UPLC-Q-TOF-MS Based Analysis of Chemical Constituents of Seeds and Peels of <i>Alpinia oxyphylla</i>

WANG Maoyuan; WANG Zhunian; WANG Qinglong; YANG Qing; ZHANG Min; YAN Xiaoxia

doi:10.3969/j.issn.1000-2561.2022.05.021

Chinese Journal of Tropical Crops >

2022 , Vol. 43 >Issue 5: 1064 - 1075

DOI: https://doi.org/10.3969/j.issn.1000-2561.2022.05.021

Post-harvest Treatment & Quality Safety

UPLC-Q-TOF-MS Based Analysis of Chemical Constituents of Seeds and Peels of Alpinia oxyphylla

WANG Maoyuan ¹^,³^,⁴ ,
WANG Zhunian ^,¹^,³^,⁴^,^* ,
WANG Qinglong ¹^,² ,
YANG Qing ¹^,³^,⁴ ,
ZHANG Min ⁵ ,
YAN Xiaoxia ¹^,³^,⁴

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1. Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
2. National Tropical Plants Germplasm Resource Center, Haikou, Hainan 571101, China
3. Tropical Wild Plant Gene Resource, Ministry of Agriculture & Rural Affairs, Danzhou, Hainan 571737, China
4. Hainan Provincial Engineering Research Center for Tropical Medicinal Plant, Danzhou, Hainan 571737, China
5. Key Laboratoory of Southem Subtropical Plant Diversity, Shenzhen Fairy Lake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen, Guangdong 518004

* WANG Zhunian,E-mail： 13807596560@163.com.

Received date: 2021-02-19

Revised date: 2021-12-29

Online published: 2022-05-30

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Abstract

The purpose of this paper was to analyze and identify the nonvolatile components of the ethanol extract in the seeds and peels of Alpinia oxyphylla. The extracts were prepared by the refluxing methods, high-resolution mass spectrometric data of the extract were collected by the ultra performance liquid chromatography coupled with quadrupole-time of flight-tandem mass spectrometry (UPLC-Q-TOF-MS). The main components in the extracts were separated by the gradient elution method with waters ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 μm), the mobile phase consisted of 0.1% formic acid and 0.01% formic acid acetonitrile, with the flow rate of 0.3 mL/min. Mass spectrometry was applied for the qualitative analysis with ESI ion source. Through molecular feature extraction (MFE), Masslynx software was then used to process the original MS data. Furthermore, the structures of the compounds were speculated according to the MS fragmentation patterns and confirmed by comparison with reference standards. Thirty-two and fifty compounds were identified from the seeds and peels respectively, including flavonoids, phenolic acids, saccharides, fatty acids and terpenes. 58 compounds were isolated from the plant for the first time. The main constituents of the seeds were phenolic acids (11 compounds), which were representative constituents of the seeds, accounting for one third of the total identified structural compounds of the seeds. On the contrary, there were few phenolic acids in the peels. The main constituents of the peels were flavonoids (19 compounds) of various kinds, accounting for one third of the total identified structural compounds of the peels. On the contrary, there was only one flavonoid identified in the seeds. A total of 4 components were found both in the nonvolatile component of the ethanol extract in the seeds and peels, including monosaccharides of D-gluconic acid and D-glucopyranuronic acid, disaccharide of sucrose, flavonoid of chysin. The results showed that the nonvolatile components of the seeds were lesser than that of the peels. The chemical constituents of the seeds and peels were found to be quite different. The main constituents of the seeds were phenolic acids, and the main constituents of the fruit were flavonoids and terpenoids. It should be treated differently in comprehensive utilization and quality control of the seeds and peels of A. oxyphylla.

Key words： UPLC-Q-TOF-MS; seeds and peels of Alpinia oxyphylla; chemical constituents

Cite this article

WANG Maoyuan , WANG Zhunian , WANG Qinglong , YANG Qing , ZHANG Min , YAN Xiaoxia . UPLC-Q-TOF-MS Based Analysis of Chemical Constituents of Seeds and Peels of Alpinia oxyphylla[J]. Chinese Journal of Tropical Crops, 2022 , 43(5) : 1064 -1075 . DOI: 10.3969/j.issn.1000-2561.2022.05.021

