A metabolomics identification method for wild Cordyceps sinensis and its substitutes

Abstract

The present invention relates to a metabolomics identification method for wild Cordyceps sinensis and its substitutes, using the metabolites of Cordyceps sinensis as characteristic markers to identify Cordyceps sinensis varieties. By using the characteristic markers of the present invention, it is not only possible to identify wild Cordyceps sinensis from different sources, but also to identify wild Cordyceps sinensis and substitutes.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to an identification technology of Cordyceps sinensis from different sources. Background Art

[0002] Cordyceps sinensis is a fungus (sperm)-larvae (residue) complex. When the spores of the ergot fungus Cordyceps sinensis infect the larvae of the bat moth family, the fungus gradually grows in the larvae, turning the larvae into zombies, standing upright with their heads up, until the fungus breaks out of the insect body and grows out of the ground before it can be picked. There are more than 750 species of Cordyceps sinensis that have been identified so far, and Cordyceps sinensis is the most prominent and widely studied species in the Cordyceps genus. It is also the only species in the genus included in the Chinese Pharmacopoeia. According to research, the record of Cordyceps sinensis began in the Qing Dynasty, and it has a history of medicinal and tonic use for hundreds of years. It is one of the most precious traditional Chinese medicines, and its main effects are to nourish the kidneys and lungs, stop bleeding and resolve phlegm. Modern research shows that Cordyceps sinensis also has multiple pharmacological effects such as anti-fatigue, anti-inflammatory, antioxidant, anti-tumor, immunomodulatory, and improvement of metabolic diseases.

[0003] Wild Cordyceps sinensis not only has a complex life cycle, but also has a harsh growth environment. In China, it is only distributed in the Qinghai-Tibet Plateau and its surrounding areas (the lowest altitude is 3000m), and its natural resources are very limited. However, the huge market demand has destroyed its resources, and its price is comparable to gold. Therefore, many researchers are committed to the study of artificial cultivation of Cordyceps sinensis, and large-scale production has been achieved. Guo Linong et al. compared the DNA barcodes of the artificially cultivated variety with those of wild Cordyceps sinensis and confirmed that the artificially cultivated variety is Cordyceps sinensis. Chen Gang et al. compared the fingerprint feature maps of the artificially cultivated variety and wild Cordyceps sinensis based on nuclear magnetic resonance technology and found that the chemical compositions of the two are highly similar. In addition, many studies have also shown that the main chemical components of the artificially cultivated variety, such as adenosine, amino acids, cordycepic acid, sterols, etc., are basically the same as those of wild Cordyceps sinensis. However, there has been no comprehensive comparison of the pharmacological activities of artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis, and whether the pharmacological effects of the two are consistent has not yet been clarified.

[0004] In order to alleviate the problem of the shortage of Cordyceps sinensis resources, there are many alternatives available on the market. Previously, the alternative cultivated varieties circulating on the market were mainly commercial preparations made from multiple strains isolated from Cordyceps sinensis through in vitro fermentation culture, such as Bailing Capsules and Jinshuibao Capsules. However, this also led to the emergence of many counterfeit products, which has a great harm to the rights and health of consumers. Not only that, because it has not been fully clarified whether the efficacy of the substitutes is consistent with that of wild Cordyceps sinensis, most consumers still prefer to buy wild Cordyceps sinensis, leading to a series of vicious cycles.

[0005] International research on Cordyceps sinensis is mostly focused on pharmacological effects, and there are few studies on characteristic markers in its quality evaluation standards. Domestic scholars have conducted continuous and in-depth research on Cordyceps sinensis, including its artificial breeding technology, formation mechanism, authentication technology, chemical composition analysis, pharmacological effects, etc., and have achieved certain research results. Some pharmacological studies at home and abroad use the whole water-soluble extract of Cordyceps sinensis to study its mechanism of action on certain tumor cells or certain disease models; another part of the study selects a certain specific chemical component contained in Cordyceps sinensis to study its mechanism of action on certain tumor cells or certain disease models. There are limited studies on the screening of characteristic markers in these studies, and the number of varieties and structural information of substances that can be detected by their detection methods are limited. From another perspective, if the characteristic marker system has not been fully established, the credibility of the Cordyceps sinensis and its active ingredients under study is questionable. Moreover, the early research on active ingredients often tends to focus on the research of a specific chemical component, ignoring the synergistic effect of the overall substance groups contained in Cordyceps sinensis, such as the metabolome, lipidome, and proteome.

