药学学报

No.	Source	Mode	t_R/min	Compound	Formula	MS² fragment	EZW	Pe	Et	Bu	Aq	IG
Phenylethanol glycosides
1	L	[M-H]^-	7.92	Osmanthuside H	C₁₉H₂₈O₁₁	299.111 7, 149.044 8, 119.035 1, 89.024 7	+				+
2	L	[M-H]^-	5.56	3, 4-Dihydroxyphenyl ethanol-O-glucoside	C₁₄H₂₀O₈	153.055 1, 135.043 9, 123.045 0, 89.024 9	+				+
3^S	L	[M-H]^-	6.87	Salidroside	C₁₄H₂₀O₇	137.061 1, 119.050 1, 89.025 1, 59.016 0	+		+	+	+
4	L	[M-H]^-	16.01	Verbascoside	C₂₉H₃₆O₁₅	461.166 2, 179.034 6, 161.024 0, 135.046 0	+		+	+
Phenylethanol
5	L	[M-H]^-	5.11	3, 4-Dihydroxyphenylethanol	C₈H₁₀O₃	123.045 1, 109.030 2, 93.035 3, 81.035 8	+		+		+
Iridoids
6	L	[M-H]^-	10.04	Nuezhengalaside	C₁₈H₂₈O₉	255.062 9, 207.101 0, 119.029 0, 89.023 4	+
7	L	[M-H]^-	10.00	10-Hydroxyoleosidedimethylester	C₁₈H₂₆O₁₂	387.136 9, 225.075 1, 177.019 5, 101.023 1	+		+		+
8	L	[M-H]^-	25.6	Lucidumoside C	C₂₇H₃₆O₁₄	151.077 6, 59.014 9	+				+
9	L	[M-H]^-	6.12	Nuezhenidic acid	C₁₇H₂₄O₁₄	433.098 4, 271.046 0, 227.055 0, 151.040 2	+					+
10	L	[M-H]^-	14.9	Neonuezhenide	C₃₁H₄₂O₁₈	539.172 2, 469.132 0, 315.107 9, 135.044 6	+				+
11	L	[M-H]^-	9.61	Oleoside-11-methyl ester	C₁₇H₂₄O₁₁	223.060 8, 179.056 1, 119.034 9, 89.024 8	+		+	+	+	+
12	L	[M-H]^-	14.54	Oleuropeinic acid	C₂₅H₃₀O₁₅	363.107 8, 331.088 0, 209.046 1, 151.039 5	+		+	+	+	+
13^S	L	[M-H]^-	17.54	Specneuzhenide	C₃₁H₄₂O₁₇	523.180 1, 453.137 8, 299.111 4, 223.059 6	+		+	+	+	+
14	L	[M-H]^-	13.88	10-Hydroxyoleuropein	C₂₅H₃₂O₁₄	537.159 0, 403.129 7, 393.116 8, 291.093 8	+				+
15	L	[M-H]^-	21.84	Oleuropein	C₂₅H₃₂O₁₃	403.121 6, 377.123 4, 345.098 0, 307.080 2	+		+		+	+
Flavonoids
16	L	[M-H]^-	14.42	Rutoside	C₂₇H₃₀O₁₆	299.016 3, 283.012 4, 235.034 4, 177.019 6	+				+
17^S	L & E	[M-H]^-	32.15	Apigenin	C₁₅H₁₀O₅	227.032 8, 151.003 8, 149.024 2, 117.013 1	+		+	+
18^S	L & E	[M-H]^-	26.38	Luteolin	C₁₅H₁₀O₆	199.039 7, 175.039 3, 151.003 1, 133.029 1	+		+	+
[M+H]⁺	287.054 4, 161.023 5, 153.018 2, 135.043 8
19	E	[M-H]^-	12.76	Isoquercitrin	C₂₁H₂₀O₁₂	301.034 6, 299.018 4, 136.987 2	+			+
20	E	[M-H]^-	15.28	Luteoloside/quercitroside	C₂₁H₂₀O₁₁	285.039 2	+			+	+
[M+H]⁺	287.053 1
21	E	[M-H]^-	18.49	Apigenin-7-O-glucoside	C₂₁H₂₀O₁₀	269.043 9	+		+		+
22	L & E	[M+H]⁺	14.45	Quercetin	C₁₅H₁₀O₇	285.037 3, 197.026 5, 155.047 4, 137.057 3	+				+
23	L	[M+H]⁺	12.47	Taxifoliol	C₁₅H₁₂O₇	287.055 2, 167.032 4, 137.023 8, 109.030 4	+		+
24	E	[M+H]⁺	31.76	Hesperidin	C₂₈H₃₄O₁₅	593.185 6, 207.065 6, 193.049 6, 165.055 1	+				+
25	E	[M+H]⁺	31.76	Buddleoside	C₂₈H₃₂O₁₄	165.053 9, 151.038 2	+				+
Phenolic acid
26	E	[M-H]^-	6.32	Ethyl protocatechuate	C₉H₁₀O₄	163.039 7, 135.043 8, 119.048 9, 72.994 6	+		+		+
27	L	[M-H]^-	11.37	4-Hydroxycinnamic acid	C₉H₈O₃	119.050 4, 117.0341, 93.035 4, 65.040 9	+		+			+
28	E	[M-H]^-	6.55	4-Hydroxybenzoic acid	C₇H₆O₃	119.011 3, 109.028 3, 93.034 6, 91.018 5	+			+
29	L	[M-H]^-	4.45	Vanillic acid	C₈H₈O₄	149.022 6, 123.044 7, 109.028 9, 93.034 6	+		+		+	+
30	E	[M-H]^-	4.95	Protocatechuic acid	C₇H₆O₄	109.029 8, 95.050 3, 91.019 4	+		+
31	E	[M-H]^-	8.88	Cryptochlorogenic acid	C₁₆H₁₈O₉	191.055 6, 179.034 5, 173.045 1, 135.044 9	+			+
32	E	[M-H]^-	20.17	Cynarin	C₂₅H₂₄O₁₂	353.086 1, 191.054 6, 173.045 5, 135.045 1	+		+
Triterpenes
33	E	[M-H]^-	38.54	Eclalbasaponin II	C₃₆H₅₈O₉	587.397 6	+		+
34	E	[M-H]^-	35.3	Eclalbasaponin III/ecliptasaponin B	C₄₈H₇₈O₁₉	911.290 6, 837.452 4, 795.454 4	+
35	L	[M+H]⁺	45.04	Ethyl oleanolate/ethyl ursolate	C₃₂H₅₂O₃	467.321 4, 425.368 2, 185.135 4	+	+	+
36	L	[M+H]⁺	44.09	Acetyl oleanolic acid/acetyl ursolic acid	C₃₂H₅₀O₄	439.361 9	+	+
37	L	[M+H]⁺	46.47	Betulin	C₃₀H₅₀O	409.380 6, 219.212 0, 205.192 0, 81.072 2	+	+
38^S	L & E	[M+H]⁺	44.25	Oleanolic acid / ursolic acid	C₃₀H₄₈O₃	439.355 9, 411.362 2, 393.354 0, 153.090 1	+	+	+
39	L	[M+H]⁺	43.31	19α-Hydroxyl-3-acetyl-ursolic acid	C₃₂H₅₀O₅	497.380 1, 455.348 7, 265.171 3, 229.204 1	+	+
40	E	[M+H]⁺	41.37	3-Keto-16α-hydroxy-12-ene-28- oleanolic acid	C₃₀H₄₆O₄	425.340 1, 407.329 7, 317.209 1, 271.205 4	+	+	+
41	L	[M+H]⁺	41.49	2α-Hydroxyl oleanolic acid/ 2α-hydroxyl ursolic acid	C₃₀H₄₈O₄	455.353 5, 409.346 2, 391.335 3, 249.184 6	+	+	+
42	L	[M+H]⁺	43.02	3β-O-trans-p-Coumaroyl-2α- hydroxyloleanolic acid	C₃₉H₅₄O₆	455.351 9, 437.339 7, 411.324 4, 261.184 0	+		+			+
43	L	[M+H]⁺	39.53	Tormentic acid	C₃₀H₄₈O₅	471.340 8, 453.336 7, 443.349 2, 425.341 2	+		+
Coumestans
44	E	[M+H]⁺	17.97	Demethylwedelolactone/ isodemethylwedelolactone	C₁₅H₈O₇	283.023 1, 273.037 7, 255.027 6, 127.058 3	+			+
45	E	[M+H]⁺	28.12	Wedelolactone	C₁₆H₁₀O₇	297.037 7, 287.054 9, 269.044 0, 231.064 1	+		+
Others
46	L	[M-H]^-	0.84	Mannitol	C₃₂H₅₂O₃	163.060 8, 101.025 2, 89.025 3, 71.015 4	+			+
47	E	[M+H]⁺	2.71	Methyl gallate	C₈H₈O₅	167.033 4, 155.034 4, 137.023 3, 109.026 8	+
48	E	[M+H]⁺	13.16	Loliolide	C₁₁H₁₆O₃	179.106 1, 151.111 5, 137.098 7, 109.104 4	+		+
49	L	[M+H]⁺	3.47	5-Hydroxymethylfurfural	C₆H₆O₃	109.029 5, 97.032 5, 81.036 8, 53.043 8	+			+		+

