Metabolomics of Fuzi-Gancao in CCl4 induced acute liver injury and its regulatory effect on bile acid profile in rats

doi:10.3748/wjg.v27.i40.6888

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World Journal of Gastroenterology

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Basic Study

World J Gastroenterol. Oct 28, 2021; 27(40): 6888-6907
Published online Oct 28, 2021. doi: 10.3748/wjg.v27.i40.6888

Table 1 Alanine aminotransferase and aspartate aminotransferase in serum of rats (n = 8)

Group	ALT (U/L)	AST (U/L)
Normal control	43.6 ± 8.7	131.6 ± 25.1
Model control	187.3 ± 29.6^a	285.1 ± 31.5^a
Positive control	86.7 ± 13.7^c	184.2 ± 37.8^c
F-G (15 g/kg)	151.2 ± 24.3	246.2 ± 39.8
F-G (30 g/kg)	119.4 ± 19.5^c	224.0 ± 34.1^b

^aP < 0.01 for the model control group and normal control group.

^bP < 0.05.

^cP < 0.01 for each treatment group and model control group, respectively. F-G: Fuzi-Gancao; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

Table 2 Calibration curve, R², and concentration range of nine metabolites

Compound	Standard curve	R²	Range (ng/mL)
L-Leu	Y = 0.0627X - 4.9797	0.9952	100-10000
5-HT	Y = 0.0385X - 0.0269	0.9939	0.8-80
N-phe	Y = 0.0229X - 4.9120	0.9958	250-25000
L-Try	Y = 0.0048X - 2.4673	0.9926	600-60000
L-Kyn	Y = 0.0331X + 0.0123	0.9960	5-500
5-HTP	Y = 0.0492X - 0.0001	0.9983	0.2-20
CA	Y = 0.00004X - 0.0010	0.9959	40-4000
GSH	Y = 0.0032X - 0.2362	0.9975	150-20000
GSSG	Y = 0.0001X + 0.0010	0.9949	50-10000

L-Leu: L-leucine; 5-HT: 5-Hydroxytryptamine; N-phe: N-phenylacetylglycine; L-Try: L-tryptophan; L-Kyn: L-kynurenine; 5-HTP: 5-hydroxytryptophan; CA: Cholic acid; GSH: Glutathione; GSSG: Glutathione oxidized.

Table 3 Accuracy and precision of the developed ultra performance liquid chromatography-tandem mass spectrometry method (n = 6)

Ingredient	Concentration (ng/mL)	Accuracy %	Precision %	Repeatability %	Stability %
L-Leu	750	100.35 ± 3.20	3.19	3.69	0.41
	1000	105.20 ± 6.45	6.13	6.91	1.69
	5000	95.53 ± 4.55	4.76	4.98	1.85
5-HT	6	99.78 ± 8.61	8.63	8.10	2.78
	8	101.78 ± 4.32	4.24	2.87	3.80
	40	101.76 ± 4.50	4.42	4.24	2.14
N-phe	1800	100.29 ± 4.32	4.31	4.72	3.07
	2500	98.97 ± 4.13	4.17	3.71	3.18
	12500	92.24 ± 5.12	5.55	5.95	1.09
L-Try	4500	97.33 ± 3.25	3.34	3.75	0.71
	6000	95.45 ± 4.12	4.32	4.22	3.35
	30000	94.89 ± 4.77	5.03	4.22	4.00
L-Kyn	37	99.61 ± 4.66	4.68	5.00	2.98
	50	102.43 ± 4.15	4.05	4.41	1.73
	250	99.76 ± 4.11	4.12	4.15	2.09
5-HTP	300	102.45 ± 4.51	4.40	3.98	3.65
	400	95.20 ± 8.80	9.24	7.50	6.16
	2000	101.77 ± 8.11	7.97	7.89	7.36
CA	1.5	99.88 ± 3.70	3.70	3.83	1.47
	2	97.00 ± 3.19	3.29	2.97	2.47
	10	96.76 ± 5.30	5.48	4.17	5.22
GSH	1500	101.91 ± 4.65	4.56	5.19	1.45
	2000	96.05 ± 3.98	4.14	4.63	0.89
	10000	97.51 ± 5.88	6.03	4.45	2.45
GSSG	750	99.64 ± 3.97	3.98	4.43	1.56
	1000	100.8 ± 6.45	6.40	7.23	4.89
	5000	98.39 ± 4.54	4.61	3.90	3.80