益智（Alpinia oxyphylla Miq.）为姜科山姜属植物,是我国著名的四大南药之一,也是海南省道地药材,在广西、广东、福建、云南等省（区）也有分布^[1]。益智以干燥成熟果实入药,商品名为益智仁、益智子,为我国历版药典收载,具有暖肾固精缩尿,温脾止泻摄唾等功效,用于肾虚遗尿,小便频数,遗精白浊,脾寒泄泻,腹中冷痛,口多唾涎等症^[2]。益智是民间常用药材,也是卫计委公布的药食同源原料,被广泛应用在我国许多著名方剂、中成药或保健品中,如缩泉丸^[3-4]、遗溺汤、补原丸、益智酒^[5]、益智调味酱^[6-7]、益智茶^[8]等。已有研究表明益智含有挥发油类、二苯庚烷类、黄酮类、萜类、甾醇及其苷类等化学成分^[9⇓⇓-12],具有保护神经、抗氧化应激、抗糖尿病肾病、改善肠胃功能、镇静催眠、抗肿瘤、抗菌等功效^{[10,13⇓⇓⇓⇓ -18]}。益智在现代临床上有去壳（果皮）和带壳两种使用方法,然而历版中国药典记载益智（果实）作为饮片炮制时,需除去外壳（果皮）,以捣碎的种子或种子团残瓣入药或进一步炮制使用,约占干燥果实29%~31%的果皮作为废弃物被丢弃,存在益智药材资源浪费的问题^[19]。近年来,关于益智种子和果皮化学成分的比较研究多集中在挥发油^[20-21]、总黄酮^[22]、总多糖^[23]和低聚糖^[24]等主要成分的含量对比分析,而采用HPLC技术对益智种子和果皮化学成分的对比分析较少^[25],尚未弄清二者的化学物质组成和区别,尤其是非挥发性成分组成及对比更鲜见报道。因此,为科学发掘益智资源的利用价值,有必要明晰益智种子和果皮的非挥发性化学物质组成和区别。基于此,在前期研究基础上^[26],笔者首次采用UPLC-Q-TOF-MS技术建立与现有中药质量控制发展水平相符合的检测方法,完成对益智种子和果皮乙醇提取物非挥发性化学成分的定性分析,通过对比分析传统药用部位益智种子和废弃物益智果皮非挥发性化学成分类别和组成,以阐明益智资源的内涵,为明确其非传统药用部位的开发利用价值提供科学依据,同时也为益智炮制和临床使用及其相关产品的开发,完善质量标准提供参考。

1 材料与方法

1.1 材料

1.1.1 植物材料

益智果实于2018年5月采摘于中国热带农业科学院热带作物品种资源研究所农业农村部热带药用植物种质圃,经王祝年研究员鉴定为姜科山姜属植物益智（Alpinia oxyphylla Miq.）的干燥成熟果实。人工剥离,分别获得益智种子和益智果皮。

1.1.2 仪器与试剂

Xevo G2-XS QTof液质联用仪,美国Waters公司;Secura513-1CN精密天平,德国Sartorius公司;KQ3200DE超声波清洗器,昆山市超声仪器有限公司;5810R高速冷冻离心机,德国Eppendorf Centrifuge公司;索氏提取器,四川蜀玻玻璃仪器有限公司;Masslynx V4.1工作站,美国Waters公司;Waters UNIFI^®科学信息系统,美国Waters公司。

乙腈（质谱纯）,美国Fisher公司;甲酸（质谱纯）,德国Merck Millipore公司;蒸馏水,广州屈臣氏食品饮料有限公司;KG2211W离心管（1.5 mL）,美国KiRGEN公司;一次性无菌注射器,丰临医疗器械有限公司;Pall GHPAcrodisc^® Syringe滤膜（0.2 μm、13 mm）,美国Pall公司;Labmed移液枪头,美国Labmed Biotech公司;2 mL样品瓶,美国Waters公司;Oasis HLB 3cc (60 mg) Extraction Cartridges,美国Waters公司。

1.2 方法

1.2.1 供试品溶液制备

益智种子和益智果皮经粉碎后,分别精密称取粉末1 g,加石油醚200 mL,于60℃水浴锅提取3次,每次30 min。提取残渣用冰水冷却、抽滤、除去石油醚。再将残渣分别用70%乙醇50 mL于80℃回流提取3次,每次30 min,合并3次的提取液。分别取益智种子和益智果皮70%乙醇提取液1 mL,冷冻离心（12 000 r/min,10 min,4℃）,静置1~2 min,取上清500 μL过HLB固相萃取柱,先用1 mL 5% MeOH洗脱,再用1 mL95% MeOH洗脱,收集95% MeOH洗脱液,过0.2 μm微孔滤膜,转至2 mL透明样品瓶,标记为益智种子（Z）和益智果皮（K）,4℃冰箱保存备用。

1.2.2 UPLC-Q-TOF-MS分析

取1.3.1制备的益智样品进行分析。

UPLC色谱条件：色谱柱为Waters ACQUITY UPLC HSS T3（100 mm×2.1 mm,1.8 μm）;流动相：0.1%甲酸水溶液（A）-0.01%甲酸乙腈溶液（B）,梯度洗脱（0~1 min,1%~25% B;1~7 min,25%~55% B;7~15 min,55%~99% B;15~17 min,99%~99% B;17~20 min,99%~1% B;）;流量：0.3 mL/min;柱温40℃;进样量1 μL。

质谱条件：电喷雾离子源（ESI）,离子源温度100℃;正、负离子模式的毛细管电压3.0、2.0 kV;锥孔电压40 V;低能量碰撞电压（CE）6 V,高能量碰撞电压20~60 V;去溶剂化温度450℃;去溶剂化气体流量600 L/h;锥孔气体流量50 L/h;扫描范围m/z 50~1200,扫描时间0.2 s,检测时间20 min。