No.	Source	Mode	t_R/min	Compound	Formula	MS² fragment	EZW	Pe	Et	Bu	Aq	IG
Phenylethanol glycosides
1	L	[M-H]^-	7.92	Osmanthuside H	C₁₉H₂₈O₁₁	299.111 7, 149.044 8, 119.035 1, 89.024 7	+				+
2	L	[M-H]^-	5.56	3, 4-Dihydroxyphenyl ethanol-O-glucoside	C₁₄H₂₀O₈	153.055 1, 135.043 9, 123.045 0, 89.024 9	+				+
3^S	L	[M-H]^-	6.87	Salidroside	C₁₄H₂₀O₇	137.061 1, 119.050 1, 89.025 1, 59.016 0	+		+	+	+
4	L	[M-H]^-	16.01	Verbascoside	C₂₉H₃₆O₁₅	461.166 2, 179.034 6, 161.024 0, 135.046 0	+		+	+
Phenylethanol
5	L	[M-H]^-	5.11	3, 4-Dihydroxyphenylethanol	C₈H₁₀O₃	123.045 1, 109.030 2, 93.035 3, 81.035 8	+		+		+
Iridoids
6	L	[M-H]^-	10.04	Nuezhengalaside	C₁₈H₂₈O₉	255.062 9, 207.101 0, 119.029 0, 89.023 4	+
7	L	[M-H]^-	10.00	10-Hydroxyoleosidedimethylester	C₁₈H₂₆O₁₂	387.136 9, 225.075 1, 177.019 5, 101.023 1	+		+		+
8	L	[M-H]^-	25.6	Lucidumoside C	C₂₇H₃₆O₁₄	151.077 6, 59.014 9	+				+
9	L	[M-H]^-	6.12	Nuezhenidic acid	C₁₇H₂₄O₁₄	433.098 4, 271.046 0, 227.055 0, 151.040 2	+					+
10	L	[M-H]^-	14.9	Neonuezhenide	C₃₁H₄₂O₁₈	539.172 2, 469.132 0, 315.107 9, 135.044 6	+				+
11	L	[M-H]^-	9.61	Oleoside-11-methyl ester	C₁₇H₂₄O₁₁	223.060 8, 179.056 1, 119.034 9, 89.024 8	+		+	+	+	+
12	L	[M-H]^-	14.54	Oleuropeinic acid	C₂₅H₃₀O₁₅	363.107 8, 331.088 0, 209.046 1, 151.039 5	+		+	+	+	+
13^S	L	[M-H]^-	17.54	Specneuzhenide	C₃₁H₄₂O₁₇	523.180 1, 453.137 8, 299.111 4, 223.059 6	+		+	+	+	+
14	L	[M-H]^-	13.88	10-Hydroxyoleuropein	C₂₅H₃₂O₁₄	537.159 0, 403.129 7, 393.116 8, 291.093 8	+				+
15	L	[M-H]^-	21.84	Oleuropein	C₂₅H₃₂O₁₃	403.121 6, 377.123 4, 345.098 0, 307.080 2	+		+		+	+
Flavonoids
16	L	[M-H]^-	14.42	Rutoside	C₂₇H₃₀O₁₆	299.016 3, 283.012 4, 235.034 4, 177.019 6	+				+
17^S	L & E	[M-H]^-	32.15	Apigenin	C₁₅H₁₀O₅	227.032 8, 151.003 8, 149.024 2, 117.013 1	+		+	+
18^S	L & E	[M-H]^-	26.38	Luteolin	C₁₅H₁₀O₆	199.039 7, 175.039 3, 151.003 1, 133.029 1	+		+	+
[M+H]⁺	287.054 4, 161.023 5, 153.018 2, 135.043 8
19	E	[M-H]^-	12.76	Isoquercitrin	C₂₁H₂₀O₁₂	301.034 6, 299.018 4, 136.987 2	+			+
20	E	[M-H]^-	15.28	Luteoloside/quercitroside	C₂₁H₂₀O₁₁	285.039 2	+			+	+
[M+H]⁺	287.053 1
21	E	[M-H]^-	18.49	Apigenin-7-O-glucoside	C₂₁H₂₀O₁₀	269.043 9	+		+		+
22	L & E	[M+H]⁺	14.45	Quercetin	C₁₅H₁₀O₇	285.037 3, 197.026 5, 155.047 4, 137.057 3	+				+
23	L	[M+H]⁺	12.47	Taxifoliol	C₁₅H₁₂O₇	287.055 2, 167.032 4, 137.023 8, 109.030 4	+		+
24	E	[M+H]⁺	31.76	Hesperidin	C₂₈H₃₄O₁₅	593.185 6, 207.065 6, 193.049 6, 165.055 1	+				+
25	E	[M+H]⁺	31.76	Buddleoside	C₂₈H₃₂O₁₄	165.053 9, 151.038 2	+				+
Phenolic acid
26	E	[M-H]^-	6.32	Ethyl protocatechuate	C₉H₁₀O₄	163.039 7, 135.043 8, 119.048 9, 72.994 6	+		+		+
27	L	[M-H]^-	11.37	4-Hydroxycinnamic acid	C₉H₈O₃	119.050 4, 117.0341, 93.035 4, 65.040 9	+		+			+
28	E	[M-H]^-	6.55	4-Hydroxybenzoic acid	C₇H₆O₃	119.011 3, 109.028 3, 93.034 6, 91.018 5	+			+
29	L	[M-H]^-	4.45	Vanillic acid	C₈H₈O₄	149.022 6, 123.044 7, 109.028 9, 93.034 6	+		+		+	+
30	E	[M-H]^-	4.95	Protocatechuic acid	C₇H₆O₄	109.029 8, 95.050 3, 91.019 4	+		+
31	E	[M-H]^-	8.88	Cryptochlorogenic acid	C₁₆H₁₈O₉	191.055 6, 179.034 5, 173.045 1, 135.044 9	+			+
32	E	[M-H]^-	20.17	Cynarin	C₂₅H₂₄O₁₂	353.086 1, 191.054 6, 173.045 5, 135.045 1	+		+
Triterpenes
33	E	[M-H]^-	38.54	Eclalbasaponin II	C₃₆H₅₈O₉	587.397 6	+		+
34	E	[M-H]^-	35.3	Eclalbasaponin III/ecliptasaponin B	C₄₈H₇₈O₁₉	911.290 6, 837.452 4, 795.454 4	+
35	L	[M+H]⁺	45.04	Ethyl oleanolate/ethyl ursolate	C₃₂H₅₂O₃	467.321 4, 425.368 2, 185.135 4	+	+	+
36	L	[M+H]⁺	44.09	Acetyl oleanolic acid/acetyl ursolic acid	C₃₂H₅₀O₄	439.361 9	+	+
37	L	[M+H]⁺	46.47	Betulin	C₃₀H₅₀O	409.380 6, 219.212 0, 205.192 0, 81.072 2	+	+
38^S	L & E	[M+H]⁺	44.25	Oleanolic acid / ursolic acid	C₃₀H₄₈O₃	439.355 9, 411.362 2, 393.354 0, 153.090 1	+	+	+
39	L	[M+H]⁺	43.31	19α-Hydroxyl-3-acetyl-ursolic acid	C₃₂H₅₀O₅	497.380 1, 455.348 7, 265.171 3, 229.204 1	+	+
40	E	[M+H]⁺	41.37	3-Keto-16α-hydroxy-12-ene-28- oleanolic acid	C₃₀H₄₆O₄	425.340 1, 407.329 7, 317.209 1, 271.205 4	+	+	+
41	L	[M+H]⁺	41.49	2α-Hydroxyl oleanolic acid/ 2α-hydroxyl ursolic acid	C₃₀H₄₈O₄	455.353 5, 409.346 2, 391.335 3, 249.184 6	+	+	+
42	L	[M+H]⁺	43.02	3β-O-trans-p-Coumaroyl-2α- hydroxyloleanolic acid	C₃₉H₅₄O₆	455.351 9, 437.339 7, 411.324 4, 261.184 0	+		+			+
43	L	[M+H]⁺	39.53	Tormentic acid	C₃₀H₄₈O₅	471.340 8, 453.336 7, 443.349 2, 425.341 2	+		+
Coumestans
44	E	[M+H]⁺	17.97	Demethylwedelolactone/ isodemethylwedelolactone	C₁₅H₈O₇	283.023 1, 273.037 7, 255.027 6, 127.058 3	+			+
45	E	[M+H]⁺	28.12	Wedelolactone	C₁₆H₁₀O₇	297.037 7, 287.054 9, 269.044 0, 231.064 1	+		+
Others
46	L	[M-H]^-	0.84	Mannitol	C₃₂H₅₂O₃	163.060 8, 101.025 2, 89.025 3, 71.015 4	+			+
47	E	[M+H]⁺	2.71	Methyl gallate	C₈H₈O₅	167.033 4, 155.034 4, 137.023 3, 109.026 8	+
48	E	[M+H]⁺	13.16	Loliolide	C₁₁H₁₆O₃	179.106 1, 151.111 5, 137.098 7, 109.104 4	+		+
49	L	[M+H]⁺	3.47	5-Hydroxymethylfurfural	C₆H₆O₃	109.029 5, 97.032 5, 81.036 8, 53.043 8	+			+		+

Compound	-Cdocker energy /kcal·mol^-1	-Cdocker interaction energy/kcal·mol^-1	Hydrogen bond interaction	Hydrophobic interaction
Rapamycin	2.10	63.45	ASP37, GLN53, VAL55, ILE56, TYR82	TYR26, PHE36, PHE46, VAL55, TRP59, TYR82, HIS87, ILE90, ILE91
Everolimus	-21.33	44.40	TYR26, LYS47, GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, TRP59, TYR82, PHE99
Temsirolimus	-16.42	60.89	ASP37, LYS52, TYR82	LYS52, VAL55, ILE56, TYR82, HIS87
Rutoside	-11.20	36.34	GLU54	TYR26, PHE46
Luteolin	23.31	26.50	PHE46, GLU54	VAL55, ILE56
Apigenin-7-O-glucoside	-14.08	35.60	ASP37, GLN53, VAL55, ILE56, TYR82	HIS87
4-Hydroxycinnamic acid	17.96	20.88	GLU54, TRP59	VAL55, TRP59
5-Hydroxymethylfurfural	11.20	44.74	PHE36, ASP37, PHE46, GLN53, GLU54, TYR82	PHE46, VAL55, TRP59, ILE90
Verbascoside	1.03	27.83	ASP37, LYS47, GLU54	ASP37, LYS47, GLU54
Wedelolactone	0.08	23.77	ILE56, TRP59	VAL55, ILE56, TRP59
Demethylwedelolactone	1.85	23.43	ILE56	PHE46, VAL55, ILE56, TYR82
Oleoside-11-methyl ester	14.24	17.26	GLU54	VAL55, ILE56
Oleuropein	3.24	26.23	TYR26, TYR82, HIS87	TYR26, VAL55, TRP59
Eclalbasaponin III	3.18	20.41	GLU54, ILE56, TYR82	-
Ursolic acid	-1.41	26.22	GLU54, HIS87	TYR82, HIS87, ILE90
2α-Hydroxyl oleanolic acid	-9.58	38.72	GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, HIS87
Ecliptasaponin B	17.50	20.09	GLU54	TYR26, VAL55, ILE56

