Polyphenol oxidase properties, anti-urease, and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Figures & data

Table 1. Purification profile of PPO from D. lotus.

Purification steps	Volume (mL)	Protein (mg/mL)	Activity (U/mL)	Specific activity (U/mg protein)	Purification (fold)
Crude extract	300	4.5	3.05	35.25	1.0
Acetone precipitation	15	1.5	6.77	105.70	3
Affinity chromatography	10	0.8	12.85	528.5	15

Figure 1. Purification of D. lotus PPO by Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column.

Figure 2. (a) SDS-PAGE of purified D. lotus PPO. Gel stained with Coomassie Brilliant Blue R-250. M: molecular weight marker; 1: crude extract; 2: supernatant obtained after the acetone precipitation step; 3: PPO purified by affinity chromatography. (b) Native electrophoresis of purified D. lotus PPO. Gel revealed with L-DOPA. 1: crude extract; 2: supernatant obtained after the acetone precipitation step; 3: PPO purified by affinity chromatography.

Table 2. Substrate specificity of D. lotus polyphenol oxidase.

Substrate	Relative activity (%)
4-Methylcatechol	100
3-(3,4-Dihydroxyphenyl) propionic acid (DHPPA)	5
p-Hydroxyphenyl propionic acid (PHPPA)	3
L-3,4-Dihydroxyphenylalanine (L-DOPA)	11
Catechol	68
L-Tyrosine	2

Figure 3. (a). pH activity profile for PPO from D. lotus. The effect of pH on PPO activity was measured for pH 3.0–9.0. The buffers used were: glycine-HCl (50 mM, pH 3.0), acetate (50 mM, pH 4.0 and 5.0), phosphate (50 mM, pH 6.0 and 7.0) and Tris-HCl (50 mM, pH 8.0 and 9.0). (b) Effect of temperature on PPO activity from D. lotus. The dependence of PPO activity on temperature was determined under standard assay conditions between 10 and 80°C. The relative activity percentage was calculated based on the maximum activity taken as 100%; 4-methylcatechol was used as substrate.

Table 3. Effects of various metal ions on PPO activity of D. lotus.

Metal ion	Residual activity (%, 1 mM)	Residual activity (%, 5 mM)
None	100	100
K^+	117.4	91.5
Na^+	87.9	67.8
Ba^2+	88.9	53.9
Ca^2+	97.4	70.4
Ni^2+	81.6	59.5
Cd^2+	97.4	51.2
Zn^2+	115.3	94.3
Co^2+	95.8	46.4
Mg^2+	65.3	57.8
Hg^2+	1.1	0.0
Fe^2+	118.9	83.6
Mn^2+	87.4	55.5
Sr^2+	122.1	89.7
Al^3+	103.7	17.8

Table 4. Apparent IC₅₀ of some inhibitors of PPO from D. lotus.

Inhibitor	IC50 (mM)
Sodium azide	5.781
Ascorbic acid	0.000121
Benzoic acid	0.374
Sodium metabisulphite	0.1185

Table 5. Percentage inhibition and IC₅₀ values of urease and AChE of D. lotus aquatic extract.

	Inhibition of AChE activity %		Inhibition of urease activity %
Sample	(30.00 mg/mL)	IC50	(12.00 mg/mL)	IC50
Diospyros lotus L.^a	88.91 ± 0.69	16.75 ± 0.11	99.97 ± 0.59	1.55 ± 0.05
Donepezil (62.5 µg/mL)^b	79.58 ± 0.49	25.22 ± 0.38	—	—
Thiourea (50 µg/mL)^b	—	—	90.06 ± 0.43	15.09 ± 0.13

^aIC₅₀ values given in terms of mg/mL.

^bIC₅₀ values given in terms of µg/mL.