Method for optimally extracting fresh notoginsenoside by using compound enzyme

Abstract

The invention discloses a method for optimally extracting fresh notoginsenoside by using a compound enzyme, and relates to the technical field of extraction and preparation of plant active ingredients. According to the invention, the synergistic effect of a compound enzyme system is researched, the dissolution amounts of three characteristic active components of fresh notoginsenoside R1, ginsenoside Rg1 and ginsenoside Rb1 are synchronously detected by adopting a high performance liquid chromatography, and the optimal proportion of each enzyme component in the compound enzymolysis system is optimized by combining a single factor test and a Box-Behnken design system; and a fresh notoginsenoside efficient extraction system based on a multi-enzyme synergistic effect is established. The optimal process is as follows: the extraction enzymolysis temperature is 53.6 DEG C, the enzymolysis PH is 5.0, the mass fraction is 5.2%, and the ratio is 0.5: 1.5: 1.5: 1. The method for extracting the fresh notoginsenoside through the compound enzyme has the advantages of being high in extraction rate, easy and convenient to operate, environmentally friendly, safe and the like, is suitable for industrial production and has good application and popularization prospects.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction and preparation technology, specifically to a method for optimizing the extraction of fresh Panax notoginseng saponins using a compound enzyme. Background Technology

[0002] Fresh Panax notoginseng, the fresh rhizome of the Panax notoginseng plant (family Araliaceae), is rich in various active ingredients and holds a unique position in traditional Chinese medicine. Saponins are one of the main active components of fresh Panax notoginseng, possessing diverse pharmacological activities, such as regulatory effects on the cardiovascular, cerebrovascular, and central nervous systems, as well as antioxidant and anti-inflammatory properties. Traditional methods for extracting Panax notoginseng saponins, including solvent extraction, distillation, and extraction, suffer from low extraction efficiency, significant component loss, and environmental pollution. Compared to traditional methods, enzymatic hydrolysis can improve the extraction rate under mild conditions and reduce damage to the saponin structure. Currently, enzymatic hydrolysis has achieved some success, with suitable enzymes screened, hydrolysis conditions optimized, and its combination with other technologies explored. However, existing research on the enzymatic extraction of fresh Panax notoginseng is limited, and most methods employ single enzyme treatments, failing to fully leverage the synergistic effects of multiple enzymes. Therefore, developing a highly efficient method for extracting Panax notoginseng saponins based on the synergistic effects of multiple enzymes is of great significance. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a method for optimizing the extraction of Panax notoginseng saponins using a compound enzyme. This method improves the extraction rate of Panax notoginseng saponins through the synergistic effect of multiple enzymes, thereby enhancing extraction efficiency and product quality, and is suitable for industrial production.

[0004] To achieve the above objectives, the present invention provides a method for optimizing the extraction of saponins from fresh Panax notoginseng using a compound enzyme, comprising the following steps:

[0005] 1. Raw material pretreatment: Wash the fresh Panax notoginseng thoroughly and remove surface moisture; use a high-speed universal grinder to grind the fresh Panax notoginseng to below 20 mesh and set aside;

[0006] 2. Preparation of buffer solution and enzyme solution

[0007] 2.1 Prepare a citrate-sodium citrate buffer solution with a pH of 5.0;

[0008] 2.2 Weigh the enzymes according to the following proportions:

[0009] Cellulase: Pectinase: β-amylase: β-glucosase = 0.5:1.5:1.5:1

[0010] 2.3 Mix the above enzymes and add them to the buffer solution to prepare a 5.2% (w / w) complex enzyme solution.

