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Article

Quality Marker Discovery and Quality Evaluation of Eucommia ulmoides Pollen Using UPLC-QTOF-MS Combined with a DPPH-HPLC Antioxidant Activity Screening Method

by
Fengqian Guo
†,
Yichun Yang
†,
Yu Duan
,
Chun Li
,
Huimin Gao
,
Hongyu Liu
,
Qiping Cui
,
Zhongyuan Guo
,
Xiaoqian Liu
* and
Zhimin Wang
*
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
*
Authors to whom correspondence should be addressed.
†
The authors contributed equally to this work.
Molecules 2023, 28(13), 5288; https://doi.org/10.3390/molecules28135288
Submission received: 11 May 2023 / Revised: 30 June 2023 / Accepted: 3 July 2023 / Published: 7 July 2023
(This article belongs to the Special Issue Application of Analytical Methods in Food, Drug, and Natural Products Research: Volume II)
Browse Figures
Versions Notes

Abstract

:
Pollen, as an important component of Eucommia ulmoides (EUP), is rich in nutrients and is receiving increasing attention. At present, there are no reports on research related to the chemical composition and quality standards of EUP, and there are significant quality differences and counterfeit phenomena in the market. This study used a UPLC-QTOF-MS system to identify 49 chemical components in EUP for the first time. In the second step, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-HPLC antioxidant activity screening technology was used to identify the main active components of EUP, quercetin-3-O-sophoroside (QSH), quercetin-3-O-sambubioside (QSB), and quercetin 3-O-neohesperidoside (QNH), and their purification, preparation, and structure identification were carried out. Third, molecular docking was used to predict the activity of these components. Fourth, the intracellular ROS generation model of RAW264.7 induced by H2O2 was used to verify and evaluate the activity of candidate active ingredients to determine their feasibility as Q-markers. Finally, a quality control method for EUP was constructed using the three selected components as Q-markers. The identification of chemical components and the discovery, prediction, and confirmation of characteristic Q-markers in EUP provide important references for better research on EUP and the effective evaluation and control of its quality. This approach provides a new model for the quality control of novel foods or dietary supplements.

1. Introduction

Eucommia ulmoides Oliver (EU), also known as Du-Zhong in China and Tuchong in Japan, is a traditional medicinal plant that originated in China and is widely distributed in central and southwest China, including Henan, Hunan, Jiangxi, and Shanxi provinces [1]. The bark of E. ulmoides (EUB) has been used in traditional Chinese medicine for more than 2000 years. It possesses the pharmacological effects of nourishing the liver and kidney, strengthening the muscles and bones, and preventing miscarriage [2]. The favorable antioxidant activities of E. ulmoides have been demonstrated in biological in vivo experiments, with validity against oxidative stress in gastric mucosal injury, chronic hepatotoxicity, diabetes complications, lead-induction, obesity, I/R induced renal and hepatic toxicity, etc., demonstrated [3,4,5,6,7]. In addition to its medical benefits, EU has a high value in developing commercial products. Male flowers of EU (EUF) and EU seed oil have been approved as novel raw food materials by the National Health Commission (NHC) of China. At present, Eucommia ulmoides flower natural health-care tea [8] and Eucommia ulmoides leaf (EUL) vinegar [9] are common in the market.
The bark, leaves, stems, fruit, and flowers of EU possess an extensive range of pharmacological effects, such as anti-inflammatory [10,11,12], neuroprotective [13,14], and anti-hyperlipidemic [15,16]; treating secondary hypertension [17,18,19]; immunomodulatory effects [20]; and anti-hyperglycemic activities [21].
Natural products have attracted considerable attention as significant resources for preventing oxidative stress-related diseases. Pollen has been recognized as an excellent functional food and feed ingredient [22,23,24] as well as a good source of different bioactive compounds [25,26,27]. The dominant presence and high content of protein, trace elements, minerals, and active ingredients in pollen, as a part of EUF, highlight it as an ideal natural supplement [28]. Previous animal experiments have shown that EUP has antioxidant, antihypertensive, and lipid-lowering effects [29,30]. It is considered to have high development value and broad application prospects in medicine and the healthcare industry. With the increasing attention paid to EUP, the demand for EUP in the market is also gradually expanding. According to research, there are uneven levels of pollen quality and counterfeit products on the market. There have been no reports on its chemical composition and quality control so far. It is necessary to establish appropriate methods to control its quality.
Pollen has significant antioxidant effects and can prevent the occurrence of related diseases by inhibiting the oxidation process [27,31,32]. This study aimed to establish a quality evaluation method for EUP based on antioxidant activity. First, based on an analysis of the chemical characteristics of different parts of EU using (UPLC)–electrospray ionization (ESI) tandem mass spectrometry (QTOF/MS), this paper searched for the characteristic components of EUP. Further, based on DPPH high-performance liquid chromatography (HPLC) antioxidant activity measurement technology, the main active components in EUP were rapidly screened, and the target compounds were separated and prepared. The activity of these components was predicted using molecular docking. Then, their antioxidant activity was verified based on the oxidative damage induced by H2O2 in vitro to confirm their qualification as Q-markers. Finally, these Q-markers were used to establish a rapid and effective quality control method that can evaluate the quality of EUP and its related health products.

2. Results

2.1. UPLC-ESI-TOF/MS Analysis

In this research, the chemical components in different parts of EU were investigated using UPLC-QTOF-MS/MS, and peak identification was performed. A total of 74 compounds (as shown in Figure 1 and Figure S1 and Table 1), including 21 lignins, 17 iridoids, 17 phenylpropanoids, 13 flavonoids, and 6 other components, were identified or tentatively assigned using UNIFY 1.7 software (Waters Corporation, Milford, CT, USA) by the matching of empirical molecular formulae, quasi-molecular ions, and fragment ions or comparing their characteristic high-resolution mass data with the data from previous publications [33,34,35,36]. The mass error for the molecular ions of all identified compounds was within ± 10 ppm and based peak ion (BPI) diagrams in the negative and positive ion modes are displayed (Figure 1 and Figure S1). The distribution of the compounds was as follows: 62 compounds in male flowers; 49 compounds in pollen; 48 compounds in bark; and 60 compounds in leaves. EUP had 3 lignans, 14 phenylpropanoids, 13 cyclic ether terpenes, 13 flavonoids, and 6 other compounds.

2.1.1. Identification of Lignins

A total of 21 lignins were identified in EUB, EUL, EUF, and EUP. Lignins were the most numerous components in the identified small molecules. When lignins were bombarded with energy, characteristic fragment ions were produced by the loss of a series of glycosyl groups and the internal cleavage of the lignins. For example, (+)-pinoresinol di-O-β-d-glucopyranoside exhibited [M + HCOO]− ions at m/z 727.2450 in the negative mode. The fragment ions of [M – H − Glc]− and [M − H − Glc − Glc]− were detected at m/z 519.19 and m/z 357.13. The fragment ion at m/z 342.1103 and m/z 151.04 was obtained by internal lignin cleavage.

2.1.2. Identification of Iridoids

Iridoids are distributed in various parts of EU, and 17 iridoids were identified in the positive and negative ion modes. Neutral fragments, such as glucose, glucose residues, H2O, CO2, CH3OH, and CH3COOH, represent fragments that are commonly cleaved from the central iridoid core and form [M − H − Glc]−, [M − H − Glc]−, [M − H − Glc − H2O]−, [M − H − Glc − O2]−, [M − H − Glc − H2O − CO2]−, and other ions. For example, the mass spectrometry cleavage products of geniposidic acid yielded the quasi-molecular ion peak at m/z 373.1132. Then, losses of CO2, H2O, Glc, etc., formed fragment peaks at m/z 211.06, 193.05, 167.08, and 149.06. The formation of fragment ions at m/z 123.04 originated from the rearrangement of γ-H after the rearrangement of the parent ring.

2.1.3. Identification of Phenylpropanoids

A total of 17 phenylpropanoids were identified in EUB, EUL, EUF, and EUP, including caffeic acid, chlorogenic acid, isochlorogenic acid A, protocatechuic acid, syringin, etc. Using caffeic acid as an example, the quasi-molecular ion peak at m/z 179.0344 continuously lost CO2 and H2O to form peaks at m/z 135.04 and m/z 117.03.

