Method for constructing fingerprint on the basis of ganoderma lucidum antioxidant active components and use thereof

Abstract

The present invention relates to the technical field of traditional Chinese medicine component research, and specifically to a method for constructing a fingerprint on the basis of Ganoderma lucidum antioxidant active components and the use thereof. First, a method for ultrasonic-assisted acidolysis of Ganoderma lucidum crude polysaccharide under the guidance of free radical scavenging activity is constructed. The method is highly efficient, exhibits good operability, and can better reflect the free radical scavenging activity of a Ganoderma lucidum crude polysaccharide depolymerized product. Hydrophilic interaction liquid chromatography–charged aerosol detection–electrospray ionization mass spectrometry is used to perform acidolysis and analysis on 52 batches of Ganoderma lucidum polysaccharides from different sources and further, in light of grey relational analysis and partial least squares regression analysis, 12 active common peaks are selected, and a Ganoderma lucidum antioxidant active component-based fingerprint is established. Finally, a principal component analysis method and a partial least squares discriminant analysis method are used to analyze Ganoderma lucidum antioxidant active component-based fingerprints of standard Ganoderma lucidum, Ganoderma sinense, Ganoderma applanatum, Ganoderma atrum and sample Ganoderma lucidum, thereby distinguishing between Ganoderma species.

Description

A method for constructing fingerprint profiles based on the antioxidant active components of Ganoderma lucidum and its application. Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component research technology, specifically to a method for constructing fingerprint spectra based on the antioxidant active components of Ganoderma lucidum and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Reishi mushroom (Ganoderma lucidum), also known as the auspicious herb, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It is a traditional and precious Chinese herbal medicine used for strengthening the body and replenishing vital energy, possessing both edible and medicinal functions, and is considered a "longevity herb." Reishi polysaccharides are one of the main active ingredients of reishi, exhibiting various activities such as lowering blood lipids, lowering blood sugar, anti-oxidation, scavenging free radicals, anti-aging, anti-tumor, and enhancing immunity. Reishi polysaccharides have been included as a major quality control indicator for reishi medicinal materials in the 2020 edition of the *Chinese Pharmacopoeia*. Polysaccharides are composed of monosaccharides linked by glycosidic bonds. Their pharmacological effects are related not only to the composition and content of polysaccharides but also to structural characteristics and physicochemical properties such as the linkage mode and sequence between monosaccharides. However, the 2020 edition of the *Chinese Pharmacopoeia* uses the "anthrone-sulfuric acid method" to determine polysaccharide content as a quality control indicator for reishi, which lacks specificity and makes it difficult to identify genuine reishi medicinal materials and control their quality.

[0004] In recent years, Ganoderma lucidum and its extracts have been increasingly used in traditional Chinese medicine, health products, and food. The 2020 edition of the Chinese Pharmacopoeia defines Ganoderma lucidum as the dried fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang. Unscrupulous merchants use adulteration and other fraudulent methods to obtain illegal profits, seriously affecting consumers' rights. Therefore, developing simple, effective, and practical methods for evaluating the quality of Ganoderma lucidum polysaccharides is of great significance for improving the quality standards of Ganoderma lucidum and promoting the healthy and sustainable development of the Ganoderma lucidum industry. Summary of the Invention

[0005] To overcome the above problems, this invention provides a method for constructing fingerprint spectra based on the antioxidant active ingredients of Ganoderma lucidum and its application.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

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

[0008] (1) After pulverizing the dried fruiting bodies of different varieties of Ganoderma lucidum, water extraction and alcohol precipitation were carried out to obtain crude polysaccharide of Ganoderma lucidum.

[0009] (2) Ganoderma lucidum crude polysaccharide was hydrolyzed under limited conditions to obtain Ganoderma lucidum polysaccharide hydrolysis products;

[0010] (3) The chemical components contained in the hydrolysate of Ganoderma lucidum polysaccharide were measured and determined by hydrophilic chromatography-electrospray detector-electrospray mass spectrometry (UPLC-CAD-ESI-Q-TOF / MS), and 29 chemical components were preliminarily identified.

[0011] (4) The antioxidant activity of Ganoderma lucidum crude polysaccharide was compared with the peak area of ​​the 29 chemical components screened in step (3) by grey relational analysis, and the 29 chemical components were identified as antioxidant active components.

