HPLC法同时测定甘薯叶中5种酚酸和异槲皮苷

傅志丰; 周鹤; 石燕; 卢坚雯

doi:10.13386/j.issn1002-0306.2022050099

HPLC法同时测定甘薯叶中5种酚酸和异槲皮苷

doi: 10.13386/j.issn1002-0306.2022050099

傅志丰^1,,
周鹤¹,
石燕²,
卢坚雯^3, ,

1.
九江市检验检测认证中心食品药品检验检测分中心，江西九江 332000
2.
南昌大学食品科学与技术国家重点实验室，江西南昌 330047
3.
宜春市硒资源开发利用中心，江西宜春 336000

详细信息

作者简介:
傅志丰（1991−），男，硕士，工程师，研究方向：食品检测技术，E-mail：1062830538@qq.com

通讯作者:
卢坚雯（1971−），女，本科，高级农艺师，研究方向：农产品质量安全检测技术研究，E-mail：1139817128@qq.com

中图分类号: TS255.7
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出版历程
- 收稿日期: 2022-05-11
- 网络出版日期: 2023-02-01
- 刊出日期: 2023-03-01

Simultaneous Determination of Five Phenolic Acids and Isoquercitrin in Sweet Potato (Ipomoea batatas (L.) Lam) Leaves by HPLC

FU Zhifeng^1
,,
ZHOU He¹,
SHI Yan²,
LU Jianwen^{3
, ,}

1.
Food and Drug Inspection Sub Center of Jiujiang Inspection, Testing and Certification Center, Jiujiang 332000, China
2.
Stare Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
3.
Selenium Resources Development and Utilization Center of Yichun, Yichun 336000, China

摘要

摘要: 咖啡酸、绿原酸、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸和异槲皮苷是甘薯叶中的主要活性成分，本研究建立高效液相色谱（high performance liquid chromatography, HPLC）同时测定这6种成分的方法。通过对检测波长、色谱柱、洗脱溶剂、洗脱流速等参数优化得到的最佳分析条件为：ZORBAX SB-C₁₈（4.6 mm×250 mm，5 μm）为分析柱，以0.1%甲酸水溶液（A）和乙腈（B）为流动相，采用梯度洗脱：0～12 min，10%~25% B；12～18 min，25% B，流速0.8 mL/min，检测波长326 nm，柱温30 ℃。咖啡酸、绿原酸、3,4-二咖啡酰奎宁酸、3,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸和异槲皮苷的线性范围分别为0.25~25.0、0.5~50.0、5.0~100.0、10.0~200.0、5.0~100.0和0.5~50.0 μg/mL。该方法分析时间短（18 min），线性度（R²≥0.998）、稳定性（RSD≤1.97%）、精密度（RSD≤1.56%）和加标回收率（95.14%~103.22%）好。对江西10个不同产地甘薯叶目标成分分析发现，宜春丰城的甘薯叶具有最高的总酚含量。
- HPLC /
- 咖啡酰奎宁酸 /
- 异槲皮苷 /
- 同时测定 /
- 甘薯叶
Abstract: In this study, a reversed-phase high performance liquid-chromatography coupled to ultraviolet detection (RP-HPLC/UV) method for simultaneous determination of six major bioactive constituents (caffeic acid, chlorogenic acid, 3,4-dicaffeoyl quinic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid and isoquercetin) in sweet potato leaves was developed and validated after optimization of various chromatographic conditions and other experimental parameters. The chromatographic separation was achieved on an Agilent ZORBAX SB-C₁₈ reserved-phase column (4.6 mm×250 mm, 5 μm) using a gradient elution program with a mobile phase consisting of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) (0~12 min, 10%~25% B; 12~18 min, 25% B), at a flow rate of 0.8 mL/min, an operating temperature of 30 ℃, and a wavelength of 326 nm. The calibration curves were linear in the range of 0.25~25.0, 0.5~50.0, 5.0~100.0, 10.0~200.0, 5.0~100.0, 0.5~50.0 μg/mL for caffeic acid, chlorogenic acid, 3,4-dicaffeoyl quinic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid and isoquercetin, respectively. The present method was fast (18 min), and demonstrated acceptable values for linearity (R²≥0.998), stability (RSD≤1.97%), recovery (95.14%~103.22%) and precision (RSD≤1.56%). Additionally, the new method was successfully applied to determine components in sweet potato leaves from ten different regions of Jiangxi Province, and the results indicated that the sample harvested in Fengcheng city of Yichun contained higher total phenol content than those collected in other regions.
- HPLC /
- caffeoylquinic acids /
- isoquercitrin /
- simultaneous determination /
- sweet potato leaves

