Liquorice exosome-like extract as well as extraction and application thereof

Abstract

The invention relates to the technical field of plant exosome-like extracts, in particular to a liquorice exosome-like extract as well as extraction and application thereof. The method comprises the following steps: firstly, removing impurities such as cells, cell debris, organelles, large vesicles and the like in homogenate by adopting a combination mode of low-speed centrifugation and medium-speed centrifugation, and further purifying the homogenate by adopting high-speed centrifugation after the low-speed centrifugation and the medium-speed centrifugation are completed, so as to obtain the exosome-like extract. And the setting of the increasing rotation speed is beneficial to finer separation of exosome-like particles with different sizes and densities, so that the purity and quality of the extract are improved. According to the method, all centrifugal operations are carried out in a low-temperature environment of 4 DEG C; the low-temperature condition is beneficial to maintaining the biological activity and stability of the sample, and the problems of protein denaturation, enzyme inactivation and the like caused by temperature rise are reduced, so that the quality and performance of the exosome-like extract are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of plant exosome-like extracts, and in particular to a licorice exosome-like extract and its extraction and application. Background Art

[0002] Licorice (Glycyrrhiza uralensis Fisch.), a perennial herb of the genus Glycyrrhiza in the Leguminosae family, is a commonly used traditional Chinese medicine, with the effects of invigorating the spleen and replenishing qi, moistening the lungs and relieving cough, clearing heat and detoxifying, and coordinating various medicines. As a traditional Chinese medicine material (CMM) and a food-medicinal homology material (FAM) in China, its glycyrrhizic acid and isoflavonoid compounds have been proven to have significant antioxidant, anti-inflammatory and photoprotective properties, but its traditional extracts have problems such as poor stability and low transdermal absorption rate.

[0003] Exosome-like vesicles are a type of nanovesicles secreted by cells with a lipid bilayer membrane structure, rich in contents such as proteins, lipids, nucleic acids, etc. Due to their wide sources and stable structures, they have received extensive attention in disease diagnosis, prognosis evaluation, immunotherapy and other aspects in recent years. The existing extraction methods of plant exosome-like vesicles are not unified and the efficiency is low, which limits their effects in practical applications; and it is urgent to find a raw material with significant curative effects and low prices. Summary of the Invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a licorice exosome-like extract and its extraction and application. The exosome-like vesicles derived from licorice are based on traditional Chinese medicine active substances, have rich contents and the advantages of greenness, and show good biocompatibility and low toxicity. Based on this, extracting exosome-like vesicles from licorice and studying their biological effects in antioxidant, anti-inflammatory, anti-ultraviolet and anti-blue light aspects have important scientific research value and industrialization prospects.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first purpose of the present invention is to provide a method for extracting a licorice exosome-like extract, comprising the following steps:

[0007] Filter the licorice juice and then perform gradient centrifugation treatment in sequence, and then perform post-treatment to obtain the exosome-like extract.

[0008] In an embodiment of the present invention, the licorice juice is prepared by the following method:

[0009] Wash the licorice root and add it to PBS solution, and then perform wall-breaking treatment to obtain the licorice juice filtrate.

[0010] In one embodiment of the present invention, the mass ratio of licorice root to PBS solution is 1:1 to 5.

[0011] In one embodiment of the present invention, the gradient centrifugation is carried out successively by low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation.

[0012] In one embodiment of the present invention, during the low-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 700 to 3000 × g, and the time is 5 to 60 min;

[0013] During the medium-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 4000 to 6000 × g, and the time is 15 to 120 min;

[0014] During the high-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 10000 to 14000 × g, and the time is 2 to 240 min.

[0015] In one embodiment of the present invention, the post-treatment is filtration using a 0.22 to 0.25 μm filter membrane.

[0016] In one embodiment of the present invention, the gradient centrifugation is specifically as follows:

[0017] The filtered licorice juice is subjected to the first low-speed centrifugation, the supernatant is collected and subjected to the first high-speed centrifugation, the supernatant is collected and added with Exosome Concentration Solution, allowed to stand, and then subjected to the second high-speed centrifugation. After completion, the precipitate is collected, resuspended with PBS, and then subjected to the third high-speed centrifugation. The supernatant is collected and added with Exosome Purication Filter, and then subjected to the second low-speed centrifugation.

[0018] In one embodiment of the present invention, during the first low-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 700 to 3000 × g, and the time is 5 to 60 min;

[0019] During the first high-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 10000 to 14000 × g, and the time is 2 to 240 min;

[0020] During the second high-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 10000 to 14000 × g, and the time is 2 to 240 min;

[0021] During the third high-speed centrifugation process, the temperature is 0 to 4 °C, the centrifugal force is 10000 to 14000 × g, and the time is 2 to 240 min;

[0022] During the second low-speed centrifugation process, the temperature is 0-4°C, the centrifugal force is 700-3000×g, and the time is 5-60 min.

