In-vivo study of synthetic and natural food colors effect on biochemical and immunity parameters

SHAKOOR, Sadaf; ISMAIL, Amin; SABRAN, Mohd Redzwan; MOHTARRUDIN, Norhafizah; KAKA, Ubedullah; NADEEM, Muhammad

doi:10.1590/fst.41420

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Food Science and Technology

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Original Article • Food Sci. Technol (Campinas) 42 • 2022 • https://doi.org/10.1590/fst.41420 copy

In-vivo study of synthetic and natural food colors effect on biochemical and immunity parameters

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Incorporation of the coloring agent, tartrazine and curcumin affects healthy physiological system leading to changes in hematology, biochemistry and enzymatic activity. Consequences of oral administration of tartrazine and curcumin were explored for 15, 30 and 45 days in female adult rat. Two doses were based on the admissible daily intake (ADI) of 9.6 and 96 (high) mg/kg/body weight for tartrazine, 3.85 and 38.5 mg/kg/body weight for curcumin. Phytochemicals such as saponins, glycosides, alkaloids, flavonoids, tannin, carbohydrates and phenolics were present in coloring agents. The renal function tests and lipid profile showed alterations in the values of uric acid, urea, total protein, albumin and cholesterol in all treatment groups. Additionally, the levels of liver enzymes fluctuated after 45 days of treatment with curcumin and tartrazine. Histopathological changes were also recorded in liver and kidney. In contrast, blood biochemistry depicted the significant reduction of RBCs, Hb, MCH, glucose, WBCs, MCHC and LDH at 15, 30 and 45 days. Besides, IgG, IgM and overall antibody GMT at 45 days were significantly increased. Study indicated that ADI and doses up to 10 times of the ADI of food colors intake exerted adverse effects on immune response and altered the biochemical parameters.

Keywords:
food colors; hematology; antibody; toxicology; biochemistry; immunity

	Moisture %	Ash %	Fiber %	Protein %	Fat %
Control	25.8	5.75	2.75	19.68	4
Low Tartrazine	27.1	5.3	1.6	17.5	4.18
High Tartrazine	10.23	7.26	3.05	21.87	4.05
Low Curcumin	8.43	5.94	3.4	21.85	3.5
High Curcumin	8.45	5.3	3.85	20.23	5.1

Compounds	Water Extract					Ethanolic Extract					Methanolic Extract
	C	LT	HT	LC	HC	C	LT	HT	LC	HC	C	LT	HT	LC	HC
Alkaloids	-ve	+ve	+ve	+ve	+ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Amino acids	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Anthocyanins	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Anthraquinones	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	+ve	-ve	-ve	-ve	-ve	+ve
Carbohydrates	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Cumarins	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve
Fixed oils	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Flavonoids	+ve	+ve	+ve	+ve	+ve	+ve	-ve	-ve	-ve	-ve	+ve	-ve	-ve	-ve	-ve
Glycosides	-ve	-ve	-ve	-ve	-ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve
Phenols	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Proteins	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Resins	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Saponins	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve	+ve
Steroids	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve
Tannins	+ve	+ve	+ve	+ve	+ve	-ve	-ve	-ve	-ve	-ve	+ve	+ve	+ve	-ve	-ve
Terpenoids	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve	-ve

Compounds	Water Extract					Ethanolic Extract					Methanolic Extract
	C	LT	HT	LC	HC	C	LT	HT	LC	HC	C	LT	HT	LC	HC
Carbohydrate mg/dL	2686	3669	3354	3404	3467	2867	3691	3691	3336	3550	2965	3557	2909	3503	3691
Tannins g/mL	293.5	291	262.5	267	270	273.5	288	279	279.5	291	282	288.5	287.5	295.5	293
Phenolics µg/mL	60.6	82.5	57.6	53.6	61.0	109.7	109.5	111.9	99.3	112.9	102.4	108.5	108.6	107.3	114.2
Alkaloids mg/mL	297	84.6	52.6	20.6	82.3	518	78.33	321.33	8.67	16	502.67	160	126.67	228	19
Flavonoids µg/mL	122.4	76.5	60.2	262	169.5	241.5	256.7	283.9	210.2	403.5	182.7	225.8	268.4	219.7	403.5