[0012] (5) Partial least squares regression was used to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharide with 29 chemical components. Twelve chemical components were obtained and used as common peaks to establish a fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum.

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

[0014] Obtain fingerprints of Ganoderma lucidum samples based on its antioxidant active components;

[0015] Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the fingerprint spectra of Ganoderma lucidum (red Ganoderma), Ganoderma sinense (purple Ganoderma), Ganoderma lingulata (tree tongue Ganoderma), Ganoderma lucidum (black Ganoderma), and sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum, so as to identify the Ganoderma lucidum varieties.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention relates to the field of traditional Chinese medicine component research technology, specifically to a method for constructing fingerprint spectra based on the antioxidant active components of Ganoderma lucidum and its application. First, a method for ultrasonic-assisted acid hydrolysis of crude Ganoderma lucidum polysaccharides guided by free radical scavenging activity was constructed. This method is efficient, easy to operate, and can effectively reflect the free radical scavenging activity of the depolymerized Ganoderma lucidum polysaccharides. Hydrophilic chromatography-electrospray ionization detector-electrospray mass spectrometry was used to acid hydrolyze and analyze 52 batches of Ganoderma lucidum polysaccharides from different sources. Further, combined with grey relational analysis and partial least squares regression analysis, 12 common activity peaks were screened, establishing fingerprint spectra based on the antioxidant active components of Ganoderma lucidum. Finally, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the fingerprint spectra based on the antioxidant active components of Ganoderma lucidum for standard Ganoderma lucidum, Ganoderma sinense, Ganoderma applanatum, Ganoderma lucidum var. truncatum, and sample Ganoderma lucidum, respectively, achieving the identification of Ganoderma lucidum varieties. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 shows the effect of different varieties of Ganoderma lucidum polysaccharides on scavenging hydroxyl free radicals;

[0020] Figure 2 illustrates the hydrolysis of Ganoderma lucidum crude polysaccharide under defined conditions. A represents the scavenging rate of hydroxyl radicals by the depolymerized Ganoderma lucidum polysaccharide at different acid concentrations; B represents the variation of the chromatographic peak area of ​​representative depolymerized Ganoderma lucidum polysaccharide with acid concentration at different acid concentrations; C represents the scavenging rate of hydroxyl radicals by the depolymerized Ganoderma lucidum polysaccharide at different ultrasonic powers; D represents the trend of the variation of the area of ​​representative characteristic peaks in the depolymerized Ganoderma lucidum polysaccharide at different ultrasonic powers; E represents the scavenging rate of hydroxyl radicals by the depolymerized Ganoderma lucidum polysaccharide at different temperatures; F represents the variation of the chromatographic peak area of ​​representative depolymerized Ganoderma lucidum polysaccharide at different temperatures with acid concentration; G represents the scavenging rate of hydroxyl radicals by the depolymerized Ganoderma lucidum polysaccharide at different acid hydrolysis times; H represents the variation of the chromatographic peak area of ​​representative depolymerized Ganoderma lucidum polysaccharide at different acid hydrolysis times with acid concentration; Legend 1-8 in B, D, F, and H represents chromatographic peaks 1-8 of the depolymerized Ganoderma lucidum polysaccharide.

[0021] Figure 3 shows the chromatogram of the depolymerized product of Ganoderma lucidum crude polysaccharide sample;

[0022] Figure 4 shows the mass spectrum analysis of Ganoderma lucidum disaccharide, where A is the primary mass spectrum of the disaccharide and B is the secondary mass spectrum of the disaccharide.

[0023] Figure 5 shows the mass spectrum analysis of Ganoderma lucidum trisaccharides, where A is the primary mass spectrum of the trisaccharides and B is the secondary mass spectrum of the trisaccharides.

[0024] Figure 6 shows the liquid phase diagrams of 52 batches of samples;

[0025] Figure 7 shows the PLSR screening results;

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

[0027] Figure 9 shows the peak areas of four characteristic peaks in different varieties of Ganoderma lucidum, where A is the peak area of ​​Peak17, B is the peak area of ​​Peak20, C is the peak area of ​​Peak25, and D is the peak area of ​​Peak29. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate 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 spectrum based on the antioxidant active ingredients of Ganoderma lucidum, comprising the following steps:

[0031] (1) After pulverizing the dried fruiting bodies of different varieties of Ganoderma lucidum, water extraction and alcohol precipitation were carried out to obtain crude polysaccharide of Ganoderma lucidum.

