Extraction method and application of hippophae rhamnoides leaf polyphenol

Abstract

The invention relates to an extraction method and application of hippophae rhamnoides leaf polyphenol, and belongs to the technical field of food functional component extraction. According to the method, the hippophae rhamnoides leaf polyphenol is extracted by innovatively adopting a wall-breaking-step extraction system combining enzymatic pretreatment with ultrasonic induction, and the method is specifically characterized in that hippophae rhamnoides leaf powder is used as a substrate, and normal-temperature enzymolysis wall-breaking treatment is performed. Furthermore, a sound field is combined to promote non-thermal dissociation of secondary metabolites in the hippophae rhamnoides leaf powder, and a continuous process of'normal-temperature enzymolysis of cellulase-ultrasonic extraction 'is constructed. Then, adding an ethanol solution, and placing in a water bath for reaction extraction; and centrifugally freeze-drying to obtain the crude polyphenol of the sea buckthorn leaves. An antibacterial experiment shows that the hippophae rhamnoides leaf polyphenol crude extract has a remarkable antibacterial effect on food-borne pathogenic bacteria such as vibrio parahaemolyticus and aeromonas hydrophila, the technical scheme effectively keeps the biological activity of polyphenol, meanwhile, the solvent consumption and the oxidative degradation rate of heat-sensitive components are lower than those of a traditional process, and the method is suitable for development of natural preservatives and functional foods.

Description

Technical Field

[0001] The invention relates to the technical field of seabuckthorn leaf polyphenol extraction, in particular to a seabuckthorn leaf polyphenol extraction method and application. Background Art

[0002] As plant secondary metabolites, polyphenols have significant biological activities and pharmacological functions, and their extraction technology and functional application research have attracted much attention. HippophaerhamnoidesL As a plant with both medicinal and edible properties, seabuckthorn polyphenols are rich in polyphenols such as flavonoids and phenolic acids. Studies have shown that seabuckthorn polyphenols have functional properties such as free radical scavenging and lipid metabolism regulation. However, their antimicrobial activity has not been systematically studied, making them valuable for development in the fields of food preservation and medicine.

[0003] Currently, plant polyphenol extraction primarily relies on two techniques: solvent extraction and enzymatic hydrolysis. While the traditional ethanol reflux method is simple to operate, it suffers from significant technical drawbacks: the extraction process requires a high temperature environment of 75-85°C, which can lead to oxidative degradation of heat-sensitive polyphenols; the high consumption of organic solvents and the complex subsequent purification process, which do not conform to the principles of green chemistry; and the low energy efficiency and high equipment operating costs associated with industrial production. While enzymatic hydrolysis offers advantages such as mild conditions and high selectivity, it is limited by technical bottlenecks such as the high cost of enzyme preparations (accounting for approximately 35%-40% of the total extraction cost) and a narrow window of optimal process parameters (temperature fluctuations of ±2°C or pH deviations of ±0.5 can significantly affect enzyme activity), making large-scale application difficult.

[0004] In the field of antimicrobial agent development, while chemically synthesized preservatives pose potential toxic side effects, botanical antimicrobials have become a research hotspot due to their safety and environmental friendliness. Existing literature suggests that seabuckthorn polyphenols have inhibitory effects on foodborne pathogens such as Staphylococcus aureus and Escherichia coli, but the mechanisms of action are not fully understood. In particular, the lack of systematic research on the influence of polyphenol extraction processes on their antimicrobial activity has led to a lack of clarity regarding their functional components, hindering their industrial application in areas such as food preservation and medical dressings.

[0005] Existing techniques, such as supercritical CO2 extraction (e.g., CN201810312022.X), rely on high-pressure equipment, are costly, and can easily degrade heat-sensitive components. Traditional enzymatic hydrolysis (e.g., CN200610095283.8), while mild, has limited extraction efficiency when used alone. This invention innovatively combines enzymatic hydrolysis with ultrasonic technology to achieve efficient cell wall disruption in seabuckthorn leaves at room temperature, significantly increasing polyphenol yield while preserving bioactivity. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a method and application for extracting polyphenols from seabuckthorn leaves, so as to solve the problems of the prior art such as high temperature extraction easily leading to polyphenol degradation, high cost of enzymatic hydrolysis, and low extraction rate of polyphenols from seabuckthorn leaves. To achieve the above object of the invention, the present invention provides a method for extracting polyphenols from seabuckthorn leaves, comprising the following steps: A method for extracting seabuckthorn leaf polyphenols comprises the following steps: S1. Seabuckthorn leaf pretreatment: Completely dried seabuckthorn leaves are crushed to obtain seabuckthorn leaf powder; S2. Enzymatic hydrolysis: Mix seabuckthorn leaf powder with cellulase solution at a solid-to-liquid ratio of 1:15-1:25 (g / ml). Incubate at room temperature for 1-1.5 hours, then inactivate the enzyme in a boiling water bath for 5-10 minutes. S3. Ultrasonic-assisted extraction: The inactivated mixture was placed in an ultrasonic field for ultrasonic extraction for 50-70 min to obtain a crude extract; S4. Ethanol extraction: Add 55-65% ethanol to the crude extract and heat in a water bath at 65-75°C for 1.5-2.5 h. S5. Isolation and purification: The extract is centrifuged, the supernatant is collected, the organic solvent is removed by rotary evaporation, and then the sea buckthorn leaf polyphenol powder is obtained by vacuum freeze-drying.

