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

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 MS 2 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.

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. erefore, in this paper, the secondary metabolites of F. velutipes were investigated to provide a reference for research on the chemical composition of F. velutipes.

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

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. e filtrate was lyophilized to obtain 87.2 g extract. e yield was 43.6%. 50 mg extract of F. velutipes was weighed, and then the sample was managed according to Yang et al. [19].

Mass Spectrometry Conditions.
Ultraperformance liquid chromatography (Waters 2D UPLC, USA) tandem Q-Exactive high-resolution mass spectrometer ( ermo Fisher Scientific, USA) was used to separate and detect the metabolites. e 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 MS 2 resolution. e 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°C and 350°C, respectively.

Total Ion Chromatogram.
e total ion current chromatogram of F. velutipes is shown in Figure 1.

Results of Metabolites' Identification.
e metabolites of F. velutipes were analyzed by UPLC-Q-Exactive-Orbitrap MS, the structural identification of compounds in F. velutipes was based on the retention time, MS data, and MS 2 data compared with the BGI high-resolution accurate mass plant metabolome database (plant metabolite: 2500 + ), mzCloud database (compound: 17000 + ), and mzVault database. e identified metabolites were classified into three grades (level 1, level 2, and level 3) according to the comparison results. e credibility sequence is as follows: level 1 > level 2 > level 3. e detailed results are shown in Table 1. 26 compounds were preliminarily identified in F. velutipes, including 3 phenylpropanoids, 7 flavonoids, 1 steroid, and 15 organic acids. e 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. MS 2 spectrum of compounds 9 and 10 is shown in Figures 4 and 5 [23]; combined with the retention time and accurate molecular weight, it was speculated that compound 11 may be bufalin.

Structural Analysis of Organic Acids.
Organic acids generally respond in the negative ion mode to produce ion [M − H] − . e organic acids in F. velutipes were mostly fatty acids. ey were prone to break apart and lose groups such as (CH 2 ) n and COOH [24]. In this paper, organic acids in F. velutipes mainly produce fragments that lose H 2 O and CO 2 . e structural analysis of some organic acid compounds is as follows. Combined with the retention time, accurate molecular weight, and the data of [25], it was speculated that compound 12 may be DL-malic acid. e structural analysis of other organic acids is similar to that of compound 12.

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 H 2 O 2 -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. ese flavonoids have many pharmacological effects such as antitumor, antiinflammatory, and antioxidation. Luteolin has been showing numerous therapeutic activities such as anticancer, antiinflammatory, 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,    Journal of Food Quality 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 collageninduced 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]. erefore, 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.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors declare that there are no conflicts of interest.  Journal of Food Quality 7