[0032] (2) Ganoderma lucidum crude polysaccharide was hydrolyzed under limited conditions to obtain Ganoderma lucidum polysaccharide hydrolysis products;

[0033] (3) The chemical components contained in the hydrolysate of Ganoderma lucidum polysaccharide were measured and determined by hydrophilic chromatography-electrospray detector-electrospray mass spectrometry (UPLC-CAD-ESI-Q-TOF / MS), and 29 chemical components were preliminarily identified.

[0034] (4) The antioxidant activity of Ganoderma lucidum crude polysaccharide was compared with the peak area of ​​the 29 chemical components screened in step (3) by grey relational analysis, and the 29 chemical components were identified as antioxidant active components.

[0035] (5) Partial least squares regression was used to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharide with 29 chemical components. Twelve chemical components were obtained and used as common peaks to establish a fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum.

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

[0037] S1. After pulverizing the dried fruiting bodies of different varieties of Ganoderma lucidum, pass them through a 30-50 mesh sieve, add deionized water at 85-95℃, and extract them by ultrasonic extraction at 85-95℃ for 35-45 minutes.

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

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

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

[0041] Preferably, in step S2, when centrifuging to obtain the supernatant, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugation 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 centrifuging to collect the precipitate, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugation time is 12-18 min, preferably 15 min.

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

[0046] In one or more embodiments, the condition defined in step (2) is:

[0047] Ganoderma lucidum crude polysaccharide was dissolved in water to obtain Ganoderma lucidum crude polysaccharide solution;

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

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

[0050] Preferably, the ultrasonic power is 245-350W, and more preferably 315W;

[0051] The temperature of the ultrasound is 70–100℃, preferably 90℃;

[0052] The ultrasound duration is 5 to 30 minutes, preferably 10 minutes.

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

[0054] Column: Xbridge TM BEH Amide column (2.1×150mm, 2.5μm); mobile phase A was an aqueous solution (0.8% formic acid + 20M ammonium formate), and mobile phase B was an acetonitrile (0.8% formic acid) solution, with 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, the flow rate during detection is 0.2–0.3 mL / min, and more preferably 0.25 mL / min.

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

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

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

[0060] Operating in both positive and negative ion modes, the sprayer pressure 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 temperature of the drying gas is 200–240 °C, preferably 200 °C; the capillary voltage is 3400–3600 V, preferably 3500 V, in positive ion mode; and 2900–3200 V, preferably 3000 V, in negative ion mode; the mass-to-charge ratio (m / z) range is 100–2000.

[0061] In one or more embodiments, in step (5), when partial least squares regression 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 the common peaks of 12 chemical components, namely peak25, peak27, peak16, peak14, peak26, peak1, peak8, peak29, peak3, peak13, peak20 and peak17.

[0063] The retention times for peak 25 were 35.0–35.5 min; peak 27 was 40.2–40.7 min; peak 16 was 26.2–26.8 min; peak 14 was 25.5–26.0 min; peak 26 was 33.8–34.3 min; peak 1 was 3.0–3.5 min; peak 8 was 18.8–19.3 min; peak 29 was 27.3–27.8 min; peak 3 was 6.8–7.3 min; peak 13 was 25.0–25.6 min; peak 20 was 27.7–28.3 min; and peak 17 was 26.6–27.2 min.

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

[0065] Obtain fingerprints of Ganoderma lucidum samples based on its antioxidant active components;

[0066] Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the fingerprint spectra of Ganoderma lucidum (red Ganoderma), Ganoderma sinense (purple Ganoderma), Ganoderma lingulata (tree tongue Ganoderma), Ganoderma lucidum (black Ganoderma), and sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum, so as to identify the Ganoderma lucidum varieties.

[0067] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to 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 Kemeio Chemical Reagent Co., Ltd.); hydrogen peroxide (analytical grade); acetonitrile (chromatographic grade, Tianjin Kangkede Technology Co., Ltd.); ferrous sulfate (99% purity, Shandong Xiya Chemical Industry Co., Ltd.); ammonium formate (chromatographic grade, Tianjin Kemeio Chemical Reagent Co., Ltd.); formic acid (UPLC grade, Tianjin Komel Co.); ultrapure water (18 MΩcm, Millipore, USA); monosaccharide standards with a purity greater than 98% were 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 lucidum samples were all purchased from Yaowanglou Market in Jinan City (Table 1), and were identified by Researcher Wang Xiao of Shandong Academy of Sciences as dried fruiting bodies of different varieties of Ganoderma lucidum.