[0007] The pH value of the cellulase solution in step S2 is 4.0-5.0, and the concentration of the cellulase solution is 0.01-0.015 mg / ml.

[0008] In step S3, the ultrasonic power is 280 W and the ultrasonic time is 60 min.

[0009] The volume fraction of the ethanol solution in step S4 is 60%, and the water bath temperature is 70°C.

[0010] In step S5, the centrifugal speed is 2500-3500 rpm, and the centrifugal time is 8-12 min.

[0011] The seabuckthorn leaf polyphenol extract prepared by the above method is used in inhibiting Vibrio parahaemolyticus, Aeromonas hydrophila or Vibrio alginolyticus.

[0012] The seabuckthorn leaf polyphenol extract prepared by the above method is used in the preparation of anti-biofilm medical dressings or antioxidant functional foods.

[0013] The present invention has the following technical effects: This invention utilizes a dual-stage "cellulase enzymatic hydrolysis-ultrasound-assisted extraction" process. Cellulase is used to decompose the cell wall structure of seabuckthorn leaves at room temperature. Combined with an ultrasonic field, this process results in a cell disruption rate exceeding 95%, accelerating the dissolution of polyphenols and significantly reducing the oxidative degradation rate of polyphenols compared to traditional high-temperature methods. Extraction is then performed with a 60% ethanol solution. After centrifugation at 3000 rpm for 10 minutes, residual solvent is removed by rotary evaporation at 45°C and vacuum freeze-drying to obtain a powdered product. Compared to existing technologies, the synergistic "cellulase pretreatment-ultrasound-assisted extraction" process in this invention increases the polyphenol yield to 85.34 mg / g, significantly improving over traditional enzymatic hydrolysis methods while reducing solvent consumption. Experiments have shown that this polyphenol exhibits significant inhibitory effects against Vibrio parahaemolyticus, Vibrio alginolyticus, and Aeromonas hydrophila, potentially expanding its application in food preservation, pharmaceutical excipients, and antibacterial skincare products. This method creates synergistic advantages in activity retention, cost control, and functionality by constructing a continuous process of "cellulase hydrolysis at room temperature-ultrasonic extraction", overcoming the difficulties of traditional technologies such as activity loss, high energy consumption, and low yield, and has significant industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the technical solutions in the implementation cases of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0015] Figure 1 、 three Contour map and response surface diagram of the interaction effects of various factors on the polyphenol content in seabuckthorn leaves Figure 2 , standard curve of gallic acid.

[0016] Figure 3 , standard curve of sea buckthorn leaf polyphenol concentration changing with ultrasonic power.

[0017] Figure 4 , standard curve of sea buckthorn leaf polyphenol concentration changing with ultrasound time.

[0018] Figure 5 , standard curve of sea buckthorn leaf polyphenol concentration changing with material-liquid ratio.

[0019] Figure 6 , standard curve of sea buckthorn leaf polyphenol concentration changing with cellulase concentration.

[0020] Figure 7 , standard curve of seabuckthorn leaf polyphenol concentration changing with ethanol concentration.

[0021] Figure 8 , standard curve of sea buckthorn leaf polyphenol concentration changing with enzymatic hydrolysis time.

[0022] Figure 9 , antibacterial experimental test results of sea buckthorn leaf polyphenols. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings used in the art. Therefore, unless otherwise defined, if there is a conflict, the present specification takes precedence.

