key: cord-0995338-7sqx5vej authors: Wang, Kanghui; Tian, Jingyuan; Li, Yueshan; Liu, Mengshi; Chao, Yingxin; Cai, Yi; Zheng, Guodong; Fang, Yi title: Identification of Components in Citri Sarcodactylis Fructus from Different Origins via UPLC-Q-Exactive Orbitrap/MS date: 2021-06-23 journal: ACS Omega DOI: 10.1021/acsomega.1c02124 sha: ffd14bf217eec2bbb52bed66f4b6eed8dc94a088 doc_id: 995338 cord_uid: 7sqx5vej [Image: see text] To systematically analyze the chemical constituents of Citri Sarcodactylis Fructus (CSF) from different origins, an efficient approach based on ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) detection for the discrimination of chemical components from of 15 batches of CSF from four main origins was used in this research. Through parent peaks, fragment peaks, fragmentation characteristics, and comparative analysis with the literature and reference standards, a total of 77 components from the methanol extracts including 18 coumarins, 24 flavonoids, seven organic acids, three limonoids, and 25 other compounds were detected and identified. Among them, 15 components have not been reported previously in the CSF. Notably, the stachydrine peak initially showed a higher content in the total ion current chromatogram. Overall, CSF produced in the Zhejiang province contained a richer variety of chemical compositions. These observations provided a theoretical basis for the further quality assessment and application of CSF. Citri Sarcodactylis Fructus (CSF, Foshou in China), the dried fruit of Citrus medica L. var. sarcodactylis Swingle, belongs to Citrus botany in Rutaceae. 1 CSF has a long cultivation history in China, which is widely distributed in Guangdong, Guangxi, Zhejiang, Sichuan, and Yunnan provinces. 2 As a medicinal and edible plant, CSF shows various pharmacological effects of antitumor, neuroprotection, antioxidation, anti-inflammatory, antimicrobial, antiblood pressure, lipid-lowering, and antianxiety. 3−11 Due to the complex compositions and diverse types of natural medicinal plants, it is difficult to separate and identify their ingredients. With a high separation efficiency, a fast scanning speed, a high throughput, a high resolution, and high sensitivity, Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) technology combines the separation capabilities of chromatography and the qualitative functions of mass spectrometry are widely used in the component analysis of complex systems of traditional Chinese medicine. 2, 12, 13 It is especially suitable for the qualitative identification of complex natural plant systems and the discovery of new compounds lacking reference substances. 14 At present, research on CSF mainly concentrated on its pharmacological activity, 6, 7 and the components mostly focused on were essential oil, 6, 7 and coumarins 15, 16 such as 5,7-dimethoxycoumarin, 7-hydroxycoumarin, scopoletin, and bergapten, as well as flavonoids such as hesperidin, 17 however, few research systematically reported on the chemical components of CSF. In addition, CSF has a wide range of production areas, and different growth environment and geographical locations may result in intermingled quality and differences in compositions. For a better comprehensive comparison, an analysis of the compositions from different origins was provided as a reference for evaluating the quality of CSF. In this work, more than 77 peaks were efficiently separated and detected within 40 min in the total ion current (TIC) chromatogram in positive ion modes via a UPLC-Q Exactive Orbitrap/MS system. The CSF extract samples were able to be isolated in the positive-ion mode, where the detection signal and resolution were both better than those in the negative-ion one. The TIC of extract samples and mixed reference standards is shown in Figure 1 . According to the peak time, standards, and relevant literature, 77 compounds were separated and identified within 40 min, and are shown in detail in Table 1 , including 18 coumarins, 24 flavonoids, seven organic acids, three limonoids, and 26 other compounds. Among them, 15 components were detected in the CSF for the first time: five flavonoids including eriocitrin, eriodictyol, astragalin, isotrifoliin, and glabrone; two coumarins including isopimpinellin and isofraxidin; one organic acid, p-hydroxycinnamic acid; and seven other compounds including oleamide, erucamide, stachydrine, coniferin, o-veratraldehyde, 6-hydroxyindole, and linderalactone. Chemical structures of 77 compounds that were tentatively identified are shown in detail in Figure 2 . This discovery provides a new direction for followup research on the quantitative analysis, component separation, and pharmacological activity of CSF. 2.1. Identification of Coumarins. Coumarins are a kind of natural compounds with a benzo-α-pyranone parent nucleus, 18 and are the primary constituents in the CSF extracts. 