1.2.3 定性分析

应用LC-MS液质联用仪采集数据,进行益智种子和益智果皮化学成分的定性分析。利用UNIFI科学信息系统,提取化学成分的一级质谱及二级碎片数据。基于强大精确质谱分析平台和ChemSpider在线检索数据库,结合文献报道信息,综合化合物相应结构特征、精确相对分子质量和质谱裂解规律,对化学组分进行识别鉴定,降低假阳性。

2 结果与分析

2.1 益智种子、果皮提取物的化学成分鉴定

基于UPLC-Q-TOF-MS技术分别在正、负离子模式下,对益智种子和益智果皮的化学成分进行定性分析。通过Masslynx软件进行初步分析及图谱信息对比,其中负离子（-）ESI-MS出峰响应较高,分离效果较好。因此,以益智种子和益智果皮的（-）ESI-MS数据导入UNIFI进行详细的化学成分鉴定,根据鉴定结果（表1、表2）,标记化合物的出峰位置,如图1所示。

表1 益智种子化学成分的UPLC-Q-TOF-MS鉴定结果（负离子模式）

Tab. 1 Identification of chemical constituents from seeds of A. oxyphylla by UPLC-Q-TOF-MS

峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ions	分子式 Formula	相对分子质量 Molecular weight/(m·z^-1)		误差 Mass error (×10^-6)	化合物 Compounds
峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ions	分子式 Formula	实测值 Observed	理论值 Theoretical	误差 Mass error (×10^-6)	化合物 Compounds
1	0.84	-	C₆H₁₂O₇	196.0582	196.0583	-0.4	D-gluconic acid
2	0.84	-	C₆H₁₀O₇	194.0425	194.0427	-0.9	D-glucopyranuronic acid
3	0.87	179.0557	C₁₂H₂₂O₁₁	342.1167	342.1162	1.3	sucrose
4	0.88	179.0557	C₁₂H₂₂O₁₁	342.1169	342.1162	1.9	α-cellobiose
5	0.93	179.0553	C₁₂H₂₂O₁₁	342.1164	342.1162	0.6	4-O-hexopyranosylhex-2-ulofuranose
6	1.97	109.0295	C₇H₁₀O₅	174.0523	174.0528	-2.2	3,4,5-trihydroxy-1-cyclohexenecarboxylic acid
7	2.21	109.0295	C₇H₆O₃	138.0311	138.0317	-4.5	salicylic acid
8	2.31	109.0295	C₁₆H₂₆O₆	314.1719	314.1729	-3.5	(1R,5S,6R)-2,7,7-trimethylbicyclo[3.1.1]hept-2-en-6-yl L-galactopyranoside
9	2.66	119.0502	C₉H₈O₃	164.0464	164.0473	-5.5	(E)-p-coumaric acid
10	2.94	-	C₉H₈O₄	180.0413	180.0423	-5.4	trans-caffeic acid
11	3.46	-	C₁₅H₂₈O₄	272.1975	272.1988	-3.9	pentadecanedioic acid
12	3.55	191.1431	C₁₃H₂₀O₂	208.1458	208.1463	-2.1	(7E,9E,11E)-7,9,11-tridecatrienoic acid
13	3.62	108.0210	C₉H₁₀O₄	182.0574	182.0579	-2.8	homovanillic acid
14	3.82	108.0210	C₁₀H₁₀O₅	210.0522	210.0528	-2.8	(2E)-5-hydroxyferulic acid
15	4.13	239.1645	C₁₅H₂₄O₆	300.1569	300.1573	-1.2	(2Z,4E)-3-methyl-5-[(1S,2R,4S)-1,2,4-trihydroxy-2- (hydroxymethyl)-6,6-dimethylcyclohexyl]-2,4-pentadienoic acid
16	4.23	189.0906	C₁₃H₁₆O₂	204.1143	204.1150	-2.9	4'-hydroxy-3'-prenylacetophenone
17	5.35	-	C₁₅H₁₀O₄	254.0576	254.0579	-1.2	chysin
18	5.73	-	C₁₆H₁₂O₅	284.0652	284.0658	-2.1	isalpinin
19	6.67	151.0754	C₁₅H₂₀O₅	280.1306	280.1311	-1.6	(+)-varitriol
20	6.84	191.1433	C₁₄H₂₀O₃	236.1408	236.1412	-1.7	1-(3,4-dimethoxyphenyl)-1-hexanone
21	7.11	151.0756	C₁₅H₂₀O₅	280.1309	280.1311	-0.7	2-[2-(2-hydroxymethyl-3-methoxy-phenyl)-vinyl]-5-methyl-tetrahydro-furan-3,4-diol
22	7.13	191.1434 235.1332	C₁₅H₂₄O₃	252.1722	252.1725	-1.3	2-(8-hydroxy-4a,8-dimethyldecahydro-2-naphthalenyl)acrylic acid
23	7.33	217.1228	C₁₅H₂₄O₃	252.1721	252.1725	-1.7	kandenol A
24	8.52	233.1537	C₁₄H₂₂O	206.1667	206.1671	-1.3	octylphenol
25	9.49	297.2432	C₁₈H₃₆O₄	316.2613	316.2614	-0.2	9,10-dihydroxystearic acid
26	9.93	233.1537	C₂₇H₄₄O₁₀	528.2926	528.2934	-1.6	3-(β-D-galactopyranosyloxy)-2-hydroxypropyl 8-{(1S,5S)-4-oxo-5-[(2Z)-2-penten-1-yl]-2-cyclopenten-1-yl}octanoate
27	10.41	277.2166	C₁₈H₃₂O₃	296.2345	296.2351	-2.2	13-hydroxyoctadecadienoic acid
28	10.47	277.2166	C₂₇H₄₆O₁₀	530.3084	530.3091	-1.3	(2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl (5xi)-6-O-[(4xi)-6-deoxy-L-lyxo-hexopyranosyl]-β-D-ribo-hexopyranoside
29	11.11	255.2325	C₂₄H₄₄O₈	460.3048	460.3036	2.4	tetracosahydrodibenzo[b,n][1,4,7,10,13,16,19,22] octaoxacyclotetracosine
30	11.58	281.2479	C₂₆H₄₆O₈	486.3212	486.3193	3.6	(2R,4aR,5S,6R)-6-hydroxy-5-[(3E)-5-hydroxy-3- methyl-3-penten-1-yl]-1,1,4a,6-tetramethyldecahydro-2-naphthalenyl β-D-allopyranoside
31	13.93	163.1121	C₂₂H₃₀	294.2344	294.2348	-1.0	4-decylbiphenyl
32	15.08	146.9646	C₁₈H₃₄O₂	282.2546	282.2559	-4.5	oleic acid