[0011] 3. Enzymatic hydrolysis

[0012] Mix each 10.00g portion of pretreated fresh Panax notoginseng powder with 100mL of compound enzyme solution; place in a 53℃ constant temperature water bath for 2 hours for enzymatic hydrolysis;

[0013] 4. Reflux extraction

[0014] After enzymatic hydrolysis, add 400 mL of 70% ethanol; heat in a reflux apparatus until the ethanol boils, and maintain reflux extraction for 2 hours; filter while hot and collect the filtrate;

[0015] 5. Repeated extraction and concentration

[0016] The filter residue was refluxed with 70% ethanol 1-2 times, and the filtrates were combined; the ethanol was removed by concentration using a rotary evaporator at 70°C.

[0017] 6. Alcohol precipitation and centrifugation

[0018] The concentrate and flocculant were mixed, stirred, and then subjected to low-temperature alcohol precipitation for 24 hours; centrifugation was performed to remove impurities; alcohol precipitation and centrifugation were then performed again.

[0019] 7. Purification and volume adjustment

[0020] Purification was performed using macroporous adsorption resin D101; first, impurities such as polysaccharides were eluted with distilled water; then, saponins were eluted with 70% ethanol at a flow rate of 60 drops / minute; the eluent was collected, concentrated and evaporated to dryness, and then diluted to 500 mL with methanol.

[0021] 8. High-performance liquid chromatography (HPLC) detection

[0022] The peak areas of Panax notoginseng saponins R1, Rg1, and Rb1 were determined; the content and extraction rate of each saponin were calculated by comparing them with the standard.

[0023] Preferably, in step 6, the ratio of extract to flocculant to 70% ethanol is 1:1:1; the centrifugation speed is 4000 rpm and the time is 15 min.

[0024] Preferably, the extraction rate formula in step 8 is as follows:

[0025] M 鲜三七 M 干三七 =10:3;

[0026] Peak area of ​​standard product: A std ;

[0027] Sample peak area: A sample ;

[0028] Volume at constant volume: V (mL);

[0029] Original medicinal material weight: M (g);

[0030] C R1 =(A sample(R1) / A std(R1) )×0.1026mg / mL;

[0031] C Rg1 =(A sample(Rg1) / A std(Rg1) )×0.4072mg / mL;

[0032] C Rb1 =(A sample(Rb1) / A std(Rb1) )×0.3955mg / mL;

[0033] R1 extraction rate (%) = [(C R1 [×V) / (M×1000)]×100%;

[0034] Rg1 extraction rate (%) = [(C Rg1 [×V) / (M×1000)]×100%;

[0035] Rb1 extraction rate (%) = [(C Rb1 [×V) / (M×1000)]×100%;

[0036] Total extraction rate (%) = [(C R1 +C Rg1 +C Rb1 [(M×1000)]×100%.

[0037] The beneficial effects of this invention are as follows: The synergistic effect of compound enzymes significantly improves the extraction rate of Panax notoginseng saponins. The enzymatic hydrolysis temperature is 53℃, and the pH is 5.0, avoiding the destruction of active ingredients by high temperature and high pressure. A citric acid buffer system is used to reduce the use of toxic reagents. The operation process is simple and suitable for large-scale production. Combined with high-performance liquid chromatography (HPLC) detection, the extraction quality is ensured to be controllable. Detailed Implementation

[0038] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0039] Example 1: A method for optimizing the extraction of saponins from fresh Panax notoginseng using a compound enzyme, comprising the following steps:

[0040] 1. Clean the purchased fresh Panax notoginseng thoroughly and remove surface moisture. Grind it into granules using a grinder and pass it through a 20-mesh sieve. Prepare a citric acid-sodium citrate buffer solution with a pH of 5.0, 5% flocculant (prepare fresh before use), and 70% ethanol. Prepare the standards: Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Rb1, with concentrations of 0.1026 mg / mL, 0.4072 mg / mL, and 0.3955 mg / mL, respectively.

[0041] 2. Weighing: Accurately weigh three portions of cellulase, pectinase, β-amylase, and β-glucase: 0.0610g of cellulase, 0.1729g of pectinase, 0.1705g of β-amylase, and 0.1211g of β-glucase, and add them to a beaker labeled 150. Then accurately weigh three portions of fresh Panax notoginseng powder, each weighing 10.00g.

