Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea

Figures & data

Figure 1. Chemical structures of apigenin (1), luteolin (2), chrysoeriol (3), quercetin (4), 8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate (5), 8α-tigloyloxyhirsutinolide-13-O-acetate (6), 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate (7), 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate (8), 2-(penta-1,3-diyn-1-yl)-5–(4-acetoxy-3-hydroxybuta-1-yn-1-yl) thiophene (9), 2-(prop-1-inyl)-5–(6-acetoxy-5-hydroxyhexa-1, 3-diinyl) thiophene (10), 2-(prop-1-inyl)-5–(5, 6-dihydroxyhexa-1,3-diinyl) thiophene (11). Chemical structure was produced using ChemDraw Professional 8.

Table 1. IC₅₀ values of extracts and compounds from V. cinerea and P. indica against CYP2A6 and CYP2A13

	CYP2A6^a		CYP2A13^a
Samples	Coincubation	Pre-incubation	Coincubation	Pre-incubation
V. cinerea
Ethanol (µg/ml)	4.00 ± 0.14^b^,^c	2.32 ± 0.01^b^,^c	3.57 ± 0.008^b	2.54 ± 0.06^b
Hexane (µg/ml)	3.31 ± 0.24^b^,^c	1.65 ± 0.05^b^,^c	3.25 ± 0.22^b	2.03 ± 0.05^b
Ethyl acetate (µg/ml)	2.90 ± 0.16^b^,^c	1.57 ± 0.03^b^,^c	2.28 ± 0.09	2.07 ± 0.08
Aqueous fraction (µg/ml)	>100	ND	>100	ND
Apigenin 1 (µM)	0.90 ± 0.07^c	0.77 ± 0.16^c	0.05 ± 0.01	0.04 ± 0.01
Luteolin 2 (µM)	1.38 ± 0.18^c	1.26 ± 0.07^c	0.18 ± 0.02	0.17 ± 0.01
Chrysoeriol 3 (µM)	1.14 ± 0.10^c	0.99 ± 0.12^c	0.82 ± 0.05	0.79 ± 0.01
Quercetin 4 (µM)	2.66 ± 0.24^c	2.15 ± 0.38^c	0.80 ± 0.01	0.74 ± 0.02
Hirsutinolide 5 (µM)	22.3 ± 2.5^b^,^c	8.64 ± 0.37^b^,^c	16.44 ± 3.10^b	4.50 ± 0.18^b
Hirsutinolide 6 (µM)	37.8 ± 3.5^b^,^c	20.80 ± 1.10^b^,^c	20.86 ± 1.46^b	7.66 ± 0.98^b
Hirsutinolide 7 (µM)	32.7 ± 2.2^b^,^c	6.80 ± 0.77^b^,^c	23.26 ± 1.35^b	10.52 ± 0.49^b
Hirsutinolide 8 (µM)	64.5 ± 5.8^b^,^c	13.10 ± 2.40^b^,^c	36.11 ± 1.39^b	15.94 ± 0.67^b
P. indica
Ethanol (µg/ml)	8.32 ± 0.07^b	4.73 ± 0.70^b	8.37 ± 0.79^b	5.44 ± 0.20^b
Hexane (µg/ml)	8.38 ± 1.10^b	3.34 ± 0.17^b	5.31 ± 0.62^b	3.39 ± 0.35^b
Ethyl acetate (µg/ml)	3.52 ± 0.85	2.91 ± 0.09	5.15 ± 0.36	4.96 ± 0.10
Aqueous fraction (µg/ml)	>100	ND	90.0 ± 0.08	85.15 ± 0.11
Thiophene 9 (µM)	6.43 ± 1.29^b	2.12 ± 0.19^b	6.18 ± 0.28^b	2.29 ± 0.34^b
Thiophene 10 (µM)	4.44 ± 0.14^b	2.97 ± 0.01^b	2.94 ± 0.01^b	1.15 ± 0.88^b
Thiophene 11 (µM)	3.90 ± 0.20^b	0.18 ± 0.01^b	2.40 ± 0.33^b	1.47 ± 0.12^b

^a Each value represents mean ± SD of triplicate experiments.

^b Significant difference (p < .05) between IC₅₀ values of coincubation versus pre-incubation.

^c Data obtained from Prasopthum et al., 2015.

ND: not determined.

Table 2. Kinetics values and mode of inhibition of purified compounds from V. cinerea and P. indica against CYP2A6 and CYP2A13.