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图 1 5种酚酸和异槲皮苷的化学结构式

Figure 1. The chemical structures of 5 phenolic acids and isoquercetin

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图 2 5种酚酸和异槲皮苷的紫外扫描光谱图

Figure 2. The ultraviolet absorbance spectra of 5 phenolic acids and isoquercetin

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图 3 不同色谱柱对甘薯叶中5种酚酸和异槲皮苷分离效果的影响

Figure 3. Effect of different columns on the separation of five phenolic acids and isoquercitrin

注：A: Agilent ZORBAX SB-C₁₈柱；B: Phenomenex Luna 5u 100A C₁₈长柱；C: Phenomenex LaChrom C₁₈短柱；1. 绿原酸；2. 咖啡酸；3. 异槲皮苷；4. 3,4-二咖啡酰奎宁酸；5. 3,5-二咖啡酰奎宁酸；6. 4,5-二咖啡酰奎宁酸；图4~图6同。

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图 4 流动相系统对甘薯叶5种酚酸和异槲皮苷分离效果的影响

Figure 4. Effect of mobile phase on the seperation of five phenolic acids and isoquercitrin

注：A：乙腈/0.1%甲酸水；B：甲醇/0.1%甲酸水。

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图 5 流速对甘薯叶5种酚酸和异槲皮苷分离效果的影响

Figure 5. Effect of the flow rate of the mobile phase on the seperation of five phenolic acids and isoquercitrin

注：A：0.6 mL/min；B：0.8 mL/min；C：1.0 mL/min；D：1.2 mL/min。

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图 6 样品和混标溶液色谱图

Figure 6. Chromatograms of samples and standard mixture

注：A：样品；B：混标溶液。

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表 1 5种酚酸和异槲皮苷的线性范围、标准曲线、决定系数、检出限及定量限（n=7）

Table 1. Linear range, standard curve, determination coefficient, detection limit and quantification limit of five phenolic acids and isoquercitrin (n=7)

化合物	线性范围（μg/mL）	标准曲线	决定系数（R²）	检出限（μg/g DM）	定量限（μg/g DM）
咖啡酸	0.25~25.0	Y=67793X−18215	0.9997	0.9	3.0
绿原酸	0.5~50.0	Y=37913X−21415	0.9996	4.3	14.2
3,4-二咖啡酰奎宁酸	5.0~100.0	Y=40131X−105662	0.9980	11.6	38.6
3,5-二咖啡酰奎宁酸	10.0~200.0	Y=25927−110983	0.9985	18.4	61.2
4,5-二咖啡酰奎宁酸	5.0~100.0	Y=44641X−81094	0.9980	12.8	42.6
异槲皮苷	0.5~50.0	Y=18595X−14879	0.9991	12.6	41.9

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表 2 5种酚酸和异槲皮苷的加标回收率结果

Table 2. The recovery results of five phenolic acids and isoquercitrin determined

化合物	样品中含量（μg/g DM）	加入量（μg/g DM）	测得总量平均值（μg/g DM）	平均回收率（%）	RSD （%）
咖啡酸	529	420	939	97.62	1.8
		525	1058	100.76	1.1
		630	1128.4	95.14	1.0
绿原酸	2416	1900	4310.3	99.70	1.6
		2400	4784	98.67	1.8
		2900	5247	97.62	1.4
3,4-二咖啡酰奎宁酸	3332	2650	6017	101.32	2.6
		3350	6790	103.22	3.0
		4000	7426.4	102.36	2.4
3,5-二咖啡酰奎宁酸	12312	9850	21933.5	97.68	2.0
		12300	24272.5	97.24	2.9
		14800	27058.7	99.64	1.7
4,5-二咖啡酰奎宁酸	3857	3100	6895.7	98.02	3.0
		3850	7580.5	96.71	2.9
		4650	8407.5	95.91	1.6
异槲皮苷	1340	1050	2361	97.24	1.6
		1340	2628.3	96.14	2.3
		1600	2874.5	95.91	1.1

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表 3 不同产地甘薯叶中5种酚酸和异槲皮苷的含量

Table 3. Contents of five phenolic acids and isoquercitrin of sweet potato leaves from different areas

