Construction method and application of fingerprint spectrum based on ganoderma lucidum antioxidant active components

Abstract

The invention relates to the technical field of traditional Chinese medicine ingredient research, in particular to a construction method and application of a fingerprint spectrum based on ganoderma lucidum antioxidant active ingredients. Firstly, a method for ultrasonic-assisted acidolysis of the ganoderma lucidum crude polysaccharide under the guidance of free radical scavenging activity is constructed, the method is efficient and good in operability, and the free radical scavenging activity of a ganoderma lucidum crude polysaccharide depolymer can be well reflected. The method comprises the following steps: carrying out acidolysis and analysis on 52 batches of ganoderma lucidum polysaccharides from different sources by adopting hydrophilic chromatography-electrospray detector-electrospray ionization mass spectrometry, further combining grey correlation analysis and partial least squares regression analysis, screening out 12 active common peaks, and establishing the fingerprint spectrum based on the ganoderma lucidum antioxidant active components. And finally, respectively analyzing the fingerprints of the standard ganoderma lucidum, ganoderma sinense, ganoderma applanatum, ganoderma atrum and the ganoderma lucidum sample based on the ganoderma lucidum antioxidant active components by utilizing a principal component analysis method and a partial least square discriminant analysis method, so as to realize identification of the ganoderma lucidum variety.

Description

Technical Field

[0001] The present invention relates to the technical field of research on traditional Chinese medicine components, and in particular to a method for constructing a fingerprint spectrum based on anti-oxidative active components of ganoderma lucidum and its application. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ganoderma lucidum, known as Ruicao in ancient times, was first recorded in Shennong's Herbal Classic. It is a traditional precious Chinese medicine that strengthens the body and nourishes the body. It has both edible and medicinal functions and is regarded as a "longevity herb". Ganoderma lucidum polysaccharides are one of the main active ingredients of Ganoderma lucidum, and have multiple activities such as lowering blood lipids, lowering blood sugar, anti-oxidation, scavenging free radicals, anti-aging, anti-tumor, and improving immunity. Ganoderma lucidum polysaccharides have been used as the main quality control indicator of Ganoderma lucidum medicinal materials in the 2020 edition of the Chinese Pharmacopoeia. Polysaccharides are composed of monosaccharides bonded by glycosidic bonds. Their pharmacological effects are not only related to the composition and content of polysaccharides, but also to the structural characteristics and physicochemical properties such as the connection mode and connection order between monosaccharides. However, the 2020 edition of the Chinese Pharmacopoeia uses the "anthrone-sulfuric acid method" to determine the polysaccharide content as the quality control indicator of Ganoderma lucidum, which lacks specificity and makes it difficult to achieve authenticity identification and quality control of Ganoderma lucidum medicinal materials.

[0004] In recent years, the application of Ganoderma lucidum and its extracts in the fields of traditional Chinese medicine, health products and food has been increasingly expanded. The 2020 edition of the Chinese Pharmacopoeia stipulates that Ganoderma lucidum is the dried fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang. Unlawful vendors use means such as mixing and adulteration to obtain illegal profits, which seriously affects the rights and interests of consumers. Therefore, the development of a simple, effective and feasible method for evaluating the quality of Ganoderma polysaccharides is of great significance to improving the quality standards of Ganoderma lucidum and promoting the healthy and sustainable development of the Ganoderma industry. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a method for constructing a fingerprint based on the antioxidant active ingredients of Ganoderma lucidum and its application.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for constructing a fingerprint of antioxidant active ingredients of Ganoderma lucidum, comprising the following steps:

[0008] (1) Ganoderma lucidum dried fruiting bodies of different varieties are crushed and then subjected to water extraction and alcohol precipitation to obtain crude polysaccharides of Ganoderma lucidum;

[0009] (2) hydrolyzing the crude Ganoderma lucidum polysaccharide under defined conditions to obtain a Ganoderma lucidum polysaccharide hydrolyzate;

[0010] (3) UPLC-CAD-ESI-Q-TOF / MS was used to measure and determine the chemical components contained in the hydrolyzate of Ganoderma lucidum polysaccharide, and 29 chemical components were preliminarily identified;

[0011] (4) performing grey correlation analysis on the antioxidant activity of the crude polysaccharide of Ganoderma lucidum and the peak areas of the 29 chemical components screened out in step (3), and determining that the 29 chemical components are antioxidant active components;

[0012] (5) The partial least squares regression method was used to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharides with 29 chemical components, and 12 chemical components were obtained. These 12 chemical components were used as common peaks to establish a fingerprint map based on the antioxidant active components of Ganoderma lucidum.

[0013] The second aspect of the present invention provides a method for identifying ganoderma lucidum varieties, comprising:

[0014] Obtaining a fingerprint of the sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum;

[0015] The principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) methods were used to analyze the fingerprints of the antioxidant active ingredients of standard red ganoderma, purple ganoderma, tree tongue ganoderma, black ganoderma and sample ganoderma to identify the ganoderma varieties.