[0071] Table 1. Ganoderma lucidum samples for testing

[0072]

[0073] (2) Instruments:

[0074] 0.01% electronic analytical balance (SARTOURIUSBSA, USA), SBL-10DT constant temperature ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.), SCIENTZ-10N freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.), TG16-WS benchtop high-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), TecanInfinite M20 microplate reader (Tecan, Switzerland), UitiMate-3000 high performance liquid chromatograph (Thermo Fisher Scientific), IMPACT II electrospray ionization-time-of-flight mass spectrometer (Bruker, Germany).

[0075] (3) Preparation of mixed reference solution

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

[0077] (4) Chromatographic conditions:

[0078] Column: Xbridge TMBEH Amide column (2.1×150mm, 2.5μm) was used. Mobile phase A was an aqueous solution (0.8% formic acid + 20M ammonium formate), and mobile phase B was acetonitrile (0.8% formic acid) solution. Elution gradient: 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. Flow rate: 0.25 mL / min; injection volume: 10 μL; column temperature: 25℃. CAD detection parameters: gas source: N2; pressure: 61.2 Psi; filter time: 5.0 sec; nebulizer temperature: 35℃.

[0079] (5) Mass spectrometry conditions

[0080] The ESI-TOF / MS was operated in both positive and negative ion modes. The nebulizer pressure was 2.0 Bar, the dry gas (N2) flow rate was 8 mL / min, the dry 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 the Fenton reaction to make H2O2 in Fe 2+ Under the catalysis of [a specific substance], hydroxyl radicals are generated, which react with salicylic acid to produce a purple compound. When the sample solution in the system has a good ability to scavenge hydroxyl radicals, the amount of purple compound generated will decrease. 50 μL of 3 mM ferrous sulfate heptahydrate solution, 6 mM salicylic acid ethanol solution, and Ganoderma lucidum crude polysaccharide (1.0 mg / mL) were placed in 96-well plates, and finally 50 μL of 3 mM hydrogen peroxide solution was added. After reacting at 37°C in the dark for 30 min, the absorbance was measured. Each sample group included a control group, a blank group, and a sample group. The control group used ultrapure water instead of the sample. The blank group used ultrapure water instead of hydrogen peroxide, with vitamin C as a positive control. The absorbance (A) of each sample was measured in triplicate. The final scavenging rate was:

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

[0084] (7) Data Statistical Analysis

[0085] The correlation between peak area and hydroxyl radical scavenging rate of Ganoderma lucidum polysaccharides was analyzed using grey relational modeling software version 7 (Grey System Research Institute, Nanjing University of Aeronautics and Astronautics, China). Partial least squares regression (PLSR), principal component analysis (PCA), and orthogonal partial least squares analysis (OPLS-DA) were performed on different varieties of Ganoderma lucidum using SIMCA-P 14.1 software (Umetrics, Sweden). Corresponding plotting analyses were performed using ORIGIN 22.0 (OriginLab, USA).

[0086] Example 1

[0087] Extraction of crude polysaccharides:

[0088] Collected dried fruiting bodies of different Ganoderma lucidum varieties, pulverized and passed through a 40-mesh sieve. Accurately weigh 2.0 g of dried Ganoderma lucidum powder sample and place it in a 150 mL Erlenmeyer flask. Add 30 mL of 90 °C hot water and extract by ultrasonication (315 W) at 90 °C for 40 min. After cooling the extract, centrifuge (4500 rpm, 15 min). The supernatant is made up to 30 mL with ultrapure water. Add ethanol (95%, w / v) to make the final ethanol concentration in the solution 80% (v / v). After ethanol precipitation at 4 °C for 12 h, centrifuge (4500 rpm, 15 min). Evaporate the precipitate to dryness in a water bath at 80 °C, redissolve it with 5 mL of hot water (80 °C), and make up to 10 mL with pure water to obtain a crude Ganoderma lucidum polysaccharide solution. Store in a refrigerator at 4 °C for later use.