[0025] Example 1 A method for extracting polyphenols from seabuckthorn leaves comprises the following steps: S1. Seabuckthorn leaf pretreatment: completely dried seabuckthorn leaves were placed in a grinder and crushed, and passed through a 60-mesh sieve to obtain seabuckthorn leaf powder; S2. Take 1g of sea buckthorn leaf powder and put it into a test tube. According to the solid-liquid ratio of sea buckthorn leaf and cellulase solution of 1:16 (g / ml), add 16ml of 0.012 mg / ml cellulase aqueous solution, mix evenly, and react at room temperature for 1h. After the reaction, inactivate the enzyme in a boiling water bath for 5min. S3, ultrasonically extracting the seabuckthorn leaf solution inactivated by S2 at an ultrasonic power of 280 W for 60 min to obtain a crude seabuckthorn leaf polyphenol extract; S4. Add 24 ml of 60% ethanol solution to the sea buckthorn leaf solution obtained in S3 and heat in a water bath at 70°C for 2 h; S5. Centrifuge the crude seabuckthorn leaf polyphenol extract obtained in S4 at a centrifuge speed of 3000 rpm for 10 minutes, collect the supernatant and discard the precipitate, perform rotary evaporation at 45°C to remove the residual organic solvent, and then remove the aqueous solution by vacuum freeze-drying to obtain a powder, thereby obtaining a seabuckthorn leaf polyphenol sample.

[0026] Example 2 A method for extracting polyphenols from seabuckthorn leaves comprises the following steps: S1. Pretreatment of seabuckthorn leaves: Put the completely dried seabuckthorn leaves into a grinder and grind them through a 60-mesh sieve to obtain seabuckthorn leaf powder; S2. Take 1g of sea buckthorn leaf powder and put it into a test tube. According to the solid-liquid ratio of sea buckthorn leaf and cellulase solution of 1:20 (g / ml), add 10ml of 0.009 mg / ml cellulase aqueous solution, mix evenly, and react at room temperature for 1h. After the reaction, inactivate the enzyme in a boiling water bath for 5min. S3, ultrasonically extracting the seabuckthorn leaf solution inactivated by S2 at an ultrasonic power of 150 W for 30 min to obtain a crude seabuckthorn leaf polyphenol extract; S4, add 20 ml of 50% ethanol solution to the crude extract of sea buckthorn leaf polyphenols obtained in S3, and heat in a water bath at 70°C for 2 h; S5. Centrifuge the crude seabuckthorn leaf polyphenol extract obtained in S4 at a centrifuge speed of 3000 rpm for 10 min, collect the supernatant and discard the precipitate, perform rotary evaporation at 45°C to remove the residual organic solvent, and then remove the aqueous solution by vacuum freeze-drying to obtain a powder, thereby obtaining a seabuckthorn leaf polyphenol sample.

[0027] Example 3 A method for extracting polyphenols from seabuckthorn leaves comprises the following steps: S1. Pretreatment of seabuckthorn leaves: Put the completely dried seabuckthorn leaves into a grinder and grind them through a 60-mesh sieve to obtain seabuckthorn leaf powder; S2. Take 1g of sea buckthorn leaf powder and put it into a test tube. According to the solid-liquid ratio of sea buckthorn leaf to cellulase solution of 1:10 (g / ml), add 10ml of 0.015mg / ml cellulase aqueous solution, mix evenly, and react at room temperature for 1h. After the reaction, inactivate the enzyme in a boiling water bath for 5min. S3, ultrasonically extracting the seabuckthorn leaf solution inactivated by S2 at an ultrasonic power of 280 W for 50 min to obtain a crude seabuckthorn leaf polyphenol extract; S4, in S3 obtained in the crude extract of sea buckthorn leaf polyphenols was added 30 ml of 75% ethanol solution, heated in a water bath at 70 ℃ for 2 h; S5. Centrifuge the crude seabuckthorn leaf polyphenol extract obtained in S4 at a centrifuge speed of 3000 rpm for 10 min, collect the supernatant and discard the precipitate, perform rotary evaporation at 45°C to remove the residual organic solvent, and then remove the aqueous solution by vacuum freeze-drying to obtain a powder, thereby obtaining a seabuckthorn leaf polyphenol sample.

[0028] The polyphenol content in the samples obtained in Examples 1-3 was then measured using the following method: Specifically, the freeze-dried polyphenol powder was first dissolved in ultrapure water, and then the polyphenol content in sea buckthorn leaves was determined using the folin phenol colorimetric method with gallic acid as the standard.

[0029] Add 100 μL of standard solution or polyphenol sample and 900 μL of ultrapure water to the test tube, then add 1 ml of Folin-phenol reagent, let it stand for 3 minutes, shake it well, then add 5 ml of ultrapure water and 3 ml of 10% anhydrous sodium carbonate solution to the test tube, mix them, keep it away from light for 30 minutes, measure the optical density (OD) at a wavelength of 765 nm, and make a standard curve with the absorbance as the horizontal axis and the amount of gallic acid (μg) as the vertical axis, such as Figure 1 The polyphenol content in the samples was calculated using the standard curve.