[0016] The beneficial effects of the present invention are:

[0017] The present invention relates to the technical field of research on Chinese medicine components, and specifically to a method for constructing a fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum and its application. First, a method for ultrasonic-assisted acid hydrolysis of Ganoderma lucidum crude polysaccharide under the guidance of free radical scavenging activity was constructed. The method is efficient and has good operability, and can better reflect the free radical scavenging activity of the depolymerization product of Ganoderma lucidum crude polysaccharide. 52 batches of Ganoderma lucidum polysaccharides from different sources were acid-hydrolyzed and analyzed by hydrophilic chromatography-charged aerosol detector-electrospray mass spectrometry. Further combined with grey correlation analysis and partial least squares regression analysis, 12 active common peaks were screened out, and a fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum was established. Finally, the fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum of standard red ganoderma, purple ganoderma, tree tongue ganoderma, black ganoderma and sample Ganoderma lucidum was analyzed respectively by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to achieve the identification of Ganoderma lucidum varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 The effect of different varieties of Ganoderma lucidum polysaccharides in scavenging hydroxyl radicals;

[0020] Figure 2 The invention is an exploration of the hydrolysis of crude Ganoderma lucidum polysaccharide under limited conditions, wherein A is the scavenging rate of Ganoderma lucidum polysaccharide depolymers on hydroxyl radicals at different acid concentrations, B is the graph showing the variation of the chromatographic peak area of ​​representative depolymers in Ganoderma lucidum polysaccharide depolymers with acid concentration at different acid concentrations; C is the scavenging rate of Ganoderma lucidum polysaccharide depolymers on hydroxyl radicals at different ultrasonic powers; D is the graph showing the variation trend of the representative characteristic peak area in Ganoderma lucidum polysaccharide depolymers at different ultrasonic powers; E is the scavenging rate of Ganoderma lucidum polysaccharide depolymers on hydroxyl radicals at different temperatures; F is the graph showing the variation of the chromatographic peak area of ​​representative depolymers in Ganoderma lucidum polysaccharide depolymers with acid concentration at different temperatures; G is the scavenging rate of Ganoderma lucidum polysaccharide depolymers on hydroxyl radicals at different acid hydrolysis times, and H is the graph showing the variation of the chromatographic peak area of ​​representative depolymers in Ganoderma lucidum polysaccharide depolymers at different acid hydrolysis times with acid concentration; legends 1 to 8 in B, D, F and H represent chromatographic peaks 1 to 8 of depolymers in Ganoderma lucidum polysaccharide depolymers;

[0021] Figure 3 This is the chromatogram of the depolymerization product of the crude polysaccharide sample of Ganoderma lucidum;

[0022] Figure 4 This is a mass spectrum analysis of Ganoderma lucidum disaccharide, wherein A is the primary mass spectrum of the disaccharide, and B is the secondary mass spectrum of the disaccharide;

[0023] Figure 5This is a mass spectrum analysis of Ganoderma lucidum trisaccharide, wherein A is the primary mass spectrum of the trisaccharide, and B is the secondary mass spectrum of the trisaccharide;

[0024] Figure 6 This is the liquid phase diagram of 52 batches of samples;

[0025] Figure 7 Filter results for PLSR;

[0026] Figure 8 For multivariate statistical analysis, A is PCA analysis and B is PLS-DA analysis.

[0027] Fig. 9 Are the peak areas of the four characteristic peaks in different varieties of Ganoderma lucidum, where A is the peak area of ​​Peak 17 in different varieties of Ganoderma lucidum, B is the peak area of ​​Peak 20, C is the peak area of ​​Peak 25, and D is the peak area of ​​Peak 29. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] A first typical embodiment of the present invention provides a method for constructing a fingerprint based on the antioxidant active ingredients of Ganoderma lucidum, comprising the following steps:

[0031] (1) Ganoderma lucidum dried fruiting bodies of different varieties are crushed and then subjected to water extraction and alcohol precipitation to obtain crude polysaccharides of Ganoderma lucidum;

[0032] (2) hydrolyzing the crude Ganoderma lucidum polysaccharide under defined conditions to obtain a Ganoderma lucidum polysaccharide hydrolyzate;

[0033] (3) UPLC-CAD-ESI-Q-TOF / MS was used to measure and determine the chemical components contained in the hydrolyzate of Ganoderma lucidum polysaccharide, and 29 chemical components were preliminarily identified;

[0034] (4) performing grey correlation analysis on the antioxidant activity of the crude polysaccharide of Ganoderma lucidum and the peak areas of the 29 chemical components screened out in step (3), and determining that the 29 chemical components are antioxidant active components;

[0035] (5) The partial least squares regression method was used to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharides with 29 chemical components, and 12 chemical components were obtained. These 12 chemical components were used as common peaks to establish a fingerprint map based on the antioxidant active components of Ganoderma lucidum.

[0036] In one or more embodiments, in step (1), the method of obtaining crude Ganoderma lucidum polysaccharide by water extraction and alcohol precipitation comprises:

[0037] S1. Ganoderma lucidum dried fruiting bodies of different varieties were crushed and passed through a 30-50 mesh sieve, added with deionized water at 85-95°C, and ultrasonically extracted at 85-95°C for 35-45 min;

[0038] S2. Centrifuge to obtain the supernatant, add ethanol, let stand for 10 to 14 hours, centrifuge to collect the precipitate, and dry to obtain the crude Ganoderma lucidum polysaccharide.

