Identification of Secondary Metabolites in Flammulina velutipes by UPLC-Q-Exactive-Orbitrap MS

Abstract

Flammulina velutipes is the fourth largest edible fungus in China with high nutritional value. In this paper, ultrahigh-performance liquid chromatography tandem hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap MS) was used to identify the secondary metabolites of F. velutipes. The metabolites were identified by comparing the retention time, accurate molecular weight, and MS² data with standard databases of mzVault and mzCloud (compound: 17,000⁺) and BGI high-resolution accurate mass plant metabolome database (plant metabolite: 2500⁺). Finally, 26 secondary metabolites were preliminarily identified, including flavonoids, phenylpropanoids, organic acids, and steroids.

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1. Introduction

Flammulina velutipes is also known as golden needle mushroom and winter mushroom with high nutritional value and medicinal value. According to “Analysis of the National Statistical Survey Results of Edible Fungi in 2019,” F. velutipes is the fourth largest edible fungus in China with an output of 2.589,600 tons in 2019. F. velutipes contains a variety of nutrients, including proteins, carbohydrates, mineral elements, vitamins, and crude fibers [1]. F. velutipes contains eighteen amino acids, including eight essential amino acids, of which lysine content is 1.09%. It has been proved that lysine and its derivatives can promote children’s growth and development and enhance memory. Therefore, F. velutipes is also known as “Zengzhi mushroom” [2, 3]. It can not only be used as functional food but also has great potential in the development of medical and health products [4]. F. velutipes contains many active components, including polysaccharides, proteins, terpenoids, phenolic acids, and flavonoids [4–10]. Ishikawa et al. isolated and identified sesquiterpenoids enokipodins A-D with the cyathane skeleton from F. velutipes [7, 8]. Five flavonoids were isolated and identified from F. velutipes by Hu et al. [10], named epicatechin, phillyrin, apigenin, kaempferol, and formononetin. F. velutipes has many pharmacological effects, such as antitumor [4], regulating immunity [4, 11], improving memory [5], antibacterial [8], antioxidation [12, 13], protecting the kidney [12], protecting the liver [14], neuroprotection [15], regulating intestinal flora [16], and improving constipation [17].

Ultrahigh-performance liquid chromatography tandem hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap MS) is a new type of liquid chromatography-mass spectrometry developed in recent years; it is also one of the techniques commonly used in metabolomics with the characteristics of high resolution, good quality and precision, and strong qualitative and quantitative abilities. It is used for the qualitative analysis of Chinese medicinal materials and can realize the rapid identification of various components [18]. At present, there are few systematic studies on the secondary metabolites of F. velutipes. Therefore, in this paper, the secondary metabolites of F. velutipes were investigated to provide a reference for research on the chemical composition of F. velutipes.

2. Materials and Methods

2.1. Materials

Fruiting bodies of F. velutipes were obtained from Henan Longfeng Industrial Co., Ltd. The specimens (no. 2020-09-09) were saved at the National Research and Development Center of Edible Fungi Processing Technology, Henan University.

2.2. Reagent

d₃-Leucine, ¹³C₉-phenylalanine, d₅-tryptophan, and ¹³C₃-progesterone were used as the internal standard. Both methanol (A454-4) and acetonitrile (A996-4) were of mass spectral grade, which were purchased from Thermo Fisher Scientific, USA. Ammonium formate (17843-250G) was obtained from Honeywell Fluka, USA. Formic acid (50144-50 mL) was obtained from DIMKA, USA.

2.3. Preparation of the Sample

Dried fruiting bodies of F. velutipes were crushed by using the grinding machine. 200 g of F. velutipes powder was immersed with 50% ethanol (2000 mL) for 2 times at room temperature, each time for 3 days. The filtrate was lyophilized to obtain 87.2 g extract. The yield was 43.6%. 50 mg extract of F. velutipes was weighed, and then the sample was managed according to Yang et al. [19].

2.4. Chromatographic Conditions

The type of column was C18 Hypersil GOLD aQ (100 mm $\ast$ 2.1 mm, $1.9\mu \text{m}$). The mobile phases were 0.1% formic acid-water (liquid A) and 0.1% formic acid-acetonitrile (liquid B) with the elution gradient of 0–2 min 5% B; 2–22 min 5%–95% B; 22–27 min 95% B; 27.1–30 min 5% B. 0.3 mL/min, ${40}^{°}\text{C}$, and $5\mu \text{L}$ were used as the flow rate, column temperature, and injection volume, respectively.