Compound	-Cdocker energy /kcal·mol^-1	-Cdocker interaction energy/kcal·mol^-1	Hydrogen bond interaction	Hydrophobic interaction
Rapamycin	2.10	63.45	ASP37, GLN53, VAL55, ILE56, TYR82	TYR26, PHE36, PHE46, VAL55, TRP59, TYR82, HIS87, ILE90, ILE91
Everolimus	-21.33	44.40	TYR26, LYS47, GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, TRP59, TYR82, PHE99
Temsirolimus	-16.42	60.89	ASP37, LYS52, TYR82	LYS52, VAL55, ILE56, TYR82, HIS87
Rutoside	-11.20	36.34	GLU54	TYR26, PHE46
Luteolin	23.31	26.50	PHE46, GLU54	VAL55, ILE56
Apigenin-7-O-glucoside	-14.08	35.60	ASP37, GLN53, VAL55, ILE56, TYR82	HIS87
4-Hydroxycinnamic acid	17.96	20.88	GLU54, TRP59	VAL55, TRP59
5-Hydroxymethylfurfural	11.20	44.74	PHE36, ASP37, PHE46, GLN53, GLU54, TYR82	PHE46, VAL55, TRP59, ILE90
Verbascoside	1.03	27.83	ASP37, LYS47, GLU54	ASP37, LYS47, GLU54
Wedelolactone	0.08	23.77	ILE56, TRP59	VAL55, ILE56, TRP59
Demethylwedelolactone	1.85	23.43	ILE56	PHE46, VAL55, ILE56, TYR82
Oleoside-11-methyl ester	14.24	17.26	GLU54	VAL55, ILE56
Oleuropein	3.24	26.23	TYR26, TYR82, HIS87	TYR26, VAL55, TRP59
Eclalbasaponin III	3.18	20.41	GLU54, ILE56, TYR82	-
Ursolic acid	-1.41	26.22	GLU54, HIS87	TYR82, HIS87, ILE90
2α-Hydroxyl oleanolic acid	-9.58	38.72	GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, HIS87
Ecliptasaponin B	17.50	20.09	GLU54	TYR26, VAL55, ILE56

二至丸及不同极性部位含药血清对H₂O₂诱导的肝细胞衰老的保护作用及物质基础研究

PDF下载

冯丽 , 王乙妃 , 高梦婷 , 李鑫 , 姚卫峰 ^* , 张丽

药学学报 | 研究论文 2021,56(4): 1137-1146

收起

药学学报 | 研究论文 2021, 56(4): 1137-1146

二至丸及不同极性部位含药血清对H₂O₂诱导的肝细胞衰老的保护作用及物质基础研究

全屏

冯丽, 王乙妃, 高梦婷, 李鑫, 姚卫峰^*, 张丽

作者信息

南京中医药大学, 江苏省中药资源产业化过程协同创新中心/中药资源产业化与方剂创新药物国家地方联合工程研究中心, 江苏南京 210023

通讯作者:

*姚卫峰, Tel: 86-25-85811053, E-mail: yaowf@njucm.edu.cn

The protective effect of drug-containing serums and polar extracts of Erzhi Wan on H₂O₂-induced hepatocyte senescence

Li FENG, Yi-fei WANG, Meng-ting GAO, Xin LI, Wei-feng YAO^*, Li ZHANG

Affiliations

Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China

出版时间: 2021-04-12 doi: 10.16438/j.0513-4870.2021-0071

文章导航

摘要

收起

利用H₂O₂诱导的大鼠肝细胞(BRL)衰老模型探究二至丸及其不同极性部位(石油醚、乙酸乙酯、正丁醇、水和环烯醚萜苷类富集部位)含药血清的抗衰老作用。通过MTT法检测细胞增殖，β-半乳糖苷酶染色实验评估细胞衰老程度，流式细胞术检测细胞内活性氧(reactive oxygen species，ROS)的改变。采用快速液相系统-飞行时间质谱(ultra-fast liquid chromatography coupled with quadrupole-time of flight-mass spectrometry，UFLC-Q/TOF-MS)对二至丸及各部位化学成分进行分析，并结合分子对接技术筛选二至丸中可能的抗衰老活性成分。结果显示，600 μmol·L^-1的H₂O₂处理细胞72 h后可显著诱导细胞衰老，引起细胞增殖抑制、细胞内β-半乳糖苷酶活力和ROS水平的升高。细胞在诱导衰老前预先用二至丸含药血清处理可有效抵抗增殖抑制。除环烯醚萜苷类，二至丸及其他部位含药血清均可降低细胞内β-半乳糖苷酶活力和ROS水平。利用液质联用技术从二至丸中共鉴定出了49个化学成分并对各部位中的成分进行了比较。通过分子对接筛选出二至丸中14个可能的抗衰老活性组分。结合细胞实验、成分鉴定及分子对接结果，本实验初步推断二至丸抗衰老的有效成分为三萜类、黄酮类和苯乙醇类，为阐明二至丸抗衰老药效物质基础及作用机制提供了参考。

关键词

二至丸 / 不同极性部位 / 肝细胞衰老 / 化学成分 / 分子对接

Abstract

收起

Using a H₂O₂-induced BRL cell senescence model, we investigated the anti-aging effects of drug-containing serums of Erzhi Wan (EZW) and various polar extracts (petroleum ether, ethyl acetate, n-butanol, water, and iridoid glycoside-enriched fractions). Cell viability was detected by MTT assay. Cell senescence was evaluated with β-galactosidase staining assay. Intracellular reactive oxygen species (ROS) were measured by flow cytometry. UFLC-Q-TOF-MS/MS was used to identify chemical components in EZW and the extracts, and molecular docking technology was used to predict the anti-aging components of EZW. Results showed that treatment of cells with 600 μmol·L^-1 H₂O₂ for 72 h markedly induced cell senescence, inhibited cell proliferation and increased intracellular β-galactosidase activity and ROS levels. If cells were pretreated with drug-containing serum of EZW this induction of senescence was decreased. A total of 49 chemical compounds were identified in EZW by liquid chromatography-mass spectrometry, 14 of these were identified by molecular docking as potential active ingredients. Based on these analyses, and the in vitro experiments with polar extracts, we conclude that the anti-aging components of EZW are triterpenes, flavonoids and phenyl alcohols, providing a basis for further elucidation of the active agents and mechanism of the anti-aging effect of EZW.

Key words

Erzhi Wan / different solvent extract / hepatocyte senescence / chemical constituent / molecular docking

引用本文

冯丽, 王乙妃, 高梦婷, 李鑫, 姚卫峰, 张丽. 二至丸及不同极性部位含药血清对H₂O₂诱导的肝细胞衰老的保护作用及物质基础研究. 药学学报, 2021 , 56 (4) : 1137 -1146 . DOI: 10.16438/j.0513-4870.2021-0071

Li FENG, Yi-fei WANG, Meng-ting GAO, Xin LI, Wei-feng YAO, Li ZHANG. The protective effect of drug-containing serums and polar extracts of Erzhi Wan on H₂O₂-induced hepatocyte senescence[J]. Acta Pharmaceutica Sinica, 2021 , 56 (4) : 1137 -1146 . DOI: 10.16438/j.0513-4870.2021-0071

正文

收起

衰老是人体伴随年龄增加而发生的自身内环境稳定能力和应激能力下降, 机体结构、功能和机能退行性病变的系列不可逆转的生理、病理现象^[1]。衰老并非疾病, 但却是多种老年疾病, 如心血管疾病、神经退行性疾病、糖尿病、癌症等的致病因素^[2]。延缓机体衰老对于提升个人生活质量具有重要意义, 因此已经成为世界范围内研究的重要科学问题^[3]。中医认为机体衰老的主要原因是五脏虚衰、阴阳失衡^[4]。肝是保持人体气机正常升降出入的重要脏腑^{[5, 6]}。在古籍《灵枢·天年》中记载着“五十岁, 肝气始衰”, 明确指出肝为衰老的始动因子, 肝气衰可导致其他脏腑功能受损^[7]。近年来, 哺乳动物雷帕霉素靶蛋白/雷帕霉素机能靶蛋白(mammalian/mechanistic target of rapamycin, mTOR), 一类丝/苏氨酸蛋白激酶, 成为了研究如何抑制衰老, 提高人类寿命的重要靶点^[8-10]。

二至丸是由女贞子和墨旱莲两味中药等比组成, 临床上常用于治疗肝肾阴虚所引起的肝肾疾病。本课题组前期通过体外实验已经表明了二至丸含药血清对于衰老肾细胞的保护作用^[11]。为了进一步探索二至丸的抗肝细胞衰老作用, 本文将基于H₂O₂诱导的肝细胞衰老模型, 结合血清药理学对二至丸、其分段提取物以及特征性成分群——环烯醚萜苷类的抗衰老作用进行研究。利用LC-MS/MS技术鉴定与分析二至丸及各部位中的成分差异, 并以mTOR为对接受体, 通过分子对接技术快速筛选二至丸中可能的抗衰老成分, 为阐明二至丸抗衰老的药效物质基础和后期作用机制研究提供实验依据。

