药学学报

Storage duration/W	Storage temperature/℃	Storage state
0	-	-
2	25	Mashed
2	4	Mashed
2	25	Unmashed
2	4	Unmashed
4	25	Mashed
4	4	Mashed
4	25	Unmashed
4	4	Unmashed

Storage duration/W	Storage temperature/℃	Storage state
0	-	-
2	25	Mashed
2	4	Mashed
2	25	Unmashed
2	4	Unmashed
4	25	Mashed
4	4	Mashed
4	25	Unmashed
4	4	Unmashed

No.	t_R/min	Mode	Measured value	Theoretical value	ppm	MS/MS	Formula	Identification
1	1.43	[M-H]^-	131.046 1	131.046 2	-0.76	114.019 6, 95.025 0, 88.040 3	C₄H₈N₂O₃	Asparagine
2	1.44	[M-H]^-	132.030 1	132.030 2	-0.76	115.003 6, 114.019 6, 88.040 3, 71.013 8	C₄H₇NO₄	Aspartic acid
3	1.44	[M-H]^-	146.045 8	146.045 9	-0.55	128.035 2, 102.056 0	C₅H₉NO₄	Glutamic acid
4	1.47	[M-H]^-	195.050 9	195.051 0	-0.51	177.040 5, 129.019 3, 99.008 7	C₆H₁₂O₇	Gluconic acid
5	1.49	[M+H]⁺	127.039 0	127.038 9	0.79	109.028 4, 99.044 0, 97.028 4	C₆H₆O₃	5-Hydroxymethyl-2-furaldehyde
6	1.50	[M+H]⁺	116.070 6	116.070 6	0.00	98.984 1, 70.065 2	C₅H₉NO₂	D-Proline
7	1.56	[M-H]^-	191.056 1	191.056 1	0.00	173.045 3, 127.040 0, 85.029 4	C₇H₁₂O₆	Quinic acid
8	1.57	[M+H]⁺	112.050 5	112.050 5	0.00	95.024 0, 84.080 7, 69.044 8	C₄H₅N₃O	Cytosine
9	1.57	[M+H]⁺	166.086 2	166.086 3	-0.30	120.081 0, 103.054 6, 91.054 5	C₉H₁₁NO₂	Phenylalanine
10	1.58	[M+H]⁺	244.092 7	244.092 8	-0.41	227.067 2, 112.050 5	C₉H₁₃N₃O₅	Cytarabine
11	1.60	[M-H]^-	341.108 2	341.108 9	-2.05	179.055 5, 119.034 9, 101.024 3, 89.024 3	C₁₂H₂₂O₁₁	Trehalose
12	1.64	[M-H]^-	128.035 2	128.035 3	-0.78	82.029 7	C₅H₆NO₃	4-Oxoproline
13	1.64	[M-H]^-	133.014 1	133.014 2	-0.75	115.003 5, 89.024 4, 71.013 8	C₄H₆O₅	Malic acid
14	1.64	[M+H]⁺	130.086 3	130.086 2	0.77	84.080 7	C₆H₁₁NO₂	Pipecolic acid
15	1.65	[M-H]^-	115.003 4	115.003 7	-2.61	71.013 8	C₄H₄O₄	Fumaric acid
16	1.65	[M-H]^-	155.009 5	155.009 8	-1.94	111.019 9	C₅H₄N₂O₄	Orotic acid
17	1.90	[M-H]^-	312.109 6	312.108 9	2.34	161.044 8, 150.055 2, 113.023 4, 71.012 6	C₁₄H₁₉NO₇	Mandelic acid amide-β-glucopyranoside
18	1.95	[M-H]^-	474.163 5	474.161 7	3.80	263.078 6, 245.068 0, 221.066 8, 179.055 7, 161.044 9, 150.055 2	C₂₀H₂₉NO₁₂	Mandelic acid amide-β-gentiobioside
19	2.02	[M-H]^-	474.161 9	474.161 0	1.90	150.055 2	C₂₀H₂₉NO₁₂	Amygdalin amide
20	2.24	[M-H]^-	111.008 7	111.008 8	-0.90	83.013 9, 67.018 9	C₅H₄O₃	2-Furoic acid
21	2.25	[M+H]⁺	268.103 5	268.104 0	-1.86	136.061 8	C₁₀H₁₃N₅O₄	Adenosine
22	2.27	[M-H]^-	191.019 6	191.019 7	-0.52	173.019 6, 147.029 6, 129.019 3	C₆H₈O₇	Citric acid
23	2.28	[M+H]⁺	124.039 4	124.039 3	0.81	97.007 5, 80.049 4	C₆H₅NO₂	Nicotinic acid
24	2.42	[M+H]⁺	132.101 8	132.101 9	-0.76	86.096 4, 69.069 9	C₆H₁₃NO₂	L-Isoleucine
25	2.88	[M+H]⁺	123.055 4	123.055 2	1.63	106.073 3, 96.044 3, 80.049 5	C₆H₆N₂O	Nicotinamide
26	3.08	[M-H]^-	475.145 5	475.145 7	-0.44	431.154 1, 161.044 8, 113.022 4, 101.023 3, 89.023 2, 71.012 6	C₂₀H₂₈O₁₃	Mandelic acid-β-gentiobioside
27	3.58	[M-H]^-	431.156 3	431.155 9	0.97	269.102 9, 161.044 6, 113.022 3, 89.023 2, 71.012 6, 59.012 6	C₁₉H₂₈O₁₁	Benzyl-β-gentiobioside
28	3.69	[M-H]^-	151.039 3	151.040 1	-5.03	107.049 1, 59.012 7	C₈H₈O₃	Mandelic acid
29	4.21	[M+H]⁺	134.060 2	134.060 0	1.49	106.065 1, 79.054 2	C₈H₇NO	Oxindole
30	4.32	[M-H]^-	456.151 6	456.151 1	1.10	174.956 0, 161.044 9	C₂₀H₂₇NO₁₁	Neoamygdalin
31	4.93	[M+H]⁺	188.070 7	188.070 6	0.53	170.060 0, 142.065 1, 118.065 1	C₁₁H₉NO₂	Indoleacrylic acid
32	4.95	[M+H]⁺	146.060 1	146.060 0	0.68	118.065 1	C₉H₇NO	4-Indolecarbaldehyde
33	4.98	[M-H]^-	203.082 5	203.082 6	-0.49	186.055 6, 159.092 3, 142.066 1, 116.050 5	C₁₁H₁₂N₂O₂	Tryptophan
34	5.11	[M+H]⁺	433.221 3	433.222 1	-1.85	384.190 7, 369.168 4	C₂₄H₃₂O₇	Schisandrin
35	5.26	[M-H]^-	353.088 1	353.087 8	0.85	191.0056 1, 147.166 4	C₁₆H₁₈O₉	Chlorogenic acid
36	5.42	[M+H]⁺	296.112 2	296.112 8	-2.03	145.049 6, 134.060 0, 116.049 4	C₁₄H₁₇NO₆	Prunasin
37	5.43	[M-H]^-	456.151 6	456.151 1	1.02	221.066 2, 203.055 8, 179.055 4, 161.044 8	C₂₀H₂₇NO₁₁	Amygdalin
38	5.94	[M+H]⁺	173.107 3	173.107 3	0.00	172.995 2, 171.993 0	C₁₁H₁₂N₂	3, 5-Dimethyl-1-phenylpyrazole
39	6.46	[M-H]^-	173.081 9	173.081 9	0.00	155.071 0, 111.081 5	C₈H₁₄O₄	Suberic acid
40	6.98	[M-H]^-	221.104 4	221.104 4	0.00	165.041 6, 164.034 0	C₁₀H₁₄N₄O₂	3-Isobutyl-1-methylxanthine
41	6.98	[M+H]⁺	356.185 3	356.185 6	-0.84	340.152 2, 277.047 6, 192.101 8, 165.090 9	C₂₁H₂₅NO₄	Tetrahydropalmatine
42	7.65	[M+H]⁺	336.122 5	336.123 0	-1.49	320.091 0, 306.075 4, 278.081 5	C₂₀H₁₈NO₄	Berberine
43	7.72	[M-H]^-	253.050 9	253.050 6	1.19	224.048 5, 209.059 8	C₁₅H₁₀O₄	Daidzein
44	8.13	[M-H]^-	138.019 6	138.019 7	-0.72	108.021 6	C₆H₅NO₃	4-Nitrophenol
45	9.04	[M+H]⁺	187.038 8	187.039 0	-1.07	143.049 2, 131.049 0, 151.054 1, 88.021 5	C₁₁H₆O₃	Psoralen
46	9.52	[M-H]^-	221.081 8	221.081 9	-0.45	177.092 3, 121.029 5	C₁₂H₁₄O₄	Monobutylphthalate
47	10.07	[M-H]^-	321.113 1	321.113 2	-0.31	277.050 8, 265.050 6, 252.024 7	C₂₀H₁₈O₄	Neobavaisoflavone
48	10.11	[M-H]^-	295.228 1	295.227 9	0.68	277.217 7, 181.145 8	C₁₈H₃₂O₃	Hydroxy-octadecatrienoic acid
49	10.34	[M+H]⁺	473.144 0	473.144 2	-0.42	193.049 4, 149.023 5	C₂₄H₂₄O₁₀	1, 6-bis-O-[(2E)-3-(4-Hydroxyphenyl)-2-Propenoyl]- Β-D-Glucopyranose
50	10.67	[M+H]⁺	295.226 9	295.226 8	0.34	277.214 7	C₁₈H₃₀O₃	(9Z, 11E)-13-Oxooctadeca-9, 11-dienoicacid
51	11.07	[M+H]⁺	415.211 5	415.211 5	0.00	400.188 8, 119.085 5	C₂₄H₃₀O₆	Bis(4-ethylbenzylidene)sorbitol
52	11.18	[M+H]⁺	183.080 4	183.080 4	0.00	105.033 5	C₁₃H₁₀O	Benzophenone
53	11.30	[M-H]^-	269.045 4	269.045 5	-0.37	241.050 8, 225.055 4, 197.060 8	C₁₅H₁₀O₅	Genistein
54	11.40	[M-H]^-	255.232 6	255.232 9	-1.18	57.309 0	C₁₆H₃₂O₂	Palmitic acid
55	11.86	[M+H]⁺	339.158 2	339.159 1	-2.65	271.095 5, 219.101 3, 147.044 0, 119.049 0	C₂₁H₂₂O₄	Bavachinin
56	11.91	[M-H]^-	389.176 5	389.175 8	1.80	277.050 5, 265.050 6, 252.043 0	C₂₅H₂₆O₄	3-{3-[(2E)-3, 7-Dimethyl-2, 6-octadien-1-Yl]-4-hydroxyphenyl}-7-hydroxy-4H-chromen-4-one
57	12.79	[M+H]⁺	262.143 4	262.143 8	-1.53	149.344 1, 121.058 7	C₁₅H₁₉NO₃	MDPBP
58	12.96	[M+H]⁺	219.174 2	219.174 3	-0.46	177.127 0, 163.111 6, 123.080 5, 121.101 0	C₁₅H₂₂O	3, 5-di-tert-Butylbenzaldehyde

