Identification and Analysis of Secondary Metabolites from CH3OH/H2O Extract of Metacordyceps Neogunnii

Secondary metabolites are considered to be the major compounds in Cordyceps with anti-tumor, anti-aging and immunity-enhancing effects. The molecular structures of secondary metabolites form the basis for the development and utilization of Cordyceps. Metacordyceps neogunnii is an important Cordyceps resource, but less study has been made on the molecular structure of its secondary metabolites. In this study, gas chromatography-mass spectrometry (GC/MS) and ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) were used to analyze and identify the secondary metabolites from CH3OH/H2O extract of Metacordyceps neogunnii. The results show that a total of 22 compounds were identified by GC-MS, including 2 n-alkanes, 1 isoparaffin, 1 cycloalkane, 6 olefins and 13 esters. The predominant compounds were (E)-ocimene, (E)-β-ocimene, methyl oleate, dioctyl adipate, methyl palmitate and methyl linoleate, obtained by means of GC/MS. Five distince classes of secondary metabolites were speculated: 3 polypeptides, 2 esters, 1 isoflavone, 1 isoindrone and 3 amides, from which 10 compounds were detected using UPLC-Q-TOF/MS. (R)-N-((1-(((9H-fluorene-9-yl) methoxy) carbonyl) pyrrolidine-2-yl) methyl)-N-(2-(6-benzoylamino-9H-purin-9-yl) acetyl) glycine, benzyl (5-(2-((3-(2, 3-dihydrobenzo[b] [1, 4] dioxin-6-yl)-4-carbonyl-4H-chromene-7-yl) oxo) acetylamino) amyl) carbamate, 5, 5’-((propane-2, 2-diyl (4, 1-phenylene)) bis (oxo)) bis (2-(naphthalene-1-yl) isoindoline-1, 3-dione), 1-dodecylazepine-2-one and other compounds were the first detected in Metacordyceps neogunnii.


Introduction
Metacordyceps neogunnii (M. neogunnii ) T.C. Wen & K.D. Hyde, namely, Cordyceps gunnii (Berk.) Berk. early reported in China which is widely present in Guizhou, Hunan and Anhui provinces [1], has various effects such as analgesia, sedation, improvement of human immunity, anti-tumor, anti-aging, promoting sleep and enhancing memory [2][3][4][5]. It has chemical composition and medicinal value similar to those of Cordyceps sinensis. Furthermore, it is characterized with such merits as wide ecological amplitude and short cultivation period [6], thus more suitable for large-scale cultivation through modern submerged fermentation. Secondary metabolites of Cordyceps sinensis are considered to be major compounds with various effects, from which it is highly likely to find new bioactive substances or lead compounds for drugs [7]. However, at present, the research on M. neogunnii mainly focuses on strain identification, genomics analysis and preliminary pharmacological effects, with little on secondary metabolites [8].
Gas chromatography-mass spectrometry (GC/MS) can be used for the qualitative analysis of organic compounds, which is a powerful means to obtain molecular structure information, but it is only suitable for volatile organic compounds with low molecular weight (< 500 Da) [9]. This technology has already been applied in the analysis of volatile components of Cordyceps sinensis [10]. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), due to its extremely high sensitivity, can be used to distinguish compounds with very close molecular weight differences or even compounds with the same molecular weight but different element composition. It can be used to have a qualitative analysis of trace substances in samples with complex components, capable of accurately and rapidly obtaining molecular structure information of an organic compounds, which has been widely applied in environmental science, medicine, drug research and development and other fields [11,12]. The study of active metabolites is an inevitable link for the efficient development and utilization of M. neogunnii resources. In order to further explore the secondary metabolites of M. neogunnii and enrich the natural product library of cordyceps fungi, based on the principle of the dissolution in the similar material structure, the enrichment and separation of group components of secondary metabolites in M. neogunnii were carried out. The molecular structure of secondary metabolites was analyzed by GC/MS and UPLC-Q-TOF/MS, and the function characteristics of these compounds were briefly discussed, which provided data support and scientific basis for the further development and utilization of these compounds.

Sample preparation
M. neogunnii was pulverized to pass through a 100mesh sieve followed by desiccation in a vacuum at 60 °C for 24 h before use. Subsequently, Cordyceps power (2 g) and 60 % methanol (30 mL) were added to a 100 mL triangular flask and exhaustively extracted under ultrasonic radiation. Extract of 60 % methanol was concentrated by distillation to obtain Extract I. Extract I was extracted with equal amount of petroleum ether using a separatory funnel for 3 times. And 3 extractions of petroleum ether were merged, concentrated and purified through a 0.45μm microporous membrane to obtain the extract II (MNE PE ). In addition, the raffinate of petroleum ether (MNR PE ) was purified by centrifugation before analysis. MNE PE was analyzed with GC/MS and MNR PE analyzed by UPLC-Q-TOF/MS.