[0039] Preferably, in step S1, the solid-liquid ratio of the dried Ganoderma lucidum fruiting body powder to deionized water is 1:(14.5-15.5) g / mL, preferably 1:15 g / mL.

[0040] Preferably, in step S1, the power of ultrasonic extraction is 300-350W, preferably 315W.

[0041] Preferably, in step S2, when the supernatant is obtained by centrifugal separation, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugal separation time is 12-18 min, preferably 15 min.

[0042] Preferably, in step S2, the concentration of ethanol is 95% (w / v), and the final concentration of ethanol in the mixed solution is 78% to 82%, preferably 80% (v / v).

[0043] Preferably, in step S2, ethanol is added and the mixture is allowed to stand at 3-5°C for 10-14 hours.

[0044] Preferably, in step S2, when collecting the precipitate by centrifugation, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugal separation time is 12-18 min, preferably 15 min.

[0045] Preferably, in step S2, the drying method is: evaporating the precipitate to dryness in a water bath at 75-85°C.

[0046] In one or more embodiments, in step (2), the conditions are defined as:

[0047] dissolving the crude polysaccharide of Ganoderma lucidum in water to obtain a crude polysaccharide solution of Ganoderma lucidum;

[0048] Trifluoroacetic acid was added to the crude polysaccharide solution of Ganoderma lucidum for ultrasonic hydrolysis.

[0049] Preferably, the concentration of trifluoroacetic acid is 3 to 6 mol / L, preferably 5 mol / L;

[0050] Preferably, the power of ultrasound is 245-350W, preferably 315W;

[0051] The temperature of ultrasound is 70-100°C, preferably 90°C;

[0052] The ultrasonication time is 5 to 30 minutes, preferably 10 minutes.

[0053] In one or more embodiments, in step (3), the chromatographic conditions are:

[0054] Column: Xbridge TM BEH Amide column (2.1×150 mm, 2.5 μm); mobile phase A was aqueous solution (0.8% formic acid + 20 M ammonium formate), mobile phase B was acetonitrile (0.8% formic acid) solution, gradient elution.

[0055] Preferably, the gradient elution conditions include: 0-13 min, 91% B-90% B; 13-20 min, 90% B-80% B; 20-40 min, 80% B; 40-41 min, 80% B-74% B; 41-50 min, 74% B; 50-51 min, 74% B-60% B; 51-60 min, 60% B.

[0056] Preferably, during detection, the flow rate is 0.2-0.3 mL / min, preferably 0.25 mL / min.

[0057] Preferably, during detection, the injection volume is 9 to 12 μL, preferably 10 μL;

[0058] Preferably, during detection, the column temperature of the chromatographic column is 20-30°C, preferably 25°C.

[0059] In one or more embodiments, in step (3), the ESI-TOF / MS conditions are:

[0060] Working in positive ion mode and negative ion mode respectively, the pressure of the nebulizer is 1.8-2.2 Bar, preferably 2.0 Bar; the flow rate of the drying gas is 7-10 mL / min, preferably 8 mL / min; the drying gas temperature is 200-240°C, preferably 200°C; the capillary voltage is 3400-3600 V, preferably 3500 V, in the positive ion mode; the capillary voltage is 2900-3200 V, preferably 3000 V in the negative ion mode; the mass-to-charge ratio (m / z) range of the scan is 100-2000.

[0061] In one or more embodiments, in step (5), when the partial least squares regression method is used to screen 29 chemical components, the screening condition is VIP>1.

[0062] In one or more embodiments, in step (5), the fingerprint spectrum based on the antioxidant active ingredients of Ganoderma lucidum includes common peaks of 12 chemical components, namely peak25, peak27, peak16, peak14, peak26, peak1, peak8, peak29, peak3, peak13, peak20 and peak17;

[0063] Among them, the retention time of peak 25 is 35.0~35.5min; the retention time of peak 27 is 40.2~40.7min; the retention time of peak 16 is 26.2~26.8min; the retention time of peak 14 is 25.5~26.0min; the retention time of peak 26 is 33.8~34.3min; the retention time of peak 1 is 3.0~3.5min; the retention time of peak 8 is 18.8~19.3min; the retention time of peak 29 is 27.3~27.8min; the retention time of peak 3 is 6.8~7.3min; the retention time of peak 13 is 25.0~25.6min; the retention time of peak 20 is 27.7~28.3min; the retention time of peak 17 is 26.6~27.2min.

[0064] A second typical embodiment of the present invention provides a method for identifying Ganoderma lucidum varieties, comprising:

[0065] Obtaining a fingerprint of the sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum;

[0066] The principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) methods were used to analyze the fingerprints of the antioxidant active ingredients of standard red ganoderma, purple ganoderma, tree tongue ganoderma, black ganoderma and sample ganoderma to identify the ganoderma varieties.