No.	t_R/min	Mode	Measured value	Theoretical value	ppm	MS/MS	Formula	Identification
1	1.43	[M-H]^-	131.046 1	131.046 2	-0.76	114.019 6, 95.025 0, 88.040 3	C₄H₈N₂O₃	Asparagine
2	1.44	[M-H]^-	132.030 1	132.030 2	-0.76	115.003 6, 114.019 6, 88.040 3, 71.013 8	C₄H₇NO₄	Aspartic acid
3	1.44	[M-H]^-	146.045 8	146.045 9	-0.55	128.035 2, 102.056 0	C₅H₉NO₄	Glutamic acid
4	1.47	[M-H]^-	195.050 9	195.051 0	-0.51	177.040 5, 129.019 3, 99.008 7	C₆H₁₂O₇	Gluconic acid
5	1.49	[M+H]⁺	127.039 0	127.038 9	0.79	109.028 4, 99.044 0, 97.028 4	C₆H₆O₃	5-Hydroxymethyl-2-furaldehyde
6	1.50	[M+H]⁺	116.070 6	116.070 6	0.00	98.984 1, 70.065 2	C₅H₉NO₂	D-Proline
7	1.56	[M-H]^-	191.056 1	191.056 1	0.00	173.045 3, 127.040 0, 85.029 4	C₇H₁₂O₆	Quinic acid
8	1.57	[M+H]⁺	112.050 5	112.050 5	0.00	95.024 0, 84.080 7, 69.044 8	C₄H₅N₃O	Cytosine
9	1.57	[M+H]⁺	166.086 2	166.086 3	-0.30	120.081 0, 103.054 6, 91.054 5	C₉H₁₁NO₂	Phenylalanine
10	1.58	[M+H]⁺	244.092 7	244.092 8	-0.41	227.067 2, 112.050 5	C₉H₁₃N₃O₅	Cytarabine
11	1.60	[M-H]^-	341.108 2	341.108 9	-2.05	179.055 5, 119.034 9, 101.024 3, 89.024 3	C₁₂H₂₂O₁₁	Trehalose
12	1.64	[M-H]^-	128.035 2	128.035 3	-0.78	82.029 7	C₅H₆NO₃	4-Oxoproline
13	1.64	[M-H]^-	133.014 1	133.014 2	-0.75	115.003 5, 89.024 4, 71.013 8	C₄H₆O₅	Malic acid
14	1.64	[M+H]⁺	130.086 3	130.086 2	0.77	84.080 7	C₆H₁₁NO₂	Pipecolic acid
15	1.65	[M-H]^-	115.003 4	115.003 7	-2.61	71.013 8	C₄H₄O₄	Fumaric acid
16	1.65	[M-H]^-	155.009 5	155.009 8	-1.94	111.019 9	C₅H₄N₂O₄	Orotic acid
17	1.90	[M-H]^-	312.109 6	312.108 9	2.34	161.044 8, 150.055 2, 113.023 4, 71.012 6	C₁₄H₁₉NO₇	Mandelic acid amide-β-glucopyranoside
18	1.95	[M-H]^-	474.163 5	474.161 7	3.80	263.078 6, 245.068 0, 221.066 8, 179.055 7, 161.044 9, 150.055 2	C₂₀H₂₉NO₁₂	Mandelic acid amide-β-gentiobioside
19	2.02	[M-H]^-	474.161 9	474.161 0	1.90	150.055 2	C₂₀H₂₉NO₁₂	Amygdalin amide
20	2.24	[M-H]^-	111.008 7	111.008 8	-0.90	83.013 9, 67.018 9	C₅H₄O₃	2-Furoic acid
21	2.25	[M+H]⁺	268.103 5	268.104 0	-1.86	136.061 8	C₁₀H₁₃N₅O₄	Adenosine
22	2.27	[M-H]^-	191.019 6	191.019 7	-0.52	173.019 6, 147.029 6, 129.019 3	C₆H₈O₇	Citric acid
23	2.28	[M+H]⁺	124.039 4	124.039 3	0.81	97.007 5, 80.049 4	C₆H₅NO₂	Nicotinic acid
24	2.42	[M+H]⁺	132.101 8	132.101 9	-0.76	86.096 4, 69.069 9	C₆H₁₃NO₂	L-Isoleucine
25	2.88	[M+H]⁺	123.055 4	123.055 2	1.63	106.073 3, 96.044 3, 80.049 5	C₆H₆N₂O	Nicotinamide
26	3.08	[M-H]^-	475.145 5	475.145 7	-0.44	431.154 1, 161.044 8, 113.022 4, 101.023 3, 89.023 2, 71.012 6	C₂₀H₂₈O₁₃	Mandelic acid-β-gentiobioside
27	3.58	[M-H]^-	431.156 3	431.155 9	0.97	269.102 9, 161.044 6, 113.022 3, 89.023 2, 71.012 6, 59.012 6	C₁₉H₂₈O₁₁	Benzyl-β-gentiobioside
28	3.69	[M-H]^-	151.039 3	151.040 1	-5.03	107.049 1, 59.012 7	C₈H₈O₃	Mandelic acid
29	4.21	[M+H]⁺	134.060 2	134.060 0	1.49	106.065 1, 79.054 2	C₈H₇NO	Oxindole
30	4.32	[M-H]^-	456.151 6	456.151 1	1.10	174.956 0, 161.044 9	C₂₀H₂₇NO₁₁	Neoamygdalin
31	4.93	[M+H]⁺	188.070 7	188.070 6	0.53	170.060 0, 142.065 1, 118.065 1	C₁₁H₉NO₂	Indoleacrylic acid
32	4.95	[M+H]⁺	146.060 1	146.060 0	0.68	118.065 1	C₉H₇NO	4-Indolecarbaldehyde
33	4.98	[M-H]^-	203.082 5	203.082 6	-0.49	186.055 6, 159.092 3, 142.066 1, 116.050 5	C₁₁H₁₂N₂O₂	Tryptophan
34	5.11	[M+H]⁺	433.221 3	433.222 1	-1.85	384.190 7, 369.168 4	C₂₄H₃₂O₇	Schisandrin
35	5.26	[M-H]^-	353.088 1	353.087 8	0.85	191.0056 1, 147.166 4	C₁₆H₁₈O₉	Chlorogenic acid
36	5.42	[M+H]⁺	296.112 2	296.112 8	-2.03	145.049 6, 134.060 0, 116.049 4	C₁₄H₁₇NO₆	Prunasin
37	5.43	[M-H]^-	456.151 6	456.151 1	1.02	221.066 2, 203.055 8, 179.055 4, 161.044 8	C₂₀H₂₇NO₁₁	Amygdalin
38	5.94	[M+H]⁺	173.107 3	173.107 3	0.00	172.995 2, 171.993 0	C₁₁H₁₂N₂	3, 5-Dimethyl-1-phenylpyrazole
39	6.46	[M-H]^-	173.081 9	173.081 9	0.00	155.071 0, 111.081 5	C₈H₁₄O₄	Suberic acid
40	6.98	[M-H]^-	221.104 4	221.104 4	0.00	165.041 6, 164.034 0	C₁₀H₁₄N₄O₂	3-Isobutyl-1-methylxanthine
41	6.98	[M+H]⁺	356.185 3	356.185 6	-0.84	340.152 2, 277.047 6, 192.101 8, 165.090 9	C₂₁H₂₅NO₄	Tetrahydropalmatine
42	7.65	[M+H]⁺	336.122 5	336.123 0	-1.49	320.091 0, 306.075 4, 278.081 5	C₂₀H₁₈NO₄	Berberine
43	7.72	[M-H]^-	253.050 9	253.050 6	1.19	224.048 5, 209.059 8	C₁₅H₁₀O₄	Daidzein
44	8.13	[M-H]^-	138.019 6	138.019 7	-0.72	108.021 6	C₆H₅NO₃	4-Nitrophenol
45	9.04	[M+H]⁺	187.038 8	187.039 0	-1.07	143.049 2, 131.049 0, 151.054 1, 88.021 5	C₁₁H₆O₃	Psoralen
46	9.52	[M-H]^-	221.081 8	221.081 9	-0.45	177.092 3, 121.029 5	C₁₂H₁₄O₄	Monobutylphthalate
47	10.07	[M-H]^-	321.113 1	321.113 2	-0.31	277.050 8, 265.050 6, 252.024 7	C₂₀H₁₈O₄	Neobavaisoflavone
48	10.11	[M-H]^-	295.228 1	295.227 9	0.68	277.217 7, 181.145 8	C₁₈H₃₂O₃	Hydroxy-octadecatrienoic acid
49	10.34	[M+H]⁺	473.144 0	473.144 2	-0.42	193.049 4, 149.023 5	C₂₄H₂₄O₁₀	1, 6-bis-O-[(2E)-3-(4-Hydroxyphenyl)-2-Propenoyl]- Β-D-Glucopyranose
50	10.67	[M+H]⁺	295.226 9	295.226 8	0.34	277.214 7	C₁₈H₃₀O₃	(9Z, 11E)-13-Oxooctadeca-9, 11-dienoicacid
51	11.07	[M+H]⁺	415.211 5	415.211 5	0.00	400.188 8, 119.085 5	C₂₄H₃₀O₆	Bis(4-ethylbenzylidene)sorbitol
52	11.18	[M+H]⁺	183.080 4	183.080 4	0.00	105.033 5	C₁₃H₁₀O	Benzophenone
53	11.30	[M-H]^-	269.045 4	269.045 5	-0.37	241.050 8, 225.055 4, 197.060 8	C₁₅H₁₀O₅	Genistein
54	11.40	[M-H]^-	255.232 6	255.232 9	-1.18	57.309 0	C₁₆H₃₂O₂	Palmitic acid
55	11.86	[M+H]⁺	339.158 2	339.159 1	-2.65	271.095 5, 219.101 3, 147.044 0, 119.049 0	C₂₁H₂₂O₄	Bavachinin
56	11.91	[M-H]^-	389.176 5	389.175 8	1.80	277.050 5, 265.050 6, 252.043 0	C₂₅H₂₆O₄	3-{3-[(2E)-3, 7-Dimethyl-2, 6-octadien-1-Yl]-4-hydroxyphenyl}-7-hydroxy-4H-chromen-4-one
57	12.79	[M+H]⁺	262.143 4	262.143 8	-1.53	149.344 1, 121.058 7	C₁₅H₁₉NO₃	MDPBP
58	12.96	[M+H]⁺	219.174 2	219.174 3	-0.46	177.127 0, 163.111 6, 123.080 5, 121.101 0	C₁₅H₂₂O	3, 5-di-tert-Butylbenzaldehyde