[0006] This application is based on a broad targeted metabolomics detection method based on UPLC-electrospray ionization (ESI)-MS / MS, and proposes characteristic markers that can be used to identify wild Cordyceps sinensis in Naqu, wild Cordyceps sinensis in Yushu, Cordyceps sinensis cultivated in Yichang, and Cordyceps sinensis mycelium, thereby providing important information for the quality evaluation of Cordyceps sinensis and the improvement of artificial cultivation technology. In addition, it is also helpful to evaluate the nutritional and medicinal value of Cordyceps sinensis, and lay the foundation for the resource protection, effective development and rational application of Cordyceps sinensis. Summary of the invention

[0007] The object of the present invention is to provide a method for identifying the source of Cordyceps sinensis by utilizing metabolites.

[0008] The solution adopted by the present invention to solve the above technical problems is:

[0009] A method for identifying wild Cordyceps sinensis and its substitutes, using the metabolites of Cordyceps sinensis as characteristic markers to identify the varieties of Cordyceps sinensis; the characteristic markers include the following three groups:

[0010] (1) Characteristic markers for identifying wild Cordyceps sinensis, including: 3-ketosphingosine, 18-hydroxyoleate, N'-formylkynurenine, (±)-12-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid, (±)-15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid, (±)-18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid, 15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid, (±)-myristoylcarnitine, carnitine C14:0, carnitine C20:1 isomer 1;

[0011] (2) Characteristic markers used to identify artificially bred Cordyceps sinensis, including: glutarylcarnitine and α-cyclopyranoic acid;

[0012] (3) Characteristic markers for identifying Cordyceps sinensis mycelium, including: 1-O-palmitoyl-2-O-acetyl-sn-glycero-3-phosphocholine, 1-(1Z-hexadecene)-sn-glycero-3-phosphocholine, lysophosphatidic acid 18:2, (±)16-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, xanthuric acid, S-(indolemethylthiohydroximoyl)-L-cysteine, γ-glutamyl-L-putrescine, histidyl-threonyl-lysine, threonyl-tyrosyl -Lysine, glutamine-valine, alanyl-valine, 2-amino-4,6-piperidinediol, creatinine, indole-3-pyruvate, 3-methoxyphenylacetic acid, 5-methyltetrahydrofolate, nicotinate methyl ester, 4-acetylaminobutyryl-coenzyme A, acetaminophenol glucuronide, 5,7,22,24(28)-dehydroergosterol, 1,3-diphenylguanidine, N-butylaniline, isoxanthopterin, 3-indolepropionic acid, 1-methyl-hydantoin, α-[3-[(hydroxymethyl)nitrosoamino]propyl]-3-pyridinemethanol, methylpropyl disulfide, 8,8a-deoxyoleane.

[0013] Furthermore, the wild Cordyceps sinensis includes Cordyceps sinensis produced in Nagqu, Tibet and Cordyceps sinensis produced in Yushu, Qinghai.

[0014] Furthermore, the metabolites containing 3-ketosphingosine are wild Cordyceps sinensis produced in Nagqu, Tibet; the metabolites containing 18-hydroxyoleate and / or N'-formylkynurenine are wild Cordyceps sinensis produced in Yushu, Qinghai.

[0015] Furthermore, the artificially cultivated Cordyceps sinensis is an artificially bred Cordyceps sinensis produced by Yichang Shancheng Shuidu Cordyceps Sinensis Co., Ltd.