Table 4 Recovery of the developed ultra performance liquid chromatography-tandem mass spectrometry method (n = 6)

Ingredient	Baseline (ng/mL)	Spiked (ng/mL)	Recovery %
L-Leu	1000	250	92.53 ± 7.13
		500	93.42 ± 11.52
		750	87.53 ± 9.59
5-HT	8	2	85.37 ± 9.79
		4	90.52 ± 13.47
		6	93.05 ± 10.16
N-phe	2500	625	94.54 ± 12.15
		1250	89.46 ± 6.54
		1875	86.72 ± 8.19
L-Try	6000	1500	93.04 ± 13.23
		3000	85.42 ± 7.12
		4500	91.16 ± 12.25
L-Kyn	50	12.5	90.48 ± 6.18
		25	85.30 ± 10.42
		37.5	86.20 ± 6.13
5-HTP	2	0.5	91.49 ± 5.36
		1	94.64 ± 7.46
		1.5	93.58 ± 5.15
CA	400	100	91.83 ± 12.12
		200	93.29 ± 8.62
		300	90.22 ± 7.28
GSH	1000	250	88.17 ± 6.33
		500	91.16 ± 6.15
		750	90.85 ± 9.81
GSSG	2000	500	91.15 ± 10.08
		1000	93.92 ± 12.15
		1500	89.27 ± 10.92

Table 5 Serum metabolites in CCl₄-induced acute liver injury in rats (n = 8)

Metabolite	Model group	Control group	FG group
L-leucine	431.1 ± 54.32	331.4 ± 48.78^d	393.5 ± 53.83
N-phenylacetylglycine	5.350 ± 0.938	1.417 ± 0.351^d	2.915 ± 0.496^b^,^d
L-tryptophan	17.96 ± 3.014	14.63 ± 2.724^c	17.05 ± 2.679^a
L-kynurenine	6.270 ± 0.729	3.948 ± 0.450^d	6.646 ± 1.214^b
Cholic acid	1.342 ± 0.230	0.047 ± 0.004^d	1.001 ± 0.124^b^,^d
GSH	2.562 ± 0.433	0.991 ± 0.285^d	2.317 ± 0.632^b
GSSG	0.108 ± 0.016	0.021 ± 0.002^d	0.051 ± 0.009^b^,^d
lactate	257.8 ± 32.57	230.8 ± 37.30	185.3 ± 31.52^b^,^d
Choline	3.548 ± 0.539	2.639 ± 0.451	2.802 ± 0.413
(E)-butenedioic acid	163.6 ± 18.57	79.48 ± 8.625^d	87.82 ± 10.55^d
Hypoxanthine	0.052 ± 0.003	0.021 ± 0.002^d	0.022 ± 0.003^d
Carnitine	23.55 ± 5.024	13.52 ± 1.370^d	20.05 ± 1.323^b
Phenylalanine	289.5 ± 37.51	165.6 ± 23.76^d	261.4 ± 32.33^b
Uric acid	4.523 ± 0.863	2.769 ± 0.474^d	2.927 ± 0.488^d
Hippuric acid	95.58 ± 16.47	65.78 ± 13.92^d	76.53 ± 16.92^d
Citric acid	11.86 ± 1.531	15.62 ± 2.437^d	13.51 ± 2.002
Pantothenic acid	7.852 ± 0.953	4.602 ± 0.560^d	4.831 ± 0.693^d
Uridine	19.37 ± 2.772	6.816 ± 0.829^d	17.53 ± 2.503^b
Glucosamine-1-phosphate	0.025 ± 0.005	0.017 ± 0.004^c	0.020 ± 0.004
8-OH-dG	0.008 ± 0.001	0.007 ± 0.001^c	0.007 ± 0.001^c
C16:1 Lyso PC	3.215 ± 0.491	4.813 ± 0.503^d	6.293 ± 1.337^b^,^d
C16:0 Lyso PC	13.37 ± 2.434	16.62 ± 3.027	18.39 ± 3.316^c
C18:1 Lyso PC	16.837 ± 2.392	23.699 ± 4.427^d	26.647 ± 4.339^a^,^d
C18:0 LysoPC	11.42 ± 1.130	18.332 ± 1.833^d	16.66 ± 2.603^a^,^d

^aP < 0.05.

