酸枣仁种皮和种仁化学成分的定性及半定量分析

doi:10.3724/SP.J.1123.2023.09015

摘要/Abstract

摘要：

酸枣仁为鼠李科植物酸枣Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou的干燥成熟种子,其分为种皮、种仁两个部位,比较研究酸枣仁不同部位化学成分组成及相对含量可为合理开发利用中药酸枣仁资源提供理论依据。基于超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF/MS)技术,从种皮、种仁中共鉴定出57个化学成分。结合主成分分析(PCA)和正交偏最小二乘法判别分析(OPLS-DA)对两者进行差异成分研究,以变量投影重要度(VIP)值> 5为标准,筛选了差异成分17个,其中白桦脂酸、桦木酮酸、麦珠子酸和酸枣仁皂苷Ⅰ主要存在于种皮部位,斯皮诺素、酸枣仁皂苷A和6‴-阿魏酰斯皮诺素等13个化合物主要存在于种仁部位。通过超高效液相色谱-电雾式检测器(UPLC-CAD)结合反梯度补偿技术,建立半定量液相色谱指纹图谱,考察了6个不同结构类型的代表成分的响应一致性,其不同浓度下平均响应因子间的RSD值为7.04%,各化合物响应一致性良好,可用于酸枣仁的半定量表征分析;结果表明:种皮部位主要成分为白桦脂酸和油酸,其中白桦脂酸的含量约是种仁的7倍;种仁部位主要成分为斯皮诺素、酸枣仁皂苷A、亚油酸、白桦脂酸和油酸,其中斯皮诺素、酸枣仁皂苷A的含量分别是种皮的18倍和24倍。综上,本研究阐明了酸枣仁种皮、种仁的化学成分差异,明确了酸枣仁两个部位中各自的主要成分及其相对含量,为酸枣仁不同部位合理开发和利用奠定了基础。

关键词: 超高效液相色谱, 电雾式检测器, 质谱, 差异成分, 酸枣仁, 种皮, 种仁, 半定量分析

Abstract:

Ziziphi Spinosae Semen refers to the dried seed of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou. The seed is composed of a reddish brown coat and a yellow kernel. A comparative study was conducted to investigate differences in the chemical composition and their relative contents between the seed coat and kernel of Ziziphi Spinosae Semen. First, the chemical compounds found in the seed coat and kernel were characterized and identified using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The analytical results tentatively identified 57 chemical compounds based on reference-compound comparison, literature retrieval, and chemical-database (e. g., MassBank) searches; these compounds included 14 triterpenes, 23 flavonoids, 7 alkaloids, 6 carboxylic acids, and 7 other types of compounds. The mass error of the identified compounds was within the mass deviation range of 5×10^-6 (5 ppm). Next, two methods of multivariate statistical analysis, namely, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), were used to compare the differential compounds between the two seed parts. A total of 17 differential compounds were screened out via OPLS-DA based on a variable importance in projection (VIP) value of >5. The results revealed that betulinic acid, betulonic acid, alphitolic acid, and jujuboside Ⅰ mainly existed in the seed coat whereas the 13 other compounds, such as spinosin, jujuboside A, and 6‴-feruloylspinosin, mainly existed in the seed kernel. Therefore, these 17 differential compounds can be used to distinguish between the two seed parts. Finally, a semiquantitative method was established using UPLC and a charged aerosol detector (CAD) with inverse gradient compensation in the mobile phase. Six representative compounds with different types were selected to examine the CAD response consistency: magnoflorine (alkaloid), spinosin (flavone), 6‴-feruloylspinosin (flavone), jujuboside A (triterpenoid saponin), jujuboside B (triterpenoid saponin), and betulinic acid (triterpenoid acid). The results showed that the relative standard deviation (RSD) of the average response factors at different levels of these six compounds was 7.04% and that their response intensities were similar. Moreover, each compound in the fingerprint demonstrated good response consistency, and the peak areas obtained directly reflected the contents of each compound. Based on the semiquantitative fingerprints obtained, betulinic acid and oleic acid were considered the main components of the seed coat. The betulinic acid content in the seed coat was approximately 7 times higher than that in the seed kernel. Spinosin, jujuboside A, linoleic acid, betulinic acid, and oleic acid were the main components of the seed kernel. The spinosin content in the seed kernel was 18 times higher than that in the seed coat. In addition, the jujuboside A content in the seed kernel was 24 times higher than that in the seed coat. The proposed method can accurately determine the main components and compare the relative contents of these components in different seed parts. In summary, this study identified the differences in chemical components between the seed coat and kernel of Ziziphi Spinosae Semen and clarified the main components and their relative contents in these parts. The findings can not only provide a basis for the identification of chemical compounds and quality research on different parts of Ziziphi Spinosae Semen but also promote the development and utilization of this traditional Chinese medicine.

Key words: ultra performance liquid chromatography (UPLC), charged aerosol detector (CAD), mass spectrometry (MS), difference markers, Ziziphi Spinosae Semen, seed coat, seed kernel, semiquantitative analysis

中图分类号:

O658

石宇浩, 南易, 郑伟, 姚兰, 梁海珍, 陈晓娟, 宋娟, 张洁, 贾德贤, 王谦, 马百平. 酸枣仁种皮和种仁化学成分的定性及半定量分析[J]. 色谱, 2024, 42(3): 234-244.

SHI Yuhao, NAN Yi, ZHENG Wei, YAO Lan, LIANG Haizhen, CHEN Xiaojuan, SONG Juan, ZHANG Jie, JIA Dexian, WANG Qian, MA Baiping. Qualitative and semiquantitative analyses of the chemical components of the seed coat and kernel of Ziziphi Spinosae Semen[J]. Chinese Journal of Chromatography, 2024, 42(3): 234-244.

