Morchella esculenta extract as well as extraction method and application thereof

Abstract

The invention discloses a morchella esculenta extract as well as an extraction method and application thereof, and belongs to the technical field of plant medicine product separation. The invention relates to a specific extraction method of phenolic acid of morchella, through the synergistic effect of low-temperature plasma pretreatment, targeted enzymolysis and deep eutectic solvent extraction, the extraction rate of phenolic acid is remarkably improved, particularly, specific extraction of chlorogenic acid can be realized, and the purity of chlorogenic acid is greater than 85%. The process is environment-friendly and efficient, and the obtained product has important application value in the fields of inflammation resistance and oxidation resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of separation of plant pharmaceutical products, and specifically relates to morel mushroom extract, its extraction method and uses. Background Art

[0002] Morel mushroom is a precious edible mushroom variety, named after the uneven surface of its cap, which resembles a sheep's stomach. Morel mushroom has high nutritional value and is rich in various bioactive substances such as protein, phenolic acids, polysaccharides, and sterols. In particular, phenolic acids have effects such as antibacterial, anti-inflammatory, antioxidant, anti-cancer, blood sugar and lipid lowering, and treatment of cardiovascular diseases. Although morel mushroom phenolic acids have good physiological activities, their extraction efficiency has been relatively low, restricting the research on the structure-activity relationship of morel mushroom phenolic acids and their in-depth development and utilization. Existing technologies mostly focus on the extraction of polysaccharides, umami substances or antibacterial components, and there is less research on the specific extraction of phenolic acids in morel mushrooms.

[0003] Morel mushroom is rich in various active ingredients such as polysaccharides, polyphenols, and sterols. Currently, the extraction technologies for morel mushroom mainly focus on the extraction of polysaccharides and total polyphenols, but there is less research on the specific extraction of phenolic acids (such as chlorogenic acid, caffeic acid, etc.). For example, although patent CN113150181B involves the extraction of morel mushroom polysaccharides, it does not solve the problem of specific extraction of phenolic acids. Among them, chlorogenic acid has various pharmacological effects such as cholagogic, antibacterial, antiviral, blood pressure lowering, increasing white blood cells, and exciting the central nervous system. Chlorogenic acid is a natural specific inhibitor of hyaluronidase (HAase) and Gle-6-p transferase, and is also a scavenger of selective hydroxyl radicals and peroxyl radicals, and can be used as an important raw material for industries such as health products, foods, drugs, and cosmetics.

[0004] The existing technologies for extracting morel mushroom phenolic acids have the following defects: First, the targeting is insufficient. Conventional solvent extraction methods (such as ethanol, methanol) can extract polyphenolic substances, but cannot selectively enrich phenolic acids, and the solvents are highly toxic. Second, the efficiency of cell wall disruption is low. It is difficult to effectively release bound phenolic acids from the cell wall of morel mushroom using traditional enzymatic hydrolysis (cellulase, pectinase). Third, the environmental friendliness is poor. The use of high-concentration organic solvents increases environmental pollution and purification costs.

[0005] Therefore, there is an urgent need to develop a phenolic acid specific extraction process with high efficiency, environmental friendliness, and strong targeting. Summary of the Invention

[0006] Regarding the above-mentioned extraction method of phenolic acids from Morchella, it has technical problems such as insufficient pertinence, low efficiency of cell wall destruction, and difficulty in specifically releasing phenolic acids by traditional enzymatic hydrolysis. The present invention proposes a specific extraction method of phenolic acids from Morchella. The method of the present invention significantly improves the extraction efficiency of phenolic acids and the purity of chlorogenic acid through the synergistic effect of low-temperature plasma pretreatment, targeted enzymatic hydrolysis, and deep eutectic solvent extraction. This process is environmentally friendly and efficient, and the obtained product has important application value in the fields of anti-inflammatory and antioxidant.

[0007] On the one hand, the present invention provides an extract of Morchella, and the extract of Morchella includes phenolic acids, and the purity of chlorogenic acid in the phenolic acids is greater than 85%.

