Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase

Figures & data

Table I.  The reversible inhibition activity of extracts of Aceraceous plants.

Acer Linn	Raw material (g)	Yield of extract (mg)	IC50 for the overall reaction (μg/mL)
1. A. platanoides	1.00	87.9	0.60
2. A. tataricum	1.00	47.8	1.81
3. A. truncatum	1.00	55.6	1.30
4. A. mono	1.00	87.7	10.0
5. A. sacharinum ‘Laciniatum wieri’	1.00	105	3.25
6. A. triflorum	1.00	84.2	2.05
7. A. capillipes	1.00	57.8	1.50
8. A. dasyphylla	1.00	36.7	3.75
9. A. dervidii	1.00	39.8	10.0
10. A.pseudoplatanus	1.00	59.9	1.76
11. A. negundo ‘Vureomarginatum’	1.00	29.8	35.4
12. A. stenolobum	1.00	37.1	6.88
13. A. negundo	1.00	67.2	1.35
14. A. griseum	1.00	114	18.8
15. A. palmatum	1.00	134	2.95
16. A. campestre	1.00	68.0	0.41

Figure 1 Fast-binding inhibition of FAS by the extracts of above six species of Aceraceous plants:(A) Acer tataricum, (B) Acer platanoides,(C) Acer capillipes,(D) Acer pseudoplatanus, (E) Acer negundo, (F) Acer campestre. (•) the overall reaction, (♦) ketoacyl reduction reaction, and (▴) enoyl reduction reaction. The reaction system contains 0.1 M potassium phosphate buffer, pH 7.0; 1.0 mM EDTA; 1.0 mM DTT; 3μM acetyl-CoA; 10μM malonyl-CoA; 32μM NADPH and FAS 20 μg in a total volume of 2.0 mL, 37°C, by following the decrease of NADPH at 340 nm within 1.5 min.

Table II.  IC₅₀ values of extracts of six species of Aceraceous plants for the FAS overall reaction and ketoacyl reduction reaction, and k_obs (min⁻¹) values.

Acer linn	IC50 for the overall reaction (μg/mL)	IC50 for the β-ketoacyl reduction reaction(μg/mL)	kobs (min^− 1)
A. platanoides	0.60	2.35	10.6 × 10^− 3
A. tataricum	1.81	7.56	1.5 × 10^− 3
A. capillipes	1.50	2.07	8.5 × 10^− 3
A. pseudoplatanus	1.76	4.16	7.6 × 10^− 3
A. negundo	1.35	6.54	4.8 × 10^− 3
A. campestre	0.41	1.21	7.2 × 10^− 3

A 5 μL sample of the EtoAc extract was added to the assay mixture, followed by 20 μg FAS starting the reaction in a total volume of 2 mL. The remaining activity of FAS was assayed as A_i, and the control activity with 5 μl solvent instead of extract was assayed as A_o. The reversible inhibition was calculated by the formula: I = (1 − A_i/A_o) × 100%.

Figure 2 Kinetic time courses of inhibition of the overall reaction in the presence of the extracts of six species of Acer:(○) Acer platanoides, (▴) Acer pseudoplatanus, (*) Acer tataricum,( × ) Acer campestre, (♦) Acer truncatum, and (▪) Acer capillipes. The insert is a plot of ln R.A.(relative activity) versus time calculated from Figure1 data (Acer platanoides). The FAS solution (0.6μM) was mixed with the extract (0.5 μg/mL) and the aliquots were assayed for remaining activity at the indicated time intervals.

Figure 3 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by A. platanoides extracts. The concentration of extract in the reaction system was 0 (0), 0. 25 μg/mL (1), 0.5 μg/mL (2), and 0.75 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of NADPH and Malonyl-CoA were 32 and 10μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Figure 4 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by A. platanoides extract. The concentration of extract in the reaction system was 0 (0), 0. 25 μg/mL (1), 0.5 μg/mL (2), and 0.75 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of Malonyl-CoA and Acetyl-CoA were 10 and 2.5μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Figure 5 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by A. platanoides extract. The concentration of extract in the reaction system was 0 (0), 0. 25 μg/mL (1), 0.5 μg/mL (2), and 0.75 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of NADPH and Acetyl-CoA were 32 and 2.5μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Figure 6 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by A. campestre extract. The concentration of extract in the reaction system was 0 (0), 0. 2 μg/mL (1), 0.4 μg/mL (2), and 0.5 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of NADPH and Malonyl-CoA were 32 and 10μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Figure 7 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by A. campestre extracts.The concentration of extracts in the reaction system was 0 (0), 0. 2 μg/mL (1), 0.4 μg/mL (2), and 0.5 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of NADPH and Acetyl-CoA were 32 and 2.5μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Figure 8 Lineweaver-Burk plot of the inhibition of the overall reaction of FAS by extracts.The concentration of extracts in the reaction system was 0 (0), 0. 2 μg/mL (1), 0.4 μg/mL (2), and 0.5 μg/mL (3). The FAS concentration was 0.012μM, and the fixed concentrations of Malonyl-CoA and Acetyl-CoA were 10 and 2.5μM, respectively. The reaction rate was the concentration of NADPH per min consumed in the overall reaction.

Table III.  The inhibition types for the extracts from Aceraceous plants against the FAS overall reaction.

	A. platanoides		A. campestre		A. truncatum		EGCG
Substrate	type	Ki(μg/ml)	type	Ki(μg/ml)	type	Ki(μg/ml)	type	Ki(μg/ml)
Acetyl-CoA	C	0.066	N	0.36	C + N	2.61	C + N	16.9
Malonyl-CoA	C + N	0.20	C + N	1.31	C	1.87	N	21.1
NADPH	C + N	0.048	C + N	0.84	U	2.43	C + N	9.16

C, competitive inhibition;N, noncompetitive inhibition;and U, uncompetitive inhibition. The K_i values for EGCG and A. truncatum are cited from (Zhang et al., 2006), and (Zhao et al.,2006), respectively.

Table IV.  The IC₅₀ of the extracts of five species of Acer against various cancer cells.

Species	CAES-17	BGC-823	MCF-7	BEL-7402	PC-3M
	(IC50) (μg/mL)
Acer tataricum	43	22	9	24	25
Acer platanoides	45	36	17	23	24
Acer capillipes	42	18	21	13	11
Acer campestre	62	39	46	24	62
Acer pseudoplatanus	57	46	36	56	61