图/表 10

参考文献

[1]	Chinese Pharmacopoeia Commission. Pharmacopoeia of People’s Republic of China, Part I. Beijing: China Medical Science Press, 2020: 382
[1]	国家药典委员会. 中华人民共和国药典, 一部. 北京: 中国医药科技出版社, 2020: 382
[2]	Ma J J, Liu P, Ma B P. International Journal of Pharmaceutical Research, 2011, 38(3): 206
[2]	马进杰, 刘萍, 马百平. 国际药学研究杂志, 2011, 38(3): 206
[3]	Yan Y, Shen C X, Pei X P, et al. J Food Compos Anal, 2020, 94: 103635
[4]	Zhang F X, Li M, Qiao L R, et al. J Pharmaceut Biomed, 2016, 122: 59
[5]	Johannah L S, Xiao J N, Jerome S, et al. Phytomedicine, 2017, 34: 38
[6]	Johannah L S, Anna H, Hailey M, et al. Complement Ther Med, 2021, 57: 102657
[7]	Liang Y, Yang X, Zhang X J, et al. J EthnoPharmacol, 2016, 191: 307
[8]	Liu J, Zhai W M, Yang Y X, et al. Pharmacol Biochem Be, 2015, 128: 41
[9]	Huang Y S, Jia Y H, Sun X G, et al. Traditional Chinese Drug Research & Clinical Pharmacology, 2011, 22(1): 51
[9]	黄宜生, 贾钰华, 孙学刚, 等. 中药新药与临床药理, 2011, 22(1): 51
[10]	Wang W S, Huang Q J, Chen Y, et al. Pharmacol Res, 2021, 170: 105723
[11]	Huang X X, Mao Y N, Li H, et al. China Modern Pharmacy, 2021, 23(12): 2077
[11]	黄晓欣, 毛怡宁, 李虹, 等. 中国现代中药, 2021, 23(12): 2077
[12]	Li Z, Du H, Xie Y J, et al. Journal of Chinese Herbal Medicine, 2021, 52(16): 4811
[12]	李泽, 杜鹤, 解玉军, 等. 中草药, 2021, 52(16): 4811
[13]	Wang J, Lin X, Ding S C. Chinese Traditional Patent Medicine, 1996(5): 23
[13]	王健, 林晓, 丁少纯. 中成药, 1996(5): 23
[14]	Xiao F Q, Liu H, Yang Y L, et al. Food Machinery, 2022, 38(4): 54
[14]	肖凤琴, 刘晖, 杨亦柳, 等. 食品与机械, 2022, 38(4): 54
[15]	Gao H N, Yin Y, Yan G J. China Modern Medicine, 2021, 28(6): 8
[15]	高红宁, 殷奕, 严国俊. 中国当代医药, 2021, 28(6): 8
[16]	Cohen R D, Liu Y, Gong X, et al. J Chromatogr A, 2012, 1229: 172
[17]	Gamache P H, McCarthy R S, Freeto S M, et al.LC GC N AM, 2005, 23: 150
[18]	Zheng W, Zhou M, Chai R P, et al. Phytochem Anal, 2022, 33(7): 1
[19]	Zhou J, Tang W W, Chen J. Pharmaceutical and Clinical Research, 2020, 28(5): 321
[19]	周洁, 汤维维, 陈君. 药学与临床研究, 2020, 28(5): 321
[20]	Zhang W G, Lang Y F, Yao Y, et al. Journal of Chinese Herbal Medicine, 2023, 54(24): 7977
[20]	张武岗, 郎一帆, 姚云, 等. 中草药, 2023, 54(24): 7977
[21]	Cheng S H, Yao L, Zhou W B, et al. China Journal of Traditional Chinese Medicine and Pharmacy, 2022, 37(8): 4702
[21]	程硕涵, 姚兰, 周文斌, 等. 中华中医药杂志, 2022, 37(8): 4702
[22]	Tong J, Liu H B, Liu Y W, et al. Acta Pharmaceutica Sinica, 2023, 58(3): 750
[22]	童俊, 刘红兵, 刘颖伟, 等. 药学学报, 2023, 58(3): 750
[23]	Liang Y L, Li Y H, Lei Y, et al. Central South Pharmacy, 2022, 20(4): 823
[23]	梁雨璐, 李忆红, 雷艳, 等. 中南药学, 2022, 20(4): 823
[24]	Li M, Zhang F X, Wei Z C, et al. J Pharm Biomed Anal, 2020, 193: 113756
[25]	Wang W J, Luo J G, Kong L Y. China Journal of Chinese Materia Medica, 2009, 34(21): 2768
[25]	王卫记, 罗建光, 孔令义. 中国中药杂志, 2009, 34(21): 2768
[26]	Lai L C, Lin Y Y, Chen F L, et al. Journal of Chinese Herbal Medicine, 2016, 47(20): 3578
[26]	赖丽嫦, 林裕英, 陈丰连, 等. 中草药, 2016, 47(20): 3578
[27]	Mao Y N, Wu W Q, Kang Y, et al. China Journal of Chinese Materia Medica, 2018, 43(24): 4884
[27]	毛怡宁, 武文奇, 康莹, 等. 中国中药杂志, 2018, 43(24): 4884
[28]	Ou Z, Bai M Y Z, Yang B, et al. Food and Nutrition in China, 2023, 29(9): 22
[28]	欧珠, 白玛玉珍, 杨斌, 等. 中国食物与营养, 2023, 29(9): 22
[29]	Wu Y, Zhang J, Chen M, et al. Chem Nat Comp, 2015, 51: 247
[30]	Woo W S, Kang S S, Wagner H, et al. Phytochemistry, 1980, 19(12): 2791
[31]	Wang X M, Liu J, Shi J L, et al. Modern Chinese Medicine, 2021, 23(11): 1949
[31]	王晓玫, 刘洁, 石晋丽, 等. 中国现代中药, 2021, 23(11): 1949
[32]	Guo S, Tang Y P, Guan J A, et al. Chinese Chem Lett, 2009, 20(2): 197
[33]	A J Y, He J, Sun R B. Acta Pharmaceutica Sinica, 2018, 53(6): 929
[33]	阿基业, 何骏, 孙润彬. 药学学报, 2018, 53(6): 929
[34]	Maniyamma A, Ajesh V, Uzini D D, et al. J Biochem Mol Toxicol, 2022, 36(12): e23206

