Comprehensive analysis of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. fruits based on UPLC–MS/MS and GC–MS: A rapid qualitative analysis

Abstract Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. fruits (ESF), as a natural edible fruit, has long been popularized. However, few studies have conducted comprehensive chemical analyses of it. This study aimed to assess nonvolatile, volatile, and fatty oil components of ESF and to preliminarily explore the antioxidant activities. The qualitative and quantitative analyses of volatile and fatty oil components of ESF from 15 different regions were performed by the gas chromatography–mass spectrometry (GC–MS). Totally, 37 and 28 compounds were identified from volatile oil and fatty oil, respectively. The ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UPLC–QTOF–MS/MS) was used to accurately detect 43 compounds of nonvolatile components. The volatile and fatty oil components and nonvolatile components of ESF were used as samples to determine the antioxidant activity of 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) in vitro. The components of ESF had antioxidant activity, and the nonvolatile components had stronger antioxidant activity. The results revealed that the proposed method, which is of great significance for the screening of new active ingredients, is valuable for the identification of pharmaceutical component and further development of food industry.

compounds in ESF have immunostimulatory activities (Gerontakos et al., 2021;Graczyk et al., 2021).A previous study found that the antioxidant activity of ESF could be related to the contents of polysaccharides (Zhao et al., 2013).However, there is no comprehensive analysis of the nonvolatile, volatile, and fatty oil components of ESF and their antioxidant activities; thus, it is essential to further explore such components.The gas chromatography-mass spectrometry (GC-MS) can analyze volatile components, label compounds quantitatively, and combine with stoichiometric methods to distinguish plants growing in different regions (Bai et al., 2021).The ultra-high-performance liquid chromatography-quadrupole timeof-flight mass spectrometry (UPLC-QTOF-MS/MS) is an efficient technique in the chromatographic separation, and it has been successfully employed for its fast, high-resolution separation with the satisfactory sensitivity.GC-MS and UPLC-MS/MS technologies have been widely used for the separation and rapid identification of compounds in natural plants (Liu et al., 2022;Pan et al., 2019).
In order to analyze and evaluate the volatile and fatty oil components and quality of ESF from different production areas and assess the composition and structural cracking principle of nonvolatile compounds, a new rapid and sensitive UPLC-MS/MS method for the detection of major or trace components was, for the first time, proposed in this study.In addition, GC-MS was used to obtain fingerprints and relative area percentage from different origin of ESF volatile and fatty oil components.This qualitative and quantitative methods based on UPLC-MS/MS and GC-MS can be utilized for the quality assessment of ESF.Hence, this study may provide a reliable basis for ESF to a certain extent and for its further rational development and utilization.

| Materials
Totally, 15 batches of dried ESF were collected from different regions from August to October 2022, which were mainly produced in Heilongjiang, Jilin, and Liaoning provinces in China (Table 1, Figure S1).After picking the ripe fruits, wash them in tap water and ultrapure water to remove impurities, and then dry them in a cool place.They were identified as dried fruits of ES by Professor Zhenyue Wang from the School of Pharmacy, Chinese Medicine Resource Center, Heilongjiang University of Chinese Medicine (Harbin, China).

| GC-MS analysis
The HP-5 MS elastic quartz capillary column (30 m × 250 μm × 0.25 μm) was utilized for GC-MS analysis.In the programmed temperature condition, the temperature of volatile oil increased from 50 to 250°C at 5°C/min.The temperature of fatty oil was kept at 80°C for 1 min, then it was heated from 80 to 250°C at 10°C/min, and was kept at 250°C for 10 min.The running time of volatile oil was 40 min and that of fatty oil was 28 min.
The temperature of the injector used was 250°C, the carrier gas was high purity helium (99.999%), and the flow rate was 3.0 mL/ min.The column pressure was 9.785 psi, the solvent delay time was 6 min, the sample size was 1 μL, and the injector operated was in split mode, with a ratio of 40:1.The ion source was EI ion source, the electron energy was 70 eV, and the mass range was m/z 50 ~ 550.The temperatures of ion source, transmission line, and quadrupole were 230°C, 280°C, and 150°C, respectively.The mass spectrum retrieval standard library was NIST14.L standard spectrum library.