[0089] Example 2

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

[0091] The ability of different Ganoderma lucidum polysaccharides to scavenge hydroxyl radicals was determined using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Figure 1. The positive control showed a scavenging rate of 99.8182% at 1 mg / mL. The scavenging rates of hydroxyl radicals by different Ganoderma lucidum polysaccharides at 1 mg / mL ranged from 20% to 50%. Among them, Ganoderma lucidum showed the highest hydroxyl radical scavenging rate at 43.2840%, while Ganoderma applanatum showed the lowest at 23.8062%. All four Ganoderma lucidum varieties possessed a certain hydroxyl radical scavenging ability, but the hydroxyl radical scavenging rates of polysaccharides varied among the different varieties.

[0092] Example 3

[0093] Acid hydrolysis is a common method for hydrolyzing polysaccharides. The reaction breaks the glycosidic bonds of polysaccharides, yielding monosaccharides, oligosaccharides, or polysaccharide fragments. Acid hydrolysis is low-cost, simple to operate, and has the advantages of good volatility and relatively mild hydrolysis. However, the acid hydrolysis process alone is lengthy and inefficient. Ultrasonic technology generates energy with localized high temperature, high pressure, and cavitation effects, which can promote chemical reactions. This energy can break or weaken intermolecular and intramolecular hydrogen bonds in polysaccharides, promoting acid penetration into the polysaccharide structure, thus effectively catalyzing polysaccharide hydrolysis. Furthermore, ultrasound-assisted hydrolysis can shorten degradation time and improve experimental efficiency. Ultrasonic-assisted technology has been used in research on the rapid depolymerization of polysaccharides in food and natural products. 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] An exploration of hydrolysis of Ganoderma lucidum crude polysaccharides under specific conditions:

[0095] 700 μL of crude polysaccharide solution was placed in a liquid chromatography vial, and the same volume of trifluoroacetic acid (TFA) solution of different concentrations (3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L) was added. The vials were placed in an ultrasonic cleaner and hydrolyzed at different ultrasonic powers (245 W, 280 W, 315 W, 350 W), different temperatures (70℃, 80℃, 90℃, 100℃), and different times (5 min, 10 min, 15 min, 20 min, 25 min, 30 min). The hydrolysate was freeze-dried and finally washed with methanol and dried under nitrogen to remove TFA residues. The final product was a partially hydrolyzed Ganoderma lucidum polysaccharide.

[0096] result:

[0097] ① The effect of acid concentration:

[0098] The effect of TFA concentration on the degree of acid hydrolysis of Ganoderma lucidum polysaccharides was investigated. Figure 2A shows the scavenging rate of hydroxyl radicals by the Ganoderma lucidum polysaccharide depolymers at different acid concentrations, and Figure 2B shows the change of peak area of ​​representative depolymers as a function of acid concentration. It can be seen that when the acid concentration is in the range of 3–6 mol / mL, the scavenging rate of hydroxyl radicals by the sample first increases and then decreases with increasing TFA concentration. Simultaneously, Figure 2B shows that the peak area of ​​each representative chromatographic peak in the Ganoderma lucidum polysaccharide depolymers first increases and then decreases with increasing acid concentration. When the TFA concentration is 5 mol / mL, the hydroxyl radical scavenging rate of the sample is the best (Figure 2A), and the peak area of ​​each representative depolymer is also the highest (Figure 2B). The reason for this is likely that as the concentration of TFA gradually increases from low to high, the concentration of reducing monosaccharides and oligosaccharides produced by hydrolysis increases, thus gradually increasing the scavenging rate of hydroxyl radicals. However, when the acid concentration reaches a certain level, it promotes the degradation of Ganoderma lucidum polysaccharides into smaller structural fragments, leading to a significant decrease in the concentration of reducing monosaccharides and oligosaccharides. Simultaneously, these polysaccharides are easily converted into furfural and its derivatives, and the reducing functional groups are destroyed, resulting in a decrease in hydroxyl radical scavenging activity. Therefore, a TFA concentration of 5 mol / mL was selected for the acid hydrolysis of Ganoderma lucidum polysaccharides.