2.1.4. Identification of Flavonoids

A total of 13 flavonoids were identified in different parts of EU. Rutin is the combination of rutinose and the glycoside of quercetin. Its mass spectrometry cleavage products were represented by a quasi-molecular ion peak at m/z 609.1461 within the primary mass spectrum. The fragment ions of [M − H − Glc]− were detected at m/z 301.03. The aglycon fragment was a retro-Diels–Alder (RDA) fragment that generated a fragment at m/z 151.00. In addition, the m/z 301.03 fragment lost one molecule of H2O and CO to generate a fragment ion at m/z 255.03. The easy-to-lose neutral molecule CO produced a fragment ion at m/z 227.03.

2.2. HPLC-DPPH Analysis

The chromatogram of the 50% methanol extraction of EUP spiking with DPPH at 254 nm showed that five compounds, 1–5, in the 50% methanol extraction of EUP possessed antioxidant activity (see Table 2, Figure 2 and Figure 3). Compounds 2–4 had larger UV absorption at 254 nm before the DPPH reaction, which significantly decreased or even disappeared after the reaction, indicating that these three compounds showed higher antioxidant capacities compared to other components. The ESI-MS results indicate that compounds 2–5 had phenolic hydroxyl structures, which were considered to be the main reason for their DPPH radical-scavenging ability.

2.3. Preparation and Identification of the Target Antioxidants

Based on the results of the UPLC-Q-TOF-MS and DPPH-HPLC antioxidant testing, three components with significant antioxidant activity and relatively high contents were selected as Q-markers.

2.3.1. Preparation of Q-Markers by Semi-Prep-HPLC

In order to improve the peak shape and resolution, this study investigated the effects of adding different concentrations of acid (0.2% phosphoric acid and 0.4% phosphoric acid) in the aqueous phase. The results showed that a good separation effect could be acquired with 0.2% phosphoric acid. Then, elution solvents (5%, 10%, and 12% acetonitrile–0.2% phosphoric acid (v/v)) and injection volumes (50 μL, 100 μL, and 200 μL) were investigated to improve the resolution and shorten the separation preparation time in the gradient elution mode. Based on the results, 12% acetonitrile–0.2% phosphoric acid (v/v) was selected as the best ratio, and 50 μL was the best injection volume. Under the optimized separation conditions, the retention times of the Q-markers in the 50% methanol extract of EUP were 28.64, 34.52, and 36.37 min, respectively.

2.3.2. Structural Identification

As Table 2 shown, compound 2 loses a glucose residue to form a fragment ion peak 465 [M + H − Glc]+ and continues to lose a glucose residue to form a fragment ion peak 303 [M + H − Glc]+ in the positive ion mode. As shown in Table 2, compound 2 loses a glucose residue to form fragment ion peak 465 [M + H − Glc]+ and continues to lose a glucose residue to form fragment ion peak 303 [M + H − Glc]+ in the positive ion mode. Compound 2 could be tentatively identified as quercetin-3-O-sophoroside (QSH) by comparing their chromatographic characteristics, absorption spectra, and previous articles. The monosaccharides of the samples were identified as d-xylose and d-glucose in compound 3 and as l-rhamnose and d-glucose in compound 4 by comparing their retention times with those of the monosaccharide standards. Combined with the results of the mass spectrometry, quercetin di-glycoside (compound 3) was quercetin-d-xylosyl-d-glucoside, and compound 4 was quercetin-l-rhamnosyl-d-glucoside. The structures of the three Q-markers were confirmed via the NMR of purified flavonoids (Table 3, Figure 4 and Figures S3–S6).
  • Compound 2: yellow powder. ESI-MS (m/z): 627.1585 [M + H]+ (positive), 625.1428 [M − H]− (negative), C27H30O17 (Cal: 626.1483). Compound 2 was identified as 5,7,3′,4′-tetrahydroxyflavone, known as quercetin-3-O-sophoroside, by an analysis of the 1H-1H correlation spectroscopy (COSY), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear multiple-bond correlation (HMBC) spectra (Figures S3 and S4). A large coupling constant (J = 7.3 Hz, 7.9 Hz) for the anomeric proton (δH 5.70, δH 4.60) of the glucose in the 1H-NMR spectrum suggested a β-configuration in glucose. In the HMBC spectrum, δH 5.70 (H-1 of 3-O-Glc) correlated with δC 133.4 (C-3), δC 76.97(C-3″), and δC 98.46(C-1″), and δH 4.60 (H-1 of Glc) correlated with δC 83.14(C-2″) and δC 74.82(C-3‴). The glucose C-2″ signal appeared at δC 83.14, while that of C-2‴ appeared at δC 74.82, suggesting that the inter glycosidic linkage was glucose-(1→2)-glucose. The obtained NMR data are consistent with those of previous research [37,38]. QSH was isolated from EUP for the first time.
  • Compound 3: light yellow powder. ESI-MS (m/z): 597.1471 [M + H]+ (positive), 595.1340 [M − H]− (negative), C26H28O16 (Cal: 596.1378). Compound 3 was also identified as 5,7,3′,4’-tetrahydroxyflavone, known as quercetin-3-O-sambubioside, by comparison with previously reported spectral data [39]. The β-configuration of the glucopyranosyl group was indicated based on the large coupling constants (J1,3 = 7.7 Hz) of the anomeric protons. In the HMBC spectrum (Figures S2 and S4), the correlation of δH 5.68 (H-1 of 3-O-glc) with δC 133.31 (C-3) and the correlation of δH 4.55 (H-1 of Xyl) with δC 82.25 (C-2″) were observed, which indicated that the sequence of the saccharide chain of C-3 was xylosyl-(1→2)-glucopyranosyl-(1→3). QSB was also isolated from EUP for the first time.
  • Compound 4: light yellow powder. ESI-MS (m/z): 611.1620 [M + H]+ (positive), 609.1487 [M − H]− (negative), C27H30O16 (Cal: 610.1534). Compound 4 was identified as quercetin 3-O-neohesperidoside (QNH) by comparison with previously reported spectral data [40,41]. The β-configuration of the glucopyranosyl group was indicated based on the large coupling constants (J1,3 > 7.0 Hz) of the anomeric protons. In the HMBC spectrum (Figures S3 and S6), the correlation of δH 5.64 (H-1 of 3-O-Glc) with δC 133.30 (C-3) and δC 77.74 (C-3″) and the correlation of δH 5.07 (H-1, Rha) with δC 77.84 (C-2″) were observed, which indicated that the sequence of the saccharide chain of C-3 was rhamnopyranosyl-(1→2)-glucopyranosyl-(1→3). QNH was isolated from EU for the first time.
In addition, the results of the HPLC peak-area normalization showed that the purities of the three compounds were above 95%.

2.4. Results of the Molecular Docking

The interaction energy states a total for all of the types of interactions, such as the van der Waals force, hydrogen bonding, the charge effect, hydrophobic interactions, etc. Figure 5 exhibits the 2D interaction diagrams for the separated compounds. As displayed, all three compounds had higher binding affinity values than quercetin. QSH exhibited the highest interaction energy of 71.67 kcal/mol with the carbon–hydrogen bonds of Ser602, Val604, Leu365, Val463, Ser503, Arg415, Gly462, Gly509, Gly603, and Gly364, followed by QNH, which showed an interaction energy of 61.62 kcal/mol with the carbon–hydrogen bonds of Arg380, Arg415, Gly603, Gly364, Val604, Leu365, Val463, and Gly509, while QSB showed the lowest interaction energy of 54.67 kcal/mol with the carbon–hydrogen bonds of Arg415, Arg483, Gly462, and Ser363. The interaction energy of quercetin is only 34.0138.