No.	t_R in Fig. 1/min	Formula	Precursor ion	Mass	Theoretical mass	Error/ 10^-6	Fragment ions (m/z)	Compound	Seed coat	Seed kernel
1	2.25	C₁₆H₁₈O₉	[M-H]^-	353.0867	353.0873	-1.7	335.0876, 191.0549, 179.0344, 161.0211, 135.0441	neochlorogenic acid^[19]	+	+
2	3.30	C₁₆H₁₈O₉	[M-H]^-	353.0870	353.0873	-0.9	191.0550, 179.0347, 161.0232, 85.0329	chlorogenic acid^[19]	+	+
3	3.52	C₁₆H₁₈O₉	[M-H]^-	353.0869	353.0873	-1.1	191.0557, 179.0339, 173.0450, 161.0248, 155.0330, 135.0444,	cryptochlorogenic acid^[19]	+	+
							93.0338
4	3.67	C₁₉H₂₄NO₃	[M]⁺	314.1760	314.1756	1.3	314.1755, 269.1203, 237.0095, 211.1090, 175.0743, 145.0653,	magnocurarine isomer^[20]	+	+
							116.9765, 107.0503
5	4.26	C₁₇H₁₉NO₃	[M+H]⁺	286.1447	286.1443	1.4	286.1440, 269.1181, 237.0911, 209.0965, 191.0856, 175.0756,	aconitine^[3]	+	+
							143.0495, 107.0496
6	4.66	C₂₀H₂₆NO₄	[M]⁺	344.1860	344.1862	-0.6	299.1278, 267.0997, 239.1145, 237.0904, 207.9867, 175.0752,	tembetarin^[19,21]a,c	+	+
							137.0577
7	4.73	C₂₇H₃₀O₁₅	[M-H]^-	593.1514	593.1506	1.4	473.1082, 395.0771, 383.0767, 353.0657, 325.0708, 297.0758	vicenin-Ⅱ^[4,22]	+	+
8	4.84	C₂₀H₂₄NO₄	[M]⁺	342.1703	342.1705	-0.6	297.1129, 282.0894, 265.0867, 237.0914, 219.0803, 58.0668	magnoflorine^b	+	+
9	5.51	C₁₉H₂₄NO₃	[M]⁺	314.1761	314.1756	1.6	314.1755, 269.1185, 237.0928, 209.0956, 175.0746, 143.0493	magnocurarine^[20,23]	+	+
10	6.00	C₄₂H₄₈O₂₃	[M+H]⁺	921.2681	921.2665	1.7	759.2183, 741.1762, 639.1682, 447.1330, 429.1185, 411.1058,	6‴-(4‴-O-glc)-vanilloylspinosin^[4]	-	+
							381.0920, 351.0924, 327.0807, 151.0363
11	6.08	C₂₇H₃₀O₁₅	[M+H]⁺	595.1649	595.1663	-2.4	433.1125, 415.1020, 397.0916, 379.0811, 367.0818, 337.0724,	meloside A^[4]	-	+
							313.0710, 283.0609
12	6.29	C₃₂H₃₈O₁₉	[M+H]⁺	727.2081	727.2086	-0.7	595.1814, 581.1440, 449.1064, 287.0556	camelliaside B^[4,23]	+	+
13	6.40	C₂₈H₃₂O₁₅	[M+H]⁺	609.1804	609.1819	-2.5	447.1285, 429.1191, 411.1051, 393.0976, 351.0873, 327.0871,	isospinosin^[3]	+	+
							297.0765, 285.0767
14	6.49	C₂₈H₃₂O₁₅	[M+H]⁺	609.1813	609.1819	-1.0	489.1479, 447.1290, 429.1180, 411.1070, 393.1081, 381.0967,	spinosin^b	+	+
							351.0864, 327.0868, 297.0757
15	6.56	C₂₁H₂₀O₁₀	[M-H]^-	431.0972	431.0978	-1.4	323.0573, 311.0557, 307.0622, 293.0428, 283.0594, 281.0456,	isovitexin^[24]	+	+
							269.0444
16	6.67	C₂₇H₃₀O₁₅	[M-H]^-	593.1511	593.1506	0.8	593.1526, 285.0394	kaempferol-3-O-rutoside isomer^[3,25]	+	+
17	6.73	C₂₉H₃₆O₁₅	[M-H]^-	623.1985	623.1976	1.4	461.1664, 179.0341, 161.0236	acteoside^[26]a,c	+	+
18	6.81	C₂₉H₃₆O₁₅	[M-H]^-	623.1982	623.1976	1.0	461.0904, 179.0342, 161.0236	isoacteoside^[26]	+	+
19	6.87	C₂₂H₂₂O₁₀	[M+H]⁺	447.1292	447.1291	0.2	429.1295, 411.1142, 327.0872, 297.0773, 174.0549	swertisin^[27]	+	+
20	7.29	C₂₅H₂₄O₁₂	[M-H]^-	515.1195	515.1190	1.0	353.0880, 191.0553, 179.0340, 173.0448, 135.0443	isochlorogenic acid B^[28]	+	+
21	7.40	C₂₇H₃₀O₁₅	[M-H]^-	593.1508	593.1506	0.3	285.0388, 284.0316, 255.0291	kaempferol-3-O-rutoside^[3,25]	-	+
22	7.53	C₂₅H₂₄O₁₂	[M-H]^-	515.1190	515.1190	0	353.0858, 191.0552, 179.0340, 173.0458, 135.0439	isochlorogenic acid A^[28]	+	+
23	7.70	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2816	944.2824	-0.9	944.2839, 824.2661, 447.1273, 429.1159, 327.0866, 297.1133	6‴-O-(3-Glc-indole-acetyl)spinosin or isomer^[4,11]	-	+
24	7.75	C₃₇H₃₈O₁₈	[M+H]⁺	771.2140	771.2136	0.5	651.1650, 609.1619, 433.1139, 415.1023, 379.0705, 337.0701,	isovitexin-2″-O-(6-feruloyl)-glucopyranoside^[4,27]	+	+
							313.0726, 177.0555
25	7.82	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2815	944.2824	0.1	944.2797, 824.2675, 447.1271, 429.1161, 411.1082, 327.0849,	6‴-O-(3-Glc-indole-acetyl)spinosin or isomer^[4,11]	-	+
							297.0788
26	8.15	C₂₅H₂₄O₁₂	[M-H]^-	515.1185	515.1190	-1.0	353.0862, 191.0551, 179.0338, 173.0447, 135.0443	isochlorogenic acid C^[28]	+	+
27	8.18	C₃₉H₄₂O₁₉	[M+H]⁺	815.2397	815.2399	-0.3	695.2109, 609.1945, 447.1345, 429.1194, 411.1042, 393.0956,	6‴-sinapoylspinosin^[3,27]	-	+
							327.0868, 297.0854, 207.0659, 163.0387
28	8.20	C₄₃H₅₂O₁₉	[M+H]⁺	873.3188	873.3181	0.8	855.3101, 735.2551, 447.1412, 429.1191, 411.1068, 393.0933,	6‴-dihydrophaseoylspinosin^[3]	-	+