[0042] 3. Enzyme treatment: Add 100 mL of citric acid-sodium citrate buffer solution with a pH of 5.0 to a beaker and place it in a constant temperature water bath at 53°C for 2 hours for enzyme treatment.

[0043] 4. Reflux extraction: After enzyme treatment, pour the mixture into a reflux reflux apparatus, add 400 mL of 70% ethanol, heat until the ethanol boils, and maintain reflux extraction for 2 hours.

[0044] 5. Filtration: After reflux, filter while hot and collect the filtrate.

[0045] 6. Repeat extraction: Reflux the filter residue with 70% ethanol 1-2 times and combine the filtrates.

[0046] 7. Concentration: Concentrate the filtrate using a rotary evaporator at 70°C to remove ethanol.

[0047] 8. First alcohol precipitation: Slowly add the concentrated extract to the mixture of flocculant and 70% ethanol (extract: flocculant: 70% ethanol = 1:1:1) while stirring. After the addition is complete, place the mixture in a low-temperature environment for alcohol precipitation for 24 hours.

[0048] 9. Centrifugation: Centrifuge the solution after the first alcohol precipitation for 15 minutes, rotor number 1, speed 4000 rpm, and then perform the second alcohol precipitation.

[0049] 10. Purification: Centrifuge the extract after the second alcohol precipitation, and then perform macroporous adsorption. First, wash away impurities such as polysaccharides with distilled water, and then wash away saponins with 70% ethanol, controlling the flow rate at 60 drops / min.

[0050] 11. Make up to volume: Collect the eluent, concentrate and evaporate to dryness, dilute and dissolve with methanol, and transfer to a 500 mL volumetric flask and make up to volume.

[0051] 12. Determination of saponins using high performance liquid chromatography.

[0052] 13. Plot the standard curve and calculate the extraction rate:

[0053] Fresh Panax notoginseng : Dried Panax notoginseng = 10 : 3

[0054] Standard peak area: Astd

[0055] Sample peak area: Asample

[0056] Volumetric volume: V (mL)

[0057] Raw material weight: M (g)

[0058] CR1=(Asample(R1) / Astd(R1))×0.1026mg / mL

[0059] CRg1=(Asample(Rg1) / Astd(Rg1))×0.4072mg / mL

[0060] CRb1=(Asample(Rb1) / Astd(Rb1))×0.3955mg / mL

[0061] R1 extraction rate (%) = [(CR1×V) / (M×1000)]×100%

[0062] Rg1 extraction rate (%) = [(CRg1×V) / (M×1000)]×100%

[0063] Rb1 extraction rate (%) = [(CRb1×V) / (M×1000)]×100%

[0064] Total extraction rate (%) = [(CR1+CRg1+CRb1)×V / (M×1000)]×100%.

[0065] Experimental results and analysis in this embodiment:

[0066] 1. Experimental Results

[0067] Table 1

[0068]

[0069] 2. Results Analysis

[0070] Comparing the three optimized experiments, under the conditions of 53℃, pH 5.0, mass fraction 5.2%, and ratio 0.5:1.5:1.5:1, the extraction rates of total saponins of Panax notoginseng were 15.7032%, 15.6132%, and 15.5491%, respectively, with an average extraction rate of 15.6128%, which is 1-2 percentage points higher than the previous values.

[0071] In this embodiment, under the conditions of 53℃, pH 5.0, mass fraction 5.2%, and ratio 0.5:1.5:1.5:1, the average extraction rate of total saponins from Panax notoginseng was 15.6128%, which is 3-4 percentage points higher than that of traditional methods for extracting dried Panax notoginseng rhizomes and 1-2 percentage points higher than that of traditional methods for extracting fresh Panax notoginseng rhizomes. This also confirms that the extraction efficiency of saponins from fresh Panax notoginseng assisted by compound enzymes is higher than that of traditional methods for extracting dried Panax notoginseng and traditional methods for extracting fresh Panax notoginseng.

