A method for constructing a fingerprint spectrum based on ganoderma lucidum antioxidant active ingredients and application thereof

Abstract

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 and partial least squares discriminant analysis 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.

Description

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 The effect of different varieties of Ganoderma lucidum polysaccharides on scavenging hydroxyl free radicals;

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

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

[0022] Figure 4 Mass spectrum analysis of Ganoderma disaccharides, where A is the primary mass spectrum of the disaccharide and B is the secondary mass spectrum of the disaccharide;

[0023] Figure 5Mass 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 The liquid phase chromatograms are for 52 batches of samples;

[0025] Figure 7 Filter the results for PLSR;

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

[0027] Figure 9 The peak areas are 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, 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 test samples

[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 TM BEH 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 polysaccharides from different varieties of Ganoderma lucidum to scavenge hydroxyl free radicals was determined using an enzyme-linked immunosorbent assay (ELISA) reader. The results are as follows: Figure 1 As shown, the scavenging rate of the positive control at 1 mg / mL was 99.8182%, and the scavenging rates of hydroxyl radicals by different varieties of Ganoderma lucidum polysaccharides at 1 mg / mL ranged from 20% to 50%. Among them, Ganoderma lucidum had the highest hydroxyl radical scavenging rate at 43.2840%, while Ganoderma applanatum had the lowest at 23.8062%. All four varieties of Ganoderma lucidum possessed a certain hydroxyl radical scavenging ability, but the hydroxyl radical scavenging rates of polysaccharides varied among 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 2 A represents the scavenging rate of hydroxyl radicals by Ganoderma lucidum polysaccharide depolymers at different acid concentrations. Figure 2 B is a graph showing the variation of the peak area of ​​a representative depolymerized product in Ganoderma lucidum polysaccharide depolymerization with acid concentration. It can be seen that when the acid concentration is in the range of 3–6 mol / mL, the scavenging rate of the sample for hydroxyl radicals first increases and then decreases with increasing TFA concentration. Meanwhile, from... Figure 2 As can be seen from B, the peak areas of each representative chromatographic peak in the Ganoderma lucidum polysaccharide depolymerization product showed a trend of first increasing and then decreasing with increasing acid concentration. The hydroxyl radical scavenging rate of the sample was best when the TFA concentration was 5 mol / mL. Figure 2 A), and the peak areas of each representative depolymerization product are also the highest. Figure 2(B) 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 the scavenging activity against hydroxyl radicals. 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] like Figure 2 As shown in Figure C, with increasing ultrasonic power, the scavenging rate of hydroxyl radicals by the sample initially increases and then decreases, with the highest scavenging rate occurring at an ultrasonic power of 315W. From... Figure 2 The trend diagram of the representative characteristic peak area in the depolymerized Ganoderma lucidum polysaccharides under different ultrasonic powers also shows that: with the increase of ultrasonic power, the peak area of ​​the representative characteristic peak first increases and then decreases, with the peak area being the highest when the ultrasonic power is 315W. It is preliminarily inferred 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 will destroy the reducing groups such as carbonyl and aldehyde groups contained in the monosaccharides and oligosaccharides, resulting in a decrease in hydroxyl radical scavenging activity. Therefore, an ultrasonic power of 315W was selected 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 as follows: Figure 2 Figures E and F show that, as temperature increases, the free radical scavenging activity and representative chromatographic peak area of ​​the Ganoderma lucidum polysaccharide depolymerization product both show a trend of first increasing and then decreasing. At 90℃, both the free radical scavenging activity and the chromatographic peak area reach their maximum values. When the temperature reaches 100℃, the movement of polysaccharide molecules intensifies, the combined effects of temperature, ultrasound, and acid hydrolysis are strengthened, the degree of polysaccharide degradation increases, the reducing sugar structure with hydroxyl free radical scavenging activity is destroyed, and the free radical scavenging rate is significantly reduced. Therefore, 90℃ is selected as the optimal acid hydrolysis temperature.

