Extraction method and application of active ingredients of American ginseng

Abstract

The invention relates to the technical field of American ginseng active component preparation, in particular to an American ginseng active component extraction method and application thereof. The method for extracting the active ingredients of American ginseng comprises the following steps: extracting the active ingredients by taking American ginseng as a raw material and adopting a micelle medium-microwave-ultrasonic synergistic extraction method; the active ingredients comprise one or more of flavone, polysaccharide and total saponins. The American ginseng active component prepared by the extraction method can be prepared into soybean protein isolate-crude polysaccharide embedded ginsenoside-flavone micro powder by an anti-solvent recrystallization method. Experiments prove that the American ginseng active component and the active component micro powder provided by the invention have the effects of reducing blood fat and blood sugar and enhancing oxidation resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of active ingredients of American ginseng, and in particular relates to a method for extracting active ingredients of American ginseng and application thereof. Background Art

[0002] In traditional Chinese medicine, American ginseng is cool in nature and has the effects of replenishing qi and nourishing yin, clearing away heat and promoting the production of body fluid. It has been used in clinical practice for many years and is often used to treat or assist in the treatment of lung deficiency, chronic cough, hemoptysis caused by lung atrophy, and damage to yang due to fever. As a medicinal and edible plant, American ginseng has great potential in the fields of health care and food.

[0003] At present, the commonly used extraction methods of traditional Chinese medicine include reflux, boiling, filtration, etc., which use many solvents, are time-consuming, costly, and have low extraction efficiency, causing pollution to the environment. In addition, traditional extraction methods easily lead to hydrolysis of saponin compounds, thereby reducing the efficacy. Although technologies such as two-phase extraction, ionic liquid extraction, and ultra-high pressure extraction can effectively separate the effective ingredients of medicinal materials, they have the disadvantages of expensive equipment, easy environmental pollution, and difficulty in popularization and application. How to reduce environmental pollution, reduce solvent consumption, and reduce energy consumption is the trend of current extraction technology development. As a raw material for food, health products, medicine and cosmetics, it is very necessary to adopt a safe and environmentally friendly extraction method. At present, there are problems such as long extraction time and low extraction efficiency for the extraction of active ingredients of American ginseng. Products prepared with active ingredients of American ginseng have problems such as poor effect. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for extracting the active ingredients of American ginseng and its application. The extraction method provided by the present invention is an extraction method using efficient, low-pollution, green micellar medium-microwave-ultrasound synergistic extraction, and the developed product is a micro-powder product with strong blood sugar-lowering, blood lipid-lowering and antioxidant abilities and high bioavailability, which improves the medicinal value and market value of American ginseng and provides technical support for the development of the American ginseng industry.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The invention provides a method for extracting active ingredients of American ginseng. The active ingredients include one or more of flavonoids, polysaccharides and total saponins. American ginseng is used as a raw material and a micellar medium-microwave-ultrasound synergistic extraction method is adopted to extract the active ingredients.

[0007] The beneficial effects of adopting the above technical scheme include: the present invention uses surfactants to replace traditional volatile, highly toxic, flammable and other organic solvents as micellar media, which has the advantages of safety and non-toxicity. Based on the amphiphilicity of surfactants, it is possible to achieve simultaneous extraction of multiple different types of target molecules, thereby achieving efficient extraction of multiple different types of drug molecules. The micellar medium-microwave ultrasound-synergistic extraction method shortens the extraction time and improves the extraction efficiency through the combined extraction of ultrasound and microwave.

[0008] Specifically, the following steps may be included: adding American ginseng powder to a solution containing a surfactant, and extracting by microwave-ultrasonic synergistic extraction. The surfactant is sodium dodecyl sulfate (SDS). Preferably, the concentration of the surfactant in the solution containing the surfactant is 0.5%; in the extraction system, the liquid-to-solid ratio is 19-20mL:1g; the extraction conditions include: ultrasonic power of 300-310W, microwave power of 290-300W, and extraction time of 30s. Optimally, the concentration of the surfactant is 0.5%, the liquid-to-solid ratio is 20mL:1g, the ultrasonic power is 300W, the microwave power is 300W, and the extraction time is 30s.

[0009] The beneficial effects of adopting the above technical solution include: adopting the above conditions can make the yield of active ingredients higher.

[0010] The present invention provides a method for preparing American ginseng active ingredient micropowder, comprising the following steps: preparing by anti-solvent recrystallization method. Further, the method may comprise the following steps: using a total saponin-flavonol solution as an anti-solvent phase, using a crude polysaccharide-soy protein aqueous solution as a solvent phase, dripping the crude polysaccharide-soy protein aqueous solution into the total saponin-flavonol solution, centrifuging, and obtaining a precipitate.

[0011] Furthermore, the preparation method of the total saponin-flavonol solution comprises the following steps: dissolving the mixed powder of ginsenosides and flavonoids in anhydrous ethanol to obtain the total saponin-flavonol solution; the preparation method of the mixed powder of ginsenosides and flavonoids comprises the following steps: extracting the active ingredients of American ginseng by the above-mentioned extraction method, cooling the extracted crude extract, centrifuging to obtain supernatant 1, taking supernatant 1 for alcohol precipitation, centrifuging again to obtain supernatant 2 and precipitate; rotary evaporating the supernatant 2, freeze-drying to obtain the mixed powder of ginsenosides and flavonoids.

[0012] Furthermore, the preparation method of the crude polysaccharide-soy protein aqueous solution includes the following steps: mixing the crude polysaccharide powder from which the protein is removed, soy protein and water to obtain the crude polysaccharide-soy protein aqueous solution; the preparation method of the crude polysaccharide powder from which the protein is removed includes the following steps: extracting the active ingredients of American ginseng by the above-mentioned extraction method, cooling the obtained crude extract, centrifuging to obtain supernatant 1, taking supernatant 1 for alcohol precipitation, centrifuging again to obtain supernatant 2 and precipitate; rinsing the precipitate with ethanol, drying to obtain crude polysaccharide powder, and removing the protein from the crude polysaccharide powder.

[0013] Specifically, the following steps may be included:

[0014] (1) Extracting the active ingredients of American ginseng: adding American ginseng powder to a solution containing a surfactant, and extracting by microwave-ultrasound synergistic extraction to obtain a crude extract;

[0015] (2) cooling the crude extract of step (1), centrifuging to obtain supernatant 1, taking supernatant 1 for alcohol precipitation, centrifuging again to obtain supernatant 2 and precipitate; rotary evaporating supernatant 2, freeze-drying to obtain a mixed powder of ginsenosides and flavonoids; washing the precipitate with ethanol, drying to obtain a crude polysaccharide powder, and removing protein from the crude polysaccharide powder;

[0016] (3) dissolving the mixed powder of ginsenoside and flavonoids in anhydrous ethanol to obtain a total saponin-flavonol solution; mixing the crude polysaccharide powder from which protein has been removed, soy protein, and water to obtain a crude polysaccharide-soy protein aqueous solution;

[0017] (4) Using the total saponin-flavonol solution as the anti-solvent phase and the crude polysaccharide-soy protein solution as the solvent phase, the crude polysaccharide-soy protein solution is dropped into the total saponin-flavonol solution, centrifuged, and the precipitate is obtained and freeze-dried.

[0018] The beneficial effects of adopting the above technical solution include: the American ginseng active ingredient powder provided by the present invention is a soybean protein isolate-coarse polysaccharide embedded ginsenoside-flavonoid powder. The above preparation method has the advantages of reliable and stable process. The American ginseng active ingredient powder prepared by the above method has the advantages of good stability, good dispersibility, good thermal stability, and not easy to be destroyed.

[0019] Furthermore, the mass ratio of crude polysaccharide powder to soy protein is 1:1.

[0020] Furthermore, the concentration of the antisolvent phase is 15-16 mg / mL, the concentration of the solvent phase is 15 mg / mL, and the volume ratio of the antisolvent phase to the solvent phase is 39:1 to 40:1.

[0021] Furthermore, the crude polysaccharide-soy protein solution was dripped into the total saponin-flavonol solution at a rate of 1 mL / min, and the total saponin-flavonol solution was stirred at 5000 r / min while dripping.

[0022] The beneficial effects of adopting the above technical solution include: adopting the above parameters is conducive to improving the encapsulation rate and drug loading.

[0023] The present invention provides an application of an active ingredient of American ginseng in preparing a blood sugar lowering product; the present invention provides an application of an active ingredient of American ginseng in preparing a blood lipid lowering product; the present invention provides an application of an active ingredient of American ginseng in preparing a product with antioxidant capacity; the active ingredient of American ginseng is prepared by the above-mentioned extraction method.

[0024] The present invention provides an application of American ginseng active ingredient powder in the preparation of a blood sugar lowering product; the present invention provides an application of American ginseng active ingredient powder in the preparation of a blood lipid lowering product; the present invention provides an application of American ginseng active ingredient powder in the preparation of a product with antioxidant capacity; the American ginseng active ingredient powder is prepared by the above method.

[0025] The products include but are not limited to food, health products, medicines, cosmetics and other products.

[0026] The beneficial effects of adopting the above technical scheme include: experimental verification shows that the American ginseng active ingredients and American ginseng active ingredient micropowder provided by the present invention can increase the binding rate of sodium cholate, increase the inhibitory effect of pancreatic lipase activity, increase the solubility inhibition rate of cholesterol micelles, increase the inhibitory effect of α-glucosidase activity, increase the inhibitory effect of α-amylase activity, enhance the ability to remove free radicals, and have the effects of improving blood lipid lowering, blood sugar lowering, and enhancing antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the effect of different surfactants on the yields of the three substances in Example 1.

[0028] Figure 2 The results of the single factor experiment in Example 1, wherein A is the effect of the concentration of surfactant SDS on the yields of the three substances, B is the effect of the solid-liquid ratio on the yields of the three substances, C is the effect of ultrasonic power on the yields of the three substances, D is the effect of microwave power on the yields of the three substances, and E is the effect of extraction time on the yields of the three substances.

[0029] Figure 3 This is a Pareto diagram of the impact of five factors on the yield, where A is total flavonoids, B is crude polysaccharides, and C is total saponins.

[0030] Figure 4 This is a 3D diagram of the flavonoid response surface. Figure 5 This is a 3D diagram of the polysaccharide response surface. Figure 6 This is the 3D response surface diagram of total saponins.

[0031] Figure 7In the figure, A shows the effect of different extraction methods on the extraction rate of three active ingredients in American ginseng; B shows the photos of crude polysaccharide before deproteinization and crude polysaccharide after deproteinization with 10% TCA solution, wherein the left picture is the photo of crude polysaccharide before deproteinization, and the right picture is the photo of crude polysaccharide after deproteinization with 10% TCA solution.

[0032] Figure 8 This is the HPLC chromatogram of American ginseng polysaccharide derivatives.

[0033] Fig. 9 These are the results of the single-factor experimental study in Example 2, where A is the effect of the anti-solvent phase concentration (total saponin-flavonol solution concentration) on the encapsulation efficiency and drug loading, B is the effect of the solvent phase concentration (polysaccharide-soy protein aqueous solution) on the encapsulation efficiency and drug loading, and C is the effect of the volume ratio of the anti-solvent phase to the solvent phase on the encapsulation efficiency and drug loading.

[0034] Fig.10 3D response surface diagram of encapsulation efficiency. Fig.11 3D response surface diagram of drug loading.

[0035] Fig.12 In the figure, A is the Tyndall effect of the micropowder of the active ingredient of American ginseng; B is the particle size spectrum of the micropowder; C is the FT-IR graph of crude polysaccharide, American ginseng ginsenoside extract, soy protein, and micropowder of the active ingredient of American ginseng.

[0036] Fig.13 It is a DSC detection curve, wherein A is the active ingredient powder of American ginseng, and B is the ginsenoside extract of American ginseng.

[0037] Fig.14 This is the TG test curve of American ginsenoside extract and American ginseng active ingredient powder.

[0038] Fig.15 Are the results of in vitro simulated gastric juice digestion, where A is the effect of gastric juice simulated digestion test on the content of total saponins and total flavonoids in ginseng, and B is the effect of intestinal juice simulated digestion test on the content of total saponins and total flavonoids in ginseng.

[0039] Fig.16 The experimental results of Example 3, wherein A is the inhibitory effect of American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on pancreatic lipase activity; B is the inhibitory effect of American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on micelle solubility; C is the IC 50=value; D is the inhibitory effect of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on α-glucosidase; E is the inhibitory effect of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on α-amylase; F is the scavenging effect of Vc control solution, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on DPPH free radicals; G is the scavenging effect of Vc control solution, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on ADPS + Free radical scavenging effect. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0041] The present invention uses American ginseng as raw material, and screens the optimal extraction process of total flavonoids, crude polysaccharides and total saponins in American ginseng by micellar medium-microwave-ultrasound synergistic extraction. The extraction process is optimized by single factor test, Plackett-Burman experiment and response surface experimental design. Then, the extracted crude polysaccharide and saponin extract are combined with soy protein isolate, and the American ginseng active ingredient powder is prepared by anti-solvent recrystallization method. The blood sugar lowering, blood lipid lowering and antioxidant ability of the American ginseng active ingredient powder are studied. The main research contents and results are as follows:

[0042] Through single factor experiment, the optimal experimental conditions were found to be 0.5% sodium dodecyl sulfate (SDS) concentration, 20mL:1g liquid-to-solid ratio, 300W ultrasonic power, 300W microwave power, and 30s extraction time; through Plackett-Burman test, it was concluded that SDS concentration, liquid-to-solid ratio, ultrasonic power, and microwave power were significant factors. On this basis, response surface experimental design was carried out, and the factor with the highest total saponin yield was determined as the optimal parameter prediction result. When the surfactant concentration was 0.5%, the liquid-to-solid ratio was 19.93mL:1g, the ultrasonic power was 304.29W, and the microwave power was 297.62W, the total flavonoids, crude polysaccharides, and total saponin yields were 0.21%, 7.69%, and 14.33%, respectively. Based on the requirements of production practice, the optimal production process parameters are: surfactant concentration of 0.5%, liquid-to-solid ratio of 20mL / g, ultrasonic power of 300W, microwave power of 300W, total flavonoids, crude polysaccharides, and total saponin yields of 0.22%±0.03%, 6.8%±0.99%, and 13.57%±1.16%, respectively. The error is within 5.0%, and the reaction surface fitting method established by this method is correct and reliable. The polysaccharides obtained by micellar medium-microwave-ultrasonic synergistic extraction method are measured by PMP pre-column derivatization combined with high performance liquid chromatography to determine the monosaccharides in American ginseng crude polysaccharides: rhamnose, xylose, mannose, ribose, arabinose, and galacturonic acid. The high content of galacturonic acid in American ginseng polysaccharides indicates that American ginseng polysaccharides have strong antioxidant activity.