[0023] The second object of the present invention is to provide a glycyrrhiza exosome-like extract prepared by the above method.

[0024] The third object of the present invention is to provide an application of a glycyrrhiza exosome-like extract in the preparation of a functional product, and the functional product has antioxidant ability, anti-inflammatory ability, anti-ultraviolet ability or anti-blue light ability.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention first adopts a combined method of low-speed centrifugation and medium-speed centrifugation to remove impurities such as cells, cell debris, cell organelles and large vesicles in the homogenate. The selection of the rotation speed range of low-speed centrifugation and medium-speed centrifugation aims to effectively separate impurities of different sizes and densities by using different centrifugal forces. The homogenate can be preliminarily purified by low-speed centrifugation and medium-speed centrifugation, creating favorable conditions for the subsequent purification of the exosome-like extract. After the low-speed and medium-speed centrifugation are completed, high-speed centrifugation is used to further purify the homogenate to obtain the exosome-like extract. The centrifugal force range of high-speed centrifugation is set to 10000-14000×g; at different stages of high-speed centrifugation, the same centrifugal rotation speed can be adopted according to actual needs, or an increasing centrifugal rotation speed (preferably an increasing rotation speed) can be adopted. The setting of the increasing rotation speed helps to more finely separate exosome-like particles of different sizes and densities, improving the purity and quality of the extract. In the present invention, all centrifugation operations are carried out in a low-temperature environment of 4°C; the low-temperature condition helps to maintain the biological activity and stability of the sample, reducing problems such as protein denaturation and enzyme inactivation caused by temperature rise, thereby ensuring the quality and performance of the exosome-like extract. Description of the Drawings

[0027] Figure 1 It is a test chart of the anti-ultraviolet ability and the anti-blue light ability of the glycyrrhiza exosome-like extract prepared in Example 1 and Example 2. Detailed Embodiments

[0028] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0029] In the following embodiments, unless otherwise specified, the reagents used are commercially available reagents, and the detection means and methods used are conventional detection means and methods in the art.

[0030] Example 1

[0031] This example provides a preparation method of a glycyrrhiza exosome-like extract, which specifically includes the following steps:

[0032] (A1) Preparation of licorice juice: Select licorice roots, carefully wash them with distilled water to ensure that there is no stain residue on the surface. Add the washed licorice roots to PBS solution pre-cooled at 4 °C in advance. Subsequently, perform cell wall breaking and pulverization treatment on the mixture, and then filter the obtained juice with medical gauze to obtain a roughly filtered juice. Aliquot the roughly filtered juice into 10 mL centrifuge tubes and place them in an ice box for standby;

[0033] (A2) Pre-cool the centrifuge in advance. Place the collected roughly filtered juice in the centrifuge. At 4 °C, first centrifuge at 1000×g for 10 minutes, collect the supernatant into a new centrifuge tube, then put it back into the centrifuge again. Under the same condition of 4 °C, centrifuge at 4000×g for 30 minutes, and collect the supernatant again; finally, put the supernatant into the centrifuge for the third time. Under the same condition of 4 °C, centrifuge at 10000×g for 60 minutes, collect the obtained supernatant, and filter it through a 0.22 μm filter membrane to finally obtain a licorice exosome-like extract for standby.

[0034] Example 2

[0035] This example provides a method for preparing a licorice exosome-like extract, which specifically includes the following steps:

[0036] (A1) Preparation of licorice juice: Select licorice roots, carefully wash them with distilled water to ensure that there is no stain residue on the surface. Add the washed licorice roots to PBS solution pre-cooled at 4 °C in advance. Subsequently, perform cell wall breaking and pulverization treatment on the mixture, and then filter the obtained juice with medical gauze to obtain a roughly filtered juice. Aliquot the roughly filtered juice into 10 mL centrifuge tubes and place them in an ice box for standby;