[0067] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0068] (1) Reagents and materials:

[0069] Anhydrous ethanol (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.); salicylic acid (analytical grade, Anhui Zesheng Technology Co., Ltd.); trifluoroacetic acid (analytical grade, Tianjin Komeo Chemical Reagent Co., Ltd.); hydrogen peroxide (analytical grade); acetonitrile (chromatographic grade, Tianjin Concord Technology Co., Ltd.); ferrous sulfate (purity 99%, Shandong Xiya Chemical Industry Co., Ltd.; ammonium formate (chromatographic grade, Tianjin Komeo Chemical Reagent Co., Ltd.); formic acid (UPLC grade, Tianjin Komel Company); ultrapure water (18MΩcm, Millipore Corporation, USA); monosaccharide standards with a purity greater than 98% and all from Shanghai Yuanye Biotechnology Co., Ltd., including D-(+)-glucose, L-rhamnose, D-galactose, D-mannose, D-arabinose, D-(+)-xylose, and L-(+)-fucose.

[0070] The dried Ganoderma samples were purchased from Yaowanglou Market in Jinan (Table 1), and were identified by Wang Xiao, a researcher at Shandong Academy of Sciences, as dried fruiting bodies of different varieties of Ganoderma.

[0071] Table 1 Ganoderma lucidum test samples

[0072]

[0073] (2) Instruments:

[0074] 1 / 10,000 electronic analytical balance (SARTOURIUSBSA, USA), SBL-10DT constant temperature ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.), SCIENTZ-10N freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.), TG16-WS desktop high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), Tecan Infinite M20 microplate reader (Tecan, Switzerland), UitiMate-3000 high performance liquid chromatograph (ThermoFisher), IMPACT II electrospray ionization-time-of-flight mass spectrometer (Bruker, Germany).

[0075] (3) Preparation of mixed reference solution

[0076] Accurately weigh 1.0 mg of each of rhamnose, fucose, xylose, arabinose, mannose, glucose and galactose reference substances, dissolve them in water and make up to 10 mL to prepare a mixed reference substance solution with a mass concentration of 0.1 mg / mL.

[0077] (4) Chromatographic conditions:

[0078] Column: Xbridge TM BEH Amide column (2.1×150mm, 2.5μm), mobile phase A is aqueous solution (0.8% formic acid + 20M ammonium formate), mobile phase B is acetonitrile (0.8% formic acid) solution. Elution gradient: 0-13min, 91% B-90% B; 13-20min, 90% B-80% B; 20-40min, 80% B; 40-41min, 80% B-74% B; 41-50min, 74% B; 50-51min, 74% B-60% B; 51-60min, 60% B. Flow rate: 0.25mL / min; injection volume: 10μL; column temperature: 25℃. CAD detection parameters: gas source is N2, pressure 61.2Psi, filter 5.0sec, nebulizer temperature 35℃.

[0079] (5) Mass spectrometry conditions

[0080] ESI-TOF / MS operated in positive ion mode and negative ion mode respectively. The nebulizer pressure was 2.0 Bar, the drying gas (N2) flow rate was 8 mL / min, the drying gas temperature was 220 °C, the capillary voltage was 3500 V in positive ion mode and 3000 V in negative ion mode, and the mass-to-charge ratio (m / z) range of the scan was 100-2000.

[0081] (6) Hydroxyl radical scavenging activity

[0082] Using Fenton reaction, H2O2 2+ Hydroxyl radicals are generated under the catalytic action of salicylic acid, and purple compounds will be produced. When the sample solution in the system has a good ability to scavenge hydroxyl radicals, the generated purple compounds will be reduced. Take 50μL of 3mM ferrous sulfate heptahydrate solution, 6mM salicylic acid ethanol solution, and Ganoderma lucidum crude polysaccharide (1.0mg / mL) respectively and place them in a 96-well plate. Finally, add 50μL of 3mM hydrogen peroxide solution. After reacting in the dark for 30 minutes at 37°C, measure the absorbance. A control group, a blank group, and a sample group were set up in each group of samples, and ultrapure water was used instead of the sample in the control group. Ultrapure water was used instead of hydrogen peroxide in the blank group, and vitamin (VC) was used as the positive control. The measurement was repeated 3 times in parallel to measure the absorbance A of each sample. The final clearance rate is:

[0083] Clearance rate (%) = [A control - (A sample - A blank) / A control] × 100

[0084] (7) Data Statistical Analysis

[0085] Grey correlation modeling software version 7 (Grey System Institute, Nanjing University of Aeronautics and Astronautics, China) was used to analyze the correlation between the peak area of ​​each peak and the scavenging rate of hydroxyl radicals by Ganoderma lucidum polysaccharides. SIMCA-P 14.1 (Umetrics, Sweden) software was used to perform partial least squares regression analysis (PLSR), principal component analysis (PCA), and orthogonal partial least squares analysis (OPLS-DA) on different varieties of Ganoderma lucidum. ORIGIN 22.0 (OriginLab, USA) was used for corresponding drawing analysis.

[0086] Example 1

[0087] Extraction of crude polysaccharides:

[0088] Take the collected dried fruiting bodies of different varieties of Ganoderma lucidum, crush them and pass through a 40-mesh sieve. Accurately weigh 2.0g of dried Ganoderma lucidum powder sample and place it in a 150mL conical flask, add 30mL of 90℃ hot water, and extract it by ultrasonic (315W) at 90℃ for 40min. After cooling the extract, centrifuge (4500r, 15min), make up the supernatant to 30mL with ultrapure water, add ethanol (95%, w / v) to it, so that the final concentration of ethanol in the solution is 80% (v / v), precipitate at 4℃ for 12h and then centrifuge (4500r, 15min), evaporate the precipitate in a water bath at 80℃, re-dissolve it with 5mL of hot water (80℃), and dilute it to 10mL with pure water to obtain a Ganoderma lucidum crude polysaccharide solution. Store it in a refrigerator (4℃) for use.