[0043] Response surface design was used to optimize the anti-solvent recrystallization process for preparing micro-powder of active ingredients of American ginseng. Through single factor experiments, the optimal extraction parameters were determined as follows: the concentration of total saponin-flavonol solution in the anti-solvent phase was 15 mg / mL, the concentration of polysaccharide-soy protein aqueous solution in the solvent phase was 15 mg / mL, and the volume ratio of anti-solvent phase to solvent phase was 40:1. Then, the response surface Box-Behnken method was used to design a three-factor three-level experiment. The final actual optimal process conditions were: anti-solvent phase concentration of 16 mg / mL, solvent phase concentration of 15 mg / mL and anti-solvent phase to solvent phase volume ratio of 39:1. The prepared American ginseng active ingredient powder was fluffy and slightly yellow, and the Tyndall phenomenon occurred after dissolution. The average particle size of the powder was 318.6 nm; the total saponin loading was 0.97% ± 0.07%, and the encapsulation efficiency was 89% ± 1.03%; the crude polysaccharide loading was 0.64% ± 0.05%, and the encapsulation efficiency was 82.69% ± 2.8%; the total flavonoid loading was 0.51% ± 0.05%, and the encapsulation efficiency was 82.73% ± 2.8%.

[0044] After 1 hour of simulated digestion with gastric juice, 63.19%±2.19% of ginsenosides were degraded and 60%±1.2% of total flavonoids were degraded. 21.9%±1.65% of ginsenosides and 20%±2.8% of total flavonoids in the micropowder of American ginseng active ingredients were degraded. After 3 hours of simulated digestion with intestinal juice, 62.10%±4.29% of ginsenosides and 34.67%±2.31% of total flavonoids were degraded; 19.24%±2.93% of ginsenosides and 17.14%±2.86% of total flavonoids in the micropowder of American ginseng active ingredients were degraded. When performing infrared spectroscopy detection at 750-1250cm -1 There is a clear difference in the absorbance of the fingerprint area between the active ingredient powder of American ginseng and the saponin extract. This shows that the active ingredient powder of American ginseng is wrapped by soybean protein and crude polysaccharide, and the process is reliable and stable.

[0045] When DSC was used for melting test, the starting temperature of the active ingredient powder of American ginseng was 169.53℃, the ending temperature was 181.51℃, and the melting enthalpy was 52.13J / g; the starting temperature of the American ginseng ginsenoside extract was 153.27℃, the ending temperature was 172.95℃, and the melting enthalpy was 674.2J / g. In the TG experiment, the weight loss temperature of the active ingredient powder of American ginseng was 208.44℃, and the weight loss temperature of the American ginseng ginsenoside extract was 150.97℃.

[0046] In the pancreatic lipase activity inhibition experiment, the IC 50 The value is 16.21 mg / mL, and the IC of the crude extract after extraction is 50 The value is 12.69mg / mL, and the IC value of American ginseng active ingredient powder is 50 The value is 7.29mg / mL. In the cholesterol micelle solubility inhibition activity experiment, the IC 50 The value is 15.11 mg / mL, and the IC of crude extract after extraction is 50 The value is 12.11mg / mL, the IC value of American ginseng active ingredient powder 50 The value was 6.32 mg / mL. Therefore, the inhibitory activity was in the following order: American ginseng active ingredient powder > crude extract > American ginseng original powder.

[0047] The in vitro antioxidant capacity of scavenging DPPH and ABTS+ free radicals was studied. In the DPPH in vitro antioxidant test, the IC 50 The value is 0.079mg / mL, and the IC value of American ginseng powder is 50 The value is 36.867 mg / mL, and the IC of the crude extract after extraction is 50 The value is 25.077mg / mL, and the IC value of American ginseng active ingredient powder is 50 The value is 14.293 mg / mL. In the ABTS+ antioxidant experiment, the IC50 The value is 0.0514mg / mL, and the IC value of American ginseng powder is 50 The value is 45.883 mg / mL, and the IC of crude extract after extraction is 50 The value is 35.270mg / mL, the IC value of American ginseng active ingredient powder 50 The value is 16.008 mg / mL. The ability to remove free radicals: Vc>American ginseng active ingredient powder> crude extract after extraction>American ginseng original powder.

[0048] The study of hypoglycemic efficacy inhibited the activity of α-glucosidase and α-amylase. The results of the α-glucosidase inhibition experiment showed that acarbose IC 50 Value 0.026mg / mL, American ginseng powder IC 50 The value is 13.17 mg / mL, and the IC of crude extract after extraction is 50 The value is 6.26mg / mL, and the IC value of American ginseng active ingredient powder is 50 The value is 3.33mg / mL. The results of the α-amylase inhibition test showed that acarbose IC 50 The value is 0.037mg / mL, and the IC value of American ginseng powder is 50 The value is 17.26 mg / mL, and the IC value of crude extract after extraction is 50 The value is 11.43mg / mL, the IC value of American ginseng active ingredient powder 50 The value is 4.34 mg / mL. Therefore, the order of the inhibitory ability of hypoglycemic activity is: acarbose>American ginseng active ingredient powder>extracted crude extract>American ginseng original powder.

[0049] In summary, the present invention has studied and determined the optimal extraction process of micellar medium-microwave-ultrasound synergistic extraction of total saponins, crude polysaccharides and total flavonoids in American ginseng, and prepared them into American ginseng active ingredient powder. Physical and chemical indicators have proved that the powder has the advantages of small particles, good dispersibility, good lipid-lowering and blood sugar-lowering activity and strong antioxidant capacity. The present invention provides technical support for the future development of deep processing of American ginseng and the development of American ginseng food and medicine.

[0050] In the present invention, unless otherwise specified, the raw materials, reagents, instruments, etc. used are all existing commercial products, and those skilled in the art can obtain them through commercial channels. Unless otherwise specified, the methods used in the present invention are conventional methods in the art.

[0051] American ginseng was purchased from Heilongjiang Dongdu Ginseng Industry Co., Ltd. Ginsenoside Rg1 standard, ginsenoside Re standard, ginsenoside Rb1 standard, ginsenoside Rg2 standard, ginsenoside Ro standard, ginsenoside Rd standard were all purchased from China Food and Drug Inspection Institute; ginsenoside Rh1 standard and ginsenoside Rb2 standard were purchased from Beijing Solaibao Technology Co., Ltd.; ginsenoside Rc standard, pseudoginsenoside F1 standard, ginsenoside Rg3 standard, pseudoginsenoside CK standard, ginsenoside S-Rh2 standard, ginsenoside R-Rh2 standard were purchased from Shanghai Chunyou Biotechnology Co., Ltd.; the purity of the above standards was 99.8%. Phosphate buffer, ABTS + and DPPH were purchased from Sinopharm Chemical Reagent Co., Ltd. 4-Nitrophenyl laurate was purchased from Shanghai Chemical Reagent Company. α-Glucosidase and Triton X-100 were purchased from Shanghai McLean Biotechnology Co., Ltd. BCA protein content kit was purchased from Laimo Biotechnology (Shanghai) Co., Ltd. Microwave light wave ultrasonic extractor (model SCI ENTZ-I IDM) was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. Laser particle size analyzer (model 90PLUSPALS) was purchased from NanoBrook Instruments, USA. In the present invention, unless otherwise specified, the percentages are all mass percentages.

[0052] In the present invention, American ginseng active ingredient powder, ginsenoside soy protein isolate powder, soy protein isolate-coarse polysaccharide-encapsulated ginsenoside-flavonoid powder all have the same meaning. For the convenience of description, the above names can also be referred to as American ginseng powder.

[0053] In the present invention, American ginsenoside extract, ginsenoside and flavonoid powder, and total saponin-flavonoids have the same meaning.

[0054] In the present invention, the polysaccharide soybean protein aqueous solution and the soybean protein-crude polysaccharide aqueous solution have the same meaning.

[0055] Example 1 Micellar medium-microwave-ultrasound synergistic extraction of American ginseng and study of its efficacy

[0056] 1.1 Experimental methods

[0057] 1.1.1 Preparation of American ginseng powder: freeze-dry the American ginseng and then grind it using an ultrafine grinder. The grinding conditions include: grinding at 10°C for 10 min, grinding to 50-100 μm, and preparing American ginseng powder (i.e., American ginseng raw powder) for later use.

[0058] 1.1.2 Determination of flavonoid content: When sodium nitrite is present in the solution and the pH of the solution is neutral or weakly alkaline, flavonoid compounds will form chelates with the aluminum salts contained therein. After adding NaOH, the solution will appear red-orange. Within a certain concentration range, the flavonoid content is proportional to the absorbance value.

[0059] (1) Prepare rutin standard stock solution: Dissolve 10 mg of rutin standard in 60% ethanol solution and make up to 100 mL. Prepare a standard stock solution with a concentration of 0.1 mg / mL.

[0060] (2) Detection of total flavonoid content: Take rutin reference substance, take 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 mL and place it in a 10 mL colorimetric tube, add 60% ethanol to dissolve it, so that the total amount is 5 mL. Add 0.3 mL of 5% sodium nitrite solution, shake to mix, let it stand for 8 minutes, then add 0.3 mL of 10% aluminum nitrate solution, shake evenly, let it stand for 10 minutes, then add 4 mL of 4% sodium hydroxide solution to it, and make up to volume. Let it stand for 10 minutes, measure the absorbance at 500 nm, and draw a standard curve of rutin concentration and absorbance.

[0061] (3) Preparation of samples: Weigh 0.5 g of American ginseng sample (i.e., the American ginseng powder prepared in Example 1.1.1) and place in a 50-mL volumetric flask, add 30 mL of 60% ethanol solution, perform ultrasound at 280 W, 60° C. for 90 min, make up to volume with 60% ethanol, centrifuge until clear, filter and set aside.

[0062] (4) Determination of sample solution: Take 1.0 mL of the solution to be tested, measure its absorbance according to the method described in (2) above, and calculate the total flavonoid content according to formula 1-1:

[0063]

[0064] Where: W is the total flavonoid content (%); C is the mass concentration of the sample solution (g / mL); V is the volume of the sample solution (mL); D is the dilution factor of the solution; M1 is the sampling volume (mg).

[0065] 1.1.3 Determination of polysaccharide content

[0066] (1) Preparation of standard curve: Prepare a standard aqueous solution of glucose at a concentration of 1 mg / mL. Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL into 6 10 mL stoppered test tubes, fill to 1 mL with distilled water, add 1.0 mL of 5% phenol, add 3 mL of concentrated sulfuric acid, shake for 5 minutes, let stand for 10 minutes, heat in a water bath at 100°C for 20 minutes, then cool to room temperature and measure the absorbance at 490 nm.

[0067] (2) Determination of sample solution: Take 0.5 g of American ginseng sample (i.e., the American ginseng powder prepared in Example 1.1.1), place it in a volumetric flask, add anhydrous ethanol, extract it with an ultrasonic extractor at 280W and 46°C for 40 minutes, then centrifuge it at 8000r / min for 15 minutes, retain the supernatant, add 50mL of water to the remaining residue, and then ultrasonically extract it 3 times, each time for 30 minutes, and centrifuge it in a centrifuge at 4000r / min for 10 minutes each time. Combine the supernatants of the three times, pour them into a 250mL volumetric flask, add water to the scale, shake well, and obtain the sample determination solution. Then accurately draw 1mL of the sample determination solution into the volumetric flask, add 1mL of 5% phenol solution and 3mL of sulfuric acid solution respectively, shake and mix well, place it in a 100°C water bath and heat it for 10 minutes, cool it to room temperature, and measure the absorbance at 490nm.

[0068] (3) Calculation of polysaccharide content

[0069]

[0070] Wherein: W is the crude polysaccharide content (%); C is the mass concentration of the sample solution (g / mL); V is the volume of the sample solution (mL); D is the dilution factor of the solution, in milliliters (mL); M2 is the sample mass (mg).

[0071] 1.1.4 Determination of total saponin content

[0072] (1) Extraction of total saponins from American ginseng: Weigh 1.0 g of American ginseng powder (i.e., the original American ginseng powder prepared in Example 1.1.1), add 50.0 mL of water-saturated n-butanol, heat in a water bath and reflux for 1.5 h, shake well and filter. Evaporate the filtrate to dryness, add an appropriate amount of 50% methanol to the residue to dissolve, dilute to a 100 mL volumetric flask, shake well and filter, and take the filtrate.

[0073] (2) Determination of total saponin content:

[0074] Preparation of reference solution: Accurately weigh several ginsenosides (Rg1, Re, Rb1, Rg2, Rc, Rh1, Ro, Rb2, Rd, Rg3, CK, S-Rh2, R-Rh2) of the standard substance, dissolve them in methanol solution, make up to volume, and filter. Chromatographic conditions: The chromatographic column is Waters C 18(250mm×4.6mm, 5μm) column; detection wavelength is 203nm; column temperature is 40℃; flow rate is 1.0mL / min; mobile phase is acetonitrile solution; gradient elution program is: 0-25min, acetonitrile volume fraction is 20%; 25-60min, acetonitrile volume fraction increases from 20% to 40%; 60-90min, acetonitrile volume fraction increases from 40% to 55%; 90-120min, acetonitrile volume fraction is 55%. The injection volume is 20μL, the peak area is determined according to the standard curve method, and the content of American ginsenosides in the sample is calculated based on the standard curve.

[0075] (3) Yield of total saponins in American ginseng: The yield of total saponins is the sum of all saponins, and the yield is as shown in Formula 1-3.

[0076]

[0077] Wherein: W—ginsenoside content (%); C—mass concentration of sample solution (g / mL); V—volume of sample solution (mL); D—solution dilution factor, in milliliters (mL); M3—sample mass (mg).

[0078] 1.1.5 Screening of surfactants: The present invention screens different types of surfactants, including ionic surfactants: sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS) and cetyltrimethylammonium bromide (CTAB); non-ionic surfactants: Span 85, polyether F-127, polyethylene glycol 4000 and Triton X-100. Weigh 1.00 g of each American ginseng sample (i.e., American ginseng powder prepared in Example 1.1.1), place in a flask, add surfactant aqueous solution (i.e., micellar medium solution) at a liquid-to-solid ratio of 20 mL: 1 g, that is, the volume mass ratio of surfactant aqueous solution to American ginseng original powder is 20 mL: 1 g, and the mass concentration of surfactant in surfactant aqueous solution is 0.3%. Use microwave light wave ultrasonic extractor for extraction, ultrasonic (power of 300 W) and microwave (power of 300 W) synergistic extraction for 30 s, 8000 r·min -1 After centrifugation for 10 min, the supernatant was taken. Each group of experiments was repeated 3 times, and the average value and its standard deviation were recorded and calculated.

[0079] 1.1.6 Single-factor experiment on the extraction of three active ingredients of American ginseng by surfactant: the selected factors were SDS concentration (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%); liquid-to-solid ratio (10mL:1g, 15mL:1g, 20mL / 1g, 25mL:1g, 30mL:1g), where the liquid-to-solid ratio was the volume mass ratio of the surfactant solution to the American ginseng raw powder; ultrasonic power (100W, 200W, 300W, 400W, 500W); microwave power (100W, 200W, 300W, 400W, 500W); extraction time (10s, 20s, 30s, 40s, 50s).