[0016] Furthermore, the Cordyceps sinensis mycelium is a commercial preparation of Bailing Capsule.

[0017] Furthermore, the characteristic markers are detected and analyzed using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS / MS).

[0018] Furthermore, the above method comprises the following steps:

[0019] (1) Sample pretreatment: grind the sample to be identified into powder, or take bacterial powder for later use, extract it with an organic solvent, and then centrifuge to obtain the supernatant;

[0020] (2) Using LC-ESI-MS / MS to perform extensive targeted metabolome detection on samples and obtain metabolite composition information;

[0021] (3) Compare the metabolite components with the characteristic markers to determine the corresponding Cordyceps sinensis variety.

[0022] Furthermore, in the broad targeted metabolome detection, the chromatographic column may be Waters ACQUITY HSS T3 C 18 Column (2.1×100 mm, 1.8 μm).

[0023] Furthermore, in the broadly targeted metabolome detection, the mobile phase A used in the chromatographic column may be an aqueous formic acid solution with a mass concentration of 0.01% to 10%; the mobile phase B may be an acetonitrile solution with a formic acid mass concentration of 0.01% to 10%.

[0024] Furthermore, in the broadly targeted metabolome detection, the chromatographic column temperature is 30-60°C, preferably 240°C, the mobile phase flow rate is 0.25-0.5 ml / min, preferably 0.35 ml / min; and the injection volume is 1-5 μL, preferably 2 μL.

[0025] The present invention discloses a detection method for the authentication and quality evaluation of characteristic markers of wild Cordyceps sinensis and its substitutes based on high performance liquid chromatography-mass spectrometry (HPLC-MS / MS), and verifies the effectiveness of the method in the improvement of quality evaluation schemes. By adopting the characteristic markers described in the present invention, it is not only possible to distinguish wild Cordyceps sinensis from different sources, but also to distinguish wild Cordyceps sinensis and substitutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1are metabolites detected in NC, YC, CC and BL, wherein A is the number of metabolites detected in NC, YC, CC and BL by ESI+ and ESI- respectively; B is the Venn diagram of metabolites detected in NC, YC, CC and BL; the CC is an artificially bred product of Cordyceps sinensis; the NC is a wild product of Cordyceps sinensis from Nagqu, Tibet; the YC is a wild product of Cordyceps sinensis from Yushu, Qinghai; the BL is a Cordyceps mycelium preparation Bailing Capsule; wherein CC and BL are substitutes for wild products. DETAILED DESCRIPTION

[0027] This example introduces the process of detecting and analyzing metabolites in four biological samples, including wild Cordyceps sinensis [NC (produced in Nagqu) and YC (produced in Yushu)], cultivated Cordyceps sinensis [CC (produced in Yichang)], and Cordyceps sinensis mycelium preparation Bailing Capsule (BL), using a broad targeted metabolomics research method based on liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS). The following description is only for those skilled in the art to better understand the present invention. It can be understood that the content of the present invention is not limited to the following examples.

[0028] 1. Experimental Instrument Description

[0029] Ultra-high performance liquid chromatography Shim-pack UFLC SHIMADZU CBM30A, purchased from Shimadzu, Japan; triple quadrupole tandem mass spectrometer Triple Quad 6500+, purchased from AB SCIEX, USA; centrifuge 5424R, purchased from Eppendorf, Germany; constant temperature metal mixer MU-G02-0448, purchased from Hangzhou Miou Instrument Co., Ltd., China; 1 / 100,000 electronic balance MS105DM, purchased from Mettler Toledo Instrument Co., Ltd., China; centrifugal concentrator CentriVap, purchased from LABCONCO, USA; vortex mixer VORTEX-5, purchased from Kyllin-Bell, China; ultrasonic cleaner KQ5200E, purchased from Kunshan Ultrasonic Instrument Co., Ltd., China; pipette Research plus, purchased from Eppendorf, Germany.