Days	Food color Exposure	Body weight gain (%)	Water Intake (mL)	Feed Intake (g)
15	Control	0.99 ± 0.49 ^e	161.20 ± 4.34 ^a	118.20 ± 6.81 ^a
	LT	0.68 ± 0.30 ^e	153.26 ± 9.26 ^abcde	103.20 ± 2.52 ^e
	HT	0.84 ± 0.58 ^e	157.33 ± 4.23 ^ab	106.73 ± 9.25 ^bcde
	LC	1.68 ± 0.59 ^e	156.93 ± 5.63 ^ab	107.93 ± 4.47 ^bcde
	HC	0.66 ± 0.76 ^e	154.13 ± 6.56 ^abcd	104.53 ± 3.02 ^cde
30	Control	5.81 ± 1.27 ^de	154.26 ± 10.59 ^abc	120.30 ± 6.90 ^a
	LT	16.28 ± 2.44 ^abc	135.80 ± 19.57 ^fg	109.96 ± 5.49 ^bc
	HT	13.69 ± 3.13 ^bcd	137.6 ± 19.31 ^efg	106.00 ± 5.72 ^bcde
	LC	13.35 ± 5.74 ^bcd	140.43 ± 21.90 ^cdefg	109.80 ± 5.81 ^bcd
	HC	16.57 ± 1.59 ^ab	143.10 ± 15.48 ^bcdef	110.73 ± 6.37 ^b
45	Control	7.85 ± 1.65 ^cde	151.42 ± 9.55 ^abcdef	111.88 ± 8.00 ^b
	LT	11.48 ± 6.08 ^bcd	133.35 ± 22.18 ^g	106.88 ± 6.00 ^bcde
	HT	16.09 ± 2.33 ^abc	137.82 ± 18.25 ^defg	102.97 ± 6.85 ^e
	LC	17.45 ± 3.06 ^ab	138.24 ± 19.2 ^cdefg	106.57 ± 6.56 ^bcde
	HC	23.44 ± 3.70 ^a	136.08 ± 19.08 ^fg	103.71 ± 5.25 ^de