[0080] 1.1.7 Plackett-Burman test: On the basis of the single-factor experiment, the factors and levels of the Plackett-Burman test design were determined, and the five factors (X1: surfactant concentration, X2: liquid-to-solid ratio, X3: ultrasonic power, X4: microwave power, X5: extraction time) of total flavonoids yield (Y1), crude polysaccharide yield (Y2), and total ginsenoside yield (Y3) were evaluated by the Plackett-Burman test to screen out significant factors, with a total of 12 independent experiments.

[0081] 1.1.8 Response surface test: Based on the screening results of the five factors by the Plackett-Burman test, the response surface Design Expert 8.06 software was used to optimize the product with the screened key factors as independent variables X1, X2, X3, and X4, and the total flavonoids, crude polysaccharides, and total saponin yields in the product as response values. See Table 1-1.

[0082] Table 1-1 Experimental level factors of response surface analysis of micellar medium-microwave-ultrasound synergistic extraction method

[0083]

[0084] 1.1.9 Comparison of different extraction processes: The three target active ingredients were extracted by ultrasound-assisted, microwave-assisted, surfactant-microwave-assisted, and surfactant-ultrasound-assisted extraction methods, and compared with the micellar medium-microwave-ultrasound synergistic extraction method of the present invention to objectively evaluate the application prospects of this technology in industry.

[0085] (1) Ultrasonic-assisted extraction method: Weigh 1 g of American ginseng powder and put it in a 50 mL conical flask. Add 20 mL of 70% ethanol solution, ultrasonic power 300 W, extraction time 30 s. Take the supernatant of the mixture obtained after extraction, centrifuge at 8000 rpm / min for 10 min, take the supernatant, and detect total saponins according to the operation method in 1.1.4.

[0086] (2) Microwave-assisted extraction method: Weigh 1 g of American ginseng powder, put it in a 50 mL conical flask, add 20 mL of 70% ethanol solution, microwave power 300 W, extraction time 30 s. Centrifuge the mixture obtained after extraction at 8000 rpm / min for 10 min, take the supernatant, and detect total saponins according to the operation method in 1.1.4.

[0087] (3) Surfactant-microwave assisted extraction method: Weigh 1g of American ginseng powder and put it in a 50mL conical flask. Add 20mL of 0.5% SDS, microwave power 300W, extraction time 30s. Centrifuge the mixture obtained after extraction at 8000rpm / min for 10min, take the supernatant and detect total saponins according to the operation method in 1.1.4.

[0088] (4) Surfactant-ultrasound-assisted extraction method: Weigh 1 g of American ginseng powder and put it in a 50 mL conical flask. Add 20 mL of 0.5% SDS solution, ultrasonic power 300 W, extraction time 30 s. Centrifuge the mixture obtained after extraction at 8000 rpm / min for 10 min, take the supernatant and detect total saponins according to the operation method in 1.1.4.

[0089] 1.2 Experimental Results

[0090] 1.2.1 Flavonoids and polysaccharides standard curve

[0091] The standard curve equation of flavonoids is y = 0.1196x + 0.0076, R 2 =0.9993, where x is the concentration of rutin (mg / mL) and y is the absorbance; the standard curve equation of polysaccharide is y=0.2315x+0.0089, R 2 =0.9981, where x is the glucose concentration (mg / mL) and y is the absorbance.

[0092] 1.2.2 Establishment of ginsenoside standard curve: The standard curves of saponins Rg1, Re, Rb1, Rg2, Rc, Rh1, Ro, Rb2, Rd, Rg3, S-Rh2, R-Rh2 and pseudo-ginsenosides F1 and CK are shown in Table 1-2. Where x is the concentration of different saponins (mg / mL) and y is the corresponding liquid chromatography peak area.

[0093] Table 1-2 Standard curves of 14 saponins

[0094]

[0095]

[0096] 1.2.3 Effect and analysis of surfactant type on extraction: Different surfactants have different destructive effects on cell membranes and thus different yields. Figure 1 It can be seen that the best total flavonoids, crude polysaccharides and total saponins yields can be obtained by using SDS aqueous solution for extraction, which are 0.376%±0.075%, 5.95%±0.24% and 9.39%±0.25% respectively. Therefore, the present invention subsequently selected anionic surfactants using sodium dodecyl sulfate (SDS) as solvent to conduct a systematic study on it.

[0097] 1.2.4 Single factor experimental results and analysis

[0098] (1) Effect of surfactant SDS concentration on extraction effect: The effect of surfactant SDS concentration on extraction effect was tested under the conditions of ultrasonic power 300W, microwave power 300W, liquid-to-solid ratio 20mL:1g, and extraction time 30s. The experimental results are as follows Figure 2 As shown in A, when the mass fraction of SDS is too small, the solvent cannot form micelles and cannot play a solubilizing effect. When the concentration reaches 0.5%, the yields of total flavonoids, crude polysaccharides, and total saponins are 0.41% ± 0.091%, 6.8% ± 0.07%, and 7.00% ± 0.26%, respectively, which are the maximum yields of each component. When the concentration exceeds 0.5%, the yields of total flavonoids, crude polysaccharides, and total saponins gradually decrease. The inventors believe that this is because the mass fraction of SDS is too large and the viscosity of the solvent increases. Therefore, the optimal concentration is 0.5% SDS aqueous solution.

[0099] (2) Effect of liquid-to-solid ratio on extraction effect Results and analysis: The effect of liquid-to-solid ratio on extraction effect was tested under the conditions of SDS concentration of 0.5%, ultrasonic power of 300W, microwave power of 300W, and extraction time of 30s. Figure 2 As shown in B, as the liquid-to-solid ratio increases, the external concentration is low, and the osmotic pressure inside and outside the cell increases, the overflow of the intracellular solute will be promoted, and the yield of total saponins will increase accordingly. When the liquid-to-solid ratio is 20mL:1g, the total flavonoids, crude polysaccharides, and total saponin yields are 0.30%±0.061%, 5.12%±0.07%, and 11.04%±1.22%, respectively. When the liquid-to-solid ratio exceeds 20mL:1g, the total flavonoids, crude polysaccharides, and total saponin yields gradually decrease. The inventors believe that this may be due to the decrease in the solute concentration of the extract, which hinders the solute oscillation and causes the yield to decrease. Taking into account the effect of extraction conditions on the saponin yield, the optimal liquid-to-solid ratio condition is 20mL:1g (i.e. Figure 2 The solid-liquid ratio in B is 1:20).

[0100] (3) Effect of ultrasonic power on extraction effect Results and analysis: The effect of ultrasonic power on extraction effect was tested under the conditions of SDS concentration of 0.5%, microwave power of 300W, liquid-to-solid ratio of 20mL:1g, and extraction time of 30s. Figure 2 As shown in C, with the increase of ultrasonic power, the yield of total saponins increases, the cavitation ability of ultrasound is enhanced, and the dissolution of ginsenosides in cells is promoted. When the ultrasonic power is 300W, the yields of total flavonoids, crude polysaccharides, and total saponins are 0.35% ± 0.06%, 5.04% ± 0.98%, and 10.17% ± 0.69%, respectively. When the ultrasonic power exceeds 300W, the yields of total flavonoids, crude polysaccharides, and total saponins gradually decrease. Considering the influence of extraction conditions, the optimal ultrasonic power is 300W.

[0101] (4) Effect of microwave on extraction effect Results and analysis: The effect of microwave on extraction effect was tested under the conditions of SDS concentration of 0.5%, ultrasonic power of 300W, liquid-to-solid ratio of 20mL:1g, and extraction time of 30s. Figure 2 As shown in D, with the increase of microwave power, the yield of total saponins increases. With the increase of power, the intracellular temperature increases, the intracellular pressure increases, and the cell wall is easily squeezed, which promotes the overflow of ginsenosides in the cell. When the microwave power is 300W, the total flavonoids, crude polysaccharides, and total saponins are 0.32% ± 0.06%, 4.96% ± 0.69%, and 10.69% ± 1.03% respectively. When the microwave power exceeds 300W, the total flavonoids, crude polysaccharides, and total saponins gradually decrease. Considering the effect of extraction conditions on saponin yield, the optimal microwave power is 300W.

[0102] (5) Effect of extraction time on extraction effect Results and analysis: The effect of extraction time on extraction effect was tested under the conditions of SDS concentration of 0.5%, ultrasonic power of 300W, microwave power of 300W, and liquid-to-solid ratio of 20mL:1g. The experimental results are as follows Figure 2 As shown in Figure E, as the extraction time increases, the yield of total saponins increases. When the extraction time is 30s, the total flavonoids, crude polysaccharides, and total saponins are 0.42% ± 0.07%, 5.98% ± 0.07%, and 10.64% ± 0.67% respectively. When the extraction time exceeds 30s, the total flavonoids, crude polysaccharides, and total saponins gradually decrease. Considering the effect of extraction conditions on saponin yield, the optimal extraction time is 30s.

[0103] 1.2.5 Plackett-Burman test results: According to the results of the single factor test, the yields of total flavonoids (Y1), crude polysaccharides (Y2), and total ginsenosides (Y3) were used as experimental response values. The design of the Plackett-Burman test and the results are shown in Table 1-3.

[0104] Table 1-3Plackett-Burman test design and results

[0105]

[0106] According to the Plackett-Burman experimental design variance analysis of total flavonoids, crude polysaccharides, and total saponins, the regression models of total flavonoid yield (Y1), crude polysaccharides yield (Y2), and total saponins yield (Y3) are all extremely significant (P<0.01), which shows that there is an obvious linear relationship between the variables, indicating that the regression is effective and the experimental design is credible. At the same time, the R 2 91.40%, 97.6%, 98.12%, R 2 Adj The results are 84.24%, 94.63% and 95.76% respectively, indicating that the model has a good fitting effect. In the Y1 model, the variable factors X1 and X2 have significant effects, in the Y2 model, the variable factors X1 and X2 have extremely significant effects, in the Y3 model, the variable factors X1 and X2 have extremely significant effects, and the variable factors C, D and E have significant effects. Therefore, the surfactant SDS concentration (X1), liquid-to-solid ratio (X2), ultrasonic power (X3) and microwave power (X4) are selected as the four factors for the next response surface test design.

[0107] like Figure 3 As shown in Figure A, the concentration of surfactant SDS (X1) and liquid-to-solid ratio (X2) are very significant factors affecting the yield of total flavonoids from American ginseng. Figure 3 As shown in B, the surfactant SDS concentration (X1) and liquid-to-solid ratio (X2) are very significant factors affecting the yield of American ginseng crude polysaccharides. Figure 3 As shown in C, the surfactant SDS concentration (X1) and liquid-to-solid ratio (X2) are extremely significant factors in affecting the yield of total saponins of American ginseng, and the ultrasonic power (X3), microwave power (X4) and extraction time (X5) are significant factors; all of which are consistent with the variance analysis of the Plackett-Burman experimental design. Since ginsenosides have higher medicinal value than crude polysaccharides and total flavonoids, the factors of surfactant SDS concentration (X1), liquid-to-solid ratio (X2), ultrasonic power (C), and microwave power (X4) are selected as the key factors for the next step of the response surface experimental design of total flavonoids, crude polysaccharides, and total saponins.

[0108] 1.2.6 Response surface experiment results and analysis

[0109] (1) Response surface Box-Behnken experimental design

[0110] The response surface Box-Behnken method was used to design a 4-factor 3-level experimental model with four factors, namely, surfactant SDS concentration (X1), liquid-to-solid ratio (X2), ultrasonic power (X3), and microwave power (X4), as independent variables, and total flavonoid yield (Y1), crude polysaccharide yield (Y2), and total saponin yield (Y3) as response values. The design scheme and experimental results are shown in Tables 1-4 and 1-5. The quadratic regression equation of the model is as follows:

[0111]

[0112]

[0113]

[0114] Table 1-4 Response surface test design and results

[0115]

[0116] Table 1-5 Response surface design scheme and results

[0117]

[0118] (2) Response surface interaction analysis: From the variance analysis results in Table 1-6, it can be seen that the model is significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the quadratic regression equation model is reliable and the fit is credible. Model determination coefficient R 2 =92.85%, indicating that the regression equation simulates this experiment well with a small error. Adjusted determination coefficient R 2 Adj = 85.69%, the software's predicted value is related to the actual value and has a high correlation. The primary phase of the equation is X1, X3, X1X2, X1X3, and the secondary phase is and The effect on yield is very significant. The influence was significant. The interaction between factors X1 and X2 was significant, the interaction between factors X1 and X3 was significant, and the other factors were not significant. The F value showed that the order of influence of each factor on the total flavonoid yield was X3>X1>X4>X2, that is, ultrasonic power>SDS concentration>microwave power>liquid-to-solid ratio.

[0119] Table 1-6 Results of variance analysis

[0120]

[0121]

[0122] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01)

[0123] As shown in Table 1-7, the model is significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the quadratic regression equation model is reliable and the fit is credible. 2 =94.78%, indicating that the regression equation simulates this experiment well with a small error. Adjusted determination coefficient R 2 Adj =88.15%, the software's predicted value is related to the actual value and has a high correlation. Factors X4, X1X2, X1 2 , and The yield was significantly affected, and factors X1, X2 and X4 had significant effects on the yield. The interaction between factors X1 and X2 was significant, and the interaction between factors X2 and X4 was significant. The other factors were not significant. The F value showed that the order of the influence of each factor on the yield of crude polysaccharides was X4>X1>X2>X3, that is, microwave power>SDS concentration>liquid-to-solid ratio>ultrasonic power.

[0124] Table 1-7 Results of variance analysis

[0125]

[0126] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01)

[0127] As shown in Table 1-8, the model is significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the quadratic regression equation model is reliable and the fit is credible. 2 =96.22%, indicating that the regression equation simulates this experiment well with a small error. Adjusted determination coefficient R 2 Adj =92.44%, the predicted value of the software is related to the actual value and has a high correlation, which shows that the linear regression equation simulates the test well and the test result has a small error. Factor X1 2 , and The yield was significantly affected by factors X1 and X4, while the other factors were not significant. The interaction between factors X1 and X4 was significant. The F value showed that the order of the influence of each factor on the yield of total saponins was X3>X4>X2>X1, that is, ultrasonic power>microwave power>liquid-to-solid ratio>surfactant SDS concentration.