Compound	VIP value	P value	FC value
Fumaric acid	1.03	3.61E-13	3.34
Asparagine	1.67	4.74E-08	2.13
Indoleacrylic acid	1.00	7.66E-08	2.10
3, 5-di-tert-Butylbenzaldehyde	4.03	0.006 295	11.72
Amygdalin	2.31	0.010 183	7.16
Nicotinic acid	3.02	0.012 177	2.10
MDPBP	1.11	0.021 144	4.20
Gluconic acid	1.94	0.025 285	3.18
Glutamic acid	2.36	0.029 055	2.86
Palmitic acid	1.83	0.041 540	0.28
Nicotinamide	1.75	0.049 483	0.43

Compound	VIP value	P value	FC value
Fumaric acid	1.03	3.61E-13	3.34
Asparagine	1.67	4.74E-08	2.13
Indoleacrylic acid	1.00	7.66E-08	2.10
3, 5-di-tert-Butylbenzaldehyde	4.03	0.006 295	11.72
Amygdalin	2.31	0.010 183	7.16
Nicotinic acid	3.02	0.012 177	2.10
MDPBP	1.11	0.021 144	4.20
Gluconic acid	1.94	0.025 285	3.18
Glutamic acid	2.36	0.029 055	2.86
Palmitic acid	1.83	0.041 540	0.28
Nicotinamide	1.75	0.049 483	0.43

Compound	VIP value	P value	FC value
Nicotinamide	4.12	2.08E-08	0.28
Cytarabine	1.26	3.30E-08	0.42
Tryptophan	1.36	9.03E-08	0.42
Malic acid	2.31	6.87E-07	0.41
Fumaric acid	1.53	7.68E-07	0.40
Amygdalin	1.45	2.67E-06	0.43
1, 6-bis-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]-β-D-Glucopyranose	1.20	3.85E-06	0.41
Glutamic acid	1.50	1.17E-05	0.28
Gluconic acid	2.13	0.000 101	0.10
Prunasin	1.27	0.000 753	0.15
Adenosine	1.69	0.010 515	5.65
Proline	2.64	0.012 215	6.48
Bavachinin	1.21	0.028 265	5.94

Compound	VIP value	P value	FC value
Nicotinamide	4.12	2.08E-08	0.28
Cytarabine	1.26	3.30E-08	0.42
Tryptophan	1.36	9.03E-08	0.42
Malic acid	2.31	6.87E-07	0.41
Fumaric acid	1.53	7.68E-07	0.40
Amygdalin	1.45	2.67E-06	0.43
1, 6-bis-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]-β-D-Glucopyranose	1.20	3.85E-06	0.41
Glutamic acid	1.50	1.17E-05	0.28
Gluconic acid	2.13	0.000 101	0.10
Prunasin	1.27	0.000 753	0.15
Adenosine	1.69	0.010 515	5.65
Proline	2.64	0.012 215	6.48
Bavachinin	1.21	0.028 265	5.94

Compound	0 W vs 2 W		2 W vs 4 W
Prunasin	1.82	1.26E-11	3.32		1.02	0.010 102	3.07
Tryptophan	1.14	1.09E-06	4.62	1.02	0.030 633	3.67
Amygdalin	1.18	3.89E-05	5.31	1.37	0.047 550	2.25
Fumaric acid	1.22	0.000 586	2.34	1.18	0.002 866	2.07
Gluconic acid	1.27	0.001 359	2.15	1.04	0.040 550	3.28
Adenosine	2.44	0.002 163	2.89	-	-	-
Pyroglutamic acid	1.37	0.002 780	2.10	2.33	0.040 751	5.96
Bavachinin	1.10	0.004 820	0.49	-	-	-
Proline	1.30	0.017 430	2.16	-	-	-
Oxindole	1.34	0.018 239	2.10	-	-	-
Glutamic acid	2.53	0.033 580	6.88	1.22	0.042 699	8.38
Palmitic acid	1.24	0.045 508	0.30	4.29	0.034 588	0.27
Cytosine	-	-	-	1.55	0.011 431	0.33

Compound	0 W vs 2 W		2 W vs 4 W
Prunasin	1.82	1.26E-11	3.32		1.02	0.010 102	3.07
Tryptophan	1.14	1.09E-06	4.62	1.02	0.030 633	3.67
Amygdalin	1.18	3.89E-05	5.31	1.37	0.047 550	2.25
Fumaric acid	1.22	0.000 586	2.34	1.18	0.002 866	2.07
Gluconic acid	1.27	0.001 359	2.15	1.04	0.040 550	3.28
Adenosine	2.44	0.002 163	2.89	-	-	-
Pyroglutamic acid	1.37	0.002 780	2.10	2.33	0.040 751	5.96
Bavachinin	1.10	0.004 820	0.49	-	-	-
Proline	1.30	0.017 430	2.16	-	-	-
Oxindole	1.34	0.018 239	2.10	-	-	-
Glutamic acid	2.53	0.033 580	6.88	1.22	0.042 699	8.38
Palmitic acid	1.24	0.045 508	0.30	4.29	0.034 588	0.27
Cytosine	-	-	-	1.55	0.011 431	0.33

基于UPLC-MS/MS技术和代谢组学方法的苦杏仁“临方捣碎”的潜在质量标志物筛选

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程遥 ¹^,^# , 毕玥琳 ²^,^# , 冯欣 ² , 王佳琪 ² , 徐浩然 ² , 张童画 ² , 于庚原 ² , 张晨宁 ² , 王景红 ¹^,^* , 孙毅坤 ²^,^*

药学学报 | 研究论文 2022,57(10): 3195-3202

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药学学报 | 研究论文 2022, 57(10): 3195-3202