样品产地	样品含量（mg/g DM）
样品产地	咖啡酸	绿原酸	3,4-二咖啡酰奎宁酸	3,5-二咖啡酰奎宁酸	4,5-二咖啡酰奎宁酸	异槲皮苷	总计含量
产地1	0.29± 0.02^cd	0.68± 0.09^a	1.55± 0.19^b	8.25± 0.32^d	2.73± 0.39^c	0.63± 0.04^c	14.13
产地2	0.22± 0.01^b	1.10± 0.12^bc	1.52± 0.18^b	8.34± 0.30^d	2.73± 0.26^c	0.67± 0.03^c	14.58
产地3	0.24± 0.02^bc	1.65± 0.14^de	0.61± 0.05^a	8.16± 0.32^d	0.99± 0.05^a	0.44± 0.02^b	12.09
产地4	0.51± 0.04^f	2.99± 0.18^g	3.04± 0.33^d	18.25± 0.20^f	4.49± 0.28^e	0.96± 0.04^e	30.24
产地5	0.19± 0.02^b	0.86± 0.10^ab	2.26± 0.23^c	3.27± 0.19^a	1.97± 0.10^b	1.03± 0.05^e	9.59
产地6	0.31± 0.02^d	1.89± 0.17^e	0.35± 0.02^a	5.40± 0.20^b	1.85± 0.10^b	0.32± 0.01^a	10.15
产地7	0.12± 0.01^a	2.73± 0.14^g	0.23± 0.01^a	4.75± 0.20^b	1.22± 0.08^a	0.40± 0.01^ab	9.45
产地8	0.53± 0.03^f	2.42± 0.12^f	3.33± 0.25^d	12.31± 0.43^e	3.86± 0.43^d	1.34± 0.07^f	23.79
产地9	0.44± 0.02^e	1.86± 0.16^e	2.31± 0.22^c	4.91± 0.20^b	0.85± 0.04^a	0.82± 0.03^d	11.19
产地10	0.41± 0.02^e	1.36± 0.12^cd	2.02± 0.18^c	7.47± 0.30^c	1.29± 0.09^a	0.62± 0.02^c	13.17
注：产地1：吉安新干县；产地2：吉安吉安县；产地3：新余渝水区；产地4：宜春丰城市；产地5：宜春袁州区；产地6：上饶横峰县；产地7：鹰潭贵溪市；产地8：南昌南昌县；产地9：景德镇浮梁县；产地10：九江永修县。同列不同小写字母表示样品间差异显著（P<0.05）。

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参考文献(33)