[0128] Table 1-8 Results of variance analysis

[0129]

[0130] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01)

[0131] like Figure 4 As shown in the figure, the elliptical contour lines indicate that the surfactant concentration of factor X1 and the liquid-to-solid ratio of factor X2 have a significant interaction. In the 3D graph of the surfactant concentration of factor X1 and the liquid-to-solid ratio of factor X2, each factor shows a trend of first rising and then falling. This is because when the SDS mass fraction is too small, the solvent cannot form micelles and cannot play a solubilizing effect; with the increase of the SDS mass fraction, the micelles in the solvent fully interact with American ginseng to promote the diffusion of flavonoids into the solvent; but when the SDS mass fraction is too large, the viscosity of the solvent increases, reducing the diffusion rate of flavonoids in the solvent; when other conditions remain unchanged, with the increase of the liquid-to-solid ratio, the flavonoid yield shows a trend of first rising and then falling. This is because the liquid-to-solid ratio increases, the external concentration is low, and the osmotic pressure inside and outside the cell increases, which will promote the overflow of intracellular solutes. However, as the liquid-to-solid ratio continues to increase, the solute concentration of the extract decreases, hindering the solute oscillation, resulting in a decrease in the yield. The elliptical contour lines indicate that the surfactant concentration of factor X1 and the ultrasonic power of factor X3 have a significant interaction. Consistent with the above analysis results, in the 3D graph of factor X1 surfactant concentration and factor X3 ultrasonic power, each factor shows a trend of first increasing and then decreasing. This is because when the SDS mass fraction is too small, the solvent cannot form micelles and cannot play a solubilizing effect; with the increase of SDS mass fraction, the micelles in the solvent fully interact with American ginseng to promote the diffusion of flavonoids into the solvent; but when the SDS mass fraction is too large, the viscosity of the solvent increases, reducing the diffusion rate of flavonoids in the solvent, thereby causing a decrease in the total flavonoid yield; when other conditions remain unchanged, with the increase of ultrasonic power, the flavonoid yield shows a trend of first increasing and then decreasing. This is because the high-power ultrasonic wave diffuses through the solvent solute, ruptures the cell wall, and enhances the physical effect, resulting in an increase in the total flavonoid yield; but when the ultrasonic power is too large, some flavonoids in the solution will be degraded, resulting in a decrease in the total flavonoid yield.

[0132] like Figure 5As shown in the figure, the elliptical contour lines indicate that the surfactant concentration of factor X1 and the liquid-to-solid ratio of factor X2 have a significant interaction. In the 3D graph of the surfactant concentration of factor X1 and the liquid-to-solid ratio of factor X2, each factor shows a trend of first increasing and then decreasing. This is because when the SDS mass fraction is too small, the solvent cannot form micelles and cannot play a solubilizing effect; with the increase of the SDS mass fraction, the micelles in the solvent fully react with American ginseng to promote the diffusion of polysaccharide compounds into the solvent; but when the SDS mass fraction is too large, the viscosity of the solvent increases, resulting in a decrease in the yield of crude polysaccharides; when other conditions remain unchanged, with the increase of the liquid-to-solid ratio, the flavonoid yield shows a trend of first increasing and then decreasing. This is because the liquid-to-solid ratio increases, the external concentration is low, and the osmotic pressure inside and outside the cell increases, which will promote the overflow of intracellular solutes. However, as the liquid-to-solid ratio continues to increase, the solute concentration of the extract decreases, hindering the solute oscillation and causing the yield to decrease. The elliptical contour lines indicate that the liquid-to-solid ratio of factor X2 and the microwave power of factor X4 have a significant interaction. In the 3D graph of factor X2 liquid-to-solid ratio and factor X4 microwave power, each factor shows a trend of first rising and then falling. With the increase of liquid-to-solid ratio, the yield of crude polysaccharides shows a trend of first rising and then falling. This is because the liquid-to-solid ratio increases, the external concentration is low, and the osmotic pressure inside and outside the cell increases, which will promote the overflow of intracellular solutes. However, as the liquid-to-solid ratio continues to increase, the solute concentration of the extract decreases, hindering the solute oscillation and causing the yield to decrease. When other conditions remain unchanged, with the increase of microwave power, the yield of crude polysaccharides shows a trend of first rising and then falling. This is because as the power increases, the intracellular temperature increases, the intracellular pressure increases, and it is easy to squeeze the cell wall, promoting the overflow of crude polysaccharides in the cell, while excessive microwave power causes the temperature to rise rapidly and destroys the structure.

[0133] like Figure 6 As shown in the figure, the elliptical contour lines indicate that the surfactant concentration of factor X1 and the microwave power of factor X4 have significant interactions. In the 3D graph of the liquid-to-solid ratio of factor X2 and the microwave power of factor X4, each factor shows a trend of first rising and then falling. This is because when the mass fraction of SDS is too small, the solvent cannot form micelles and cannot play a solubilizing effect, resulting in a small amount of total saponins dissolved; with the increase of the mass fraction of SDS, the micelles in the solvent fully interact with American ginseng to promote the diffusion of total saponins into the solvent; but when the mass fraction of SDS is too large, the viscosity of the solvent increases, reducing the diffusion rate of total saponins in the solvent, which leads to a decrease in the yield. When other conditions remain unchanged, with the increase of microwave power, the total saponin yield shows a trend of first rising and then falling. This is because with the increase of power, the intracellular temperature increases, the intracellular pressure increases, and it is easy to squeeze the cell wall, promoting the overflow of total saponins in the cell, while the excessive microwave power causes the temperature to rise rapidly and destroys the structure.

[0134] 1.2.7 Model validation results: Using Design-Expert 8.06 software, the best extraction conditions were predicted to affect the total saponin content of American ginseng through optimization, and the factor with the highest total saponin yield was determined as the optimal parameter. The prediction results showed that when the surfactant concentration was 0.5%, the liquid-to-solid ratio was 19.93 mL / g, the ultrasonic power was 304.29 W, and the microwave power was 297.62 W, ​​the total flavonoids, crude polysaccharides, and total saponin yields were 0.21%, 7.69%, and 14.33%, respectively. Based on the requirements of production practice, the preferred production process parameters are: surfactant concentration of 0.5%, liquid-to-solid ratio of 20 mL / g, ultrasonic power of 300 W, microwave power of 300 W, and the total flavonoids, crude polysaccharides, and total saponin yields were 0.22% ± 0.03%, 6.8% ± 0.99%, and 13.57% ± 1.16%, respectively. The error is within 5.0%, and the response surface fitting method established by this method is correct and reliable.

[0135] 1.2.8 Comparison of different extraction processes: This experiment compared the effects of ultrasound-assisted ethanol solution extraction, microwave-assisted ethanol solution, surfactant microwave-assisted extraction, and surfactant ultrasonic-assisted extraction on the extraction efficiency of three target compounds contained in American ginseng. The effects of different extraction methods on the yield (i.e., extraction rate) of the three chemical substances are as follows: Figure 7 As shown in A. Figure 7 It can be seen from Figure A that the yield of total flavonoids extracted by ultrasound-assisted ethanol extraction was 0.251%±0.075%, the yield of crude polysaccharides was 3.51%±0.25%, and the yield of total saponins was 7.54%±0.25%; the yield of total flavonoids extracted by microwave-assisted ethanol extraction was 0.286%±0.083%, the yield of crude polysaccharides was 4.26%±0.23%, and the yield of total saponins was 7.63%±0.26%; The yield of total flavonoids, crude polysaccharides and total saponins by SDS-microwave-assisted extraction was 0.305%±0.098%, 5.13%±0.26% and 8.26%±0.23% respectively; the yield of total flavonoids, crude polysaccharides and total saponins by SDS-ultrasound-assisted extraction was 0.299%±0.087%, 4.98%±0.28% and 8.35%±0.28% respectively.

[0136] Compared with the single-factor experimental results of surfactant extraction of the three active components of American ginseng (after response surface optimization, the yield of total flavonoids in the synergistic extraction of the three active components of American ginseng by surfactant SDS-microwave-ultrasound was 0.22%±0.03%, the yield of crude polysaccharides was 6.8%±0.99%, and the yield of total saponins was 13.57%±1.16%), it can be seen that the synergistic extraction of American ginseng by surfactant SDS-microwave-ultrasound has obvious advantages, and the order of strength of the methods for extracting the three active components of American ginseng is: surfactant SDS-microwave-ultrasound synergistic extraction method > surfactant SDS ultrasound synergistic extraction method > surfactant SDS microwave extraction method > microwave-assisted ethanol extraction method > ultrasound-assisted ethanol extraction method.

[0137] In summary, the present embodiment found that the best experimental conditions were SDS concentration of 0.5%, liquid-to-solid ratio of 20mL:1g, ultrasonic power of 300W, microwave power of 300W, and extraction time of 30s through single factor experiment; the conclusion was drawn through Plackett-Burman test that SDS concentration, liquid-to-solid ratio, ultrasonic power and microwave power were significant factors. On this basis, response surface experimental design was carried out, and the factor with the highest total saponin yield was determined as the optimal parameter prediction result. When the surfactant concentration was 0.5%, the liquid-to-solid ratio was 19.93mL:1g, the ultrasonic power was 304.29W, and the microwave power was 297.62W, the total flavonoids, crude polysaccharides, and total saponin yields were 0.21%, 7.69%, and 14.33%, respectively.

[0138] Based on the requirements of production practice, the optimal production process parameters are: surfactant concentration of 0.5%, liquid-to-solid ratio of 20mL:1g, ultrasonic power of 300W, microwave power of 300W, total flavonoids, crude polysaccharides, and total saponin yields of 0.22%±0.03%, 6.8%±0.99%, and 13.57%±1.16%, respectively. The error is within 5.0%, and the reaction surface fitting method established by this method is correct and credible. The crude extract obtained under this condition was used for subsequent experiments.

[0139] This example compares the effects of several extraction techniques, including ultrasound-assisted ethanol solution, microwave-assisted ethanol solution, surfactant microwave-assisted extraction, and surfactant-ultrasound-assisted extraction, on the extraction efficiency of three target compounds contained in American ginseng. The results show that the micellar medium-microwave-ultrasound synergistic extraction method has the best effect and the highest extraction efficiency.

[0140] Example 2 Response surface design method to optimize anti-solvent recrystallization to prepare American ginseng active ingredient powder

[0141] The present invention uses soybean protein isolate as an embedding material, uses an anti-solvent method to prepare American ginseng active ingredient micropowder, optimizes the process through single factor and response surface experiments, and then uses a laser particle size analyzer to detect the particle size and Zeta potential of the American ginseng active ingredient micropowder to clarify the dispersibility of the micropowder in water. The American ginseng active ingredient micropowder product with strong antioxidant ability, high bioavailability, stability, and not easy to be destroyed is developed.

[0142] 2.1 Experimental methods

[0143] 2.1.1 Extraction of saponins and crude polysaccharides from American ginseng after extraction: After the crude extract after extraction (i.e., the crude extract after extraction prepared under the conditions optimized by response surface in Example 1) was cooled to room temperature, centrifuged at 8000r / min for 15min, supernatant 1 was taken, and anhydrous ethanol was added at a volume ratio of 1:4 of supernatant 1 to anhydrous ethanol for low-temperature alcohol precipitation overnight, and then centrifuged for 15min to obtain a precipitate (i.e., crude polysaccharide) and supernatant 2; supernatant 2 was rotary evaporated at 60°C, and lyophilized to obtain a mixed powder of ginsenosides and flavonoids. The precipitate was rinsed with hot ethanol (generally 50-60°C) for multiple times, and freeze-dried after removing SDS to obtain a crude polysaccharide powder.

[0144] 2.1.2 The crude polysaccharide prepared in 2.1.1 is deproteinized by TCA method, comprising the following steps: 1.0 g of the crude polysaccharide powder prepared in 2.1.1 is dissolved in 100 mL of water to prepare a crude polysaccharide solution of American ginseng; 50 mL of the crude polysaccharide solution of American ginseng is accurately weighed, and added to a 150 mL conical flask, 5% trichloroacetic acid (TCA) solution is added, stirred, and placed in a refrigerator for 24 hours, and then centrifuged for 20 minutes at 4000 r / min. The supernatant (i.e., the crude polysaccharide solution after deproteinization) is taken, and the protein content, protein removal rate, and polysaccharide retention rate are calculated according to the following method.

[0145] The protein content was detected using a BCA protein content kit, including the following steps:

[0146] Reagent preparation: Under alkaline conditions, cysteine, cystine, tryptophan, tyrosine and peptide bonds in proteins can transfer Cu 2+ Reduction to Cu + ; Two molecules of BCA and Cu + Combine to form a purple complex with absorption peaks at 540-595nm, with the strongest absorption peak at 562nm. Mix reagent A and reagent B in a volume ratio of 50:1, preheat in a 60℃ water bath for 30 minutes to obtain a working solution. Add reagents in the container in order according to Table 2-1.

[0147] Table 2-1 Add reagents in the container in sequence

[0148]

[0149] Calculate protein content

[0150]

[0151] Where: W: sample mass (g); C 标准品 : 0.5 mg / mL; D: dilution multiple.

[0152] Calculation of polysaccharide protein removal rate

[0153]

[0154] Calculation of polysaccharide retention

[0155]

[0156] 2.1.3 Analysis of monosaccharide composition of polysaccharides: Reference: Zhang Y, Pan X, Ran S, et al. Purification, structural elucidation and anti-inflammatory activity invitro of polysaccharides from Smilax china L. [J]. International Journal of Biological Macromolecules, 2019, 139: 233-243. The monosaccharide composition of polysaccharides was determined by ion chromatography.

[0157] 2.1.4 Preparation of American ginseng active ingredient powder by anti-solvent recrystallization method: This experiment uses the mixed powder of ginsenosides and flavonoids (referred to as total saponins-flavonoids) prepared by method 2.1.1 as raw material, and uses the anti-solvent recrystallization method to prepare American ginseng active ingredient powder. Through experimental screening, anhydrous ethanol was selected as the anti-solvent and the aqueous solution was used as the solvent. The specific method includes the following steps: weighing the total saponins-flavonoids and ultrasonically dissolving them in an appropriate amount of anhydrous ethanol, fully dissolving them, and obtaining a total saponin-flavonol solution; ultrasonically dissolving soybean protein and crude polysaccharide in an appropriate amount of pure water, the mass ratio of soybean protein to crude polysaccharide is 1:1, and the crude polysaccharide is obtained by drying the crude polysaccharide after removing the protein in Example 2.1.2 to obtain a crude polysaccharide-soy protein solution, and slowly dripping the crude polysaccharide-soy protein solution at a rate of 1mL / min into the total saponin-flavonol solution in magnetic stirring (5000r / min). Finally, the precipitation was freeze-dried by centrifugation at 4000r / min for 10min to obtain the American ginseng active ingredient powder.

[0158] 2.1.5 Single factor experimental design of micro powder preparation process: This experiment selected encapsulation efficiency and drug loading as the measurement indicators of single factor experiment. The concentration of anti-solvent phase (i.e. the concentration of total saponin-flavonol solution) was 5, 10, 15, 20, 25 mg / mL respectively; the concentration of solvent phase was 5, 10, 15, 20, 25 mg / mL respectively, the concentration of solvent phase refers to the mass concentration of soy protein + crude polysaccharide in soy protein-crude polysaccharide aqueous solution (i.e. the concentration of the sum of the mass of soy protein and crude polysaccharide), and the mass ratio of soy protein to crude polysaccharide was 1:1; the anti-solvent phase: solvent phase (the volume ratio of total saponin-flavonol solution to soy protein-crude polysaccharide aqueous solution) was 10mL:1mL, 20mL:1mL, 30mL:1mL, 40mL:1mL, 50mL:1mL respectively.

[0159] 2.1.6 Response surface experimental design of micropowder preparation process: Based on the single factor experiment, the anti-solvent phase concentration, solvent phase concentration, and anti-solvent phase: solvent phase ratio were selected to investigate the influence of three factors on the micropowder preparation process of American ginseng active ingredient. The response surface Box-Behnken design8.0 was used to perform the response surface experiment. There were 3 factors and 3 levels, for a total of 17 experiments. The Box-Behnken test factors and levels are shown in Table 2-2, and the experimental design scheme is shown in Table 2-3.