Extraction of volatile oil
Volatile oil was obtained from ESF (200.2 g) by reflux condensation for 5 h, according to the Chinese Pharmacopeia 2020 (Committee, 2020).
Volatile oil was dried over Na 2 SO 4 , centrifuged at 13,000 rpm for 10 min, and stored at 4°C until further analysis.Following the same procedure, all 15 components of ESF were acquired.

Extraction and methyl esterification of fatty oil
ESF (30.04 g) was weighed and 450 mL n-hexane was added at the ratio of 1:15 (M/V).Under the condition of ultrasonic power of 250 W, ultrasonic extraction was carried out for 30 min.After vacuum filtration, the fatty oil was obtained by rotating evaporation in water bath (60°C) until no n-hexane was emitted.Then, 4 mL of 0.6 mol/L potassium hydroxide solution, methanol, and n-hexane were added, respectively.After the mixture was evenly mixed and bathed at 60°C for 30 min, 10 mL distilled water was added and stratified.The upper layer was dried with Na 2 SO 4 , centrifuged at 13,000 rpm for 10 min, and stored at 4°C for further analysis.Following the same procedure, all 15 components of ESF were acquired.

Extraction of nonvolatile compounds from ESF
ESF (20.02 g) was randomly weighed, 30 mL of 70% methanol at a ratio of 1:15 (M/V) was added, stirred and mixed, ultrasonically extracted for 1 h, leached at room temperature, and centrifuged at 12,000 rpm for 10 min, in which the supernatant was the aqueous extract of ESF.   2 and 3).By comparing the GC-MS retention time of 15 chromatograms, the obtained mass spectra were matched with the standard mass spectra in the NIST14.L library and the literature.

| Determination of antioxidant activities of volatile components using DPPH assay
Notably, 37 and 28 compounds were identified in volatile oil and fatty oil, respectively.
The peak area of more than half of the components of ESF in volatile and fatty oils accounted for more than 70% of the total peak area of each sample, indicating that the identified compounds could represent the main components of ESF in volatile and fatty oils (Tables 4   and 5).Moreover, α-bisabolol accounted for most of the chemical components of ESF detected in volatile oil.In components of ESF in fatty oil, 10-octadecenoic acid methyl ester accounted for the most of chemical components.
Relative area percentage of common peaks of the components of ESF in volatile oil showed that the contents of α-bisabolol and β-bisabolene were the highest in S12 and S15, which were 45.34% and 8.052%, respectively.In the components of ESF in fatty oil, 10-octadecenoic acid methyl ester exhibited to have the highest content in S15 (69.53%) and S5 (63.01%).The results revealed that the main components of volatile substance were similar in different regions, while the content was different.
It was indicated that S1, S2, S15, S10, S13, S4, S6, S14, S9, and S12 belonged to the same category, and S3, S7, S8, and S5 belong to the same category in volatile oil (Figure 2a).The distance between the two categories was only 2, confirming that the components of ESF in volatile oil from these two production areas were similar with a relatively satisfactory quality, and S11 belonged to the same category independently in volatile oil.Similarly, S1, S10, S3, S7, S9, S14, S2, S6, S8, S12, S15, and S11 belonged to the same category, and S5 and S13 belong to the same category in fatty oil (Figure 2b).The distance between these two categories was only 3, proving that the quality of the components of ESF in fatty oil from these two categories was relatively satisfactory.Furthermore, S4 belonged to the same category independently, and the distance between the other two categories was 40, indicating that the quality of the components of ESF in fatty oil in this area was relatively poor.

| Nonvolatile components of ESF
According to the exact fragmentation rules of fragment ions and literature, 43 compounds were identified (Figure 3, Table 6), which were mainly triterpene and phenylpropanoid (Hu et al., 2022;Liu et al., 2021).

