Preventive Effect of Flavonoid Extract from the Peel of Gonggan (Citrus reticulata Blanco Var. Gonggan) on CCl₄-Induced Acute Liver Injury in Mice

Figures & data

Table 1 Sequences of Primers Used in This Study

Gene Name	Sequence
SOD1	Forward: 5'-AACCAGTTGTGTTGTCAGGAC-3'
	Reverse: 5'-CCACCATGTTTCTTAGAGTGAGG-3'
SOD2	Forward: 5'-CAGACCTGCCTTACGACTATGG-3'
	Reverse: 5'-CTCGGTGGCGTTGAGATTGTT-3'
CAT	Forward: 5'-AGCGACCAGATGAAGCAGTG-3'
	Reverse: 5'-TCCGCTCTCTGTCAAAGTGTG-3'
γ-GCS	Forward: 5'-CTTCCCTCCCTTCGGATCG-3'
	Reverse: 5'-GTCCACAGAGATGCAGTGAAA-3'
GSH-Px	Forward: 5'-AATGTCGCGTCTCTCTGAGG-3'
	Reverse: 5'-TCCGAACTGATTGCACGGG-3'
Nrf2	Forward: 5'-CTGAACTCCTGGACGGGACTA-3'
	Reverse: 5'-CGGTGGGTCTCCGTAAATGG-3'
IL-6	Forward: 5'-CTGCAAGAGACTTCCATCCAG-3'
	Reverse: 5'-AGTGGTATAGACAGGTCTGTTGG-3'
TNF-α	Forward: 5'-GAGGCCAAGCCCTGGTATG-3'
	Reverse: 5'-CGGGCCGATTGATCTCAGC-3'
GAPDH	Forward: 5'-TGGCCTTCCGTGTTCCTAC-3'
	Reverse: 5'-GAGTTGCTGTTGAAGTCGCA-3'

Abbreviations: SOD1, copper/zinc superoxide dismutase; SOD2, manganese superoxide dismutase; CAT, catalase; γ-GCS, γ-glutamylcysteine synthetase; GSH-Px, glutathione peroxidase; Nrf2, nuclear factor erythroid 2-related factor 2; IL-6, interleukin- 6; TNF-α, tumor necrosis factor-α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 1 Flavonoids in GPFE. The detection wavelength is 285 nm. (A) standard chromatograms; (B) flavonoids constituents of GPFE: 1. narirutin, 2. hesperidin, 3. nobiletin, 4. tangeretin, 5. 5-demethylnobiletin.

Figure 2 Histopathological observation of liver tissues sections in mice of the different groups after staining with hematoxylin and eosin (H&E).

Table 2 The Liver Injury Degree in Mice

Group	Liver Index (%)	ALT (U/L)	AST(U/L)
Normal	4.07 ± 0.10^a	34.1±7.4^a	12.6±2.1^a
Model	5.02 ± 0.29^b	162.9±11.9^b	40.2±2.6^b
Silymarin	4.58 ± 0.34^c	57.5±10.8^c	14.2±2.9^a
GPFE-H	4.67 ± 0.18^c	81.6±11.2^d	21.2±2.2^c
GPFE-L	4.88 ±0.22^bc	112.1±7.9^e	27.1±4.2^d

Notes: Values are presented as the mean ± standard deviation (n = 8/group). Silymarin, mice treated with 200 mg/kg silymarin; GPFE-H, mice treated with 300 mg/kg GPFE; GPFE-L, mice treated with 150 mg/kg GPFE. ^a–eMean values with different letters in the same column are significantly different (P < 0.05) based on Tukey’s test.

Table 3 Liver Tissue Levels of ALT, AST, SOD, CAT and MDA in Mice from Each Group

Group	SOD (U/mgprot)	CAT (U/mgprot)	MDA (nmol/mgprot)
Normal	650±26^a	6.73±0.42^a	0.88±0.11^a
Model	266±37^b	4.39±0.47^b	2.35±0.30^b
Silymarin	567±40^c	5.86±0.23^c	1.37±0.16^c
GPFE-H	595±15^c	6.04±0.26^c	1.31±0.19^c
GPFE-L	509±36^d	5.16±0.44^d	1.76±0.11^d

Notes: Values are presented as the mean ± standard deviation (n = 8/group). Silymarin, mice treated with 200 mg/kg silymarin; GPFE-H, mice treated with 300 mg/kg GPFE; GPFE-L, mice treated with 150 mg/kg GPFE. ^a–dMean values with different letters in the same column are significantly different (P < 0.05) based on Tukey’s test.

Table 4 Serum Levels of TNF-α, IL-1β, IL-6, and IFN-γ in Mice of Each Groups

Group	IL-1β (pg/mL)	IL-6 (pg/mL)	TNF-α (pg/mL)	IFN-γ (pg/mL)
Normal	47.3±3.7^a	89.1±10.1^a	333±13^a	426±16^a
Model	79.6±2.6^b	149.8±11.2^b	531±34^b	662±47^b
Silymarin	60.6±2.6^c	98.1±7.4^ac	373±14^c	459±39^ac
GPFE-H	52.8±4.3^d	97.0±6.8^ac	445±17^d	459±29^ac
GPFE-L	64.5±2.8^c	101.6±4.9^c	475±12^e	505±32^c

Notes: Values are presented as the mean ± standard deviation (n = 8/group). Silymarin, mice treated with 200 mg/kg silymarin; GPFE-H, mice treated with 300 mg/kg GPFE; GPFE-L, mice treated with 150 mg/kg GPFE. ^a–eMean values with different letters in the same column are significantly different (P < 0.05) based on Tukey’s test.

Figure 3 mRNA expression levels of SOD1, SOD2, CAT, γ-GCS, GSH-Px, Nrf2, IL-6 and TNF-α in liver tissues of the different groups were investigated by qPCR. The data are shown as mean ± SD (n = 8). ^a–eMean values with different letters are significant difference (p < 0.05) according to analysis of variance.