[0160] Table 2-2 Box-Behnken test factors and levels for optimization of micro powder preparation process

[0161]

[0162] Table 2-3 Response surface experimental design scheme

[0163]

[0164] 2.1.7 Calculation of drug loading and encapsulation efficiency of American ginseng active ingredient micropowder

[0165] Weigh 0.1g of American ginseng active ingredient powder, add 10mL of anhydrous ethanol, and adjust the volume to 25mL of anhydrous ethanol after ultrasonication for 30min. Centrifuge at 8000r / min for 15min to obtain a precipitate and a supernatant solution. Take the supernatant solution, detect the drug loading and encapsulation efficiency of the total flavonoids in the sample solution, and substitute them into formula 2-4 and public 2-7 to calculate the drug loading and encapsulation efficiency of the total flavonoids in the American ginseng active ingredient powder. The precipitate after centrifugation is re-dissolved with distilled water, the concentration of the crude polysaccharide in the sample solution is detected, and it is substituted into formula 2-5 and public 2-8 to calculate the drug loading and encapsulation efficiency of the crude polysaccharide in the American ginseng active ingredient powder. Take the supernatant solution and use the experimental method of Example 1 to measure the total saponin concentration, and substitute it into formula 2-6 and formula 2-9 to calculate the drug loading and encapsulation efficiency of the total saponins in the American ginseng active ingredient powder.

[0166]

[0167] 2.1.8 Characterization of the physicochemical properties of American ginseng active ingredient powder

[0168] (1) Detection of powder potential: After diluting the active ingredient powder of American ginseng, place it in a laser particle size analyzer for potential detection.

[0169] (2) Particle size detection: After diluting the active ingredient powder of American ginseng, the particle size is detected using a laser particle size analyzer.

[0170] (3) Differential scanning calorimetry: Take the active ingredient powder of American ginseng and the ginsenoside extract of American ginseng with the same saponin mass, place them in a crucible, and use a differential scanning calorimeter to detect. The DSC detection conditions are: temperature range 20-300℃, heating rate 10℃ / min. This experiment needs to be tested under nitrogen protection. Use a thermogravimetric analyzer to test its thermal stability.

[0171] (4) Thermogravimetric analysis: Thermogravimetric analysis equipment was used to detect thermal stability and composition. TG test conditions: temperature range 40-800°C, heating rate 10°C / min.

[0172] 2.1.9 In vitro digestion experiment

[0173] (1) In vitro simulated gastric juice digestion experiment: Take two 100mL conical flasks, add 25mL artificial gastric juice containing protease in turn, the protease content in the artificial gastric juice is 10g / L, adjust the pH between 2.0-2.5, add 700mg of American ginseng active ingredient powder and 100mg American ginseng original powder with the same saponin content in two conical flasks, simulate the gastric digestion process, and shake in a water bath at 37°C, 150rmp for 1h. After the reaction is completed, centrifuge at 8000r / min for 10min, and take the supernatant as gastric digestion juice. Determine the content of ginsenosides after in vitro digestion according to the method of Example 1, and substitute the results into the standard curve for calculation. Compare the destruction of American ginseng active ingredient powder and American ginseng original powder in gastric juice.

[0174] (2) In vitro simulated intestinal digestion experiment: Take two 100mL conical flasks, add 25mL of artificial intestinal juice containing pancreatin in turn, the content of pancreatin in the artificial intestinal juice is 10g / L, adjust the pH to 7.0, add 700mg of American ginseng active ingredient powder and 100mg of American ginseng original powder with the same saponin content in two conical flasks respectively, simulate the intestinal digestion process, shake at 37°C, 150rmp, water bath for 3h, after the reaction is completed, centrifuge at 8000r / min for 10min, and take the supernatant as intestinal digestion juice. Determine the content of ginsenosides after in vitro digestion according to the method of Example 1, substitute the results into the standard curve for calculation, and use HPLC to detect the drug concentration of American ginseng active ingredient powder and American ginseng original powder. Compare the destruction of American ginseng active ingredient powder and American ginseng original powder in intestinal juice.

[0175] 2.2 Experimental Results

[0176] 2.2.1 Results of polysaccharide deproteinization: Photos of crude polysaccharide before deproteinization and crude polysaccharide after deproteinization with 10% TCA solution are shown in the figure below. Figure 7 As shown in B. The protein content of crude polysaccharide before deproteinization was 2.76%, and the protein content of crude polysaccharide after deproteinization with 10% TCA solution was 1.03%, the protein removal rate was 63%, and the polysaccharide retention rate was 79.2%.

[0177] 2.2.2 Analysis of monosaccharide composition of American ginseng polysaccharide: The monosaccharide composition of American ginseng polysaccharide was compared with the HPLC chromatograms of the derivatives of 9 standard monosaccharides (mannose, ribose, rhamnose, glucose, galactose, xylose, arabinose, glucuronic acid, galacturonic acid). According to the retention time and peak area, it was determined that American ginseng polysaccharide is mainly composed of 6 monosaccharides, namely mannose, ribose, rhamnose, xylose, arabinose, and galacturonic acid. Figure 8 As shown, the galacturonic acid content in American ginseng polysaccharides is high, indicating that American ginseng polysaccharides have strong antioxidant activity.

[0178] 2.2.3 Results of single factor experiment

[0179] (1) Effect of antisolvent phase concentration (total saponin-flavonol solution concentration) on encapsulation efficiency and drug loading

[0180] like Fig. 9As shown in A, when the antisolvent phase concentration is between 5-25 mg / mL, the concentration of the antisolvent phase increases with different trends. When the concentration reaches 15 mg / mL, the encapsulation efficiency is 85.21% ± 2.8%, and the drug loading is 0.89% ± 0.09%. The encapsulation efficiency and drug loading both reach their maximum values. After that, the antisolvent phase concentration continues to increase, and the encapsulation efficiency and drug loading gradually decrease. Supersaturation in the system is one of the main reasons affecting the encapsulation efficiency and drug loading. An increase in the actual concentration of the drug will lead to an increase in supersaturation in the system. In order to prepare micropowders with a larger encapsulation efficiency and drug loading as much as possible, the optimal value of the antisolvent phase concentration is selected as 15 mg / mL.

[0181] (2) Effect of solvent phase concentration (polysaccharide-soy protein aqueous solution) on encapsulation efficiency and drug loading

[0182] Effect of solvent phase concentration (polysaccharide-soy protein aqueous solution) on encapsulation efficiency and drug loading Fig. 9 As shown in B. When the solvent phase concentration is between 5-25 mg / mL, the concentration of the solvent phase increases in different trends. When the concentration reaches 15 mg / mL, the encapsulation rate is 88.29% ± 1.9%, and the drug loading is 0.89% ± 0.05%. The encapsulation rate and drug loading both reach their maximum values. After that, the encapsulation rate and drug loading gradually decrease as the solvent phase concentration continues to increase. In order to prepare nanopowders with a larger encapsulation rate and drug loading as much as possible, the optimal value of the anti-solvent phase concentration is selected as 15 mg / mL.

[0183] (3) Effect of the volume ratio of antisolvent phase to solvent phase on encapsulation efficiency and drug loading

[0184] from Fig. 9 It can be seen from Figure C that within the range of (10-50):1, at 40:1, the encapsulation efficiency is 85.22%±2.3%, the drug loading is 0.89%±0.09%, and the encapsulation efficiency and drug loading both reach the maximum value. After that, the solvent phase concentration continues to increase, and the encapsulation efficiency and drug loading gradually decrease. This phenomenon may be due to the increase in volume ratio, which leads to a simultaneous decrease in the equilibrium concentration and actual concentration of the American ginseng extract in the mixed system, and the supersaturation of the mixed system. When the volume ratio increases from 10:1 to 40:1, the actual concentration of the system decreases by more than the equilibrium concentration, and the supersaturation in the mixed system increases. Therefore, with the increase in volume ratio, the encapsulation efficiency and drug loading of the micropowder show an increasing trend. However, when the volume ratio is greater than 40:1, the supersaturation decreases. Therefore, in the process of increasing the volume ratio from 40:1 to 50:1, the encapsulation efficiency and drug loading of the American ginseng active ingredient micropowder show a decreasing trend. According to the above experimental results, the optimal value of the volume ratio of the antisolvent to the main solvent is 40:1.

[0185] 2.2.4 Response surface experimental design and results

[0186] (1) Response surface Box-Behnken experimental design

[0187] According to the results of the single factor test, it was found that the concentration of total saponin-flavonol solution in the anti-solvent phase, the concentration of polysaccharide-soy protein aqueous solution in the solvent phase, and the volume ratio of the anti-solvent phase to the solvent significantly affected the drug loading and encapsulation efficiency. Therefore, the drug loading (Y1) and encapsulation efficiency (Y2) were selected as the response values, and the concentration of total saponin-flavonol solution in the anti-solvent phase (X1), the concentration of polysaccharide-soy protein aqueous solution in the solvent phase (X2), and the volume ratio of the anti-solvent phase to the solvent (X3) were used as independent variable factors to conduct a response surface test with 3 factors and 3 levels. The factor level coding table is shown in Table 2-4, and the response surface test plan and results are shown in Table 2-5. The quadratic regression equation is as follows:

[0188] Encapsulation efficiency (Y1) = 91.14 + 3.06A + 3.84B - 3.13C - 5.60AB - 1.28AC - 4.68BC - 8.80A 2 -7.05B 2 -3.52C 2

[0189] Drug loading (Y2) = 1.09 + 0.12A - 9.375 × 10 -3 B-0.024C-0.060AB-0.080AC-0.057BC-0.28A 2 -0.19B 2 -0.13C 2

[0190] Table 2-4 Experimental parameters for optimizing the preparation process of micronized powder of active ingredients of American ginseng

[0191]

[0192] Table 2-5 Factor level coding table

[0193]

[0194]

[0195] (2) Response surface interaction analysis

[0196] As shown in Table 2-6, the model is significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the quadratic regression equation model is reliable and the fit is credible. Model determination coefficient R 2 =92.75%, indicating that the regression equation simulates this experiment well with a small error. Adjusted determination coefficient R 2 Adj=83.43%, the predicted value of the software is related to the actual value and has a high correlation, which shows that the linear regression equation simulates the test well and the error of the test result is small. It has a very significant effect on the encapsulation rate. Factors X1, X2, X3, X1X2, X1X3, and X2X3 have a significant effect on the encapsulation rate. The interaction between factors X1 and X2 is significant, the interaction between factors X2 and X3 is significant, and the interaction between factors X1 and X3 is significant. The remaining factors are not significant. The F value shows that the order of the influence of each factor on the total saponin yield is X2>X3>X1, that is, the solvent phase concentration (polysaccharide-soy protein aqueous solution)>the volume ratio of the anti-solvent phase to the solvent phase>the anti-solvent phase concentration (total saponin-flavonol solution concentration).

[0197] Table 2-6 Results of variance analysis

[0198]

[0199] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01)

[0200] Depend on Fig.10It can be seen that the contour maps of the total saponin-flavonol solution concentration of factor X1 and the polysaccharide-soy protein aqueous solution concentration of factor X2 are both elliptical, and the interaction is significant, which is consistent with the analysis results in Table 2-6. In the interactive 3D response surface diagram of factor X1 and factor X2, under the same conditions, the drug loading first increases and then gradually decreases due to the increase in the concentration of the total saponin-flavonol solution. This is mainly because there are still impurities such as sugars in the total saponin-flavonol solution. When the concentration of the total saponin-flavonol solution increases, the viscosity of the solution also increases, thereby affecting the dispersion and recrystallization of the isolated protein during the dripping process and the ability to encapsulate saponins; when other conditions remain unchanged, with the increase in the concentration of the soybean protein polysaccharide solution, the drug loading shows a trend of first increasing and then decreasing. This is because with the increase in the concentration of the soybean protein isolate dripping, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of the soybean protein isolate after dripping, and reduces the encapsulation ability of saponins. The contour maps of the total saponin-flavonol solution concentration of factor X1 and the volume ratio of factor X3 are both elliptical, with significant interactions, which is consistent with the analysis results in Table 2-6. In the interactive 3D response surface diagram of factor X1 and factor X3, when other conditions remain unchanged, with the increase of the concentration of the alcohol solution of saponins, the drug loading of the American ginseng active ingredient micropowder first increases and then decreases. This is because the purified ginsenosides still contain some impurities such as sugars. With the increase of the concentration of the alcohol solution of saponins, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of soy protein isolate after dripping, and reduces the encapsulation ability of ginsenosides; under the same other conditions, the drug loading increases with the increase of the volume ratio, and then gradually decreases. This is mainly because, with the increase of the volume ratio of the alcohol solution, the content of soy protein isolate in the solution will gradually decrease, and the lower content of isolated protein is not conducive to the recrystallization of protein, nor to the embedding of saponins. The contour maps of the polysaccharide-soy protein aqueous solution concentration of factor X2 and the volume ratio of factor X3 are both elliptical, with significant interactions, which is consistent with the analysis results in Table 2-6. In the interactive 3D response surface diagram of factor X2 and factor X3, when other conditions remain unchanged, with the increase of the concentration of soybean protein polysaccharide solution, the drug loading shows a trend of first increasing and then decreasing. This is because with the increase of the concentration of soybean protein isolate dripping, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of soybean protein isolate after dripping, and reduces the encapsulation ability of saponin; under the same other conditions, the drug loading increases with the increase of volume ratio, and then gradually decreases. This is mainly because, with the increase of the volume ratio of alcohol solution, the content of soybean protein isolate in the solution will gradually decrease, and the lower content of isolated protein is not conducive to the recrystallization of protein, nor to the embedding of saponin substances.

[0201] As shown in Table 2-7, the model is significant (P<0.01) and the lack of fit term is not significant (P>0.05), indicating that the quadratic regression equation model is reliable and the fit is credible. Model determination coefficient R2 =98.27%, indicating that the regression equation simulates this experiment well with a small error. Adjusted determination coefficient R 2 Adj =96.06%, the software's predicted value is related to the actual value and has a high correlation, which shows that the linear regression equation simulates the test well and the test result has a small error. It has a very significant effect on drug loading, factors X1X2 and X2X3 have a significant effect on drug loading, factors X1 and X2 interact significantly, factors X2 and X3 interact significantly, factors X1 and X3 interact significantly, and the remaining factors are not significant. From the size of the F value, it can be seen that the order of the influence of each factor on drug loading is X1>X3>X2, that is, the concentration of the antisolvent phase (total saponin-flavonol solution concentration)>the volume ratio of the antisolvent phase to the solvent phase>the concentration of the solvent phase (polysaccharide-soy protein aqueous solution concentration).