[0103] ④ Effect of acid hydrolysis time

[0104] The effect of acidolysis time was investigated under optimized acid concentration, ultrasonic power, and temperature conditions. Results Figure 2 As shown in G and H. From Figure 2G shows that, within the timeframe of 5–30 minutes, the hydroxyl radical scavenging rate initially increased and then slowly decreased with increasing sonication time. Furthermore, from… Figure 2 As can be seen from H, the peak areas of each characteristic peak fluctuate to some extent with the increase of ultrasonic time. To ensure that the depolymerized 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] 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. Acid hydrolysis samples of Ganoderma lucidum polysaccharides were prepared according to optimized acid hydrolysis conditions. Liquid chromatography analysis was performed according to the aforementioned chromatographic conditions (4), 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 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 the depolymerized Ganoderma lucidum polysaccharides from different varieties are shown below. Figure 3 As shown.

[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]

[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 the disaccharide. - m / z387.1138[M+COOH] - Fragment ions, with a m / z of 341.1084 [MH]. - 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). 12The ion fragments following O6, with a molecular weight of 161.0598, are obtained by losing one sugar residue and then one water molecule from the parent ion. The fragment ion with m / z 455.1003 is 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 using this method. Figure 4 It is marked in (A).

[0113] Analysis of the mass spectrum of Ganoderma lucidum trisaccharides, such as... Figure 5 As shown, the first-order mass spectrum contains 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.

[0114] Example 5

[0115] Spectral-effect correlation analysis and fingerprint spectrum establishment:

[0116] (1) Grey relational analysis

[0117] Fifty-two batches of Ganoderma lucidum crude polysaccharide solutions were prepared into sample solutions using optimized acid hydrolysis conditions. Liquid chromatography analysis was then performed on these 52 batches of samples, and the final liquid chromatography chromatograms for these 52 batches are shown below. Figure 6 As shown, the liquid chromatography chromatogram was analyzed, and 29 common peaks were identified. The peak areas of these 29 common peaks were integrated, and a grey relational analysis was performed on the hydroxyl radical scavenging rate of the Ganoderma lucidum sample and the peak areas of the common peaks. The results are shown in Table 3. It can be observed from the table that the correlation between the peak area 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 that there is a correlation between the two; the larger the r, the stronger the correlation. The 29 peaks with r > 0.8 all showed high correlation, indicating that the hydroxyl radical scavenging rate of Ganoderma lucidum polysaccharides is the result of the synergistic effect of different chemical components.

[0118] Table 3. Grey Relational Analysis Results

[0119]

[0120] (2) Partial Least Squares Regression Analysis

[0121] Partial least squares regression (PLSR) generally assumes that when VIP > 1, the independent variable is significantly important in explaining the dependent variable. This study used PLSR to correlate the antioxidant activity of Ganoderma lucidum crude polysaccharide with 29 chemical components, such as... Figure 7 As shown, 12 common active peaks were screened using PLSR, namely 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.

[0122] (3) Multivariate statistical analysis

[0123] 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. Figure 8 As shown in Figure A, the distances between samples of different Ganoderma lucidum varieties are relatively large, indicating significant differences in the polysaccharide content among different varieties. The results indicate that PCA analysis can distinguish between different Ganoderma lucidum varieties to a certain extent. Using the common peak of the active polysaccharide fingerprint as a variable, supervised PLS-DA analysis was performed on samples of different Ganoderma lucidum varieties, as shown in Figure A. Figure 8 As shown in Figure B, the Y matrix explains 0.971, the X matrix explains 0.973, and the predictive power Q2 is 0.954, indicating that the model has strong predictive power.

[0124] Importance projection (VIP) analysis Figure 8 C) indicates that Peak29 (DP3), Peak 20 (unknown), Peak 25 (unknown), and Peak 17 (unknown) (VIP>1) contribute significantly to the differentiation. The content distribution of these four components in different varieties is shown below. Figure 9 As shown in the figure, the content of peak17 (unknown), peak20 (unknown), and peak25 (unknown) in Ganoderma lucidum were relatively high; the content of peak29 (DP3) in Ganoderma lucidum var. cinnamon was relatively high; the content of peak25 (unknown) in Ganoderma lucidum var. aurantiacus was relatively low; and the content of peak20 (unknown) in Ganoderma lucidum var. purpurea was relatively low. 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 identifying different varieties of Ganoderma lucidum.