表2 益智果皮化学成分的UPLC/Q-TOF-MS鉴定结果（负离子模式）

Tab. 2 Identification of chemical constituents from peels of A. oxyphylla by UPLC-Q-TOF-MS

峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ion	分子式 Formula	相对分子质量 Molecular weight/(m·z^-1)		误差 Mass error (×10^-6)		化合物 Compound
峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ion	分子式 Formula	实测值 Observed	理论值 Theoretical	误差 Mass error (×10^-6)		化合物 Compound
1	0.82	-	C₆H₁₄O₆	182.0779	182.0790	-6.5		D-galactitol
2	0.83	103.0030	C₆H₁₂O₇	196.0575	196.0583	-4.3		D-gluconic acid
3	0.84	103.0030	C₆H₁₀O₇	194.0418	194.0427	-4.3		D-glucopyranuronic acid
4	0.88	161.0444 179.0551	C₁₂H₂₂O₁₁	342.1154	342.1162	-2.2		sucrose
5	0.93	191.0186	C₁₂H₁₈O₁₂	354.0791	354.0798	-2.2		4-O-[(5xi)-4-deoxy-β-L-threo-hexopyranuronosyl]-α-D-galactopyranuronic acid
6	2.00	289.0715 425.0867	C₃₀H₂₆O₁₂	578.1434	578.1424	1.6		procyanidin B1
7	2.07	300.0277	C₃₃H₄₀O₂₀	756.2124	756.2113	1.5		quercetin-3-O-α-L-rhamnopyranosyl(1-2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside
8	2.16	289.0711 301.0344	C₂₇H₂₈O₁₇	624.1323	624.1326	-0.5		luteolin-7-O-β-D-glucuronopyranosyl-(1-6)-β- D-glucopyranoside
9	2.19	300.0264	C₂₇H₃₀O₁₆	610.1521	610.1534	-2.1		quercetin-3-O-α-L-rhamnopyranosyl(1-6)-β-D-glucopyranoside
10	2.27	243.0293 300.0277	C₂₇H₃₀O₁₆	610.1544	610.1534	1.7		quercetin-5-O-α-L-rhamnopyranosyl(1-6)-β-D-glucopyranoside
11	2.30	285.0399	C₂₇H₂₈O₁₆	608.1380	608.1377	0.5		apigenin-7-O-β-D-glucopyranoside
12	2.42	151.0030 285.0387 300.0268 301.0349	C₂₁H₁₈O₁₃	478.0743	478.0747	-0.9		miquelianin
13	2.44	284.0316 300.0268	C₂₁H₂₀O₁₂	464.0950	464.0955	-1.1		quercetin-3-O-β-D-glucopyranoside
14	2.49	284.0320 285.0396	C₂₇H₃₀O₁₅	594.1582	594.1585	-0.5		kaempferol-3-O-rutinoside
15	2.56	284.0320 285.0395 545.1298	C₂₉H₃₀O₁₇	650.1482	650.1483	-0.2		malonylapiin
16	2.70	285.0401 284.0322	C₂₁H₁₈O₁₂	462.0798	462.0798	-0.1		kaempferol-3-glucuronide
17	2.70	285.0401	C₂₁H₂₀O₁₁	448.1003	448.1006	-0.6		luteolin-7-O-glucoside
18	3.31	151.0391	C₂₁H₂₈O₇	392.1822	392.1835	-3.3		1-(3,4-Dimethoxyphenyl)-1-methoxy-3-(2,4,6-trimethoxyphenyl)-2-propanol
19	3.35	268.0366	C₂₁H₂₀O₁₀	432.1042	432.1056	-3.4		3,5,7-trihydroxy-flavone-3-O-β-D-glucoside
20	3.71	165.0549 195.0655	C₂₁H₂₆O₇	390.1673	390.1679	-1.5		2,5-dimethoxybenzyl 3-(3,4,5-trimethoxyphenyl)propanoate
21	3.90	253.0498	C₂₁H₂₀O₉	416.1094	416.1107	-2.9		7,4'-dihydroxy-flavone-7-O-β-D-glucoside
22	3.99	151.0387 290.1668	C₂₂H₃₀O₇	406.1980	406.1992	-2.8		1,1'-[oxybis(2,1-ethanediyloxy-2,1-ethanediyloxy)]bis(2-methoxybenzene)
23	4.06	151.0028	C₁₅H₁₀O₇	302.0422	302.0427	-1.4		quercetin