2.5. Cytotoxicity Assay and the Antioxidant Effect

Three different concentrations (25, 50, and 100 μM) were studied in our experiments, and the results are summarized in Figure 6A. As shown, all compounds exhibited cell viability at the tested concentrations. The results demonstrated that the maximum concentration of 100 μM could be used for subsequent antioxidant experiments. A concentration-dependent study of viability losses was investigated in RAW264.7 cells induced by H2O2. After treatment with increasing concentrations of H2O2 for 4 h, the cell viability was determined using the CCK 8 method. As shown in Figure S7, gradual reductions in cell viability were found with increasing concentrations of H2O2. Based on the results, RAW264.7 cells were treated with 1.0 mM H2O2 for 4.0 h, and the cell viability was about 50.52 ± 1.48%. Finally, we chose a concentration of 1.0 mM for further experiments.
The effects of QSH, QSB, and QNH on the intracellular ROS levels of RAW264.7 cells are shown in Figure 6B. Treatment with 1.0 mM H2O2 significantly increased the intracellular ROS levels. As indicated, all tested compounds exhibited significant protective effects against H2O2-induced oxidation damage, even at the lowest concentration, compared to the positive control (quercetin). This was consistent with the results of the molecular docking.

2.6. Development and Validation of the Quality Standard

2.6.1. Optimization of the Extraction

The extraction solvent, solid–liquid ratio, and extraction time have important effects on the extraction of target constituents in EUP. In order to obtain the proper extraction efficiency of QSH, QSB, and QNH, single-factor tests were performed for the extraction time (15, 30, 45, and 60 min), the extraction solvent (methanol, 50% methanol, ethanol, and 50% ethanol (v/v)), and the solid–liquid ratio (1:50 g·mL−1, 1:100 g·mL−1, and 1:250 g·mL−1). Finally, by comparing the extraction yields of the three constituents in a 50% methanol solvent, the best extraction method for UHPLC-QTOF-MS was 0.5 g of the sample powder extracted with 50% methanol (25 mL) on an ultrasonic machine for 30 min.

2.6.2. Optimization of Chromatographic Conditions

To establish an efficient and accurate content determination method, the chromatographic column type, mobile phase composition, and current speed were optimized. Then, we found that when the Halo Phenyl-Hexyl column was selected, and acetonitrile–0.2% phosphoric acid solution was used as the mobile phase, with a flow rate of 0.5 mL·min−1, the Q-markers could be resolved well, the symmetry and shape of the peaks were good, and the elution time was short.

2.6.3. Validation of the Analytical Method

We developed a simultaneous HPLC analysis method using the three markers as indicators for the efficient quality control of EUP. The assay was tested with several parameters, including linearity, stability, precision, repeatability, and recovery. The coefficient of determination (r2), which evaluates linearity, showed excellent linearity from 0.9999 to 1.0000 for all markers based on the prepared calibration curve (QSH, y = 665.43x + 0.18; QSB, y = 591.50x + 0.77; QNH, y = 666.92x + 0.60), and the linear ranges were 0.0128 to 1.27 mg·mL−1, 0.0157 to 0.786 mg·mL−1, and 0.0117 to 0.392 mg·mL−1, respectively. All RSD values of the repeatability, precision, and stability of the investigated markers were <1.55%. The recoveries (%) of compounds 1–3 ranged from 98% to 102% for each concentration level, and the RSDs were less than 2%. These results demonstrated that the sensitivity and applicability of the optimized HPLC-PDA were feasible for the quantitation analysis of the three Q-markers in pollen.

2.6.4. Sample Analysis

The contents of the three Q-markers from the EUP samples of different batches are given in Table 4. The results showed that the contents of the Q-markers in S33 and S34 were 0; that is, they did not contain these three components, which proved that these two batches of samples were fake, and the microscopic identification results also proved this. Samples from other batches contained Q-markers, and the content distribution ranges of QSH, QSB, and QNH were 9.12 to 14.74 mg/g, 7.29 to 10.52 mg/g, and 2.05 to 4.05 mg/g, respectively. This shows that this method can scientifically and accurately identify the authenticity of EUP, and it is necessary to establish a quality evaluation method for EUP.
Four varieties and mixed pollen were tested, and it was found that the contents of the three quality markers in the EUP of different varieties were within the normal ranges (Figure 7A). In addition, pollen from different producing areas was compared, and there were no significant differences in the contents of quality markers in pollen from five places (see Figure 7B).

3. Discussion

EU has a long history of application as a traditional Chinese medicine in China. So far, more than 200 compounds have been isolated and identified from EU. There are many articles on the chemical composition and quality evaluation of Eucommia ulmoides leaves, bark, and male flowers. EUP, as a non-medicinal part of EU that is rich in nutrients, trace elements, and minerals [28], has been increasingly studied in recent years. However, the quality control of EUP has not been well established due to the lack of quality markers (Q-markers). The chemical components in EUP were identified using UPLC-QTOF-MS and compared with their relative peak areas in EUB, EUL, and EUF. The results suggested that flavonoids are its characteristic components.
Oxidative stress has been shown to participate in a wide range of diseases, including cardiovascular disease [42], Alzheimer’s disease [43], male infertility [44,45], and cancer [46]. The application of antioxidants can alleviate oxidative stress-induced disease progression. Considering safety, the discovery of natural antioxidants has received increasing attention in recent years. As a new functional food ingredient, EUP has significant antioxidant activity, which may be related to its high content of polyphenolic compounds. Despite the employment of many methods in the extraction of antioxidants from EU, the traditional strategy is time-consuming, cumbersome, and less efficient for screening. The DPPH-HPLC active component detection method is convenient, fast, and highly accurate, and it is widely used for screening antioxidant components in plants [47,48,49]. After interaction with DPPH, the UV absorption of free radical-scavenging compounds decreased or disappeared, and identity confirmation could be achieved using the UPLC–DAD–TOF/MS technique.
Using this method, five components with antioxidant activity in EUP were selected, all of which had phenolic hydroxyl structures, which were considered to be the main reason for their DPPH radical-scavenging ability. QSH, QSB, and QNH had larger UV absorption at 254 nm before the DPPH reaction, which significantly decreased or even disappeared after the reaction, indicating that these three compounds showed higher antioxidant capacities compared to other components [39].
Kelch-like ECH-associated protein 1 (Keap1), an adaptor of the E3 ligase complex that promotes the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2), is a master transcriptional regulator in the antioxidative response. The KEAP1–Nrf2 signaling pathway senses reactive oxygen species and regulates cellular oxidative stress. Inhibiting KEAP1 to activate the Nrf2 antioxidant response has been proposed as a promising strategy to treat chronic diseases caused by oxidative stress [50,51,52]. The higher the binding energy with KEAP1, the better the antioxidant capacity of the compound.
Three flavonoids were successively isolated and purified from a 50% methanol extract of EUP for the first time under the guidance of the DPPH-HPLC method. The molecular docking results and in vitro cell experiments both indicated that QSH, QSB, and QNH have significant antioxidant activity, which is the same as the results reported in the literature [37]. Semi-quantitative results showed that the peak-area ratio of the three compounds in pollen was significantly higher than that in other organs. Considering that they are also characteristic components of EUP, selecting these three components as indicators to evaluate the quality of EUP is considered scientific and reasonable. EU has many varieties, such as Huazhong 11, Huazhong 22, Huazhong 23, Huazhong 24, etc. Different varieties and environments may affect the composition of bioactive chemicals in EUP. This study only explored the two key factors that may affect the quality of pollen, namely, origin and variety, but not other factors that may affect the quality of pollen, such as the harvest time and original processing methods, which will continue to be studied in the future.