| Analysis of triterpenoids
To date, no systematic characterization of triterpenoid in ESF by UPLC-MS/MS has been reported.A total of 16 triterpenoid saponins have been identified in this study.According to their structural characteristics, they were mainly oleanolic acid type.
In the positive and negative ion modes, the additional ions of triterpenoid saponin were mainly

| Analysis of phenylpropanoids
Six of the nine phenylpropanoid compounds identified were lignans with the same parent nucleus.These lignans generally break CO (28 Da), OCH 3 (31 Da), and some glycosyl.In addition, the 7 and

| Determination of antioxidant activity
Several studies have characterized the nonvolatile, volatile, and fatty oil components in plants by GC-MS and UPLC-MS/MS, accompanied by antioxidant activities of components in plants (Ali et al., 2022;Al-Nemari et al., 2020;Castillo et al., 2023;Duan et al., 2022;Hefny Gad et al., 2021).A previous study demonstrated that phenolic acids, represented by chlorogenic acid and caffeic acid, are the main reason for the antioxidant effect of ESF (Kim et al., 2015).However, the antioxidant effects of triterpene and phenylpropanoid in the nonvolatile components of ESF and the volatile and fatty oil components of ESF have not yet been studied.
The changes of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging abilities of the volatile and fatty oil of ESF with the concentration are shown in Figure 5.The scavenging ability of fatty oil on DPPH free radical was significantly stronger than that of volatile oil, and with the increase of concentration, the scavenging ability of fatty oil from different origins on DPPH free radical was gradually enhanced.S1, S7, S10, S3, S9, and S14 exhibited to have weaker than other production areas, and S15 had the strongest removal capacity.
When the concentration of fatty oil in S15 reached 8.031 mg/mL, the scavenging rate was 82.04%, while the largest component in S15 was 10-octadecenoic acid methyl ester.Moreover, this ingredient has been confirmed to have antioxidant activity, and it was speculated that this ingredient might have a certain relationship with the antioxidant activities of the components of ESF in fatty oil.The free radical scavenging abilities of the components of ESF in volatile oil from different production areas were not the same.Furthermore, S8, S7, S5, S3, and S11 also increased with the elevation of concentration before reaching 1.982 mg/mL, which did not show regularity.The scavenging abilities of the components of ESF in volatile oil from other production areas were enhanced with the increase of volatile oil concentration before reaching 4.022 mg/mL, and weakened to varying degrees when the concentration was greater than 4.022 mg/mL.Besides, S12 had the strongest scavenging ability, and the scavenging rate was 71.10% when the concentration of volatile oil reached 4.022 mg/mL.α-Bisabolol, which accounted for the  the production of human neutrophils, thus producing antioxidant effects (Schepetkin et al., 2016).
Many fatty acid compounds can be obtained from food and used as a natural antioxidant, such as palmitic acid methyl ester (PAME), which dilates blood vessels and plays a significant role in brain damage caused by asphyxia cardiac arrest, elevated cholesterol, and cancer (Ichihara, 2021;Lee et al., 2019).Arachidic acid methyl ester plays a significant role for the prevention of gallstones by acting as a cholesterol solvent (Gilat et al., 2001).Methyl linoleate serves as an emulsifier in cosmetics and plays a direct role in the epidermal osmotic barrier, thus achieving an antioxidant effect (Qin et al., 2007).
In our study, ESF volatile oil and fatty oil have antioxidant effects, and its main components are α-bisabolol and 10-octadecenoic acid methyl ester.α-Bisabolol was found to slow ROS production and inhibit the deposition of beta-amyloid protein (Aβ) peptide induced by Alzheimer's disease in Candida albicans and N-formyl-methionylleucyl-phenylalanine(fMLP).Restoration of mitochondrial membrane potential (MMP) leads to antioxidant effects (Braga et al., 2009;Eddin et al., 2022;Gger et al., 2018).10-Octadecenoic acid methyl  components of ESF.Based on NIST14.L mass spectrometry database and precise molecular weight, 37 and 28 compounds were identified and analyzed from volatile oil and fatty oil of ESF, respectively, from different regions.The cluster analysis results of volatile oil showed that the distance between S11 and the other two categories was 16, and the cluster analysis results of fatty oil showed that the distance between S4 and the other two categories was 40, and the quality of oil from these two regions was significantly different from that of other producing areas.
In addition, 43 compounds were identified and analyzed from the nonvolatile components of ESF, and the cracking principles of some identified compounds were studied.DPPH antioxidant assay further verified that nonvolatile and volatile components of ESF might be associated with antioxidant activity.It has been suggested that ESF could be developed as a natural and potentially effective drug or functional food, however, its pharmacological action and related mechanisms need additional in vivo studies.
and fatty oil components of ESF Total ion chromatograms of volatile oil and fatty oil of 15 batches of ESF were collected under optimized chromatographic conditions (Figure 1, Tables 1 TIC chromatograms of volatile ESF components (top) and fatty ESF components (bottom) from different regions (S1-S15).
pathway of simple lignans was deduced by compound 27.The quasi-molecular ion peak of [M + Na] + was m/z 543.1832 (C 26 H 32 O 11 ), and one Glc was lost.The fragment ion m/z 359.1475 (C 20 H 22 O 6 ) and the loss of two neutral fragments H 2 O led to achieve the fragment m/z 323.1310 (C 20 H 18 O 4 ).Therefore, compound 27 was preliminarily identified as tetracentronside B, and the cleavage pathway is shown in Figure 4c.In the positive ion mode, compound 30 was represented, and the possible cleavage pathway of cyclolignans was predicted.The quasimolecular ion peak of [M + Na] + was m/z 545.1993 (C 26 H 34 O 11 ), and one Glc was lost.Fracture occurs at the 7 and 7′ positions and the loss of C 7 H 8 O results in the fragment ion m/z 219.1025 (C 11 H 16 O 3 ).Therefore, compound 30 was preliminarily identified as isolariciresinol-4-Oβdglucopyranoside, and the cleavage pathway is illustrated in Figure 4d.
-"indicates that the value is not detected or the relative content is too low."*" indicates a statistical difference."**" indicates a significant statistical difference."***" indicates an extremely significant statistical difference.TA B L E 4 (Continued) TA B L E 5 Relative area percentage of common peaks in fatty ESF components from different regions (S1-S15).-" indicates that the relative content is not detected or is too low."*" indicates a statistical difference."**" indicates a significant statistical difference."***" indicates a extremely significant statistical difference.F I G U R E 2 Cluster analysis of volatile ESF components (a) and fatty ESF components (b) from different regions (S1-S15).F I G U R E 3 The BPI chromatograms of nonvolatile ESF components were detected at 7-22 min in positive ion mode (a) and at 1-17 min in negative ion mode (b).
ester has been shown to lower blood cholesterol, have antifungal properties, and antioxidant effects(Belakhdar et al., 2015;Kewlani et al., 2022).However, there are few researches on the specific mechanism of its antioxidant.The study on the antioxidant activity of ESF volatile oil and fatty oil with terpene compounds and fatty acid compounds as the main components can be used as a new direction of ESF as a natural antioxidant in the food industry for the preparation of different health products.Moderate development of ESF can also provide another idea for the waste caused by excessive exploitation of ES roots and rhizome.4| CON CLUS IONSIn this study, rapid and sensitive UPLC-QTOF-MS/MS plus GC-MS methods were developed for the analysis of nonvolatile and volatile F I G U R E 5 (a) DPPH radical scavenging standard curve.(b) Scavenging effects of volatile oil and fatty oil with different concentrations on DPPH free radical (S1-S15).