24	4.38	253.0498	C₁₅H₁₀O₅	270.0518	270.0528	-3.4		apigenin
25	4.66	179.0704	C₂₁H₂₆O₆	374.1723	374.1729	-1.6		hexahydrocurcumin
26	4.99	-	C₁₅H₁₀O₆	286.0474	286.0477	-1.3		luteolin
27	5.35	-	C₁₅H₁₀O₄	254.0566	254.0579	-4.2		chysin
28	5.55	99.0444 235.0972	C₂₁H₂₄O₆	372.1567	372.1573	-1.7		tetrahydrocurcumin
29	5.77	277.2158 321.2065	C₁₉H₃₂O₅	340.2242	340.2250	-2.4		3α-hydroxy-3,5-dihydromonacolin L acid
30	5.88	206.0934 323.2216	C₂₀H₃₂O₆	368.2186	368.2199	-3.6		(5E)-7-{4-Hydroxy-2-[(1E)-3-hydroxy-1-octen-1-yl]-6-oxotetrahydro-2H-pyran-3-yl}-5-heptenoic acid
31	6.61	295.1906 307.1914	C₁₈H₃₀O₅	326.2086	326.2093	-2.2		penisporolide A
峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ion	分子式 Formula	相对分子质量 Molecular weight/(m·z^-1)		误差 Mass error (×10^-6)	化合物 Compound
峰号 No.	保留时间 t_R/min	特征碎片 Fragmentation ion	分子式 Formula	实测值 Observed	理论值 Theoretical	误差 Mass error (×10^-6)	化合物 Compound
32	6.70	289.2163 307.2269	C₂₀H₃₂O₅	352.2240	352.2250	-2.6	(-)-prostaglandin E2
33	6.77	337.2015	C₂₀H₃₀O₇	382.1985	382.1992	-1.8	cytosporin E
34	6.94	291.1964 335.1860	C₁₉H₃₀O₆	354.2045	354.2042	0.8	3,4-bis[(6-hydroxyhexyl)oxy]benzoic acid
35	7.06	291.1964	C₂₀H₃₀O₇	382.1990	382.1992	-0.3	3-(hexanoyloxy)-2-butanyl 3,4,5-trimethoxybenzoate
36	7.13	-	C₁₅H₁₂O₄	256.0729	256.0736	-2.6	liquiritigenin
37	7.49	99.0443	C₁₆H₂₆O₄	282.1822	282.1831	-3.2	1,10-decanediyl bisacrylate
38	7.80	277.2165 307.2268	C₂₀H₃₂O₅	352.2241	352.2250	-2.5	(5E)-7-{3-[(1E)-3-hydroxy-1-octen-1-yl]-2,6-dioxabicyclo[3.1.1]hept-4-yl}-5-heptenoic acid
39	8.42	261.1853	C₂₀H₂₈O₄	332.1982	332.1988	-1.6	carnosic acid
40	8.52	259.2058	C₁₉H₃₀O₄	322.2138	322.2144	-2.0	6-decylubiquinone
41	8.58	287.2008	C₂₀H₃₀O₅	350.2086	350.2093	-2.0	(-)-andrographolide
42	8.69	279.1591 305.2114	C₂₀H₃₀O₅	350.2087	350.2093	-1.9	florlide A
43	8.76	261.1852	C₁₈H₂₈O₄	308.1982	308.1988	-1.7	5-O-methyl embelin
44	9.51	265.1465 289.2154	C₂₀H₃₀O₄	334.2132	334.2144	-3.7	leukotriene B5
45	9.88	271.2050 289.2154	C₂₀H₃₀O₄	334.2143	334.2144	-0.3	(3R,3aS,4S,8aS)-3-hydroxy-3-isopropyl-6,8a-dimethyl-8-oxo-1,2,3,3a,4,5,8,8a-octahydro-4-azulenyl (2E)-2-methyl-2-butenoate
46	9.98	255.2328 397.1349 653.3759	C₃₂H₆₀O₁₆	700.3889	700.3881	1.1	tetradecylα-D-galactopyranosyl-(1-4)-α-D-galactopyranosyl-(1-4)-α-D-galactopyranoside
47	10.14	319.1970	C₂₀H₂₈O₆	364.1883	364.1886	-0.9	rabdoternin A
48	12.33	151.0391	C₂₉H₄₀O₆	484.2779	484.2825	-9.5	stigmatellin Y
49	14.09	-	C₁₈H₃₂O₂	280.2388	280.2402	-5.2	linolic acid
50	14.26	151.0391 376.1511	C₃₆H₆₂O₉	638.4382	638.4394	-1.9	ginsenoside F1