4. Materials and Methods

4.1. Materials and Apparatus

The EUP was collected from Xuchang, Henan province; Hanzhong, Shanxi province; Longnan, Gansu province; and Zhangjiajie, Hunan province, China. The EUL, EUF, and EUB samples were collected from Xuchang Henan province. Samples S1–S34 were authenticated by Professor Zhimin Wang at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, P.R. China. Chemical reference substances (CRSs), including aucubin, geniposidic acid, chlorogenic acid, asperuloside, quercetin-3-O-sophoroside, quercetin-3-O-sambubioside, quercetin-3-O-neohesperidoside, rutin, quercetin, asperuloside, hyperoside, genipin acid, chlorogenic acid, caffeic acid, gallic acid, luteolin, oleanolic acid, neochlorogenic acid, cryptogenic acid, naringin, naringenin, scutellarin, ursolic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and genipin, were purchased from Herb Purify Biological Technology Co., Ltd., Chengdu, China. The purity of all CRSs was over 98%. DPPH was purchased from Coolaber Science & Technology Co., Ltd., Beijing, China. Acetonitrile and methanol were purchased from Fisher, Waltham, MA, USA, and were chromatographically pure. The water was distilled water, and other analysis-grade reagents were purchased from Sino Pharm Chemical Reagent Co., Shanghai, China. Fetal bovine serum was purchased from Beijing Pulilai Gene Technology Co., Ltd. (Beijing, China). Streptomycin, phosphate-buffered saline, Dulbecco’s modified Eagle medium (DMEM), and penicillin were purchased from Beijing Dongdu Kaiyuan Biotechnology Co., Ltd. (Beijing, China). The RAW264.7 cell line was purchased from the Chinese Type Culture Collection. An HC-2518 high-speed centrifuge (Anhui ustic zonka scientific instruments Co., Ltd., Hefei, China), a Xevo g2-s QTOF mass spectrometer, a Waters ACQUZTY UPLC system (Waters Technologies, Shanghai, China), an HC-2518 fragmentation voltage (Anhui Zhongke Zhongjia Science Co., Ltd., Hefei, China), a KQ-250DE CNC Ultrasonic Cleaner (Kun Shan Ultrasonic Instruments Co., Ltd., Kunshan, China), an Ultimate3000 high-performance liquid chromatography system (ThermoFisher Co., Ltd., Waltham, MA, USA), an LC3000 preparative HPLC system (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China), and a Halo Phenyl-Hexyl column (4.6 × 150 mm, 2.7 μm; Waters Technologies, Milfordcity, MA, USA) were used.

4.2. Preparation of Sample

About 0.5 g of the sample powder was precisely weighed. Then, 25 mL of 70% methanol was precisely added, weighed, extracted via sonication (250 W, 40 kHz) for 30 min at room temperature, and cooled. The weight loss comprised 70% methanol, and the solution was shaken well and filtered. The filtrate was centrifuged at 12,000 r·min−1 for 10 min, and the supernatant was filtered through a 0.22 µm-microporous membrane and stored at 4 °C in a refrigerator.

4.3. UPLC-ESI-TOF/MS Analysis

The extracts of EUP were analyzed using UPLC-ESI-TOF/MS, which consisted of a Waters ACQUZTY UPLC system (Waters Technologies, Milford, MA, USA) coupled to a Xevo G2-S QT mass spectrometer (Waters Technologies, USA). An ACQUITY UPLC® BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Technologies, USA) was used during the analysis, and the temperature of the column was maintained at 40 °C. The flow rate was 0.3 mL/min, the injection volume was 2 μL, and the determination wavelength was set at 190–400 nm. The mobile phases were composed of A (water containing 0.1% acetic acid (v/v)) and B (acetonitrile). The linear gradient program was as follows: 0–5 min, 98% A; 5–35 min, 98–5% A; 35–40 min, 5% A. Mass spectrometry was carried out in the scan mode from 50 m/z to 1200 m/z using both negative and positive modes at 450 °C with a corona discharge at ±6.0 kV. The ESI-MS conditions were as follows: the capillary voltage was set to 2.0 kV; the temperature was 120 °C; the drying gas flow was 10.0 L/min; and the nebulizing gas pressure was 45 psi.

4.4. Screening Active Compounds by HPLC-DPPH

First, an HPLC analysis of eight standards—geniposidic acid, chlorogenic acid, asperuloside, geniposide, QSH, QSB, QNH, and rutin—was optimized to obtain a baseline separation. This analysis was carried out using a U3000 system equipped with a diode array detector (PDA) system, a column oven, and an automatic injector. The injection volume was 10 μL. A Halo Phenyl-Hexyl column (2.7 μm, 4.6 × 150 mm) was used during the analysis, and the temperature of the column was 30 °C. The flow rate was 0.5 mL/min, the injection volume was 10 μL, and the determination wavelength was set at 254 nm. The mobile phases were composed of A (acetonitrile) and B (water containing 0.2% phosphoric acid (v/v)). The linear gradient program was as follows: 0–2 min, 1% A; 8–18 min, 1–7% A; 18–60 min, 7–17% A. There was a 10-min-post run to re-equilibrate the column for each run. First, the sample solution was analyzed by HPLC to obtain as much chemical information as possible. A DPPH-radical solution was freshly prepared in methanol. The EUP extract was mixed with a DPPH-methanol solution (2 mg/mL) at a ratio of 1:1 (v/v). After incubation in the dark at 25 °C for 60 min, the mixture was centrifuged at 12,000 rpm for 10 min, and then the supernatant was analyzed by the same chromatographic condition. By comparing the chromatographic profiles of DPPH-reacted samples and control samples, the main antioxidants in the EUP extract could be screened.
The EUP extract was analyzed by HPLC–ESI-Q-TOF-MS. The chromatographic conditions were the same as those in Section 4.4, except for replacing phosphoric acid with formic acid. The mass spectrometry conditions were the same as those in Section 4.3.

4.5. Preparation and Characterization of the Main Active Components

4.5.1. Analytical Condition

An LC3000-prep-HPLC system was used to accomplish the preparation and characterization of the active components screened from EUP. A Kromasil 100-5 C18 column (250 × 100 mm, 10 mm; Waters Technologies, USA) was used; the flow rate was maintained at 2.0 mL/min; the injection volume was 50 mL; the determination wavelengths were set at 254 nm and 353 nm; and the mobile phases were composed of A (methanol) and B (water containing 0.2% acetic acid (v/v)). The linear gradient program was as follows: 0~10 min, 20→36% A; 10~32 min, 36% A; 32~33 min, 36→80% A; 33~41 min, 80% A; 41~41.5 min, 80→20% A; 41.5~48 min, 20% A. For the GC analysis, an Optima 5MS capillary chromatographic column (320 μm × 0.25 μm, 30 m, MACHEREY-NAGEL) and an FID detector were used. The temperature program was as follows: an initial temperature of 170 °C for 3 min; a temperature increase of 2 °C/min to 230 °C for 5 min; carrier gas: N2; injection temperature: 250 °C; detector temperature: 300 °C; nitrogen gas flow: 1 mL/min; hydrogen gas flow: 30 mL/min; airflow: 50 mL/min; injection volume: 2.0 μL; injection method: split injection; split ratio: 60:1.

4.5.2. The Derivatization Procedures of Quercetin Di-Glycoside

The pretreatment of samples and the derivatization of monosaccharides were carried out according to previous studies [53,54].

4.5.3. Nuclear Magnetic Resonance Spectroscopy (NMR)

The sample was dissolved in DMSO-d6 (0.5 mL). The 13C-NMR, 1H-NMR, COSY, TOCSY, DEPT, HMQC, and HMBC spectra were recorded at 298 K using a JNM-ECP 600 MHz NMR spectrometer.

4.6. Molecular Docking

To confirm the interactions between the core antioxidant targets and the components, molecular docking was conducted by selecting the key oxidative stress protein KEAP1, with a high median degree value, as a receptor and the three isolated compounds as the ligands. The non-mutated tertiary structure of the targeted protein (KEAP1, PDB code: 4XMB) was initially downloaded in the PDB format from the Protein Data Bank (https://www.rcsb.org/, accessed on 26 September 2022). The 3D chemical structures of the candidate compounds were drawn and saved in the SDF format. All the documents were converted to the PDB format for subsequent molecular docking. In the Discovery Studio 2020 software, the water molecules were deleted from the ligands, nonpolar hydrogen was added, and the Gasteiger charge was calculated. The potential core ligands were subjected to the energy minimization treatment, and the ligand atom type was obtained after a calculation. The Discovery Studio 2020 software was used for the calculation of the docking of semisoft molecules.

4.7. Antioxidant Activity Evaluation of Each Compound

4.7.1. Cell Culture and Cell Viability Assay

The RAW264.7 cell line was purchased from the Chinese Type Culture Collection (NICR, Beijing, China). The cell lines were grown in DMEM with 10% FBS and 1% P/S and were incubated at 37 °C in 5% CO2. To determine cell viability, RAW264.7 cells were seeded into a 96-well plate at a density of 1 × 104 cells/well, followed by treatment with compounds at 25, 50, and 100 μM. The results were expressed as the mean cell survival, normalized to the control, as determined using a CCK-8 assay, according to the manufacturer’s protocols.