[0202] Table 2-7 Results of variance analysis

[0203]

[0204] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01)

[0205] Depend on Fig.11It can be seen that the contour maps of the total saponin-flavonol solution concentration of factor X1 and the polysaccharide-soy protein aqueous solution concentration of factor X2 are both elliptical, and the interaction is significant, which is consistent with the analysis results in Table 2-7. In the interactive 3D response surface diagram of factor X1 and factor X2, when other conditions remain unchanged, with the increase of the concentration of the alcohol solution of saponin, the drug loading of the American ginseng active ingredient micropowder first increases and then decreases. This is because the purified ginsenosides still contain some impurities such as sugars. With the increase of the concentration of the alcohol solution of saponin, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of the soy protein isolate after dripping, and reduces the drug loading capacity of ginsenosides; when other conditions remain unchanged, with the increase of the concentration of the soy protein polysaccharide solution, the drug loading shows a trend of first increasing and then decreasing. This is because with the increase of the concentration of the soy protein isolate dripping, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of the soy protein isolate after dripping, and reduces the drug loading capacity of saponins. The contour maps of total saponin-flavonol solution concentration of factor X1 and volume ratio of factor X3 are both elliptical, and the interaction is significant, which is consistent with the analysis results in Table 2-7. In the interactive 3D response surface diagram of factor X1 and factor X3, when other conditions remain unchanged, with the increase of the concentration of saponin alcohol solution, the drug loading of American ginseng active ingredient micropowder shows a trend of first rising and then falling. This is because the purified ginsenosides still contain some impurities such as sugars. With the increase of the concentration of saponin alcohol solution, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of soy protein isolate after dripping, and reduces the drug loading capacity of ginsenosides; when other conditions remain unchanged, with the increase of volume ratio, the drug loading shows a trend of first rising and then falling. This is because with the continuous increase of the volume ratio of alcohol solution, the concentration of soy protein isolate in the solution continues to decrease. Too low soy protein isolate concentration is not conducive to protein recrystallization, nor is it conducive to the encapsulation of ginsenosides in the solution, which reduces the drug loading capacity of saponins. The contour maps of the polysaccharide-soy protein aqueous solution concentration of factor X2 and the volume ratio of the antisolvent phase to the solvent phase of factor X3 are both elliptical, and the interaction is significant, which is consistent with the analysis results in Table 2-7. In the interactive 3D response surface diagram of factor X2 and factor X3, when other conditions remain unchanged, with the increase of the concentration of soybean protein polysaccharide solution, the drug loading capacity shows a trend of first increasing and then decreasing. This is because with the increase of the concentration of soybean protein isolate dripping, the viscosity of the solution increases, which is not conducive to the dispersion and recrystallization of soybean protein isolate after dripping, and reduces the drug loading capacity of saponin; when other conditions remain unchanged, with the increase of volume ratio, the drug loading capacity shows a trend of first increasing and then decreasing. This is because with the continuous increase of the volume ratio of alcohol solution, the concentration of soybean protein isolate in the solution continues to decrease. Too low soybean protein isolate concentration is not conducive to protein recrystallization, nor is it conducive to the encapsulation of ginsenosides in the solution, which reduces the drug loading capacity of saponins.

[0206] (3) Verification model

[0207] The response surface design method was used to optimize the optimal conditions for the preparation of American ginseng active ingredient micropowder by anti-solvent recrystallization. The optimal process conditions were predicted based on the maximum drug loading condition, which were: the total saponin-flavonol solution concentration in the anti-solvent phase (X1) was 16.21 mg / mL, the polysaccharide-soy protein aqueous solution concentration in the solvent phase (X2) was 14.80 mg / mL, the volume ratio of the anti-solvent phase to the solvent phase (X3) was 38.45:1, the encapsulation efficiency was 91.67%, and the drug loading was 1.10%. According to the actual operation needs, the actual optimal process conditions were: the total saponin-flavonol solution concentration in the anti-solvent phase (X1) was 16 mg / mL, the polysaccharide-soy protein aqueous solution concentration in the solvent phase (X2) was 15 mg / mL, and the volume ratio of the anti-solvent phase to the solvent phase (X3) was 39:1, the encapsulation efficiency was 89%±1.03%, and the drug loading was 0.97%±0.07%. The error was within 5.0%, indicating that the process model fitted by the response surface was accurate and reliable. Fig.12 As shown in A, the response surface design method is used to optimize the anti-solvent recrystallization to prepare the active ingredient powder of American ginseng. The powder is fluffy and slightly yellow. After dissolving in water, it is a clear and transparent liquid. Under the irradiation of laser, the Tyndall phenomenon occurs.

[0208] 2.2.5 Drug loading capacity: Under the optimal process conditions, i.e., the concentration of total saponin-flavonol solution in the anti-solvent phase was 16 mg / mL, the concentration of polysaccharide-soy protein aqueous solution in the solvent phase was 15 mg / mL, and the volume ratio of the anti-solvent phase to the solvent phase was 39:1, the drug loadings of total saponin, crude polysaccharide, and total flavonoids were 0.97%±0.07%, 0.64%±0.05%, and 0.51%±0.05%, respectively.

[0209] 2.2.6 Encapsulation efficiency: Under the optimal process conditions, namely, the concentration of total saponin-flavonol solution in the anti-solvent phase was 16 mg / mL, the concentration of polysaccharide-soy protein aqueous solution in the solvent phase was 15 mg / mL, and the volume ratio of the anti-solvent phase to the solvent phase was 39:1, the encapsulation rates of total saponins, crude polysaccharides, and total flavonoids were 89%±1.03%, 82.69%±2.8%, and 82.73%±2.8%, respectively.

[0210] 2.2.7 Characterization results and analysis of the physicochemical properties of American ginseng active ingredient powder

[0211] (1) Potential detection results: The absolute value of the potential of the micropowder is -33.05 mV, which is consistent with the curve, indicating that the micropowder solution of the active ingredient of American ginseng has good stability.

[0212] (2) Particle size test results of American ginseng active ingredient powder: The particle size of American ginseng active ingredient powder after anti-solvent recrystallization was tested using a laser particle size analyzer. Each sample was tested three times. The particle size distribution of American ginseng active ingredient powder by laser particle size analysis is shown in Fig.12In the sample B, the average particle size is 318.6 nm, the particle size distribution is narrow, and the dispersibility is good, which indicates that the active ingredient powder of American ginseng has been successfully prepared into nanometer level by anti-solvent recrystallization method.

[0213] (3) Infrared spectroscopy (FT-IR) detection: The results are as follows Fig.12 As shown in C, at 750-1250cm -1 The micro powder is different from the American ginseng saponin extract, and the absorbance in the fingerprint area is obviously different. This shows that the micro powder is wrapped by soybean protein and crude polysaccharide, and the process is reliable and stable.

[0214] (4)DSC analysis: Fig.13 The DSC detection curves are of the American ginseng ginsenoside extract (i.e., the ginsenoside and flavonoid powder in Example 2.1.1) and the American ginseng active ingredient powder (i.e., the American ginseng active ingredient powder prepared under the response surface optimization conditions). When DSC is used for melting test, as the temperature is raised, the American ginseng ginsenoside extract and the prepared American ginseng active ingredient powder will show obvious endothermic phenomenon in the melting range. The American ginseng active ingredient powder has a starting temperature of 169.53°C, an ending temperature of 181.51°C, and a melting enthalpy of 52.13 J / g; the American ginseng ginsenoside extract has a starting temperature of 153.27°C, an ending temperature of 172.95°C, and a melting enthalpy of 674.2 J / g. This shows that the prepared American ginseng active ingredient powder has higher thermal stability than the original powder.

[0215] (5) Thermogravimetric analysis: Fig.14 It is the thermogravimetric detection spectrum of American ginseng ginsenoside extract and American ginseng active ingredient powder (i.e. ginsenoside soy protein isolate powder). Fig.14 It can be seen that the endothermic peaks of the American ginseng ginsenoside extract and the American ginseng active ingredient powder have a slow decline process before 200°C, and then drop sharply from 200°C to 500°C, and gradually level off after 500°C. The weight loss temperature of the American ginseng active ingredient powder is 208.44°C, and the weight loss temperature of the American ginseng ginsenoside extract is 150.97°C, indicating that the American ginseng active ingredient powder is more stable than the American ginseng ginsenoside extract.

[0216] 2.2.8 In vitro digestion test results and analysis

[0217] (1) Results and analysis of in vitro simulated gastric juice digestion

[0218] Depend on Fig.15It can be seen from Figure A that after 1h of simulated digestion with gastric juice, the total ginsenosides in the original American ginseng powder (i.e., the American ginseng powder prepared in Example 1.1.1) were 2.58±0.15mg and 0.15±0.03mg, respectively, which means that 63.19%±2.19% of the ginsenosides were degraded and 60%±1.2% of the total flavonoids were degraded. The total ginsenosides in the American ginseng active ingredient powder were 5.33±0.38mg and 0.07±0.01mg, respectively, which means that 21.9%±1.65% of the ginsenosides were degraded and 20%±2.8% of the total flavonoids were degraded. Proteases and the like can have a destructive effect on ginsenosides and soy protein. However, it can be seen that the American ginseng active ingredient powder can effectively protect the sensitive substances, ginsenosides and flavonoids, from being destroyed in simulated gastric juice. This shows that the American ginseng active ingredient powder is not easily destroyed and has good stability.

[0219] (2) Results and analysis of in vitro simulated intestinal digestion

[0220] Depend on Fig.15 From Figure B, it can be seen that after 3 hours of simulated digestion by intestinal fluid, the total ginsenosides and flavonoids of American ginseng powder were 2.59±0.19mg and 0.16±0.01mg respectively, i.e., 62.10%±4.29% of ginsenosides and 34.67%±2.31% of flavonoids were degraded; the total ginsenosides and flavonoids of American ginseng powder were 5.62±0.21mg and 0.29±0.01mg respectively, i.e., 19.24%±2.93% of ginsenosides and 17.14%±2.86% of flavonoids were degraded, because the intestinal fluid is milder than the gastric fluid environment, and there are fewer proteases, which have little damage to the protein carrier. It can be seen that the active ingredient powder of American ginseng can effectively protect the sensitive substances, ginsenosides and flavonoids, from being destroyed in the simulated intestinal fluid. This shows that the active ingredient powder of American ginseng is not easily destroyed and has good stability.

[0221] Crude polysaccharide and American ginseng ginsenoside extracts were prepared by micellar medium-microwave-ultrasound synergistic extraction method. The crude polysaccharide was deproteinized with 10% TCA solution. The protein content of the crude polysaccharide was 2.76% before deproteinization and 1.03% after deproteinization with 10% TCA solution. The protein removal rate was 63% and the polysaccharide retention rate was 79.2%.

[0222] In this embodiment, the response surface design is used to optimize the process of preparing the active ingredient powder of American ginseng by anti-solvent recrystallization. Through single factor experiments, the optimal extraction parameters are determined as follows: the total saponin-flavonol solution concentration (X1) of the anti-solvent phase is 15 mg / mL, the polysaccharide-soy protein aqueous solution concentration (X2) of the solvent phase is 15 mg / mL, and the volume ratio of the anti-solvent phase to the solvent phase (X3) is 40: 1. Then, the response surface Box-Behnken method is used to design a 3-factor 3-level experiment. The final actual optimal process conditions are: the anti-solvent phase concentration (X1) is 16 mg / mL, the solvent phase concentration (X2) is 15 mg / mL, and the volume ratio of the anti-solvent phase to the solvent phase (X3) is 39: 1. Therefore, the American ginseng active ingredient powder obtained under the above optimal conditions is used for subsequent experiments.

[0223] The prepared American ginseng active ingredient powder is fluffy and slightly yellow, and the Tyndall phenomenon occurs after dissolution. The average particle size of the powder is 318.6nm; the total saponin loading is 0.97%±0.07%, and the encapsulation efficiency is 89%±1.03%; the crude polysaccharide loading is 0.64%±0.05%, and the encapsulation efficiency is 82.69%±2.8%; the total flavonoid loading is 0.51%±0.05%, and the encapsulation efficiency is 82.73%±2.8%.

[0224] After 1 hour of simulated digestion with gastric juice, 63.19%±2.19% of ginsenosides were degraded and 60%±1.2 of total flavonoids were degraded. 21.9%±1.65% of ginsenosides and 20%±2.8% of total flavonoids of American ginseng active ingredient micro-powder were degraded. After 3 hours of simulated digestion with intestinal juice, 62.10%±4.29% of ginsenosides and 34.67%±2.31% of total flavonoids were degraded; 19.24%±2.93% of ginsenosides and 17.14%±2.86% of total flavonoids of American ginseng active ingredient micro-powder were degraded. When performing infrared spectroscopy detection at 750-1250cm -1 There is a significant difference in the absorbance of the fingerprint area between the American ginseng active ingredient powder and the saponin extract. This shows that the American ginseng active ingredient powder is wrapped by soy protein and crude polysaccharides, and the process is reliable and stable. When DSC was used for melting test, the starting temperature of the American ginseng active ingredient powder was 169.53℃, the ending temperature was 181.51℃, and the melting enthalpy was 52.13J / g; the starting temperature of the American ginseng ginsenoside extract was 153.27℃, the ending temperature was 172.95℃, and the melting enthalpy was 674.2J / g. The weight loss temperature of the American ginseng active ingredient powder was 208.44℃, and the weight loss temperature of the American ginseng ginsenoside extract was 150.97℃, indicating that the American ginseng active ingredient powder is more stable than the American ginseng ginsenoside extract.

[0225] Example 3 In vitro study on hypolipidemic, hypoglycemic and anti-oxidative properties

[0226] 3.1 Experimental methods

[0227] 3.1.1 Preparation of standard curves for sodium glycocholate, sodium taurocholate and sodium cholate: Take 2 mL of each standard solution of sodium glycocholate, sodium taurocholate and sodium cholate of different concentrations (0.03, 0.06, 0.12, 0.18, 0.24, 0.30, 0.36, 0.42 and 0.48 mmol / L) in a volumetric flask, add 6 mL of 60% sulfuric acid, incubate in a water bath at 70°C for 20 min, then in an ice bath for 5 min, and measure the absorbance at a wavelength of 387 nm using a UV spectrophotometer.

[0228] 3.1.2 Determination of bile salt binding rate: Take 0.5 g of American ginseng raw powder (i.e., American ginseng powder prepared in Example 1.1.1), American ginseng crude extract (i.e., the crude extract after extraction prepared under the conditions optimized by response surface in Example 1), and American ginseng active ingredient powder (i.e., American ginseng active ingredient powder prepared under the conditions optimized by response surface), add 3 mL of 10 mg / mL pepsin (dissolved in 0.1 mol / L phosphate buffer at pH 6.5) and 1 mL of 0.01 mol / L hydrochloric acid, place the sample in a constant temperature water bath at 37°C, and continue to oscillate digestion for 1 hour to simulate the effect of gastric digestion. After that, the pH of the solution was adjusted to 6.3 with 0.1 mol / L NaOH solution, and then 4 mL of 10 mg / mL trypsin (dissolved in 0.1 mol / L phosphate buffer at pH 6.5) was added. The sample was placed in a constant temperature water bath at 37°C and digested with shaking for 1 hour to simulate the effect of intestinal digestion. Finally, 4 mL of 0.4 mmol / L sodium glycocholate and 4 mL of 0.5 mmol / L sodium taurocholate were added to the sample. The sample was placed in a constant temperature water bath at 37°C and digested with shaking for 1 hour. The mixed solution was transferred to a centrifuge tube and centrifuged at 10,000 r / min for 20 minutes. The supernatant was filtered with a 0.45 μm aqueous needle filter, and the absorbance was measured at a wavelength of 387 nm using an ultraviolet spectrophotometer to calculate the content of sodium glycocholate, sodium taurocholate, and sodium cholate. The calculation method for the cholate binding rate is as follows:

[0229]

[0230] 3.1.3 Determination of pancreatic lipase activity inhibition rate

[0231] Preparation of the main solution: 0.08% (w / v) 4-nitrophenyl laurate (p-PNL) solution: Weigh 0.4 g p-PNL, add it to 5 mmol / L sodium acetate solution containing 1% Triton X-100, boil it in a boiling water bath for 1 min to help it dissolve, cool to room temperature, make up to 50 mL with 5 mmol / L sodium acetate solution, and store it in a refrigerator at 4°C.