15, 17 Twenty coumarins were identified in the CSF extracts. In the positive mode, these kinds of compounds may Furanocoumarins can be divided into linear furanocoumarin and angular furanocoumarin. 21 Due to the construction features of furanocoumarins, it is easy for them to continuously lose CO (28 Da) and CO 2 (44 Da) groups, so the fragment ions of these kinds of compounds in the second MS were characterized by m/z 203 and m/z 158. 22 23 Furthermore, compound 51 exhibited a molecular ion at m/ z 217.0495 [M + H] + in the positive mode. The second order spectrum shows fragment ions at m/z 202.0261 and m/z 173.0598, which correspond to the loss of CH 3 and CO of the parent ion, respectively. Therefore, compound 51 was identified as bergapten 15 (the fragmentation pattern is shown in Figure 3f ). In addition, compound 49 was confirmed as 5,7dimethoxycoumarin, which was known as a common Figure 3h ). Compounds 57 and 61 were, respectively, identified as nobiletin and tangeretin in the light of standard references. The comprehensive fragmentation pattern of compound 57 is shown in Figure 3g . Besides the above PMFs, other flavonoids were also identified in this work. Based on the literature, it is easy for flavonoids to lose some neutral fragments of CO, CO 2 , H 2 O, and CH 3 in the MS/MS, subsequently the retro-Diels−Alder (RDA) reaction occurred. Under the same collision energy, flavonoid glycosides easily lose their linked sugar molecules, afterward the RDA reaction occurs as well. 22, 26 Glycoside is composed of aglycone and sugar. Analogously, diosmin is composed of diosmetin, a rhamnose, and a glucose. S9 S10 S11 S12 S13 S14 S15 179.0341, 177.0545, 153.0181, 147 .0440, and 137.0599 were formed for the both compounds. Among them, m/z 285.0760 was generated by the removal of a H 2 O molecule from the parent ion, furthermore, the parent ion lost its B ring and m/z 177.0545 was obtained. Fragment ions at m/z 153.0181, 147.0390, and 137.0752 were attributed to the RDA reaction of the flavonoid skeleton, which was the typical fragmentation method of the dihydroflavonoids. In accordance with reference standards and second MS fragmentation information above, compound 40 was finally determined as hesperetin and compound 41 was determined as hesperidin. The comprehensive fragmentation pattern of compound 40 is shown in Figure 3d. 2.3. Identification of Limonoids. Combined with earlier literature, limonoids are rich in citrus and show antitumor, anti-inflammatory, antibacterial, antiviral, antioxidant, and liver protection effects. 2, 4, 9, 17, 22 Three limonoids were detected from the CSF extracts: limonin, nomilin, and obacunone. Compound 62 exhibited a quasimolecular ion at m/z 455.2058 [M + H] + . In the second MS, under high-energy collision, the parent ion of compound 62 can lose a H 2 O molecule or a CO 2 or a HCOOH to obtain fragments 437.1962, 411.2162, and 409.2010, respectively, so that compound 62 was identified as obacunone. 27 Compounds 54 and 59 were detected as limonin and nomilin by comparing the reference substances and peak times. 2.4. Identification of Organic Acid. Seven organic acids, including vanillin acid, sinapic acid, ferulic acid, isoferulic acid, p-hydroxycinnamic acid, and α-linolenic acid, were identified in this study under positive-ion full MS scanning. These kinds of compounds are known to remove some neutral molecules of H 2 O, HCOOH, and CO 2 in the positive mode. 28 (17 Da) in the mass spectrometry during the cleavage process, which result from the unique neutral fragment loss of amino acids. 