[0008] On the other hand, the present invention proposes a method for extracting phenolic acids from an extract of Morchella. The method for extracting phenolic acids from Morchella includes the following steps:

[0009] S1. Pretreatment: Treat Morchella with low-temperature plasma;

[0010] S2. Targeted enzymatic hydrolysis: Add cellulase, pectinase, and β-glucosidase, and perform enzymatic hydrolysis at pH 4.5 - 5.5 and a temperature of 40 - 50 °C for 2 - 4 hours;

[0011] S3. Deep eutectic solvent extraction: Use a deep eutectic solvent as an extraction medium and perform ultrasonic-assisted extraction at 50 - 60 °C;

[0012] S4. Purification: Purify the extract by macroporous resin adsorption to obtain an extract of phenolic acids from Morchella.

[0013] Further, in step S1, the Morchella can be crushed and passed through a 100-mesh sieve; or directly cut into pieces for use.

[0014] Further, in step S1, when performing the low-temperature plasma treatment, the plasma is argon plasma.

[0015] Further, the low-temperature plasma treatment includes: using an argon low-temperature plasma reactor, treating at a temperature below 40 °C for a treatment time of 5 - 15 min to further destroy the cell wall and ensure the full release of phenolic acids.

[0016] Further, in step S2, the concentration of the cellulase is 0.5 - 2 U / g.

[0017] Further, in step S2, the concentration of the pectinase is 1 - 2 U / g.

[0018] Further, in step S2, the concentration of the β-glucosidase is 1 - 3 U / g.

[0019] Further, in step S3, the deep eutectic solvent is a mixed solvent with a choline to lactic acid molar ratio of 1:2 - 1:3.

[0020] Further, in step S3, the power of the ultrasonic wave is 300 W, the frequency is 40 kHz, and the ultrasonic time is 30 - 60 minutes.

[0021] Further, in step S4, the macroporous resin is at least one of macroporous adsorption resins of AB - 8, D101, D100, S - 8, LS - 600, HPD100, HPD300, NKA - 2, NKA - 9, or HZ806 type.

[0022] The present invention also provides morchella phenolic acid obtained by the above - mentioned method.

[0023] The present invention provides the application of the morchella extract prepared by the above - mentioned method in the preparation of anti - inflammatory and antioxidant functional foods or drugs.

[0024] The beneficial effects of the present invention are as follows: Through the synergistic effects of low - temperature plasma pretreatment, targeted enzymatic hydrolysis, and deep eutectic solvent extraction, the present invention significantly improves the extraction efficiency and purity of phenolic acids. Compared with traditional solvents, the extraction rate of phenolic acids is increased by 60%. The deep eutectic solvent replaces ethanol or methanol, reducing toxicity and solvent residues. β - glucosidase releases bound phenolic acids, significantly increasing the extraction rate of the target product. After purification by macroporous resin, the purity of chlorogenic acid is specifically increased, and the purity is greater than 85%. The morchella extract obtained by the present invention has important application values in the fields of anti - inflammation and antioxidant in functional foods (such as antioxidant beverages, anti - inflammatory food additives); and the pharmaceutical field (such as ulcerative colitis treatment preparations). Detailed Embodiments

[0025] For ease of understanding, the present application will be described more comprehensively below, and preferred embodiments of the present application are given. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive. For technologies or conditions not specified in the embodiments, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through the market.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0027] Determination method for total phenolic acids content in Morchella esculenta:

[0028] 1) Weigh 0.01 g of tannic acid and make up the volume to 10 mL with distilled water to prepare a 1000 ppm tannic acid solution. Then dilute the tannic acid stock solution with distilled water to 0, 50, 100, 150, 200, 250 ppm tannic acid dilution solutions. Use the Folin-phenol assay. Pipette 50 μL of different concentration tannic acid dilution solutions into 5 mL centrifuge tubes, then add 50 μL of Folin-phenol reagent, shake well and react for 3 min (with color change), then add 1 mL of 0.7 mol / L Na2CO3 (experimental group), using distilled water instead of Folin-phenol reagent as the blank. After reacting at room temperature in the dark for 30 min, pipette 200 μL of samples with different dilution concentrations into a 96-well microplate, repeat 3 times; pipette 200 μL of the blank group solution into a 96-well microplate, and measure the absorbance at a wavelength of 750 nm. Plot the standard curve with the dilution concentration as the abscissa and the absorbance as the ordinate;

