In vitro cytoprotection of modified casein hydrolysates by plastein reaction on rat hepatocyte cells

Figures & data

Table 1. Antioxidant properties of casein hydrolysate and its modified or fractionated products.^a

Propiedades antioxidantes de hidrolizado de caseína y sus productos modificados o fraccionados.

Peptide samples^b	Scavenging activity on DPPH radicals (%)^c	Reducing power	Scavenging activity on hydroxyl radicals (%)
Casein hydrolysate	38.7 ± 0.5 ^A	0.497 ± 0.002 ^A	17.2 ± 0.2 ^A
Mix1	39.7 ± 0.4 ^AB	0.521 ± 0.004 ^B	21.1 ± 0.1 ^B
Mix2	39.7 ± 0.4 ^AB	0.529 ± 0.002 ^B	21.1 ± 0.1 ^B
MCH	42.9 ± 1.3 ^C	0.557 ± 0.011 ^C	21.4 ± 0.3 ^B
MCH1	49.2 ± 0.2 ^E	0.634 ± 0.008 ^F	22.3 ± 1.9 ^B
MCH2	46.7 ± 0.9 ^D	0.662 ± 0.007 ^G	29.7 ± 2.8 ^D
MCH1-SF	56.6 ± 0.3 ^G	0.725 ± 0.004 ^H	22.6 ± 0.4 ^B
MCH1-PF	41.9 ± 0.6 ^C	0.581 ± 0.007 ^D	21.9 ± 0.5 ^B
MCH2-SF	52.0 ± 0.4 ^F	0.722 ± 0.007 ^H	34.4 ± 0.2 ^E
MCH2-PF	40.7 ± 0.4 ^B	0.608 ± 0.002 ^E	25.0 ± 0.7 ^C

^aDifferent capital letters after the values as superscripts in same column indicate that one-way ANOVA of the means is significantly different (P < 0.05).^bMCH, MCH1, and MCH2, modified casein hydrolysates without amino acid addition, with Phe and Tyr addition, respectively; SF and PF, soluble and precipitated fractions by ethanol-water solvent in 6:4 (v/v). Mix1 or Mix2, mixture of casein hydrolysate and Phe or Tyr, respectively.

Table 2. Amino acids compositions (%, in molar) of casein hydrolysate and four fractionated hydrolysates by ethanol-water solvent.^a

Composicion de aminoácidos (%, en moles) de hidrolizado de caseína y cuatro hidrolizados fraccionados con disolvente etanol-agua.

Amino acids	Casein hydrolysate	MCH1-SF	MCH1-PF	MCH2-SF	MCH2-PF
Asp	7.11	6.24	6.45	6.39	6.32
Thr	4.75	4.19	4.32	4.23	4.30
Ser	7.17	5.93	6.97	6.16	6.82
Glu	20.07	17.59	18.44	17.96	18.11
Gly	3.24	2.77	2.87	2.87	2.82
Ala	4.24	3.65	3.77	3.81	3.64
Cys	1.47	1.69	1.40	1.54	1.85
Val	6.23	5.67	5.40	5.51	5.70
Met	1.63	1.17	0.16	0.81	1.32
Ile	3.94	3.22	3.26	3.26	3.42
Leu	8.68	7.77	7.61	7.78	7.56
Tyr	3.82	3.54	3.33	14.49	13.70
Phe	3.54	14.81	14.60	3.08	2.88
Lys	7.03	6.31	6.29	6.46	6.31
His	2.59	2.40	2.40	2.27	2.52
Arg	2.49	2.23	2.27	2.24	2.26
Pro	12.00	10.82	10.47	11.14	10.46
Total	100.00	100.0	100.0	100.0	100.0
THAA	42.45	49.49	48.44	49.08	47.37

^aTHAA, total hydrophobic amino acids; MCH1 and MCH2, modified casein hydrolysates in the presence of Phe and Tyr, respectively; SF and PF, soluble and precipitated fraction by ethanol-water solvent in 6:4 (v/v).

Table 3. Protective effects of the investigated hydrolysates on rat hepatocytes against H₂O₂-induced injury.^a

Los efectos protectores de los hidrolizados investigados en hepatocitos de rata frente a la lesión inducida por H₂O_2.