^bP < 0.01 compared with the negative control group.

^cP < 0.05.

^dP < 0.01 compared with the model control group. GSH: Glutathione; GSSG: Glutathione oxidized.

Table 6 Variable importance in the projection values of serum metabolites and effects of CCl₄ on their changes (model group vs negative group)

Var ID (primary)	M1-VIP	Variation trend
GSSG	1.19631	↑↑
Cholic acid	1.18964	↑↑
Uridine	1.18609	↑↑
N-phenylacetylglycine	1.16986	↑↑
Hypoxanthine	1.13469	↑↑
(E)-butenedioic acid	1.1299	↑↑
GSH	1.12862	↑↑
C18:0LPC	1.12325	↓↓
Pantothenic acid	1.08663	↑↑
Carnitine	1.08264	↑↑
Phenylalanine	1.07794	↑↑
Hippuric acid	1.06083	↑↑
C16:1LPC	1.05866	↓↓
Uric acid	1.01621	↑↑
C18:1LPC	0.943612	↓↓
L-kynurenine	0.919692	↑
L-leucine	0.90164	↑↑
Glucosamine-1-phosphate	0.893372	↑
Citric acid	0.866932	↓↓
L-tryptophan	0.842517	↑
8-OH-dG	0.711595	↑
Lactate	0.704198	NS
Choline	0.639509	NS
C16:0LPC	0.514772	NS

↑ represents a P value ≤ 0.05 for the increase in mean content; ↑↑ represents a P value ≤ 0.01 for the increase in mean content; ↓ represents a P value ≤ 0.05 for the mean content decrease; ↓↓ represents a P value ≤ 0.01 for the mean content decrease; NS: The mean concentrations of metabolites in the model group were not statistically significant; GSSG: Glutathione oxidized; GSH: Glutathione; VIP: Variable importance in the projection.

Table 7 Variable importance in the projection values of serum metabolites and effects of Fuzi-Gancao on their changes (Fuzi-Gancao group vs model group)

Var ID (primary)	M1-VIP	Variation trend
Hypoxanthine	1.37706	↓↓
GSSG	1.36516	↓↓
Pantothenic acid	1.28548	↓↓
C18:1LPC	1.27880	↑↑
C16:1LPC	1.25881	↑↑
N-phenylacetylglycine	1.22022	↓↓
Lactate	1.21835	↓↓
Hippuric acid	1.21598	↓↓
Uric acid	1.21291	↓↓
(E)-butenedioic acid	1.21107	↓↓
Cholic acid	1.17091	↓↓
C18:0LPC	1.15078	↑↑
C16:0LPC	1.00088	↑
Choline	0.906949	NS
L-kynurenine	0.893225	↑↑
Uridine	0.810356	NS
8-OH-dG	0.777736	NS
Citric acid	0.633654	NS
GSH	0.512313	NS
L-leucine	0.497685	NS
Carnitine	0.447434	NS
Phenylalanine	0.427897	NS
Glucosamine-1-phosphate	0.312245	NS
L-tryptophan	0.0800812	NS

↑ represents a P value ≤ 0.05 for the increase in mean content; ↑↑ represents a P value ≤ 0.01 for the increase in mean content; ↓ represents a P value ≤ 0.05 for the mean content decrease; ↓↓ represents a P value ≤ 0.01 for the mean content decrease; NS: No statistically significant change in the mean concentration of metabolites in the Fuzi-Gancao group; GSSG: Glutathione oxidized; GSH: Glutathione; VIP: Variable importance in the projection.