材料与方法

收起

药物与试剂 含药血清为实验室自制。PBS (美国Hyclone公司, 批号AE29456443); DMEM (美国Gibco公司, 批号8120286); 0.25%胰蛋白酶(美国Gibco公司, 批号2120736); 胎牛血清(FBS) (杭州四季青公司, 批号11011-8611); 链霉素-青霉素(双抗, 上海碧云天生物技术有限公司, 批号2185224); MTT (上海阿拉丁试剂有限公司, 批号C2025115); H₂O₂ (美国Sigma公司, 批号STBH9407); 细胞衰老β-半乳糖苷酶染色试剂盒(上海碧云天生物技术有限公司, 批号043020200526); 二氯二氢荧光素-乙酰乙酸酯(2′, 7′-dichlorodihydrofluorescein diacetate, DCFH-DA) 活性氧检测试剂盒(上海碧云天生物技术有限公司, 批号041720200930)。女贞子和墨旱莲均购自铜陵禾田中药饮片股份有限公司, 由南京中医药大学中医药吴啟南教授鉴定分别为木犀科植物女贞(Ligustrum lucidum Ait.) 的干燥成熟果实和菊科植物鲤肠(Eclipta prostrata L.) 的干燥地上部分。

仪器 Shimadzu快速液相色谱仪(日本岛津公司, 包括LC-20AD XR型泵, SIL-20A XR型自动进样器, CTO-20AC型柱温箱); Triple-TOF^TM 5600⁺型质谱仪(美国AB Sciex公司, 包括Analyst TF 1.6数据采集工作站和Peak View 1.2数据分析软件); CO₂恒温培养箱(美国Thermo Fisher公司); 超净工作台(苏州苏净公司); Dmi8倒置荧光显微镜(德国Leica公司); 光谱扫描多功能酶标仪(澳大利亚Tecan公司); 细胞计数仪Contess^®II FL (美国Thermo Fisher公司); 旋转蒸发仪(巩义宇翔仪器有限公司); 真空干燥箱(上海和晟仪器科技有限公司); 流式细胞仪(美国BD公司)。

动物与细胞 实验方案经南京中医药大学实验动物福利与伦理委员会审查(批准号: 201906A029); 清洁级SD大鼠购于浙江省医学科学院, 生产许可证号SCXK (浙) 2019-0002, 雄性, 体重220~260 g, 饲养于南京中医药大学实验动物中心屏障环境设施中。大鼠肝(BRL) 细胞株购于中国科学院典型培养物保藏委员会细胞库。

二至丸样品及提取物的制备 按照文献^[12]优化的炮制工艺制备酒女贞子, 超微粉碎。取墨旱莲药材适量, 加入10倍量水(w/v) 煎煮2次, 每次1 h, 过滤后合并滤液, 浓缩减压干燥得浸膏。按生药量1∶1混合酒女贞子超微粉与墨旱莲浸膏。按照文献^[11]提取石油醚、乙酸乙酯、正丁醇和水提部位, 即取二至丸样品适量, 加少许水混匀, 按质量1∶1加入硅藻土拌匀, 减压干燥。加入10倍量(w/v) 石油醚回流提取至无色, 每次1 h, 过滤, 合并滤液, 减压干燥成浸膏。将滤渣于通风橱挥干, 按照石油醚部位提取步骤依次用乙酸乙酯、正丁醇和水提取, 最终得各部位浸膏。环烯醚萜苷类成分在二至丸中的含量较高, 且其中的特女贞苷是《中国药典》中二至丸质量评价的指标成分, 故单独提取此类成分以研究其生物活性, 其提取方法参考文献^{[13, 14]}, 即取女贞子适量, 粉碎, 加入14倍量(w/v) 70%乙醇回流提取3次, 每次2.5 h, 过滤浓缩, 滤液上D101型大孔树脂, 用5倍柱体积(bed volume, BV) 水洗脱, 5 BV 50%乙醇以2 BV·h^-1流速洗脱, 收集乙醇洗脱液, 减压回收乙醇, 50 ℃减压干燥得浸膏。

含药血清的制备 用0.5% CMC-Na溶液配制二至丸灌胃溶液使浓度为0.36 g·mL^-1。同法, 根据得率, 按同等生药量配制不同部位灌胃溶液。SD雄性大鼠随机分成7组(空白组、二至丸组和不同极性部位组), 每组8只。各组按10 mL·kg^-1给予灌胃溶液, 空白对照组给予相同剂量的0.9%生理盐水, 每日2次, 连续7天。于末次灌胃1 h后腹主动脉采血, 分离血清, 56 ℃水浴灭活30 min, 用0.22 μm滤膜过滤后分装, 储存-20 ℃备用。鉴于前期研究 ^{[11, 15]}基础, 本实验以20%的大鼠血清进行研究, 按照DMEM培养基∶大鼠血清∶双抗= 79∶20∶1比例混匀即得浓度为20%的空白血清或含药血清, 保存于4 ℃备用。

细胞培养及传代 购得的BRL细胞株经复苏后用含10% FBS和1%双抗的DMEM完全培养基于37 ℃、5% CO₂恒温培养箱中培养。细胞每2天更换一次新鲜培养基, 当细胞密度达到80%~90%时, 用0.25%胰蛋白酶消化, 按照1∶4的比例传代, 均取对数生长期细胞进行实验。

MTT实验 取第6代对数生长期BRL细胞按3×10³细胞/孔均匀地接种于96孔板中, 设置对照组和实验组, 每组复6孔。用完全培养基稀释H₂O₂至浓度为200、400、600、1 000和2 000 μmol·L^-1。待细胞完全贴壁后, 实验组分别加入相应浓度的H₂O₂溶液100 μL处理48 h或72 h, 对照组加入等体积的新鲜培养基, 每24 h换液一次。结束后每孔避光加入MTT (5 mg·mL^-1) 20 μL, 继续培养4 h。吸弃上清液后, 每孔加入DMSO 150 μL, 轻摇10 min, 用酶标仪在490 nm处测定各孔的吸光度值。细胞存活率计算公式为: 细胞存活率(%) = (A_样品组/A_对照组)×100%

H₂O₂干预浓度及时间筛选 将BRL细胞按5×10⁴细胞/孔均匀地接种于6孔板中。根据MTT实验结果, 设置对照组和H₂O₂不同浓度组(200、400和600 μmol·L^-1), 处理48 h或72 h, 每组复6孔。待培养结束后, 每组3孔按β-半乳糖苷酶染色试剂盒说明进行细胞染色, 于37 ℃无CO₂培养箱中避光孵育24 h。第二天用倒置显微镜每孔随机取3个视野观测细胞, 计算细胞阳性染色率。另外各组3孔按照活性氧检测试剂盒说明先收集细胞再装载探针, 再在CO₂培养箱中培养20 min, PBS洗涤细胞2次后, 用PBS重悬细胞。然后用流式细胞仪检测, 参数设置为FITC。相对荧光强度为样品组平均荧光强度与对照组平均荧光强度的比值。

二至丸含药血清对细胞增殖的影响 将BRL细胞按1×10³细胞/孔均匀地接种于96孔板中, 每组复6孔。设置对照组(A组)、后造模组(B组)、预防组(C组)、先造模组(D组)、治疗组(E组)。待细胞贴壁后, B组和C组分别先用完全培养基或20%二至丸含药血清培养48 h, 后用600 μmol·L^-1 H₂O₂处理72 h; 而D组和E组先用600 μmol·L^-1 H₂O₂处理72 h, 后再用完全培养基或20%二至丸含药血清培养48 h。对照组(A组) 始终以完全培养基培养。结束后MTT法检测细胞增殖。

二至丸及不同极性部位含药血清抗衰老作用研究 将BRL细胞按5×10⁴细胞/孔均匀地接种于6孔板中, 设置对照组、模型组和给药组(二至丸及不同极性部位含药血清组), 每组复6孔。模型组和给药组先分别用20%空白血清或含药血清培养48 h, 后用600 μmol·L^-1 H₂O₂处理72 h。对照组始终以完全培养基培养。培养结束后各组3孔进行β-半乳糖苷酶染色, 另外3孔检测细胞内ROS水平。

LC-MS/MS样品处理 取二至丸粉末0.8 g, 精密称定, 置具塞锥形瓶中, 精密加入80%甲醇25 mL, 密塞, 称定重量。超声(功率600 W, 频率40 kHz) 30 min, 放冷, 再称定重量, 用80%甲醇补足减失重量, 摇匀。取适量14 000 r·min^-1离心10 min, 上清进样分析。不同部位浸膏根据得率按与二至丸相同生药量称取, 同法制备。分别取齐墩果酸、熊果酸、木犀草素、芹菜素、特女贞苷和红景天苷对照品适量, 精密称定, 加80%甲醇制成浓度为100 μg·mL^-1的混合溶液。

LC-MS/MS分析条件 色谱条件为Poroshell 120 EC-C18色谱柱(100 mm×2.1 mm, 2.7 μm), 流动相0.1%甲酸水(A)-0.1%甲酸乙腈溶液(B) 梯度洗脱(0~10 min, 2%~15% B; 10~30 min, 15%~25% B; 30~35 min, 25%~35% B; 35~40 min, 35%~75% B; 40~46 min, 75%~95% B; 46~48 min, 95%~2% B), 流速0.3 mL·min^-1, 柱温30 ℃, 进样量4 μL。质谱条件为电喷雾离子源(electrospray ionization, ESI), 正负离子扫描方式, 扫描范围m/z 50~1 500。正离子检测方式为气帘气40 psi (1 psi ≈ 6.9 kPa), 雾化气55 psi, 辅助气55 psi, 电喷雾电压5.5 kV, 离子源温度550 ℃, 去簇电压100 V, 母离子碰撞电压10 V, 子离子碰撞电压40 V, 碰撞电压差20 V; 负离子检测方式除电喷雾电压为-4.5 kV, 其余参数同正离子检测仅电压值为负。

分子对接 将二至丸中鉴定到的化学成分保存为mol2格式文件, 然后将化合物导入Discovery Studio 4.0软件中加CHARMm力场进行能量优化, 保存作为配体分子集。参考文献^[16], 选取靶蛋白mTOR (INSG) 为受体。利用Discovery Studio 4.0软件对蛋白晶体进行删除水分子和经Prepare Protein工具处理。然后定义蛋白为受体, 以原配体位置为中心修改sphere球半径为14.5, 以此作为结合口袋并保留蛋白质A链用于后续分子对接。从Drug Bank网站(https://www.drugbank.ca) 中搜索与mTOR靶点有关的已上市小分子药物, 将2个抑制剂坦西莫司和依维莫司以及原配体作为参考配体集, 处理方法同配体分子。利用CDOCKER模块进行对, 将对接参数Pose Cluster Radius设为0.5 Å (1 Å = 10^-10 m), 其余均为默认值。以对照药的平均-Cdocker energy值的80%为阈值, 筛选配体分子集中的化合物。用Analyze Ligands Pose模块分析对照药和配体化合物与靶点的作用模式, 进一步根据配体与靶蛋白相互作用的残基至少有3个及以上与对照药相同原则筛选活性化合物。