[0099] ② The effect of ultrasonic power:

[0100] As shown in Figure 2C, with increasing ultrasonic power, the scavenging rate of hydroxyl radicals in the sample initially increased and then decreased, reaching its highest level at an ultrasonic power of 315W. Figure 2D, showing the trend of representative characteristic peak areas in the depolymerized Ganoderma lucidum polysaccharides under different ultrasonic powers, also indicates that with increasing ultrasonic power, the peak areas of the representative characteristic peaks initially increased and then decreased, reaching their highest level at 315W. Preliminary inference is that appropriate ultrasonic power can accelerate the depolymerization of Ganoderma lucidum polysaccharides into reducing monosaccharides and oligosaccharides, thereby improving their hydroxyl radical scavenging activity. However, when the ultrasonic power reaches a certain level, it can damage the reducing groups such as carbonyl and aldehyde groups contained in the monosaccharides and oligosaccharides, leading to a decrease in hydroxyl radical scavenging activity. Therefore, an ultrasonic power of 315W was chosen for subsequent research.

[0101] ③ The effect of acidolysis temperature:

[0102] The effects of different temperatures (70℃, 80℃, 90℃, and 100℃) on the degree of acid hydrolysis and free radical scavenging activity of Ganoderma lucidum polysaccharides were investigated. The results are shown in Figures 2E and 2F. As can be seen from the figures, with increasing temperature, the free radical scavenging activity and the area of ​​representative chromatographic peaks of the depolymerized Ganoderma lucidum polysaccharides both showed a trend of first increasing and then decreasing. At 90℃, both the free radical scavenging activity and the chromatographic peak area reached their maximum values. When the temperature reached 100℃, 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 free radical scavenging activity was destroyed, and the free radical scavenging rate decreased significantly. Therefore, 90℃ was selected as the optimal acid hydrolysis temperature.

[0103] ④ Effect of acid hydrolysis time

[0104] Under optimized acid concentration, ultrasonic power, and temperature conditions, the effect of acidolysis time was investigated, and the results are shown in Figures 2G and 2H. Figure 2G shows that within the time range of 5–30 min, the hydroxyl radical scavenging rate initially increased and then slowly decreased with increasing ultrasonic time. Furthermore, Figure 2H shows that the peak areas of each characteristic peak fluctuated with increasing ultrasonic time. To ensure good free radical scavenging activity of the depolymerized Ganoderma lucidum polysaccharide while improving acidolysis efficiency, a final ultrasonic acidolysis time of 10 min was selected.

[0105] Example 4

[0106] ESI-TOF / MS identification of partially acid-hydrolyzed polymers of Ganoderma lucidum polysaccharides

[0107] Different varieties of Ganoderma lucidum crude polysaccharide solutions were prepared according to the method in Example 1. Ganoderma lucidum polysaccharide acid hydrolysis samples were prepared according to the optimized acid hydrolysis conditions. Liquid chromatography analysis was performed according to the chromatographic conditions described in (4) above, and chromatograms were obtained using a CAD detector. First, a preliminary experiment was conducted using one Ganoderma lucidum crude polysaccharide acid hydrolysis sample. It was found that well separated chromatographic peaks could be obtained using the optimized acid hydrolysis conditions. Subsequently, acid hydrolysis samples of four varieties of Ganoderma lucidum polysaccharides were prepared and corresponding liquid chromatography analyses were performed. The chromatograms of depolymerized Ganoderma lucidum polysaccharides of different varieties are shown in Figure 3.

[0108] The eluent was fed into ESI-Q-TOF / MS for high-resolution mass spectrometry analysis. Using UPLC-CAD-ESI-Q-TOF / MS, and through comparative analysis with literature, 29 compounds were preliminarily identified, 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] The mass spectrum of Ganoderma lucidum disaccharide was analyzed, as shown in Figure 4. Fragment ions at m / z 341.1084 [MH]- and m / z 387.1138 [M+COOH]- were observed in the MS primary mass spectrum of the disaccharide. Simultaneously, fragment ions at m / z 341.1084 [MH]- were also observed. - The MS / MS mass spectrum of the parent ion shows ion fragments at m / z 179.0539 and m / z 161.0598, with m / z 179.0539 representing the parent ion having lost a sugar residue (C6H). 12 The ion fragments following O6, 161.0598, are obtained by losing one sugar residue and then one water molecule from the parent ion. m / z 455.1003 is the fragment ion obtained by adding a parent ion with a mass of 113.9919. The compound added is unclear, but this molecular weight appears in most monosaccharide and oligosaccharide fragment ions obtained by this method, so it is marked in Figure 4(A).