							351.0852, 327.0861, 247.1341, 207.0649, 163.0397
29	8.30	C₃₇H₃₈O₁₇	[M+H]⁺	755.2174	755.2187	-1.7	635.1722, 609.1754, 447.1285, 429.1180, 351.0857, 327.0863,	6‴-p-coumaloylspinosin^[3]	-	+
							309.0979, 147.0442
30	8.39	C₃₈H₄₀O₁₈	[M+H]⁺	785.2289	785.2293	-0.5	665.1870, 609.1771, 447.1184, 429.1184, 411.1092, 393.0977,	6‴-feruloylspinosin^b	+	+
							381.0981, 351.0871, 327.0869, 207.0765, 177.0551
31	8.74	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2810	944.2824	-1.5	944.2797, 782.2302, 764.2184, 489.1389, 393.0977, 327.0861	3‴-(N-β-D-Glucopyranosyl)-2‴,3‴-dihydro-2‴-	-	+
								oxo-indol-3‴-yl-acetate spinosin or isomer^[29]
32	8.84	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2811	944.2824	-1.4	944.2799, 782.2269, 764.2170, 602.1631, 489.1395, 393.0974,	6‴-(N-β-D-glucopyranosyl)-2‴,3‴-dihydro-2‴-	-	+
							327.0861	oxo-indol-3‴-yl-acetate spinosin or isomer^[29]
33	9.37	C₁₈H₁₉NO₂	[M+H]⁺	282.1490	282.1494	-1.4	265.1223, 250.0998, 235.0762, 219.0824, 191.0863	N-nornuciferine^[4]	+	+
34	9.71	C₃₈H₄₀O₁₈	[M+H]⁺	785.2310	785.2293	2.2	665.1846, 447.1279, 429.1184, 411.1117, 327.0863, 177.0545	6‴-feruloylspinosin isomer^[3,4,27]	-	+
35	10.11	C₅₄H₅₈NO₂₅	[M+HCOO]^-	1164.3208	1164.3196	1.0	1118.3135, 942.2715, 762.2023, 663.1707, 293.0443	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin or	-	+
								isomer^[4]
36	10.18	C₅₄H₅₈NO₂₅	[M+HCOO]^-	1164.3210	1164.3196	1.2	1118.3145, 942.2654, 783.2103, 762.2061, 663.1730, 293.0453	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin^[4]	-	+
37	10.42	C₄₈H₅₀O₂₁	[M-H]^-	961.2802	961.2766	3.75	943.2595, 931.2581, 307.0561	6‴-p-coumaroyl-6‴-sinapoyl spinosin^[11,30]	+	+
38	11.01	C₅₈H₉₆O₂₇	[M+HCOO]^-	1269.6135	1269.6115	1.6	1223.6045, 919.4577, 787.4113, 625.3592, 479.3006, 255.2336	protojujuboside B^[4,11]	+	+
39	11.56	C₅₃H₈₆O₂₂	[M-H]^-	1073.5532	1073.5537	-0.5	911.5039, 749.4492, 603.3906, 471.3479, 279.2314	jujuboside Ⅰ^[4]	+	+
40	11.63	C₃₁H₄₂N₄O₄	[M+H]⁺	535.3268	535.3284	-3.0	148.1116	sanjoinine A^[3]	+	+
41	11.90	C₅₃H₈₆O₂₂	[M+HCOO]^-	1119.5603	1119.5587	1.4	1073.5677, 749.4483, 603.3909, 471.3471, 279.2328	jujuboside Ⅰ isomer^[4]	+	+
42	12.48	C₅₈H₉₄O₂₆	[M+HCOO]^-	1251.6025	1251.6010	1.1	1073.5540, 911.5007, 749.4480, 603.3908, 471.3503	jujuboside A^b	+	+
43	12.71	C₅₈H₉₄O₂₆	[M+HCOO]^-	1251.6018	1251.6010	0.6	1073.5531, 911.4987, 749.4526, 279.2318	jujuboside A isomer^[3,12]	+	+
44	12.94	C₅₂H₈₄O₂₁	[M+HCOO]^-	1089.5496	1089.5482	1.3	1043.5425, 911.5007, 749.4480, 603.3908, 471.3448	jujuboside B^b	+	+
45	13.10	C₅₅H₈₆O₂₄	[M-H]^-	1129.5452	1129.5431	1.9	1085.5536, 1043.5443, 911.5009, 749.4488, 603.3905,	malonyl-jujuboside B^a,c	+	+
							471.3534
46	13.36	C₅₄H₈₆O₂₂	[M+HCOO]^-	1131.5596	1131.5587	0.8	1043.5497, 911.5013, 749.4485, 603.3876, 471.1332	acetyl-jujuboside B^[4,11]	+	+
47	15.33	C₂₇H₄₉O₁₂P	[M-H]^-	595.2886	595.2883	0.5	595.2848, 279.2317, 241.0104	1-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-	+	+
								(1'-myo-inositol)^a,c
48	15.98	C₄₃H₇₉O₁₃P	[M-H]^-	833.5173	833.5180	-0.8	571.2925, 371.0077, 281.2468, 255.2321, 241.0103, 152.9950,	1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-	-	+
							96.9687	glycero-3-phospho-(1'-myo-inositol) or isomer^a,c
49	16.53	C₃₀H₄₈O₄	[M-H]^-	471.3479	471.3474	1.1	471.3474, 279.2320, 183.0129	alphitolic acid^[4,31]	+	+
50	17.74	C₃₀H₄₆O₄	[M-H]^-	469.3318	469.3318	0	451.3236, 391.2265, 279.2319	zizyberanalic acid^[32]	+	+
51	18.26	C₃₀H₄₆O₅	[M-H]^-	485.3273	485.3267	1.2	439.3222, 423.3263	ceanothic acid^[31]	+	+
52	18.73	C₃₀H₄₆O₄	[M-H]^-	469.3320	469.3318	0.4	391.2245, 279.2321	zizyberanalic acid isomer^[32]	+	+
53	18.94	C₂₇H₅₃O₁₂P	[M-H]^-	599.3203	599.3196	1.2	315.0501, 283.2631, 255.2322, 241.0107, 223.0011, 152.9950,	1-octadecanoyl-sn-glycero-3-phospho-(1'-myo-	-	+
							78.9684	inositol)^a,c
54	21.70	C₃₀H₄₈O₃	[M-H]^-	455.3525	455.3525	0	455.3525, 407.3291	betulinic acid^b	+	+
55	23.42	C₁₈H₃₂O₂	[M-H]^-	279.2324	279.2324	0	279.2322	linoleic acid^[4]	+	+
56	23.90	C₃₀H₄₆O3	[M-H]^-	453.3370	453.3369	0.2	325.1949, 293.1786, 279.2320, 255.2321	betulonic acid^[4]	+	+
57	24.81	C₁₈H₃₄O₂	[M-H]^-	281.2476	281.2481	-1.8	281.2487	oleic acid^[4]	+	+