2.5. Mass Spectrometry Conditions

Ultraperformance liquid chromatography (Waters 2D UPLC, USA) tandem Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, USA) was used to separate and detect the metabolites. The mass spectrometry parameters were set according to Yang et al. [19]. In brief, 150–1500 and 70,000 were used as the mass range and MS resolution, respectively. 35,000 was used as MS² resolution. The fragmentation energy was 20, 40, and 60 eV. Sheath gas flow rate and aux gas flow rate were 40 and 10, respectively. Spray voltage ($KV$) of the positive ion mode and negative ion mode was 3.80 and 3.20, respectively. Ion capillary temperature and aux gas heater temperature were ${320}^{°}\text{C}$ and ${350}^{°}\text{C}$, respectively.

2.6. Data Analysis

BGI high-resolution accurate mass plant metabolome database (plant metabolite: 2500⁺), mzCloud database (compound: 17000⁺), and mzVault database were used to identify the metabolites.

3. Results

3.1. Total Ion Chromatogram

The total ion current chromatogram of F. velutipes is shown in Figure 1.

[figure omitted; refer to PDF]

Compound 2 was deprotonated in the negative ion mode to produce ion m/z 285 [M − H]⁻ and then underwent RDA cracking to obtain two fragment ions m/z 133 [M − H-C₇H₄O₄]⁻ and m/z 151 [M − H-C₈H₆O₂]⁻. It was speculated that compound 2 may be luteolin. Possible MS fragmentation pathway of luteolin is shown in Figure 3. The cracking process of compound 4 is similar to that of compound 2 with fragment ions of m/z 269 [M − H]⁻, m/z 117 [M − H-C₇H₄O₄]⁻, and m/z 151 [M − H-C₈H₆O]⁻. It was speculated that compound 4 may be apigenin.

[figure omitted; refer to PDF]

Compound 5 was protonated in the positive ion mode to produce the ion m/z 301 [M + H]⁺, then lost methyl to obtain m/z 286, and further lost the CO group to obtain m/z 258 [M + H–CH₃–CO]⁺. It was speculated that compound 5 may be diosmetin.

Both compounds 6 and 7 contain a methoxy group, the quasi-molecular ion was m/z 299 [M − H]⁻ and m/z 283 [M − H]⁻, respectively, and then they lost the methyl unit to produce the ion [M − H-CH₃]⁻ of m/z 284 and m/z 268, respectively. It was speculated that compounds 6 and 7 were hispidulin and acacetin, respectively.

3.2.2. Structural Analysis of Phenylpropanoids

Compound 8 was protonated in the positive ion mode to produce ion m/z 193 [M + H]⁺ and then produced two fragment ions m/z 165 and m/z 137; they may be [M + H–CO]⁺ and [M + H–2CO]⁺; the cracking process of compound 8 is consistent with that of coumarins [22]. It was speculated that compound 8 may be 5,7-dihydroxy-4-methylcoumarin.

Compounds 9 and 10 had the same quasi-molecular ion m/z 515 [M − H]⁻, and both had characteristic fragment ions m/z 191 [quininic acid-H]⁻ and m/z 173 [quininic acid-H-H₂O]⁻. It was speculated that they were chlorogenic acids. The replacement position of caffeic acid can be determined according to the strength of the fragment ions [18]. Combined with the retention time, it was speculated that compounds 9 and 10 may be isochlorogenic acid B and isochlorogenic acid C, respectively. MS² spectrum of compounds 9 and 10 is shown in Figures 4 and 5, respectively.

[figure omitted; refer to PDF][figure omitted; refer to PDF]

3.2.3. Structural Analysis of Steroids

Compound 11 was protonated in the positive ion mode to obtain the ion m/z 387 [M + H]⁺ and then continuously lost the H₂O group to obtain fragment ions m/z 369 [M + H–H₂O]⁺ and 351 [M + H–2H₂O]⁺ [23]; combined with the retention time and accurate molecular weight, it was speculated that compound 11 may be bufalin.

3.2.4. Structural Analysis of Organic Acids

Organic acids generally respond in the negative ion mode to produce ion [M − H]⁻. The organic acids in F. velutipes were mostly fatty acids. They were prone to break apart and lose groups such as (CH₂)_n and COOH [24]. In this paper, organic acids in F. velutipes mainly produce fragments that lose H₂O and CO₂. The structural analysis of some organic acid compounds is as follows.