[0030] The measurement results showed that the mass of polyphenols extracted from 1 g of sea buckthorn leaves by the extraction method in Example 1 was approximately 85.34 mg; the mass of polyphenols extracted from 1 g of sea buckthorn leaves by the extraction method in Example 2 was approximately 76.42 mg; and the mass of polyphenols extracted from 1 g of sea buckthorn leaves by the extraction method in Example 3 was approximately 80.51 mg.

[0031] By comparison, it was found that the extraction method of Example 1 extracted the most polyphenols from 1 g of sea buckthorn leaves, making it the optimal example.

[0032] Response surface optimization experiment: According to the results of the single-factor experiment, the polyphenol content of seabuckthorn leaves was used as the response value, and the three factors that had a greater impact on the polyphenol content, namely, ethanol concentration, enzyme concentration, and ultrasonic time, were selected as independent variables. A three-factor three-level experimental design was conducted using Design-Expert 13 software. The experimental factors and levels are shown in Table 1: Table 1 Response surface test factors and levels Based on the results of the single-factor experiment, a response surface optimization experiment design was conducted. The experimental factor combinations are shown in Table 2.

[0033] Table 2 Response surface experimental design and results Serial number Aethanol concentration B enzyme concentration C Ultrasound time D. Sea buckthorn leaf polyphenol content (mg / g) 1 40 0.9 50 71.0748 2 60 0.9 50 74.2796 3 40 1.5 50 79.9345 4 60 1.5 50 82.0951 5 40 1.2 40 76.1544 6 60 1.2 40 77.4596 7 40 1.2 60 75.3507 8 60 1.2 60 83.5981 9 50 0.9 40 74.2748 10 50 1.5 40 84.9067 11 50 0.9 60 74.9654 12 50 1.5 60 85.5402 13 50 1.2 50 89.1298 14 50 1.2 50 87.2947 15 50 1.2 50 89.3671 16 50 1.2 50 87.0395 17 50 1.2 50 89.9395 Combined with the response surface test results in Table 2, a response surface quadratic regression model was established, and the regression experimental equation was determined to be Y=88.55+1.86A+4.74B+0.8324C-0.2611AB+1.74AC-0.0143BC-6.74A²-4.96B²-3.67C² As shown in Table 3, the model's P value is < 0.0001, indicating that the model is highly significant. The lack-of-fit term is not significant (P = 0.3824 > 0.05), indicating a good fit. The coefficient of determination (R²) is 0.9784, indicating a high correlation between the actual and predicted values. Therefore, this model can well reflect the relationship between various factors and the response during seabuckthorn leaf polyphenol extraction and predict the optimal process conditions. The P values indicate that ethanol concentration (A), enzyme concentration (B), and all quadratic terms have highly significant effects on seabuckthorn leaf polyphenol extraction, while the remaining factors are non-significant (P > 0.05). The F values indicate that the order of influence of the three factors on seabuckthorn leaf polyphenol extraction is: B > A > C, i.e., enzyme concentration > ethanol concentration > ultrasonic time.

[0034] Design-Expert 13 was used to create contour lines and 3D surface diagrams of the interaction between various factors in this experiment on the extraction of seabuckthorn leaf polyphenols. Figure 1 In the 3D surface diagram of the interaction in this response surface experiment design, the most distorted 3D surface diagram is the interaction diagram of ethanol concentration (A) and enzyme concentration (B), indicating that the interaction between ethanol concentration (A) and enzyme concentration (B) has the most significant effect on the extraction of seabuckthorn leaf polyphenols. This result is consistent with the results of the Figure 3 The results of the significance test of the partial regression coefficient are consistent with Table 3 Analysis of variance of regression model factor coefficient sum of squares degrees of freedom mean square error F-number P-value Significance Model 615.04 9 68.34 35.28 <0.0001 ** A +1.86 27.82 1 27.82 14.36 0.0068 ** B +4.74 179.38 1 179.38 92.59 <0.0001 ** C +0.8324 5.54 1 5.54 2.86 0.1346 AB -0.2611 0.2726 1 0.2726 0.1407 0.7187 AC +1.74 12.05 1 12.05 6.22 0.0414 BC -0.0143 0.0008 1 0.0008 0.0004 0.9842 A² -6.74 191.54 1 191.54 98.87 <0.0001 ** B² -4.96 103.73 1 103.73 53.55 0.0002 ** C² -3.67 56.67 1 56.67 29.25 0.0010 ** residual 13.56 7 1.94 Lack of Fit 6.77 3 2.26 1.33 0.3824 Pure error 6.79 4 1.70 sum 628.60 16 #timg# 0.9784 #timg# 0.9507 Note: ** indicates a very significant difference (p<0.01) Using the regression equation optimal solution analysis function in Design-Expert 13 software, we determined that the optimal combination for extracting seabuckthorn leaf polyphenols was 60% ethanol concentration, 1.2% enzyme concentration, and 60 minutes of ultrasonication. Under these conditions, the seabuckthorn leaf polyphenol yield was 82.57 mg / g. Based on actual operations, we selected 60% ethanol concentration, 1.5% enzyme concentration, and 50 minutes of ultrasonication. Under these conditions, the average seabuckthorn leaf polyphenol content was measured to be 85.34 mg / g, which is similar to the model's predicted value, indicating that this model can be used to optimize the process for extracting seabuckthorn leaf polyphenols.