4.7.2. Detection of Intracellular ROS Generation

Intracellular ROS levels were measured using the fluorescent probe DCFH-DA, which, after crossing the plasma membrane, is hydrolyzed to DCFH and oxidized to the fluorescent product DCF. Sample stock solutions were prepared in DMSO. RAW264.7 cells were grown in 96-well plates at 1 × 104 cells/well and cultured for 24 h at 37 °C with 5% CO2. RAW264.7 cells were incubated with a DMEM culture. The sample solutions were added to the cell culture for 24 h. Each treatment was exposed to a medium containing H2O2 (1.0 mM) for another 4 h, while the medium in the control group was only replaced with a complete medium for another 4 h. Next, the treated RAW264.7 cells (1 × 104/well in 96-well plates) were incubated in a cell medium containing 5 μM DCFH-DA for 30 min, followed by three washes using a serum-free cell-culture medium. The fluorescence intensity was measured using a Varioskan Flash full-wavelength multifunctional microplate reader with excitation at 485 nm and emission at 530 nm.

4.8. Development and Validation of the Quality Standard

4.8.1. Chromatographic Conditions

The extracts of EUP were analyzed using UHPLC. A Halo Phenyl-Hexyl column (2.7 μm, 4.6 × 150 mm) was used during the analysis, and the temperature of the column was set at 30 °C. The flow rate was 0.5 mL·min−1, the injection volume was 10 μL, and the determination wavelength was set to 254 nm. The mobile phases were composed of A (acetonitrile) and B (water containing 0.2% phosphoric acid (v/v)). The linear gradient program was as follows: 0–2 min, 1% A; 8–18 min, 1–7% A; 18–60 min, 7–17% A.

4.8.2. Validation of the Method

The quantification method of the three Q-markers was validated with respect to linearity, stability, precision, repeatability, and recovery in accordance with the guidelines for the validation of analytical methods of the Chinese Pharmacopoeia (fourth part) (Chinese Pharmacopoeia, 2020). Serial dilutions of mixed standards were used to establish the standard curves, and the linear regression equation correlation coefficient and linear range were calculated. For precision, the solutions were examined in triplicate for 3 consecutive days. To validate the repeatability, six samples of EUP were accurately weighed and prepared independently, according to the optimal conditions above, and then analyzed. The same sample solution was taken and determined at 0, 2, 4, 8, 12, 24, and 48 h after its fresh preparation, according to the above chromatographic conditions, to evaluate the stability. Recovery experiments were used to assess the accuracy of the method. Standards at three different concentration levels, including low (80%), median (100%), and high (120%) levels, were added to samples with known content. Each experiment was repeated three times, and the spiked samples were analyzed using UHPLC-PDA to evaluate the recoveries. The recoveries were calculated using the following formula: recovery (%) = (detected amount − original amount)/spiked amount × 100%.

4.9. Statistical Analysis

The obtained data were analyzed with SPSS (version 21) software using a one-way ANOVA, followed by an LSD post hoc test. The data were represented as means ± SDs, and p < 0.05 indicated statistical significance.

5. Conclusions

The chemical composition of EUP was investigated for the first time, providing a foundation for further in-depth research. Three characteristic active ingredients in EUP had strong and clear effects on DPPH while significantly reducing the production of ROS in RAW264.7 cells induced by H2O2. To evaluate and monitor the quality of EUP more scientifically, this study took the discovery and determination of Q-markers as the main finding and established a fast, sensitive, and characteristic evaluation method for the first time. We believe that this work can provide a new quality assessment model and a demonstration for the further development and utilization of EUP in the food or nutrition industries. This study only discussed two key factors that may affect pollen quality, namely, origin and variety, without exploring other factors that may affect pollen quality, such as the harvest time and original processing methods, which will continue to be studied in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28135288/s1, Figure S1: UPLC/QTOF-MS based peak ion diagram (BPI) of different parts of EU.; Figure S2: Chromatogram for preparing liquid phase; Figure S3: Correlations and Key HMBCs of compounds 2, 3 and 4; Figure S4: 1H/13C HMBC spectrum of the compound 2 dissolved in DMSO-d6; Figure S5: 1H/13C HMBC spectrum of the compound 3 dissolved in DMSO-d6; Figure S6: 1H/13C HMBC spectrum of the compound 4 dissolved in DMSO-d6; Figure S7: Viability losses in RAW264.7 cells induced by various concentration of H2O2.

Author Contributions

F.G. and Y.Y. were involved in experimental design, performing most of the experiments, Writing Draft, and writing—reviewing and editing. Y.D. verified the experimental results. C.L. verified the results of the structural identification, and reviewed and edited the manuscript. H.G. verified the results, and reviewed and edited the manuscript; H.L. and Q.C. realized the analytical measurements. Z.G. performed Molecular docking. Z.W. and X.L. were involved in conceptualization, supervision, writing—reviewing and editing. All authors contributed to the preparation of the figures. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Scientific and Technological Innovation Project of the China Academy of Chinese Medical Sciences (grant numbers CI2021A04308 and CI2021A04407) and the China National Key R & D Projects (grant numbers 2017YFC1701900).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest associated with this publication.

Sample Availability

Samples of compounds 2–4 are available from the authors.

Abbreviations

EUEucommia ulmoides Oliver
EUPthe pollen of Eucommia ulmoides Oliver
EUFMale flowers of Eucommia ulmoides Oliver
DPPH2,2-Diphenyl-1-picrylhydrazyl
HPLChigh-performance liquid chromatography
NMRnuclear magnetic resonance
QSHquercetin-3-O-sophoroside
QSBquercetin-3-O-sambubioside
QNHquercetin 3-O-neohesperidoside
NHCthe National Health Commission