基于UPLC-MS/MS技术和代谢组学方法的苦杏仁“临方捣碎”的潜在质量标志物筛选

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程遥¹^,^#, 毕玥琳²^,^#, 冯欣², 王佳琪², 徐浩然², 张童画², 于庚原², 张晨宁², 王景红¹^,^*, 孙毅坤²^,^*

作者信息

1.中国中医科学院望京医院药学部, 北京 100102

2.北京中医药大学中药学院, 北京 102488

通讯作者:

*王景红, Tel: 13801091033, E-mail: wangjinghong2002@126.com;

孙毅坤, Tel: 15010706091, E-mail: sunyk@bucm.edu.cn

Identification of potential Q-markers of Semen Armeniacae Amarum based on UPLC-MS/MS and metabonomics

Yao CHENG¹, Yue-lin BI², Xin FENG², Jia-qi WANG², Hao-ran XU², Tong-hua ZHANG², Geng-yuan YU², Chen-ning ZHANG², Jing-hong WANG¹^,^*, Yi-kun SUN²^,^*

Affiliations

1. Department of Pharmacy, Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China

2. College of Traditional Chinese Medicine, Beijing University of Traditional Chinese Medicine, Beijing 102488, China

出版时间: 2022-10-12 doi: 10.16438/j.0513-4870.2022-0581

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摘要

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苦杏仁在储藏过程中容易出现走油变质等情况, 故苦杏仁在临床使用时常要求“临方捣碎”。为进一步确认苦杏仁“临方捣碎”的科学理论价值, 确定苦杏仁的最佳储存条件, 本研究采用UPLC-MS/MS技术对不同储存状态(捣碎和未捣碎)、储存时间(0、2和4周) 和储存温度(25 ℃和4 ℃) 下的苦杏仁进行化学成分比较研究。经文献比对、课题组前期工作积累及Compound Discoverer软件检索, 共从苦杏仁提取液中鉴定出58种物质。同时, 发现不同储存状态、储存时间和储存温度的苦杏仁化学成分含量具有明显的统计学差异。研究结果表明, 未捣碎储存相较于捣碎储存的苦杏仁化学成分组成稳定; 4 ℃储存的相较于25 ℃储存的苦杏仁化学组成更稳定; 储存时间越短, 其化学成分变化越少。苦杏仁的主要药效成分苦杏仁苷在以上三种储存条件下均表现出统计学差异, 可作为苦杏仁“临方捣碎”的潜在质量标志物。

关键词

苦杏仁 / 临方捣碎 / UPLC-MS/MS / 差异化合物 / 代谢组学

Abstract

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Bitter almonds (Semen Armeniacae Amarum) are prone to oil deterioration during storage, so they often require mashing prior to clinical use. To confirm the medical value of bitter almonds "being mashed when used" and to determine the optimal storage conditions for bitter almonds, UPLC-MS/MS was used to perform a comparative study of the chemical composition of bitter almonds in different storage states (mashed and unmashed), storage times (0, 2 and 4 weeks), and storage temperatures (25 ℃ and 4 ℃). A total of 58 substances were identified in bitter almond extracts through literature review, this group's previous work, and a Compound Discoverer software search. Statistically significant differences were found in the chemical composition and content of bitter almonds in different storage states, storage times, and storage temperatures. The results show that the chemical composition of bitter almonds stored unmashed was more stable than that of bitter almonds stored mashed; the chemical composition of bitter almonds stored at 4 ℃ was more stable than that of bitter almonds stored at 25 ℃; and the shorter the storage time, the less the chemical composition changed. Amygdalin, the main medicinal component of bitter almonds, showed statistically significant differences in content under the above three storage conditions, which can be used as a potential quality marker for bitter almonds.

Key words

Semen Armeniacae Amarum / mashed when used / UPLC-MS/MS / differential compound / metabolomics

引用本文

程遥, 毕玥琳, 冯欣, 王佳琪, 徐浩然, 张童画, 于庚原, 张晨宁, 王景红, 孙毅坤. 基于UPLC-MS/MS技术和代谢组学方法的苦杏仁“临方捣碎”的潜在质量标志物筛选. 药学学报, 2022 , 57 (10) : 3195 -3202 . DOI: 10.16438/j.0513-4870.2022-0581

Yao CHENG, Yue-lin BI, Xin FENG, Jia-qi WANG, Hao-ran XU, Tong-hua ZHANG, Geng-yuan YU, Chen-ning ZHANG, Jing-hong WANG, Yi-kun SUN. Identification of potential Q-markers of Semen Armeniacae Amarum based on UPLC-MS/MS and metabonomics[J]. Acta Pharmaceutica Sinica, 2022 , 57 (10) : 3195 -3202 . DOI: 10.16438/j.0513-4870.2022-0581

正文

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苦杏仁(Semen Armeniacae Amarum) 为蔷薇科(Rosaceae) 植物山杏(Prunus armeniaca L. var. ansu Maxim.)、西伯利亚杏(Prunus sibirica L.)、东北杏(Prunus mandshurica Koehne.) 或杏(Prunus armeniaca L.) 的干燥成熟种子^[1], 主要产于我国内蒙古、吉林、辽宁等地。苦杏仁具有降气、镇咳平喘和润肠通便的作用, 临床上常与麻黄结为药对, 用于治疗小儿哮喘及流行性感冒等疾病^[2]。苦杏仁中含有大量油脂^[3], 在储藏过程中极易走油变质, 导致苦杏仁苷和苦杏仁油含量降低, 影响其临床疗效^[4]。

2020版《中华人民共和国药典》规定苦杏仁在临床调剂应用时需“临方捣碎”。“临方捣碎”指中药饮片在调配制剂之前需进行捣碎处理, 使中药饮片中的有效成分溶出度更高。这种操作方法便于中药饮片的储存运输, 能在最大程度上保存药性^[5]。然而, 目前对苦杏仁的研究主要集中于其药理作用及其成分鉴定上, 对其“临方捣碎”的科学研究极少。故本研究模拟临床应用中苦杏仁的储存情况, 收集不同储存状态、储存时间和储存温度下的样品, 并采用超高效液相色谱-四极杆-静电轨道场离子阱质谱仪(UPLC-Q-exactive-orbitrap-MS) 及植物代谢组学方法筛选其差异化合物, 为苦杏仁的最佳保存方式及最佳炮制方式提供科学依据。

材料与方法

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仪器 Thermo Scientific Q Exactive质谱仪、Xcalibur工作站、Vanquish Duo UHPLC System for Dual LC Workflows和Pico 17高速离心机(美国Thermo Scientific公司) 液相系统; CPA225D电子天平(德国Sartorius公司); KQ5200DA超声波清洗机(中国昆山舒美公司); 恒温恒湿箱(上海一恒科学仪器有限公司)。

药物及主要试剂 质谱级乙腈(中国赛默飞世尔科技有限公司, 批号: 151902)、质谱级甲酸(美国ROE公司, 批号: 214911); 色谱用水为屈臣氏饮用水(批号: 20211222); 苦杏仁(北京四方中药饮片有限公司, 批号: 20072304)。

样品制备 需捣碎储存的苦杏仁使用研钵进行手动捣碎, 并过4号筛, 放置于表 1所示条件湿度为60%的恒温恒湿箱中至相应周数时取出并制备样品溶液; 临方捣碎的苦杏仁放置于以上相同条件的恒温恒湿箱中至相应周数时取出, 并在制备样品溶液前进行相同的捣碎处理。分别取不同储存条件下的样品精密称定0.25 g置于锥形瓶中, 加入75%甲醇10 mL, 超声提取30 min, 5 000 r·min^-1离心10 min, 取上清液, 氮气干燥, 用75% 甲醇5 mL复溶, 稀释10倍后, 13 000 r·min^-1离心15 min, 取上清液即得样品溶液。

色谱条件 Waters ACQUITY UPLC BEH C18色谱柱(150 mm × 2.1 mm, 1.7 µm, Milford, 美国), 柱温40 ℃, 进样量5 μL, 流速0.3 mL·min^-1, 流动相为0.1%甲酸水溶液(A) 和乙腈(B)。洗脱程序: 0~1 min, 5% B; 1~12 min, 5%~98% B; 12~13 min, 98% B; 13~14.5 min, 98%~5% B; 14.5~15 min, 5% B。

质谱条件 离子源为电喷雾离子化源(ESI); 正负离子交替扫描; 扫描模式为: 全扫描/数据依赖的二级扫描(Full scan/ddMS²), 扫描范围为100~1 300 Da, 毛细管温度为350 ℃。正模式下的喷雾电压为3 800 V, 负模式下的喷雾电压为3 200 V, 鞘气为35 arb, 辅助气为15 arb, MS²采用低、中、高的3种碰撞能量对母离子进行二级碎裂, 分别为30、50和70 V。一级质谱分辨率为70 000 FWHM (full width at half maximum), 二级质谱分辨率为17 500 FWHM。