[1]	NGUYEN H C, CHEN C C, LIN K H, et al. Bioactive compounds, antioxidants, and health benefits of sweet potato leaves[J]. Molecules,2021,26(7):1820. doi: 10.3390/molecules26071820
[2]	JANG Y, KOH E. Antioxidant content and activity in leaves and petioles of six sweet potato (Ipomoea batatas L.) and antioxidant properties of blanched leaves[J]. Food Science and Biotechnology,2019,28(2):337−345. doi: 10.1007/s10068-018-0481-3
[3]	TAIRA J, UEHARA M, TSUCHIDA E, et al. Inhibition of the β-catenin/Tcf signaling by caffeoylquinic acids in sweet potato leaf through down regulation of the Tcf-4 transcription[J]. Journal of Agricultural and Food Chemistry,2014,62(1):167−172. doi: 10.1021/jf404411r
[4]	LUO D, MU T H, SUN H N. Profiling of phenolic acids and flavonoids in sweet potato (Ipomoea batatas L.) leaves and evaluation of their anti-oxidant and hypoglycemic activities[J]. Food Bioscience,2021,39:100801. doi: 10.1016/j.fbio.2020.100801
[5]	NTCHAPDA F, TCHATCHOUANG F C, MIAFFO D, et al. Hypolipidemic and anti-atherosclerogenic effects of aqueous extract of Ipomoea batatas leaves in diet-induced hypercholesterolemic rats[J]. Journal of Integrative Medicine,2021,19(3):243−250. doi: 10.1016/j.joim.2021.02.002
[6]	ISHIDA H, SUZUNO H, SUGIYAMA N, et al. Nutritive evaluation on chemical components of leaves, stalks and stems of sweet potatoes (Ipomoea batatas poir)[J]. Food Chemistry,2000,68(3):359−367. doi: 10.1016/S0308-8146(99)00206-X
[7]	CHEN Y, FU Z F, TU Z C, et al. Influence of in vitro gastrointestinal digestion on the bioavailability and antioxidant activity of polyphenols from Ipomoea batatas leaves[J]. International Journal of Food Science & Technology,2017,52(5):1131−1137.
[8]	ZHANG C C, LIU D Q, WU L H, et al. Chemical characterization and antioxidant properties of ethanolic extract and its fractions from sweet potato (Ipomoea batatas L.) leaves[J]. Foods,2019,9(1):15. doi: 10.3390/foods9010015
[9]	NAVEED M, HEJAZI V, ABBAS M, et al. Chlorogenic acid (CGA): A pharmacological review and call for further research[J]. Biomedicine & Pharmacotherapy,2018,97:67−74.
[10]	CHEN S, LIU J, DONG G Q, et al. Flavonoids and caffeoylquinic acids in Chrysanthemum morifolium Ramat flowers: A potentially rich source of bioactive compounds[J]. Food Chemistry,2021,344:128733. doi: 10.1016/j.foodchem.2020.128733
[11]	LIAN W W, DU G H. Caffeic acid[M]//Natural small molecule drugs from plants. Springer, Singapore, 2018: 19−23.
[12]	LI C, TAN F, YANG J J, et al. Antioxidant effects of Apocynum venetum tea extracts on D-galactose-induced aging model in mice[J]. Antioxidants,2019,8(9):381. doi: 10.3390/antiox8090381
[13]	STASZOWSKA-KARKUT M, MATERSKA M. Phenolic composition, mineral content, and beneficial bioactivities of leaf extracts from black currant (Ribes nigrum L.), raspberry (Rubus idaeus), and aronia (Aronia melanocarpa)[J]. Nutrients,2020,12(2):463. doi: 10.3390/nu12020463
[14]	EL-HAWARY S S, MOHAMMED R, EL-DIN M E, et al. Comparative phytochemical analysis of five Egyptian strawberry cultivars (Fragaria×ananassa Duch.) and antidiabetic potential of festival and red merlin cultivars[J]. RSC Advances,2021,11(27):16755−16767. doi: 10.1039/D0RA10748D
[15]	FU Z F, TU Z C, ZHANG L, et al. Antioxidant activities and polyphenols of sweet potato (Ipomoea batatas L.) leaves extracted with solvents of various polarities[J]. Food Bioscience,2016,15:11−18. doi: 10.1016/j.fbio.2016.04.004
[16]	WAY M L, JONES J E, NICHOLS D S, et al. A comparison of laboratory analysis methods for total phenolic content of cider[J]. Beverages,2020,6(3):55. doi: 10.3390/beverages6030055
[17]	BAE I K, HAM H M, JEONG M H, et al. Simultaneous determination of 15 phenolic compounds and caffeine in teas and mate using RP-HPLC/UV detection: Method development and optimization of extraction process[J]. Food Chemistry,2015,172:469−475. doi: 10.1016/j.foodchem.2014.09.050
[18]	RUSSO M, FANALI C, TRIPODO G, et al. Analysis of phenolic compounds in different parts of pomegranate (Punica granatum) fruit by HPLC-PDA-ESI/MS and evaluation of their antioxidant activity: Application to different Italian varieties[J]. Analytical and Bioanalytical Chemistry,2018,410(15):3507−3520. doi: 10.1007/s00216-018-0854-8