[0035] Experimental testing of the antibacterial properties of polyphenols obtained from seabuckthorn leaves: Experimental bacteria: Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas hydrophila Experimental methods: S1. Extract preparation: Prepare 5 ml of seabuckthorn leaf polyphenol sample solution with a concentration of 1 g / ml using distilled water. S2. Preparation of bacterial suspension: Use a pipette to transfer 20 μL of Vibrio parahaemolyticus, Vibrio alginolyticus, and Aeromonas hydrophila cultured for 24 h into 100 ml of LB medium and LBS medium, respectively. Incubate in an air bath shaker at 200 rpm for 24 h to prepare bacterial suspensions with a certain bacterial concentration for later use.

[0036] S4. Antibacterial Effect Assay: Use a hole punch to punch filter paper into 6 mm diameter discs, sterilize at 121°C for 20 min, and soak in the sea buckthorn leaf polyphenol sample solution for 2 h. On a clean bench, use a pipette to aspirate 100 μL of each of the four bacterial suspensions. Using a spreading rod, evenly spread the bacterial suspension onto a solid culture medium to create a bacterial plate. Use sterile tweezers to affix the filter paper disc to the surface of the culture medium and cover the Petri dish. Use a filter paper disc soaked in 75% ethanol as a control. Place three discs equally spaced on each bacterial plate. Perform three replicates for each bacterial strain.

[0037] By comparing the sea buckthorn leaf polyphenol solution group with the 75% ethanol solution group, it was clearly found that the polyphenols in the sea buckthorn leaves had a significant inhibitory effect on the activity of the three bacteria.

[0038] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting seabuckthorn leaf polyphenols, characterized in that: The following steps are involved: S1. Seabuckthorn leaf pretreatment: Completely dried seabuckthorn leaves are crushed to obtain seabuckthorn leaf powder; S2. Enzymatic hydrolysis: Mix seabuckthorn leaf powder with cellulase solution at a solid-to-liquid ratio of 1:15-1:25 (g / ml). Incubate at room temperature for 1-1.5 hours, then inactivate the enzyme in a boiling water bath for 5-10 minutes. S3. Ultrasonic-assisted extraction: The inactivated mixture is placed in an ultrasonic field for extraction for 50-70 min to obtain a crude extract. S4. Ethanol extraction: Add 55-65% ethanol to the crude extract and heat in a water bath at 65-75°C for 1.5-2.5 h. S5. Isolation and purification: The extract is centrifuged, the supernatant is collected, the organic solvent is removed by rotary evaporation, and then the sea buckthorn leaf polyphenol powder is obtained by vacuum freeze-drying.

2. The method according to claim 1, wherein: The pH value of the cellulase solution in step S2 is 4.0-5.0, and the concentration of the cellulase solution is 0.01-0.015 mg / ml.

3. The method according to claim 1, wherein: In step S3, the ultrasonic power is 280 W and the ultrasonic time is 60 min.

4. The method according to claim 1, wherein: The volume fraction of the ethanol solution in step S4 is 60%, and the water bath temperature is 70°C.

5. The method according to claim 1, wherein: In step S5, the centrifugal speed is 2500-3500 rpm, and the centrifugal time is 8-12 min.

6. Use of the seabuckthorn leaf polyphenol extract prepared according to the method according to any one of claims 1 to 5 in inhibiting Vibrio parahaemolyticus, Aeromonas hydrophila or Vibrio alginolyticus.

7. Use of the seabuckthorn leaf polyphenol extract prepared according to the method according to any one of claims 1 to 5 in the preparation of anti-biofilm medical dressings or antioxidant functional foods.

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