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图1 益智种子（Z）和果皮（K）负离子模式的BPI色谱图

Fig. 1 Base peak chromatograms (BPI) with negative ion mode of seeds (Z) and peels (K) of A. oxyphylla

根据检测到的化合物精确相对分子质量（保留4位小数）、特征碎片、保留时间和峰型参考,综合文献报道信息及在线数据库检索结果,分析得到益智种子中32个化合物,益智果皮中50个化合物。

2.2 化合物分析

2.2.1 单糖和二糖类化合物

糖类是植物光合作用形成的初级产物,也是绝大多数天然产物生物合成的初始原料。从益智的果皮和种子中共鉴定得到7个单糖和二糖类化合物,结果见图2。其中单糖D-gluconic acid（K2,Z1）和D-glucopyranuronic acid（K3,Z2）,二糖sucrose（K4,Z3）是益智果皮和种子所共有成分,单糖D-galactitol（K1）和二糖4-O-[(5xi)-4-deoxy-β- L-threo-hexopyranuronosyl]-α-D-galactopyranuronic acid（K5）仅在益智果皮中鉴定得到。从益智种子中鉴定得到2个二糖类化合物α-cellobiose（Z4）和4-O-hexopyranosylhex-2-ulofuranose（Z5）。其中K1和K2/Z1是单糖的糖醇结构,是天然界分布很广的一类成分,具有甜味。K2、Z1为直链D-葡萄糖酸,K3、Z2为D-葡萄糖酸的六元环结构,K4和Z3是二糖蔗糖。

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图2 单糖、二糖和脂肪类化合物结构图

Fig. 2 Structure of monosaccharide, disaccharide and fats from seeds (Z) and peels (K) of A. oxyphylla

2.2.2 脂肪类化合物

脂肪类化合物又称开链化合物,是有机分子中的碳原子连接成链状,如长链烯烃、酮类、脂肪醇类和脂肪酸类等。脂肪类化合物广泛存在于植物体内,是合成复杂有机分子的前体成分。从图2可知,益智果皮中鉴定得到4个脂肪类化合物包括1,10-decanediyl bisacrylate（K37）、leukotriene B5（K44）、tetradecylα-D-galactopyranosyl-(1-4)-α-D-galactopyranosyl-(1-4)-α-D-galactopyranoside（K46）、linolic acid（K49）。其中K37、K44和K49是脂肪烯酸结构,K46是脂肪酸的糖苷结构。从益智种子中鉴定得到5个脂肪酸类化合物,其中化合物pentadecanedioic acid（Z11）和9,10- dihydroxystearic acid（Z25）是长链脂肪酸,化合物(7E,9E,11E)-7,9,11-tridecatrienoic acid（Z12）、13-hydroxyoctadecadienoic acid（Z27）和oleic acid（Z32）是多烯脂肪酸结构。

2.2.3 酚酸类化合物

酚酸类成分在植物中广泛分布,其基本结构是酚羟基取代的芳香羧酸。从益智果皮中仅鉴定得到6个酚酸类化合物（图3）,化合物1-(3,4-dimethoxyphenyl)-1-methoxy-3-(2,4, 6-trimethoxyphenyl)-2-propanol（K18）、2,5-dimethoxybenzyl 3-(3,4,5-trimethoxyphenyl) propanoate（K20）、1,1'-[oxybis(2,1-ethanediyloxy-2,1-ethanediyloxy)] bis(2-methoxybenzene)（K22）和3-(hexanoyloxy)-2-butanyl 3,4,5-trimethoxybenzoate（K35）均为多羟基取代苯酚的甲氧基衍生物,另外鉴定得到益智的特征性化合物,二苯基庚烷类成分hexahydrocurcumin（K25）和tetrahydrocurcumin（K28）。酚酸类化合物是益智种子的代表性成分,共鉴定得到11个化合物,其中化合物3,4,5-trihydroxy-1-cyclohexenecarboxylic acid（Z6）、salicylic acid（Z7）、4'-hydroxy-3'-prenylacetophenone（Z16）、(+)-varitriol（Z19）、1-(3,4-dimethoxyphenyl)-1-hexanone（Z20）、2-[2-(2-hydroxymethyl-3-methoxy-phenyl)-vinyl]-5-methyl-tetrahydro-furan-3,4-diol（Z21）和octylphenol（Z24）是苯酚类化合物的甲氧基衍生物。化合物trans-caffeic acid（Z10）、homovanillic acid（Z13）和(2E)-5-hydroxyferulic acid（Z14）是多羟基取代苯丙酸类成分。