Days	Food color Exposure	RBC (M/µL)	WBC (K/uL)	Platelates (K/UI)	Haemoglobin (g/dL)	Haematocrit (%)	MCV (fl)	MCH (PG)	MCHC (g/dL)	Neutrophiles (%)	Lymphocytes (%)	Monocytes (%)	Eosinophils (%)
15	Control	6.18 ± 0.52 ^ab	13.60 ± 4.23 ^a	786.33 ± 263.73 ^a	13.03 ± 0.84 ^abc	36.05 ± 3.92 ^abc	52.93 ± 1.16 ^fg	26.63 ± 7.53 ^a	38.98 ± 0.93 ^ab	26.16 ± 4.57 ^ab	71.66 ± 5.12 ^bcd	0.33 ± 0.51 ^bcd	1.83 ± 0.75 ^ab
	LT	5.81 ± 0.19 ^ab	9.43 ± 2.65 ^abc	663.16 ± 116.07 ^a	12.41 ± 0.61 ^abc	33.70 ± 1.06 ^abc	57.06 ± 1.76 ^abcd	19.10 ± 3.21 ^bc	36.05 ± 0.45 ^cdef	28.83 ± 2.97 ^ab	69.50 ± 3.78 ^bcd	0.50 ± 0.54 ^bcd	2.50 ± 0.83 ^a
	HT	5.52 ± 0.42 ^b	8.62 ± 2.28 ^bc	869.83 ± 164.68 ^a	11.83 ± 0.61 ^abc	34.86 ± 3.19 ^abc	56.50 ± 0.41 ^bcde	19.76 ± 1.99 ^abc	35.88 ± 0.39 ^def	29.17 ± 2.63 ^ab	65.16 ± 7.16 ^d	1.16 ± 0.75 ^ab	2.00 ± .00 ^ab
	LC	6.03 ± 0.54 ^ab	9.20 ± 2.31 ^abc	803.33 ± 222.57 ^a	13.28 ± 0.57 ^ab	35.51 ± 4.76 ^abc	55.35 ± 2.14 ^cdef	22.58 ± 2.80 ^abc	37.83 ± 0.82 ^abcde	25.66 ± 3.61 ^ab	72.83 ± 3.37 ^bcd	0.00 ± 0.00 ^d	1.50 ± 1.04 ^ab
	HC	5.90 ± 0.54 ^ab	8.05 ± 1.77 ^bc	764.33 ± 87.61 ^a	11.63 ± 1.21 ^bc	34.45 ± 5.99 ^abc	58.51 ± 2.85 ^abc	21.65 ± 1.66 ^abc	34.48 ± 2.84 ^f	31.50 ± 4.32 ^ab	70.83 ± 4.21 ^bcd	1.50 ± 0.54 ^a	2.33 ± 0.51 ^a
30	Control	6.86 ± 0.57 ^a	7.13 ± 1.42 ^c	747.00 ± 115.87 ^a	13.40 ± 0.60 ^ab	37.05 ± 1.77 ^ab	60.13 ± 1.57 ^a	26.78 ± 3.41 ^a	35.65 ± 0.66 ^ef	22.33 ± 3.14 ^b	75.83 ± 3.48 ^ab	0.00 ± 0.00 ^ad	1.83 ± 1.16 ^ab
	LT	5.91 ± 0.73^ab	10.35 ± 5.06 ^abc	858.83 ± 495.49 ^a	12.01 ± 1.61 ^abc	35.10 ± 4.41 ^abc	53.23 ± 1.47 ^fg	16.28 ± 2.87 ^bc	37.07 ± 0.92 ^bcde	34.50 ± 2.25 ^a	81.77 ± 7.26 ^a	0.16 ± 0.40 ^cd	0.67 ± .51 ^b
	HT	5.88 ± 0.46 ^ab	11.62 ± 2.39 ^abc	968.33 ± 160.98 ^a	11.40 ± 1.05 ^abc	33.60 ± 1.84 ^abc	53.30 ± 1.17 ^efg	15.61 ± 4.30 ^c	39.18 ± 0.37 ^ab	34.62 ± 2.90 ^a	64.75 ± 3.63 ^d	0.16 ± 0.40 ^cd	0.67 ± .51 ^b
	LC	6.19 ± 0.23 ^ab	9.17 ± 3.71 ^abc	724.83 ± 363.83 ^a	11.98 ± 1.86 ^abc	33.83 ± 4.45 ^abc	51.90 ± 0.63 ^g	20.15 ± 1.40 ^abc	39.90 ± 0.84 ^a	28.77 ± 2.78 ^ab	69.33 ± 2.33 ^bcd	0.16 ± 0.40 ^cd	1.33 ± 0.8 ^ab
	HC	5.92 ± 1.00 ^ab	12.17 ± 3.22 ^ab	966.66 ± 73.86 ^a	11.78 ± 0.96 ^bc	36.15 ± 2.42 ^abc	57.73 ± 1.83 ^abcd	19.81 ± 3.93 ^abc	37.93 ± 1.47 ^abcd	29.48 ± 3.93 ^ab	69.18 ± 4.02 ^bcd	0.16 ± 0.40 ^cd	2.42 ± 0.49 ^a
45	Control	6.36 ± 0.37 ^ab	7.15 ± 1.55 ^c	712.83 ± 139.72 ^a	13.96 ± 0.70 ^a	39.13 ± 2.78 ^a	59.18 ± 1.20 ^ab	23.26 ± 3.38 ^ab	36.26 ± 1.08 ^cdef	24.00 ± 5.06 ^b	73.50 ± 4.50 ^abcd	1.00 ± 0.47 ^abc	2.33 ± 0.51 ^a
	LT	5.68 ± 0.47 ^b	11.03 ± 5.73 ^abc	774.83 ± 225.10 ^a	12.21 ± 1.57 ^abc	33.71 ± 2.14 ^abc	52.91 ± 1.47 ^fg	16.23 ± 3.28 ^bc	39.51 ± 0.72 ^a	30.02 ± 2.35 ^ab	66.03 ± 2.16 ^cd	0.33 ± 0.51 ^bcd	1.33 ± 0.81 ^ab
	HT	5.57 ± 0.89 ^b	10.17 ± 3.31 ^abc	800.16 ± 106.69 ^a	11.13 ± 1.19 ^c	32.30 ± 4.20 ^abc	55.06 ± 1.91 ^defg	15.40 ± 3.01 ^c	38.51 ± 1.11 ^ab	30.00 ± 5.67 ^ab	66.45 ± 4.96 ^bcd	0.16 ± 0.40 ^cd	1.17 ± 0.75 ^ab
	LC	5.87 ± 0.56 ^ab	8.68 ± 5.73 ^bc	846.50 ± 95.41 ^a	13.08 ± 0.29 ^abc	31.86 ± 3.93 ^bc	53.71 ± 0.81 ^efg	18.71 ± 2.09 ^bc	39.51 ± 0.40 ^a	31.97 ± 8.42 ^ab	74.16 ± 3.06 ^abc	0.33 ± 0.51 ^bcd	1.67 ± 0.81 ^ab
	HC	5.35 ± 0.68 ^b	11.35 ± 3.90 ^abc	794.50 ± 61.42 ^a	11.36 ± 1.07 ^bc	29.20 ± 3.01 ^c	55.03 ± 1.57 ^defg	16.30 ± 4.70 ^bc	38.11 ± 0.65 ^abc	30.48 ± 9.65 ^ab	66.83 ± 2.83 ^bcd	0.25 ± 0.41 ^bcd	1.66 ± 1.36 ^ab