[0029] 2) Pipette 50 μL of different sample solutions into 5 mL centrifuge tubes, then add 50 μL of Folin-phenol reagent, shake well and react for 3 min (with color change), then add 1 mL of 0.7 mol / L Na2CO3. After reacting at room temperature in the dark for 30 min, pipette 200 μL of different sample solutions into a 96-well microplate, repeat 3 times, and measure the absorbance at a wavelength of 750 nm. Calculate the total phenol content of the samples according to the tannic acid standard curve.

[0030] The total phenol content of the samples is calculated according to the following formula:

[0031] X = ρ × V × N / m

[0032] Where: X is the extraction amount of total phenolic acids, mg / g; ρ is the mass concentration of total phenolic acids calculated according to the standard curve, mg / mL; V is the volume of the test solution, mL; N is the dilution factor of the test solution; m is the mass of the test sample, g.

[0033] In the present invention, before the DPPH antioxidant experiment, first measure the OD value of the blank 96-well microplate, and the OD value of the blank 96-well microplate shall not exceed 0.06. O2 – Before the antioxidant experiment, the OD value of the blank 96-well microplate shall not exceed 0.4.

[0034] Determination method for the purity of chlorogenic acid in phenolic acid extracts:

[0035] Instruments: Agilent 1260 type high performance liquid chromatograph, ultraviolet-visible spectrophotometer (Agilent Technologies, Inc., USA); chromatographic column (Agilent PoroShell 120 EC-C18 (50 mm × 4.6 mm, 2.7 μm).

[0036] Reagents: Chlorogenic acid (purity 98.9%, Shanghai Yuanye Bio-Technology Co., Ltd.), water (ultrapure water).

[0037] Mobile phase: 0.1% acetic acid (A) - methanol (B); flow rate 0.8 - 1.5 mL / min; column temperature 25 - 35 °C; detection wavelength 330 nm.

[0038] Example 1

[0039] Pretreatment: Take 100 g of Morchella esculenta, and use an argon low-temperature plasma treatment device to treat the sample. The argon flow rate is controlled at 5 L / min, the treatment time is 10 minutes, and the temperature is kept below 40 °C.

[0040] Targeted enzymatic hydrolysis: Mix the pretreated powder with a buffer solution of pH 5.0 (solid-liquid ratio 1:20), add cellulase (1.5 U / g), pectinase (1.5 U / g) and β-glucosidase (2 U / g), and carry out enzymatic hydrolysis at 45 °C for 3 hours.

[0041] Deep eutectic solvent extraction: Mix the enzymatic hydrolysis product with choline-lactic acid at a ratio of 1:2, carry out ultrasonic extraction at 60 °C (power 300 W) for 40 minutes, and centrifuge to obtain the supernatant.

[0042] Purification: The supernatant is adsorbed by AB-8 resin, eluted with 30% ethanol, concentrated and dried to obtain a phenolic acid extract. The total phenolic acid is 12.5 mg / g, and among them, the purity of chlorogenic acid is 85%.

[0043] Comparative experiment:

[0044] Traditional solvent method: Take 100 g of Morchella esculenta and extract it with an ethanol extraction solvent. The supernatant is separated and retained, the residue is added with 5 mol / L NaOH aqueous solution for re-extraction, and the combined extraction supernatant is concentrated and dried to obtain a phenolic acid extract. The purity of chlorogenic acid extracted by ethanol is only 62%, and the extraction rate is 7.8 mg / g.

[0045] The present invention: The purity of chlorogenic acid is 85%, the extraction rate is 12.5 mg / g, an increase of 60%.

[0046] Example 2

[0047] Pretreatment: Take 100 g of dry powder of Morchella esculenta, and use an argon low-temperature plasma treatment device to treat the sample. The argon flow rate is controlled at 4 L / min, the treatment time is 15 minutes, and the temperature is kept below 40 °C.