No.	t_R in Fig. 1/min	Formula	Precursor ion	Mass	Theoretical mass	Error/ 10^-6	Fragment ions (m/z)	Compound	Seed coat	Seed kernel
1	2.25	C₁₆H₁₈O₉	[M-H]^-	353.0867	353.0873	-1.7	335.0876, 191.0549, 179.0344, 161.0211, 135.0441	neochlorogenic acid^[19]	+	+
2	3.30	C₁₆H₁₈O₉	[M-H]^-	353.0870	353.0873	-0.9	191.0550, 179.0347, 161.0232, 85.0329	chlorogenic acid^[19]	+	+
3	3.52	C₁₆H₁₈O₉	[M-H]^-	353.0869	353.0873	-1.1	191.0557, 179.0339, 173.0450, 161.0248, 155.0330, 135.0444,	cryptochlorogenic acid^[19]	+	+
							93.0338
4	3.67	C₁₉H₂₄NO₃	[M]⁺	314.1760	314.1756	1.3	314.1755, 269.1203, 237.0095, 211.1090, 175.0743, 145.0653,	magnocurarine isomer^[20]	+	+
							116.9765, 107.0503
5	4.26	C₁₇H₁₉NO₃	[M+H]⁺	286.1447	286.1443	1.4	286.1440, 269.1181, 237.0911, 209.0965, 191.0856, 175.0756,	aconitine^[3]	+	+
							143.0495, 107.0496
6	4.66	C₂₀H₂₆NO₄	[M]⁺	344.1860	344.1862	-0.6	299.1278, 267.0997, 239.1145, 237.0904, 207.9867, 175.0752,	tembetarin^[19,21]a,c	+	+
							137.0577
7	4.73	C₂₇H₃₀O₁₅	[M-H]^-	593.1514	593.1506	1.4	473.1082, 395.0771, 383.0767, 353.0657, 325.0708, 297.0758	vicenin-Ⅱ^[4,22]	+	+
8	4.84	C₂₀H₂₄NO₄	[M]⁺	342.1703	342.1705	-0.6	297.1129, 282.0894, 265.0867, 237.0914, 219.0803, 58.0668	magnoflorine^b	+	+
9	5.51	C₁₉H₂₄NO₃	[M]⁺	314.1761	314.1756	1.6	314.1755, 269.1185, 237.0928, 209.0956, 175.0746, 143.0493	magnocurarine^[20,23]	+	+
10	6.00	C₄₂H₄₈O₂₃	[M+H]⁺	921.2681	921.2665	1.7	759.2183, 741.1762, 639.1682, 447.1330, 429.1185, 411.1058,	6‴-(4‴-O-glc)-vanilloylspinosin^[4]	-	+
							381.0920, 351.0924, 327.0807, 151.0363
11	6.08	C₂₇H₃₀O₁₅	[M+H]⁺	595.1649	595.1663	-2.4	433.1125, 415.1020, 397.0916, 379.0811, 367.0818, 337.0724,	meloside A^[4]	-	+
							313.0710, 283.0609
12	6.29	C₃₂H₃₈O₁₉	[M+H]⁺	727.2081	727.2086	-0.7	595.1814, 581.1440, 449.1064, 287.0556	camelliaside B^[4,23]	+	+
13	6.40	C₂₈H₃₂O₁₅	[M+H]⁺	609.1804	609.1819	-2.5	447.1285, 429.1191, 411.1051, 393.0976, 351.0873, 327.0871,	isospinosin^[3]	+	+
							297.0765, 285.0767
14	6.49	C₂₈H₃₂O₁₅	[M+H]⁺	609.1813	609.1819	-1.0	489.1479, 447.1290, 429.1180, 411.1070, 393.1081, 381.0967,	spinosin^b	+	+
							351.0864, 327.0868, 297.0757
15	6.56	C₂₁H₂₀O₁₀	[M-H]^-	431.0972	431.0978	-1.4	323.0573, 311.0557, 307.0622, 293.0428, 283.0594, 281.0456,	isovitexin^[24]	+	+
							269.0444
16	6.67	C₂₇H₃₀O₁₅	[M-H]^-	593.1511	593.1506	0.8	593.1526, 285.0394	kaempferol-3-O-rutoside isomer^[3,25]	+	+
17	6.73	C₂₉H₃₆O₁₅	[M-H]^-	623.1985	623.1976	1.4	461.1664, 179.0341, 161.0236	acteoside^[26]a,c	+	+
18	6.81	C₂₉H₃₆O₁₅	[M-H]^-	623.1982	623.1976	1.0	461.0904, 179.0342, 161.0236	isoacteoside^[26]	+	+
19	6.87	C₂₂H₂₂O₁₀	[M+H]⁺	447.1292	447.1291	0.2	429.1295, 411.1142, 327.0872, 297.0773, 174.0549	swertisin^[27]	+	+
20	7.29	C₂₅H₂₄O₁₂	[M-H]^-	515.1195	515.1190	1.0	353.0880, 191.0553, 179.0340, 173.0448, 135.0443	isochlorogenic acid B^[28]	+	+
21	7.40	C₂₇H₃₀O₁₅	[M-H]^-	593.1508	593.1506	0.3	285.0388, 284.0316, 255.0291	kaempferol-3-O-rutoside^[3,25]	-	+
22	7.53	C₂₅H₂₄O₁₂	[M-H]^-	515.1190	515.1190	0	353.0858, 191.0552, 179.0340, 173.0458, 135.0439	isochlorogenic acid A^[28]	+	+
23	7.70	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2816	944.2824	-0.9	944.2839, 824.2661, 447.1273, 429.1159, 327.0866, 297.1133	6‴-O-(3-Glc-indole-acetyl)spinosin or isomer^[4,11]	-	+
24	7.75	C₃₇H₃₈O₁₈	[M+H]⁺	771.2140	771.2136	0.5	651.1650, 609.1619, 433.1139, 415.1023, 379.0705, 337.0701,	isovitexin-2″-O-(6-feruloyl)-glucopyranoside^[4,27]	+	+
							313.0726, 177.0555
25	7.82	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2815	944.2824	0.1	944.2797, 824.2675, 447.1271, 429.1161, 411.1082, 327.0849,	6‴-O-(3-Glc-indole-acetyl)spinosin or isomer^[4,11]	-	+
							297.0788
26	8.15	C₂₅H₂₄O₁₂	[M-H]^-	515.1185	515.1190	-1.0	353.0862, 191.0551, 179.0338, 173.0447, 135.0443	isochlorogenic acid C^[28]	+	+
27	8.18	C₃₉H₄₂O₁₉	[M+H]⁺	815.2397	815.2399	-0.3	695.2109, 609.1945, 447.1345, 429.1194, 411.1042, 393.0956,	6‴-sinapoylspinosin^[3,27]	-	+
							327.0868, 297.0854, 207.0659, 163.0387
28	8.20	C₄₃H₅₂O₁₉	[M+H]⁺	873.3188	873.3181	0.8	855.3101, 735.2551, 447.1412, 429.1191, 411.1068, 393.0933,	6‴-dihydrophaseoylspinosin^[3]	-	+