[0089] Example 2

[0090] Hydroxyl radical scavenging activity of polysaccharides from different varieties of Ganoderma lucidum

[0091] The ability of different varieties of Ganoderma lucidum polysaccharides to scavenge hydroxyl free radicals was determined using an enzyme marker. Figure 1 As shown, the clearance rate of the positive control at 1 mg / mL was 99.8182%, and the clearance rate of 1 mg / mL of different varieties of Ganoderma lucidum polysaccharides for hydroxyl radicals was between 20% and 50%. Among them, the hydroxyl radical clearance rate of Ganoderma lucidum was the highest, at 43.2840%, and the clearance rate of Ganoderma lucidum was the lowest, at 23.8062%. The four varieties of Ganoderma lucidum all have a certain ability to scavenge hydroxyl radicals, but the hydroxyl radical clearance rates of polysaccharides in different varieties of Ganoderma lucidum are not the same.

[0092] Example 3

[0093] Acid hydrolysis is a common method for hydrolyzing polysaccharides. The glycosidic bonds of polysaccharides can be broken through the reaction to obtain monosaccharides, oligosaccharides or polysaccharide fragments. Acid hydrolysis has the advantages of low cost, simple operation, good volatility and relatively mild hydrolysis. However, the acid hydrolysis process alone is long and the efficiency is too low. The energy generated by ultrasonic technology has the effects of local high temperature, high pressure and cavitation, which can promote the chemical reaction. This energy can destroy or weaken the hydrogen bonds between polysaccharide molecules and intramolecular hydrogen bonds, promote the penetration of acid in the polysaccharide structure, and thus can effectively catalyze the hydrolysis of polysaccharides. In addition, ultrasound-assisted hydrolysis can shorten the degradation time and improve the efficiency of the experiment. Ultrasound-assisted technology has been used in the study of rapid depolymerization of polysaccharides in food and natural products. In order to achieve efficient, stable and controllable depolymerization of Ganoderma lucidum polysaccharides, the effect of ultrasound-assisted trifluoroacetic acid hydrolysis conditions on the depolymerization of Ganoderma lucidum polysaccharides was investigated using the chromatographic peak area of ​​representative polysaccharide hydrolysates as an indicator.

[0094] Exploration of hydrolysis of Ganoderma lucidum crude polysaccharide under limited conditions:

[0095] 700 μL of crude polysaccharide solution was taken into a liquid phase vial, and trifluoroacetic acid (TFA) solution of the same volume and different concentrations (3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L) was added respectively. It was placed in an ultrasonic cleaner and hydrolyzed at different ultrasonic powers (245 W, 280 W, 315 W, 350 W), different temperatures (70 ° C, 80 ° C, 90 ° C, 100 ° C) and different times (5 min, 10 min, 15 min, 20 min, 25 min, 30 min). The hydrolyzate was freeze-dried, and finally washed with methanol and dried with nitrogen to remove TFA residues. Finally, a partial hydrolyzate of Ganoderma lucidum polysaccharide was obtained.

[0096] result:

[0097] ①Influence of acid concentration:

[0098] The effect of TFA concentration on the acid hydrolysis of Ganoderma lucidum polysaccharide was investigated. Figure 2 A is the scavenging rate of hydroxyl radicals by Ganoderma lucidum polysaccharide depolymers at different acid concentrations. Figure 2 B is the graph showing the variation of the chromatographic peak area of ​​the representative depolymer of Ganoderma lucidum polysaccharide with the acid concentration. It can be seen that when the acid concentration is in the range of 3 to 6 mol / mL, as the TFA concentration increases, the scavenging rate of the sample for hydroxyl radicals first increases and then decreases. Figure 2 B shows that the peak areas of the representative chromatographic peaks in the depolymerization of Ganoderma lucidum polysaccharide all increase first and then decrease with the increase of acid concentration. When the TFA concentration is 5 mol / mL, the hydroxyl radical scavenging rate of the sample is the best ( Figure 2 A), and the peak area of ​​each representative depolymer is also the highest ( Figure 2B). The possible reason is that when the concentration of TFA increases from low to high, the concentration of reducing monosaccharides and oligosaccharides produced by hydrolysis increases, and thus the scavenging rate of hydroxyl radicals gradually increases; and when the acid concentration reaches a certain level, it promotes the degradation of Ganoderma lucidum polysaccharides into smaller structural fragments, resulting in a significant decrease in the concentration of reducing monosaccharides and oligosaccharides, and at the same time, it is easy to convert into furfural and its derivatives, and the reducing functional groups are destroyed, resulting in a decrease in the scavenging activity of hydroxyl radicals. Therefore, a TFA concentration of 5 mol / mL was selected for the acid hydrolysis of Ganoderma lucidum polysaccharides.