[0048] Targeted enzymatic hydrolysis: Mix the pretreated powder with a buffer solution of pH 5.0 (solid-liquid ratio 1:25), add cellulase (1.5 U / g), pectinase (1.0 U / g) and β-glucosidase (1 U / g), and carry out enzymatic hydrolysis at 45 °C for 3 hours.

[0049] Deep eutectic solvent extraction: The enzymatic hydrolysate was mixed with choline-lactic acid at a ratio of 1:2.5, and ultrasonic extraction was carried out at 60 °C (power 300 W) for 40 minutes, and the supernatant was obtained by centrifugation.

[0050] Purification: The supernatant was adsorbed by AB-8 resin, eluted with 30% ethanol, concentrated and dried to obtain a phenolic acid extract. The total phenolic acid was 12.6 mg / g, and among them, the purity of chlorogenic acid was 87%.

[0051] Example 3

[0052] Pretreatment: 100 g of dried Morchella powder was taken and treated with an argon low-temperature plasma treatment device. The argon flow rate was controlled at 5 L / min, the treatment time was 5 minutes, and the temperature was kept below 40 °C.

[0053] Targeted enzymatic hydrolysis: The pretreated powder was mixed with a buffer solution at pH 5.0 (solid-liquid ratio 1:20), and cellulase (0.5 U / g), pectinase (1.0 U / g) and β-glucosidase (2 U / g) were added, and enzymatic hydrolysis was carried out at 45 °C for 3 hours.

[0054] Deep eutectic solvent extraction: The enzymatic hydrolysate was mixed with choline-lactic acid at a ratio of 1:2, and ultrasonic extraction was carried out at 60 °C (power 300 W) for 40 minutes, and the supernatant was obtained by centrifugation.

[0055] Purification: The supernatant was adsorbed by D101 resin, eluted with 30% ethanol, concentrated and dried to obtain a phenolic acid extract. The total phenolic acid was 12 mg / g, and among them, the purity of chlorogenic acid was 86%.

[0056] Verification Example 1

[0057] Referring to the preparation method in Example 2, the low-temperature plasma treatment was not used in the pretreatment step, and ethanol extraction was used instead, and the others remained unchanged. A phenolic acid extract was prepared. The total phenolic acid was 1.05 mg / g, and among them, the purity of chlorogenic acid was 33%.

[0058] Verification Example 2

[0059] Referring to the preparation method in Example 3, β-glucosidase was not added in the targeted enzymatic hydrolysis step, and the others remained unchanged. A phenolic acid extract was prepared. The total phenolic acid was 3.7 mg / g, and among them, the purity of chlorogenic acid was 73%.

[0060] Verification Example 3

[0061] Referring to the preparation method in Example 2, the effect of macroporous resin on the purity of chlorogenic acid in the purification step was investigated, and the others remained unchanged. The results are shown in the following table.

[0062]

[0063]

[0064] After purification by macroporous resin, the purity of chlorogenic acid was significantly improved.

[0065] Example 4

[0066] Determination of in vitro antioxidant activity

[0067] Experimental method

[0068] The experimental groups were set as 3 groups: the sample group, the control group, and the blank group.

[0069] Sample group: Add 100 μL of DPPH solution (0.2 mM) to a 96-well plate, and then add 50 μL of the sample to be tested (the phenolic acid extract of Morchella esculenta prepared in Examples 1-3) at 600 μM respectively. The total volume of each well is 150 μL, so that the maximum final concentration of the sample is 200 μM.

[0070] Control group: Add 100 μL of absolute ethanol to a 96-well plate, and then add 50 μL of the sample to be tested (the phenolic acid extract of Morchella esculenta prepared in the comparative experiment) at 600 μM respectively. The total volume of each well is 150 μL.

[0071] Blank group: Add 100 μL of DPPH solution (0.2 mM) and 50 μL of DMSO in sequence.

[0072] After adding according to the above grouping, place the 96-well plate in the dark at room temperature for 30 min. After the reaction is completed, shake and mix well. Detect the OD value of each well at a wavelength of 517 nm by an enzyme-labeled instrument, and calculate the DPPH free radical scavenging rate of the sample to be tested according to formula (1).