							351.0852, 327.0861, 247.1341, 207.0649, 163.0397
29	8.30	C₃₇H₃₈O₁₇	[M+H]⁺	755.2174	755.2187	-1.7	635.1722, 609.1754, 447.1285, 429.1180, 351.0857, 327.0863,	6‴-p-coumaloylspinosin^[3]	-	+
							309.0979, 147.0442
30	8.39	C₃₈H₄₀O₁₈	[M+H]⁺	785.2289	785.2293	-0.5	665.1870, 609.1771, 447.1184, 429.1184, 411.1092, 393.0977,	6‴-feruloylspinosin^b	+	+
							381.0981, 351.0871, 327.0869, 207.0765, 177.0551
31	8.74	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2810	944.2824	-1.5	944.2797, 782.2302, 764.2184, 489.1389, 393.0977, 327.0861	3‴-(N-β-D-Glucopyranosyl)-2‴,3‴-dihydro-2‴-	-	+
								oxo-indol-3‴-yl-acetate spinosin or isomer^[29]
32	8.84	C₄₄H₄₉O₂₂N	[M+H]⁺	944.2811	944.2824	-1.4	944.2799, 782.2269, 764.2170, 602.1631, 489.1395, 393.0974,	6‴-(N-β-D-glucopyranosyl)-2‴,3‴-dihydro-2‴-	-	+
							327.0861	oxo-indol-3‴-yl-acetate spinosin or isomer^[29]
33	9.37	C₁₈H₁₉NO₂	[M+H]⁺	282.1490	282.1494	-1.4	265.1223, 250.0998, 235.0762, 219.0824, 191.0863	N-nornuciferine^[4]	+	+
34	9.71	C₃₈H₄₀O₁₈	[M+H]⁺	785.2310	785.2293	2.2	665.1846, 447.1279, 429.1184, 411.1117, 327.0863, 177.0545	6‴-feruloylspinosin isomer^[3,4,27]	-	+
35	10.11	C₅₄H₅₈NO₂₅	[M+HCOO]^-	1164.3208	1164.3196	1.0	1118.3135, 942.2715, 762.2023, 663.1707, 293.0443	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin or	-	+
								isomer^[4]
36	10.18	C₅₄H₅₈NO₂₅	[M+HCOO]^-	1164.3210	1164.3196	1.2	1118.3145, 942.2654, 783.2103, 762.2061, 663.1730, 293.0453	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin^[4]	-	+
37	10.42	C₄₈H₅₀O₂₁	[M-H]^-	961.2802	961.2766	3.75	943.2595, 931.2581, 307.0561	6‴-p-coumaroyl-6‴-sinapoyl spinosin^[11,30]	+	+
38	11.01	C₅₈H₉₆O₂₇	[M+HCOO]^-	1269.6135	1269.6115	1.6	1223.6045, 919.4577, 787.4113, 625.3592, 479.3006, 255.2336	protojujuboside B^[4,11]	+	+
39	11.56	C₅₃H₈₆O₂₂	[M-H]^-	1073.5532	1073.5537	-0.5	911.5039, 749.4492, 603.3906, 471.3479, 279.2314	jujuboside Ⅰ^[4]	+	+
40	11.63	C₃₁H₄₂N₄O₄	[M+H]⁺	535.3268	535.3284	-3.0	148.1116	sanjoinine A^[3]	+	+
41	11.90	C₅₃H₈₆O₂₂	[M+HCOO]^-	1119.5603	1119.5587	1.4	1073.5677, 749.4483, 603.3909, 471.3471, 279.2328	jujuboside Ⅰ isomer^[4]	+	+
42	12.48	C₅₈H₉₄O₂₆	[M+HCOO]^-	1251.6025	1251.6010	1.1	1073.5540, 911.5007, 749.4480, 603.3908, 471.3503	jujuboside A^b	+	+
43	12.71	C₅₈H₉₄O₂₆	[M+HCOO]^-	1251.6018	1251.6010	0.6	1073.5531, 911.4987, 749.4526, 279.2318	jujuboside A isomer^[3,12]	+	+
44	12.94	C₅₂H₈₄O₂₁	[M+HCOO]^-	1089.5496	1089.5482	1.3	1043.5425, 911.5007, 749.4480, 603.3908, 471.3448	jujuboside B^b	+	+
45	13.10	C₅₅H₈₆O₂₄	[M-H]^-	1129.5452	1129.5431	1.9	1085.5536, 1043.5443, 911.5009, 749.4488, 603.3905,	malonyl-jujuboside B^a,c	+	+
							471.3534
46	13.36	C₅₄H₈₆O₂₂	[M+HCOO]^-	1131.5596	1131.5587	0.8	1043.5497, 911.5013, 749.4485, 603.3876, 471.1332	acetyl-jujuboside B^[4,11]	+	+
47	15.33	C₂₇H₄₉O₁₂P	[M-H]^-	595.2886	595.2883	0.5	595.2848, 279.2317, 241.0104	1-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-	+	+
								(1'-myo-inositol)^a,c
48	15.98	C₄₃H₇₉O₁₃P	[M-H]^-	833.5173	833.5180	-0.8	571.2925, 371.0077, 281.2468, 255.2321, 241.0103, 152.9950,	1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-	-	+
							96.9687	glycero-3-phospho-(1'-myo-inositol) or isomer^a,c
49	16.53	C₃₀H₄₈O₄	[M-H]^-	471.3479	471.3474	1.1	471.3474, 279.2320, 183.0129	alphitolic acid^[4,31]	+	+
50	17.74	C₃₀H₄₆O₄	[M-H]^-	469.3318	469.3318	0	451.3236, 391.2265, 279.2319	zizyberanalic acid^[32]	+	+
51	18.26	C₃₀H₄₆O₅	[M-H]^-	485.3273	485.3267	1.2	439.3222, 423.3263	ceanothic acid^[31]	+	+
52	18.73	C₃₀H₄₆O₄	[M-H]^-	469.3320	469.3318	0.4	391.2245, 279.2321	zizyberanalic acid isomer^[32]	+	+
53	18.94	C₂₇H₅₃O₁₂P	[M-H]^-	599.3203	599.3196	1.2	315.0501, 283.2631, 255.2322, 241.0107, 223.0011, 152.9950,	1-octadecanoyl-sn-glycero-3-phospho-(1'-myo-	-	+
							78.9684	inositol)^a,c
54	21.70	C₃₀H₄₈O₃	[M-H]^-	455.3525	455.3525	0	455.3525, 407.3291	betulinic acid^b	+	+
55	23.42	C₁₈H₃₂O₂	[M-H]^-	279.2324	279.2324	0	279.2322	linoleic acid^[4]	+	+
56	23.90	C₃₀H₄₆O3	[M-H]^-	453.3370	453.3369	0.2	325.1949, 293.1786, 279.2320, 255.2321	betulonic acid^[4]	+	+
57	24.81	C₁₈H₃₄O₂	[M-H]^-	281.2476	281.2481	-1.8	281.2487	oleic acid^[4]	+	+