数据处理 通过查阅相关文献^[6-12]、检索Compound Discoverer 3.1.1.12软件以及课题组前期工作积累构建苦杏仁化合物数据库, 并通过对比保留时间、母离子与碎片离子相对分子质量进行色谱峰的定性鉴别。使用AnalysisBaseFileConverter软件对所得数据进行格式转化, 利用MSDIAL软件对数据进行归一化处理, 包括选择、去卷积和对齐, 主要涉及保留时间、相对分子质量和峰强度等。利用SIMCA 14.1软件对数据进行主成分分析(PCA) 和正交偏最小二乘法判别分析(OPLS-DA), 并通过200 permutation检验评估OPLS-DA模型拟合的准确性。以OPLS-DA分析所得到的投影中变量重要性(variable importance in projection, VIP)、进行独立样本t检验所得的P值以及差异倍数值(fold change, FC) 为判别条件筛选差异代谢物, 即为VIP值> 1, P < 0.05, FC < 0.5或FC > 2。

结果

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1 苦杏仁的成分鉴定

将所有样品溶液按以上方法进行UPLC-MS/MS分析, 得到正负离子模式下的基峰色谱图见图 1。将得到的准分子离子峰的保留时间、母离子及碎片离子的相对分析质量等相关信息与本研究所构建的数据库进行比对, 以鉴定苦杏仁中的化合物。共鉴定出58个化合物, 其中糖苷类9个、有机酸类8个、氨基酸类8个、杂环类8个、黄酮类5个、脂肪酸类4个及其他种类化合物16个, 化合物信息见表 2。

2 主成分分析(PCA)

无监督的主成分分析可以通过对数据的降维处理, 展示组内的重复性与组间的差异, 并发现离群点。本研究利用AnalysisBaseFileConverter、MSDIAL软件对UPLC-MS/MS技术采集的不同储存条件下苦杏仁保留时间、准分子离子峰强度等信息进行峰校正、峰对齐、归一化处理, 通过SIMCA 14.1软件对数据进行无监督模式的主成分分析, 判别不同储存条件下苦杏仁的化学成分差别。如图 2所示, 所有组分均存在于置信区间内, 同一组分样本聚集, 不同组分样本各自分离, 并未发现异常值。

3 正交偏最小二乘法判别分析(OPLS-DA)

为更清晰地表明不同因素对苦杏仁中非挥发性成分的影响, 本研究利用有监督的OPLS-DA分析来进行差异化合物的筛选。OPLS-DA分析经常被应用于代谢组学的分析, 以进行最大化组间差异并寻找其差异代谢物。本研究通过SIMCA 14.1软件针对三种储存条件下的苦杏仁化学成分进行了OPLS-DA分析, 分别进行了差异化合物的筛选, 通过构建模型所得到的R²X、R²Y通常表示其对原数据矩阵的解释能力, 而Q²则用来评价模型的预测能力, R²X、R²Y和Q²越接近1, 则表示OPLS-DA模型拟合数据效果越好。

3.1 不同储存状态对苦杏仁的影响

将数据结果分为“捣碎储存”及“临方捣碎”两组, 建立其OPLS-DA模型, 如图 3。本次建模中正离子模式下R²X = 0.612, R²Y = 0.995, Q² = 0.974, 负离子模式下R²X = 0.723, R²Y = 0.991, Q² = 0.980, 其数值均大于0.5, 说明本模型建立可靠, 可用于差异化合物的筛选。设置n = 200, 进行置换检验, 其R²、Q²均随置换保留度的下降而下降, 说明模型不存在过拟合的问题, 模型稳定性较高。继而对差异化合物进行筛选, 最终筛选出234个差异离子, 其中通过鉴定差异离子所得的差异化合物相关数据见表 3。

3.2 不同温度下储存对苦杏仁的影响

以储存于25 ℃和4 ℃环境中为分组条件进行OPLS-DA分析, 其正负离子模式下所得OPLS-DA得分图见图 4。经OPLS-DA模型建立, 正离子模式下所得R²X = 0.731, R²Y = 0.996, Q² = 0.937, 负离子模式下所得R²X = 0.872, R²Y = 0.997, Q² = 0.960, 说明模型建立可靠。进行200 permutation检验, 其结果如图 4所示, 右侧R²和Q²高于左侧的值, 且Q²回归线与纵轴交点为负值, 可见模型不存在过拟合现象, 即进行筛选差异化合物, 最终所得差异离子共503个, 其中可鉴定的共13种, 见表 4。

3.3 储存不同时长对苦杏仁的影响

将数据依据储存周数分组并分别进行OPLS-DA分析, 所得结果见图 5。经OPLS-DA分析, 正负离子模式下所得的R²X、R²Y及Q²值均大于0.5, 说明模型建立良好, 可以进行差异代谢物的分析及筛选。以迭代循环200次为前提进行置换检验, 如图 5所示模型中不存在过拟合现象, 模型具有良好的稳定性。通过筛选差异化合物, 最终于0周与2周数据中筛选出91个差异离子, 于2周与4周数据中筛选出26个差异离子, 其中可鉴定的共13种, 见表 5所示。

讨论

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苦杏仁是临床常用中药之一, 但其中含有苦杏仁苷、脂肪油、蛋白质及大量的游离氨基酸, 极容易走油变质^[3], 故临床应用时要求“临方捣碎”再进行调配, 以加大其成分溶出率。本研究即应用了UPLC-MS/MS技术对不同储存条件下的差异化合物进行了筛选和鉴定, 其中, 针对于“临方捣碎”条件共筛选出234个差异离子, 其中可鉴定出的差异化合物共11种; 在不同温度下储存的苦杏仁共筛选出503个差异离子, 其中可鉴定出的差异化合物共13种; 而储存不同时间的苦杏仁之间鉴定出的差异化合物共13种。

三种不同储存条件下筛选出的差异化合物均包括苦杏仁苷, 经比较三种储存条件下苦杏仁苷的FC值可以确定是否捣碎储存这一条件对其影响最大, 未捣碎储存的苦杏仁中苦杏仁苷的含量是捣碎储存的苦杏仁中苦杏仁苷含量的7.16倍。经查阅相关文献^[13], 苦杏仁苷较不稳定, 在苦杏仁苷酶及野黑樱苷酶的催化下会发生水解反应, 生成葡萄糖、苯甲醛和氢氰酸。其中, 氢氰酸为剧毒, 会造成人体呼吸困难, 甚至窒息死亡。苦杏仁苷具有良好的抗肿瘤、抗纤维化的药理功能, 随着苦杏仁苷的降低, 苦杏仁的药理作用会显著下降^[14]。野黑樱苷是储存不同时长及不同储存温度条件下的差异化合物。25 ℃储存的比4 ℃储存的苦杏仁中野黑樱苷的含量低(FC值为0.28)。随时间的推移, 野黑樱苷的含量发生了下降, 苦杏仁储存两周后野黑樱苷含量下降(FC值为3.32), 储存四周后较储存两周的含量下降(FC值为3.07)。野黑樱苷具有抗纤维化的作用, 在体内可作为原型成分被吸收^[15]。野黑樱苷的下降可能会影响到苦杏仁抗纤维化的药理效用。棕榈酸是在不同储存状态及不同储存时长条件下的差异化合物。临方捣碎的苦杏仁相较于捣碎储存的苦杏仁中棕榈酸含量更低。随着储存时间的增长, 棕榈酸的含量升高, 储存两周后棕榈酸含量升高3.3倍, 储存四周后棕榈酸持续升高3.7倍。棕榈酸的增多, 提示苦杏仁可能正走向走油状态, 发生酸败的概率显著上升^[16]。本研究发现“临方捣碎”苦杏仁相较于储存前捣碎苦杏仁的棕榈酸含量较低, 提示“临方捣碎”可能会延缓苦杏仁发生走油现象。苦杏仁中含有多种氨基酸, 在不同储存条件下, 氨基酸的含量也发生了改变。捣碎储存的苦杏仁相对于临方捣碎的苦杏仁精氨酸和谷氨酸的含量有所下降; 25 ℃储存的苦杏仁相对于4 ℃储存的苦杏仁谷氨酸及色氨酸的含量有所下降; 随着储存时间的延长, 谷氨酸、焦谷氨酸及色氨酸的含量也有着不同程度的降低。

“临方捣碎”的概念最早出现在梁代《本草经集注》中所文“凡汤中用完物皆擘破”^{[17, 18]}。当前, 北京市针对于需临方捣碎的中药饮片按《北京市中药饮片调剂规程》进行调剂, 其一般规定为“若用量较大, 又必需提前进行捣碎时, 其储存量不超过两周时间为宜”。而在临床应用时, 对“临方捣碎”理解不透彻的药师对饮片处理方法交代不清, 导致饮片炮制方法有误, 耽误病情的情况屡见不鲜^[19]。然而当临床使用捣碎饮片量大时, 药房则会选择使用机械提前粉碎的方法对其进行处理, 如此一来, 便容易在储存条件、储存时长等方面发生问题^[20]。“临方捣碎”中药饮片的质量控制因素包括捣碎粒度, 药品外包装, 储存温度及储存时间长短^[21]。本实验经过对苦杏仁捣碎状态、储存温度及储存时长的差异化合物研究, 初步确定苦杏仁的最佳储存方式即为于未捣碎状态, 放置于4 ℃环境中, 且储存时间尽量缩短, 为苦杏仁需“临方捣碎”这一关键炮制方法提供了科学论证及理论支持。但本研究目前并未对苦杏仁的捣碎粒度及其外包装的影响进行探究, 未来可针对以上两条件进行更深入的探索, 以对苦杏仁的临床应用提供更有力的质量监管依据。