[0030] 2. Description of medicinal materials and preparations:

[0031] Wild samples of Cordyceps sinensis from Nagqu, Tibet and Yushu, Qinghai were purchased from Hubei Tianji Pharmaceutical Co., Ltd. and Qinghai Yuanzu Biotechnology Co., Ltd., respectively. The samples of cultivated Cordyceps sinensis (batch number: 200111-I01) were produced in Hubei Yichang Cordyceps sinensis Ecological Nurturing Base (Yichang Shancheng Shuidu Cordyceps sinensis Co., Ltd.). Cordyceps sinensis fermented fungus powder preparation Bailing Capsules (Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., National Medicine Standard Z10910036, Batch Number: 1911355) were purchased from a pharmacy.

[0032] 3. Experimental Methods

[0033] 3.1 Pretreatment of Cordyceps sinensis samples

[0034] The wild Naqu Cordyceps (NC), Yushu Cordyceps (YC) and Cordyceps (CC) were freeze-dried and ground into powder, and the capsule shell of Bailing Capsule (BL) was removed and the powder was taken for later use. Each sample was weighed in 3 portions, each portion was 50±2 mg, and a steel ball was added to the weighed sample, and homogenized at 30Hz for 3min. Then, 1mL of 70% methanol was added to the homogenized centrifuge tube, and after oscillation for 5min, centrifuged at 4℃ and 12000r / min for 10min. After centrifugation, 400μL of supernatant was taken into the corresponding EP tube and placed in a -20℃ refrigerator overnight. Finally, centrifuged again at 4℃ and 12000r / min for 3min, and 200μL of supernatant was taken into the liner tube of the corresponding numbered injection bottle for subsequent LC-MS analysis.

[0035] 3.2 Conditions for extensive targeted metabolome acquisition

[0036] 3.2.1 Chromatographic conditions

[0037] Column: Waters ACQUITY HSS T3 C 18 Column (2.1×100mm, 1.8μm)

[0038] Mobile phase A: aqueous solution (0.1% formic acid)

[0039] Mobile phase B: acetonitrile solution (0.1% formic acid)

[0040] Column temperature: 40°C

[0041] Flow rate: 0.35ml / min;

[0042] Injection volume: 2 μL

[0043] The mobile phase gradient is shown in Table 1 below:

[0044] Table 1 Gradient elution program

[0045]

[0046] 3.2.2 Mass spectrometry conditions

[0047] Data acquisition was performed by UPLC coupled to a triple quadrupole tandem mass spectrometer (Triple Quad 6500+) mass spectrometer, and the acquisition conditions are shown in Table 2 below:

[0048] Table 2 Mass spectrometry conditions in positive and negative ion modes

[0049]

[0050] Each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0051] 4 Analytical methods

[0052] Based on the self-built MWDB database (metabolite database, Wuhan Metwell Biotechnology Co., Ltd.) and / or the public databases Metlin, HMDB and KEGG directly available to the public, the obtained offline data, including retention time, parent-daughter ion pair information and MS / MS spectra, were matched with the information in the database to identify the components. The relative quantification of each component was performed by integrating and correcting the chromatographic peaks in the offline data file using MultiQuant 3.0.3 software, and the peak area of ​​each chromatographic peak represented the relative content of the corresponding component.

[0053] 5 Experimental results

[0054] In the mixed QC sample consisting of NC, YC, CC and BL extracts, a total of 901 metabolites were detected. Table 3 shows that the chemical composition of Cordyceps samples includes vitamins and coenzymes, lipids, amino acids, nucleotides, carbohydrates, organic acids, and their derivatives, etc. It is basically consistent with the previously reported categories of components in Cordyceps sinensis, such as nucleosides, polysaccharides, fatty acids, proteins, amino acids and peptides.