[0099] ②The influence of ultrasonic power:

[0100] like Figure 2 As shown in C, as the ultrasonic power continues to increase, the scavenging rate of the sample for hydroxyl radicals shows a trend of first increasing and then decreasing. When the ultrasonic power is 315W, the scavenging rate of hydroxyl radicals is the highest. Figure 2 D The trend of the representative characteristic peak area in the depolymerization of Ganoderma lucidum polysaccharide under different ultrasonic powers can also be seen: with the increase of ultrasonic power, the peak area of ​​the representative characteristic peak shows a trend of first rising and then falling. When the ultrasonic power is 3.15W, the peak area of ​​the characteristic peak is the highest. It is preliminarily inferred that appropriate ultrasonic power can accelerate the depolymerization of Ganoderma lucidum polysaccharide into reducing monosaccharides and oligosaccharides, and improve its hydroxyl radical scavenging activity. When the ultrasonic power reaches a certain level, it will destroy the reducing groups such as carbonyl and aldehyde groups contained in monosaccharides and oligosaccharides, resulting in a decrease in hydroxyl radical scavenging activity. Therefore, an ultrasonic power of 3.15W was selected for subsequent research.

[0101] ③The influence of acid hydrolysis temperature:

[0102] The effects of different temperatures (70℃, 80℃, 90℃ and 100℃) on the acidolysis degree and free radical scavenging activity of Ganoderma lucidum polysaccharide were investigated. Figure 2 As shown in Figures E and F. As can be seen from the figure, with the increase of temperature, the free radical scavenging activity and representative chromatographic peak area of ​​Ganoderma lucidum polysaccharide depolymers both showed a trend of first increasing and then decreasing. At 90°C, the free radical scavenging activity and chromatographic peak area both reached their maximum values. When the temperature reached 100°C, the movement of polysaccharide molecules intensified, the combined effects of temperature, ultrasound and acid hydrolysis were strengthened, the degree of polysaccharide degradation increased, the reducing sugar structure with hydroxyl radical scavenging activity was destroyed, and the free radical scavenging rate was significantly reduced. Therefore, 90°C was selected as the optimal acid hydrolysis temperature.

[0103] ④Influence of acid hydrolysis time

[0104] Under the optimized conditions of acid concentration, ultrasonic power and temperature, the effect of acid hydrolysis time was investigated. Figure 2 As shown in G and H. Figure 2G shows that during the period of 5 to 30 minutes, as the ultrasonic time increases, the hydroxyl radical scavenging rate first increases and then slowly decreases. Figure 2 It can be seen that with the increase of ultrasonic time, the peak area of ​​each characteristic peak has a certain volatility. In order to ensure that the depolymerization of Ganoderma lucidum polysaccharide has good free radical scavenging activity and improve the acid hydrolysis efficiency, the ultrasonic acid hydrolysis time was finally selected as 10 min.

[0105] Example 4

[0106] Identification of Partial Acid Depolymerization of Ganoderma Lucidum Polysaccharide by ESI-TOF / MS

[0107] According to the method in Example 1, different varieties of Ganoderma lucidum polysaccharide solutions were prepared, and Ganoderma lucidum polysaccharide acid hydrolysis samples were prepared according to the optimized acid hydrolysis conditions. Liquid phase analysis was performed according to the above-mentioned chromatographic conditions (4), and chromatograms were obtained using a CAD detector. First, a preliminary experiment was conducted using a Ganoderma lucidum crude polysaccharide acid hydrolysis sample. It was found that the optimized acid hydrolysis conditions could obtain chromatographic peaks with better separation. Then, four varieties of Ganoderma lucidum polysaccharide acid hydrolysis samples were prepared accordingly and the corresponding liquid phase analysis was performed. The chromatograms of the depolymerized Ganoderma lucidum polysaccharides of different varieties are shown in FIG. Figure 3 shown.

[0108] The eluate was connected to ESI-Q-TOF / MS for high-resolution mass spectrometry analysis. Using UPLC-CAD-ESI-Q-TOF / MS, 29 compounds were preliminarily identified through comparative analysis with the literature, including monosaccharides, oligosaccharides, and unknown compounds containing sugar ion fragments. The results are shown in Table 2.

[0109] Table 2 ESI-Q-TOF / MS identification of Ganoderma lucidum polysaccharide depolymers

[0110]

[0111]

[0112] The mass spectrum of Ganoderma disaccharide was analyzed, such as Figure 4 As shown, m / z 341.1084 [MH] can be observed in the MS primary mass spectrum of disaccharide - 、m / z387.1138[M+COOH] - The fragment ion of - In the MS / MS spectrum of the parent ion, ion fragments of m / z 179.0539 and m / z 161.0598 can be observed, of which 179.0539 is the parent ion that lost a sugar residue (C6H 12O6), 161.0598 is the ion fragment obtained after the parent ion loses a sugar residue and then loses a molecule of water. m / z 455.1003 is the fragment ion obtained by adding the parent ion mass 113.9919. The added compound is unclear, but this molecular weight appears in the fragment ions of most monosaccharides and oligosaccharides obtained by this method. Figure 4 It is marked in (A).