[0037] (A2)Pre-cool the centrifuge in advance, place the collected coarsely filtered juice in the centrifuge, and centrifuge at 3000×g for 10 minutes at 4°C (if there is a lot of precipitate, it can be centrifuged at 3000×g for 10 minutes multiple times). Collect the supernatant into a new centrifuge tube. Then, put the supernatant obtained in the previous step back into the centrifuge and centrifuge at 10000×g for 10 minutes under the same condition of 4°C. Collect the supernatant again. Finally, add Exosome Concentration Solution reagent to the supernatant collected in this step, let it stand for 8 hours, and then centrifuge at high speed (10000×g, 60 minutes) at 4°C again. Discard the supernatant, centrifuge at high speed (10000×g, 2 minutes) at 4°C again, discard the supernatant, take an appropriate amount of PBS and gently pipette and resuspend the centrifuged precipitate. After it dissolves, centrifuge the resuspended solution at high speed (12000×g, 2 minutes) at 4°C again (this condition can be centrifuged multiple times until there is no obvious precipitate). Take the supernatant and transfer it to an Exosome Purication Filter, and centrifuge at low speed (3000×g, 10 minutes) at 4°C to finally obtain the licorice exosome-like extract for standby.

[0038] Performance measurement:

[0039] (1) Determination of antioxidant property

[0040] 1) Determination of DPPH free radical scavenging rate

[0041] a. Weigh 2.3 mg of DPPH powder and dissolve it in 50 mL of ethanol solution with a volume concentration of 95%. Ultrasonic for 15 minutes to make the DPPH solution evenly mixed. After ultrasonic, measure its absorbance at 517 nm, and adjust the absorbance of the DPPH solution between 1.2 and 1.3, and store it in the dark.

[0042] b. Take 1 mL of the licorice exosome-like extract solution (the licorice exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same below) in a test tube, add 1 mL of DPPH solution, denoted as A1; take 1 mL of the licorice exosome-like extract solution, add 1 mL of PBS, denoted as A2; take 1 mL of PBS, add 1 mL of DPPH solution, denoted as A3; take 2 mL of PBS, denoted as A0. Mix well and react in the dark for 30 minutes. The DPPH free radical scavenging ability (DPPH scavenging rate) of the licorice exosome-like extract solution is calculated by the following formula:

[0043]

[0044] Where: A1 is the absorbance of the DPPH solution containing the sample (referring to the glycyrrhiza exosome-like extract solution); A2 is the absorbance of the PBS containing the sample (referring to the glycyrrhiza exosome-like extract solution); A3 is the absorbance of the PBS containing DPPH; A0 is the absorbance of the PBS

[0045] Table 1 DPPH radical scavenging rate

[0046] Group DPPH Free Radical Scavenging Rate / % Example 1 64.55±0.2 Example 2 78.12±0.6

[0047] As can be seen from the data in Table 1, the glycyrrhiza exosome-like extracts obtained by extraction in Example 1 and Example 2 both have good ability to scavenge DPPH radicals, and among them, the ability of the extract in Example 2 to scavenge DPPH radicals is the strongest.

[0048] 2) Determination of ABTS radical scavenging rate

[0049] Mix 5 mL of 7.4 mmol / L ABTS stock solution with 88 μL of 2.6 mmol / L K2S2O8 and let it stand for 14 hours to prepare the working solution of ABTS.

[0050] Take 0.4 mL of the ABTS solution and dilute it with PBS solution to an absorbance (measuring the absorption value at 734 nm at room temperature) of 0.7 ± 0.02 as the working solution of ABTS.

[0051] Take 2 mL of the working solution of ABTS and 0.5 mL of the glycyrrhiza exosome-like extract solution (the glycyrrhiza exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same hereinafter), denoted as A1; take 2 mL of PBS and 0.5 mL of the glycyrrhiza exosome-like extract solution, denoted as A2; take 2 mL of the working solution of ABTS and 0.5 mL of PBS, denoted as A3; take 2.5 mL of PBS, denoted as A0. Mix them well and let them stand at room temperature in the dark for 10 min. At room temperature, measure the absorbance at 734 nm wavelength in parallel 3 times. The scavenging ability of the glycyrrhiza exosome-like extract solution on ABTS radicals is calculated by the following formula:

[0052]

[0053] Where: A1 is the absorbance of the sample (referring to the glycyrrhiza exosome-like extract solution) and ABTS added; A2 is the absorbance of the sample and PBS added; A3 is the absorbance of ABTS and PBS added; A0 is the absorbance of PBS.

[0054] Table 2 ABTS radical scavenging rate

[0055]

[0056]

[0057] As can be seen from the data in Table 2, the glycyrrhiza exosome-like extracts obtained in Example 1 and Example 2 both have excellent ability to scavenge ABTS free radicals, and among them, the ability of Example 2 to scavenge ABTS free radicals is the strongest.