[0232] 3 mL of pH 7.4 phosphate buffer, 3 mL of 0.08% lauric acid 4-nitrophenyl ester solution, 3 mL of 1.0 mg / mL pancreatic lipase solution, and a certain amount of American ginseng original powder, crude extract after extraction, and American ginseng active ingredient powder were mixed evenly to prepare sample solutions with concentrations of 1, 3, 6, 9, 12, and 15 mg / mL, and placed in a 37°C constant temperature water bath for 30 minutes, and then the absorbance value at 410 nm was measured and recorded as A. At the same time, the absorbance at 410 nm without adding sample was A1, and the absorbance at 410 nm with sample but without pancreatic lipase solution (replaced with phosphate buffer) was A0, and the pancreatic lipase inhibition rate was calculated according to the formula:

[0233]

[0234] Wherein, C is the inhibition rate of pancreatic lipase (%); A is the absorbance value of the sample group; A1 is the absorbance value of the blank group; A0 is the absorbance value of the control group.

[0235] 3.1.4 Determination of inhibition rate of cholesterol micelle solubility

[0236] To prepare 10 mL of cholesterol micelle solution, take 7.7 mg of cholesterol, 53.8 mg of sodium taurocholate, 77.1 mg of sodium chloride, 14.1 mg of oleic acid, and 7.5 mL of 0.02 mol pH 7.4 phosphate buffer, mix them, perform ultrasonic homogenization at 160 W for 50 min, and place them in a 37 ° C environment for 12 h. Prepare sample solutions with concentrations of 1, 3, 6, 9, 12, and 15 mg / mL of American ginseng raw powder, crude extract after extraction, and American ginseng active ingredient powder, respectively. Take equal volumes of sample solutions of different concentrations and mix them with micelle solution, then shake and culture the mixed solution at 37 ° C for 2 h, centrifuge at 14000 r / min for 20 min, collect the supernatant, and use a cholesterol determination kit to determine the cholesterol content in the micelle. The cholesterol solubility of the sample solution is A, and the cholesterol solubility without adding the sample solution is A0. As the control group, the inhibition rate is calculated as follows:

[0237]

[0238] Where: A is the absorbance value of the sample solution; A0 is the absorbance value of the control group.

[0239] 3.1.5 In vitro hypoglycemic activity studies

[0240] (1) Determination of α-glucosidase inhibition activity

[0241] PNPG can be hydrolyzed into 1-glucose and PNP under the action of α-glucosidase. PNP has a maximum absorbance at 410nm. If there is an α-glucosidase inhibitor in the sample solution, it can specifically inhibit α-glucosidase, reduce the degradation of PNPG by α-glucosidase, reduce the generation of glucose and PNP, and reduce the absorbance.

[0242] 40 μL of different mass concentrations (1, 15, 10, 15, 20 mg / mL) of American ginseng raw materials, crude extracts after extraction, and micro-powder solutions were added with 40 μL of 1U / mL α-glucosidase solution (dissolved in 25 mmol / L pH 6.8 phosphate buffer) and mixed evenly, mixed and reacted in a 37°C water bath for 10 minutes, and then added with 20 μL of 7.5 mmol / L p-NPG solution (dissolved in 0.1 mol / L pH 6.8 phosphate buffer), reacted in a 37°C water bath for 30 minutes, and then added 100 μL of 0.1 mol / L sodium carbonate solution to terminate the reaction. The absorbance value was measured at 410 nm, acarbose was used as a positive control, and an equal volume of 0.1 mol / L pH 6.8 phosphate buffer was used instead of α-glucosidase as a control group. The specific operation is shown in Table 3-1.

[0243] Table 3-1 Experimental procedures for determining the activity of α-glucosidase inhibition

[0244]

[0245] The inhibition rate formula is as follows:

[0246]

[0247] Wherein: W—α-glucosidase inhibition rate (%); A—sample group absorbance; A0—sample background group absorbance; A1—blank group absorbance.

[0248] (2) Determination of α-amylase inhibition activity

[0249] 20 μL of American ginseng powder of different mass concentrations (1, 5, 10, 15, 20 mg / mL), crude extract after extraction, and American ginseng active ingredient powder were mixed with 100 μL of 1U / mL α-amylase solution (dissolved in pH 6.8 phosphate buffer) in a 37°C water bath for 10 min, and then 300 μL of 1% soluble starch solution was added, mixed and reacted in a 37°C water bath for 10 min, and then 50 μL of DNS reagent was added, and the reaction was terminated by heating in a boiling water bath for 5 min. After cooling to room temperature, the absorbance was measured at 540 nm. Acarbose was used as a positive control, and an equal volume of 0.1 mol / L pH 6.8 phosphate buffer was used instead of α-amylase as a control group. The specific operations are shown in Table 3-2.

[0250] Table 3-2 Experimental procedures for determining the activity of α-amylase inhibition

[0251]

[0252] The inhibition rate formula is as follows:

[0253] Wherein: W—α-glucosidase inhibition rate (%); A—sample group absorbance; A0—sample background group absorbance; A1—blank group absorbance.

[0254] 3.1.6 In vitro antioxidant activity studies

[0255] (1) DPPH in vitro antioxidant test

[0256] ①Prepare DPPH test solution: prepare 0.1mmol / L DPPH anhydrous ethanol solution.

[0257] ②Vc control experiment: prepare 2.5 mg / mL Vc reference solution; then dilute it with deionized water to 0.0025, 0.005, 0.01, 0.025, 0.05, 0.075, and 0.1 mg / mL reference solutions.

[0258] ③Prepare sample solution: Weigh different masses of samples and dissolve them in 70% ethanol solution to prepare sample solutions of 0.0025, 1.0, 10, 20, 40, 60, and 80 mg / mL. Ultrasonic extraction was performed at 280 W and 45 °C for 1 h, followed by centrifugation at 8000 r / min for 20 min, and the supernatant was retained for testing.

[0259] ④ Determine DPPH scavenging ability: Take 2mL of each concentration of Vc control solution, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder solution, add 2mL of DPPH solution, mix thoroughly, seal and place in the dark for 30 minutes, measure the absorbance value of each sample at 517nm, and substitute the results into formula 3-6 to calculate the scavenging rate of each concentration of Vc control solution, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder. The scavenging rate calculation formula is as follows:

[0260]

[0261] Where: A0—absorbance of 2 mL DPPH solution and 2 mL anhydrous ethanol; A1—absorbance of 2 mL sample solution and 2 mL anhydrous ethanol; A2—absorbance of 2 mL DPPH solution and 2 mL sample solution.

[0262] (2)ABTS + In vitro antioxidant assay

[0263] ① Preparation of ABTS + Test solution: Accurately weigh 0.75mmol potassium persulfate and ABTS + 0.25mmol was placed in a 100mL volumetric flask, dissolved and fixed to volume with distilled water to prepare ABTS + After reacting at room temperature in the dark for 16 h, dilute ABTS with 1 mmol / L PBS buffer at pH 7.4. + The absorbance value of the mother solution is about 0.70±0.02 at a wavelength of 734nm, that is, ABTS is obtained. + Test solution.

[0264] ② Vc control experiment: prepare 2.5 mg / mL Vc reference solution; then dilute it with deionized water to 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, and 0.125 mg / mL reference solutions.

[0265] ③Prepare sample solution: prepare the sample into solutions of 0.0025, 1.0, 10, 20, 40, and 60 mg / mL, dissolve in 70% ethanol solution at a solid-liquid ratio of 1:25, perform ultrasonic extraction at 280 W and 45 °C for 1 h, then centrifuge at 8000 r / min for 20 min, and take the supernatant for later use.

[0266] ④ Determination of scavenging capacity: Take 4.8mL ABTS + The test solution was added with 0.2 mL of each concentration of Vc control solution, American ginseng powder, crude extract after extraction, and sample solution of American ginseng active ingredient powder, respectively. The mixture was sealed and protected from light for 30 minutes. The absorbance of each sample was detected at a wavelength of 734 nm, and the absorbance value was recorded as A. The blank was adjusted to zero with 1 mmol / L phosphate buffer with a pH value of 7.4, and the results were brought into ABTS + The clearance rate formula is used to calculate the clearance rates of Vc control solution of each concentration, American ginseng original powder, crude extract after extraction, and American ginseng active ingredient powder solution. The clearance rate calculation formula is as follows:

[0267]

[0268] 3.1.7 Calculating IC 50 Value: IC 50 The value refers to the concentration at which the sample scavenges half of the free radicals. 50 The value calculator calculated the inhibitory capacity of acarbose control solution, Vc control solution, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on α-glucosidase and α-amylase, DPPH and ABTS + Clearing ability.

[0269] 3.2 Experimental Results and Analysis

[0270] 3.2.1 Standard curve: The standard curve equation of sodium glycocholate is y=2.8473x+0.0319, R 2 =0.9973; the standard curve equation of sodium taurocholate is y=1.8793x+0.1544, R 2 =0.9914; the standard curve equation of sodium cholate is y=1.2413x+0.019, R 2 =0.9948. Wherein, x (mmol / L) is the concentration of sodium sulfocholate and sodium cholate, and y is the absorbance.

[0271] 3.2.2 Analysis of the binding rate of sodium glycocholate: The binding rate of sodium glycocholate of the original powder of American ginseng was 26.12%±2.45%, the binding rate of the crude extract of sodium cholate after extraction was 41.13%±2.16%, and the binding rate of the micro powder of the active ingredient of American ginseng was 68.45%±2.39%. It was proved that the optimization process of preparing the micro powder of the active ingredient of American ginseng by recrystallization method can improve the binding rate of sodium glycocholate and improve its lipid-lowering effect.

[0272] 3.2.3 Analysis of the binding rate of sodium taurocholate: The binding rate of sodium taurocholate of American ginseng raw powder is 29.45%±1.98%, the binding rate of sodium cholate of crude extract after extraction is 54.68%±2.46%, and the binding rate of American ginseng active ingredient powder is 75.36%±1.97%. It is proved that the optimization process of preparing American ginseng active ingredient powder by recrystallization can improve the binding rate of sodium taurocholate and improve its lipid-lowering effect.

[0273] 3.2.4 Analysis of sodium cholate binding rate: The sodium cholate binding rate of American ginseng raw powder was 30.12%±2.13%, the binding rate of crude extract after extraction was 52.13%±2.10%, and the binding rate of American ginseng active ingredient powder was 70.45%±2.15%. It was proved that the optimization process of preparing American ginseng active ingredient powder by recrystallization method can improve the binding rate of sodium cholate and improve its lipid-lowering effect.

[0274] 3.2.5 Analysis of the effect of pancreatic lipase activity inhibition rate: Fig.16As can be seen in Figure A, the inhibitory effects of American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on pancreatic lipase are concentration-dependent and increase with increasing concentration. When the concentration of American ginseng powder solution is less than 15 mg / mL, its inhibitory effect on pancreatic lipase activity increases rapidly with increasing concentration. After 18 mg / mL, the dependence of the inhibitory effect on concentration weakens. At 15 mg / mL, the inhibition rate reaches 49.15%±1.05%, and at 18 mg / mL, the inhibition rate reaches 53.91%±2.57%. The inhibitory effect of the crude extract after extraction on pancreatic lipase is significantly stronger, and it also shows a dose relationship. At a concentration of 15 mg / mL, its inhibition rate is 1.00%. The inhibition rate was 59.43%±1.28% at 18mg / mL, and reached 69.61%±1.29% at 18mg / mL; the inhibitory effect of the active ingredient powder of American ginseng on pancreatic lipase activity was the best, with an inhibition rate of 77.77%±3.16% at a concentration of 15mg / mL, and an inhibition rate of 81.69%±1.26% at 18mg / mL, proving that the optimized process of preparing the active ingredient powder of American ginseng by recrystallization method did improve the inhibitory effect of pancreatic lipase activity, and the powder made the drug more effective. The strength is: American ginseng active ingredient powder> crude extract after extraction> American ginseng original powder.

[0275] 3.2.6 Analysis of the effect of cholesterol micelle solubility inhibition rate: This experiment simulated the conditions of the human small intestine in vitro to compare the binding ability of American ginseng raw powder, crude extract after extraction, and American ginseng active ingredient powder with cholesterol. Fig.16 As can be seen from B, the three samples have different binding abilities to cholesterol, and the solubility of cholesterol decreases with increasing concentration. This indicates that all three samples can reduce the solubility of cholesterol in micellar solution. After comparing their binding abilities to cholesterol, it was found that the binding ability of American ginseng active ingredient powder to cholesterol was the strongest, with an inhibition rate of 91.69%±1.25% at a concentration of 18 mg / mL. The binding ability of the crude extract after extraction to cholesterol was stronger than that of the original American ginseng powder, reaching 67.95%±2.52% at a concentration of 18 mg / mL. It can be seen that the binding ability of the original American ginseng powder was the weakest, with an inhibition rate of 62.24%±1.70% on the solubility of cholesterol micelles at a concentration of 18 mg / mL. The strength is: American ginseng active ingredient powder > crude extract after extraction > original American ginseng powder.

[0276] 3.2.7IC 50 Calculation results and analysis: Fig.16 As shown in C, in the pancreatic lipase activity inhibition experiment, the IC 50 The value is 16.21 mg / mL, and the IC of the crude extract after extraction is 50 The value is 12.69mg / mL, and the IC value of American ginseng active ingredient powder is 50The value is 7.29mg / mL. In the cholesterol micelle solubility inhibition activity experiment, the IC 50 The value is 15.11 mg / mL, and the IC of crude extract after extraction is 50 The value is 12.11mg / mL, the IC value of American ginseng active ingredient powder 50 The value was 6.32 mg / mL. Therefore, the inhibitory activity was in the following order: American ginseng active ingredient powder > crude extract > American ginseng original powder.