统计学方法 采用Prism 8软件进行处理, 实验数据均以x±s表示。两组间数据若均符合正态分布采用非配对t-test检验; 若不都符合正态分布采用非配对Mann-Whitney U检验, P < 0.05为有显著性差异。

结果

收起

1 肝细胞衰老模型的建立

1.1 H₂O₂细胞毒性考察

由图 1可知, H₂O₂能够抑制BRL细胞增殖, 且随着H₂O₂浓度的升高抑制作用越强。对比作用时间48 h与72 h, 可以看出用200~600 μmol·L^-1 H₂O₂处理细胞并没有使细胞停止增殖, 只是随着H₂O₂浓度和作用时间的增加, 细胞的增殖速率下降, 但当H₂O₂浓度大于1 000 μmol·L^-1时, 细胞几乎停止增殖。因1 000 μmol·L^-1以上的H₂O₂对细胞增殖的抑制作用太强, 所以本研究选择200~600 μmol·L^-1的H₂O₂进行后续实验。

1.2 H₂O₂最佳干预浓度和时间

根据染色结果可知(图 2), 正常的BRL细胞经200~600 μmol·L^-1 H₂O₂处理48 h或72 h后, β-半乳糖苷酶染色阳性率均有显著升高, 并且细胞染色阳性率与H₂O₂浓度和作用时间呈一定的正相关。

利用荧光探针DCFH-DA评估细胞内ROS水平是否受到干扰。DCFH-DA可以自由穿过细胞膜且本身没有荧光, 进入细胞后可被酯酶水解生成不能通透细胞膜的DCFH。而细胞内ROS可以氧化DCFH生成有荧光的DCF, 进而可通过检测荧光强度确定细胞内ROS水平。如图 3所示, H₂O₂作用于细胞后, 细胞内的ROS水平有所上升, 且表现出一定的剂量与时间依赖性。在各个组中, 仅当H₂O₂浓度为600 μmol·L^-1, 作用时间为72 h时, 细胞内ROS水平与对照组相比具有统计学意义。故本实验选定用600 μmol·L^-1 H₂O₂处理72 h来诱导BRL细胞衰老。

2 二至丸含药血清造模前与造模后给药对细胞增殖的影响

由图 4可见, 与对照组(A组) 相比, 细胞经H₂O₂处理后(B和D组), 其生长被明显抑制。而若在H₂O₂干预细胞前, 先让细胞与二至丸含药血清作用48 h (C组), 细胞活力与对照组相比无明显差异。但在H₂O₂干预细胞后再与含药血清作用(E组), 没有改善H₂O₂对细胞的增殖抑制。故本实验选择在H₂O₂诱导细胞衰老前先用含药血清预处理48 h。

3 二至丸及不同部位含药血清抗衰老作用评价

从图 5可以看出, 模型组的细胞染色较对照组更明显, 表明H₂O₂成功诱导了肝细胞衰老。根据染色的量化结果可见, 除环烯醚萜苷类组细胞染色阳性率较模型组没有下降外, 其余组均有不同程度的改善, 其中水提部位组效果最好, 且较模型组有显著差异。

从图 6可知, 模型组较对照组荧光强度显著升高, 表明衰老细胞内ROS水平的上升。给药组中除了环烯醚萜苷类组的荧光强度与模型组相比没有显著差异外, 其余组均有显著降低, 表明不同部位含药血清均可降低衰老细胞内ROS水平。

4 化学成分分析

使用Peak View软件对正负离子模式下采集的质谱数据进行分析, 从二至丸中共鉴定到49个化合物, 包含5个苯乙醇类、10个环烯醚萜苷类、10个黄酮类、7个酚酸类、11个三萜类、2个香豆草醚类和4个其他成分。具体结果见表 1。

5 分子对接结果

靶点活性口袋的半径为14.5 Å, 坐标为-9.48、26.884、36.385 Å。原配体对接回结合口袋, 对接构象与原配体结构之间的均方根偏差(RMSD) 值为0.582 7, RMSD ≤ 2 Å, 表明对接程序参数设置合理^[17]。根据参考配体的-Cdocker energy值以及参考配体跟衰老靶点

mTOR相互作用的残基筛选二至丸中可能的活性成分, 共筛选到了4个三萜类, 3个黄酮类, 香豆草醚类和环烯醚萜苷类各2个, 苯乙醇类、酚酸和醛类各1个。表 2列出了筛选出的活性化合物, 以及它们与靶蛋白相互作用模式和相互作用的氨基酸残基。

讨论

收起

越来越多的研究表明细胞衰老对于衰老进程的驱动作用, 衰老细胞在体内的逐渐积累直接引起器官的衰竭和衰老表型的出现^{[18, 19]}。细胞衰老可以分为复制性衰老、癌源性衰老和应激性衰老等, 其中应激诱导的细胞衰老已经被广泛用于了解衰老过程和研究衰老的调控机制。H₂O₂是一种膜通透性的ROS, 它在细胞内能生成大量的羟自由基, 进而引起体内的氧化应激、炎症和免疫反应, 致使机体产生衰老相关的生理病理变化^[20]。研究表明, 非致死性浓度的H₂O₂诱导的衰老与自然衰老的特征相似^[21]。因此, 本研究选择利用H₂O₂去建立肝细胞衰老模型。许多研究表明, 线粒体ROS的产量与最大寿命呈负相关, 长寿动物的线粒体ROS产量低于短寿动物^{[22, 23]}。而β-半乳糖苷酶活力是一种经典的细胞衰老表征^[24]。因此本研究以β-半乳糖苷酶活力和ROS水平作为评价指标去建立肝细胞衰老模型以及研究二至丸及不同极性部位的抗衰老作用。

在本研究中确定了H₂O₂诱导肝细胞衰老的最佳浓度和时间。利用已建立的模型发现二至丸含药血清在造模前给药可以有效地预防H₂O₂导致的细胞增殖抑制, 但造模后给药这种效果不显著, 表明二至丸的预防作用强于治疗作用。本研究还评估了二至丸及不同部位含药血清的抗衰老作用, 结果显示除环烯醚萜苷类富集部位, 其余各部位均有一定的药效作用, 其中水提部位效果最好。Yan等^[25]研究发现二至丸水提物对CCl₄和H₂O₂诱导的肝细胞损伤具有保护作用。体内研究发现二至丸水提物对CCl₄所致小鼠急性肝损伤也具有保护作用^[26]。这些报道表明本研究利用衰老细胞模型筛选中药活性部位具有一定的可靠性。

利用LC-MS/MS技术比较分析了二至丸及各部位中的成分, 进一步根据分子对接结果以及各成分在二至丸中的含量, 推断黄酮、三萜以及苯乙醇类可能为抗衰老的有效成分。根据对接结果, 三萜类如旱莲苷III、熊果酸、2α-羟基齐墩果酸和旱莲苷B, 黄酮类如芦丁、木犀草素和芹菜素-7-O-吡喃葡萄糖苷, 苯乙醇类如毛蕊花糖苷, 可能是二至丸的有效成分。已有研究表明, 天然的三萜、黄酮类以及对羟基苯乙醇具有抗衰老作用。三萜类和苯乙醇类的抗衰老机制与它们影响了多种长寿以及抗衰老的蛋白和基因有关^[27-30]。黄酮类化合物的抗衰老机制是其具有的多个酚羟基能与自由基反应生成较稳定的半醌式自由基, 从而终止自由基链式反应^[31]。石油醚部位鉴定到的三萜类较多, 而乙酸乙酯、正丁醇和水提部位鉴定到的苯乙醇及黄酮类较多, 这揭示了各部位可能的抗衰老机制。此外, 有研究表明, 女贞子多糖能降低D-半乳糖诱导的小鼠心、肝和肾器官的衰老程度^[32]。因此水提部位显著的抗衰老作用可能还与多糖有关。

为了更为全面地筛选二至丸可能的抗衰老活性组分, 作者将二至丸中鉴定到的所有成分均与近年来关注度较高的抗衰老靶蛋白mTOR进行了对接, 后期也将对二至丸及各部位含药血清中的入血原形成分及代谢产物进行鉴定, 并对虚拟筛选结果进行进一步的生物学验证。综上, 本研究借助细胞模型、分子对接技术和液质联用技术揭示了二至丸可能的抗衰老活性成分, 为进一步的二至丸抗衰老活性成分研究和机制研究提供了依据。

作者贡献: 冯丽负责资料调研、实验和写作论文; 王乙妃、高梦婷和李鑫参与实验; 姚卫峰和张丽提出想法、设计实验、指导并修改论文。

利益冲突: 本文作者声明无利益冲突。

基金

收起

国家自然科学基金资助项目(81973445)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

Carmona

, Michan

. Biology of healthy aging and longevity[J]. Rev Invest Clin, 2016, 68: 7-16. http://europepmc.org/abstract/MED/27028172

[2]

Lopez-Otin

, Blasco

, Partridge

et al . The hallmarks of aging[J]. Cell, 2013, 153: 1194-1217.

https://doi.org/10.1016/j.cell.2013.05.039

[3]

Zhang

, Li

, Jiang

et al . ROS factors in aging hypothesis[J]. Prog Physiol Sci (生理科学进展), 2020, 51: 327-331.

[4]

Sheng

, Wei

, Yuan

et al . Study on the relationship between the signs of senile aging and TCM syndromes[J]. J Tradit Chin Med (中医杂志), 1997, 10: 621-622.