[0058] 3) Determination of hydroxyl radical scavenging rate

[0059] Take 1 mL of the glycyrrhiza exosome-like extract solution (the glycyrrhiza exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same hereinafter), and add it successively to a test tube containing 0.5 mL of 2 mmol / L FeSO4 solution and 0.5 mL of 6 mmol / L salicylic acid solution. Shake well, add 0.5 mL of 1 mmol / L H2O2, and label it as A1; use 0.5 mL of deionized water instead of 0.5 mL of 1 mmol / L H2O2 as a blank control, and label it as A2; use 1 mL of deionized water instead of the sample solution as a negative control, and label it as A0. Start, heat at a temperature of 37 °C for 30 min, take out, adjust the wavelength of the spectrophotometer to 510 nm to measure the absorbance value, and the scavenging ability of the glycyrrhiza exosome-like extract solution to hydroxyl radicals is calculated by the following formula:

[0060]

[0061] In the formula: A0 is the absorbance value of the negative control group; A1 is the absorbance value of the sample group solution; A2 is the absorbance value of the blank control group.

[0062] Table 3 Hydroxyl radical scavenging rate

[0063] Group Hydroxyl Radical Scavenging Rate / % Example 1 87.46±0.07 Example 2 99.95±0.01

[0064] As can be seen from the data in Table 3, the glycyrrhiza exosome-like extracts obtained in Example 1 and Example 2 both have excellent ability to scavenge hydroxyl radicals, and among them, the ability of Example 2 to scavenge hydroxyl radicals is the strongest.

[0065] (2) Determination of hyaluronidase inhibition rate

[0066] Prepare 0.1M glacial acetic acid solution, 0.1M sodium acetate solution, 0.5M sodium carbonate solution, 10M hydrochloric acid solution, 0.1M acetic acid buffer solution, 4000U / mL hyaluronidase solution, 12.5mM calcium chloride solution, 2mg / mL sodium hyaluronate solution (solvent: 0.1M acetic acid buffer solution), 0.4M sodium hydroxide solution, 0.9M acetylacetone solution (solvent: 0.5M sodium carbonate solution), and 0.2M p-dimethylaminobenzaldehyde solution (solvent: 10M hydrochloric acid solution) for later use. Add reagents according to Table 4, where the sample groups are glycyrrhiza exosome-like solution (glycyrrhiza exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same below) and dipotassium glycyrrhizinate solution (DPG). The dipotassium glycyrrhizinate solution serves as a positive control for the inhibitory effect on hyaluronidase activity. After the reaction, measure the absorbance at 585nm with an enzyme-labeling instrument, and calculate the inhibitory ability of the glycyrrhiza exosome-like extract solution on hyaluronidase activity. Among them, three parallel experiments are set for each group (as shown in Table 4 specifically).

[0067] Table 4 Inhibitory Experiment of Hyaluronidase Activity

[0068]

[0069] The inhibitory rate of the glycyrrhiza exosome-like extract on hyaluronidase can reflect the strength of its anti-inflammatory activity. The higher the inhibitory rate, the stronger the anti-inflammatory activity. Calculate the inhibitory rate of the glycyrrhiza exosome-like extract solution on hyaluronidase activity according to the following formula:

[0070]

[0071] In the formula: T is the OD of the sample tube; T0 is the OD of the sample control tube; C is the OD of the negative control tube; C0 is the OD of the blank control tube.

[0072] Table 5 Inhibitory Rate of Hyaluronidase

[0073] Group Hyaluronidase Inhibition Rate / % Example 1 65.96±0.3 Example 2 78.91±0.6

[0074] It can be seen from the data in Table 5 that the glycyrrhiza exosome-like extracts obtained by extraction in Example 1 and Example 2 both have good anti-inflammatory effects, and among them, the inhibitory ability of Example 2 on hyaluronidase is the strongest.

[0075] (3) Determination of Anti-ultraviolet Ability

[0076] Use ultraviolet spectrophotometry to measure the transmittance of the glycyrrhiza exosome-like extract solution (glycyrrhiza exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same below) within the wavelength range of 280 - 400nm to evaluate the sunscreen performance of the glycyrrhiza exosome-like extract.

[0077] Measure the transmittance of the sample at 280, 290, 300, 310, and 320 nm in the UVB region and at 320, 330, 340, 350, 360, 370, 380, 390, and 400 nm in the UVA region

[0078] Ultraviolet absorption rate formula: Ultraviolet absorption rate / % = (1 - transmittance) × 100%

[0079] From Figure 1 The data shows that the glycyrrhiza exosome-like extracts obtained from Example 1 and Example 2 have excellent ultraviolet resistance in the UVA and UVB regions. The average absorption rate of Example 1 in the UVA region is 91.50%, and the average absorption rate of Example 2 in the UVA region is 97.93%. Among them, the absorption rate of Example 2 is the highest. The average absorption rate of Example 1 in the UVB region is 77.76%, and the average absorption rate of Example 2 in the UVB region is 95.99%. Among them, the absorption rate of Example 2 is the highest.