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Molecules 28 05288 g001 550
Figure 1. UPLC/QTOF-MS based peak ion diagram (BPI) of different parts of EU. A, EUB; B, EUL; C, EUF; D, EUP; +, positive; −, negative.
Figure 1. UPLC/QTOF-MS based peak ion diagram (BPI) of different parts of EU. A, EUB; B, EUL; C, EUF; D, EUP; +, positive; −, negative.
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Molecules 28 05288 g002 550
Figure 2. Antioxidant components of 50% methanol extract from EUP by HPLC-DPPH. (the number corresponds to Table 2; Red square, three compounds with the highest response value changes).
Figure 2. Antioxidant components of 50% methanol extract from EUP by HPLC-DPPH. (the number corresponds to Table 2; Red square, three compounds with the highest response value changes).
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Molecules 28 05288 g003 550
Figure 3. The structure of compounds 1–5.
Figure 3. The structure of compounds 1–5.
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Figure 4. The structure of the 3 Q-markers. Compound 2: QSH; compound 3: QSB; compound 4: QNH.
Figure 4. The structure of the 3 Q-markers. Compound 2: QSH; compound 3: QSB; compound 4: QNH.
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Molecules 28 05288 g005 550
Figure 5. Interaction of (A) quercetin (control), (B) QSH, (C) QSB, and (D) QNH with KEAP1 receptors.
Figure 5. Interaction of (A) quercetin (control), (B) QSH, (C) QSB, and (D) QNH with KEAP1 receptors.
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Molecules 28 05288 g006 550
Figure 6. Cell experiment results. (A) Effects of different concentrations of QSH, QSB, and QNH on the activity of Raw264.7 cells, (B) Effects of different concentrations of QSH, QSB, and QNH on ROS production in Raw264.7 cells. DCF fluorescence was quantified in control (Con) and Raw264.7 cells incubated in the absence (M) or in the presence of QSH, QSB, or QNH for 24 h. Data are expressed as a percentage of control. Values are the mean ± SD from three independent experiments. (HPS) quercetin, (M) Model. Significant differences are denoted by symbols: ## p < 0.01, # p < 0.05 vs. control; ** p < 0.01 vs. M.
Figure 6. Cell experiment results. (A) Effects of different concentrations of QSH, QSB, and QNH on the activity of Raw264.7 cells, (B) Effects of different concentrations of QSH, QSB, and QNH on ROS production in Raw264.7 cells. DCF fluorescence was quantified in control (Con) and Raw264.7 cells incubated in the absence (M) or in the presence of QSH, QSB, or QNH for 24 h. Data are expressed as a percentage of control. Values are the mean ± SD from three independent experiments. (HPS) quercetin, (M) Model. Significant differences are denoted by symbols: ## p < 0.01, # p < 0.05 vs. control; ** p < 0.01 vs. M.
Molecules 28 05288 g006
Molecules 28 05288 g007 550
Figure 7. The influence of variety and producing areas on the quality of EUP. (A) The influence of variety on the quality of EUP. (B) The influence of producing areas on the quality of EUP.
Figure 7. The influence of variety and producing areas on the quality of EUP. (A) The influence of variety on the quality of EUP. (B) The influence of producing areas on the quality of EUP.
Molecules 28 05288 g007
Table
Table 1. LC-MS analysis of chemical components in different organs of EU.
Table 1. LC-MS analysis of chemical components in different organs of EU.
Peak NumbertR/(min)Molecular
Weight		[M + H]+/[M + Na]+/[M + NH4]+ (Error, ppm)[M − H]−/[M + HCOO]− (Error, ppm)MS/MS
Fragments (P)		MS/MS
Fragments (N)		Molecular FormulaCompoundPart
10.85342.1162365.1049 (−1.4)387.1140 (0.35)163.0633, 119.0341, 225.0872, 164.0695191.0561, 195.0515, 129.0197, 101.0244C12H22O11IsomaltoseEUB, EUL, EUF, EUP
20.91164.0473182.0802 (−2.7) 145.0482, 131.0497, 119.0479, 149.0592 C9H8O3p-coumaric acidEUL, EUF, EUP
32.61350.1577373.1462 (−1.9)395.1553 (−0.1)135.0785, 153.0897195.0287, 153.0540, 149.0614C15H26O9Eucommiol IIEUB, EUL
42.80350.1577373.146 (−2.4)395.1556 (0.7)135.0785, 153.0897195.0287, 153.0540, 149.0614C15H26O9Eucommiol II isomerEUB, EUL
53.44346.1264369.1149 (3.3) 311.1122, 149.0590, 131.0484 C15H22O9Aucubin *EUL, EUF, EUP
63.44392.1319 391.124 (−0.1) 295.1032, 345.1188, 227.0565, 183.0664C16H24O11ReptosideEUB, EUL, EUF, EUP
73.74390.1162413.1052 (−1.9)389.1078 (−1.5)193.0480, 175.0373, 147.0429371.0977, 227.0510, 165.0563C16H22O11ScandosideEUB, EUL, EUF, EUP
84.07154.0266155.0339 (−3.4)153.0192 (2.7)137.0238109.0312C7H6O4Protocatechuic acidEUB, EUL, EUF, EUP
96.75212.0685213.0756 (−3.3)211.0613 (3.09)195.0647, 177.0538149.0613, 193.0501C10H12O5C-veratroylglycolEUB, EUL, EUF, EUP
106.98374.1213397.1092 (−4.7)373.1132 (−0.7)357.1173, 177.0544, 149.0593, 195.0649211.0606, 193.0627, 167.0706, 149.0600, 123.0443C16H22O10Geniposidic acid *EUB, EUL, EUF, EUP
116.99404.1319 449.1295 (−0.0)353.0863, 211.0611, 149.0608, 373.1136, 353.0863 C17H24O11Deacetyl asperulosidic acid methyl esterEUB, EUL, EUF, EUP
128.56290.079291.0855 (−4.7) 147.0428, 139.0374, 131.0479 C15H14O6CatechinEUL, EUF, EUP
138.72432.1268450.1606 (−1.22)431.1179 (−2.4)175.0388, 147.0435, 119.0485, 193.0522373.1125, 257.1029, 211.0611, 251.0562C18H24O12Asperuloside acid *EUL, EUF, EUP
148.77354.0951 353.0854 (−2.4) 191.0565, 173.0457, 307.0816, 133.0295C16H18O9Chlorogenic acid *EUB, EUL, EUF
158.79332.1107355.1018 (3.7) 181.0477, 179.0325 C14H20O9KoaburasideEUB, EUL, EUF, EUP
168.87700.2579718.2905 (−2.4)745.2551 (1.2)341.1376, 323.1269, 217.0853, 137.0584699.2483, 583.1983, 537.1973, 375.1438, 341.1375, 359.1476, 195.0661, 137.0586C32H44O17Olivil 4,4″-di-O-b-d-glucopyranosideEUB, EUF
178.93180.0423181.0499 (−1.0)179.0344 (−0.2)163.1225135.0445, 161.0405, 117.0338C9H8O4Caffeic acid *EUB, EUL, EUF, EUP
189.45414.1162437.1047 (−2.9)459.1142 (−0.7)175.0375, 163.0736, 131.0478353.0867, 251.0597C18H22O11Asperuloside *EUL, EUF, EUP
199.45368.1107391.0988 (−4.3)413.1072 (−2.9)353.0867, 147.0453 C17H20O9Methyl chlorogenateEUL, EUF, EUP
209.60536.1894537.1969 (−0.6)535.1795 (0.7)357.1323, 375.1438, 323.0546373.1265, 343.1180, 285.1060, 520.1627C26H32O12(+)-1-Hydroxypinoresinol 4′-O-b-d-glucopyranosideEUB, EUL