Days	Food color Exposure	ALT (U/L)	AST (U/L)	ALP (U/L)	LDH (U/L)
15	Control	33.33 ± 9.75^abc	89.83 ± 5.19 ^c	40.05 ± 5.45 ^ab	962.66 ± 182.62 ^a
	LT	27.83 ± 7.27 ^abc	80.16 ± 8.03 ^c	35.22 ± 7.38 ^abcd	646.33 ± 194.71 ^ab
	HT	24.5 ± 3.14 ^abc	98.33 ± 14.90 ^bc	23.16 ± 12.25 ^abcd	680.00 ± 227.55 ^ab
	LC	35.5 ± 25.46 ^abc	86.66 ± 14.47 ^c	38.66 ± 22.72 ^abc	727.00 ± 116.37 ^ab
	HC	40.66 ± 21.74 ^abc	97.50 ± 11.41 ^bc	15.50 ± 1.87 ^cd	539.83 ± 163.41 ^ab
30	Control	24.83 ± 4.66 ^abc	91.83 ± 30.49 ^c	45.8 ± 23.79 ^a	620.33 ± 361.00 ^ab
	LT	18.33 ± 3.44 ^bc	92.5 ± 23.60 ^c	28.25 ± 13.08 ^abcd	850.00 ± 218.43 ^ab
	HT	15.66 ± 3.38 ^c	99.25 ± 29.11 ^bc	21.50 ± 8.66 ^abcd	505.16 ± 192.69 ^b
	LC	24.66 ± 3.93 ^abc	77.00 ± 29.61 ^c	20.25 ± 4.16 ^abcd	726.00 ± 277.68 ^ab
	HC	39.66 ± 25.76 ^abc	114.41 ± 73.22 ^abc	24.5 ± 5.07 ^abcd	749.50 ± 152.05 ^ab
45	Control	26.83 ± 6.79 ^abc	89.00 ± 27.01 ^c	36.50 ± 21.71 ^abcd	660.00 ± 317.61 ^ab
	LT	47.16 ± 7.33 ^a	105.50 ± 34.14 ^bc	21.25 ± 9.98 ^bcd	581.00 ± 181.80 ^ab
	HT	48.5 ± 9.58 ^a	173.50 ± 34.14 ^a	14.00 ± 3.40 ^d	810.00 ± 125.51 ^ab
	LC	41.66 ± 7.03 ^ab	155.75 ± 25.50 ^ab	24.00 ± 10.41 ^abcd	814.33 ± 93.70 ^ab
	HC	46.5 ± 6.97 ^a	152.25 ± 28.59 ^ab	17.00 ± 6.81 ^bcd	940.00 ± 169.24 ^a

