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No.	Metabolite	HMDB ID	Formula	Measured m/z	t_R /min	MS/MS	Adduct	P value
1	Sulfite	HMDB0000240	H₂O₃S	80.965 2	5.34	79.957 6, 71.014 7, 59.014 3	[M-H]^-	0.043 4	0.002 7
2	Glyceric acid	HMDB0000139	C₃H₆O₄	105.025 2	1.44	87.009 8, 79.957 3, 57.034 5	[M-H]^-	0.000 7	0.029 8
3	Acetylenedicarboxylic acid	HMDB0247933	C₄H₂O₄	112.985 6	11.22	96.960 6, 79.957 7, 68.996 9	[M-H]^-	0.001 0	0.000 6
4	3-Ethylphenol	HMDB0059873	C₈H₁₀O	121.064 9	6.10	119.050 3, 107.050 6, 79.958 0	[M-H]^-	0.002 7	0.039 7
5	Nicotinic acid	HMDB0001488	C₆H₅NO₂	122.973 7	0.95	112.981 8, 104.956 4, 92.927 9	[M-H]^-	0.005 0	0.013 5
6	3-Methylcatechol	HMDB0301753	C₇H₈O₂	123.045 5	8.49	105.035 8, 79.957 8	[M-H]^-	0.006 1	0.029 6
7	L-Aspartic acid	HMDB0000191	C₄H₇NO₄	132.066 4	8.84	150.078 0, 132.030 8, 119.050 3	[M-H]^-	0.012 2	0.008 7
8	Indole-3-carboxaldehyde	HMDB0029737	C₉H₇NO	144.046 8	6.28	122.047 6, 114.055 7, 74.025 9	[M-H]^-	0.024 3	0.043 3
9	Norepinephrine	HMDB0000216	C₈H₁₁NO₃	168.067 6	6.48	109.063 9, 80.965 6	[M-H]^-	0.005 6	0.000 4
10	Indoleacetic acid	HMDB0000197	C₁₀H₉NO₂	174.057 6	5.58	129.090 5, 106.045 2	[M-H]^-	0.004 2	0.029 4
11	L-Tyrosine	HMDB0000158	C₉H₁₁NO₃	180.066 6	5.36	124.076 6, 106.051 0, 80.965 5	[M-H]^-	0.020 1	0.000 4
12	D-Glucuronic acid	HMDB0000127	C₆H₁₀O₇	193.021 5	0.91	161.045 4, 111.022 7, 73.028 9	[M-H]^-	0.020 6	0.000 6
13	Tryptophan	HMDB0000929	C₁₁H₁₂N₂O₂	203.047 2	1.21	159.083 7, 134.055 1, 116.047 5	[M-H]^-	0.013 4	0.000 3
14	2-Methyl-2-[(1-oxo-2-propenyl) amino]-1-propanesulfonic acid	HMDB0031200	C₇H₁₃NO₄S	206.046 9	5.83	112.985 9, 96.960 7, 80.965 8	[M-H]^-	0.036 7	0.019 9
15	Undecanedioic acid	HMDB0000888	C₁₁H₂₀O₄	215.111 6	5.17	142.050 6, 112.041 0, 84.045 8	[M-H]^-	0.023 6	0.040 5
16	N-Acetyl-L-tyrosine	HMDB0000866	C₁₁H₁₃NO₄	222.078 0	5.37	163.044 2, 145.066 8, 109.040 3	[M-H]^-	0.006 7	0.000 5
17	6-Benzylaminopurine	HMDB0039238	C₁₂H₁₁N₅	224.092 8	6.15	197.083 9, 134.042 1, 107.036 4	[M-H]^-	0.033 0	0.045 8
18	γ-Glutamyl-S-methylcysteine	HMDB0031985	C₉H₁₆N₂O₅S	263.067 8	4.70	162.055 9, 118.032 2, 96.962 1	[M-H]^-	0.015 8	0.012 3
19	Adenosine	HMDB0000050	C₁₀H₁₃N₅O₄	275.107 6	4.32	180.994 7, 162.056 3, 118.035 1	[M-H]^-	0.025 0	0.049 4
20	Hexadecanedioic acid	HMDB0000672	C₁₆H₃₀O₄	285.206 3	10.57	267.218 0, 221.177 7, 199.694 7	[M-H]^-	0.008 2	0.014 8
21	Enterolactone	HMDB0006101	C₁₈H₁₈O₄	297.114 6	4.66	253.122 4, 121.065 0, 107.049 9	[M-H]^-	0.021 8	0.028 1
22	Fructosyl-lysine	HMDB0252495	C₁₂H₂₄N₂O₇	307.151 1	4.26	245.114 3, 215.103 7, 145.098 3	[M-H]^-	0.020 2	0.011 0
23	Glutamyl-tyrosine	HMDB0011741	C₁₄H₁₈N₂O₆	309.109 9	4.65	259.152 5, 215.093 7, 123.053 0	[M-H]^-	0.013 0	0.000 2
24	N-Glycolylneuraminate	HMDB0000833	C₁₁H₁₉NO₁₀	324.122 2	7.97	306.083 1, 159.029 3	[M-H]^-	0.010 1	0.003 2
25	2-Dodecylbenzenesulfonic acid	HMDB0031031	C₁₈H₃₀O₃S	325.184 4	14.35	260.376 7, 169.161 0, 197.029 3	[M-H]^-	0.016 8	0.021 8
26	Acetaminophen glucuronide	HMDB0010316	C₁₄H₁₇NO₈	326.090 4	0.91	272.039 6, 196.101 9, 101.061 1	[M-H]^-	0.031 8	0.006 3
27	Deoxycholate	HMDB0000626	C₂₄H₄₀O₄	391.286 5	10.66	327.268 9, 345.278 3	[M-H]^-	0.004 9	0.003 0
28	Glycoursodeoxycholic acid	HMDB0000708	C₂₄H₃₈O₃	433.296 8	13.98	373.274 8, 269.191 2	[M+HAc-H]^-	0.030 6	0.022 9
29	Cholic acid	HMDB0000619	C₂₄H₄₀O₅	815.562 2	8.30	429.237 6, 403.250 3, 96.960 7	[2M-H]^-	0.021 0	0.041 4
30	L-2, 3-Diaminopropionic acid	HMDB0002006	C₃H₈N₂O₂	105.070 7	10.93	95.084 9, 81.069 4, 55.054 0	[M+H]⁺	0.029 8	0.025 7
31	Proline	HMDB0000162	C₅H₉NO₂	116.067 8	7.83	110.070 4, 84.078 5, 70.063 4	[M+H]⁺	0.014 0	0.020 3
32	Isocaproic acid	HMDB0000689	C₆H₁₂O₂	117.091 7	17.91	107.072 7, 91.055 0, 77.039 6	[M+H]⁺	0.009 1	0.044 6
33	L-Valine	HMDB0000883	C₅H₁₁NO₂	118.090 6	0.84	106.066 0, 94.065 8, 77.039 5	[M+H]⁺	0.022 5	0.014 1
34	Leucinic acid	HMDB0000665	C₆H₁₂O₃	115.076 4	17.17	97.062 4, 87.045 6, 59.053 3	[M+H-H₂O]⁺	0.003 2	0.037 1
35	Tyramine	HMDB0000306	C₈H₁₁NO	121.061 2	10.09	115.074 3, 87.045 0, 59.051 4	[M+H]⁺	0.001 7	0.027 3
36	4-Acetylbutyric acid	HMDB0061881	C₆H₁₀O₃	131.084 5	14.46	105.068 6, 93.068 8, 81.068 9	[M+H]⁺	0.039 2	0.040 4
37	Ornithine	HMDB0000214	C₅H₁₂N₂O₂	133.098 0	14.28	109.099 0, 81.068 6, 59.053 0	[M+H]⁺	0.013 1	0.004 0
38	Phenylethanolamine	HMDB0062626	C₈H₁₂NO	138.092 3	4.08	122.096 5, 105.630 1, 77.108 2	[M+H-H₂O]⁺	0.028 4	0.003 0
39	5-Hydroxylysine	HMDB0000450	C₆H₁₄N₂O₃	145.101 7	9.32	105.068 4, 81.069 0, 67.053 6	[M+H]⁺	0.049 5	0.044 7
40	L-Glutamine	HMDB0000148	C₅H₉NO₄	148.076 6	7.48	112.894 1, 142.937 0	[M+H]⁺	0.047 8	0.001 0
41	Indole-3-acetaldehyde	HMDB0001190	C₁₀H₉NO	160.100 6	8.02	142.064 3, 118.065 2, 130.065 1	[M+H]⁺	0.038 3	0.038 0
42	Tryptamine	HMDB0000303	C₁₀H₁₂N₂	161.129 0	7.96	158.964 0, 121.087 0, 58.066 2	[M+H]⁺	0.047 7	0.002 1
43	Indole-3-carboxylic acid	HMDB0003320	C₉H₇NO₂	162.056 2	10.99	144.043 9, 55.053 9	[M+H]⁺	0.007 6	0.028 0
44	Phenacemide	HMDB0015253	C₉H₁₀N₂O₂	165.093 9	12.86	119.048 2, 91.054 1, 77.038 3	[M+H]⁺	0.031 1	0.027 4
45	Phenylalanine	HMDB0000159	C₉H₁₁NO₂	166.086 2	7.48	120.036 8, 103.025 9, 92.994 1	[M+H]⁺	0.014 7	0.035 0
46	Pyridoxamine	HMDB0001431	C₈H₁₂N₂O₂	169.101 3	8.32	95.078 4, 84.077 1, 70.062 1	[M+H]⁺	0.003 3	0.032 0
47	N-Methyltryptamine	HMDB0004370	C₁₁H₁₄N₂	175.133 9	17.17	144.066 5, 117.056 5, 91.043 7	[M+H]⁺	0.001 1	0.015 2
48	Geraniol	HMDB0005812	C₁₀H₁₈O	177.114 5	17.15	155.143 6, 137.133 0, 109.101 7	[M+H]⁺	0.021 4	0.021 4
49	Indole-3-propionic acid	HMDB0002302	C₁₁H₁₁NO₂	190.107 2	6.38	130.064 7, 172.077 3	[M+H]⁺	0.043 0	0.047 3
50	Metyrosine	HMDB0014903	C₁₀H₁₃NO₃	196.101 4	6.06	129.099 0, 95.081 0, 70.063 4	[M+H]⁺	0.049 9	0.010 4
51	Alanyl-leucine	HMDB0028691	C₉H₁₈N₂O₃	203.136 0	7.22	159.111 4, 119.081 0	[M+H-H₂O]⁺	0.010 0	0.001 8
52	Glutamyl-glycine	HMDB0028819	C₇H₁₂N₂O₅	205.157 8	13.15	187.062 7, 102.053 0, 85.025 9	[M+H]⁺	0.037 2	0.044 0
53	Dihydrojasmonic acid	HMDB0033601	C₁₂H₂₀O₃	213.150 1	17.16	163.105 7, 131.082 3, 107.081 9	[M+H]⁺	0.004 0	0.016 7
54	Melatonin	HMDB0001389	C₁₃H₁₆N₂O₂	233.165 9	17.91	191.180 4, 174.091 3	[M+H]⁺	0.002 3	0.043 5
55	Valyl-isoleucine	HMDB0029130	C₁₁H₂₂N₂O₃	231.159 6	17.15	187.063 1, 164.094 3, 119.081 6	[M+H]⁺	0.003 0	0.018 9