[0112] The mass spectrum of Ganoderma lucidum trisaccharides was analyzed, as shown in Figure 5. The primary mass spectrum showed m / z 503.1603 [MH]. - m / z 549.1659 [M+COOH] - Fragment ions, with m / z 503.1603 [MH] - The parent ion undergoes glycosidic bond cleavage, losing a hexose residue (162n), yielding an ionic fragment with m / z 179.0157. Simultaneously, the parent ion undergoes ring-opening cleavage, yielding fragment ion 383.1274. 2.4 A3), 221.0637 ( 2.4 A2) ion fragments.

[0113] Example 5

[0114] Spectral-effect correlation analysis and fingerprint spectrum establishment:

[0115] (1) Grey relational analysis

[0116] Fifty-two batches of Ganoderma lucidum crude polysaccharide solutions were prepared into sample solutions using optimized acid hydrolysis conditions. Liquid chromatography (LC) analysis was performed on these 52 batches of samples, resulting in LC chromatograms, as shown in Figure 6. Analysis of the LC chromatograms identified 29 common peaks. Integrating these 29 common peaks yielded their corresponding peak areas. Grey relational analysis was performed between the hydroxyl radical scavenging rate of the Ganoderma lucidum samples and the peak areas of the common peaks. The results are shown in Table 3. The table shows that the correlation between the peak areas of the selected characteristic peaks and the hydroxyl radical scavenging rate is greater than 0.6. When the correlation coefficient r > 0.6, it indicates a correlation between the two; the larger the r, the stronger the correlation. The 29 peaks with r > 0.8 all exhibited high correlations, indicating that the hydroxyl radical scavenging rate of Ganoderma lucidum polysaccharides is the result of the synergistic effect of different chemical components.

[0117] Table 3. Grey Relational Analysis Results

[0118]

[0119] (2) Partial Least Squares Regression Analysis

[0120] Partial least squares regression (PLSR) generally assumes that when VIP > 1, the independent variable has significant importance in explaining the dependent variable. PLSR was used to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharide with 29 chemical components, as shown in Figure 7. PLSR screened out 12 common activity peaks: peak25, peak27, peak16, peak14, peak26, peak1, peak8, peak29, peak3, peak13, peak20, and peak17. Using these 12 chemical components as common peaks, a fingerprint spectrum based on the antioxidant active components of Ganoderma lucidum was established.

[0121] (3) Multivariate statistical analysis

[0122] Unsupervised PCA analysis of the bioactive polysaccharide fingerprints of different Ganoderma lucidum varieties was performed using SIMCA-P (14.1, Umetrics, Sweden) software, allowing for a more intuitive observation of the differences between these varieties. As shown in Figure 8A, the distances between samples from different Ganoderma lucidum varieties are relatively large, indicating significant differences in polysaccharide content. The results demonstrate that PCA analysis can distinguish between different Ganoderma lucidum varieties to a certain extent. Supervised PLS-DA analysis of different Ganoderma lucidum varieties was then performed using the common peaks of the bioactive polysaccharide fingerprints as variables, as shown in Figure 8B. The explained value of the Y matrix was 0.971, the explained value of the X matrix was 0.973, and the predictive power Q2 was 0.954, indicating strong predictive ability of the model.

[0123] Importance projection (VIP) analysis (Figure 8C) showed that Peak29 (DP3), Peak 20 (unknown), Peak 25 (unknown), and Peak 17 (unknown) (VIP>1) contributed significantly to differentiation. The content distribution of these four components in different varieties is shown in Figure 9. Specifically, *Ganoderma lucidum* contained higher levels of Peak17 (unknown), Peak20 (unknown), and Peak 25 (unknown); *Ganoderma sinense* contained a higher level of Peak29 (DP3); *Ganoderma rubrum* contained a lower level of Peak25 (unknown); and *Ganoderma purpurea* contained a lower level of Peak20 (unknown). One-way ANOVA showed significant differences among the four components in different varieties (P<0.05), indicating that they are likely to serve as quality markers for different varieties of *Ganoderma*.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum based on the antioxidant active ingredients of Ganoderma lucidum, characterized in that, Includes the following steps: (1) After pulverizing the dried fruiting bodies of different varieties of Ganoderma lucidum, water extraction and alcohol precipitation were carried out to obtain crude polysaccharide of Ganoderma lucidum. (2) Ganoderma lucidum crude polysaccharide was hydrolyzed under limited conditions to obtain Ganoderma lucidum polysaccharide hydrolysis products; (3) The chemical components contained in the hydrolysate of Ganoderma lucidum polysaccharide were measured and determined by hydrophilic chromatography-electrospray detector-electrospray mass spectrometry (UPLC-CAD-ESI-Q-TOF / MS), and 29 chemical components were preliminarily identified. (4) The antioxidant properties of Ganoderma lucidum crude polysaccharide and the peak areas of the 29 chemical components screened in step (3) were analyzed by grey relational analysis to determine that the 29 chemical components were antioxidant active ingredients. (5) Partial least squares regression 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 spectrum based on the antioxidant active components of Ganoderma lucidum.