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图3 酚酸类化合物结构图

Fig. 3 Structure of phenolic acids from seeds (Z) and peels (K) of A. oxyphylla

2.2.4 萜类化合物

萜类化合物是一类以五个碳为基本单元,组成的骨架庞杂,种类繁多的化合物。根据碳原子数的多少分为单萜（10个碳）、倍半萜（15个碳）、二萜（20个碳）和三萜（30个碳）。从益智果皮和种子中共鉴定得到12个萜类化合物（图4）,其中从益智果皮中鉴定的萜类化合物包括7个二萜类化合物：cytosporin E（K33）、carnosic acid（K39）、(-)-andrographolide（K41）、florlide A（K42）、(3R,3aS,4S,8aS)-3-hydroxy-3-isopropyl-6,8a-dimethyl-8-oxo-1,2,3,3a,4,5,8,8a-octahydro-4-azulenyl (2E)-2-methyl-2-butenoate（K45）和rabdoternin A（K47）均为复杂环状二萜类成分,并且鉴定得到1个三萜类化合物ginsenoside F1（K50）,是达玛烷型三萜的葡萄糖苷。益智种子中的萜类成分包括单萜：(1R,5S,6R)-2,7,7-trimethylbicyclo[3.1.1]hept-2-en-6-yl L-galactopyranoside（Z8）、二萜：(2Z,4E)-3-methyl-5-[(1S,2R,4S)-1,2,4-trihydroxy-2-(hydroxymethyl)-6,6-dimethylcyclohexyl]-2,4-pentadienoic acid（Z15）、2-(8-hydroxy-4a,8-dimethyldecahydro-2-naphthalenyl)acrylic acid（Z22）和kandenol A（Z23）,其中化合物Z22和Z23是双环倍半萜。从益智种子中仅鉴定得到一个半日花烷型二萜的葡萄糖苷(2R,4aR,5S,6R)- 6-hydroxy-5-[(3E)-5-hydroxy-3-methyl-3-penten-1-yl]-1,1,4a,6-tetramethyldecahydro-2-naphthalenyl β-D-allopyranoside（Z30）。

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图4 萜类化合物结构图

Fig. 4 Structure of terpenes from seeds (Z) and peels (K) of A. oxyphylla

2.2.5 黄酮类化合物

黄酮类化合物是自然界最为重要的一类天然有机化合物, 其结构以C₆-C₃-C₆为基本骨架。从益智的果皮和种子中共鉴定得到20个黄酮类化合物, 结果见图5。从益智的果皮中分离鉴定得到19个黄酮类化合物：其中quercetin-3-O-α-L-rhamnopyranosyl(1-2)-β-D- glucopyranoside-7-O-α-L-rhamnopyranoside（K7）、 quercetin-3-O-α-L-rhamnopyranosyl(1-6)-β-D-glucopyranoside（K9）、 quercetin-5-O-α-L-rhamnopyranosyl(1-6)-α-D-glucopyranoside（K10）, miquelianin
（K12）、 quercetin-3-O-β-D-glucopyranoside（K13）和quercetin（K23）均为槲皮素的葡萄糖、鼠李糖和葡萄糖酸的糖苷衍生物及苷元;luteolin-7-O-β-D-glucuronopyranosyl-(1-6)-β-D-glucopyranoside（K8）、 luteolin-7-O-glucoside（K17）和luteolin（K26）是木犀草素的葡萄糖、葡萄糖酸的糖苷和苷元;apigenin-7-O-β-D-glucopyranoside（K11）、 malonylapiin（K15）和apigenin（K24）是芹菜素的糖苷衍生物和苷元; kaempferol-3-O-rutinoside（K14）和kaempferol-3- glucuronide（K16）是山奈酚的糖苷类化合物;liquiritigenin（K36）为二氢黄酮;procyanidin B1（K6）为花青素;另有3,5,7-trihydroxy-flavone- 3-O-β-D-glucoside（K19）、 7,4'-dihydroxy-flavone- 7-O-β-D-glucoside（K21）和chysin（K27）。从益智种子中鉴定得到2个黄酮类化合物： chysin（Z17,K27）和isalpinin（Z18）, 其中chysin（Z17, K27）是种子和果皮的共有成分。

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图5 黄酮类化合物结构图

Fig. 5 Structure of flavonoids from seeds (Z) and peels (K) of A. oxyphylla

2.2.6 其他类化合物

如图6所示,从益智果皮中鉴定得到其他成分有蒽酮类6-decylubiquinone（K40）和5-O-methyl embelin（K43）,以及脂肪链与环氧环酮以及苯环结合形成的化合物(5E)-7-{4-hydroxy-2-[(1E)-3-hydroxy-1-octen-1-yl]-6-oxotetrahydro-2H-pyran-3-yl}-5-heptenoic acid（K30）、(-)-prostaglandin E2（K32）、3, 4-Bis[(6- hydroxyhexyl)oxy]benzoic acid（K34）、(5E)-7-{3- [(1E)-3-hydroxy-1-octen-1-yl]-2, 6-dioxabicyclo[3.1.1]hept-4-yl}-5-heptenoic acid（K38）、3α-hydroxy- 3, 5-dihydromonacolin L acid （K29）、stigmatellin Y（K48）和penisporolide A（K31）。如图6所示,从益智种子中鉴定得到的其他类成分为：联苯类化合物4-decylbiphenyl（Z31）,大环醚类化合物tetracosahydrodibenzo[b,n][1,4,7,10,13,16,19,22]octaoxacyclotetracosine（Z29）,以及环状脂肪烯的糖苷衍生物3-(β-D-Galactopyranosyloxy)-2-hydroxypropyl 8-{(1S,5S)-4-oxo-5-[(2Z)-2-penten-1-yl]- 2-cyclopenten-1-yl}octanoate（Z26）、(2E,6E)-3, 7, 11-trimethyl-2, 6, 10-dodecatrien-1-yl (5xi)-6-O-[(4xi)-6-deoxy-L-lyxo-hexopyranosyl]-β-D-ribo-hexopyranoside（Z28）。