No.	Metabolite	HMDB ID	Formula	Measured m/z	t_R /min	MS/MS	Adduct	P value
1	Sulfite	HMDB0000240	H₂O₃S	80.965 2	5.34	79.957 6, 71.014 7, 59.014 3	[M-H]^-	0.043 4	0.002 7
2	Glyceric acid	HMDB0000139	C₃H₆O₄	105.025 2	1.44	87.009 8, 79.957 3, 57.034 5	[M-H]^-	0.000 7	0.029 8
3	Acetylenedicarboxylic acid	HMDB0247933	C₄H₂O₄	112.985 6	11.22	96.960 6, 79.957 7, 68.996 9	[M-H]^-	0.001 0	0.000 6
4	3-Ethylphenol	HMDB0059873	C₈H₁₀O	121.064 9	6.10	119.050 3, 107.050 6, 79.958 0	[M-H]^-	0.002 7	0.039 7
5	Nicotinic acid	HMDB0001488	C₆H₅NO₂	122.973 7	0.95	112.981 8, 104.956 4, 92.927 9	[M-H]^-	0.005 0	0.013 5
6	3-Methylcatechol	HMDB0301753	C₇H₈O₂	123.045 5	8.49	105.035 8, 79.957 8	[M-H]^-	0.006 1	0.029 6
7	L-Aspartic acid	HMDB0000191	C₄H₇NO₄	132.066 4	8.84	150.078 0, 132.030 8, 119.050 3	[M-H]^-	0.012 2	0.008 7
8	Indole-3-carboxaldehyde	HMDB0029737	C₉H₇NO	144.046 8	6.28	122.047 6, 114.055 7, 74.025 9	[M-H]^-	0.024 3	0.043 3
9	Norepinephrine	HMDB0000216	C₈H₁₁NO₃	168.067 6	6.48	109.063 9, 80.965 6	[M-H]^-	0.005 6	0.000 4
10	Indoleacetic acid	HMDB0000197	C₁₀H₉NO₂	174.057 6	5.58	129.090 5, 106.045 2	[M-H]^-	0.004 2	0.029 4
11	L-Tyrosine	HMDB0000158	C₉H₁₁NO₃	180.066 6	5.36	124.076 6, 106.051 0, 80.965 5	[M-H]^-	0.020 1	0.000 4
12	D-Glucuronic acid	HMDB0000127	C₆H₁₀O₇	193.021 5	0.91	161.045 4, 111.022 7, 73.028 9	[M-H]^-	0.020 6	0.000 6
13	Tryptophan	HMDB0000929	C₁₁H₁₂N₂O₂	203.047 2	1.21	159.083 7, 134.055 1, 116.047 5	[M-H]^-	0.013 4	0.000 3
14	2-Methyl-2-[(1-oxo-2-propenyl) amino]-1-propanesulfonic acid	HMDB0031200	C₇H₁₃NO₄S	206.046 9	5.83	112.985 9, 96.960 7, 80.965 8	[M-H]^-	0.036 7	0.019 9
15	Undecanedioic acid	HMDB0000888	C₁₁H₂₀O₄	215.111 6	5.17	142.050 6, 112.041 0, 84.045 8	[M-H]^-	0.023 6	0.040 5
16	N-Acetyl-L-tyrosine	HMDB0000866	C₁₁H₁₃NO₄	222.078 0	5.37	163.044 2, 145.066 8, 109.040 3	[M-H]^-	0.006 7	0.000 5
17	6-Benzylaminopurine	HMDB0039238	C₁₂H₁₁N₅	224.092 8	6.15	197.083 9, 134.042 1, 107.036 4	[M-H]^-	0.033 0	0.045 8
18	γ-Glutamyl-S-methylcysteine	HMDB0031985	C₉H₁₆N₂O₅S	263.067 8	4.70	162.055 9, 118.032 2, 96.962 1	[M-H]^-	0.015 8	0.012 3
19	Adenosine	HMDB0000050	C₁₀H₁₃N₅O₄	275.107 6	4.32	180.994 7, 162.056 3, 118.035 1	[M-H]^-	0.025 0	0.049 4
20	Hexadecanedioic acid	HMDB0000672	C₁₆H₃₀O₄	285.206 3	10.57	267.218 0, 221.177 7, 199.694 7	[M-H]^-	0.008 2	0.014 8
21	Enterolactone	HMDB0006101	C₁₈H₁₈O₄	297.114 6	4.66	253.122 4, 121.065 0, 107.049 9	[M-H]^-	0.021 8	0.028 1
22	Fructosyl-lysine	HMDB0252495	C₁₂H₂₄N₂O₇	307.151 1	4.26	245.114 3, 215.103 7, 145.098 3	[M-H]^-	0.020 2	0.011 0
23	Glutamyl-tyrosine	HMDB0011741	C₁₄H₁₈N₂O₆	309.109 9	4.65	259.152 5, 215.093 7, 123.053 0	[M-H]^-	0.013 0	0.000 2
24	N-Glycolylneuraminate	HMDB0000833	C₁₁H₁₉NO₁₀	324.122 2	7.97	306.083 1, 159.029 3	[M-H]^-	0.010 1	0.003 2
25	2-Dodecylbenzenesulfonic acid	HMDB0031031	C₁₈H₃₀O₃S	325.184 4	14.35	260.376 7, 169.161 0, 197.029 3	[M-H]^-	0.016 8	0.021 8
26	Acetaminophen glucuronide	HMDB0010316	C₁₄H₁₇NO₈	326.090 4	0.91	272.039 6, 196.101 9, 101.061 1	[M-H]^-	0.031 8	0.006 3
27	Deoxycholate	HMDB0000626	C₂₄H₄₀O₄	391.286 5	10.66	327.268 9, 345.278 3	[M-H]^-	0.004 9	0.003 0
28	Glycoursodeoxycholic acid	HMDB0000708	C₂₄H₃₈O₃	433.296 8	13.98	373.274 8, 269.191 2	[M+HAc-H]^-	0.030 6	0.022 9
29	Cholic acid	HMDB0000619	C₂₄H₄₀O₅	815.562 2	8.30	429.237 6, 403.250 3, 96.960 7	[2M-H]^-	0.021 0	0.041 4
30	L-2, 3-Diaminopropionic acid	HMDB0002006	C₃H₈N₂O₂	105.070 7	10.93	95.084 9, 81.069 4, 55.054 0	[M+H]⁺	0.029 8	0.025 7
31	Proline	HMDB0000162	C₅H₉NO₂	116.067 8	7.83	110.070 4, 84.078 5, 70.063 4	[M+H]⁺	0.014 0	0.020 3
32	Isocaproic acid	HMDB0000689	C₆H₁₂O₂	117.091 7	17.91	107.072 7, 91.055 0, 77.039 6	[M+H]⁺	0.009 1	0.044 6
33	L-Valine	HMDB0000883	C₅H₁₁NO₂	118.090 6	0.84	106.066 0, 94.065 8, 77.039 5	[M+H]⁺	0.022 5	0.014 1
34	Leucinic acid	HMDB0000665	C₆H₁₂O₃	115.076 4	17.17	97.062 4, 87.045 6, 59.053 3	[M+H-H₂O]⁺	0.003 2	0.037 1
35	Tyramine	HMDB0000306	C₈H₁₁NO	121.061 2	10.09	115.074 3, 87.045 0, 59.051 4	[M+H]⁺	0.001 7	0.027 3
36	4-Acetylbutyric acid	HMDB0061881	C₆H₁₀O₃	131.084 5	14.46	105.068 6, 93.068 8, 81.068 9	[M+H]⁺	0.039 2	0.040 4
37	Ornithine	HMDB0000214	C₅H₁₂N₂O₂	133.098 0	14.28	109.099 0, 81.068 6, 59.053 0	[M+H]⁺	0.013 1	0.004 0
38	Phenylethanolamine	HMDB0062626	C₈H₁₂NO	138.092 3	4.08	122.096 5, 105.630 1, 77.108 2	[M+H-H₂O]⁺	0.028 4	0.003 0
39	5-Hydroxylysine	HMDB0000450	C₆H₁₄N₂O₃	145.101 7	9.32	105.068 4, 81.069 0, 67.053 6	[M+H]⁺	0.049 5	0.044 7
40	L-Glutamine	HMDB0000148	C₅H₉NO₄	148.076 6	7.48	112.894 1, 142.937 0	[M+H]⁺	0.047 8	0.001 0
41	Indole-3-acetaldehyde	HMDB0001190	C₁₀H₉NO	160.100 6	8.02	142.064 3, 118.065 2, 130.065 1	[M+H]⁺	0.038 3	0.038 0
42	Tryptamine	HMDB0000303	C₁₀H₁₂N₂	161.129 0	7.96	158.964 0, 121.087 0, 58.066 2	[M+H]⁺	0.047 7	0.002 1
43	Indole-3-carboxylic acid	HMDB0003320	C₉H₇NO₂	162.056 2	10.99	144.043 9, 55.053 9	[M+H]⁺	0.007 6	0.028 0
44	Phenacemide	HMDB0015253	C₉H₁₀N₂O₂	165.093 9	12.86	119.048 2, 91.054 1, 77.038 3	[M+H]⁺	0.031 1	0.027 4
45	Phenylalanine	HMDB0000159	C₉H₁₁NO₂	166.086 2	7.48	120.036 8, 103.025 9, 92.994 1	[M+H]⁺	0.014 7	0.035 0
46	Pyridoxamine	HMDB0001431	C₈H₁₂N₂O₂	169.101 3	8.32	95.078 4, 84.077 1, 70.062 1	[M+H]⁺	0.003 3	0.032 0
47	N-Methyltryptamine	HMDB0004370	C₁₁H₁₄N₂	175.133 9	17.17	144.066 5, 117.056 5, 91.043 7	[M+H]⁺	0.001 1	0.015 2
48	Geraniol	HMDB0005812	C₁₀H₁₈O	177.114 5	17.15	155.143 6, 137.133 0, 109.101 7	[M+H]⁺	0.021 4	0.021 4
49	Indole-3-propionic acid	HMDB0002302	C₁₁H₁₁NO₂	190.107 2	6.38	130.064 7, 172.077 3	[M+H]⁺	0.043 0	0.047 3
50	Metyrosine	HMDB0014903	C₁₀H₁₃NO₃	196.101 4	6.06	129.099 0, 95.081 0, 70.063 4	[M+H]⁺	0.049 9	0.010 4
51	Alanyl-leucine	HMDB0028691	C₉H₁₈N₂O₃	203.136 0	7.22	159.111 4, 119.081 0	[M+H-H₂O]⁺	0.010 0	0.001 8
52	Glutamyl-glycine	HMDB0028819	C₇H₁₂N₂O₅	205.157 8	13.15	187.062 7, 102.053 0, 85.025 9	[M+H]⁺	0.037 2	0.044 0
53	Dihydrojasmonic acid	HMDB0033601	C₁₂H₂₀O₃	213.150 1	17.16	163.105 7, 131.082 3, 107.081 9	[M+H]⁺	0.004 0	0.016 7
54	Melatonin	HMDB0001389	C₁₃H₁₆N₂O₂	233.165 9	17.91	191.180 4, 174.091 3	[M+H]⁺	0.002 3	0.043 5
55	Valyl-isoleucine	HMDB0029130	C₁₁H₂₂N₂O₃	231.159 6	17.15	187.063 1, 164.094 3, 119.081 6	[M+H]⁺	0.003 0	0.018 9