[0277] 3.2.8 Evaluation of α-glucosidase activity inhibition effect: Fig.16 As can be seen from Figure D, the four solutions have different inhibitory effects on α-glucosidase, and are dose-dependent. As the mass concentration of the solution increases, the inhibition rates of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on α-glucosidase continue to increase. At different concentrations of 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, and 25 mg / mL, the inhibition rates of American ginseng powder on α-glucosidase were 7.11%±1.41%, 9.42%±0.9%, 20.89%±1.38%, 38.23%±2.17%, 58.42%±3.53%, 63.43%±3.60%, respectively. %±2.86%, 66.07%±1.00%; the inhibition rates of crude extracts on α-glucosidase after extraction were 10.21%±1.74%, 25.15%±1.57%, 35.33%±1.07%, 59.43%±3.12%, 65.68%±3.66%, 74.44%±1.65%, 77.2%±1.55%, respectively; the inhibition rates of American ginseng active ingredient powder on α-glucosidase were The production rates were 20.8%±4.51%, 32.21%±2%, 55.44%±0.84%, 65.74%±4.57%, 67.75%±3.4%, 79.9%±2.93%, and 85.24%±0.62%, respectively; the concentrations of acarbose were 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, and 1.0mg / mL Under the conditions of 4040 × 240 nm, the inhibition rates of α-glucosidase were 31.05%±1.96%, 44.72%±2.18%, 65.97%±3.18%, 74.39%±3.75%, 92.04%±2.24% and 92.67%±2.21%, respectively; therefore, in the experiment of α-glucosidase activity inhibition, the inhibition rate was as follows: acarbose>American ginseng active ingredient powder>crude extract after extraction>American ginseng original powder.

[0278] 3.2.9 Evaluation of α-amylase activity inhibition effect: Fig.16As can be seen from Figure E, the four solutions have different inhibitory effects on α-amylase and are dose-dependent. As the mass concentration of the solution increases, the inhibition rates of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on α-amylase continue to increase. At different concentrations of 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, and 25 mg / mL, the inhibition rates of American ginseng powder on α-amylase were 4.54%±1.1%, 8.32%±0.99%, 14.42%±1.11%, 35.00%±2.26%, 47.12%±2.24%, and 57.70%±2.60%, respectively. %±2.28%; 62.45%±1.96%; the inhibition rates of crude extracts on α-amylase after extraction were 11.43%±1.05%, 18.68%±1.2%, 30.5%±1.00%, 51.47%±1.16%, 58.25%±1.38%, 71.31%±1.21%; 76.85%±1.58%; the inhibition rates of American ginseng active ingredient powder on α-amylase were The results were as follows: 19.73% ± 1.46%, 29.12% ± 0.76%, 41.28% ± 1.43%, 56.9% ± 2.01%, 68.53% ± 2.01%, 76.54% ± 3.77%; 85.04% ± 1.43%; acarbose at concentrations of 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, At 0.5 mg / mL, the inhibition rates of α-amylase were 24.94%±1.98%, 38.02%±2.28%, 51.44%±1.56%, 67.1%±0.77%, 87.87%±2%, and 93.2%±2.22%, respectively. Therefore, in the α-amylase activity inhibition experiment, the inhibition rate was: acarbose>American ginseng active ingredient powder>crude extract after extraction>American ginseng original powder.

[0279] 3.2.10 Results and analysis of in vitro antioxidant experiments

[0280] (1) Results and analysis of DPPH in vitro antioxidant test: As shown in Figure 16F, the R% values ​​of the seven concentrations of American ginseng powder were 0.01% ± 0.001%, 2.19% ± 0.24%, 14.16% ± 0.9%, 34.19% ± 0.64%, 55.5% ± 4.71%, 61.73% ± 2.47%, and 77.31% ± 1.95%. The R% values ​​of the seven concentrations of aqueous solutions of the crude extract after extraction were 0.0113% ± 0.0015, 3.34% ± 1.22%, 19.17% ± 1.44%, 42.22% ± 2.45%, 61.81% ± 3.2%, 73.64% ± 2.59%, and 83.01% ± 3.1%. The R% values ​​of the seven concentrations of the aqueous solution of the active ingredient powder of American ginseng were 0.021% ± 0.03%, 5.38% ± 0.78%, 23.47% ± 2.64%, 58.6% ± 2.76%, 80.04% ± 3.31%, 83.74% ± 3.31%, 88.31% ± 2.53%. The R% values ​​of the seven concentrations of the Vc aqueous solution were 0.82% ± 0.97%, 1.56% ± 0.28%, 8.62% ± 0.97%, 19.83% ± 0.49%, 46.67% ± 1.15%, 74.18% ± 1.76%, 88.16% ± 0.61%. In the DPPH in vitro antioxidant experiment, the strength of the ability to remove free radicals was: Vc> American ginseng active ingredient powder> crude extract after extraction> American ginseng original powder.

[0281] (2)ABTS + In vitro antioxidant test results and analysis: Fig.16As shown in G, the R% values ​​of the 7 concentrations of American ginseng raw powder are 0.015% ± 0.045%, 2.07% ± 0.16%, 12.88% ± 0.63%, 32.93% ± 3.22%, 48.41% ± 2.56%, 58.65% ± 2.00%, 71.30% ± 1.11%. The R% values ​​of the 7 concentrations of aqueous solutions of the crude extract after extraction are 0.018% ± 0.05%, 3.69% ± 0.78%, 17.17% ± 2.01%, 32.85% ± 2.41%, 55.31% ± 3.36%, 61.47% ± 2.75%, 76.95% ± 1.59%. The R% values ​​of the seven concentrations of the aqueous solution of the American ginseng active ingredient powder were 0.037% ± 0.006%, 5.81% ± 0.90%, 24.07% ± 2.13%, 55.30% ± 1.74%, 75.01% ± 2.69%, 81.18% ± 1.97%, 84.98% ± 0.84%. The R% values ​​of the seven concentrations of the Vc aqueous solution were 0.43% ± 0.07%, 1.16% ± 0.17%, 6.65% ± 0.35%, 23.77% ± 1.50%, 58.09% ± 2.32%, 72.58% ± 2.68%, 79.15% ± 0.26%. Therefore, in ABTS + In the in vitro antioxidant experiment, the ability to remove free radicals was as follows: Vc>American ginseng active ingredient powder>extract after extraction>American ginseng active ingredient powder.

[0282] 3.2.11IC 50 Calculation results and analysis of values: As shown in Table 3-3, the activities of α-glucosidase and α-amylase were studied for hypoglycemic efficacy. The results of the α-glucosidase inhibition rate experiment showed that acarbose IC 50 Value 0.026mg / mL, American ginseng powder IC 50 The value is 13.17 mg / mL, and the IC of crude extract after extraction is 50 The value is 6.26mg / mL, and the IC value of American ginseng active ingredient powder is 50 The value is 3.33mg / mL. The results of the α-amylase inhibition test showed that acarbose IC 50 The value is 0.037mg / mL, and the IC value of American ginseng powder is 50 The value is 17.26 mg / mL, and the IC value of crude extract after extraction is 50 The value is 11.43mg / mL, the IC value of American ginseng active ingredient powder 50 The value is 4.34 mg / mL. Therefore, the order of the inhibitory ability of hypoglycemic activity is: acarbose>American ginseng active ingredient powder>extracted crude extract>American ginseng original powder.

[0283] As shown in Table 3-4, the research on the removal of DPPH and ABTS+ In vitro antioxidant capacity of free radicals. In the DPPH in vitro antioxidant test, the IC 50 The value is 0.079mg / mL, and the IC value of American ginseng powder is 50 The value is 36.867 mg / mL, and the IC of the crude extract after extraction is 50 The value is 25.077mg / mL, and the IC value of American ginseng active ingredient powder is 50 The value is 14.293 mg / mL. + In the in vitro antioxidant test, the IC 50 The value is 0.0514mg / mL, and the IC value of American ginseng powder is 50 The value is 45.883 mg / mL, and the IC of crude extract after extraction is 50 The value is 35.270 mg / mL, and the IC 50 The value is 16.008 mg / mL. The ability to remove free radicals: acarbose>American ginseng active ingredient powder> crude extract after extraction>American ginseng original powder.

[0284] Table 3-3 IC values ​​of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on the inhibition of α-glucosidase and α-amylase activities 50 Value (mg / mL)

[0285]

[0286] Table 3-4 Effects of acarbose, American ginseng powder, crude extract after extraction, and American ginseng active ingredient powder on DPPH and ABTS + IC of clearance 50 Value (mg / mL)

[0287]

[0288] The present invention studies the binding rate of sodium glycocholate, sodium taurocholate and sodium cholate, and obtains that the binding rate of sodium glycocholate of American ginseng raw powder is 26.12%±2.45%, the binding rate of sodium glycocholate of crude extract after extraction is 41.13%±2.16%, and the binding rate of sodium glycocholate of American ginseng active ingredient micropowder is 68.45%±2.39%, indicating that the binding rate of American ginseng active ingredient micropowder to sodium cholate is the best. The binding rate of sodium taurocholate of American ginseng raw powder is 29.45%±1.98%, the binding rate of sodium taurocholate of crude extract after extraction is 54.68%±2.46%, and the binding rate of sodium taurocholate of American ginseng active ingredient micropowder is 75.36%±1.97%. The sodium cholate binding rate of American ginseng raw powder was 30.12%±2.13%, the sodium cholate binding rate of crude extract after extraction was 52.13±2.10%, and the sodium cholate binding rate of American ginseng active ingredient powder was 70.45%±2.15%. When the concentration of American ginseng raw powder solution was 18mg / mL, the inhibition rate reached 53.91%±2.57%; the crude extract after extraction had a significantly stronger inhibitory effect on pancreatic lipase, and also showed a dose relationship, with an inhibition rate of 69.61%±1.29% at a concentration of 18mg / mL; the American ginseng active ingredient powder had the best inhibitory effect on pancreatic lipase activity, with an inhibition rate of 81.69%±1.26% at 18mg / mL, proving that the optimized process of preparing American ginseng active ingredient powder by recrystallization method did improve the inhibitory effect of pancreatic lipase activity.

[0289] The binding ability of the active ingredient powder of American ginseng to cholesterol is the strongest, and the inhibition rate reaches 91.69%±1.25% at a concentration of 18 mg / mL. The binding ability of the crude extract after extraction to cholesterol is stronger than that of the original American ginseng powder, reaching 67.95%±2.52% at a concentration of 18 mg / mL, and the binding ability of the original American ginseng powder is the weakest. The experimental results show that the optimization process of preparing the active ingredient powder of American ginseng by recrystallization method improves the cholesterol-solid micelle inhibition effect, thereby improving the ability to lower blood lipids. Because the active ingredient powder of American ginseng makes the powder particles smaller and easier to be digested and absorbed by the human body, the blood lipid-lowering effect is better.

[0290] The results of the in vitro hypoglycemic experiment showed that the inhibitory strengths on the activities of α-glucosidase and α-amylase were as follows: acarbose > American ginseng active ingredient powder > crude extract after extraction > American ginseng original powder.

[0291] The activity of α-glucosidase and α-amylase was studied to investigate the hypoglycemic effect. The results of the α-glucosidase inhibition test showed that the IC 50 The value is 3.33mg / mL. The results of the α-amylase inhibition test show that the active ingredient of American ginseng powder IC 50The value was 4.34 mg / mL, indicating that the preparation of American ginseng active ingredient powder by anti-solvent recrystallization optimized by response surface design method had a good hypoglycemic effect.

[0292] The results of in vitro antioxidant experiments showed that the + The scavenging ability is as follows: Vc>American ginseng active ingredient powder> crude extract after extraction> American ginseng original powder. + In the DPPH in vitro antioxidant test, the IC 50 The value is 14.293 mg / mL. + In the in vitro antioxidant test, the IC 50 The value was 16.008 mg / mL. This showed that the preparation of American ginseng active ingredient powder by anti-solvent recrystallization optimized by response surface design method had good antioxidant capacity.

[0293] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting active ingredients from American ginseng, characterized in that: The following steps are involved: American ginseng is used as a raw material and a micellar medium-microwave-ultrasound synergistic extraction method is adopted to extract active ingredients; the active ingredients include one or more of flavonoids, polysaccharides and total saponins.

2. The extraction method according to claim 1, characterized in that The following steps are involved: American ginseng powder is added to a solution containing a surfactant and extracted by a microwave-ultrasonic synergistic extraction method; in the solution containing a surfactant, the concentration of the surfactant is 0.5%; the liquid-to-solid ratio of the extraction system is (19-20) mL: 1 g; the ultrasonic power is 300-310 W; the microwave power is 290-300 W; and the extraction time is 30 s.

3. The extraction method according to claim 2, characterized in that The concentration of surfactant was 0.5%, the liquid-to-solid ratio was 20 mL: 1 g, the ultrasonic power was 300 W, the microwave power was 300 W, and the extraction time was 30 s.

4. The extraction method according to any one of claims 1 to 3, characterized in that The surfactant is sodium lauryl sulfate.

5. A method for preparing American ginseng active ingredient powder, characterized in that: The following steps are involved: The total saponin-flavonol solution is used as the anti-solvent phase, the crude polysaccharide-soy protein aqueous solution is used as the solvent phase, the crude polysaccharide-soy protein aqueous solution is dropped into the total saponin-flavonol solution, and centrifuged to obtain a precipitate.

6. The preparation method according to claim 5, characterized in that: The preparation method of the total saponin-flavonol solution comprises the following steps: dissolving the mixed powder of ginsenosides and flavonoids in anhydrous ethanol to obtain the total saponin-flavonol solution; the preparation method of the mixed powder of ginsenosides and flavonoids comprises the following steps: extracting the active ingredients of American ginseng by the extraction method described in any one of claims 1 to 4, cooling the extracted crude extract, centrifuging to obtain supernatant 1, taking supernatant 1 for alcohol precipitation, centrifuging again to obtain supernatant 2 and precipitate; rotary evaporating the supernatant 2, freeze-drying to obtain the mixed powder of ginsenosides and flavonoids.

7. The preparation method according to claim 5, characterized in that: The method for preparing a crude polysaccharide-soy protein aqueous solution comprises the following steps: mixing crude polysaccharide powder from which protein has been removed, soy protein and water to obtain a crude polysaccharide-soy protein aqueous solution; the method for preparing the crude polysaccharide powder from which protein has been removed comprises the following steps: extracting the active ingredients of American ginseng by the extraction method described in any one of claims 1 to 4, cooling the extracted crude extract, centrifuging to obtain supernatant 1, taking supernatant 1 for alcohol precipitation, centrifuging again to obtain supernatant 2 and a precipitate; washing the precipitate with ethanol, drying to obtain crude polysaccharide powder, and removing protein from the crude polysaccharide powder.

8. The preparation method according to any one of claims 5 to 7, characterized in that: The concentration of the antisolvent phase is 15-16 mg / mL, the concentration of the solvent phase is 15 mg / mL, and the volume ratio of the antisolvent phase to the solvent phase is 39:1 to 40:

1.

9. The active ingredient of American ginseng is used in any one or more of (1) to (3), characterized in that: The American ginseng active ingredient is prepared by the extraction method according to any one of claims 1 to 4; (1) preparing blood sugar lowering products; (2) preparing blood lipid lowering products; (3) Prepare products with antioxidant capacity.

10. The active ingredient powder of American ginseng is used in any one or more of (1) to (3), characterized in that: The American ginseng active ingredient powder is prepared by the preparation method according to any one of claims 5 to 8; (1) preparing blood sugar lowering products; (2) preparing blood lipid lowering products; (3) Prepare products with antioxidant capacity.

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