F
Scavenging effects of nonvolatile components with different concentrations on DPPH free radical.

| Determination of antioxidant activities of nonvolatile components using DPPH assay
Qualitative analysis results of volatile ESF components.
− , and [M + HCOO] − , and the nuclear parent fragment was obtained by breaking or continuously breaking O-glycosyl or glycosyl.It TA B L E 2 Qualitative analysis results of fatty ESF components.
positions of the benzene ring are prone to fracture, resulting in benzyl cleavage.If there is hydroxyl group on the side chain benzene ring, it can form OCH 2 O (46 Da) characteristic fragments with the methoxy group.With compound 24 as the representative in the positive ion mode, the possible cleavage pathway of the bisepoxylignans was speculated.The quasi-molecular ion peak of [M + Na] + possible cleavage pathway of monoepoxylignans was speculated.The quasi-molecular ion peak of [M-H] − was m/z 521.2093 (C 26 H 34 O 11 ), and one Glc was lost to obtain the fragment ion m/z 359.1555 (C 20 H 24 O 6 ).Losing another OCH 3 was resulted in the fragment ion m/z 329.1437 (C 19 H 22 O 5 ), with two possible fragments.Therefore, compound 22 was preliminarily identified as lariciresinol-4′-Oβd-glucoside, and the cleavage pathway is displayed in Figure 4b.TA B L E 3 TA B L E 4 Relative area percentage of common peaks in volatile ESF components from different regions (S1-S15).
Characterization of non-volatile ESF components by UPLC-MS/MS.
TA B L E 6