Label	Pathway	Total metabolite	Hits	P value	Impact
1	Tryptophan metabolism	41	6	2.035 1E^-5	0.224 21
2	Arginine biosynthesis	14	3	1.014 8E^-3	0.177 66
3	Phenylalanine, tyrosine and tryptophan biosynthesis	4	2	1.311 2E^-3	1
4	Phenylalanine metabolism	8	2	5.894 5E^-3	0.357 14
5	Arginine and proline metabolism	36	3	1.596 6E^-2	0.181 39

Label	Pathway	Total metabolite	Hits	P value	Impact
1	Tryptophan metabolism	41	6	2.035 1E^-5	0.224 21
2	Arginine biosynthesis	14	3	1.014 8E^-3	0.177 66
3	Phenylalanine, tyrosine and tryptophan biosynthesis	4	2	1.311 2E^-3	1
4	Phenylalanine metabolism	8	2	5.894 5E^-3	0.357 14
5	Arginine and proline metabolism	36	3	1.596 6E^-2	0.181 39

非靶向代谢组学对栀子苷给药后大鼠盲肠内容物中差异代谢物分析

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罗苗苗 ¹^,²^,^# , 许洪展 ²^,³^,^# , 何镜 ² , 刘筱婧 ² , 赵紫歆 ² , 姜云云 ¹^,⁴^,^* , 周婷婷 ²^,^*

药学学报 | 专题报道: 以多学科交叉探寻中药现代化发展之路 2025,60(3): 655-666

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药学学报 | 专题报道: 以多学科交叉探寻中药现代化发展之路 2025, 60(3): 655-666

非靶向代谢组学对栀子苷给药后大鼠盲肠内容物中差异代谢物分析

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罗苗苗¹^,²^,^#, 许洪展²^,³^,^#, 何镜², 刘筱婧², 赵紫歆², 姜云云¹^,⁴^,^*, 周婷婷²^,^*

作者信息

1.安徽医科大学药学院, 安徽合肥 230032

2.中国人民解放军海军军医大学药学系, 上海市药物 (中药) 代谢产物研究重点实验室, 上海 200433

3.沈阳药科大学药学院, 辽宁沈阳 117004

4.中国人民解放军第904医院, 江苏无锡 214000

通讯作者:

^*姜云云, Tel: 18921150318, E-mail: 20791374@qq.com

周婷婷, Tel: 13636382618, E-mail: tingting_zoo@163.com

Untargeted metabolomics analysis of differential metabolites in cecal contents of rats after geniposide administration

Miao-miao LUO¹^,², Hong-zhan XU²^,³, Jing HE², Xiao-jing LIU², Zi-xin ZHAO², Yun-yun JIANG¹^,⁴^,^*, Ting-ting ZHOU²^,^*

Affiliations

1. School of Pharmacy, Anhui Medical University, Hefei 230032, China

2. Shanghai Key Laboratory of Drug (Traditional Chinese Medicine) Metabolite Research, Department of Pharmacy, PLA Naval Medical University, Shanghai 200433, China

3. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 117004, China

4. The 904 Hospital of the Chinese People's Liberation Army, Wuxi 214000, China

出版时间: 2025-03-12 doi: 10.16438/j.0513-4870.2024-1068

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摘要

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本研究运用非靶向代谢组学结合16S rRNA高通量测序技术筛选栀子苷潜在的肠道菌群介导的抗抑郁活性效应物质。首先采用慢性不可预见性温和刺激(chronic unpredictable mild stimulation, CUMS) 联合孤养方式构建大鼠抑郁模型。再利用超高效液相色谱-四极杆飞行时间质谱(UHPLC-Q/TOF-MS) 技术, 对大鼠盲肠内容物进行非靶向代谢组学分析, 筛选其差异代谢物。通过16S rRNA高通量测序找出盲肠内容物中差异显著的菌属, 然后对差异代谢物与肠道菌属进行相关分析。对比正常对照组、抑郁模型组以及栀子苷治疗组的代谢轮廓, 发现与正常组相比, 模型组中有147种代谢物下调, 381种代谢物上调; 与模型组相比, 栀子苷治疗组中212种代谢物上调, 288种代谢物下调。然后结合差异倍数(fold change, FC) > 2、P < 0.05及变量投影重要性(variable importance in the projection, VIP) > 1的筛选条件、二级图谱及数据库比对, 最终确定55种在三组之间差异显著的代谢物。代谢通路富集分析结果显示, 筛选的差异代谢物主要涉及5条代谢途径, 包括色氨酸代谢、精氨酸生物合成、苯丙氨酸与酪氨酸及色氨酸的生物合成、苯丙氨酸代谢和精氨酸与脯氨酸的代谢。16S rRNA测序结果, 进一步揭示了栀子苷可能通过影响肠道菌群介导的色氨酸代谢途径发挥抗抑郁作用, 为肠-脑轴在抑郁症的治疗策略的研究提供了新的依据。所有动物实验经海军军医大学生物医学研究伦理委员会批准。

关键词

抑郁症 / 盲肠内容物 / 非靶向代谢组学 / 栀子苷 / 肠道菌群

Abstract

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In the study, we employed an untargeted metabolomic approach in conjunction with 16S rRNA high-throughput sequencing to identify potential antidepressant active effectors mediated by gut-flora among geniposides. Firstly, the rat depressed model was constructed using chronic unpredictable mild stress stimulation (CUMS) combined with orphaned model. Then the cecal contents of the rats were analyzed by untargeted metabolomics using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) in order to identify differential metabolites. The intestinal genera exhibiting significant differences in the cecal contents were identified through 16S rRNA high-throughput sequencing, and correlation analysis was subsequently conducted between the differential metabolites and the intestinal genera. The comparison of the metabolic profiles between the normal control group, the depression model group, and the geniposide treatment group revealed that 147 metabolites were down-regulated and 381 metabolites were up-regulated in the model group compared with the normal group. Furthermore, 212 metabolites were up-regulated and 288 metabolites were down-regulated in the geniposide treatment group compared with the model group. Subsequently, the combination of the screening conditions of FC (fold change) > 2, P < 0.05 and VIP (variable importance in the projection) > 1, secondary mapping and database comparison led to the identification of 55 metabolites with significant differences among the three groups. The results of the metabolic pathway enrichment analysis indicated that the identified differential metabolites were primarily involved in five metabolic pathways, namely tryptophan metabolism, arginine biosynthesis, phenylalanine biosynthesis with tyrosine and tryptophan, phenylalanine metabolism, and arginine metabolism with proline. The 16S rRNA sequencing results further indicated that Gardenia jasminoides extract may exert its antidepressant effects, thereby providing a new basis for the study of the gut-brain axis in the therapeutic strategy of depression. All animal experiments were conducted with the approval of the Biomedical Research Ethics Committee of the Naval Medical University.