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图6 其他类化合物结构图

Fig. 6 Structure of other compounds from seeds (Z) and peels (K) of A. oxyphylla

3 讨论

通过文献分析发现,大多研究以益智挥发油为主要活性物质和研究对象^[27⇓-29],其含量较高的成分有圆柚酮、p-聚伞花烃、香橙烯、姜酮等。随着柱层析等分离手段的多样化,益智中的多种成分如倍半萜类、二苯庚烷类、黄酮类、挥发油类、甾体及其苷类等化学成分也被分离鉴定出来^[10],但未见关于益智种子和果皮非挥发性物质全面系统分析的报道,本研究基于UPLC-Q-TOF-MS技术对益智种子和果皮乙醇提取物非挥发性成分进行了分析和鉴定,从益智种子和果皮中分别鉴定了32个和50个化合物,其中有58个化合物为首次从益智中鉴定得出。

通过对比益智种子和果皮乙醇提取物非挥发性成分发现,二者的组成成分类型基本相似,都含有黄酮类、酚酸类、糖类、脂肪酸类和萜类化合物等5类成分,但主要成分差异较大：其中益智种子的主要成分为酚酸类（11个）化合物,也是益智种子的代表性成分,占总鉴定结构化合物总数的三分之一,而益智果皮中的酚酸类成分较少,仅鉴定得到6个酚酸类化合物;益智果皮主要成分为黄酮类（19个）化合物,占总鉴定结构的化合物总数近三分之一,且种类丰富,而种子中仅鉴定得到1个黄酮类化合物,吴德玲等^[20]也曾报道益智果皮总黄酮和总多糖的含量明显高于益智种子;在益智果皮中发现了益智特征性化合物二苯基庚烷类（2个）成分,分别是hexahydrocurcumin（K25）和tetrahydrocurcumin（K28）,其中K28首次从益智中分离到,而在种子中并未发现,推测有可能二苯基庚烷类化合物更多的富集在果皮中,梁木恒等^[18]曾报道益智果皮中的二苯基庚烷类化合物相较于益智种子更为丰富;在果皮中还首次发现了1个三萜化合物Ginsenoside F1（K50）,为达玛烷型的葡萄糖苷,从种子中也首次鉴定得到一个半日花烷型二萜的葡萄糖苷（Z30）。此外,虽然种子和果皮的组成成分类型相似,但二者具体类型的化合物组成存在较大的差异：二者样品中仅有4个共有成分,分别是2个单糖类化合物D-gluconic acid、D-glucopyranuronic acid,1个二糖类化合物sucrose和1个黄酮类化合物chysin。本研究结果与此前的研究有较大差异,这可能跟提取方法和检测方法有关。

研究发现益智种子非挥发性化学物质较少,而益智果皮中含有的化学物质种类较多,尤以黄酮类成分为主,在后期对益智资源综合开发利用时,应特别予以关注。研究益智总黄酮的提取工艺和方法,探究其开发利用的可行性;此外,益智不同部位均有一定的利用价值^[16,19,30],在充分利用益智仁的同时,深入挖掘益智非传统药用部位潜在开发利用价值,如可以考虑从果皮中提取糖类成分,形成果胶产品等,深入研究其他可利用部位的价值,变废为宝,以实现对其可利用部位的综合利用。

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Abstract

Cite this article

1 材料与方法

1.1 材料

1.1.1 植物材料

1.1.2 仪器与试剂

1.2 方法

1.2.1 供试品溶液制备

1.2.2 UPLC-Q-TOF-MS分析

1.2.3 定性分析

2 结果与分析

2.1 益智种子、果皮提取物的化学成分鉴定

表1 益智种子化学成分的UPLC-Q-TOF-MS鉴定结果（负离子模式）

表2 益智果皮化学成分的UPLC/Q-TOF-MS鉴定结果（负离子模式）

图1 益智种子（Z）和果皮（K）负离子模式的BPI色谱图

2.2 化合物分析

2.2.1 单糖和二糖类化合物

图2 单糖、二糖和脂肪类化合物结构图

2.2.2 脂肪类化合物

2.2.3 酚酸类化合物

图3 酚酸类化合物结构图

2.2.4 萜类化合物

图4 萜类化合物结构图

2.2.5 黄酮类化合物

图5 黄酮类化合物结构图

2.2.6 其他类化合物

图6 其他类化合物结构图

3 讨论

References