[19]	MUSTAFA A M, ANGELONI S, ABOUELENEIN D, et al. A new HPLC-MS/MS method for the simultaneous determination of 36 polyphenols in blueberry, strawberry and their commercial products and determination of antioxidant activity[J]. Food Chemistry,2022,367:130743. doi: 10.1016/j.foodchem.2021.130743
[20]	钟伟. 红薯叶中多酚类物质的抗氧化及抗肿瘤细胞增殖作用研究[D]. 广州: 华南理工大学, 2015 ZHONG W. Research evaluation of antioxidant and antitumour activities of Ipomoea batatas leaves[D]. Guangzhou: South China University of Technology, 2015
[21]	Q2 (R1) Validation of analytical procedures: Text and methodology[S].2005.
[22]	覃日宏, 陈汝旭, 盘涌, 等. HPLC法同时测定五指毛桃中6个黄酮类成分的含量[J]. 药物分析杂志,2020,40(12):2244−2249. [QIN R H, CHEN R X, PAN Y, et al. Simultaneous determination of six flavonoids in Ficushirta Vahl. by HPLC[J]. Chinese Journal of Pharmaceutical Analysis,2020,40(12):2244−2249. doi: 10.16155/j.0254-1793.2020.12.19
[23]	TSOLMON B, FANG Y, YANG T, et al. Structural identification and UPLC-ESI-QTOF-MS-MS analysis of flavonoids in the aquatic plant Landoltia punctata and their in vitro and in vivo antioxidant activities[J]. Food Chemistry,2021,343:128392. doi: 10.1016/j.foodchem.2020.128392
[24]	LIU J, MU T H, SUN H N, et al. Optimization of ultrasonic-microwave synergistic extraction of flavonoids from sweet potato leaves by response surface methodology[J]. Journal of Food Processing and Preservation,2019,43(5):e13928. doi: 10.1111/jfpp.13928
[25]	马帅, 王纪华, 高媛, 等. 超高效液相色谱-串联质谱法同时测定5个产地花椰菜和西兰花中的23种酚酸类化合物[J]. 食品科学,2018,39(4):176−187. [MA S, WANG J H, GAO Y, et al. Simultaneous determination of twenty-three phenolic acids in Cauliflower (Brassica oleracea L. var. botrytis L.) and Broccoli (B. oleracea L. var. italica) from five producing places by ultra performance liquid chromatography-tandem mass spectrometry[J]. Food Science,2018,39(4):176−187. doi: 10.7506/spkx1002-6630-201804027
[26]	ZHANG A, WAN L, WU C Y, et al. Simultaneous determination of 14 phenolic compounds in grape canes by HPLC-DAD-UV using wavelength switching detection[J]. Molecules,2013,18(11):14241−14257. doi: 10.3390/molecules181114241
[27]	HUANG R T, LU Y F, INBARAJ B S, et al. Determination of phenolic acids and flavonoids in Rhinacanthus nasutus (L.) kurz by high-performance-liquid-chromatography with photodiode-array detection and tandem mass spectrometry[J]. Journal of Functional Foods,2015,12:498−508. doi: 10.1016/j.jff.2014.12.002
[28]	段云飞, 吴光斌, 叶洪, 等. HPLC法同时测定采后莲雾果实7种有机酸的含量[J]. 食品科学,2021,42(4):175−180. [DUAN Y F, WU G B, YE H, et al. Simultaneous determination of seven organic acids in wax apple (Syzygium samarangenese [Blume] Merrill & L. M. Perry) fruit during postharvest storage by high performance liquid chromatography[J]. Food Science,2021,42(4):175−180. doi: 10.7506/spkx1002-6630-20191025-287
[29]	朱亚珠. 甘薯叶中咖啡酰奎尼酸类物质的分离纯化和高效液相色谱法分析[J]. 食品工业科技,2015,36(10):73−77. [ZHU Y Z. Purification of caffeoylquinic acids from sweet potato leaves and their analysis by high performance liquid chromatography[J]. Science and Technology of Food Industry,2015,36(10):73−77. doi: 10.13386/j.issn1002-0306.2015.10.006
[30]	MAKORI S I, MU T H, SUN H N. Total polyphenol content, antioxidant activity, and individual phenolic composition of different edible parts of 4 sweet potato cultivars[J]. Natural Product Communications, 2020, 15(7): 1934578X20936931.
[31]	SUÁREZ S, MU T H, SUN H N, et al. Antioxidant activity, nutritional, and phenolic composition of sweet potato leaves as affected by harvesting period[J]. International Journal of Food Properties,2020,23(1):178−188. doi: 10.1080/10942912.2020.1716796
[32]	XIANG G, YANG H Y, YANG L, et al. Multivariate statistical analysis of tobacco of different origin, grade and variety according to polyphenols and organic acids[J]. Microchemical Journal,2010,95(2):198−206. doi: 10.1016/j.microc.2009.12.001
[33]	ZHANG D Y, WAN Y, HAO J Y, et al. Evaluation of the alkaloid, polyphenols, and antioxidant contents of various mulberry cultivars from different planting areas in eastern China[J]. Industrial Crops and Products,2018,122:298−307. doi: 10.1016/j.indcrop.2018.05.065