Sample analysis
The processed MNE PE was analyzed by GC/MS. The injection volume was 0.5μL, the injector temperature was set at 290 °C, the split ratio was 5:1, and the chromatographic column was HP-5MS elasticity quartz capillary column (30 m×250μm×0.25μm). The carrier gas was high purity nitrogen (99.999 %) at a flow rate of 1.0 mL/min, the oven was programmed to start at 60 °C, increased to 160 o C with 5 °C /min, hold for 1 min, then increased from 160 °C to 190 °C with 5 °C /min, hold for 1 min, increased from 190 °C to 280 °C with 3 °C /min, hold for 9 min. The solvent delay was 3.00min, the ion source temperature was set at 230 °C with the electron energy at 70 eV and the mass range at m/z 50-500, Electron Impact mode. The MSD ChemStation software was used to process the data, and the mass spectra obtained were identified by comparing with the NIST08 standard spectra, the compounds were quantitatively analyzed by normalization method.
The processed MNE PE was analyzed by UPLC-Q-TOF/MS. (ACQUITY HSS T3 column (2.1 mm×100 mm, 1.8μm) was used with mobile phase consisted of 0.1 % formic acid water-0.1 % acetonitrile at the flow rate of 0.3mL/min, the column temperature was set at 30 °C and the injection volume was 1μL. Gradient elution conditions: 0~5 min, 95 % A; 5~50 min, 95 %A; 50~55 min, 5 %A; 55~57 min, 5 %A; 57~60 min, 95 %A. Electrospray ionization source, detected in positive ion mode. The ion source temperature was set at 120 °C; the desolvation gas temperature at 80 °C with a desolvation gas flow of 900L/h; the collision energy was 3 eV; the capillary voltage was set at 3.1 kV, the cone voltage was up to 35 V; the cone gas flow was set to 50L/h; the scan range of primary mass spectrometry parent ion was from 50 to 1200 m/z; the scan range of second-order mass spectrometry fragment ion was from 50 to 1000 m/z). Compounds were identified using Massfragment in Masslynx software and its molecular weight and molecular structure formula were summarized according to relevant literature. Massfragment can automatically identify ion fragments of compounds by using a series of unique chemical intelligence algorithms.

Analysis of MNEPE
The compounds of MNE PE were analyzed and identified by GC/MS. 22 compounds were identified, including 9 hydrocarbons and 13 ester compounds, with the relative contents of 7.24 % and 92.80 %, respectively. Total ion chromatography of MNE PE and the main compounds were summarized in Table 1.

Hydrocarbon compounds in MNEPE
Hydrocarbon compounds in MNE PE include 2 normal alkanes, 1 isoparaffin and 6 olefins, with relative content distribution of 0.23 %, 0.24 % and 6.77 %, respectively (As seen in Table 1). An alkane is an organic compound, existing in higher eukaryotes such as mammals, insects and birds, and has effects of antidesiccation, nerve protection, signal transmission and regulation [13]. N-alkanes belong to the biomarker compound of fungi [14]. 2,6,10,14-Tetramethylhexadecane (Peak 8), also known as phytane, is one kind of regular isoprenoid alkanes and has the characteristics of a biomarker compound as well. The detection of these compounds is of great significance for the study of the origin and genetic properties of entomogenous fungi. RT retention time, MF molecular formula, SI similarity with NIST08 standard spectra, RC relative content by peak area The major olefins in MNE PE were terpenes with a relative content of 6.62 %, mainly including 4 compounds (Table 1), namely, D-limonene (Peak 1), Ocimene (Peak 2 & 3) and Allo-Ocimene (Peak 4). Dlimonene is a natural monoterpene, widely existing in citrus, vegetables and spices, and has anti-inflammatory, antibacterial, anti-tumor and other effects [15]. Its anticancer mechanism mainly lies in chemical prevention, inhibition of cell cycle, induction of cell apoptosis, and inhibition of tumor metastasis and invasion [16]. Its role in cancer prevention has attracted extensive attention; so far it has been widely used in many industries and fields such as food, pesticide, chemical industry and medicin e [17]. Ocimene is an acyclic monoterpene with an aromatic odor, which can be used in the production of flavors and fragrances. It has three types, containing α, cis-β and trans-β, and the isomer is allo-ocimene. Ocimene is also an important communication signal molecule, but the current research on ocimene and β-ocimene is not sufficient enough, and the mechanisms of its medicinal functions remain to be further explored [18].

Esters in MNEPE
MNE PE contained 13 esters with a relative content of 92.80 %, consisting of phthalates, the fatty acid methyl ester and other esters. Phthalates, detected as ioctyl hexanedioate (Peak 9, 0.90 %), diisobutyl phthalate (Peak 11, 54.47 %) and dibutyl phthalate (DBP, Peak 22, 1.74 %), are mainly plasticizers. DBP is widely used in plastic products, cosmetics, lubricants and other fields. Strain fermentation culture was carried out with DBP by some researchers by using cordyceps militaris and paecilomyces verticillatus as raw materials. The results showed that 6 % DBP was capable of increasing the biomass of bacterial strains. The total production of cordycepin was significantly higher than that of the control group without organic phase [19]. It is important to increase the content of cordycepin for using mycelium as medicine and functional food. Therefore, DBP should be further developed as the microbial production of secondary metabolites.
There are 6 kinds of the fatty acid methyl ester ( Table  1)  , and a small quantity of saturated fatty acids, such as palmitic acid, stearic acid and arachidic acid, which are consistent with the research results of our previous work [20]. Linoleic acid is ω-6 series polyunsaturated fatty acid, which has the effects of reducing cholesterol and inhibiting tumor cells, and can be converted into arachidonic acid to generate prostaglandins through an unsaturated action [21]. Oleic acid is ω-9 series monounsaturated fatty acid, which has the effects of regulating the concentration ratio of high and low density lipoprotein cholesterol in serum, preventing cardiovascular diseases, and reducing cholecystitis and cholelithiasis [22]. Although being a saturated fatty acid, stearic acid is rapidly converted into oleic acid in vivo without affecting the concentration of cholesterol.