219.85538.205556.2395 (−0.2)583.2031 (0.7)341.1374, 345.1319, 137.0583, 311.0537375.1444, 337.0927, 345.1324C26H34O12(−)-Olivil 4′-O-b-d-glucopyranosideEUB, EUL, EUF, EUP
229.95698.2422699.2481 (−2.7)743.2379 (−2.6)519.1858, 375.1422, 327.1207535.1737, 373.1261, 343.1265, 325.1095C32H42O17(+)-1-Hydroxypinoresinol 4′,4″-di-O-b-d-glucopyranosideEUB
239.99388.1369 433.1347 (−0.2) 375.1310, 207.0664, 175.0360, 371.0965C17H24O10Geniposide *EUB, EUF, EUP
249.99342.1315 387.1294 (−0.7) 165.0559, 123.0445, 147.0444C16H22O8(E)-ConiferinEUB, EUF, EUP
259.99180.0786181.0862 (−1.5)225.0765 (0.9)149.0593, 163.0743, 131.0480147.0460, 123.0454, 103.0151C10H12O3Pinusolidic acidEUP, EUF
2610.06536.1894537.1959 (−2.4) 375.1430, 357.1324, 519.1857 C26H32O12(+)-1-Hydroxypinoresinol 4″-O-b-d-glucopyranosideEUB
2710.21538.205556.2386 (−1.4)583.2023 (−0.6)341.1373, 297.1113, 165.0677, 137.0585375.1412, 277.1275, 507.1518, 123.0449C26H34O12(−)-Olivil 4″-O-b-d-glucopyranosideEUB
2810.31342.1315 387.1288 (−0.8) 179.0518, 297.1067, 147.0453C16H22O8ConiferinEUB, EUF, EUP
2910.44626.1483627.1556 (−0.8)625.1404 (−0.1)465.1027, 303.0501, 285.0387, 247.0596, 153.0175463.0869, 445.0765, 300.0279, 301.0325, 271.0241C27H30O17Baimaside *EUL, EUF, EUP
3010.55682.2473700.2802 (−2.1)727.2450 (0.1)357.1324, 235.0955681.2398, 519.1866, 357.1339, 342.1103, 151.0398, 136.162C32H42O16(+)-Pinoresinol di-O-b-d-glucopyranoside *EUB
3110.55682.2473700.2796 (−2.9)727.2444 (−0.7)341.1380, 175.0742, 187.0725, 323.1268519.1871, 357.1338, 327.1210C32H42O16(+)Dehydrodiconiferyl 4,γ-di-O-b-d-glucopyranosideEUL, EUB, EUF
3210.59372.142 417.1399 (0.7) 179.0567, 162.0322C17H24O9SyringinEUL, EUF, EUP
3310.671086.996 1085.336 (0.2) 669.2391, 505.1715, 413.1081, 207.0661, 195.0657, 179.0563C48H62O28Ulmoidoside AEUB, EUL
3410.71682.2473700.2815 (−0.2)727.2444 (−0.7)311.1260, 323.0528, 571.1435339.1228, 519.1867, 501.1761, 309.1124C32H42O16(+)Dehydrodiconiferyl 4,γ-di-O-b-d-glucopyranosideEUL, EUB, EUF
3510.8712.2579730.2915 (−1.0)757.2547 (−1.1)713.2574, 151.0378, 519.1867, 235.0946491.1912, 545.1787, 387.1438, 372.1197C33H44O17(+)-Medioresinol di-O-β-d-glucopyranosideEUB, EUL, EUF
3610.83226.0841249.0732 (−2.8)225.0767 (1.8)209.0804, 163.0743, 149.0948, 227.0906211.0611, 207.0662, 179.0350C11H14O5Genipin *EUB, EUF
3710.83596.1378597.1473 (2.7)595.1305 (1.0)303.0500, 153.0178, 465.1030463.0869, 445.0771, 301.0322, 271.0247, 243.0295C26H28O16Quercetin 3-O-sambubioside *EUL, EUF, EUP
3810.89568.2156586.2477 (−3.9)613.2128 (−0.7)533.2036, 341.1376, 167.0690, 191.0697405.1723, 537.2082, 371.1327, 531.1871, 207.0664C27H36O13Citrusin BEUL, EUB, EUF
3910.96610.1534611.1624 (−2.0)609.1456 (0.1)465.1025, 303.0501, 153.0177, 285.0384463.0876, 445.0754, 301.0319, 151.0036C27H30O16Quercetin 3-O-neohesperidoside *EUF, EUP
4011.04418.1628419.1685 (−5.0)417.1545 (−1.1)401.1592, 371.1122403.1431, 387.1094C22H26O8(+)-SyringaresinolEUB, EUL
4111.04742.2684760.3028 (0.0)787.2652 (−1.1)401.1591, 265.1058, 151.0375579.2074, 417.1550, 551.1768, 403.1431, 387.1077C34H46O18LiriodendrinEUL, EUB, EUF
4211.27610.1534611.1618 (1.0)609.1461 (0.9)303.0496, 465.1016, 245.0456301.0320, 271.0241, 255.0297, 243.0295, 227.0342, 151.0031C27H30O16Rutin *EUL, EUF, EUP
4311.45258.0258259.0599 (−2.9)303.0508 (−1.1)260.0669 C14H10O5AlternariolEUF, EUP, EUL
4411.51580.1792581.1516 (1.7)579.1355 (−0.9)449.1081, 287.0601, 153.0118463.0932, 284.0333, 255.0316, 227.0357C26H28O15Kaempherol-3-O-sambubiosideEUL, EUF, EUP
4511.52464.0955465.1021 (−2.6)463.0879 (0.5) 301.0335, 271.0299, 151.0038, 145.0291C21H20O12Isoquercitrin *EUL, EUF, EUP
4611.55375.1438 421.1497 (−1.9) 360.1202, 227.0345, 271.0247, 345.1341C24H20O7(+) Cyclo-olivilEUL, EUF, EUP
4711.78376.1522 375.1432 (−3.1) 225.0763, 308.1137, 327.1245, 357.1348, 343.1181C20H24O7(−)-olivilEUB, EUL
4811.79550.205568.2383 (−1.1)595.2026 (−0.14)435.1639, 329.1002, 321.1070373.1267, 467.1566, 195.0661C27H34O12Eucommia AEUL, EUB, EUF
4911.88908.3314926.3583 (−2.9)953.3279 (−1.8)549.1991, 387.1425, 181.0482745.2667, 583.2174, 387.1436, 195.0660C43H56O21Hedyotol C-4″,4‴-di-O-b-d-glucopyranosideEUB, EUL, EUF
5011.91460.1006 505.0981 (−0.2) 445.0748, 443.0583, 177.0163, 145.0291, 151.0037C22H20O11WogonosideEUL, EUF, EUP
5112.20968.3525986.3816 (−3.7)1013.347 (−3.7)775.2781, 549.1991, 417.1506, 417.1527745.2667, 643.2385, 353.0870, 805.2880, 893.2989C45H60O23Guaiacylglycerol-b-syringaresinol ether-4″,4″-di-O-b-d-glucopyranosideEUB, EUL
5212.22516.1628517.1329 (−3.3)515.1186 (−0.7)499.1232, 287.0548, 135.0430353.0876, 191.0557C25H24O12Isochlorogenic acid A *EUB, EUL, EUF
5312.24448.1006 447.0927 (−0.1) 285.0390, 151.0032, 227.0347C21H20O11Astragalin *EUL, EUF, EUP
5412.34520.1945538.2272 (−3.0)519.1864 (−0.5)357.1323, 165.0685357.1335, 342.1096, 136.0161C26H32O11(+)-Medioresinol di-O-b-d-glucopyranosideEUB, EUL, EUF
5512.54580.2156598.2487 (−2.1)579.2074 (0.6)417.1513, 247.0657417.1551, 387.1456, 551.1286C28H36O13(−)-Syringaresinol-O-b-d-glucopyranosideEUB, EUL, EUF
5612.69516.1628517.1328 (−3.5)515.1193 (−0.7)499.1225, 163.0384, 179.0892, 135.0422353.0886, 191.0561, 161.0237C25H24O12Isochlorogenic acid C *EUB, EUL, EUF, EUP
5712.81188.1049211.0942 (−2.0)187.0973 (0.0)135.0794, 153.0897, 107.0840125.0973, 169.0862, 141.0919, 123.0813C9H16O4EucommiolEUB, EUF, EUP
5813.16374.1366375.1433 (−2.9)373.1286 (−0.3)339.1217, 233.0795,358.1068, 327.0871, 313.1084, 345.0982C20H22O7(+)-1-HydroxypinoresinolEUB, EUL
5913.28284.0685302.1025 (0.6)283.0606 (−2.1)193.0478, 183.0309147.0442, 136.0165, 125.0234C16H12O5Oroxylin AEUF, EUP
6013.55196.1099219.1001 (4.1)241.1082 (2.5)161.0595, 149.0576, 163.0358163.0386, 145.0274C11H16O3LoliolideEUB, EUL, EUF, EUP
6114.32302.0427303.0498 (−2.2)301.0345 (−1.1)153.0171, 285.0373, 195.0272151.0038, 285.0397, 271.0232C15H10O7Quercetin *EUL, EUF, EUP
6215.52272.0685 271.0607 (−0.2) 151.0072, 119.0528, 93.0365, 177.0216, 227.0727C15H12O5NaringeninEUL, EUF, EUP
6315.53270.0528271.0597 (−3.5)269.0453 (−1.1)145.0630, 179.0329177.0193, 145.0536C15H10O5BaicaleinEUL, EUF, EUP
6415.76286.0477287.0558 (0.7)285.0396 (−1.1)153.0180, 179.0321227.0337, 151.0026, 145.9311C15H10O6KaempferolEUL, EUF, EUP
6521.27172.1099 195.0999 (1.0)95.0472, 121.0259 C9H16O31-DeoxyeucommiolEUL, EUF, EUP