[0073] DPPH free radical scavenging rate (%) = [1 - (ODsample - ODcontrol) / ODblank] × 100%

[0074] Experimental results and analysis

[0075]

[0076] For the phenolic acid extract of Morchella esculenta prepared by the method of the present invention, compared with the phenolic acid extract of Morchella esculenta provided by the conventional solvent control group, its DPPH free radical scavenging rate was significantly improved, showing high antioxidant activity.

[0077] Evaluation of anti-inflammatory efficacy

[0078] Experimental method

[0079] An anti-inflammatory efficacy test was carried out using a lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophage model. Methods: Macrophages Raw264.7 were cultured in vitro and treated with LPS at different concentrations (0.1 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L). The optimal stimulation response conditions of LPS were determined by combining the cell viability and the degree of change in inflammation-related factors (NO, TNF-α), etc. Based on this stimulation condition (5 mg / L LPS), the efficacy evaluation of anti-inflammatory standard substances and test active substances was carried out through the change in the level of inflammatory factors before and after treatment with the test substance.

[0080] Evaluation method: After the cells were seeded for 24 h, the test substance was treated for 1 h, then 5 mg / L LPS was added, and the cells were cultured in an incubator at 37 °C and 5% CO2 for 24 h. The cell culture supernatant was collected, and the contents of inflammation-related factors NO and TNF-α were analyzed using a nitric oxide kit and an ELISA kit, respectively. At the same time, the corresponding blank group was evaluated, and each experiment was independently repeated 3 times.

[0081]

[0082] For the evaluation of the LPS-induced RAW264.7 mouse macrophage model, the phenolic acid extract of Morchella esculenta prepared by the method of the present invention had lower contents of NO and TNF-α inflammatory factors compared with the phenolic acid extract of Morchella esculenta provided by the conventional solvent control group, showing high anti-inflammatory activity.

[0083] The above-described embodiments only represent the implementation modes of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. An extract of Morchella esculenta, characterized in that, The morel extract contains phenolic acids, and the purity of chlorogenic acid in the phenolic acids is greater than 85%.

2. The extraction method of the morel extract according to claim 1, characterized in that, It includes the following steps: S1. Pretreatment: Treat the morel with low-temperature plasma. S2. Targeted enzymatic hydrolysis: Add cellulase, pectinase and β-glucosidase, and perform enzymatic hydrolysis at pH 4.5 - 5.5 and a temperature of 40 - 50 °C for 2 - 4 hours. S3. Deep eutectic solvent extraction: Use a deep eutectic solvent as the extraction medium and perform ultrasonic-assisted extraction at 50 - 60 °C. S4. Purification: Purify the extract by macroporous resin adsorption to obtain the phenolic acid extract of morel.

3. The method according to claim 1, characterized in that In step S1, the morel can be crushed and passed through a 100-mesh sieve; or directly cut into pieces for use.

4. The method according to claim 1, wherein The low-temperature plasma treatment in step S1 includes: using an argon low-temperature plasma reactor and treating for 5 - 15 minutes at a temperature below 40 °C.

5. The method according to claim 1, characterized in that, In step S2, the concentration of cellulase is 0.5 - 2 U / g; the concentration of pectinase is 1 - 2 U / g; the concentration of β-glucosidase is 1 - 3 U / g.

6. The method according to claim 1, wherein In step S3, the deep eutectic solvent is a mixed solvent with a molar ratio of choline to lactic acid of 1:2 - 1:

3.

7. The method according to claim 1, wherein In step S3, the power of the ultrasonic wave is 300 W, the frequency is 40 kHz, and the ultrasonic time is 30 - 60 minutes.

8. The method according to claim 1, wherein In step S4, the macroporous resin is at least one of macroporous adsorption resins of AB-8, D101, D100, S-8, LS-600, HPD100, HPD300, NKA-2, NKA-9 or HZ806 type.

9. Use of the phenolic acid extract prepared by any of the methods of claims 1 - 8 in the preparation of anti-inflammatory and antioxidant functional foods or drugs.

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