Samples ^b	Addition level (mg/mL)	Cell viability (%)	LDH leakage (U/L)	MDA content (µmol/g protein)
Casein hydrolysate	0.5	41.1 ± 0.6 ^B	537 ± 13 ^J	27.3 ± 0.1 ^K
	1.0	46.5 ± 0.7 ^DE	460 ± 1 ^G	25.3 ± 0.7 ^J
	2.0	48.4 ± 0.2 ^EF	432 ± 5 ^F	23.4 ± 0.3 ^H
Mix1	0.5	37.2 ± 0.7 ^A	550 ± 3 ^K	25.6 ± 0.4 ^J
	1.0	42.2 ± 0.3 ^BC	541 ± 4 ^J	23.1 ± 0.2 ^H
	2.0	46.6 ± 1.0 ^DE	392 ± 4 ^D	21.5 ± 0.5 ^G
Mix2	0.5	44.6 ± 0.3 ^CD	501 ± 2 ^I	24.2 ± 0.4 ^I
	1.0	48.4 ± 2.6 ^EF	480 ± 5 ^H	20.8 ± 0.5 ^F
	2.0	51.5 ±0.6 ^GH	412 ± 4 ^E	19.1 ± 0.2 ^BC
MCH1-SF	0.5	48.2 ± 0.4 ^EF	551 ± 2 ^K	22.9 ± 0.4 ^H
	1.0	49.4 ± 0.9 ^FG	498 ± 4 ^I	20.9 ± 0.3 ^F
	2.0	54.2 ± 0.9 ^I	318 ± 2 ^B	18.9 ± 0.4 ^B
MCH1-PF	0.5	41.9 ± 1.0 ^B	543 ± 2 ^JK	24.1 ± 0.1 ^I
	1.0	49.1 ± 0.7 ^EFG	400 ± 3 ^D	23.0 ± 0.2 ^H
	2.0	53.2 ± 1.1 ^HI	375 ± 2 ^C	20.0 ± 0.2 ^DE
MCH2-SF	0.5	47.7 ± 0.3 ^EF	435 ± 2 F	21.9 ± 0.3 ^G
	1.0	48.8 ± 0.7 ^EF	392 ± 1 ^D	19.5 ± 0.2 ^CD
	2.0	56.9 ± 0.8 ^J	294 ± 13 ^A	16.5 ± 0.2 ^A
MCH2-PF	0.5	39.9 ± 1.1 ^B	462 ± 5 ^G	23.5 ± 0.1 ^H
	1.0	44.5 ± 0.9 ^CD	400 ± 2 ^D	20.1 ± 0.2 ^E
	2.0	47.2 ± 0.5 ^EF	372 ± 3 ^C	18.6 ± 0.5 ^B

^aLDH and MDA, lactate dehydrogenase and malondialdehyde. Cell viability and LDH leakage for the cells in model group are (29.0 ± 0.4)% and (568 ± 6) U/L, while MDA content for the cells in control and model group is (8.9 ± 0.6) and (27.9 ± 0.3) µmol/g protein, respectively. Different capital letters after the values as superscripts in same column indicate that one-way ANOVA of the means is significantly different (P < 0.05).^bMCH1 and MCH2, modified casein hydrolysates in the presence of Phe and Tyr, respectively; SF and PF, soluble and precipitated fraction by ethanol-water solvent in 6:4 (v/v). Mix1 or Mix2, mixture of casein hydrolysate and Phe or Tyr, respectively.

Table 4. Protective effects of the investigated casein hydrolysates on rat hepatocytes against galactosamine-induced injury.^a

Efectos protectores de los hidrolizados de caseína investigados en hepatocitos de rata frente a la lesión inducida por galactosamina.