29 By comparing the secondary fragment ion peaks under the full MS scanning, combined with the literature and the OTCML database, the compounds 1, 3, 5, 7, and 11 were decided as valine, tyrosine, proline, leucine, and tryptophan, respectively. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 30 Notably, compound 2 had a fairly high peak in the TIC chromatogram, and was detected as stachydrine. The carbon atom connected to the carboxyl group and the nitrogen atom in the stachydrine molecule had high reactivity, and was prone to electron transfer and dehydrogenation to produce a double bond. Then, the single bond between the methylene group and the nitrogen atom was broken to eventually form fragment ions at m/z 84.0813 and m/z 58.0659. The comprehensive fragmentation pattern of compound 2 is shown in Figure 3a . In addition, in combination with the literature 31 and structural information provided by MS, compounds 74 and 77 were identified as oleamide and erucamide, which were rarely reported in the form of compounds in natural products. 2.6. Discussion. In general, all origins contained mostly the same chemical components, such as coumarins. On the whole, the coumarins extracted from different origins of CSF were abundant. According to this study and combined with relevant literature, 15 the coumarins are the main chemical components of CSF, thus the coumarins were well preserved by means of using the extraction method in this work. The chemical component differences among CSF from different origins are shown in Table 2 . As for the flavonoids, other CSFs were more abundant than that of CSF-Guang. In addition, eriocitrin, eriodictyol, and glabrone were only detected in the Guangxi province, while none of the other origins were detected, which needs a further verification. Among them, eriocitrin and eriodictyol are common in citrus, while they have not been reported previously in the CSF. Overall, CSF produced in the Zhejiang province contained a richer variety of chemical components. More noticeably, according to previous studies, the research on the compositions of CSF mostly focused on its coumarins, whereas few reported on the polymethoxyflavonoids. 15 In this work, a total of seven polymethoxyflavonoids have been found, including nobliletin, tangeretin, 5-demethylnobiletin, etc., which have shown different pharmacological activities such as antitumor, antibacterial, antineuritis, antioxidant, cardiovascular protection, and antihyperlipidemic effects. 2, 10, 12, 22, 32 In this paper, UPLC-Q-Exactive Orbitrap/MS technology was used for the first time to establish a qualitative analysis method for the chemical composition of different origins of CSF. A total of 77 chemical components in the CSF extracts were successfully isolated and identified, 15 of which were first detected. In the four main origins, CSF-Jin contained a richer variety of chemical components. In summary, this research provided an efficient and rapid method for qualitative analysis and quality control of chemical components in CSF. an online degasser, a quaternary pump, and a column temperature compartment, and it was combined with a Q Exactive Orbitrap tandem mass spectrometer (Thermo Scientific, USA) by means of an electrospray ionization interface. 4.3. Preparation of Samples and Standards. 4.3.1. Preparation of Samples. Approximately 0.5 g of 15 batches of CSF were, respectively, weighed, which were passed through the 40-mesh screen, added to 25 mL of methanol, and subjected to ultrasonic extraction (320 W, 40 kHz) for 30 min. Different batches of the CSF filtrate were, respectively, concentrated to 1 mL using a rotary evaporator and then transferred to an automatic injection bottle after passing through a 0.22 μm PTFE membrane for UPLC-Q Exactive Orbitrap-MS analysis. High-resolution MS source parameters include a 3.5 kV spray voltage, a 320°C capillary temperature, a 30 unit sheath gas, a 10 unit auxiliary gas, a 300°C auxiliary gas heater temperature, and a 5 unit sweep gas, and were used in positive ion mode. Data acquired from 70−1000 Da in full MS scan mode were processed via Metworks software. All operations above were controlled by Xcalibur software. Committee of National Pharmacopoeia. 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