作者贡献: 第一作者程遥、毕玥琳负责实验部分、论文撰写; 冯欣、王佳琪、徐浩然负责样品的储存及前处理; 张童画、于庚原负责文献整理和格式校正; 张晨宁、王景红、孙毅坤负责论文修改。

利益冲突: 本文无利益冲突。

基金

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中国西藏自治区科技攻关项目(XZ201801-GA-16)

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2022年第57卷第10期

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doi: 10.16438/j.0513-4870.2022-0581

接收时间：2022-05-12
首发时间：2025-12-24
出版时间：2022-10-12

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收稿日期：2022-05-12
修回日期：2022-08-02

基金

中国西藏自治区科技攻关项目(XZ201801-GA-16)

作者信息

1.中国中医科学院望京医院药学部, 北京 100102

2.北京中医药大学中药学院, 北京 102488

通讯作者:

*王景红, Tel: 13801091033, E-mail: wangjinghong2002@126.com;

孙毅坤, Tel: 15010706091, E-mail: sunyk@bucm.edu.cn

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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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Storage duration/W	Storage temperature/℃	Storage state
0	-	-
2	25	Mashed
2	4	Mashed
2	25	Unmashed
2	4	Unmashed
4	25	Mashed
4	4	Mashed
4	25	Unmashed
4	4	Unmashed

Storage duration/W

Storage temperature/℃

Storage state

Mashed

Unmashed

Mashed

Unmashed

No.	t_R/min	Mode	Measured value	Theoretical value	ppm	MS/MS	Formula	Identification
1	1.43	[M-H]^-	131.046 1	131.046 2	-0.76	114.019 6, 95.025 0, 88.040 3	C₄H₈N₂O₃	Asparagine
2	1.44	[M-H]^-	132.030 1	132.030 2	-0.76	115.003 6, 114.019 6, 88.040 3, 71.013 8	C₄H₇NO₄	Aspartic acid
3	1.44	[M-H]^-	146.045 8	146.045 9	-0.55	128.035 2, 102.056 0	C₅H₉NO₄	Glutamic acid
4	1.47	[M-H]^-	195.050 9	195.051 0	-0.51	177.040 5, 129.019 3, 99.008 7	C₆H₁₂O₇	Gluconic acid
5	1.49	[M+H]⁺	127.039 0	127.038 9	0.79	109.028 4, 99.044 0, 97.028 4	C₆H₆O₃	5-Hydroxymethyl-2-furaldehyde
6	1.50	[M+H]⁺	116.070 6	116.070 6	0.00	98.984 1, 70.065 2	C₅H₉NO₂	D-Proline
7	1.56	[M-H]^-	191.056 1	191.056 1	0.00	173.045 3, 127.040 0, 85.029 4	C₇H₁₂O₆	Quinic acid
8	1.57	[M+H]⁺	112.050 5	112.050 5	0.00	95.024 0, 84.080 7, 69.044 8	C₄H₅N₃O	Cytosine
9	1.57	[M+H]⁺	166.086 2	166.086 3	-0.30	120.081 0, 103.054 6, 91.054 5	C₉H₁₁NO₂	Phenylalanine
10	1.58	[M+H]⁺	244.092 7	244.092 8	-0.41	227.067 2, 112.050 5	C₉H₁₃N₃O₅	Cytarabine
11	1.60	[M-H]^-	341.108 2	341.108 9	-2.05	179.055 5, 119.034 9, 101.024 3, 89.024 3	C₁₂H₂₂O₁₁	Trehalose
12	1.64	[M-H]^-	128.035 2	128.035 3	-0.78	82.029 7	C₅H₆NO₃	4-Oxoproline
13	1.64	[M-H]^-	133.014 1	133.014 2	-0.75	115.003 5, 89.024 4, 71.013 8	C₄H₆O₅	Malic acid
14	1.64	[M+H]⁺	130.086 3	130.086 2	0.77	84.080 7	C₆H₁₁NO₂	Pipecolic acid
15	1.65	[M-H]^-	115.003 4	115.003 7	-2.61	71.013 8	C₄H₄O₄	Fumaric acid
16	1.65	[M-H]^-	155.009 5	155.009 8	-1.94	111.019 9	C₅H₄N₂O₄	Orotic acid
17	1.90	[M-H]^-	312.109 6	312.108 9	2.34	161.044 8, 150.055 2, 113.023 4, 71.012 6	C₁₄H₁₉NO₇	Mandelic acid amide-β-glucopyranoside
18	1.95	[M-H]^-	474.163 5	474.161 7	3.80	263.078 6, 245.068 0, 221.066 8, 179.055 7, 161.044 9, 150.055 2	C₂₀H₂₉NO₁₂	Mandelic acid amide-β-gentiobioside
19	2.02	[M-H]^-	474.161 9	474.161 0	1.90	150.055 2	C₂₀H₂₉NO₁₂	Amygdalin amide
20	2.24	[M-H]^-	111.008 7	111.008 8	-0.90	83.013 9, 67.018 9	C₅H₄O₃	2-Furoic acid
21	2.25	[M+H]⁺	268.103 5	268.104 0	-1.86	136.061 8	C₁₀H₁₃N₅O₄	Adenosine
22	2.27	[M-H]^-	191.019 6	191.019 7	-0.52	173.019 6, 147.029 6, 129.019 3	C₆H₈O₇	Citric acid
23	2.28	[M+H]⁺	124.039 4	124.039 3	0.81	97.007 5, 80.049 4	C₆H₅NO₂	Nicotinic acid
24	2.42	[M+H]⁺	132.101 8	132.101 9	-0.76	86.096 4, 69.069 9	C₆H₁₃NO₂	L-Isoleucine
25	2.88	[M+H]⁺	123.055 4	123.055 2	1.63	106.073 3, 96.044 3, 80.049 5	C₆H₆N₂O	Nicotinamide
26	3.08	[M-H]^-	475.145 5	475.145 7	-0.44	431.154 1, 161.044 8, 113.022 4, 101.023 3, 89.023 2, 71.012 6	C₂₀H₂₈O₁₃	Mandelic acid-β-gentiobioside
27	3.58	[M-H]^-	431.156 3	431.155 9	0.97	269.102 9, 161.044 6, 113.022 3, 89.023 2, 71.012 6, 59.012 6	C₁₉H₂₈O₁₁	Benzyl-β-gentiobioside
28	3.69	[M-H]^-	151.039 3	151.040 1	-5.03	107.049 1, 59.012 7	C₈H₈O₃	Mandelic acid
29	4.21	[M+H]⁺	134.060 2	134.060 0	1.49	106.065 1, 79.054 2	C₈H₇NO	Oxindole
30	4.32	[M-H]^-	456.151 6	456.151 1	1.10	174.956 0, 161.044 9	C₂₀H₂₇NO₁₁	Neoamygdalin
31	4.93	[M+H]⁺	188.070 7	188.070 6	0.53	170.060 0, 142.065 1, 118.065 1	C₁₁H₉NO₂	Indoleacrylic acid
32	4.95	[M+H]⁺	146.060 1	146.060 0	0.68	118.065 1	C₉H₇NO	4-Indolecarbaldehyde
33	4.98	[M-H]^-	203.082 5	203.082 6	-0.49	186.055 6, 159.092 3, 142.066 1, 116.050 5	C₁₁H₁₂N₂O₂	Tryptophan
34	5.11	[M+H]⁺	433.221 3	433.222 1	-1.85	384.190 7, 369.168 4	C₂₄H₃₂O₇	Schisandrin
35	5.26	[M-H]^-	353.088 1	353.087 8	0.85	191.0056 1, 147.166 4	C₁₆H₁₈O₉	Chlorogenic acid
36	5.42	[M+H]⁺	296.112 2	296.112 8	-2.03	145.049 6, 134.060 0, 116.049 4	C₁₄H₁₇NO₆	Prunasin
37	5.43	[M-H]^-	456.151 6	456.151 1	1.02	221.066 2, 203.055 8, 179.055 4, 161.044 8	C₂₀H₂₇NO₁₁	Amygdalin
38	5.94	[M+H]⁺	173.107 3	173.107 3	0.00	172.995 2, 171.993 0	C₁₁H₁₂N₂	3, 5-Dimethyl-1-phenylpyrazole
39	6.46	[M-H]^-	173.081 9	173.081 9	0.00	155.071 0, 111.081 5	C₈H₁₄O₄	Suberic acid
40	6.98	[M-H]^-	221.104 4	221.104 4	0.00	165.041 6, 164.034 0	C₁₀H₁₄N₄O₂	3-Isobutyl-1-methylxanthine
41	6.98	[M+H]⁺	356.185 3	356.185 6	-0.84	340.152 2, 277.047 6, 192.101 8, 165.090 9	C₂₁H₂₅NO₄	Tetrahydropalmatine
42	7.65	[M+H]⁺	336.122 5	336.123 0	-1.49	320.091 0, 306.075 4, 278.081 5	C₂₀H₁₈NO₄	Berberine
43	7.72	[M-H]^-	253.050 9	253.050 6	1.19	224.048 5, 209.059 8	C₁₅H₁₀O₄	Daidzein
44	8.13	[M-H]^-	138.019 6	138.019 7	-0.72	108.021 6	C₆H₅NO₃	4-Nitrophenol
45	9.04	[M+H]⁺	187.038 8	187.039 0	-1.07	143.049 2, 131.049 0, 151.054 1, 88.021 5	C₁₁H₆O₃	Psoralen
46	9.52	[M-H]^-	221.081 8	221.081 9	-0.45	177.092 3, 121.029 5	C₁₂H₁₄O₄	Monobutylphthalate
47	10.07	[M-H]^-	321.113 1	321.113 2	-0.31	277.050 8, 265.050 6, 252.024 7	C₂₀H₁₈O₄	Neobavaisoflavone
48	10.11	[M-H]^-	295.228 1	295.227 9	0.68	277.217 7, 181.145 8	C₁₈H₃₂O₃	Hydroxy-octadecatrienoic acid
49	10.34	[M+H]⁺	473.144 0	473.144 2	-0.42	193.049 4, 149.023 5	C₂₄H₂₄O₁₀	1, 6-bis-O-[(2E)-3-(4-Hydroxyphenyl)-2-Propenoyl]- Β-D-Glucopyranose
50	10.67	[M+H]⁺	295.226 9	295.226 8	0.34	277.214 7	C₁₈H₃₀O₃	(9Z, 11E)-13-Oxooctadeca-9, 11-dienoicacid
51	11.07	[M+H]⁺	415.211 5	415.211 5	0.00	400.188 8, 119.085 5	C₂₄H₃₀O₆	Bis(4-ethylbenzylidene)sorbitol
52	11.18	[M+H]⁺	183.080 4	183.080 4	0.00	105.033 5	C₁₃H₁₀O	Benzophenone
53	11.30	[M-H]^-	269.045 4	269.045 5	-0.37	241.050 8, 225.055 4, 197.060 8	C₁₅H₁₀O₅	Genistein
54	11.40	[M-H]^-	255.232 6	255.232 9	-1.18	57.309 0	C₁₆H₃₂O₂	Palmitic acid
55	11.86	[M+H]⁺	339.158 2	339.159 1	-2.65	271.095 5, 219.101 3, 147.044 0, 119.049 0	C₂₁H₂₂O₄	Bavachinin
56	11.91	[M-H]^-	389.176 5	389.175 8	1.80	277.050 5, 265.050 6, 252.043 0	C₂₅H₂₆O₄	3-{3-[(2E)-3, 7-Dimethyl-2, 6-octadien-1-Yl]-4-hydroxyphenyl}-7-hydroxy-4H-chromen-4-one
57	12.79	[M+H]⁺	262.143 4	262.143 8	-1.53	149.344 1, 121.058 7	C₁₅H₁₉NO₃	MDPBP
58	12.96	[M+H]⁺	219.174 2	219.174 3	-0.46	177.127 0, 163.111 6, 123.080 5, 121.101 0	C₁₅H₂₂O	3, 5-di-tert-Butylbenzaldehyde