6625.83278.1518301.1405 (−3.6)277.1444 (1.5)149.0217, 121.0270121.0289C16H22O41,2-benzenedicarboxylic acid bis(2-methylpropyl) esterEUB, EUL, EUF, EUP
6728.17392.1471410.1811 (−1.0) 313.0743, 185.0803 C20H24O8Erythro-dihydroxydehydrodiconiferylEUB, EUL, EUF, EUP
6828.17184.0736185.0810 (−2.1) 111.0070, 113.0218 C9H12O4EucommidiolEUB, EUL, EUF, EUP
6929.24456.3604457.3662 (−4.3)455.3526 (0.2)411.3613, 393.3506277.2171, 407.1728, 377.1420C30H48O3Betulinic acidEUB, EUL, EUF, EUP
7029.32456.3604457.3699 (3.8)455.3531 (−1.3)439.3572, 393.3507, 411.3617, 203.1787277.2171, 407.3311C30H48O3Ursolic acid *EUL, EUF, EUP
7130.83256.2402257.2474 (−2.6) 239.2364 C16H32O2Palmitic acidEUB, EUL, EUF, EUP
7232.21426.3862427.3927 (3.0) 409.3821, 191.1783, 203.1777, 149.1315 C30H50OUlmoprenolEUB, EUF, EUP
7332.76282.2559283.263 (−2.5)281.2484 (1.2)265.2521, 137.1313, 123.1159181.1241, 163.1133C18H34O2Oleic acidEUB, EUL, EUF, EUP
7436.14576.439599.4269 (−3.1)621.4368 (0.3)397.3818, 423.3231, 175.1460473.2820, 283.1105C35H60O6DaucosterolEUL, EUF, EUP
Note: * means determined by comparison with the reference sample; tR: retention time.
Table
Table 2. Antioxidant components from EUP.
Table 2. Antioxidant components from EUP.
Peak Number tR (min)CompoundMolecular FormulaMolecular Weight[M + H]+/ [M + Na]+/[M + NH4]+ (Error, ppm)[M − H]−/[M + HCOO]− (Error, ppm)MS/MS
Fragments (P)		MS/MS
Fragments (N)
120.72Geniposidic acid *C16H22O10374.1213397.1111 (0.1)373.1131 (−1.0)353.0553, 293.0344, 217.0472211.0602, 149.0606, 123.0446
248.65Quercetin 3-O-sophoroside *C27H30O17626.1483627.1556 (−0.8)625.1406 (0.2)303.0506, 465.1033, 285.0383271.0370, 301.0448, 463.0997
352.75Quercetin 3-O-sambubioside *C26H28O16596.1378597.1453 (−0.4)595.1296 (−0.5)303.0551, 465.1052, 285.0411301.0448, 271.0336, 463.1041, 445.0971
453.62Quercetin 3-O-neohesperidoside *C27H30O16610.1534611.1602 (−1.7)609.1453 (−0.4)303.0504, 465.1023, 279.1604300.0720, 301.0319, 271.0251, 445.0784
559.24Isoquercitrin *C21H20O12464.0955 463.0876 (−0.1) 300.0259, 301.0337, 271.0238, 191.9362
Note: * means determined by comparison with the reference sample; tR: retention time.
Table
Table 3. 1H (600 MHz) and 13C NMR (150 MHz) data of compounds 2–4 in DMSO-d6.
Table 3. 1H (600 MHz) and 13C NMR (150 MHz) data of compounds 2–4 in DMSO-d6.
Position Compound 2 Compound 3 Compound 4
δCδC
Ref.		δH (J in Hz)δH
Ref.		δCδC
Ref.		δH (J in Hz)δH
Ref.		δCδC
Ref.		δH (J in Hz)δH
Ref.
2156.66157.07 155.61156.70 156.52156.32
3133.44133.69 133.31133.90 133.3133.50
4177.88178.35 177.63178.30 177.71177.58
5161.7161.715-OH: 12.68 s 161.64162.30 161.68161.4212.66 s12.67 s
699.0399.796.19 d6.21 d99.32100.106.11 d6.19 d99.0798.786.18 d6.19 d
7164.43164.587-OH: 10.85 s 165.63165.10 164.49164.0210.83 s10.84 s
893.8593.276.40 d6.40 d93.9795.606.31 d6.40 d93.8893.446.38 d6.39 d
9155.99157.43 156.74157.50 156.68156.74
10104.37103.59 103.92104.90 104.43103.87
1′121.57121.63 121.5122.10 121.62121.96
2′115.81114.747.55 d (2.2)7.68 d115.67116.207.51 d (8.4)7.56 d115.53115.847.52 d7.53 d
3′145.24144.553′-OH: 9.71 s 145.41149.50 145.27144.959.72 s9.17 s
4′148.92148.394′-OH: 9.21 s 149.14151.10 148.79148.189.16 s9.73 s
5′116.5116.346.87 d (8.5)6.90 d116.33119.306.80 d (8.4)6.85 d116.42116.166.82 d6.83 d
6′122.25121.637.61 dd (8.5, 2.2)7.55 dd122.27122.607.62 d (8.4, 2.2)7.66 d122.09121.157.59 dd7.60 dd
1″98.4698.455.70 d (7.3)5.37 d98.3995.505.68 d (6.7)5.72 d98.898.485.64 d (7.7)5.65 d (7.6)
2″83.1481.493.46 82.2581.70 77.9176.76
3″76.9774.15 76.5277.50 77.7477.34
4″70.0769.533.03 70.0469.40 70.769.86
5″77.1876.50 77.2676.103.43 77.8477.15
6″61.1760.91 61.0160.503.51 61.3960.52
1‴104.60104.344.60 d (7.9)4.77 d104.92104.404.55 d (7.3)4.58 d100.92100.765.01 d5.08
2‴74.8276.493.50 74.373.80 71.0170.36
3‴76.9776.853.44 76.5276.70 71.0770.83
4‴70.0069.64 69.8469.30 72.3171.90
5‴77.9376.66 66.0265.603.65 68.6768.57
6‴61.0960.99 17.6317.410.760.77
Table
Table 4. The contents of Q-markers in thirty-four batches of EUP, as determined using the UHPLC/PDA method.
Table 4. The contents of Q-markers in thirty-four batches of EUP, as determined using the UHPLC/PDA method.
NumberInformationQSH (mg/g)QSB (mg/g)QNH (mg/g)
S1HN202110.517.423.2
S22021032311.267.763.76
S32021032811.968.063.59
S42021033113.538.843.21
S52021032411.547.893.86
S62021032513.888.943.00
S72021032711.337.94.05
S82021040413.258.773.49
S92021032812.178.163.53
S102021041013.628.843.64
S112021041113.969.062.82
S122021041311.427.853.08
S132021040910.127.293.13
S14Z2021033013.448.773.17
S15Z2021032312.318.223.01
S16Z2021032512.958.582.88
S17Z2021032614.59.263.12
S18Z2021033113.428.753.28
S19HNYY2021032812.178.163.53
S20HNYL20200412.58.573.69
S21SXLY20200412.177.72.48
S22GSLN2020049.327.492.29
S23SXHZ20200410.138.552.81
S24HNZJJ202210.437.842.86
S25SXHZ20229.138.722.64
S262019043012.677.773.80
S27201904079.1210.522.95
S28201904049.959.132.66
S29H1113.48.612.64
S30H2213.678.872.88
S31H2311.617.932.12
S32H2414.749.362.05
S33 *YHSW221207-10.000.000.00
S34 *BZXH20220.000.000.00
Note: * The sample was fake.
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Guo, F.; Yang, Y.; Duan, Y.; Li, C.; Gao, H.; Liu, H.; Cui, Q.; Guo, Z.; Liu, X.; Wang, Z. Quality Marker Discovery and Quality Evaluation of Eucommia ulmoides Pollen Using UPLC-QTOF-MS Combined with a DPPH-HPLC Antioxidant Activity Screening Method. Molecules 2023, 28, 5288. https://doi.org/10.3390/molecules28135288

AMA Style

Guo F, Yang Y, Duan Y, Li C, Gao H, Liu H, Cui Q, Guo Z, Liu X, Wang Z. Quality Marker Discovery and Quality Evaluation of Eucommia ulmoides Pollen Using UPLC-QTOF-MS Combined with a DPPH-HPLC Antioxidant Activity Screening Method. Molecules. 2023; 28(13):5288. https://doi.org/10.3390/molecules28135288

Chicago/Turabian Style

Guo, Fengqian, Yichun Yang, Yu Duan, Chun Li, Huimin Gao, Hongyu Liu, Qiping Cui, Zhongyuan Guo, Xiaoqian Liu, and Zhimin Wang. 2023. "Quality Marker Discovery and Quality Evaluation of Eucommia ulmoides Pollen Using UPLC-QTOF-MS Combined with a DPPH-HPLC Antioxidant Activity Screening Method" Molecules 28, no. 13: 5288. https://doi.org/10.3390/molecules28135288

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