Days	Food color Exposure	BUN (mg/dL)	CREATINE (mg/dL)	URIC ACID (mg/dL)	ALBUMIN (g/dL)	BILIRUBIN (mg/dL)	GLOBULIN (g/dL)	A/G RATIO	TOTAL PROTEIN (g/dL)
15	Control	46.81 ± 5.58 ^abcd	0.30 ± 0.02 ^d	1.65 ± 0.25 ^b	4.30 ± 1.00 ^bcd	0.25 ± 0.04 ^c	3.65 ± 0.37 ^bc	1.01 ± 0.12 ^b	8.36 ± 0.44 ^b
	LT	42.0 ± 17.22 ^bcd	0.46 ± 0.05 ^cd	0.42 ± 0.17 ^cd	3.42 ± 0.68 ^def	0.15 ± 0.08 ^c	3.60 ± 0.33 ^bc	1.12 ± 0.04 ^ab	7.50 ± 0.48 ^abcd
	HT	59.25 ± 5.68 ^abc	0.71 ± 0.37 ^abcd	0.75 ± 0.26 ^bcd	3.22 ± 0.82 ^def	0.17 ± 0.04 ^c	2.90 ± 0.38 ^c	1.43 ± 0.33 ^ab	7.72 ± 0.73 ^abc
	LC	40.5 ± 15.41 ^bcd	0.60 ± 0.18 ^bcd	0.22 ± 0.10 ^d	4.56 ± 0.24 ^bc	0.15 ± 0.05 ^c	4.05 ± 0.61 ^b	1.09 ± 0.12 ^b	8.35 ± 0.86 ^ab
	HC	29.25 ± 3.79 ^d	0.43 ± 0.05 ^cd	0.55 ± 0.17 ^bcd	3.46 ± 0.46 ^cdef	0.10 ± 0.00 ^c	3.92 ± 0.52 ^b	1.47 ± 0.68 ^ab	7.30 ± 0.97 ^abcd
30	Control	45.66 ± 10.21 ^abcd	0.92 ± 0.47 ^abc	3.07 ± 0.86 ^a	4.30 ± 1.00 ^bcde	0.67 ± 0.29 ^ab	4.22 ± 0.44 ^b	1.18 ± 0.40 ^ab	8.26 ± 1.28 ^ab
	LT	61.25 ± 10.21 ^ab	1.13 ± 0.31 ^ab	1.30 ± 0.93 ^bcd	3.41 ± 0.49 ^def	0.37 ± 0.31 ^bc	3.48 ± 0.44 ^bc	1.30 ± 0.23 ^ab	8.65 ± 0.63 ^a
	HT	59.41 ± 14.15 ^abc	0.39 ± 0.04 ^cd	1.60 ± 0.57 ^b	3.21 ± 0.87 ^ef	0.17 ± 0.10 ^c	3.28 ± .88 ^bc	1.75 ± 0.22 ^a	6.48 ± 0.99 ^cd
	LC	33.00 ± 9.82 ^d	1.18 ± 0.48 ^a	1.03 ± 0.57 ^bcd	4.10 ± 0.23 ^bcde	0.25 ± 0.05 ^c	3.55 ± 0.70 ^bc	1.22 ± 0.23 ^ab	6.83 ± 0.60 ^cd
	HC	34.25 ± 6.19 ^cd	0.68 ± 0.48 ^abcd	1.15 ± 0.59 ^bcd	4.50 ± 0.20 ^bcd	0.20 ± 0.06 ^c	3.37 ± .42 ^bc	1.53 ± 0.39 ^ab	6.60 ± 0.31 ^cd
45	Control	48.71 ± 12.48 ^abcd	0.32 ± 0.19 ^d	3.22 ± 1.11 ^a	5.00 ± 0.36 ^ab	0.73 ± 0.36 ^a	4.15 ± 0.37 ^b	1.09 ± 0.13 ^b	8.50 ± 0.73 ^a
	LT	63.75 ± 17.02 ^ab	0.55 ± 0.15 ^cd	1.42 ± 0.35 ^bc	3.20 ± 0.18 ^ef	0.17 ± 0.04 ^c	3.58 ± 0.31 ^bc	1.31 ± 0.11 ^ab	7.00 ± 0.55 ^bcd
	HT	70.00 ± 12.55 ^a	0.79 ± 0.15 ^abcd	1.39 ± 0.36 ^bc	2.98 ± 0.26 ^f	0.17 ± 0.04 ^c	3.45 ± 0.33 ^bc	1.27 ± 0.52 ^ab	6.20 ± 0.17 ^d
	LC	48.75 ± 7.13 ^abcd	0.55 ± 0.12 ^cd	0.35 ± 0.10 ^cd	6.02 ± 0.45 ^a	0.20 ± 0.07 ^c	3.45 ± 0.33 ^bc	1.14 ± 0.07 ^ab	6.72 ± 0.57 ^cd
	HC	70.25 ± 17.69 ^a	0.52 ± 0.10 ^cd	1.00 ± 0.50 ^bcd	3.63 ± 0.69 ^cdef	0.22 ± 0.04 ^c	5.25 ± 034 ^a	1.44 ± 0.36 ^ab	6.43 ± 0.27 ^cd