[5]

Wang

. Regulating Qi by the liver is the prerequisite for nourishing the kidney, strengthening the spleen and anti-aging[J]. Inf Tradit Chin Med (中医药信息), 2012, 29: 8-9.

[6]

Liu

, Zhan

, Zhao

et al . Study on process of aging induced by oxidative stress in normal human body based on theory of liver[J]. Chin Arch Tradit Chin Med (中华中医药学刊), 2019, 37: 1352-1355.

[7]

Guan

, Gao

, Wang

et al . Exploration on the relation of five internal organs and senility[J]. Acta Chin Med (中医学报), 2015, 30: 1433-1435. http://en.cnki.com.cn/Article_en/CJFDTOTAL-HNZK201510018.htm

[8]

, Liu

. mTOR signaling in aging and aging-associated diseases[J]. Prog Biochem Biophys (生物化学与生物物理进展), 2014, 41: 257-265. http://www.researchgate.net/publication/286494977_mTOR_Signaling_in_Aging_and_Aging-associated_Diseases

[9]

Johnson

, Rabinovitch

, Kaeberlein

. mTOR is a key modulator of ageing and age-related disease[J]. Nature, 2013, 493: 338-345.

https://doi.org/10.1038/nature11861

[10]

Kennedy

, Lamming

. The mechanistic target of rapamycin: the grand conducTOR of metabolism and aging[J]. Cell Metab, 2016, 23: 990-1003.

https://doi.org/10.1016/j.cmet.2016.05.009

[11]

Liu QN. The Protective Function and Cell Metabolomics Study of Erzhiwan on Ageing Kidney Cells (二至丸对衰老肾细胞的保护作用及细胞代谢组学研究)[D]. Nangjing: Nanjing University of Chinese Medicine, 2018.

[12]

, Liu

, Zhang

et al . Optimization of processing technology of Ligustri Lucidi Fructus stewed with wine based on analytic[J]. Chin Tradit Herb Drugs (中草药), 2016, 47: 2832-2837. http://www.zhangqiaokeyan.com/academic-journal-cn_chinese-traditional-herbal-drugs_thesis/0201219011834.html

[13]

Shen

, Ma

, Zhang

et al . Optimization of purification process of iridoid total glycosides from Ligustri Lucidi Fructus[J]. Chin Tradit Pat Med (中成药), 2018, 40: 1403-1406.

[14]

Yang

, Yi

, Ma

et al . Optimization of extraction process for iridoid glycosides from Ligustrum lucidum by multi-index orthogonal test[J]. China Pharm (中国药房), 2016, 27: 3970-3973. http://en.cnki.com.cn/Article_en/CJFDTotal-ZGYA201628027.htm

[15]

Xu J. Protective Effect of Erzhiwan on Senescent Kidney Cells-based on "Homogeny of Liver and Kidney" and Cell Metabolomics (基于"肝肾同源"理论和细胞代谢组学研究二至丸对衰老肾细胞的保护作用)[D]. Nangjing: Nanjing University of Chinese Medicine, 2019.

[16]

, Zhang

, Ye

et al . Screening of anti-aging active ingredients and mechanism analysis based on molecular docking technology[J]. China J Chin Mater Med (中国中药杂志), 2016, 41: 2522-2526. http://europepmc.org/abstract/MED/28905579

[17]

Lin

, Li

et al . Screening of active anti-myocardial ischemia components of Panax notoginseng based on molecular docking technology[J]. China J Chin Mater Med (中国中药杂志), 2020, 45: 2560-2567. http://www.researchgate.net/publication/342748015_Screening_of_active_anti-myocardial_ischemia_components_of_Panax_notoginseng_based_on_molecular_docking_technology

[18]

Naina

, Kanherkar

, Stair

et al . Cellular senescence as the causal nexus of aging[J]. Front Genet, 2016, 7: 13 http://pubmedcentralcanada.ca/pmcc/articles/PMC4751276/

[19]

Baker

, Childs

, Durik

et al . Naturally occurring p16^Ink4a-positive cells shorten healthy lifespan[J]. Nature, 2016, 530: 184-189.

https://doi.org/10.1038/nature16932

[20]

Zhou

, Yan

, Gao

et al . Study on aging animal models and its application in activity screening of anti-aging drugs[J]. Chin Tradit Herb Drugs (中草药), 2017, 48: 1061-1071. http://www.researchgate.net/publication/321667955_Study_on_aging_animal_models_and_its_application_in_activity_screening_of_anti-aging_drugs

[21]

Chen

, Ozanne

, Hales

. Methods of cellular senescence induction using oxidative stress[J]. Methods Mol Biol, 2013, 1048: 135-144. http://www.ncbi.nlm.nih.gov/pubmed/17634582

[22]

Biala

, Dhingra

, Kirshenbaum

. Mitochondrial dynamics: orchestrating the journey to advanced age[J]. J Mol Cell Cardiol, 2015, 83: 37-43.

https://doi.org/10.1016/j.yjmcc.2015.04.015

[23]

Lopez-Lluch

, Hunt

, Jones

et al . Calorie restriction induces mitochondrial biogenesis and bioen-ergetic efficiency[J]. Proc Natl Acad Sci U S A, 2006, 103: 1768-1773.

https://doi.org/10.1073/pnas.0510452103

[24]

Lee

, Han

, Im

et al . Senescence-associated beta-galactosidase is lysosomal beta-galactosidase[J]. Aging Cell, 2006, 5: 187-195.

https://doi.org/10.1111/j.1474-9726.2006.00199.x

[25]

Yan

, Ding

, Zhang

. Protective effects of aqueous extract of Erzhi pill on hepatocytes injured by CCl₄ or H₂O₂[J]. J Pharm Pract (药学实践杂志), 2010, 28: 433-436. http://www.zhangqiaokeyan.com/academic-journal-cn_journal-pharmaceutical-practice_thesis/0201241553498.html

[26]

Yan

, Ding

, Zhang

. Protective effect of aqueous extract of Erzhi pill against acute hepatic injury induced by xarbon tetrachloride[J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2010, 16: 131-134. http://en.cnki.com.cn/Article_en/CJFDTotal-ZSFX201014042.htm

[27]

Cañuelo

, Esteban

, Peragón

. Gene expression profiling to investigate tyrosol-induced lifespan extension in Caenorhabditis elegans[J]. Eur J Nutr, 2016, 55: 639-650.

https://doi.org/10.1007/s00394-015-0884-3

[28]

Mukherjee

, Lekli

, Gurusamy

et al . Expression of the longevity proteins by both red and white wines and their cardioprotective components, resveratrol, tyrosol, and hydroxytyrosol[J]. Free Radic Biol Med, 2009, 46: 573-578.

https://doi.org/10.1016/j.freeradbiomed.2008.11.005

[29]

Staats

, Wagner

, Lüersen

. Dietary ursolic acid improves health span and life span in male Drosophila melanogaster[J]. Biofactors, 2019, 45: 169-186.

https://doi.org/10.1002/biof.1467

[30]

Bahrami

, Bakhtiari

. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level[J]. Biomed Pharmacother, 2016, 82: 8-14.

https://doi.org/10.1016/j.biopha.2016.04.047

[31]

Huang

, Luo

, Bin

. Study on the relation between structure and antioxygenic activity of flavonoid[J]. J Guangdong Univ Technol (广东工业大学学报), 2000, 2: 71-75. http://en.cnki.com.cn/Article_en/CJFDTOTAL-GDGX200002016.htm

[32]

Zhang

, Ge

, Xu

et al . Antisenile effect of polysaccharides from Fructus Ligustri Lucidi[J]. Chin J Pharmacol Toxicol (中国药理学与毒理学杂志), 2006, 20: 108-111. http://en.cnki.com.cn/Article_en/CJFDTOTAL-YLBS200602004.htm

2021年第56卷第4期

PDF下载

141

引用本文

BibTeX

文章信息

doi: 10.16438/j.0513-4870.2021-0071

接收时间：2021-01-13
首发时间：2025-12-18
出版时间：2021-04-12

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2021-01-13
修回日期：2021-02-22

基金

国家自然科学基金资助项目(81973445)

作者信息

南京中医药大学, 江苏省中药资源产业化过程协同创新中心/中药资源产业化与方剂创新药物国家地方联合工程研究中心, 江苏南京 210023

通讯作者:

*姚卫峰, Tel: 86-25-85811053, E-mail: yaowf@njucm.edu.cn

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2021-0071

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

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

关闭全屏

BibTeX
EndNote
RefWorks
TxT

No.	Source	Mode	t_R/min	Compound	Formula	MS² fragment	EZW	Pe	Et	Bu	Aq	IG
Phenylethanol glycosides
1	L	[M-H]^-	7.92	Osmanthuside H	C₁₉H₂₈O₁₁	299.111 7, 149.044 8, 119.035 1, 89.024 7	+				+
2	L	[M-H]^-	5.56	3, 4-Dihydroxyphenyl ethanol-O-glucoside	C₁₄H₂₀O₈	153.055 1, 135.043 9, 123.045 0, 89.024 9	+				+
3^S	L	[M-H]^-	6.87	Salidroside	C₁₄H₂₀O₇	137.061 1, 119.050 1, 89.025 1, 59.016 0	+		+	+	+
4	L	[M-H]^-	16.01	Verbascoside	C₂₉H₃₆O₁₅	461.166 2, 179.034 6, 161.024 0, 135.046 0	+		+	+
Phenylethanol
5	L	[M-H]^-	5.11	3, 4-Dihydroxyphenylethanol	C₈H₁₀O₃	123.045 1, 109.030 2, 93.035 3, 81.035 8	+		+		+
Iridoids
6	L	[M-H]^-	10.04	Nuezhengalaside	C₁₈H₂₈O₉	255.062 9, 207.101 0, 119.029 0, 89.023 4	+
7	L	[M-H]^-	10.00	10-Hydroxyoleosidedimethylester	C₁₈H₂₆O₁₂	387.136 9, 225.075 1, 177.019 5, 101.023 1	+		+		+
8	L	[M-H]^-	25.6	Lucidumoside C	C₂₇H₃₆O₁₄	151.077 6, 59.014 9	+				+
9	L	[M-H]^-	6.12	Nuezhenidic acid	C₁₇H₂₄O₁₄	433.098 4, 271.046 0, 227.055 0, 151.040 2	+					+
10	L	[M-H]^-	14.9	Neonuezhenide	C₃₁H₄₂O₁₈	539.172 2, 469.132 0, 315.107 9, 135.044 6	+				+
11	L	[M-H]^-	9.61	Oleoside-11-methyl ester	C₁₇H₂₄O₁₁	223.060 8, 179.056 1, 119.034 9, 89.024 8	+		+	+	+	+
12	L	[M-H]^-	14.54	Oleuropeinic acid	C₂₅H₃₀O₁₅	363.107 8, 331.088 0, 209.046 1, 151.039 5	+		+	+	+	+
13^S	L	[M-H]^-	17.54	Specneuzhenide	C₃₁H₄₂O₁₇	523.180 1, 453.137 8, 299.111 4, 223.059 6	+		+	+	+	+
14	L	[M-H]^-	13.88	10-Hydroxyoleuropein	C₂₅H₃₂O₁₄	537.159 0, 403.129 7, 393.116 8, 291.093 8	+				+
15	L	[M-H]^-	21.84	Oleuropein	C₂₅H₃₂O₁₃	403.121 6, 377.123 4, 345.098 0, 307.080 2	+		+		+	+
Flavonoids
16	L	[M-H]^-	14.42	Rutoside	C₂₇H₃₀O₁₆	299.016 3, 283.012 4, 235.034 4, 177.019 6	+				+
17^S	L & E	[M-H]^-	32.15	Apigenin	C₁₅H₁₀O₅	227.032 8, 151.003 8, 149.024 2, 117.013 1	+		+	+
18^S	L & E	[M-H]^-	26.38	Luteolin	C₁₅H₁₀O₆	199.039 7, 175.039 3, 151.003 1, 133.029 1	+		+	+
[M+H]⁺	287.054 4, 161.023 5, 153.018 2, 135.043 8
19	E	[M-H]^-	12.76	Isoquercitrin	C₂₁H₂₀O₁₂	301.034 6, 299.018 4, 136.987 2	+			+
20	E	[M-H]^-	15.28	Luteoloside/quercitroside	C₂₁H₂₀O₁₁	285.039 2	+			+	+
[M+H]⁺	287.053 1
21	E	[M-H]^-	18.49	Apigenin-7-O-glucoside	C₂₁H₂₀O₁₀	269.043 9	+		+		+
22	L & E	[M+H]⁺	14.45	Quercetin	C₁₅H₁₀O₇	285.037 3, 197.026 5, 155.047 4, 137.057 3	+				+
23	L	[M+H]⁺	12.47	Taxifoliol	C₁₅H₁₂O₇	287.055 2, 167.032 4, 137.023 8, 109.030 4	+		+
24	E	[M+H]⁺	31.76	Hesperidin	C₂₈H₃₄O₁₅	593.185 6, 207.065 6, 193.049 6, 165.055 1	+				+
25	E	[M+H]⁺	31.76	Buddleoside	C₂₈H₃₂O₁₄	165.053 9, 151.038 2	+				+
Phenolic acid
26	E	[M-H]^-	6.32	Ethyl protocatechuate	C₉H₁₀O₄	163.039 7, 135.043 8, 119.048 9, 72.994 6	+		+		+
27	L	[M-H]^-	11.37	4-Hydroxycinnamic acid	C₉H₈O₃	119.050 4, 117.0341, 93.035 4, 65.040 9	+		+			+
28	E	[M-H]^-	6.55	4-Hydroxybenzoic acid	C₇H₆O₃	119.011 3, 109.028 3, 93.034 6, 91.018 5	+			+
29	L	[M-H]^-	4.45	Vanillic acid	C₈H₈O₄	149.022 6, 123.044 7, 109.028 9, 93.034 6	+		+		+	+
30	E	[M-H]^-	4.95	Protocatechuic acid	C₇H₆O₄	109.029 8, 95.050 3, 91.019 4	+		+
31	E	[M-H]^-	8.88	Cryptochlorogenic acid	C₁₆H₁₈O₉	191.055 6, 179.034 5, 173.045 1, 135.044 9	+			+
32	E	[M-H]^-	20.17	Cynarin	C₂₅H₂₄O₁₂	353.086 1, 191.054 6, 173.045 5, 135.045 1	+		+
Triterpenes
33	E	[M-H]^-	38.54	Eclalbasaponin II	C₃₆H₅₈O₉	587.397 6	+		+
34	E	[M-H]^-	35.3	Eclalbasaponin III/ecliptasaponin B	C₄₈H₇₈O₁₉	911.290 6, 837.452 4, 795.454 4	+
35	L	[M+H]⁺	45.04	Ethyl oleanolate/ethyl ursolate	C₃₂H₅₂O₃	467.321 4, 425.368 2, 185.135 4	+	+	+
36	L	[M+H]⁺	44.09	Acetyl oleanolic acid/acetyl ursolic acid	C₃₂H₅₀O₄	439.361 9	+	+
37	L	[M+H]⁺	46.47	Betulin	C₃₀H₅₀O	409.380 6, 219.212 0, 205.192 0, 81.072 2	+	+
38^S	L & E	[M+H]⁺	44.25	Oleanolic acid / ursolic acid	C₃₀H₄₈O₃	439.355 9, 411.362 2, 393.354 0, 153.090 1	+	+	+
39	L	[M+H]⁺	43.31	19α-Hydroxyl-3-acetyl-ursolic acid	C₃₂H₅₀O₅	497.380 1, 455.348 7, 265.171 3, 229.204 1	+	+
40	E	[M+H]⁺	41.37	3-Keto-16α-hydroxy-12-ene-28- oleanolic acid	C₃₀H₄₆O₄	425.340 1, 407.329 7, 317.209 1, 271.205 4	+	+	+
41	L	[M+H]⁺	41.49	2α-Hydroxyl oleanolic acid/ 2α-hydroxyl ursolic acid	C₃₀H₄₈O₄	455.353 5, 409.346 2, 391.335 3, 249.184 6	+	+	+
42	L	[M+H]⁺	43.02	3β-O-trans-p-Coumaroyl-2α- hydroxyloleanolic acid	C₃₉H₅₄O₆	455.351 9, 437.339 7, 411.324 4, 261.184 0	+		+			+
43	L	[M+H]⁺	39.53	Tormentic acid	C₃₀H₄₈O₅	471.340 8, 453.336 7, 443.349 2, 425.341 2	+		+
Coumestans
44	E	[M+H]⁺	17.97	Demethylwedelolactone/ isodemethylwedelolactone	C₁₅H₈O₇	283.023 1, 273.037 7, 255.027 6, 127.058 3	+			+
45	E	[M+H]⁺	28.12	Wedelolactone	C₁₆H₁₀O₇	297.037 7, 287.054 9, 269.044 0, 231.064 1	+		+
Others
46	L	[M-H]^-	0.84	Mannitol	C₃₂H₅₂O₃	163.060 8, 101.025 2, 89.025 3, 71.015 4	+			+
47	E	[M+H]⁺	2.71	Methyl gallate	C₈H₈O₅	167.033 4, 155.034 4, 137.023 3, 109.026 8	+
48	E	[M+H]⁺	13.16	Loliolide	C₁₁H₁₆O₃	179.106 1, 151.111 5, 137.098 7, 109.104 4	+		+
49	L	[M+H]⁺	3.47	5-Hydroxymethylfurfural	C₆H₆O₃	109.029 5, 97.032 5, 81.036 8, 53.043 8	+			+		+

No.