[0113] The mass spectrum of Ganoderma lucidum trisaccharide was analyzed, such as Figure 5 As shown, m / z 503.1603 [MH] appears in the primary mass spectrum - 、m / z549.1659[M+COOH] - The fragment ion is m / z 503.1603 [MH] - As the parent ion, the glycosidic bond breaks, the hexose residue (162n) is lost, and the ion fragment of m / z 179.0157 is obtained. At the same time, the parent ion undergoes ring-opening cleavage to obtain the fragment ion 383.1274 ( 2,4 A3), 221.0637( 2,4 A2) ion fragments.

[0114] Example 5

[0115] Spectrum-effect correlation analysis and fingerprint establishment:

[0116] (1) Grey correlation analysis

[0117] 52 batches of Ganoderma lucidum crude polysaccharide solutions were prepared into sample solutions using optimized acid hydrolysis conditions, and the 52 batches of samples were subjected to liquid phase analysis, and finally the liquid phase diagrams of the 52 batches of samples were obtained, as shown in FIG. Figure 6 As shown in Table 3, the liquid phase diagram was analyzed, and 29 common peaks were screened out. The 29 common peaks were integrated to obtain the corresponding peak areas. The hydroxyl radical scavenging rate of the Ganoderma lucidum sample and the peak area of ​​the common peak were subjected to grey correlation analysis, and the results are shown in Table 3. It can be observed from the table that the correlation between the peak area of ​​the screened characteristic peaks and the hydroxyl radical scavenging rate is greater than 0.6. When the correlation r>0.6, it indicates that there is a correlation between the two. The larger the r, the stronger the correlation between the two. The correlation of the 29 chromatographic peaks with r>0.8 is relatively high, indicating that the hydroxyl radical scavenging rate of Ganoderma lucidum polysaccharide is the result of the synergistic effect of different chemical components.

[0118] Table 3 Grey correlation results

[0119]

[0120] (2) Partial least squares regression analysis

[0121] Partial least squares regression (PLSR) generally believes that when VIP>1, the independent variable is significantly important in explaining the dependent variable. The antioxidant activity of Ganoderma lucidum polysaccharide was associated with 29 chemical components using partial least squares regression, such as Figure 7 As shown, 12 common active peaks were screened out by PLSR, namely peak25, peak27, peak16, peak14, peak26, peak1, peak8, peak29, peak3, peak13, peak20 and peak17. These 12 chemical components were used as common peaks to establish a fingerprint map based on the antioxidant active components of Ganoderma lucidum.

[0122] (3) Multivariate statistical analysis

[0123] Using SIMCA-P (14.1, Umetrics, Sweden) software to perform unsupervised PCA analysis on the active polysaccharide fingerprints of different varieties of Ganoderma lucidum can more intuitively observe the differences between different varieties of Ganoderma lucidum. Figure 8 As can be seen in A, the distances between samples of different varieties of Ganoderma are relatively far, indicating that the polysaccharides contained in different varieties of Ganoderma are significantly different. The results show that PCA analysis can distinguish samples of different varieties of Ganoderma to a certain extent. Using the common peak of active polysaccharide fingerprint as a variable, supervised PLS-DA was used to analyze samples of different varieties of Ganoderma. Figure 8 B. Among them, the explanation rate of the Y matrix is ​​0.971, the explanation rate of the X matrix is ​​0.973, and the prediction ability Q2 value is 0.954, indicating that the model has strong prediction ability.

[0124] Variable Importance Projection (VIP) analysis ( Figure 8 C) shows that Peak 29 (DP3), peak 20 (unknown), peak 25 (unknown), and peak 17 (unknown) (VIP>1) contribute more to the differentiation. The content distribution of these four components in different varieties is as follows Fig. 9 As shown. Among them, the contents of peak 17 (unknown), peak 20 (unknown), and peak 25 (unknown) in Ganoderma lucidum were all high; the content of peak 29 (DP3) in Ganoderma lucidum was high; the content of peak 25 (unknown) in Ganoderma lucidum was low; and the content of peak 20 (unknown) in Ganoderma lucidum was low. The one-way ANOVA showed that there were significant differences among the four components in different varieties (P<0.05), indicating that they are expected to be used as quality markers for the identification of different varieties of Ganoderma lucidum.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of antioxidant active ingredients of Ganoderma lucidum, characterized in that: The following steps are involved: (1) Ganoderma lucidum dried fruiting bodies of different varieties are crushed and then subjected to water extraction and alcohol precipitation to obtain crude polysaccharides of Ganoderma lucidum; (2) hydrolyzing the crude Ganoderma lucidum polysaccharide under defined conditions to obtain a Ganoderma lucidum polysaccharide hydrolyzate; (3) UPLC-CAD-ESI-Q-TOF / MS was used to measure and determine the chemical components contained in the hydrolyzate of Ganoderma lucidum polysaccharide, and 29 chemical components were preliminarily identified; (4) performing grey correlation analysis on the antioxidant activity of the crude polysaccharide of Ganoderma lucidum and the peak areas of the 29 chemical components screened out in step (3), and determining that the 29 chemical components are antioxidant active components; (5) The partial least squares regression method was used to screen 29 chemical components and obtain 12 chemical components. These 12 chemical components were used as common peaks to establish a fingerprint map based on the antioxidant active components of Ganoderma lucidum.