Key words

depression / cecal content / untargeted metabolomics / geniposide / gut microbiota

引用本文

罗苗苗, 许洪展, 何镜, 刘筱婧, 赵紫歆, 姜云云, 周婷婷. 非靶向代谢组学对栀子苷给药后大鼠盲肠内容物中差异代谢物分析. 药学学报, 2025 , 60 (3) : 655 -666 . DOI: 10.16438/j.0513-4870.2024-1068

Miao-miao LUO, Hong-zhan XU, Jing HE, Xiao-jing LIU, Zi-xin ZHAO, Yun-yun JIANG, Ting-ting ZHOU. Untargeted metabolomics analysis of differential metabolites in cecal contents of rats after geniposide administration[J]. Acta Pharmaceutica Sinica, 2025 , 60 (3) : 655 -666 . DOI: 10.16438/j.0513-4870.2024-1068

正文

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随着工作与生活节奏的加快, 个体所面临的压力强度显著提升, 导致抑郁症的发病率逐年攀升, 严重威胁着人们的身心健康。其发病机制复杂, 治疗周期长, 临床表现主要为情绪低落、思维迟缓、意志活动减退、认知功能障碍以及失眠、乏力、食欲减退等躯体症状。根据世界卫生组织的数据, 抑郁症是全球精神和身体残疾的主要原因, 约3.8%的人口患有抑郁症, 是造成全球疾病负担的主要因素(WHO 2023)。目前关于抑郁症发病机制的研究理论有很多, 主要包括: 单胺神经递质及受体假说^[1]、下丘脑-垂体-肾上腺(hypothalamic-pituitary-adrenal axis, HPA) 轴失调假说^[2]、神经营养假说^[3]、细胞因子假说^[4]及氧化应激假说^[5]等。目前临床常用的抗抑郁药物在一定程度上能够缓解抑郁症状, 但仍存在不良反应大、有效率不高、起效较慢等问题。因此, 研究者们开始探索新的治疗途径。

代谢组学是继基因组、转录组和蛋白质组之后新发展起来的一门组学技术, 可对生物体内所有低分子量的代谢产物进行定性和定量分析。作为系统生物学最下游的“组学”, 与基因组、转录组和蛋白组互相补充, 代谢物水平的改变在代谢层面上放大了基因组、转录组和蛋白组的细微变化, 使之更容易检测到。小分子代谢物作为基因表达后的最终产物, 可以直接并准确地反映机体的病理生理状态^[6]。代谢组学在抑郁症相关研究中的应用也在不断增多^[7], 为中药新药治疗抑郁症寻找新的靶点提供了更多的途径。

近年来, 肠道菌群与抑郁症之间的潜在联系引起了研究者们的广泛关注^[8]。

肠道内寄生着数万亿的肠道微生物, 这些微生物构成肠道内的微环境, 参与人体的消化、免疫和代谢等生命活动, 与人们的身体健康息息相关^[9]。临床和实验证据表明, 肠道微生物、肠道和大脑可能通过多种直接和间接途径进行双向交流, 这些交流信号在从肠道到神经系统的过程中被检测和转导, 且涉及神经、免疫、内分泌、代谢等多种机制^[10]。肠道菌群含有丰富的代谢酶, 其结构的变化会导致代谢产物的轮廓发生相应的改变, 而菌群本身也可以直接调节肠脑通讯, 且分泌的物质可以改变常用药物的结构和药效^[11]。肠脑双相通信在许多疾病中的作用都已被证实, 肠道微生物群在调节大脑功能和包括抑郁症在内的多种精神疾病的发病机制中发挥着重要作用^[12], Yao等^[13]发现肠道微生物可以通过调节海马NLRP3介导的神经炎症减轻慢性乙醇导致的抑郁样行为; Liao等^[14]发现通过调节肠道微生物的丰度可以减轻小鼠阿尔茨海默病的临床表现。因此, 肠道菌群可能作为治疗抑郁症的新靶点。关于肠道菌群及其代谢物的研究, 啮齿类动物一般选择盲肠内容物^[15]。盲肠位于小肠与结肠的连接部位, 且具有一端封闭的结构, 其内容物能够储存更长的时间, 使盲肠内的细菌数量更多且种类丰富, 是肠道菌群的“汇集地”, 有助于更全面地了解肠道菌群的组成和特征。因此, 盲肠成为研究肠道菌群及其代谢的理想部位。

本研究采用非靶向代谢组学的方法对大鼠盲肠内容物进行定性分析, 结合16S rRNA测序结果, 发现栀子苷通过肠道菌介导发挥抗抑郁作用物质基础与色氨酸细菌代谢相关, 并锁定了潜在的效应物质, 为肠道菌在治疗抑郁症中的作用提供了更多依据。

材料与方法

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仪器 Agilent 1290/6520 Accurate-Mass Q-TOF LC/MS系统(美国Agilent公司); FA224电子天平(上海舜宇恒平科学仪器有限公司); QUINTIX125D-1CN分析天平、BT224S电子天平(德国Sartorius公司); Auto Vap S60氮吹仪(美国ATR-Tech公司); 氮气发生器(上海析维医疗科技有限公司); KQ-800DE数控超声波清洗器(昆山市超声仪器有限公司); Micro21R冷冻离心机(美国Thermo-Fisher科技公司); JXFSTPRP-48L全自动样品快速研磨仪(上海净信实业发展有限公司); S0200-230V-EU涡旋混合仪(美国莱伯特公司); SCIENTZ-10N/C冷冻干燥机(宁波新芝生物科技股份有限公司); DP-01真空泵(天津市东康科技有限公司); DYCZ-21电泳槽、DYY-6C电泳仪电源(北京市六一仪器厂); T100^TM Thermal Cyeler PCR仪(美国生命医学产品有限公司); -80 ℃超低温冰箱(日本Panasonic公司); 4 ℃冰箱、-20 ℃冰箱(青岛海尔股份有限公司)。

试剂与药物 4-氯苯丙氨酸(C6506) 购于美国Sigma公司; 色谱级甲醇(L1243235239) 和乙腈(L1231229229) 购于美国Thermo-Fisher科技公司; LCMS级甲酸(73C1512FE) 购于美国ACS公司; 栀子苷(纯度98.0%, AF20062302) 购于中国成都埃法生物科技有限公司; 羧甲基纤维素钠(E528BA0031) 购于中国上海生工生物工程有限公司; 蔗糖(分析纯, P1833713) 购于上海泰坦科技股份有限公司; 乌来糖(N28GS169692) 购于中国大连美仑生物科技有限公司; Magnetic Stool DNA Kit (GDP712-01) 购于北京天根生化科技有限公司; 磷酸盐缓冲溶液(NO240005008) 购于北京索莱宝公司。

动物实验造模及给药 18只体重为220 ± 20 g的SPF级雄性SD大鼠, 由杭州医学院提供, 实验动物生产许可证号: SCXK (浙) 2019-0002。本研究中所有动物实验经海军军医大学生物医学研究伦理委员会批准, 在海军军医大学动物实验中心进行。所有大鼠均饲养于标准动物房, 每日接受12 h光照(7:00~19:00), 室温为22 ± 2 ℃, 湿度为55% ± 5%。18只大鼠随机分为3组, 每组6只, 分别为空白对照组(C)、抑郁模型组(M) 和栀子苷治疗组(G)。所有大鼠适应性饲养一周, 期间均给予正常饮水和饲料。本研究采用慢性不可预见性轻度应激(CUMS) 联合单笼饲养的方式建立SD大鼠抑郁模型^{[16, 17]}。在7周CUMS刺激后, 根据课题组前期研究的剂量优化结果, G组按50 mg·kg^-1剂量每日给予栀子苷灌胃^[18], C组和M组每日灌胃等体积的羧甲基纤维素钠溶液。

动物行为学测试 大鼠七周刺激造模结束及给药两周后测行为学对其抑郁状态进行评价。行为学测试方法参考前期研究工作^{[16, 19]}, 并做适当修改。测试内容主要包括: 强迫游泳测试(forced swimming test, FST)、悬尾测试(tail suspension test, TST) 以及糖水偏爱测试(sucrose preference test, SPT)。

样本采集 大鼠末次给药后禁食12 h, 按0.005 mL·g^-1的剂量腹腔注射20%乌拉坦麻醉。解剖后分离盲肠, 取出其内容物分装于2 mL离心管内, 立即置于-80 ℃冰箱保存。

UHPLC-Q/TOF-MS分析

样本前处理从冰箱取出盲肠内容物, 冻干, 匀质化。称取20.0 mg冻干粉末置于一新的2 mL研磨管中。加入400 μL蛋白沉淀剂(乙腈-甲醇-水, 6∶3∶1) 和18 μL内标溶液(10 μg·mL^-1 4-Cl-Phe甲醇溶液)。用自动匀浆机匀浆2 min。匀浆后冰浴超声20 min。4 ℃条件下12 500 r·min^-1离心10 min。吸取上清液300 μL移至新的1.5 mL离心管中, 并使用温和氮气吹干。剩余残渣用50%乙腈60 μL复溶, 涡旋2 min, 4 ℃、12 500 r·min^-1离心10 min, 取40 μL上清液装于放有内衬管的进样小瓶中等待进样分析。从每一份样品中取出10 μL上清液混合均匀, 制备质控(quality control, QC) 样品。

色谱条件使用色谱柱为Waters Atlantis^TM T3 (100 mm × 2.1 mm, 3 μm, 美国Waters公司)。柱温设定为40 ℃, 采用梯度洗脱方式, 流动相组成为A相(0.1%甲酸水溶液) 和B相(0.1%甲酸乙腈溶液), 流速为0.4 mL·min^-1, 进样量为1 μL。梯度洗脱程序如下: 2~16 min, 95%~5% A; 16~18 min, 5% A; 18~18.1 min, 5%~95% A; 18.1~20 min, 95% A。每进3个样本后, 进一针QC样品以监测仪器运行过程中的稳定性。