No.

t_R/min

Mode

Measured value

Theoretical value

ppm

MS/MS

Formula

Identification

1.43

[M-H]^-

131.046 1

131.046 2

-0.76

114.019 6, 95.025 0, 88.040 3

C₄H₈N₂O₃

Asparagine

1.44

[M-H]^-

132.030 1

132.030 2

-0.76

115.003 6, 114.019 6, 88.040 3, 71.013 8

C₄H₇NO₄

Aspartic acid

1.44

[M-H]^-

146.045 8

146.045 9

-0.55

128.035 2, 102.056 0

C₅H₉NO₄

Glutamic acid

1.47

[M-H]^-

195.050 9

195.051 0

-0.51

177.040 5, 129.019 3, 99.008 7

C₆H₁₂O₇

Gluconic acid

1.49

[M+H]⁺

127.039 0

127.038 9

0.79

109.028 4, 99.044 0, 97.028 4

C₆H₆O₃

5-Hydroxymethyl-2-furaldehyde

1.50

[M+H]⁺

116.070 6

0.00

98.984 1, 70.065 2

C₅H₉NO₂

D-Proline

1.56

[M-H]^-

191.056 1

0.00

173.045 3, 127.040 0, 85.029 4

C₇H₁₂O₆

Quinic acid

1.57

[M+H]⁺

112.050 5

0.00

95.024 0, 84.080 7, 69.044 8

C₄H₅N₃O

Cytosine

1.57

[M+H]⁺

166.086 2

166.086 3

-0.30

120.081 0, 103.054 6, 91.054 5

C₉H₁₁NO₂

Phenylalanine

1.58

[M+H]⁺

244.092 7

244.092 8

-0.41

227.067 2, 112.050 5

C₉H₁₃N₃O₅

Cytarabine

1.60

[M-H]^-

341.108 2

341.108 9

-2.05

179.055 5, 119.034 9, 101.024 3, 89.024 3

C₁₂H₂₂O₁₁

Trehalose

1.64

[M-H]^-

128.035 2

128.035 3

-0.78

82.029 7

C₅H₆NO₃

4-Oxoproline

1.64

[M-H]^-

133.014 1

133.014 2

-0.75

115.003 5, 89.024 4, 71.013 8

C₄H₆O₅

Malic acid

1.64

[M+H]⁺

130.086 3

130.086 2

0.77

84.080 7

C₆H₁₁NO₂

Pipecolic acid

1.65

[M-H]^-

115.003 4

115.003 7

-2.61

71.013 8

C₄H₄O₄

Fumaric acid

1.65

[M-H]^-

155.009 5

155.009 8

-1.94

111.019 9

C₅H₄N₂O₄

Orotic acid

1.90

[M-H]^-

312.109 6

312.108 9

2.34

161.044 8, 150.055 2, 113.023 4, 71.012 6

C₁₄H₁₉NO₇

Mandelic acid amide-β-glucopyranoside

1.95

[M-H]^-

474.163 5

474.161 7

3.80

263.078 6, 245.068 0, 221.066 8, 179.055 7, 161.044 9, 150.055 2

C₂₀H₂₉NO₁₂

Mandelic acid amide-β-gentiobioside

2.02

[M-H]^-

474.161 9

474.161 0

1.90

150.055 2

C₂₀H₂₉NO₁₂

Amygdalin amide

2.24

[M-H]^-

111.008 7

111.008 8

-0.90

83.013 9, 67.018 9

C₅H₄O₃

2-Furoic acid

2.25

[M+H]⁺

268.103 5

268.104 0

-1.86

136.061 8

C₁₀H₁₃N₅O₄

Adenosine

2.27

[M-H]^-

191.019 6

191.019 7

-0.52

173.019 6, 147.029 6, 129.019 3

C₆H₈O₇

Citric acid

2.28

[M+H]⁺

124.039 4

124.039 3

0.81

97.007 5, 80.049 4

C₆H₅NO₂

Nicotinic acid

2.42

[M+H]⁺

132.101 8

132.101 9

-0.76

86.096 4, 69.069 9

C₆H₁₃NO₂

L-Isoleucine

2.88

[M+H]⁺

123.055 4

123.055 2

1.63

106.073 3, 96.044 3, 80.049 5

C₆H₆N₂O

Nicotinamide

3.08

[M-H]^-

475.145 5

475.145 7

-0.44

431.154 1, 161.044 8, 113.022 4, 101.023 3, 89.023 2, 71.012 6

C₂₀H₂₈O₁₃

Mandelic acid-β-gentiobioside

3.58

[M-H]^-

431.156 3

431.155 9

0.97

269.102 9, 161.044 6, 113.022 3, 89.023 2, 71.012 6, 59.012 6

C₁₉H₂₈O₁₁

Benzyl-β-gentiobioside

3.69

[M-H]^-

151.039 3

151.040 1

-5.03

107.049 1, 59.012 7

C₈H₈O₃

Mandelic acid

4.21

[M+H]⁺

134.060 2

134.060 0

1.49

106.065 1, 79.054 2

C₈H₇NO

Oxindole

4.32

[M-H]^-

456.151 6

456.151 1

1.10

174.956 0, 161.044 9

C₂₀H₂₇NO₁₁

Neoamygdalin

4.93

[M+H]⁺

188.070 7

188.070 6

0.53

170.060 0, 142.065 1, 118.065 1

C₁₁H₉NO₂

Indoleacrylic acid

4.95

[M+H]⁺

146.060 1

146.060 0

0.68

118.065 1

C₉H₇NO

4-Indolecarbaldehyde

4.98

[M-H]^-

203.082 5

203.082 6

-0.49

186.055 6, 159.092 3, 142.066 1, 116.050 5

C₁₁H₁₂N₂O₂

Tryptophan

5.11

[M+H]⁺

433.221 3

433.222 1

-1.85

384.190 7, 369.168 4

C₂₄H₃₂O₇

Schisandrin

5.26

[M-H]^-

353.088 1

353.087 8

0.85

191.0056 1, 147.166 4

C₁₆H₁₈O₉

Chlorogenic acid

5.42

[M+H]⁺

296.112 2

296.112 8

-2.03

145.049 6, 134.060 0, 116.049 4

C₁₄H₁₇NO₆

Prunasin

5.43

[M-H]^-

456.151 6

456.151 1

1.02

221.066 2, 203.055 8, 179.055 4, 161.044 8

C₂₀H₂₇NO₁₁

Amygdalin

5.94

[M+H]⁺

173.107 3

0.00

172.995 2, 171.993 0

C₁₁H₁₂N₂

3, 5-Dimethyl-1-phenylpyrazole

6.46

[M-H]^-

173.081 9

0.00

155.071 0, 111.081 5

C₈H₁₄O₄

Suberic acid

6.98

[M-H]^-

221.104 4

0.00

165.041 6, 164.034 0

C₁₀H₁₄N₄O₂

3-Isobutyl-1-methylxanthine

6.98

[M+H]⁺

356.185 3

356.185 6

-0.84

340.152 2, 277.047 6, 192.101 8, 165.090 9

C₂₁H₂₅NO₄

Tetrahydropalmatine

7.65

[M+H]⁺

336.122 5

336.123 0

-1.49

320.091 0, 306.075 4, 278.081 5

C₂₀H₁₈NO₄

Berberine

7.72

[M-H]^-

253.050 9

253.050 6

1.19

224.048 5, 209.059 8

C₁₅H₁₀O₄

Daidzein

8.13

[M-H]^-

138.019 6

138.019 7

-0.72

108.021 6

C₆H₅NO₃

4-Nitrophenol

9.04

[M+H]⁺

187.038 8

187.039 0

-1.07

143.049 2, 131.049 0, 151.054 1, 88.021 5

C₁₁H₆O₃

Psoralen

9.52

[M-H]^-

221.081 8

221.081 9

-0.45

177.092 3, 121.029 5

C₁₂H₁₄O₄

Monobutylphthalate

10.07

[M-H]^-

321.113 1

321.113 2

-0.31

