Biochemical properties of partially purified polyphenol oxidase and phenolic compounds of Prunus spinosa L. subsp. dasyphylla as measured by HPLC-UV

Figures & data

Table 1. RP-HPLC-UV validation parameters of the phenolic compounds in P. spinosa L.

No.	Compound	R^2	% RSD (Retention time)	% RSD(Area)	LOD^a	LOQ^a
1	Gallic acid	0.999	0.210	1.941	0.022	0.067
2	Protocatechuic acid	0.999	0.871	1.920	0.042	0.128
3	p-OH benzoic acid	0.998	0.351	3.055	0.036	0.109
4	Catechin	0.997	0.492	4.279	0.040	0.121
5	Vanillic acid	1.000	0.828	2.066	0.025	0.075
6	Caffeic acid	0.998	0.179	4.039	0.062	0.187
7	Syringic acid	1.000	0.550	0.848	0.009	0.027
8	Epicatechin	0.999	0.429	3.819	0.030	0.090
9	p-Coumaric acid	0.999	0.204	1.562	0.010	0.030
10	Ferulic acid	0.999	0.222	1.301	0.011	0.033
11	Rutin	1.000	0.234	3.139	0.041	0.123
12	Daidzein	0.998	0.174	1.545	0.018	0.054
13	t-Cinnamic acid	1.000	0.262	1.071	0.014	0.042
14	Luteolin	0.994	0.229	5.833	0.043	0.130

^a: Values are expressed as mg/L.

Figure 1. Native PAGE electrophoresis gel revealed by incubating with 100 mM of L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate of polyphenol oxidase. 1: Crude enzyme after acetone precipitation. Each isoenzyme band was marked.

Table 2. Substrate specificity and activity (U/mg protein) of P. spinosa polyphenol oxidase.

Substrate	Type of phenolic compound	Activity (U/mg protein)	Wavelength (nm)
p-Coumaric acid	Monohydroxy	3	500
PHPPA	Monohydroxy	5	500
L-Tyrosine	Monohydroxy	33	500
Gallic acid	Trihydroxy	38	500
Quercetin	Pentahydroxy	155	500
Epicatechin	Pentahydroxy	908	500
Catechin	Pentahydroxy	1133	500
Hydrocaffeic acid	Dihydroxy	1840	500
Catechol	Dihydroxy	2240	500
4-Methylcatechol	Dihydroxy	3795	494

Table 3. Some biochemical parameters of P. spinosa polyphenol oxidase towards various substrates.

Substrate	pH	Temperature (°C)	Km (mM)	Vmax (U/mg protein)
4-Methylcatechol	7.0	40	0.97	4753
Catechol	7.0	30	2.35	3964
Hydrocaffeic acid	4.0-6.0	60	2.49	7628
Catechin	5.0	60	4.26	1686
Epicatechin	5.0	60	6.04	2093

Figure 2. The Michaelis–Menten equation of diphenolase activity of P. spinosa for various substrates. (A) 4-methylcatechol (0.05–0.80 mM), (B) catechol (0.10–1.50 mM), (C) hydrocaffeic acid (0.15–5.00 mM), (D) catechin (1.25–15.00 mM), and (E) epicatechin (0.31–10.00 mM) substrates were analysed as 1/specific activity (1/V) versus 1/substrate concentration (1/[S]). The Michaelis–Menten constants (K_m) and maximum velocity (V_max) were obtained from the Lineweaver–Burk plots.

Figure 3. pH-activity profile for P. spinosa polyphenol oxidase in the presence of various substrates; (A) 4-methylcatechol, (B) catechol, (C) hydrocaffeic acid, (D) catechin, and (E) epicatechin, and (F) pH stability of P. spinosa polyphenol oxidase at 4°C, at pH 4.0, 5.0, 6.0, and 7.0, incubated in the presence of 4-methylcatechol as a substrate for 24 days.

Figure 4. Optimum temperature of P. spinosa polyphenol oxidase in the presence of various substrates; (A) 4-methylcatechol, (B) catechol, (C) hydrocaffeic acid, (D) catechin, and (E) epicatechin, and (F) thermal stability of P. spinosa polyphenol oxidase at 30, 40, and 60°C, at pH 7.0, for 60 min.

Table 4. The IC₅₀ values of several inhibitors on the diphenolase activity of P. spinosa polyphenol oxidase using 4-MTC as substrate.

Inhibitor	IC50 (mM)
Sodium metabisulphite	0.01 ± 0.00
Ascorbic acid	0.04 ± 0.00
Sodium azide	50.02 ± 1.01
Benzoic acid	5.92 ± 0.28
Thiourea	3.95 ± 0.32
L-cysteine	23.25 ± 1.45

Figure 5. Inhibition effect of common polyphenol oxidase inhibitors on the diphenolase activity of P. spinosa. (A) Ascorbic acid (12.50–200 µM), (B) sodium azide (6.25–200 mM), (C) benzoic acid (1.56–50 mM), (D) sodium metabisulphite (3.13–50 µM), (E) thiourea (0.78–25 mM), and (F) L-cysteine (6.25–200 mM).

Table 5. Effect of various metal ions on P. spinosa polyphenol oxidase activity in the presence of 4-MTC as substrate.

Metal ion	Residual activity (%)
	1 mM	5 mM	10 mM
Control	100	100	100
Na^+	103.74	71.63	64.19
K^+	84.79	77.85	66.40
Mg^2+	90.92	89.37	88.94
Ca^2+	94.13	64.50	35.16
Ba^2+	76.51	28.59	15.92
Mn^2+	94.16	55.92	31.27
Co^2+	146.33	91.78	77.96
Ni^2+	103.05	89.07	70.81
Zn^2+	119.24	95.86	92.65
Cd^+2	88.60	49.73	32.62
Hg^2+	10.29	2.95	0.67
Al^3+	110.82	21.54	8.36

Table 6. Phenolic acid content of Prunus spinosa L. plum.

	Standard	Plum (µg phenolic/g sample)
1	Gallic acid	N.D.
2	Protocatechuic acid	0.20 ± 0.06
3	p-OH benzoic acid	0.22 ± 0.09
4	Catechin	N.D.
5	Vanillic acid	1.56 ± 0.13
6	Caffeic acid	N.D.
7	Syringic acid	0.21 ± 0.05
8	Epicatechin	0.60 ± 0.10
9	p-Coumaric acid	0.59 ± 0.07
10	Ferulic acid	N.D.
11	Rutin	N.D.
12	Daidzein	N.D.
13	t-Cinnamic acid	N.D.
14	Luteolin	0.94 ± 0.12

N.D.: Not determined.

Figure 6. Chromatograms of phenolic acid standards (1. gallic acid; 2. protocatechuic acid; 3. p-OH benzoic acid; 4. catechin; 5. vanillic acid; 6. caffeic acid; 7. syringic acid; 8. epicatechin; 9. p-coumaric acid; 10. ferulic acid; 11. rutin; 12. daidzein; 13. t-cinnamic acid; 14. luteolin).