Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa

Figures & data

Figure 1. Chemical structures of isolated geranyl compounds (1–8) from the fruits of P. tomentosa.

Figure 2. (A) Inhibitory effect of compound (1) and positive control (NaVO₄) on PTP1B activity. (B) Determination of the reversible inhibitory mechanism of compound 1 on PTP1B. Data represent the results of three independent experiments performed in triplicates for each sample.

Figure 3. (A–D) Kinetic assays of PTP1B inhibition, caused by compounds 1 and 6. (A and B) Lineweaver–Burk plots were constructed for the inhibition of PTP1B. The plots are expressed as 1/velocity (1/V) versus 1/substrate (1/[S]) with or without inhibitors. Insets (I) and (II) represents the secondary plots of the slopes and the intercepts of the straight lines versus concentrations of compound 1 and 6. (C and D) Dixon plots for inhibition of PTP1B by compound 1 and 6, respectively.

Table 1. Inhibitory activities on PTP1B and α-glucosidase of isolated compounds.

	PTP1B			α-Glucosidase
Compounds	IC50 (μM)^a	Ki (μM)^b	Type of inhibition	IC50 (μM)^a	Ki (μM)^b	Type of inhibition
1	1.9 ± 0.1	0.8 ± 0.1	Mixed Type I	30.7 ± 1.5	26.7 ± 1.4	Noncompetitive
2	3.9 ± 0.3	3.2 ± 0.2	Mixed Type I	18.4 ± 0.9	17.2 ± 1.0	Noncompetitive
3	7.8 ± 0.6	8.1 ± 0.6	Mixed Type I	19.6 ± 1.1	18.1 ± 0.9	Noncompetitive
4	5.9 ± 0.4	6.1 ± 0.5	Mixed Type I	6.5 ± 0.5	7.1 ± 0.6	Noncompetitive
5	3.8 ± 0.3	3.5 ± 0.3	Mixed Type I	78.9 ± 2.1	72.6 ± 2.3	Noncompetitive
6	4.9 ± 0.5	3.8 ± 0.3	Mixed Type I	17.8 ± 1.1	16.9 ± 1.2	Noncompetitive
7	8.2 ± 0.6	7.9 ± 0.4	Mixed Type I	25.8 ± 1.2	24.4 ± 1.3	Noncompetitive
8	6.6 ± 0.5	6.2 ± 0.4	Mixed Type I	2.2 ± 0.2	3.6 ± 0.3	Noncompetitive
NaVO4^d	32.6 ± 1.5	NT^c	NT^c	>200	NT^c	NT^c
Voglibose^d	>200	NT^c	NT^c	24.5 ± 1.2	NT^c	NT^c

^a All compounds were examined in as set of experiments repeated three times; IC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss.

^b Values of inhibition constant.

^c NT: not tested.

^d Positive control.

Figure 4. (A) Inhibitory effects of compounds (1–8) on α-glucosidase activity. (B and C) Linweaver–Burk plots of compounds 4 and 8 for the inhibition of α-glucosidase catalyzed hydrolysis of p-nitrophenyl glucopyranoside. (D and E) Dixon plots of inhibition of α-glucosidase by compounds 4 and 8, respectively, which were used for determination of K_i values.

Table 2. Effect of different concentrations of compound 1 on V_max, K_m, and the K_ik to K_iv ratio using PTP1B and α-glucosidase.

Enzyme	[I] µM	Vmax	Km	Kik/Kiv
PTP1B^a	0	0.016	0.712	//
	0.78	0.012	1.193	2.660
	1.56	0.009	1.779	3.335
	3.13	0.007	2.248	3.694
α-Glucosidase^b	0	0.157	157.68	//
	12.5	0.117	154.47	12.42
	25	0.082	153.76	19.34
	50	0.054	153.91	27.48

V_max and K_m values were calculated according to Lineweaver–Burk from the data shown in Figures 3, 4 and supplemental. K_ik/K_iv ratio was calculated according to Yang et al.²¹

^a p-Nitrophenyl phosphate (pNPP) as substrate.

^b p-Nitrophenyl glucopyranoside (pNPG) as substrate.

Yang X, Du Z, Pu J, et al. Classification of difference between inhibition constants of an inhibitor to facilitate identifying the inhibition type. J Enzyme Inhib Med Chem 2013;28:205–13.

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