2. The construction method as described in claim 1, characterized in that, In step (1), the method for obtaining crude Ganoderma lucidum polysaccharides by water extraction and alcohol precipitation includes: S1. After pulverizing the dried fruiting bodies of different varieties of Ganoderma lucidum, pass them through a 30-50 mesh sieve, add deionized water at 85-95℃, and extract them by ultrasonic extraction at 85-95℃ for 35-45 minutes. S2. Centrifuge to obtain the supernatant, add ethanol, let stand for 10-14 hours, centrifuge to collect the precipitate, and dry to obtain Ganoderma lucidum crude polysaccharide.

3. The construction method as described in claim 2, characterized in that, In step S1, the ratio of dried Ganoderma lucidum fruiting body powder to deionized water is 1:(14.5~15.5)g / mL, preferably 1:15g / mL; Alternatively, in step S1, the power of ultrasonic extraction is 300-350W, preferably 315W; Alternatively, in step S2, when centrifuging to obtain the supernatant, the rotation speed is 4300-4800 rpm, preferably 4500 rpm; the centrifugation time is 12-18 min, preferably 15 min. Alternatively, 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). Alternatively, in step S2, ethanol is added and the mixture is allowed to stand at 3–5°C for 10–14 hours. Alternatively, in step S2, when centrifuging to collect the precipitate, 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 to evaporate the precipitate in a water bath at 75-85°C.

4. The construction method as described in claim 1, characterized in that, In step (2), the limiting conditions are: Ganoderma lucidum crude polysaccharide was dissolved in water to obtain Ganoderma lucidum crude polysaccharide solution; Trifluoroacetic acid was added to the crude polysaccharide solution of Ganoderma lucidum, and the solution was subjected to ultrasonic hydrolysis.

5. The construction method as described in claim 4, characterized in that, The concentration of trifluoroacetic acid is 3–6 mol / L, preferably 5 mol / L; Alternatively, the ultrasonic power is 245–350W, preferably 315W; the ultrasonic temperature is 70–100℃, preferably 90℃; and the ultrasonic time is 5–30min, preferably 10min.

6. The construction method as described in claim 1, characterized in that, In step (3), the chromatographic conditions are as follows: Column: Xbridge TM BEH Amide column (2.1×150mm, 2.5μm); mobile phase A was an aqueous solution (0.8% formic acid + 20M ammonium formate), mobile phase B was an 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, the flow rate during detection is 0.2–0.3 mL / min, more preferably 0.25 mL / min; Preferably, the injection volume during detection is 9–12 μL, more preferably 10 μL; Preferably, during detection, the column temperature of the chromatographic column is 20–30°C, more preferably 25°C.

7. The construction method as described in claim 1, characterized in that, In step (3), the ESI-TOF / MS conditions are: Operating in both positive and negative ion modes, the sprayer pressure 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 temperature of the drying gas is 200–240 °C, preferably 200 °C; the capillary voltage is 3400–3600 V, preferably 3500 V, in positive ion mode; and 2900–3200 V, preferably 3000 V, in negative ion mode; the mass-to-charge ratio (m / z) range is 100–2000.

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

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

10. A method for identifying a variety of Ganoderma lucidum, characterized in that, include: Obtain fingerprints of Ganoderma lucidum samples based on its antioxidant active components; Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the fingerprint spectra of Ganoderma lucidum (red Ganoderma), Ganoderma sinense (purple Ganoderma), Ganoderma lingulata (tree tongue Ganoderma), Ganoderma lucidum (black Ganoderma), and sample Ganoderma lucidum based on the antioxidant active components of Ganoderma lucidum, so as to identify the Ganoderma lucidum varieties.

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