[0080] (4) Measurement of blue light blocking ability

[0081] The ultraviolet spectrophotometry was used to measure the transmittance of the glycyrrhiza exosome-like extract solution (the glycyrrhiza exosome-like extract solutions prepared in Example 1 and Example 2 respectively, the same below) within the wavelength range of 400 - 500 nm to evaluate the blue light blocking performance of the glycyrrhiza exosome-like extract. The effects of blue light of different wavelengths on the eyes may be different. For example, short-wave blue light (400 - 450 nm) may be more harmful than long-wave blue light (450 - 500 nm).

[0082] Therefore, measure the transmittance of the sample at 400, 410, 420, 430, 440, and 450 nm in the short-wave blue light region and at 450, 460, 470, 480, 490, and 500 nm in the long-wave blue light region.

[0083] Blue light blocking rate formula: Blue light blocking rate / % = (1 - transmittance) × 100%

[0084] From Figure 1 The data shows that the glycyrrhiza exosome-like extracts obtained from Example 1 and Example 2 have excellent blue light blocking ability. The average absorption rate of Example 1 in the short-wave blue light region is 86.65%, and the average absorption rate of Example 2 in the short-wave blue light region is 92.44%. Among them, the absorption rate of Example 2 is the highest. The average absorption rate of Example 1 in the long-wave blue light region is 38.04%, and the average absorption rate of Example 2 in the long-wave blue light region is 43.42%. Among them, the absorption rate of Example 2 is the highest.

[0085] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those who are familiar with the technology in this field can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the interpretation of the present invention should be within the protection scope of the present invention.

Claims

1. A method for extracting a licorice exosome-like extract, characterized in that, Including the following steps: Filter the licorice juice and then perform gradient centrifugation treatment in sequence, and then obtain exosome-like extract through post-treatment.

2. The extraction method of a glycyrrhiza exosome-like extract according to claim 1, characterized in that, The licorice juice is prepared by the following method: Wash the licorice roots, add them to PBS solution, and then perform cell wall breaking treatment to obtain the filtrate of licorice juice.

3. The extraction method of a glycyrrhizin exosome-like extract according to claim 2, characterized in that, The mass ratio of the licorice roots to the PBS solution is 1:1 - 5.

4. The extraction method of a glycyrrhiza exosome-like extract according to claim 1, wherein The gradient centrifugation is to perform low-speed centrifugation treatment, medium-speed centrifugation treatment and high-speed centrifugation treatment in sequence.

5. The extraction method of a glycyrrhiza exosome-like extract according to claim 4, characterized in that, During the low-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 700 - 3000×g, and the time is 5 - 60 min; During the medium-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 4000 - 6000×g, and the time is 15 - 120 min; During the high-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 10000 - 14000×g, and the time is 2 - 240 min.

6. The extraction method of a licorice exosome-like extract according to claim 4, characterized in that, The post-treatment is to perform filtration treatment with a 0.22 - 0.25 μm filter membrane.

7. The extraction method of a glycyrrhiza exosome-like extract according to claim 1, characterized in that, The gradient centrifugation is specifically as follows: Perform the first low-speed centrifugation treatment on the filtered licorice juice, collect the supernatant and perform the first high-speed centrifugation treatment, collect the supernatant, add Exosome Concentration Solution, let it stand, and then perform the second high-speed centrifugation treatment. After completion, add and collect the precipitate, resuspend it with PBS, and then perform the third high-speed centrifugation treatment. Collect the supernatant, add ExosomePurication Filter, and then perform the second low-speed centrifugation treatment.

8. The extraction method of a licorice exosome-like extract according to claim 7, characterized in that, During the first low-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 700 - 3000×g, and the time is 5 - 60 min; During the first high-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 10000 - 14000×g, and the time is 2 - 240 min; During the second high-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 10000 - 14000×g, and the time is 2 - 240 min; During the third high-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 10000 - 14000×g, and the time is 2 - 240 min; During the second low-speed centrifugation treatment, the temperature is 0 - 4 °C, the centrifugal force is 700 - 3000×g, and the time is 5 - 60 min.

9. A glycyrrhiza exosome-like extract, characterized in that, Prepared by the method according to any one of claims 1 - 8.

10. Use of the glycyrrhiza exosome-like extract according to claim 9 in the preparation of a functional product, characterized in that, The functional product has antioxidant ability, anti-inflammatory ability, anti-ultraviolet ability or anti-blue light ability.

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