2. The construction method according to claim 1, characterized in that: In step (1), the method of obtaining crude Ganoderma lucidum polysaccharide by water extraction and alcohol precipitation comprises: S1. Ganoderma lucidum dried fruiting bodies of different varieties were crushed and passed through a 30-50 mesh sieve, added with deionized water at 85-95°C, and ultrasonically extracted at 85-95°C for 35-45 min; S2. Centrifuge to obtain the supernatant, add ethanol, let stand for 10 to 14 hours, centrifuge to collect the precipitate, and dry to obtain the crude Ganoderma lucidum polysaccharide.

3. The construction method according to claim 2, characterized in that: In step S1, the solid-liquid ratio of the dried Ganoderma lucidum fruiting body powder to deionized water is 1:(14.5-15.5) g / mL, preferably 1:15 g / mL; Or, in step S1, the power of ultrasonic extraction is 300-350W, preferably 315W; Or, in step S2, when the supernatant is obtained by centrifugal separation, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugal separation time is 12-18 min, preferably 15 min; Or, in step S2, the concentration of ethanol is 95% (w / v), and the final concentration of ethanol in the mixed solution is 78% to 82%, preferably 80% (v / v); Or, in step S2, ethanol is added and allowed to stand at 3 to 5° C. for 10 to 14 hours; Or, in step S2, when collecting the precipitate by centrifugation, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugation time is 12-18 min, preferably 15 min; Preferably, in step S2, the drying method is: evaporating the precipitate to dryness in a water bath at 75-85°C.

4. The construction method according to claim 1, characterized in that: In step (2), the conditions are as follows: dissolving the crude ganoderma polysaccharide in water to obtain a crude ganoderma polysaccharide solution; Trifluoroacetic acid was added to the crude polysaccharide solution of Ganoderma lucidum for ultrasonic hydrolysis.

5. The construction method according to claim 4, characterized in that: The concentration of trifluoroacetic acid is 3 to 6 mol / L, preferably 5 mol / L; Or, the power of ultrasound is 245-350 W, preferably 315 W; the temperature of ultrasound is 70-100° C., preferably 90° C.; the time of ultrasound is 5-30 min, preferably 10 min.

6. The construction method according to claim 1, characterized in that: In step (3), the chromatographic conditions are: Column: Xbridge TM BEH Amide column (2.1×150 mm, 2.5 μm); mobile phase A was aqueous solution (0.8% formic acid + 20 M ammonium formate), mobile phase B was acetonitrile (0.8% formic acid) solution, gradient elution; Preferably, the gradient elution conditions include: 0-13 min, 91% B-90% B; 13-20 min, 90% B-80% B; 20-40 min, 80% B; 40-41 min, 80% B-74% B; 41-50 min, 74% B; 50-51 min, 74% B-60% B; 51-60 min, 60% B; Preferably, during detection, the flow rate is 0.2 to 0.3 mL / min, preferably 0.25 mL / min; Preferably, during detection, the injection volume is 9 to 12 μL, preferably 10 μL; Preferably, during detection, the column temperature of the chromatographic column is 20-30°C, preferably 25°C.

7. The construction method according to claim 1, characterized in that: In step (3), the ESI-TOF / MS conditions are: Working in positive ion mode and negative ion mode respectively, the pressure of the nebulizer is 1.8-2.2 Bar, preferably 2.0 Bar; the flow rate of the drying gas is 7-10 mL / min, preferably 8 mL / min; the drying gas temperature is 200-240°C, preferably 200°C; the capillary voltage is 3400-3600 V, preferably 3500 V, in the positive ion mode; the capillary voltage is 2900-3200 V, preferably 3000 V in the negative ion mode; the mass-to-charge ratio (m / z) range of the scan is 100-2000.

8. The construction method according to claim 1, characterized in that: In step (5), when partial least squares regression method is used to screen 29 chemical components, the screening condition is VIP>1.

9. The construction method according to claim 1, characterized in that: In step (5), the fingerprint spectrum based on the antioxidant active ingredients of Ganoderma lucidum includes common peaks of 12 chemical components, namely peak25, peak27, peak16, peak14, peak26, peak1, peak8, peak29, peak3, peak13, peak20 and peak17; Among them, the retention time of peak 25 is 35.0~35.5min; the retention time of peak 27 is 40.2~40.7min; the retention time of peak 16 is 26.2~26.8min; the retention time of peak 14 is 25.5~26.0min; the retention time of peak 26 is 33.8~34.3min; the retention time of peak 1 is 3.0~3.5min; the retention time of peak 8 is 18.8~19.3min; the retention time of peak 29 is 27.3~27.8min; the retention time of peak 3 is 6.8~7.3min; the retention time of peak 13 is 25.0~25.6min; the retention time of peak 20 is 27.7~28.3min; the retention time of peak 17 is 26.6~27.2min.

10. A method for identifying Ganoderma lucidum species, characterized in that: include: Obtaining a fingerprint of the sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum; The principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) methods were used to analyze the fingerprints of the antioxidant active ingredients of standard red ganoderma, purple ganoderma, tree tongue ganoderma, black ganoderma and sample ganoderma to identify the ganoderma varieties.

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