Source

Mode

t_R/min

Compound

Formula

MS² fragment

EZW

Phenylethanol glycosides

[M-H]^-

7.92

Osmanthuside H

C₁₉H₂₈O₁₁

299.111 7, 149.044 8, 119.035 1, 89.024 7

[M-H]^-

5.56

3, 4-Dihydroxyphenyl ethanol-O-glucoside

C₁₄H₂₀O₈

153.055 1, 135.043 9, 123.045 0, 89.024 9

3^S

[M-H]^-

6.87

Salidroside

C₁₄H₂₀O₇

137.061 1, 119.050 1, 89.025 1, 59.016 0

[M-H]^-

16.01

Verbascoside

C₂₉H₃₆O₁₅

461.166 2, 179.034 6, 161.024 0, 135.046 0

Phenylethanol

[M-H]^-

5.11

3, 4-Dihydroxyphenylethanol

C₈H₁₀O₃

123.045 1, 109.030 2, 93.035 3, 81.035 8

Iridoids

[M-H]^-

10.04

Nuezhengalaside

C₁₈H₂₈O₉

255.062 9, 207.101 0, 119.029 0, 89.023 4

[M-H]^-

10.00

10-Hydroxyoleosidedimethylester

C₁₈H₂₆O₁₂

387.136 9, 225.075 1, 177.019 5, 101.023 1

[M-H]^-

25.6

Lucidumoside C

C₂₇H₃₆O₁₄

151.077 6, 59.014 9

[M-H]^-

6.12

Nuezhenidic acid

C₁₇H₂₄O₁₄

433.098 4, 271.046 0, 227.055 0, 151.040 2

[M-H]^-

14.9

Neonuezhenide

C₃₁H₄₂O₁₈

539.172 2, 469.132 0, 315.107 9, 135.044 6

[M-H]^-

9.61

Oleoside-11-methyl ester

C₁₇H₂₄O₁₁

223.060 8, 179.056 1, 119.034 9, 89.024 8

[M-H]^-

14.54

Oleuropeinic acid

C₂₅H₃₀O₁₅

363.107 8, 331.088 0, 209.046 1, 151.039 5

13^S

[M-H]^-

17.54

Specneuzhenide

C₃₁H₄₂O₁₇

523.180 1, 453.137 8, 299.111 4, 223.059 6

[M-H]^-

13.88

10-Hydroxyoleuropein

C₂₅H₃₂O₁₄

537.159 0, 403.129 7, 393.116 8, 291.093 8

[M-H]^-

21.84

Oleuropein

C₂₅H₃₂O₁₃

403.121 6, 377.123 4, 345.098 0, 307.080 2

Flavonoids

[M-H]^-

14.42

Rutoside

C₂₇H₃₀O₁₆

299.016 3, 283.012 4, 235.034 4, 177.019 6

17^S

L & E

[M-H]^-

32.15

Apigenin

C₁₅H₁₀O₅

227.032 8, 151.003 8, 149.024 2, 117.013 1

18^S

L & E

[M-H]^-

26.38

Luteolin

C₁₅H₁₀O₆

199.039 7, 175.039 3, 151.003 1, 133.029 1

[M+H]⁺

287.054 4, 161.023 5, 153.018 2, 135.043 8

[M-H]^-

12.76

Isoquercitrin

C₂₁H₂₀O₁₂

301.034 6, 299.018 4, 136.987 2

[M-H]^-

15.28

Luteoloside/quercitroside

C₂₁H₂₀O₁₁

285.039 2

[M+H]⁺

287.053 1

[M-H]^-

18.49

Apigenin-7-O-glucoside

C₂₁H₂₀O₁₀

269.043 9

L & E

[M+H]⁺

14.45

Quercetin

C₁₅H₁₀O₇

285.037 3, 197.026 5, 155.047 4, 137.057 3

[M+H]⁺

12.47

Taxifoliol

C₁₅H₁₂O₇

287.055 2, 167.032 4, 137.023 8, 109.030 4

[M+H]⁺

31.76

Hesperidin

C₂₈H₃₄O₁₅

593.185 6, 207.065 6, 193.049 6, 165.055 1

[M+H]⁺

31.76

Buddleoside

C₂₈H₃₂O₁₄

165.053 9, 151.038 2

Phenolic acid

[M-H]^-

6.32

Ethyl protocatechuate

C₉H₁₀O₄

163.039 7, 135.043 8, 119.048 9, 72.994 6

[M-H]^-

11.37

4-Hydroxycinnamic acid

C₉H₈O₃

119.050 4, 117.0341, 93.035 4, 65.040 9

[M-H]^-

6.55

4-Hydroxybenzoic acid

C₇H₆O₃

119.011 3, 109.028 3, 93.034 6, 91.018 5

[M-H]^-

4.45

Vanillic acid

C₈H₈O₄

149.022 6, 123.044 7, 109.028 9, 93.034 6

[M-H]^-

4.95

Protocatechuic acid

C₇H₆O₄

109.029 8, 95.050 3, 91.019 4

[M-H]^-

8.88

Cryptochlorogenic acid

C₁₆H₁₈O₉

191.055 6, 179.034 5, 173.045 1, 135.044 9

[M-H]^-

20.17

Cynarin

C₂₅H₂₄O₁₂

353.086 1, 191.054 6, 173.045 5, 135.045 1

Triterpenes

[M-H]^-

38.54

Eclalbasaponin II

C₃₆H₅₈O₉

587.397 6

[M-H]^-

35.3

Eclalbasaponin III/ecliptasaponin B

C₄₈H₇₈O₁₉

911.290 6, 837.452 4, 795.454 4

[M+H]⁺

45.04

Ethyl oleanolate/ethyl ursolate

C₃₂H₅₂O₃

467.321 4, 425.368 2, 185.135 4

[M+H]⁺

44.09

Acetyl oleanolic acid/acetyl ursolic acid

C₃₂H₅₀O₄

439.361 9

[M+H]⁺

46.47

Betulin

C₃₀H₅₀O

409.380 6, 219.212 0, 205.192 0, 81.072 2

38^S

L & E

[M+H]⁺

44.25

Oleanolic acid / ursolic acid

C₃₀H₄₈O₃

439.355 9, 411.362 2, 393.354 0, 153.090 1

[M+H]⁺

43.31

19α-Hydroxyl-3-acetyl-ursolic acid

C₃₂H₅₀O₅

497.380 1, 455.348 7, 265.171 3, 229.204 1

[M+H]⁺

41.37

3-Keto-16α-hydroxy-12-ene-28- oleanolic acid

C₃₀H₄₆O₄

425.340 1, 407.329 7, 317.209 1, 271.205 4

[M+H]⁺

41.49

2α-Hydroxyl oleanolic acid/ 2α-hydroxyl ursolic acid

C₃₀H₄₈O₄

455.353 5, 409.346 2, 391.335 3, 249.184 6

[M+H]⁺

43.02

3β-O-trans-p-Coumaroyl-2α- hydroxyloleanolic acid

C₃₉H₅₄O₆

455.351 9, 437.339 7, 411.324 4, 261.184 0

[M+H]⁺

39.53

Tormentic acid

C₃₀H₄₈O₅

471.340 8, 453.336 7, 443.349 2, 425.341 2

Coumestans

[M+H]⁺

17.97

Demethylwedelolactone/ isodemethylwedelolactone

C₁₅H₈O₇

283.023 1, 273.037 7, 255.027 6, 127.058 3

[M+H]⁺

28.12

Wedelolactone

C₁₆H₁₀O₇

297.037 7, 287.054 9, 269.044 0, 231.064 1

Others

[M-H]^-

0.84

Mannitol

C₃₂H₅₂O₃

163.060 8, 101.025 2, 89.025 3, 71.015 4

[M+H]⁺

2.71

Methyl gallate

C₈H₈O₅

167.033 4, 155.034 4, 137.023 3, 109.026 8

[M+H]⁺

13.16

Loliolide

C₁₁H₁₆O₃

179.106 1, 151.111 5, 137.098 7, 109.104 4

[M+H]⁺

3.47

5-Hydroxymethylfurfural

C₆H₆O₃

109.029 5, 97.032 5, 81.036 8, 53.043 8

Compound	-Cdocker energy /kcal·mol^-1	-Cdocker interaction energy/kcal·mol^-1	Hydrogen bond interaction	Hydrophobic interaction
Rapamycin	2.10	63.45	ASP37, GLN53, VAL55, ILE56, TYR82	TYR26, PHE36, PHE46, VAL55, TRP59, TYR82, HIS87, ILE90, ILE91
Everolimus	-21.33	44.40	TYR26, LYS47, GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, TRP59, TYR82, PHE99
Temsirolimus	-16.42	60.89	ASP37, LYS52, TYR82	LYS52, VAL55, ILE56, TYR82, HIS87
Rutoside	-11.20	36.34	GLU54	TYR26, PHE46
Luteolin	23.31	26.50	PHE46, GLU54	VAL55, ILE56
Apigenin-7-O-glucoside	-14.08	35.60	ASP37, GLN53, VAL55, ILE56, TYR82	HIS87
4-Hydroxycinnamic acid	17.96	20.88	GLU54, TRP59	VAL55, TRP59
5-Hydroxymethylfurfural	11.20	44.74	PHE36, ASP37, PHE46, GLN53, GLU54, TYR82	PHE46, VAL55, TRP59, ILE90
Verbascoside	1.03	27.83	ASP37, LYS47, GLU54	ASP37, LYS47, GLU54
Wedelolactone	0.08	23.77	ILE56, TRP59	VAL55, ILE56, TRP59
Demethylwedelolactone	1.85	23.43	ILE56	PHE46, VAL55, ILE56, TYR82
Oleoside-11-methyl ester	14.24	17.26	GLU54	VAL55, ILE56
Oleuropein	3.24	26.23	TYR26, TYR82, HIS87	TYR26, VAL55, TRP59
Eclalbasaponin III	3.18	20.41	GLU54, ILE56, TYR82	-
Ursolic acid	-1.41	26.22	GLU54, HIS87	TYR82, HIS87, ILE90
2α-Hydroxyl oleanolic acid	-9.58	38.72	GLN53, GLU54, VAL55, TYR82	VAL55, ILE56, HIS87
Ecliptasaponin B	17.50	20.09	GLU54	TYR26, VAL55, ILE56

Compound

-Cdocker energy /kcal·mol^-1

-Cdocker interaction energy/kcal·mol^-1

Hydrogen bond interaction

Hydrophobic interaction

Rapamycin

2.10

63.45

ASP37, GLN53, VAL55, ILE56, TYR82

TYR26, PHE36, PHE46, VAL55, TRP59, TYR82, HIS87, ILE90, ILE91

Everolimus

-21.33

44.40

TYR26, LYS47, GLN53, GLU54, VAL55, TYR82

VAL55, ILE56, TRP59, TYR82, PHE99

Temsirolimus

-16.42

60.89

ASP37, LYS52, TYR82

LYS52, VAL55, ILE56, TYR82, HIS87

Rutoside

-11.20

36.34

GLU54

TYR26, PHE46

Luteolin

23.31

26.50

PHE46, GLU54

VAL55, ILE56

Apigenin-7-O-glucoside

-14.08

35.60

ASP37, GLN53, VAL55, ILE56, TYR82

HIS87

4-Hydroxycinnamic acid

17.96

20.88

GLU54, TRP59

VAL55, TRP59

5-Hydroxymethylfurfural

11.20

44.74

PHE36, ASP37, PHE46, GLN53, GLU54, TYR82

PHE46, VAL55, TRP59, ILE90

Verbascoside

1.03

27.83

ASP37, LYS47, GLU54

Wedelolactone

0.08

23.77

ILE56, TRP59

VAL55, ILE56, TRP59

Demethylwedelolactone

1.85

23.43

ILE56

PHE46, VAL55, ILE56, TYR82

Oleoside-11-methyl ester

14.24

17.26

GLU54

VAL55, ILE56

Oleuropein

3.24

26.23

TYR26, TYR82, HIS87

TYR26, VAL55, TRP59

Eclalbasaponin III

3.18

20.41

GLU54, ILE56, TYR82

Ursolic acid

-1.41

26.22

GLU54, HIS87

TYR82, HIS87, ILE90

2α-Hydroxyl oleanolic acid

-9.58

38.72

GLN53, GLU54, VAL55, TYR82

VAL55, ILE56, HIS87

Ecliptasaponin B

17.50

20.09

GLU54

TYR26, VAL55, ILE56