Analysis of MNRPE
The compounds of MNR PE were analyzed and identified with UPLC-Q-TOF/MS. MassLynx 4.1 was used to analyze the total ion current in the positive ion mode. 10 compounds and their fragmentation pathway were analyzed and deduced combined with ion fragmentation, retention time, accurate mass-to-charge ratio, online database Chemspider and references.    According to its fragmentation rule, it is preliminarily speculated that the compound may be L-tyrosyl-L-valyl-L-alanyl-L-glutamic acid, the molecular formula was C 22 H 32 N 4 O 8 and the molecular weight was 480.2220. Its possible fragmentation pathway in Figure 4.  According to its fragmentation rule, it was preliminarily speculated that the compound may be Benzyl (5-(2-((3-(2,3-dihydrobenzo[b] [1,4] dioxin-6-yl)-4-oxo-4Hchromene-7-yl) oxy) acetamido) pentyl) carbamate, with molecular formula being C 32 H 32 N 2 O 8 and molecular weight being 572.2159. Its possible fragmentation pathway in Figure 6.     Figure 9.
The polypeptide, with a small molecular weight, can control the growth, development, immune regulation and metabolism of the human body. It is characterized with the sensitiveness to tumors, little drug resistance and low cost [23]. Although the biotechnological technology of synthetic peptides is relatively mature, the peptides extracted from Cordyceps are used as raw materials to synthesize isomerized peptides, which belong to a novel anti-tumor polypeptide [24,25]. Zhu et al [26] isolated and purified the peptides with high acidic amino acid residues from the mycelium of Paecilomyces gunnii, which had a strong analgesic effect but without morphine dependence. Given the strong analgesic effect and nonaddiction of secondary metabolites of polypeptides in M. neogunnii, to develop new analgesic and detoxification drugs will be an important direction for the development and utilization of M. neogunnii in the future.
Isoflavone compound, with C 6 -C 3 -C 6 as the mother stone, is one of the effective components of many traditional Chinese medicines and has been included into The Chinese Pharmacopoeia [27]. Isoflavone has a variety of activities such as cardiovascular protection [28], anti-tumor [29], osteoporosis prevention [30], nerve protection, neurodegeneration resistance [31], blood sugar reduction [32], and immune system regulation [33]. Its anti-cancer properties are prominent, and it can inhibit the growth and spread of cancer cells, effective to cancer cells only. Wang et al [34] confirmed that isoflavone compounds had an inhibitory effect on the proliferation of prostate cancer cell PC-3.
Isoindrones are benzo heterocyclic compounds [35] with excellent pharmaceutical activities, like analgesic, anti-inflammatory, antibacterial, anti-tumor, antidiabetes, anti-cardiovascular diseases and other activities [36,37]. The parent ring structure of isoindrone is contained in Falipamil, Lenalidomide, Indoprofen and other drugs, which plays an important role in the development and utilization of new drugs.
Amide compounds are widely found in nature. Many alkaloids, such as colchicine, febrifugine, ergotine and other molecular structures, contain amido bonds (-CON H-) [37]. Amide bond (or peptide bond) is the basic structure of protein. Hexa-lactam skeleton widely exists in natural products and small-molecule active compounds, which has an important biological significance as well as research value [38,39]. For example, compound UK224671 containing the skeleton has neurohormone 2 (NK2) receptor antagonism [37], and AM-8553 has a better effect for MDM2-p53 inhibition [38].

Conclusions
M. neogunnii, a new and importance Cordyceps resource to be developed, underwent the compound enrichment and separation with organic solvents. Secondary metabolites of M. neogunnii were separated and analyzed combined with GC/MS and UPLC-Q-TOF/MS, and related biological activities were discussed. In this paper, 22 compounds, mainly hydrocarbons and esters, were identified in MNE PE . 10 nitrogen-containing compounds, including peptides, amides, isoflavones, isoindrones, esters and other compounds, were analyzed from MNR PE .
The carbon number of each compound was greater than 16, and its molecular weight ranged from 200 to 780. Such findings have not been reported in other literatures so far. The results revealed that the secondary metabolites of M. neogunnii were diverse, reflecting that M. neogunnii contained abundant secondary metabolites with important pharmacological effects. It is necessary to further explore M. neogunnii by means of multi-layer separation analysis, which may provide the scientific basis for its rational development and utilization, as well as the reference for other Cordyceps fungi.