Human neutrophil elastase inhibitory potential of flavonoids from Campylotropis hirtella and their kinetics

Figures & data

Figure 1. Chemical structures of isolated flavonoids (1–3) from the root bark of C. hirtella and parent compounds (dalbergioidin) of 3.

Figure 2. (A) Effect of compounds on the HNE-catalyzed hydrolysis of methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (compound 1, •; compound 2, ▾; compound 3, ○; dalbergioidin, △, respectively.) (B–D) Lineweaver–Burk plot for inhibition of the HNE-catalyzed hydrolysis of substrate by compounds (1–3).

Table 1. Inhibitory effect of isolated compounds 1–8 on human leukocyte elastase.

Compounds	IC50^a value (μM)	Inhibition mode (Ki^b, μM)
1	17.9 ± 1.5	Noncompetitive (16.8 ± 1.4)
2	8.4 ± 0.8	Competitive (4.5 ± 0.6)
3	30.8 ± 1.3	Mixed (17.1 ± 2.1)
4–8	>200	NT^c
Dalbergioidin	>200	NT
Oleanolic acid^d	21.68 ± 3.2	NT

^aIC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss.

^bValues of inhibition constant.

^cNT is not tested.

^dOleanolic acid was used as a positive control.

Table 2. Peak assignments, mass spectral data, and cited references of flavonoids in the root bark of C. hirtella from the positive and negative mode using HPLC-DAD-ESI/MS.

Compounds	tR (min) at 280 nm	Exact mass M (calc.)	m/z for [M ± H]^+/−	Major fragment ions m/z (rel. intensity)	UV λmax (nm)	Refs.
1	43.75	438.204	439.3 [M + H]	439.3 (100), 315.3 (47)	295	Shou et al.12
2	40.14	440.183	439.4 [M − H]	421.4 (100)	259	Shou et al.12
3	42.61	424.188	425.2 [M + H]	301.3 (100), 175.2 (54)	287	Shou et al.12
4	36.36	384.157	385.2 [M + H]	219.3 (100), 137.2 (69)	287	Shou et al.16
5	44.79	422.172	423.3 [M + H]	299.0 (100)	262	Shou et al.12
6	45.93	406.178	408.2 [M + H]	408.2 (100), 284.2 (36)	262	Kang et al.23, Shou et al.12
7	46.71	438.204	439.2 [M + H]	383 (100), 203.3 (42)	294	Shou et al.12
8	47.42	422.209	423.4 [M + H]	287 (100)	292	Kang et al.23, Shou et al.12

Figure 3. (A) Typical HPLC-DAD chromatogram of the methanol extracts of C. hirtella detected at 280 nm. (B) Mass fragmentation patterns of identified peaks that accord with compound number. (a) compound 1; (b) compound 2; (c) compound 3; (d) compound 4; (e) compound 5; (f) compound 6; (g) compound 7; (h) compound 8.

Figure 4. Inhibition of HNE by compound 2. (A) The hydrolytic activity of HNE as function of enzyme concentration at different concentrations of compound 2. (B) Time-dependent inhibition of HNE in the presence of compound 2 with varying pre-incubation times. (C) Pre-incubation time dependence of the fractional velocity of the enzyme-catalyzed reaction in the presence of varying concentration of compound 2. (D) Dependence of k_obs on the concentration of compound 2. The k_obs values determined in (C) were fitted to Equation (3).

Figure 5. Scheme for time-dependent enzyme inhibition. The upper part (k₁, k₂, k_cat) denotes turnover of the enzyme in the absence of inhibition and were calculated using traditional methods as delineated in Materials and methods section. The lower part illustrates the mechanism for a slow-binding inhibition. In simple reversible slow-binding inhibition process of the values of k₃ and k₄ are slow relative to enzyme turnover and k₅/k₆ does not occur. In enzyme isomerization, an initial binding of the inhibitor to the enzyme leads to formation of the EI complex, which undergoes an isomerization to form the new complex E*I.

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