No.	Peak No.¹⁾	t_R/min	Formula	Compound²⁾	VIP	Main presence part
1	54	21.70	C₃₀H₄₈O₃	betulinic acid	31.85	seed coat
2	14	6.49	C₂₈H₃₂O₁₅	spinosin	16.92	seed kernel
3	42	12.48	C₅₈H₉₄O₂₆	jujuboside A	16.85	seed kernel
4	30	8.39	C₃₈H₄₀O₁₈	6‴-feruloylspinosin	14.55	seed kernel
5	56	23.90	C₃₀H₄₆O₃	betulonic acid	13.06	seed coat
6	49	16.53	C₃₀H₄₈O₄	alphitolic acid	12.05	seed coat
7	44	12.94	C₅₂H₈₄O₂₁	jujuboside B	10.75	seed kernel
8	47	15.33	C₂₇H₄₉O₁₂P	1-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-(1'-myo-inositol)	10.07	seed kernel
9	45	13.10	C₅₅H₈₆O₂₄	malonyl-jujuboside B	9.77	seed kernel
10	48	15.98	C₄₃H₇₉O₁₃P	1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-	8.81	seed kernel
				(1'-myo-inositol) or isomer
11	8	4.84	C₂₀H₂₄NO₄	magnoflorine	7.99	seed kernel
12	29	8.30	C₃₇H₃₈O₁₇	6‴-p-coumaloylspinosin	7.18	seed kernel
13	39	11.56	C₅₃H₈₆O₂₂	jujuboside Ⅰ	6.49	seed coat
14	35	10.11	C₅₄H₅₈NO₂₅	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin	6.22	seed kernel
15	11	6.08	C₂₇H₃₀O₁₅	meloside A	5.78	seed kernel
16	32	8.84	C₄₄H₄₉O₂₂N	6‴-(N-β-D-glucopyranosyl)-2‴,3‴-dihydro-2‴-oxo-indol-3‴-yl-	5.66	seed kernel
				acetate spinosin or isomer
17	13	6.40	C₂₈H₃₂O₁₅	isospinosin	5.07	seed kernel