质谱条件离子源为电喷雾电离源(ESI), 喷雾电压80 V, 离子源温度325 ℃, 扫描模式为正负离子扫描, 碰撞能为20 eV, 一级扫描范围为m/z 50~1 700, 二级扫描范围为m/z 50~1 700, 雾化气温度为350 ℃, 雾化气流速为11 L·min^-1, 雾化气压力为45 psi (1 psi ≈ 6.9 kPa)。

数据处理及分析用ABF convert软件将原始数据格式转换为.abf格式, 导入MS-DIAL 4.9软件进行处理, 经过峰识别、峰对齐和保留时间校正等处理得到质荷比、保留时间和峰面积等信息, 进行标准化、归一化处理, 筛选出符合80%规则且QC样品峰面积相对标准偏差(relative standard deviation, RSD) 小于20%的代谢物。将筛选出的代谢物数据导入SIMCA 14.1软件中进行主成分分析(principal components analysis, PCA) 和正交偏最小二乘判别分析(orthogonal partial least-squares discriminant analysis, OPLS-DA)。通过多元变量分析筛选出VIP > 1的代谢物, 并进行t检验和FC分析, 最终筛选出P < 0.05、VIP > 1且FC > 2的代谢物为两组之间的差异代谢物, 将差异代谢物用METLIN、HMDB、KEGG等数据库进行比对, 最终确定差异代谢物的具体信息。将差异代谢物导入MetaboAnalyst网站中进行代谢通路分析, 选择Impact > 0.1且P < 0.05的代谢通路作为差异代谢通路。

16S rRNA 按照Magnetic Stool DNA Kit试剂盒说明书提取盲肠内容物总DNA。使用特异性引物515F (5′-GTGCCAGCMGCCGCGGTAA-3′) 和806R (5′-GGACTACHVGGGTWTCTAAT-3′) 对16S rRNA基因V3-V4可变区进行PCR扩增。测定PCR产物的浓度, 配制相同体系混匀, 使用2%的琼脂糖凝胶对PCR产物进行电泳检测, 后割胶进行回收。采用Illumina公司的TruSeq DNA PCR-Free Library Preparation Kit建库试剂盒进行文库的构建, 进行Qubit定量以及文库检测, 最后使用NovaSeq 6000进行上机测序。在Illumina NovaSeq PE250测序平台获得各组大鼠肠道菌群高通量测序的原始数据, 对原始数据拼接过滤后, 得到有效数据。利用Uparse算法(Uparse v7.0.1001) 对有效数据进行OTUs (operational taxonomic units) 聚类和物种注释及统计分析。

其他数据处理 使用GraphPad Prism 8.0.2软件(GraphPad Software, 美国) 进行数据统计分析和图表绘制。所有实验数据以均值±标准差(

$ \bar{x} $

± s) 的方式表示, 多组间比较采用单因素方差分析(analysis of variance, ANOVA), 并采用Tukey检验进一步对多组间进行两两比较, P < 0.05则认为具有统计学显著差异。

结果

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1 大鼠行为学评价

大鼠行为学结果显示(图 1), 在TST和FST实验中, 与正常组相比, CUMS模型组大鼠悬尾不动时间(P < 0.001) 和游泳不动时间(P < 0.01) 显著延长; 与模型组相比, 栀子苷治疗组大鼠的悬尾不动时间(P < 0.01) 和游泳不动时间(P < 0.05) 显著缩短。在SPT实验中, CUMS模型大鼠糖水偏爱程度较正常组显著下降(P < 0.01)。栀子苷治疗组大鼠糖水偏爱程度较模型组显著提高(P < 0.001)。模型组大鼠的悬尾不动时间和游泳不动时间偏长, 表明抑郁模型的构建是成功的, 而栀子苷给药组大鼠悬尾不动时间和游泳不动时间较短且糖水偏爱百分比较高, 表明栀子苷对于抑郁症的治疗是有效的, 其组织取材可以用于后续的实验。

2 大鼠盲肠内容物非靶向代谢组学分析

2.1 多元变量统计分析

使用SIMCA软件对预处理后的数据进行PCA分析及有监督的OPLS-DA分析, 结果如图 2所示, 表明QC样本集中, 仪器状态在检测分析中稳定, 各组间差异明显。原模型能很好地解释两组样本之间的差异, 不存在过拟合现象, 可以进行后续的差异成分寻找和分析。

2.2 差异代谢物的筛选与鉴定

选择FC > 2且P < 0.05的代谢物, 得到各两组之间代谢物的差异, 结果显示(图 3A、B), 模型M组与对照C组相比, 有147种代谢物下调、381种上调; 栀子苷治疗G组与模型M组相比, 有212种代谢物上调、288种下调。将差异代谢物的质荷比在HMDB和KEGG等开源数据库中进行检索, 并结合MS-DIAL二级图谱进行确认, 得到对应代谢物的信息, 共鉴定出55个差异代谢物(表 1), 各组间差异代谢物的含量变化(图 3D)。

2.3 差异代谢通路分析

将得到的55个差异代谢物的HMDB ID导入MetaboAnalyst (https://www.metaboanalyst.ca/) 网站中, 使用通路分析模块, 进行富集分析。以P < 0.05且Impact > 0.1为标准, 筛选符合条件的代谢通路(图 3C, 表 2), 结果显示栀子苷通过肠道内代谢物发挥抗抑郁作用可能与能量代谢、氨基酸代谢及脂质代谢有关, 主要涉及色氨酸代谢、精氨酸生物合成、苯丙氨酸与酪氨酸及色氨酸生物合成、苯丙氨酸代谢以及精氨酸和脯氨酸代谢这5条代谢途径。结合鉴定出的差异代谢物含量变化, 推测色氨酸细菌代谢通路可能为关键代谢通路(图 4)。

3 栀子苷对大鼠肠道菌群结构的影响

为确定肠道微生物群的物种组成和群落结构, 本研究采用16S rRNA高通量测序技术对正常组、模型组和栀子苷给药组大鼠的盲肠内容物样本进行分析。Alpha多样性观察的指数有: Chao、Ace、Shannon和Simpson。结果显示(图 5A), 与C组相比, M组、G组的Shannon和Simpson指数均无显著性差异; 而M组的Chao1指数和Ace指数显著降低, G组无显著性差异。与M组相比, G组的Chao1指数显著升高。说明CUMS造模使抑郁大鼠盲肠内容物中菌群多样性减少, 栀子苷给药后有所回调。Beta多样性用于对不同样本的微生物群落构成进行比较分析, 可表现样本的组间差异。主坐标分析选取贡献率最大的坐标进行作图, 样本距离越接近, 表示物种组成结构越相似, 结果所示(图 5C), C组、M组和G组之间样本差异较大, 存在明显的聚类分离现象。为了更好地显示各组丰度差异显著的微生物群, 以LDA得分≥ 4为筛选标准, 通过线性判别分析效应(LEfSe) 分析阐明了差异存在的微生物的种类与功能(图 5B), 结果表明, 在属水平上三组大鼠盲肠内容物之间存在显著性差异的菌属主要有Ligilactobacillus、Romboutsia、Akkermansia、[Eubacterium]_ruminantium、Lactobacillus、Ruminococcus、Clostridium_sensu_stricto_1。

4 盲肠内容物中菌群与差异代谢物相关性分析

将筛选得到的差异代谢物与16S rRNA测序得到差异菌属做相关性分析(图 5D), 结果显示盲肠内容物中色氨酸的含量与[Eubacterium]_ruminantium (P < 0.05) 呈正相关; 吲哚-3-丙酸的含量与Akkermansia (P < 0.01) 呈正相关; 吲哚-3-甲醛的含量与[Eubacterium]_ ruminantium (P < 0.01) 呈负相关, 与Lactobacillus (P < 0.01) 呈正相关; 吲哚-3-乙醛的含量与[Eubacterium]_ ruminantium (P < 0.05) 呈正相关; 吲哚-3-羧酸的含量与Akkermansia (P < 0.05)、Ruminococcus (P < 0.05) 呈正相关; 吲哚-3-乙酸的含量与Lactobacillus (P < 0.01) 呈正相关。Ligilactobacillus与Lactobacillus (P < 0.05) 呈正相关; Romboutsia与Clostridium_sensu_stricto_1 (P < 0.01) 呈正相关; Akkermansia与各组间吲哚-3-丙酸的含量变化(P < 0.01)、吲哚-3-羧酸的含量变化(P < 0.05) 都呈正相关, 且与吲哚-3-丙酸的产生更相关; [Eubacterium]_ruminantium与色氨酸的含量变化(P < 0.05) 和吲哚-3-乙醛的含量变化(P < 0.05) 呈正相关, 与吲哚-3-甲醛的含量变化(P < 0.01) 呈负相关; Lactobacillus与吲哚-3-甲醛的含量变化(P < 0.01)、吲哚-3-乙酸的含量变化(P < 0.01) 呈正相关; Ruminococcus与色氨酸的含量变化(P < 0.05) 呈负相关, 与吲哚-3-羧酸含量变化(P < 0.05) 呈正相关。

讨论

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本研究采用CUMS联合孤养的方式建立神经损伤的抑郁动物模型, 以SPT、FST和TST结果来评价模型是否建立成功和栀子苷的抗抑郁效果。CUMS模型以快感作为关键测量指标, 不仅可以模拟大多数抑郁症患者的临床症状, 而且能被目前的抗抑郁药治疗, 是研究抑郁症发病机制的理想模型^{[20, 21]}。对于群居型的啮齿类动物来说, 长期独居, 不与其他同伴互动, 可能导致抑郁, 采用孤养模式可以同时很好地模拟社会隔离模式所致的抑郁症状^[22]。SPT能够用于评估动物体验快感的能力, 糖水偏爱百分比的降低表明了伴有快感缺失的抑郁症^[23]。FST和TST是研究动物绝望行为的测试^{[24, 25]}, 动物在水中和悬挂时身体不动状态, 实际反映了由不可避免因素引起的类似灭绝的抑制性学习行为^[26]。