Samples^b	Addition level (mg/mL)	Cell viability (%)	LDH leakage (U/L)	MDA content (µmol/g protein)
Casein hydrolysate	0.5	36.6 ± 1.0 ^A	287 ± 4 ^J	19.2 ± 0.3 ^K
	1.0	40.3 ± 2.5 ^A	260 ± 25 ^I	17.1 ± 0.8 ^I
	2.0	47.7 ± 2.9 ^BC	212 ± 23 ^EFGH	14.7 ± 0.6 ^EFG
Mix 1	0.5	40.0 ± 2.9 ^A	225 ± 1 ^FGH	17.6 ± 0.1 ^IJ
	1.0	44.7 ± 2.0 ^B	203 ± 8 ^CDEF	15.1 ± 0.7 ^FG
	2.0	51.0 ± 5.3 ^CD	201 ± 18 ^BCDEF	15.1 ± 0.3 ^FG
Mix 2	0.5	51.9 ± 2.1 ^CD	229 ± 9 ^GH	18.0 ± 0.4 ^J
	1.0	55.4 ± 1.6 ^EEF	208 ± 5 ^DEFG	15.2 ± 0.9 ^G
	2.0	60.0 ± 0.5 ^FGH	206 ± 27 ^DEFG	14.7 ± 0.5 ^EFG
MCH1-SF	0.5	55.3 ± 2.1 ^DEF	266 ± 8 ^IJ	15.0 ± 0.3 ^FG
	1.0	58.2 ± 2.9 ^EFG	202 ± 20 ^BCDEF	13.9 ± 0.7 ^E
	2.0	77.9 ± 1.1 ^J	178 ± 3 ^AB	11.7 ± 0.2 ^ABC
MCH1-PF	0.5	54.4 ± 1.6 ^DE	262 ± 9 ^I	16.2 ± 0.3 ^H
	1.0	61.1 ± 2.7 ^GH	210 ± 19 ^DEFGH	14.6 ± 0.5 ^EFG
	2.0	62.8 ± 3.8 ^GH	194 ± 3 ^ABCDE	12.7 ± 0.1 ^D
MCH2-SF	0.5	53.8 ± 3.9 ^DE	233 ± 2 ^H	14.0 ± 0.1 ^E
	1.0	70.2 ± 0.3 ^I	192 ± 1 ^ABCDE	12.2 ± 0.2 ^BCD
	2.0	80.1 ± 2.2 ^J	175 ± 4 ^A	11.2 ± 0.1 ^A
MCH2-PF	0.5	50.3 ± 3.3 ^CD	198 ± 5 ^ABCDE	14.3 ± 0.1 ^EF
	1.0	53.1 ± 0.6 ^D	186 ± 9 ^ABCD	12.3 ± 0.2 ^CD
	2.0	63.3 ± 3.1 ^H	181 ± 2 ^ABC	11.5 ± 0.2 ^AB

^aLDH and MDA, lactate dehydrogenase and malondialdehyde. Cell viability and LDH leakage for the cells in model group are (33.5 ± 1.4)% and (289 ± 3) U/L, while MDA content for the cells in control and model group is (8.2 ± 0.3) and (19.5 ± 0.6) µmol/g protein, respectively. Different capital letters after the values as superscripts in same column indicate that one-way ANOVA of the means is significantly different (P < 0.05).^bMCH1 and MCH2, modified casein hydrolysates in the presence of Phe and Tyr, respectively; SF and PF, soluble and precipitated fraction by ethanol-water solvent in 6:4 (v/v). Mix1 or Mix2, mixture of casein hydrolysate and Phe or Tyr, respectively.

Table 5. Correlation coefficients between cell viability, cellular lactate dehydrogenase (LDH) leakage or malondialdehyde (MDA) content and scavenging activity (SA) on DPPH or hydroxyl (OH) radicals, and reducing power (RP) of the evaluated seven casein hydrolysates.

Coeficientes de correlación entre la viabilidad celular, eliminación de lactato deshidrogenasa celular (LDH) o contenido de malondialdehído (MDA) y la actividad de atrapamiento (SA) sobre los radicales hidroxilo (OH) o DPPH, poder reductora (RP) de los siete hidrolizados de caseína evaluados.

Treatment	Sample level (mg/mL)	Antioxidant properties	Cell viability	LDH Leakage	MDA content
H2O2-induced injury	0.5	SA on DPPH	0.828^*	–0.137	–0.721
		SA on OH	0.496	–0.806^*	–0.825^*
		RP	0.767^*	–0.392	–0.874^*
	1.0	SA on DPPH	0.564	–0.068	–0.531
		SA on OH	0.197	–0.546	–0.772^*
		RP	0.488	–0.334	–0.710
	2.0	SA on DPPH	0.760^*	–0.896^**	–0.608
		SA on OH	0.620	–0.831^*	–0.869^*
		RP	0.746	–0.971^**	–0.790^*
Galactosamine-induced injury	0.5	SA on DPPH	0.608	–0.173	–0.660
		SA on OH	0.546	–0.437	–0.805^*
		RP	0.726	–0.070	–0.870*
	1.0	SA on DPPH	0.646	–0.379	–0.517
		SA on OH	0.818^*	–0.663	–0.850^*
		RP	0.783^*	–0.578	–0.763^*
	2.0	SA on DPPH	0.898^**	–0.778^*	–0.698
		SA on OH	0.771^*	–0.786^*	–0.723
		RP	0.971^**	–0.936^**	–0.885^**

*P < 0.05.**P < 0.01.