277.050 8, 265.050 6, 252.024 7

C₂₀H₁₈O₄

Neobavaisoflavone

10.11

[M-H]^-

295.228 1

295.227 9

0.68

277.217 7, 181.145 8

C₁₈H₃₂O₃

Hydroxy-octadecatrienoic acid

10.34

[M+H]⁺

473.144 0

473.144 2

-0.42

193.049 4, 149.023 5

C₂₄H₂₄O₁₀

1, 6-bis-O-[(2E)-3-(4-Hydroxyphenyl)-2-Propenoyl]- Β-D-Glucopyranose

10.67

[M+H]⁺

295.226 9

295.226 8

0.34

277.214 7

C₁₈H₃₀O₃

(9Z, 11E)-13-Oxooctadeca-9, 11-dienoicacid

11.07

[M+H]⁺

415.211 5

0.00

400.188 8, 119.085 5

C₂₄H₃₀O₆

Bis(4-ethylbenzylidene)sorbitol

11.18

[M+H]⁺

183.080 4

0.00

105.033 5

C₁₃H₁₀O

Benzophenone

11.30

[M-H]^-

269.045 4

269.045 5

-0.37

241.050 8, 225.055 4, 197.060 8

C₁₅H₁₀O₅

Genistein

11.40

[M-H]^-

255.232 6

255.232 9

-1.18

57.309 0

C₁₆H₃₂O₂

Palmitic acid

11.86

[M+H]⁺

339.158 2

339.159 1

-2.65

271.095 5, 219.101 3, 147.044 0, 119.049 0

C₂₁H₂₂O₄

Bavachinin

11.91

[M-H]^-

389.176 5

389.175 8

1.80

277.050 5, 265.050 6, 252.043 0

C₂₅H₂₆O₄

3-{3-[(2E)-3, 7-Dimethyl-2, 6-octadien-1-Yl]-4-hydroxyphenyl}-7-hydroxy-4H-chromen-4-one

12.79

[M+H]⁺

262.143 4

262.143 8

-1.53

149.344 1, 121.058 7

C₁₅H₁₉NO₃

MDPBP

12.96

[M+H]⁺

219.174 2

219.174 3

-0.46

177.127 0, 163.111 6, 123.080 5, 121.101 0

C₁₅H₂₂O

3, 5-di-tert-Butylbenzaldehyde

Compound	VIP value	P value	FC value
Fumaric acid	1.03	3.61E-13	3.34
Asparagine	1.67	4.74E-08	2.13
Indoleacrylic acid	1.00	7.66E-08	2.10
3, 5-di-tert-Butylbenzaldehyde	4.03	0.006 295	11.72
Amygdalin	2.31	0.010 183	7.16
Nicotinic acid	3.02	0.012 177	2.10
MDPBP	1.11	0.021 144	4.20
Gluconic acid	1.94	0.025 285	3.18
Glutamic acid	2.36	0.029 055	2.86
Palmitic acid	1.83	0.041 540	0.28
Nicotinamide	1.75	0.049 483	0.43

Compound

VIP value

P value

FC value

Fumaric acid

1.03

3.61E-13

3.34

Asparagine

1.67

4.74E-08

2.13

Indoleacrylic acid

1.00

7.66E-08

2.10

3, 5-di-tert-Butylbenzaldehyde

4.03

0.006 295

11.72

Amygdalin

2.31

0.010 183

7.16

Nicotinic acid

3.02

0.012 177

2.10

MDPBP

1.11

0.021 144

4.20

Gluconic acid

1.94

0.025 285

3.18

Glutamic acid

2.36

0.029 055

2.86

Palmitic acid

1.83

0.041 540

0.28

Nicotinamide

1.75

0.049 483

0.43

Compound	VIP value	P value	FC value
Nicotinamide	4.12	2.08E-08	0.28
Cytarabine	1.26	3.30E-08	0.42
Tryptophan	1.36	9.03E-08	0.42
Malic acid	2.31	6.87E-07	0.41
Fumaric acid	1.53	7.68E-07	0.40
Amygdalin	1.45	2.67E-06	0.43
1, 6-bis-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]-β-D-Glucopyranose	1.20	3.85E-06	0.41
Glutamic acid	1.50	1.17E-05	0.28
Gluconic acid	2.13	0.000 101	0.10
Prunasin	1.27	0.000 753	0.15
Adenosine	1.69	0.010 515	5.65
Proline	2.64	0.012 215	6.48
Bavachinin	1.21	0.028 265	5.94

Compound

VIP value

P value

FC value

Nicotinamide

4.12

2.08E-08

0.28

Cytarabine

1.26

3.30E-08

0.42

Tryptophan

1.36

9.03E-08

0.42

Malic acid

2.31

6.87E-07

0.41

Fumaric acid

1.53

7.68E-07

0.40

Amygdalin

1.45

2.67E-06

0.43

1, 6-bis-O-[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]-β-D-Glucopyranose

1.20

3.85E-06

0.41

Glutamic acid

1.50

1.17E-05

0.28

Gluconic acid

2.13

0.000 101

0.10

Prunasin

1.27

0.000 753

0.15

Adenosine

1.69

0.010 515

5.65

Proline

2.64

0.012 215

6.48

Bavachinin

1.21

0.028 265

5.94

Compound	0 W vs 2 W		2 W vs 4 W
Prunasin	1.82	1.26E-11	3.32		1.02	0.010 102	3.07
Tryptophan	1.14	1.09E-06	4.62	1.02	0.030 633	3.67
Amygdalin	1.18	3.89E-05	5.31	1.37	0.047 550	2.25
Fumaric acid	1.22	0.000 586	2.34	1.18	0.002 866	2.07
Gluconic acid	1.27	0.001 359	2.15	1.04	0.040 550	3.28
Adenosine	2.44	0.002 163	2.89	-	-	-
Pyroglutamic acid	1.37	0.002 780	2.10	2.33	0.040 751	5.96
Bavachinin	1.10	0.004 820	0.49	-	-	-
Proline	1.30	0.017 430	2.16	-	-	-
Oxindole	1.34	0.018 239	2.10	-	-	-
Glutamic acid	2.53	0.033 580	6.88	1.22	0.042 699	8.38
Palmitic acid	1.24	0.045 508	0.30	4.29	0.034 588	0.27
Cytosine	-	-	-	1.55	0.011 431	0.33

Compound

0 W vs 2 W

2 W vs 4 W

VIP value

P value

FC value

VIP value

P value

FC value

Prunasin

1.82

1.26E-11

3.32

1.02

0.010 102

3.07

Tryptophan

1.14

1.09E-06

4.62

1.02

0.030 633

3.67

Amygdalin

1.18

3.89E-05

5.31

1.37

0.047 550

2.25

Fumaric acid

1.22

0.000 586

2.34

1.18

0.002 866

2.07

Gluconic acid

1.27

0.001 359

2.15

1.04

0.040 550

3.28

Adenosine

2.44

0.002 163

2.89

Pyroglutamic acid

1.37

0.002 780

2.10

2.33

0.040 751

5.96

Bavachinin

1.10

0.004 820

0.49

Proline

1.30

0.017 430

2.16

Oxindole

1.34

0.018 239

2.10

Glutamic acid

2.53

0.033 580

6.88

1.22

0.042 699

8.38

Palmitic acid

1.24

0.045 508

0.30

4.29

0.034 588

0.27

Cytosine

1.55

0.011 431

0.33