[0072] The method for extracting saponins from fresh Panax notoginseng using compound enzymes in this embodiment has the advantages of high extraction rate, simple operation, and environmental safety. It is suitable for industrial production and has good prospects for promotion and application.

[0073] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for optimizing the extraction of saponins from fresh Panax notoginseng using a compound enzyme, characterized in that, Includes the following steps: (1) Raw material pretreatment: Wash the fresh Panax notoginseng thoroughly and remove surface moisture; Use a high-speed universal grinder to grind the fresh Panax notoginseng to below 20 mesh, and set aside. (2) Preparation of buffer solution and enzyme solution (2.1) Prepare a citrate-sodium citrate buffer solution with a pH of 5.0; (2.2) Weigh the enzymes according to the following proportions: Cellulase: Pectinase: β-amylase: β-glucosase = 0.5:1.5:1.5:1 (2.3) Mix the above enzymes and add them to the buffer solution to prepare a 5.2% (w / w) complex enzyme solution; (3) Enzymatic hydrolysis Mix each 10.00g portion of pretreated fresh Panax notoginseng powder with 100mL of compound enzyme solution; place in a 53℃ constant temperature water bath for 2 hours for enzymatic hydrolysis; (4) Reflux extraction After enzymatic hydrolysis, add 400 mL of 70% ethanol; heat in a reflux apparatus until the ethanol boils, and maintain reflux extraction for 2 hours; filter while hot and collect the filtrate; (5) Repeated extraction and concentration The filter residue was extracted again by reflux with 70% ethanol 1-2 times, and the filtrates were combined. Ethanol was removed by concentration at 70°C using a rotary evaporator; (6) Alcohol precipitation and centrifugation The concentrate was mixed with flocculant, stirred, and then subjected to alcohol precipitation at low temperature for 24 hours; centrifuged to remove impurities; and then subjected to alcohol precipitation and centrifugation again. (7) Purification and volume adjustment Purification was performed using macroporous adsorption resin D101; first, impurities such as polysaccharides were eluted with distilled water; then, saponins were eluted with 70% ethanol at a flow rate of 60 drops / minute; the eluent was collected, concentrated and evaporated to dryness, and then diluted to 500 mL with methanol. (8) High performance liquid chromatography detection The peak areas of Panax notoginseng saponins R1, Rg1, and Rb1 were determined; the content and extraction rate of each saponin were calculated by comparing them with the standard.

2. The method for optimizing the extraction of fresh Panax notoginseng saponins using a compound enzyme according to claim 1, characterized in that, The extract in step (6) consists of flocculant and 70% ethanol in a ratio of 1:1:1; the centrifugation speed is 4000 rpm and the time is 15 min.

3. The method for optimizing the extraction of saponins from fresh Panax notoginseng using a compound enzyme according to claim 1, characterized in that, The extraction rate formula for step (8) is as follows: M 鲜三七 :M 干三七 =10:3; Peak area of ​​standard product: A std ; Sample peak area: A sample ; Volume at constant volume: V (mL); Original medicinal material weight: M (g); C R1 =(A sample(R1) / A std(R1) )×0.1026mg / mL; C Rg1 =(A sample(Rg1) / A std(Rg1) )×0.4072mg / mL; C Rb1 =(A sample(Rb1) / A std(Rb1) )×0.3955mg / mL; R1 extraction rate (%) = [(C R1 [×V) / (M×1000)]×100%; Rg1 extraction rate (%) = [(C Rg1 [×V) / (M×1000)]×100%; Rb1 extraction rate (%) = [(C Rb1 [×V) / (M×1000)]×100%; Total extraction rate (%) = [(C R1 +C Rg1 +C Rb1 [(M×1000)]×100%.

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