxychroan-4-one.
DCM – dichloromethane, EtOAc – ethyl acetate and MeOH – methanol.
Adenosine triphosphate (ATP) assay
Caco-2, HepG2, and Hek-293 cells were treated with IC80 and IC50 concentrations of the three homoisoflavonoids and three crude extracts. The ATP value for the controls was calculated to be 1, and those for treated cells were calculated as a ratio against the control (Figure 4.3). ATP
97
decreased to 91% in Caco-2, and 74% in HepG2 (p =0.0270), but a significant increase to 144% in Hek-293 (p =0.0706) after exposure to the IC80 concentration of compound 1. At the IC50 concentration, a significant reduction to 66% (p =0.0080) and 61% (0.0028) was observed in Caco-2 and HepG2, respectively, while a significant increase (p =0.0014) to 120% was seen in Hek-293. Treatment with an IC80 concentration of compound 2 decreased ATP to 95% in Caco-2, and 83% in HepG2, while an increase (p =0.0322) to 131% was observed in Hek-93.
The IC50 concentration decreased ATP in Caco-2 to 70% (p =0.0115) and 32% (p =0.0008) in HepG2, with a 122% (p =0.0396) increase for Hek-293. For treatment with the IC80 dose of compound 3 (Figure 3), a 6% and 19% decrease in ATP was observed in Caco-2 and HepG2, respectively, with a 40% (p =0.0339) increase for Hek-293. The IC50 concentration of the same compound resulted in a 19% (p =0.0229) and 29% (p =0.0103) decrease of ATP in Caco-2 and HepG2, respectively, and 30% increase in ATP for Hek-293.
Treatment with the DCM extract at IC80 concentration decreased ATP to 77% in Caco-2 and 68% (p =0.0176) in HepG2, but increased ATP to 175% (p =0.0180) in Hek-293, while IC50
treatment decreased ATP to 81% (p =0.0251) in Caco-2 and 39% (p =0.0010) in HepG2 but increased to 142% in Hek-293. The EtOAC extract at IC80 concentration decreased ATP to 95% in Caco-2 and 70% (p =0.0163) in HepG2 but increased it to 216% in Hek-293. At the IC50 concentration, ATP was reduced to 72% (p =0.0075) in Caco-2 and 65% (p =0.0044) in HepG2 but increased to 216% in Hek-293. ATP decreased to 71% (p =0.0168) in HepG2 after treatment with the MeOH extract at the IC80 concentration but increased to 105%, and 337%
(p =0.0422) in Caco-2 and Hek-293, respectively. At the IC50 concentration, ATP decreased to 72% (p =0.0051) in Caco-2 and 67% (p =0.0106) in HepG2 but increased to 167% (p =0.0087) in Hek-293.
98
Figure 4.3. Effect of IC80 and IC50 concentrations of tested samples on the intracellular ATP levels of Caco-2, HepG2 and Hek-293 cell lines.
Compound 1: 3-(4-methoxybenzyl)-5,7-dimethoxychroman-4-one, Compound 2: 3-(4-methoxybenzyl)-6- hydroxy-5,7-dimethoxychroman-4-one, Compound 3 3-(4-hydroxybenzylidene)-5,7-dihydroxychroan-4-one.
DCM – dichloromethane, EtOAc – ethyl acetate and MeOH – methanol.
Lactate dehydrogenase (LDH) release assay
Caco-2, HepG2, and Hek-293 cells were treated with IC80 and IC50 concentrations of the three homoisoflavonoids and three crude extracts (Figure 4.4). An increase in LDH level from 100%
in the control to 143%, 259% (p =0.0469) and 148% (p =0.0173) was observed in Caco-2, HepG2 and Hek-293 cell lines after treatment with compound 1 at IC80 concentration. At IC50
concentration the increase was 225% (p =0.0158) in Caco-2, 133% (p =0.0311) in HepG2 and 160% (p =0.0251) in Hek-293 cell lines. Treatment with compound 2 IC80 concentration caused significant increase (p =0.0218) in LDH released to 217% in Caco-2, and non- significant increase to 103% and 127% in HepG2 and Hek-293 cell lines, respectively. At IC50
treatment of compound 2, significant increase of 295% (p =0.0243) and 168% (p =0.0199) was observed in Caco-2 and HepG2 cell lines, and non-significant increase of 162% in Hek-
99
293. Treatment with IC80 value of compound 3 significantly reduced LDH in Caco-2 to 88%
(p =0.0498) and in HepG2 to 48% (p =0.0047) but an increase to 116% was observed in Hek- 293.
Figure 4.4. Effect of IC80 and IC50 concentrations of tested samples on the plasma membrane of Caco- 2, HepG2 and Hek-293 cell lines.
Compound 1: 3-(4-methoxybenzyl)-5,7-dimethoxychroman-4-one, Compound 2: 3-(4-methoxybenzyl)-6- hydroxy-5,7-dimethoxychroman-4-one, Compound 3 3-(4-hydroxybenzylidene)-5,7-dihydroxychroan-4-one.
DCM – dichloromethane, EtOAc – ethyl acetate and MeOH – methanol.
Treatment with the DCM extract at the IC80 concentration resulted in 132% increase in LDH for Caco-2, 87% significant decrease (p =0.0113) in HepG2, and 114% increase in Hek-293 cell lines. AT IC50 DCM extract treatment, LDH was increased to 107% in Caco-2, 106% in HepG2 and a significant increase (p =0.0067) of 117% in Hek-293. IC80 concentration of EtOAc increased LDH to 109%, 120% and 133% in Caco-2, HepG2 and Hek-293 cell lines, respectively. At IC50 treatment LDH was significantly increased (p =0.0377) to 150% in Caco- 2, significantly reduced (p =0.0190) to 72% in HepG2 and non-significant increase to 127% in
100
Hek-293. Treatment with IC80 concentration of MeOH extract significantly increase LDH in Caco-2 (p =0.0185) to 161% and in HepG2 (p =0.0174) to 164%. No change was observed in LDH level in Hek-293 at the same concentration. IC50 concentration significantly increase (p
=0.0008) LDH to 204% in Caco-2, with non-significant increase of 146% in HepG2 and a non- significant decrease to 75% in Hek-293. All tested samples showed an increase in LDH for Hek-293 at both IC80 and IC50 except for the methanol extract.