[0055] Table 3 Metabolites detected in QC samples mixed from NC, YC, CC, and BL extracts

[0056]

[0057] In ESI+ mode, 453, 466, 453, and 452 metabolites were detected in NC, YC, CC, and BL, respectively; in ESI- mode, 389, 385, 379, and 382 metabolites were detected in NC, YC, CC, and BL, respectively ( Figure 1 A). Figure 1The Venn diagram in B shows that among the 901 metabolites detected, a total of 753 metabolites were detected in the four Cordyceps samples. In addition, there were 1, 2, 2 and 29 unique metabolites in NC, YC, CC and BL, respectively, as shown in Table 4 below.

[0058] Table 4 Unique metabolites in NC, YC, CC, and BL.

[0059]

[0060]

[0061] 6. Result Verification

[0062] In order to verify the accuracy of the selected characteristic markers, metabolite analysis of single samples was performed on four raw materials, NC, YC, CC, and BL, and the detection of characteristic markers was compared. The results are shown in Table 5. It can be seen that the characteristic markers selected in this application all show good specificity.

[0063] Table 5 Detection of characteristic markers

[0064]

[0065]

[0066] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A metabolomics identification method for wild Cordyceps sinensis and its substitutes, characterized in that: Using the metabolites of Cordyceps sinensis as characteristic markers to identify the varieties of Cordyceps sinensis; comprising the following steps: (1) Sample pretreatment: grind the sample to be identified into powder, or take bacterial powder for later use, extract it with an organic solvent, and then centrifuge to obtain the supernatant; (2) Use LC-ESI-MS / MS to perform extensive targeted metabolome detection on samples to obtain metabolite composition information; (3) Compare the metabolite components with the characteristic markers to determine the corresponding Cordyceps sinensis variety; The characteristic markers include the following three groups: (A) Characteristic markers for identifying wild Cordyceps sinensis, including: 3-ketosphingosine, 18-hydroxyoleate, N'-formylkynurenine, (±)-12-hydroxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid, (±)-15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid, (±)-18-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid, 15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid, (±)-myristoylcarnitine, carnitine C14:0, carnitine C20:1 isomer 1; (B) Characteristic markers for identifying artificially bred Cordyceps sinensis, including: glutarylcarnitine and α-cyclopyranoic acid; the artificially bred Cordyceps sinensis is produced by Yichang Shancheng Shuidu Cordyceps Sinensis Co., Ltd.; (C) Characteristic markers used to identify Cordyceps sinensis mycelium include: 1-O-palmitoyl-2-O-acetyl-sn-glycero-3-phosphocholine, 1-(1Z-hexadecene)-sn-glycero-3-phosphocholine, lysophosphatidic acid 18:2, (±)16-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, xanthuric acid, S-(indolemethylthiohydroximoyl)-L-cysteine, γ-glutamyl-L-putrescine, histidyl-threonine Aminoacyl-lysine, threonyl-tyrosyl-lysine, glutamine-valine, alanyl-valine, 2-amino-4,6-piperidinediol, creatinine, indole-3-pyruvic acid, 3-methoxyphenylacetic acid, 5-methyltetrahydrofolate, nicotinate methyl ester, 4-acetylaminobutyryl-coenzyme A, acetaminophenol glucuronide, 5,7,22,24(28)-dehydroergosterol, 1,3-diphenylguanidine, N-butylaniline, isoxanthopterin, 3-indolepropionic acid, 1-methyl-hydantoin, α-[3-[(hydroxymethyl)nitrosoamino]propyl]-3-pyridinemethanol, methylpropyl disulfide, 8,8a-deoxyoleane; the cordyceps mycelium is a commercial preparation of Bailing capsules.

2. The method according to claim 1, characterized in that The wild Cordyceps sinensis includes Cordyceps sinensis produced in Nagqu, Tibet and Cordyceps sinensis produced in Yushu, Qinghai.

3. The method according to claim 2, characterized in that The metabolites containing 3-ketosphingosine are wild Cordyceps sinensis produced in Nagqu, Tibet; the metabolites containing 18-hydroxyoleate and N'-formylkynurenine are wild Cordyceps sinensis produced in Yushu, Qinghai.

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