课题组前期研究发现, 栀子苷靶向肠道菌群-脑-肠轴发挥抗抑郁作用。在此基础上, 本研究通过非靶向代谢组学技术对正常对照组、抑郁模型组和栀子苷治疗组大鼠的盲肠内容物进行定性分析, 挖掘其中潜在的肠道菌群介导的抗抑郁效应物质, 表征了55个差异代谢物。代谢通路结果表明, 栀子苷通过肠道菌介导的抗抑郁作用可能与色氨酸代谢、精氨酸生物合成、苯丙氨酸与酪氨酸及色氨酸生物合成、苯丙氨酸代谢以及精氨酸和脯氨酸代谢有关。结合盲肠内容物中差异代谢物在各组间含量变化, 鉴定出色胺、吲哚-3-乙醛、N-乙酰神经氨酸、吲哚-3-甲醛、吲哚-3-丙酸、吲哚乙酸等与色氨酸代谢相关的关键代谢物。16S rRNA测序结果得到, 在属水平上, 栀子苷给药使Ruminococcus、Akkermansia等菌属相对丰度发生显著变化, 并上调[Eubacterium]_ruminantium、Lactobacillus等有益菌属的相对丰度, 改善肠道菌群结构, 从而影响肠道内色氨酸代谢。

色氨酸是必需芳香族氨基酸, 虽然生物体内含量低, 但它是许多微生物和机体合成代谢的前体物质, 可以显著影响哺乳动物的免疫系统和神经系统等^{[27, 28]}。其代谢途径主要分为内源性代谢和细菌代谢。在细菌代谢中, 肠道菌群可以直接影响色氨酸菌群衍生代谢物的类型和水平, 色氨酸被代谢为吲哚、吲哚酸、色胺以及吲哚酸衍生物^[29], 其中吲哚和吲哚酸衍生物是有效的生物活性物质, 作为配体可通过激活孕烷X受体(PXR) 或芳烃受体(AHR) 对靶器官产生影响, 有文献^[30]报道, 吲哚-3-乳酸作为精神益生菌的双歧杆菌的产物可能通过AHR信号激活减轻神经炎症从而达到抗抑郁功效。因此, 吲哚及其衍生物可能是色氨酸细菌代谢通路上发挥抗抑郁作用效应物质之一。

抑郁模型组大鼠盲肠内容物中色氨酸含量偏高, 可能是由于抑郁状态下肠道菌群结构破坏, 有益菌属丰度降低, 导致色氨酸转化酶受抑制, 使色氨酸堆积; 而栀子苷给药后, 对肠道菌群产生调节作用, 色氨酸转化酶增多。肠道菌群生长环境复杂, 吲哚衍生物的产生受菌群间相互作用的影响, 不仅仅来源于某种单一肠道菌的代谢。当生物体接受外界刺激或伤害导致肠道菌群结构发生改变时, 这些由肠道菌调控的代谢物含量也会发生改变, 对机体的保护作用减弱或产生损伤。有研究表明, 吲哚衍生物的产生与肠道中厚壁菌门、梭菌及丁酸盐产生菌等的相对丰度有关; 吲哚乙酸的产生可能与拟杆菌属有关; 吲哚-3-甲醛的产生可能与乳杆菌属有关, 包括嗜酸乳杆菌、鼠乳杆菌, 罗伊氏乳杆菌等^[31]; 乳酸杆菌科与双歧杆菌科通过调节肠道菌代谢物吲哚-3-丙酸产生抗流感感染作用^[32]; 鼠乳杆菌代谢相关产生的乙酸可以影响肠道细胞谱系的定型^[33]; 双歧杆菌代谢产生的吲哚-3-羧酸可以缓解对乙酰氨基酚诱导的急性肝损伤^[34]。在本研究中发现, 吲哚-3-丙酸的产生可能与阿克曼氏菌属有关; 吲哚-3-甲醛的产生可能与中真杆菌属、乳杆菌属有关; 吲哚-3-羧酸的产生可能与阿克曼氏菌属、胃瘤球菌属有关; 吲哚-3-乙酸的产生与乳杆菌属有关; 色氨酸的产生与真杆菌属有关; 吲哚-3-乙醛的产生与真杆菌属有关。因此, 有可能通过调节肠道菌群的结构影响色氨酸代谢中吲哚衍生物的产生来治疗抑郁症。

栀子苷有显著的神经系统治疗作用, 但其发挥作用的物质基础及其作用机制尚不明确。本研究通过非靶向代谢组学技术表征栀子苷发挥抗抑郁作用的潜在效应物质, 采用模式识别的方法筛选出细菌源性色氨酸代谢产物, 明确栀子苷通过肠道菌群介导发挥抗抑郁作用的物质基础, 为肠脑轴在抑郁症治疗方面的研究提供了新的思路和方向。

作者贡献: 罗苗苗和许洪展为实验主要完成者及文章主要撰写者; 何镜参与了实验方案设计及动物造模; 刘筱婧、赵紫歆参与了行为学测试及数据处理分析; 周婷婷和姜云云提出本文研究思路并指导论文撰写与修改。

利益冲突: 本文所有作者均声明不存在利益冲突。

基金

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国家自然科学基金国际(地区) 合作与交流项目(82461160264)
国家自然科学基金(82474056)
无锡市卫健委面上项目(M202310)
上海市卫生健康委卫生学科带头人计划(2022XD0037)
上海市科委科技行动创新计划(21S21902400)

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doi: 10.16438/j.0513-4870.2024-1068

接收时间：2024-10-30
首发时间：2025-11-06
出版时间：2025-03-12

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收稿日期：2024-10-30
修回日期：2025-02-08

基金

国家自然科学基金国际(地区) 合作与交流项目(82461160264)

国家自然科学基金(82474056)

无锡市卫健委面上项目(M202310)

上海市卫生健康委卫生学科带头人计划(2022XD0037)

上海市科委科技行动创新计划(21S21902400)

作者信息

1.安徽医科大学药学院, 安徽合肥 230032

2.中国人民解放军海军军医大学药学系, 上海市药物 (中药) 代谢产物研究重点实验室, 上海 200433

3.沈阳药科大学药学院, 辽宁沈阳 117004

4.中国人民解放军第904医院, 江苏无锡 214000

通讯作者:

^*姜云云, Tel: 18921150318, E-mail: 20791374@qq.com

周婷婷, Tel: 13636382618, E-mail: tingting_zoo@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