	CYP2A6^a				CYP2A13^a
Compounds	Ki (µM)	KI (µM)	Kinact	Mode	Ki (µM)	KI (µM)	Kinact	Mode
V. cinerea (µM)
Apigenin 1	0.43 ± 0.17^b	NA^b	NA^b	Mixed type^b	0.014 ± 0.002	NA	NA	Mixed type
Luteolin 2	0.80 ± 0.06^b	NA^b	NA^b	Competitive^b	0.07 ± 0.01	NA	NA	Competitive
Chrysoeriol 3	0.63 ± 0.12^b	NA^b	NA^b	Competitive^b	0.28 ± 0.01	NA	NA	Competitive
Quercetin 4	1.19 ± 0.27^b	NA^b	NA^b	Competitive^b	0.11 ± 0.01	NA	NA	Competitive
Hirsutinolide 5	15.1 ± 2.1^b	7.45 ± 0.62^b	0.05 ± 0.01^b	Mixed type^b	5.61 ± 0.99	0.92 ± 0.17	0.03 ± 0.01	Mixed type
Hirsutinolide 6	30.6 ± 1.5^b	15.4 ± 1.80^b	0.03 ± 0.01^b	Mixed type^b	7.97 ± 0.78	3.60 ± 1.05	0.06 ± 0.01	Mixed type
Hirsutinolide 7	20.4 ± 5.4^b	5.32 ± 0.29^b	0.03 ± 0.01^b	Mixed type^b	12.56 ± 1.26	8.67 ± 0.15	0.06 ± 0.01	Mixed type
Hirsutinolide 8	42.3 ± 8.4^b	7.64 ± 1.24^b	0.09 ± 0.01^b	Mixed type^b	15.76 ± 2.5	8.62 ± 0.37	0.10 ± 0.01	Mixed type
P. indica (µM)
Thiophene 9	3.23 ± 0.43	0.88 ± 0.09	0.10 ± 0.02	Mixed type	2.96 ± 0.10	0.97 ± 0.04	0.08 ± 0.01	Mixed type
Thiophene 10	2.07 ± 0.23	1.01 ± 0.07	0.11 ± 0.01	Mixed type	1.12 ± 0.09	0.86 ± 0.09	0.06 ± 0.01	Mixed type
Thiophene 11	1.80 ± 0.18	0.11 ± 0.02	0.10 ± 0.02	Mixed type	1.08 ± 0.05	0.67 ± 0.08	0.05 ± 0.01	Mixed type

^a Each value represents mean ± SD of triplicate experiments.

^b Data obtained from Prasopthum et al., 2015.

NA: not applicable.

Table 3. Effects of various trapping agents and dialysis of purified compounds from V. cinerea and P. indica on CYP2A6 and CYP2A13

	Per cent remaining activity^a
	Pre-incubation	Dialysis (−) NADPH	Dialysis (+) NADPH	5 mM semicarbazide	2 mM GSH	2000 U catalase	70 μM K3Fe(CN)6
CYP2A6
P. indica
5 µM Thiophene 9	30.14 ± 3.70	89.33 ± 7.00	20.09 ± 5.21	26.75 ± 2.39	21.63 ± 1.97	26.89 ± 3.07	22.71 ± 1.58
5 µM Thiophene 10	35.22 ± 3.22	87.48 ± 9.25	23.69 ± 2.06	21.12 ± 3.31	28.52 ± 1.72	24.09 ± 4.49	19.18 ± 1.83
5 µM Thiophene 11	12.54 ± 1.93	82.39 ± 2.49	10.08 ± 1.84	12.92 ± 3.47	8.42 ± 0.90	14.42 ± 1.41	11.23 ± 2.96
CYP2A13
V. cinerea
10 µM Hirsutinolide 5	29.28 ± 2.53	86.03 ± 6.65	18.63 ± 0.94	24.35 ± 2.54	28.20 ± 6.54	20.27 ± 6.24	24.07 ± 0.62
10 µM Hirsutinolide 6	28.35 ± 2.92	85.34 ± 4.98	22.77 ± 1.13	25.04 ± 3.81	33.60 ± 4.80	26.61 ± 3.86	25.91 ± 1.87
10 µM Hirsutinolide 7	22.16 ± 1.88	93.27 ± 2.10	21.35 ± 4.45	26.60 ± 1.25	18.29 ± 1.54	20.90 ± 5.18	21.39 ± 2.75
20 µM Hirsutinolide 8	23.89 ± 3.17	92.39 ± 4.03	18.37 ± 1.10	20.70 ± 2.92	25.79 ± 4.20	19.44 ± 1.39	21.82 ± 1.85
P. indica
5 µM Thiophene 9	28.97 ± 4.57	85.34 ± 4.98	20.92 ± 0.24	23.17 ± 3.38	23.17 ± 3.38	22.14 ± 2.39	24.86 ± 3.95
5 µM Thiophene 10	28.95 ± 1.25	93.02 ± 6.58	21.43 ± 3.95	26.98 ± 8.11	26.98 ± 8.11	22.18 ± 2.38	26.89 ± 1.75
5 µM Thiophene 11	20.17 ± 2.76	96.84 ± 0.73	19.63 ± 4.21	19.58 ± 2.73	23.48 ± 1.72	19.54 ± 2.95	18.87 ± 0.18

^a Data represent means ± SD of triplicate experiments.

Figure 2. Time- and concentration-dependent inactivation and kinetics of inhibition of CYP2A6-mediated coumarin 7-hydroxylation by 2-(prop-1-inyl)-5–(5, 6-dihydroxyhexa-1,3-diinyl) thiophene 11 (A), and of CYP2A13-mediated coumarin 7-hydroxylation by 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate 7 (B), and 2-(prop-1-inyl)-5–(5, 6-dihydroxyhexa-1,3-diinyl) thiophene 11 (C). Data are represented as mean ± SD of triplicate experiments.