Table 8 Standard curve, R², and concentration range of 11 bile acids in serum

Compound	Standard curve	R²	Range (ng/mL)
CDCA	Y = 0.0235X - 0.0056	0.9981	2.5-1000
UDCA	Y = 0.0013X + 0.2156	0.9972	1.25-500
GCA	Y = 0.0164X + 1.1577	0.9956	1.25-500
GDCA	Y = 0.0025X + 0.833	0.9982	1.25-500
TCA	Y = 0.0198X - 0.0113	0.9953	1.25-500
TUDCA	Y = 0.0332X + 0.0023	0.9985	2.5-1000
TCDCA	Y = 0.0306X + 0.00422	0.9923	2.5-1000
GCDCA	Y = 0.0133X - 0.00324	0.9915	2.5-1000
GLCA	Y = 0.0018X + 0.00242	0.9931	2.5-1000
DCA	Y = 0.0076X + 0.00614	0.9956	2.5-1000
CA	Y = 0.0916X - 0.00465	0.9987	2.5-1000

CDCA: Chenodeoxycholic acid; UDCA: Ursodeoxycholic acid; GCA: Glycocholic acid; GDCA: Glycochenodeoxycholic acid; TCA: Taurocholic acid; TUDCA: Taurocholic acid; GCDCA: Glycochenodeoxycholic acid; GLCA: Glycinic acid; DCA: Deoxycholic acid; CA: Cholic acid.

Table 9 Precision, accuracy, and recovery of bile acids in serum samples (n = 6, mean ± SD)

Compound	Concentration (ng/mL)	Recovery (%)	Intraday		Interday
Compound	Concentration (ng/mL)	Recovery (%)	Accuracy (%)	Precision (%)	Accuracy (%)	Precision (%)
CA	2.5	90.12 ± 2.16	96.44 ± 2.05	4.26	91.83 ± 3.32	5.03
	500	88.21 ± 5.34	91.65 ± 4.14	3.60	94.42 ± 4.55	4.36
	2000	85.60 ± 7.22	92.11 ± 4.11	5.42	94.54 ± 8.35	6.00
GLCA	1.25	91.52 ± 5.51	92.13 ± 2.97	5.71	98.48 ± 2.22	3.75
	250	86.74 ± 7.12	87.31 ± 1.92	4.23	93.42 ± 3.67	5.40
	1000	90.48 ± 3.31	91.55 ± 5.31	6.13	89.24 ± 4.63	4.57
GDCA	1.25	88.54 ± 2.93	90.67 ± 6.87	6.22	88.55 ± 3.87	3.28
	250	98.43 ± 1.26	97.24 ± 7.86	6.38	101.53 ± 2.54	2.76
	1000	95.56 ± 4.01	89.34 ± 2.65	2.81	89.23 ± 6.25	6.16
GCDCA	1.25	96.02 ± 3.11	93.24 ± 3.78	6.30	89.32 ± 4.32	3.19
	250	90.13 ± 3.42	97.43 ± 2.82	5.03	88.54 ± 4.75	2.10
	1000	89.32 ± 3.13	86.53 ± 4.27	2.96	89.52 ± 3.63	3.28
GCA	1.25	80.87 ± 3.83	99.31 ± 5.19	4.18	89.42 ± 1.71	2.32
	250	91.43 ± 6.38	101.33 ± 2.23	2.59	112.53 ± 5.76	2.29
	1000	92.43 ± 2.14	88.76 ± 1.46	1.20	89.16 ± 3.46	3.09
TCA	2.5	84.77 ± 2.38	103.55 ± 1.27	1.50	98.24 ± 2.14	5.51
	500	86.92 ± 4.96	89.04 ± 6.42	5.53	95.33 ± 2.86	3.82
	2000	81.34 ± 1.84	88.57 ± 4.85	4.77	97.81 ± 3.08	4.50
CDCA	1.25	80.33 ± 3.23	94.33 ± 4.62	4.39	90.49 ± 4.83	5.18
	250	86.34 ± 2.45	92.33 ± 4.93	3.70	91.49 ± 7.39	4.49
	1000	84.62 ± 3.21	92.33 ± 6.03	5.58	92.77 ± 4.83	6.18
DCA	1.25	95.54 ± 2.17	94.67 ± 4.93	5.88	104.21 ± 7.22	3.87
	250	82.56 ± 4.32	106.33 ± 7.37	4.36	99.31 ± 5.66	5.57
	1000	94.45 ± 5.34	101.33 ± 5.77	6.31	89.83 ± 4.53	4.71
UDCA	1.25	83.32 ± 1.43	91.67 ± 4.62	6.41	93.43 ± 4.63	3.38
	250	88.13 ± 5.94	95.33 ± 4.73	6.57	92.12 ± 4.49	2.84
	1000	85.26 ± 3.01	94 ± 4.58	2.90	91.79 ± 5.74	6.35
TCDCA	1.25	86.04 ± 3.36	93.67 ± 5.86	6.49	92.77 ± 4.83	3.28
	250	87.98 ± 3.11	94.67 ± 4.93	5.18	89.83 ± 4.53	2.17
	1000	83.72 ± 2.19	91.67 ± 4.62	3.05	92.77 ± 7.36	3.38
TUDCA	1.25	87.17 ± 4.61	94.67 ± 7.51	4.31	96.69 ± 9.82	2.39
	250	87.41 ± 4.09	98.67 ± 10.02	2.66	98.33 ± 8.33	2.36
	1000	99.06 ± 3.56	100.33 ± 8.5	1.23	92.45 ± 4.53	3.18