No.	Metabolite	HMDB ID	Formula	Measured m/z	t_R /min	MS/MS	Adduct	P value
1	Sulfite	HMDB0000240	H₂O₃S	80.965 2	5.34	79.957 6, 71.014 7, 59.014 3	[M-H]^-	0.043 4	0.002 7
2	Glyceric acid	HMDB0000139	C₃H₆O₄	105.025 2	1.44	87.009 8, 79.957 3, 57.034 5	[M-H]^-	0.000 7	0.029 8
3	Acetylenedicarboxylic acid	HMDB0247933	C₄H₂O₄	112.985 6	11.22	96.960 6, 79.957 7, 68.996 9	[M-H]^-	0.001 0	0.000 6
4	3-Ethylphenol	HMDB0059873	C₈H₁₀O	121.064 9	6.10	119.050 3, 107.050 6, 79.958 0	[M-H]^-	0.002 7	0.039 7
5	Nicotinic acid	HMDB0001488	C₆H₅NO₂	122.973 7	0.95	112.981 8, 104.956 4, 92.927 9	[M-H]^-	0.005 0	0.013 5
6	3-Methylcatechol	HMDB0301753	C₇H₈O₂	123.045 5	8.49	105.035 8, 79.957 8	[M-H]^-	0.006 1	0.029 6
7	L-Aspartic acid	HMDB0000191	C₄H₇NO₄	132.066 4	8.84	150.078 0, 132.030 8, 119.050 3	[M-H]^-	0.012 2	0.008 7
8	Indole-3-carboxaldehyde	HMDB0029737	C₉H₇NO	144.046 8	6.28	122.047 6, 114.055 7, 74.025 9	[M-H]^-	0.024 3	0.043 3
9	Norepinephrine	HMDB0000216	C₈H₁₁NO₃	168.067 6	6.48	109.063 9, 80.965 6	[M-H]^-	0.005 6	0.000 4
10	Indoleacetic acid	HMDB0000197	C₁₀H₉NO₂	174.057 6	5.58	129.090 5, 106.045 2	[M-H]^-	0.004 2	0.029 4
11	L-Tyrosine	HMDB0000158	C₉H₁₁NO₃	180.066 6	5.36	124.076 6, 106.051 0, 80.965 5	[M-H]^-	0.020 1	0.000 4
12	D-Glucuronic acid	HMDB0000127	C₆H₁₀O₇	193.021 5	0.91	161.045 4, 111.022 7, 73.028 9	[M-H]^-	0.020 6	0.000 6
13	Tryptophan	HMDB0000929	C₁₁H₁₂N₂O₂	203.047 2	1.21	159.083 7, 134.055 1, 116.047 5	[M-H]^-	0.013 4	0.000 3
14	2-Methyl-2-[(1-oxo-2-propenyl) amino]-1-propanesulfonic acid	HMDB0031200	C₇H₁₃NO₄S	206.046 9	5.83	112.985 9, 96.960 7, 80.965 8	[M-H]^-	0.036 7	0.019 9
15	Undecanedioic acid	HMDB0000888	C₁₁H₂₀O₄	215.111 6	5.17	142.050 6, 112.041 0, 84.045 8	[M-H]^-	0.023 6	0.040 5
16	N-Acetyl-L-tyrosine	HMDB0000866	C₁₁H₁₃NO₄	222.078 0	5.37	163.044 2, 145.066 8, 109.040 3	[M-H]^-	0.006 7	0.000 5
17	6-Benzylaminopurine	HMDB0039238	C₁₂H₁₁N₅	224.092 8	6.15	197.083 9, 134.042 1, 107.036 4	[M-H]^-	0.033 0	0.045 8
18	γ-Glutamyl-S-methylcysteine	HMDB0031985	C₉H₁₆N₂O₅S	263.067 8	4.70	162.055 9, 118.032 2, 96.962 1	[M-H]^-	0.015 8	0.012 3
19	Adenosine	HMDB0000050	C₁₀H₁₃N₅O₄	275.107 6	4.32	180.994 7, 162.056 3, 118.035 1	[M-H]^-	0.025 0	0.049 4
20	Hexadecanedioic acid	HMDB0000672	C₁₆H₃₀O₄	285.206 3	10.57	267.218 0, 221.177 7, 199.694 7	[M-H]^-	0.008 2	0.014 8
21	Enterolactone	HMDB0006101	C₁₈H₁₈O₄	297.114 6	4.66	253.122 4, 121.065 0, 107.049 9	[M-H]^-	0.021 8	0.028 1
22	Fructosyl-lysine	HMDB0252495	C₁₂H₂₄N₂O₇	307.151 1	4.26	245.114 3, 215.103 7, 145.098 3	[M-H]^-	0.020 2	0.011 0
23	Glutamyl-tyrosine	HMDB0011741	C₁₄H₁₈N₂O₆	309.109 9	4.65	259.152 5, 215.093 7, 123.053 0	[M-H]^-	0.013 0	0.000 2
24	N-Glycolylneuraminate	HMDB0000833	C₁₁H₁₉NO₁₀	324.122 2	7.97	306.083 1, 159.029 3	[M-H]^-	0.010 1	0.003 2
25	2-Dodecylbenzenesulfonic acid	HMDB0031031	C₁₈H₃₀O₃S	325.184 4	14.35	260.376 7, 169.161 0, 197.029 3	[M-H]^-	0.016 8	0.021 8
26	Acetaminophen glucuronide	HMDB0010316	C₁₄H₁₇NO₈	326.090 4	0.91	272.039 6, 196.101 9, 101.061 1	[M-H]^-	0.031 8	0.006 3
27	Deoxycholate	HMDB0000626	C₂₄H₄₀O₄	391.286 5	10.66	327.268 9, 345.278 3	[M-H]^-	0.004 9	0.003 0
28	Glycoursodeoxycholic acid	HMDB0000708	C₂₄H₃₈O₃	433.296 8	13.98	373.274 8, 269.191 2	[M+HAc-H]^-	0.030 6	0.022 9
29	Cholic acid	HMDB0000619	C₂₄H₄₀O₅	815.562 2	8.30	429.237 6, 403.250 3, 96.960 7	[2M-H]^-	0.021 0	0.041 4
30	L-2, 3-Diaminopropionic acid	HMDB0002006	C₃H₈N₂O₂	105.070 7	10.93	95.084 9, 81.069 4, 55.054 0	[M+H]⁺	0.029 8	0.025 7
31	Proline	HMDB0000162	C₅H₉NO₂	116.067 8	7.83	110.070 4, 84.078 5, 70.063 4	[M+H]⁺	0.014 0	0.020 3
32	Isocaproic acid	HMDB0000689	C₆H₁₂O₂	117.091 7	17.91	107.072 7, 91.055 0, 77.039 6	[M+H]⁺	0.009 1	0.044 6
33	L-Valine	HMDB0000883	C₅H₁₁NO₂	118.090 6	0.84	106.066 0, 94.065 8, 77.039 5	[M+H]⁺	0.022 5	0.014 1
34	Leucinic acid	HMDB0000665	C₆H₁₂O₃	115.076 4	17.17	97.062 4, 87.045 6, 59.053 3	[M+H-H₂O]⁺	0.003 2	0.037 1
35	Tyramine	HMDB0000306	C₈H₁₁NO	121.061 2	10.09	115.074 3, 87.045 0, 59.051 4	[M+H]⁺	0.001 7	0.027 3
36	4-Acetylbutyric acid	HMDB0061881	C₆H₁₀O₃	131.084 5	14.46	105.068 6, 93.068 8, 81.068 9	[M+H]⁺	0.039 2	0.040 4
37	Ornithine	HMDB0000214	C₅H₁₂N₂O₂	133.098 0	14.28	109.099 0, 81.068 6, 59.053 0	[M+H]⁺	0.013 1	0.004 0
38	Phenylethanolamine	HMDB0062626	C₈H₁₂NO	138.092 3	4.08	122.096 5, 105.630 1, 77.108 2	[M+H-H₂O]⁺	0.028 4	0.003 0
39	5-Hydroxylysine	HMDB0000450	C₆H₁₄N₂O₃	145.101 7	9.32	105.068 4, 81.069 0, 67.053 6	[M+H]⁺	0.049 5	0.044 7
40	L-Glutamine	HMDB0000148	C₅H₉NO₄	148.076 6	7.48	112.894 1, 142.937 0	[M+H]⁺	0.047 8	0.001 0
41	Indole-3-acetaldehyde	HMDB0001190	C₁₀H₉NO	160.100 6	8.02	142.064 3, 118.065 2, 130.065 1	[M+H]⁺	0.038 3	0.038 0
42	Tryptamine	HMDB0000303	C₁₀H₁₂N₂	161.129 0	7.96	158.964 0, 121.087 0, 58.066 2	[M+H]⁺	0.047 7	0.002 1
43	Indole-3-carboxylic acid	HMDB0003320	C₉H₇NO₂	162.056 2	10.99	144.043 9, 55.053 9	[M+H]⁺	0.007 6	0.028 0
44	Phenacemide	HMDB0015253	C₉H₁₀N₂O₂	165.093 9	12.86	119.048 2, 91.054 1, 77.038 3	[M+H]⁺	0.031 1	0.027 4
45	Phenylalanine	HMDB0000159	C₉H₁₁NO₂	166.086 2	7.48	120.036 8, 103.025 9, 92.994 1	[M+H]⁺	0.014 7	0.035 0
46	Pyridoxamine	HMDB0001431	C₈H₁₂N₂O₂	169.101 3	8.32	95.078 4, 84.077 1, 70.062 1	[M+H]⁺	0.003 3	0.032 0
47	N-Methyltryptamine	HMDB0004370	C₁₁H₁₄N₂	175.133 9	17.17	144.066 5, 117.056 5, 91.043 7	[M+H]⁺	0.001 1	0.015 2
48	Geraniol	HMDB0005812	C₁₀H₁₈O	177.114 5	17.15	155.143 6, 137.133 0, 109.101 7	[M+H]⁺	0.021 4	0.021 4
49	Indole-3-propionic acid	HMDB0002302	C₁₁H₁₁NO₂	190.107 2	6.38	130.064 7, 172.077 3	[M+H]⁺	0.043 0	0.047 3
50	Metyrosine	HMDB0014903	C₁₀H₁₃NO₃	196.101 4	6.06	129.099 0, 95.081 0, 70.063 4	[M+H]⁺	0.049 9	0.010 4
51	Alanyl-leucine	HMDB0028691	C₉H₁₈N₂O₃	203.136 0	7.22	159.111 4, 119.081 0	[M+H-H₂O]⁺	0.010 0	0.001 8
52	Glutamyl-glycine	HMDB0028819	C₇H₁₂N₂O₅	205.157 8	13.15	187.062 7, 102.053 0, 85.025 9	[M+H]⁺	0.037 2	0.044 0
53	Dihydrojasmonic acid	HMDB0033601	C₁₂H₂₀O₃	213.150 1	17.16	163.105 7, 131.082 3, 107.081 9	[M+H]⁺	0.004 0	0.016 7
54	Melatonin	HMDB0001389	C₁₃H₁₆N₂O₂	233.165 9	17.91	191.180 4, 174.091 3	[M+H]⁺	0.002 3	0.043 5
55	Valyl-isoleucine	HMDB0029130	C₁₁H₂₂N₂O₃	231.159 6	17.15	187.063 1, 164.094 3, 119.081 6	[M+H]⁺	0.003 0	0.018 9

No.