Compound 12 responded in the negative ion mode to produce ion m/z 133 [M − H]⁻, then lost the group H₂O to produce ion m/z 115 [M − H-H₂O]⁻, and further lost the group CO₂ to produce ion m/z 71[M − H-H₂O-CO₂]⁻. Combined with the retention time, accurate molecular weight, and the data of [25], it was speculated that compound 12 may be DL-malic acid. The structural analysis of other organic acids is similar to that of compound 12.

4. Discussion and Conclusion

Most of the compounds in F. velutipes have good biological activities. Hu et al. [15] investigated neuroprotective effects of six compounds from F. velutipes on H₂O₂-induced oxidative damage in PC12 cells, including arbutin, epicatechin, phillyrin, apigenin, kaempferol, and formononetin, and the results revealed that all components except apigenin mediate the apoptosis of PC12 cells via the endogenous pathway. In this paper, 7 flavonoids were identified by UPLC-Q-Exactive-Orbitrap MS, including linarin, luteolin, glycitin, apigenin, diosmetin, hispidulin, and acacetin. These flavonoids have many pharmacological effects such as antitumor, anti-inflammatory, and antioxidation. Luteolin has been showing numerous therapeutic activities such as anticancer, anti‐inflammatory, antioxidation, and antimicrobial [26]. Apigenin has the cytostatic and cytotoxic effects on various cancer cells, prevents atherogenesis, hypertension, cardiac hypertrophy, ischemia/reperfusion-induced heart injury, and autoimmune myocarditis, protects the chemical- and ischemia/reperfusion-induced liver injury, inhibits asthma, bleomycin-induced pulmonary fibrosis, abnormal behavior, and oxygen and glucose deprivation/reperfusion-induced neural cell apoptosis, and improves pancreatitis, type 2 diabetes and its complications, osteoporosis, and collagen-induced arthritis [27]. Acacetin has neuroprotective, cardioprotective, anticancer, anti-inflammatory, antidiabetic, and antimicrobial activities [28]. Hispidulin has diverse pharmacological effects such as anticancer, anti-inflammatory, antifungal, antiplatelet, anticonvulsant, and antiosteoporotic [29]. Linarin could suppress glioma through inhibition of NF-κB/p65 and upregulating p53 expression in vitro and in vivo [30]. Glycitin has effects of alleviating lipopolysaccharide-induced acute lung injury via inhibiting NF-κB and MAPK pathway activation in mice [31]. Diosmetin has anti-inflammatory effects on IL-4- and LPS-induced macrophage activation and the atopic dermatitis model [32]. Therefore, it is beneficial to develop flavonoids in F. velutipes.

One steroid (bufalin) was identified in F. velutipes in this paper. Bufalin is one of the main pharmacological and toxicological components of Venenum Bufonis and many traditional Chinese medicine preparations [33]. Currently, there is no report of bufalin in F. velutipes. Whether F. velutipes contains bufalin needs more research to determine.

Chen et al. [25] investigated chemical compositions in the stipe and pileus of F. filiformis by UPLC-Q/TOF-MS, 130 compounds were identified, including 33 amino acids and derivatives, 34 nucleotides and derivatives, 37 organic acids and lipids, 9 carbohydrate alcohols, 8 alkaloids, and 9 other compounds, and most of them were primary metabolites. Han et al. [34] investigated chemical compositions of F. velutipes, 11 compounds were isolated and identified, including arabinitol, ergosterol, cis-9-tricosene, uracil, nicotinamide, xanthine, glycerol, adenosine, trehalose, mannitol, and tyrosine, and most of them were primary metabolites. In this paper, 26 secondary metabolites were preliminarily identified by UPLC-Q-Exactive-Orbitrap MS in F. velutipes from Henan province, including 3 phenylpropanoids, 7 flavonoids, 1 steroid, and 15 organic acids. It provides a reference for the future separation of chemical components of F. velutipes.

Acknowledgments

This work was supported by Major Public Welfare Projects in Henan Province (201300110200), Research on Precision Nutrition and Health Food, Department of Science and Technology of Henan Province (CXJD2021006), and the Key Project in Science and Technology Agency of Henan Province (212102110019 and 202102110283).

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