[0125] 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 polysaccharides 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 ingredients of Ganoderma lucidum. The specified conditions are as follows: dissolve Ganoderma lucidum crude polysaccharide in water to obtain Ganoderma lucidum crude polysaccharide solution; add trifluoroacetic acid to the Ganoderma lucidum crude polysaccharide solution and perform ultrasonic hydrolysis; The concentration of trifluoroacetic acid is 3-6 mol / L; the ultrasonic power is 245-350 W; the ultrasonic temperature is 70-100℃; and the ultrasonic time is 5-30 min. In step (3), the chromatographic conditions are as follows: Column: Xbridge TM BEH Amide column, 2.1×150 mm, 2.5 μm; mobile phase A is 0.8% formic acid 20 M ammonium formate aqueous solution, mobile phase B is 0.8% formic acid acetonitrile solution, gradient elution; The gradient elution conditions included: 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; and 51–60 min, 60% B. During detection, the flow rate is 0.2–0.3 mL / min; the injection volume is 9–12 μL; and the column temperature is 20–30 ℃. The ESI-TOF / MS conditions were as follows: operating in both positive and negative ion modes; nebulizer pressure of 1.8–2.2 Bar; dry gas flow rate of 7–10 mL / min; dry gas temperature of 200–240 °C; capillary voltage of 3400–3600 V in positive ion mode; and capillary voltage of 2900–3200 V in negative ion mode; with a mass-to-charge ratio (m / z) range of 100–2000. 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.

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 min. S2. Centrifuge to obtain the supernatant, add ethanol, let stand for 10-14 h, 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; Alternatively, in step S1, the power of ultrasonic extraction is 300~350w; Alternatively, in step S2, when centrifuging to obtain the supernatant, the rotation speed is 4300~4800 rpm; the centrifugation time is 12~18 min. Alternatively, in step S2, the mass-volume concentration of ethanol is 95%, and the final concentration of ethanol in the mixed solution is 78%~82%; Alternatively, in step S2, ethanol is added and the mixture is allowed to stand at 3-5 °C for 10-14 h. Alternatively, in step S2, when centrifuging to collect the precipitate, the rotation speed is 4300~4800 rpm; the centrifugation time is 12~18 min.

4. The construction method as described in claim 3, characterized in that, The ratio of dried Ganoderma lucidum fruiting body powder to deionized water is 1:15 g / mL.

5. The construction method as described in claim 3, characterized in that, The power of the ultrasonic extraction was 315 W.

6. The construction method as described in claim 3, characterized in that, When centrifuging to obtain the supernatant, the rotation speed was 4500 rpm; the centrifugation time was 15 min.

7. The construction method as described in claim 3, characterized in that, The final volume concentration of ethanol in the mixed solution is 80%.

8. The construction method as described in claim 3, characterized in that, When collecting the precipitate by centrifugation, the rotation speed is 4500 rpm; the centrifugation time is 15 min.

9. The construction method as described in claim 1, characterized in that, In step S2, the drying method is to evaporate the precipitate in a water bath at 75~85 ℃.

10. The construction method as described in claim 1, characterized in that, The concentration of trifluoroacetic acid is 5 mol / L; Alternatively, the ultrasonic power is 315 W; the ultrasonic temperature is 90 ℃; and the ultrasonic time is 10 min.

11. The construction method as described in claim 1, characterized in that, The flow rate was 0.25 mL / min.

12. The construction method as described in claim 1, characterized in that, The injection volume was 10 μL.

13. The construction method as described in claim 1, characterized in that, The column temperature of the chromatographic column is 25 ℃.

14. The construction method as described in claim 1, characterized in that, The sprayer pressure is 2.0 Bar.

15. The construction method as described in claim 1, characterized in that, The flow rate of the drying gas is 8 mL / min.

16. The construction method as described in claim 1, characterized in that, The temperature of the drying gas is 200 ℃.

17. The construction method as described in claim 1, characterized in that, The capillary voltage is 3500 V in positive ion mode.

18. The construction method as described in claim 1, characterized in that, The capillary voltage is 3000 V in negative ion mode.

19. 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.

20. A method for identifying a variety of Ganoderma lucidum, characterized in that, include: The fingerprint spectrum of Ganoderma lucidum samples based on its antioxidant active ingredients was obtained using the construction method described in claim 1. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the fingerprint spectra of Ganoderma lucidum, Ganoderma sinense, Ganoderma lingulata, Ganoderma niger, and Ganoderma sinense samples based on their antioxidant active components, respectively, to identify Ganoderma lucidum varieties.

Images