Metabolite

HMDB ID

Formula

Measured m/z

t_R /min

MS/MS

Adduct

P value

C vs M

M vs G

Sulfite

HMDB0000240

H₂O₃S

80.965 2

5.34

79.957 6, 71.014 7, 59.014 3

[M-H]^-

0.043 4

0.002 7

Glyceric acid

HMDB0000139

C₃H₆O₄

105.025 2

1.44

87.009 8, 79.957 3, 57.034 5

[M-H]^-

0.000 7

0.029 8

Acetylenedicarboxylic acid

HMDB0247933

C₄H₂O₄

112.985 6

11.22

96.960 6, 79.957 7, 68.996 9

[M-H]^-

0.001 0

0.000 6

3-Ethylphenol

HMDB0059873

C₈H₁₀O

121.064 9

6.10

119.050 3, 107.050 6, 79.958 0

[M-H]^-

0.002 7

0.039 7

Nicotinic acid

HMDB0001488

C₆H₅NO₂

122.973 7

0.95

112.981 8, 104.956 4, 92.927 9

[M-H]^-

0.005 0

0.013 5

3-Methylcatechol

HMDB0301753

C₇H₈O₂

123.045 5

8.49

105.035 8, 79.957 8

[M-H]^-

0.006 1

0.029 6

L-Aspartic acid

HMDB0000191

C₄H₇NO₄

132.066 4

8.84

150.078 0, 132.030 8, 119.050 3

[M-H]^-

0.012 2

0.008 7

Indole-3-carboxaldehyde

HMDB0029737

C₉H₇NO

144.046 8

6.28

122.047 6, 114.055 7, 74.025 9

[M-H]^-

0.024 3

0.043 3

Norepinephrine

HMDB0000216

C₈H₁₁NO₃

168.067 6

6.48

109.063 9, 80.965 6

[M-H]^-

0.005 6

0.000 4

Indoleacetic acid

HMDB0000197

C₁₀H₉NO₂

174.057 6

5.58

129.090 5, 106.045 2

[M-H]^-

0.004 2

0.029 4

L-Tyrosine

HMDB0000158

C₉H₁₁NO₃

180.066 6

5.36

124.076 6, 106.051 0, 80.965 5

[M-H]^-

0.020 1

0.000 4

D-Glucuronic acid

HMDB0000127

C₆H₁₀O₇

193.021 5

0.91

161.045 4, 111.022 7, 73.028 9

[M-H]^-

0.020 6

0.000 6

Tryptophan

HMDB0000929

C₁₁H₁₂N₂O₂

203.047 2

1.21

159.083 7, 134.055 1, 116.047 5

[M-H]^-

0.013 4

0.000 3

2-Methyl-2-[(1-oxo-2-propenyl) amino]-1-propanesulfonic acid

HMDB0031200

C₇H₁₃NO₄S

206.046 9

5.83

112.985 9, 96.960 7, 80.965 8

[M-H]^-

0.036 7

0.019 9

Undecanedioic acid

HMDB0000888

C₁₁H₂₀O₄

215.111 6

5.17

142.050 6, 112.041 0, 84.045 8

[M-H]^-

0.023 6

0.040 5

N-Acetyl-L-tyrosine

HMDB0000866

C₁₁H₁₃NO₄

222.078 0

5.37

163.044 2, 145.066 8, 109.040 3

[M-H]^-

0.006 7

0.000 5

6-Benzylaminopurine

HMDB0039238

C₁₂H₁₁N₅

224.092 8

6.15

197.083 9, 134.042 1, 107.036 4

[M-H]^-

0.033 0

0.045 8

γ-Glutamyl-S-methylcysteine

HMDB0031985

C₉H₁₆N₂O₅S

263.067 8

4.70

162.055 9, 118.032 2, 96.962 1

[M-H]^-

0.015 8

0.012 3

Adenosine

HMDB0000050

C₁₀H₁₃N₅O₄

275.107 6

4.32

180.994 7, 162.056 3, 118.035 1

[M-H]^-

0.025 0

0.049 4

Hexadecanedioic acid

HMDB0000672

C₁₆H₃₀O₄

285.206 3

10.57

267.218 0, 221.177 7, 199.694 7

[M-H]^-

0.008 2

0.014 8

Enterolactone

HMDB0006101

C₁₈H₁₈O₄

297.114 6

4.66

253.122 4, 121.065 0, 107.049 9

[M-H]^-

0.021 8

0.028 1

Fructosyl-lysine

HMDB0252495

C₁₂H₂₄N₂O₇

307.151 1

4.26

245.114 3, 215.103 7, 145.098 3

[M-H]^-

0.020 2

0.011 0

Glutamyl-tyrosine

HMDB0011741

C₁₄H₁₈N₂O₆

309.109 9

4.65

259.152 5, 215.093 7, 123.053 0

[M-H]^-

0.013 0

0.000 2

N-Glycolylneuraminate

HMDB0000833

C₁₁H₁₉NO₁₀

324.122 2

7.97

306.083 1, 159.029 3

[M-H]^-

0.010 1

0.003 2

2-Dodecylbenzenesulfonic acid

HMDB0031031

C₁₈H₃₀O₃S

325.184 4

14.35

260.376 7, 169.161 0, 197.029 3

[M-H]^-

0.016 8

0.021 8

Acetaminophen glucuronide

HMDB0010316

C₁₄H₁₇NO₈

326.090 4

0.91

272.039 6, 196.101 9, 101.061 1

[M-H]^-

0.031 8

0.006 3

Deoxycholate

HMDB0000626

C₂₄H₄₀O₄

391.286 5

10.66

327.268 9, 345.278 3

[M-H]^-

0.004 9

0.003 0

Glycoursodeoxycholic acid

HMDB0000708

C₂₄H₃₈O₃

433.296 8

13.98

373.274 8, 269.191 2

[M+HAc-H]^-

0.030 6

0.022 9

Cholic acid

HMDB0000619

C₂₄H₄₀O₅

815.562 2

8.30

429.237 6, 403.250 3, 96.960 7

[2M-H]^-

0.021 0

0.041 4

L-2, 3-Diaminopropionic acid

HMDB0002006

C₃H₈N₂O₂

105.070 7

10.93

95.084 9, 81.069 4, 55.054 0

[M+H]⁺

0.029 8

0.025 7

Proline

HMDB0000162

C₅H₉NO₂

116.067 8

7.83

110.070 4, 84.078 5, 70.063 4

[M+H]⁺

0.014 0

0.020 3

Isocaproic acid

HMDB0000689

C₆H₁₂O₂

117.091 7

17.91

107.072 7, 91.055 0, 77.039 6

[M+H]⁺

0.009 1

0.044 6

L-Valine

HMDB0000883

C₅H₁₁NO₂

118.090 6

0.84

106.066 0, 94.065 8, 77.039 5

[M+H]⁺

0.022 5

0.014 1

Leucinic acid

HMDB0000665

C₆H₁₂O₃

115.076 4

17.17

97.062 4, 87.045 6, 59.053 3

[M+H-H₂O]⁺

0.003 2

0.037 1

Tyramine

HMDB0000306

C₈H₁₁NO

121.061 2

10.09

115.074 3, 87.045 0, 59.051 4

[M+H]⁺

0.001 7

0.027 3

4-Acetylbutyric acid

HMDB0061881

C₆H₁₀O₃

131.084 5

14.46

105.068 6, 93.068 8, 81.068 9

[M+H]⁺

0.039 2

0.040 4

Ornithine

HMDB0000214

C₅H₁₂N₂O₂

133.098 0

14.28

109.099 0, 81.068 6, 59.053 0

[M+H]⁺

0.013 1

0.004 0

Phenylethanolamine

HMDB0062626

C₈H₁₂NO

138.092 3

4.08

122.096 5, 105.630 1, 77.108 2

[M+H-H₂O]⁺

0.028 4

0.003 0

5-Hydroxylysine

HMDB0000450

C₆H₁₄N₂O₃

145.101 7

9.32

105.068 4, 81.069 0, 67.053 6

[M+H]⁺

0.049 5

0.044 7

L-Glutamine

HMDB0000148

C₅H₉NO₄

148.076 6

7.48

112.894 1, 142.937 0

[M+H]⁺

0.047 8

0.001 0

Indole-3-acetaldehyde

HMDB0001190

C₁₀H₉NO

160.100 6

8.02

142.064 3, 118.065 2, 130.065 1

[M+H]⁺

0.038 3

0.038 0

Tryptamine

HMDB0000303

C₁₀H₁₂N₂

161.129 0

7.96

158.964 0, 121.087 0, 58.066 2

[M+H]⁺

0.047 7

0.002 1

Indole-3-carboxylic acid

HMDB0003320

C₉H₇NO₂

162.056 2

10.99

144.043 9, 55.053 9

[M+H]⁺

0.007 6

0.028 0

Phenacemide

HMDB0015253

C₉H₁₀N₂O₂

165.093 9

12.86

119.048 2, 91.054 1, 77.038 3

[M+H]⁺

0.031 1

0.027 4

Phenylalanine

HMDB0000159

C₉H₁₁NO₂

166.086 2

7.48

120.036 8, 103.025 9, 92.994 1

[M+H]⁺

0.014 7

0.035 0

Pyridoxamine

HMDB0001431

C₈H₁₂N₂O₂

169.101 3

8.32

95.078 4, 84.077 1, 70.062 1

[M+H]⁺

0.003 3

0.032 0

N-Methyltryptamine

HMDB0004370

C₁₁H₁₄N₂

175.133 9

17.17

144.066 5, 117.056 5, 91.043 7

[M+H]⁺

0.001 1

0.015 2

Geraniol

HMDB0005812

C₁₀H₁₈O

177.114 5

17.15

155.143 6, 137.133 0, 109.101 7

[M+H]⁺

0.021 4

Indole-3-propionic acid

HMDB0002302

C₁₁H₁₁NO₂

190.107 2

6.38

130.064 7, 172.077 3

[M+H]⁺

0.043 0

0.047 3

Metyrosine

HMDB0014903

C₁₀H₁₃NO₃

196.101 4

6.06

129.099 0, 95.081 0, 70.063 4

[M+H]⁺

0.049 9

0.010 4

Alanyl-leucine

HMDB0028691

C₉H₁₈N₂O₃

203.136 0

7.22

159.111 4, 119.081 0

[M+H-H₂O]⁺

0.010 0

0.001 8

Glutamyl-glycine

HMDB0028819

C₇H₁₂N₂O₅

205.157 8

13.15

187.062 7, 102.053 0, 85.025 9

[M+H]⁺

0.037 2

0.044 0

Dihydrojasmonic acid

HMDB0033601

C₁₂H₂₀O₃

213.150 1

17.16

163.105 7, 131.082 3, 107.081 9

[M+H]⁺

0.004 0

0.016 7

Melatonin

HMDB0001389

C₁₃H₁₆N₂O₂

233.165 9

17.91

191.180 4, 174.091 3

[M+H]⁺

0.002 3

0.043 5

Valyl-isoleucine

HMDB0029130

C₁₁H₂₂N₂O₃

231.159 6

17.15

187.063 1, 164.094 3, 119.081 6

[M+H]⁺

0.003 0

0.018 9

Label	Pathway	Total metabolite	Hits	P value	Impact
1	Tryptophan metabolism	41	6	2.035 1E^-5	0.224 21
2	Arginine biosynthesis	14	3	1.014 8E^-3	0.177 66
3	Phenylalanine, tyrosine and tryptophan biosynthesis	4	2	1.311 2E^-3	1
4	Phenylalanine metabolism	8	2	5.894 5E^-3	0.357 14
5	Arginine and proline metabolism	36	3	1.596 6E^-2	0.181 39

Label

Pathway

Total metabolite

Hits

P value

Impact

Tryptophan metabolism

2.035 1E^-5

0.224 21

Arginine biosynthesis

1.014 8E^-3

0.177 66

Phenylalanine, tyrosine and tryptophan biosynthesis

1.311 2E^-3

Phenylalanine metabolism

5.894 5E^-3

0.357 14

Arginine and proline metabolism

1.596 6E^-2

0.181 39