Days	Food color Exposure	GLUCOSE (mg/dL)	CHOLESTROL (mg/dL)	TRIGLYCERIDES (mg/dL)
15	Control	84.25 ± 6.91^cde	42.75 ± 6.95 ^ab	103.83 ± 87.08 ^bcd
	LT	83.00 ± 9.44 ^cde	28.25 ± 6.72 ^c	41.66 ± 6.40 ^ef
	HT	67.75 ± 14.42 ^ef	30.00 ± 5.10 ^abc	36.33 ± 24.99 ^ef
	LC	71.75 ± 5.55 ^def	30.00 ± 3.86 ^bc	16.50 ± 4.84 ^f
	HC	70.95 ± 10.23 ^def	31.25 ± 6.58 ^bc	21.50 ± 2.73 ^ef
30	Control	93.00 ± 14.51 ^bcd	50.00 ± 9.08 ^a	94.67 ± 40.66 ^cdef
	LT	89.25 ± 8.02 ^cde	39.78 ± 2.27 ^abc	80.17 ± 42.57 ^def
	HT	56.75 ± 19.58 ^f	37.85 ± 5.25 ^abc	81.00 ± 44.06 ^def
	LC	85.00 ± 4.52 ^cde	36.75 ± 6.55 ^abc	41.50 ± 12.011 ^ef
	HC	73.25 ± 7.64 ^def	40.63 ± 7.25 ^abc	57.83 ± 13.32 ^def
45	Control	93.00 ± 4.33 ^bcd	42.50 ± 11.91 ^ab	131.83 ± 38.54 ^bcd
	LT	104.00 ± 19.06 ^abc	33.75 ± 3.51 ^bc	188.33 ± 42.01 ^ab
	HT	122.50 ± 18.22 ^a	30.00 ± 4.34 ^bc	229.50 ± 57.80 ^a
	LC	76.75 ± 10.10 ^def	28.50 ± 10.17 ^c	179.33 ± 35.36 ^abc
	HC	115.50 ± 10.38 ^ab	30.00 ± 6.90 ^bc	244.16 ± 66.89 ^a

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[1] *Corresponding author: gs45177@student.upm.edu.my