Table 10 Stability of bile acid standards in serum samples (n = 6, mean ± SD)

Compound	Concentration (ng/mL)	Concentration (mean ± SD)
Compound	Concentration (ng/mL)	Initial	Freeze-thaw	Short-term	Long-term	Post-preparation
CA	2.5	2.35 ± 0.17	2.3 ± 0.16	2.56 ± 0.18	2.33 ± 0.17	2.52 ± 0.08
	500	495.43 ± 12.15	485.52 ± 11.91	538.93 ± 13.22	511.98 ± 12.56	505.09 ± 5.54
	2000	2080.38 ± 101	2038.77 ± 98.98	2263.03 ± 109.87	2149.88 ± 104.38	2194.52 ± 99.16
GLCA	1.25	1.24 ± 0.14	1.22 ± 0.13	1.17 ± 0.15	1.21 ± 0.14	1.25 ± 0.14
	250	247.1 ± 10.76	242.15 ± 10.55	258.79 ± 11.71	255.35 ± 11.12	250.24 ± 10.9
	1000	979.04 ± 40.32	959.46 ± 39.51	985 ± 43.86	1011.75 ± 41.66	991.52 ± 40.83
GDCA	1.25	1.27 ± 0.05	1.24 ± 0.05	1.24 ± 0.06	1.19 ± 0.06	1.26 ± 0.08
	250	240.43 ± 3.98	235.62 ± 3.9	261.54 ± 4.33	248.46 ± 4.12	249.32 ± 5.42
	1000	975.71 ± 45.93	956.19 ± 45.01	1061.38 ± 49.96	1008.31 ± 47.46	1035.03 ± 14.83
GCDCA	1.25	1.27 ± 0.06	1.23 ± 0.08	1.36 ± 0.08	1.21 ± 0.09	1.21 ± 0.08
	250	243.49 ± 4.03	242.1 ± 19.01	237.25 ± 18.63	263.35 ± 20.68	250.18 ± 19.64
	1000	988.14 ± 46.51	1047.04 ± 49.29	1026.1 ± 48.31	1108.97 ± 53.62	1082.02 ± 50.94
GCA	1.25	1.19 ± 0.03	1.27 ± 0.03	1.27 ± 0.03	1.22 ± 0.03	1.21 ± 0.04
	250	237.1 ± 1.93	232.35 ± 1.9	257.91 ± 2.1	245.02 ± 2	244.16 ± 1.88
	1000	982.38 ± 30.18	962.73 ± 29.58	1068.63 ± 32.83	1015.2 ± 31.19	1014.36 ± 44.06
TCA	2.5	2.25 ± 0.17	2.21 ± 0.16	2.45 ± 0.18	2.33 ± 0.17	2.42 ± 0.08
	500	492.1 ± 28.22	482.25 ± 27.65	535.3 ± 30.69	508.54 ± 29.16	494.76 ± 23.69
	2000	2013.71 ± 105.21	1973.43 ± 103.1	2190.51 ± 114.44	2080.99 ± 108.72	2142.85 ± 26.09
CDCA	1.25	1.19 ± 0.1	1.28 ± 0.1	1.23 ± 0.11	1.24 ± 0.1	1.22 ± 0.08
	250	232.1 ± 6.9	227.45 ± 6.77	252.47 ± 7.51	239.85 ± 7.14	236.41 ± 5.54
	1000	1010.38 ± 39.44	990.17 ± 38.65	1099.09 ± 42.91	1044.13 ± 40.76	1057.77 ± 46.98