No.	Peak No.¹⁾	t_R/min	Formula	Compound²⁾	VIP	Main presence part
1	54	21.70	C₃₀H₄₈O₃	betulinic acid	31.85	seed coat
2	14	6.49	C₂₈H₃₂O₁₅	spinosin	16.92	seed kernel
3	42	12.48	C₅₈H₉₄O₂₆	jujuboside A	16.85	seed kernel
4	30	8.39	C₃₈H₄₀O₁₈	6‴-feruloylspinosin	14.55	seed kernel
5	56	23.90	C₃₀H₄₆O₃	betulonic acid	13.06	seed coat
6	49	16.53	C₃₀H₄₈O₄	alphitolic acid	12.05	seed coat
7	44	12.94	C₅₂H₈₄O₂₁	jujuboside B	10.75	seed kernel
8	47	15.33	C₂₇H₄₉O₁₂P	1-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-(1'-myo-inositol)	10.07	seed kernel
9	45	13.10	C₅₅H₈₆O₂₄	malonyl-jujuboside B	9.77	seed kernel
10	48	15.98	C₄₃H₇₉O₁₃P	1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-glycero-3-phospho-	8.81	seed kernel
				(1'-myo-inositol) or isomer
11	8	4.84	C₂₀H₂₄NO₄	magnoflorine	7.99	seed kernel
12	29	8.30	C₃₇H₃₈O₁₇	6‴-p-coumaloylspinosin	7.18	seed kernel
13	39	11.56	C₅₃H₈₆O₂₂	jujuboside Ⅰ	6.49	seed coat
14	35	10.11	C₅₄H₅₈NO₂₅	6‴-O-(3-Glc-indole-acetyl)-6‴-feruloylspinosin	6.22	seed kernel
15	11	6.08	C₂₇H₃₀O₁₅	meloside A	5.78	seed kernel
16	32	8.84	C₄₄H₄₉O₂₂N	6‴-(N-β-D-glucopyranosyl)-2‴,3‴-dihydro-2‴-oxo-indol-3‴-yl-	5.66	seed kernel
				acetate spinosin or isomer
17	13	6.40	C₂₈H₃₂O₁₅	isospinosin	5.07	seed kernel

Mass concentration/(μg/mL)	f_Magnoflorine	f_Spinosin	f_{6‴-Feruloylspinosin}	f_{Jujuboside A}	f_{Jujuboside B}	f_{Betulinic acid}	RSD/%
100	0.005242	0.005262	0.005430	0.005395	0.005767	0.006391	7.87
75	0.004873	0.005108	0.005215	0.005203	0.00556	0.006309	9.43
50	0.005095	0.005231	0.005323	0.005534	0.005833	0.005757	5.41
25	0.004915	0.005239	0.005250	0.005268	0.005787	0.005812	6.53
10	0.004299	0.005059	0.005099	0.005519	0.005632	0.00542	9.37
Mean	0.004885	0.005180	0.005264	0.005384	0.005716	0.005938	7.04