DCA	1.25	1.13 ± 0.03	1.15 ± 0.06	1.18 ± 0.13	1.23 ± 0.13	1.24 ± 0.14
	250	236.33 ± 13.65	229.67 ± 21.36	233 ± 10.15	244.65 ± 10.66	247.1 ± 10.76
	1000	967.67 ± 15.82	947.67 ± 106.82	1031 ± 115.66	982.55 ± 53.41	979.04 ± 40.32
UDCA	1.25	1.22 ± 0.06	1.19 ± 0.06	1.23 ± 0.07	1.26 ± 0.07	1.28 ± 0.07
	250	252.1 ± 6.9	247.05 ± 6.77	244.23 ± 7.51	250.52 ± 7.14	257.07 ± 5.54
	1000	931.71 ± 85.92	971.43 ± 120.15	958.15 ± 108.46	940.89 ± 109.04	1061.11 ± 99.16
TCDCA	1.25	1.22 ± 0.06	1.25 ± 0.06	1.28 ± 0.07	1.22 ± 0.06	1.19 ± 0.06
	250	251.76 ± 3.11	246.73 ± 3.05	273.87 ± 3.39	260.17 ± 3.22	261.73 ± 2.5
	1000	1013.71 ± 69.97	993.43 ± 68.57	1102.71 ± 76.11	1047.58 ± 72.31	1057.77 ± 99.16
TUDCA	1.25	1.22 ± 0.02	1.2 ± 0.02	1.23 ± 0.03	1.26 ± 0.02	1.27 ± 0.03
	250	249.1 ± 6.47	244.11 ± 6.34	270.97 ± 7.04	257.42 ± 6.68	259.14 ± 8.46
	1000	977.04 ± 18.84	957.5 ± 18.46	1062.83 ± 20.49	1009.69 ± 19.47	1006.1 ± 26.09

Table 11 Ratio of bile acids in each experimental group to corresponding composition of negative control group

Bile acid	Negative	Model	Positive	FGL	FGM	FGH
CA	1.00	2.57	1.38	2.16	1.92	1.40
GLCA	1.00	1.61	1.08	1.45	1.17	1.15
GDCA	1.00	4.45	2.43	3.53	2.78	2.14
GCDCA	1.00	1.74	1.18	1.85	1.28	1.09
GCA	1.00	1.30	1.25	1.22	1.16	1.12
TCA	1.00	1.99	1.11	1.90	1.37	1.31
CDCA	1.00	4.12	2.08	3.76	2.90	2.03
UDCA	1.00	5.68	3.17	4.87	3.72	3.62
TUDCA	1.00	6.00	3.08	4.73	4.13	3.87
TCDCA	1.00	3.24	1.57	2.92	2.63	2.03
DCA	1.00	5.16	2.40	4.48	3.84	2.95

Citation: Wang MF, Zhao SS, Thapa DM, Song YL, Xiang Z. Metabolomics of Fuzi-Gancao in CCl₄induced acute liver injury and its regulatory effect on bile acid profile in rats. World J Gastroenterol 2021; 27(40): 6888-6907
URL: https://www.wjgnet.com/1007-9327/full/v27/i40/6888.htm
DOI: https://dx.doi.org/10.3748/wjg.v27.i40.6888