Preparation method and application of fructus hippophae pomace flavone extract freeze-dried powder

Abstract

The invention relates to a preparation method and application of sea buckthorn pomace flavone extract freeze-dried powder, and belongs to the technical field of natural product extraction. The method comprises the following steps: pretreating (drying and powdering) a sea buckthorn pomace raw material, preparing a crude extract, purifying the extract, freeze-drying the extract, and finally obtaining the sea buckthorn pomace flavone extract freeze-dried powder. The by-product sea buckthorn pomace in the sea buckthorn processing process is subjected to resource utilization, the vibration type ultrafine pulverizer is adopted for pulverizing to increase the extraction surface area, the ultrasonic synergistic biological enzymolysis method is adopted for promoting release of active molecules, the efficiency is improved, energy and time are saved, no harmful solvent is used in the extraction process, and the method is environmentally friendly, simple in preparation process and high in operability; the large-scale production is easy to realize. According to the sea buckthorn pomace flavone extract freeze-dried powder prepared by the method, the total flavone extraction amount reaches 17.79-23.05 mg / g of the dry weight of sea buckthorn pomace, the purity can reach 50-65%, and the sea buckthorn pomace flavone extract freeze-dried powder has good oxidation resistance, alpha-glucosidase inhibitory activity and alpha-amylase inhibitory activity and can be used for developing products for reducing blood sugar.

Description

1. Technical Field

[0001] The invention relates to a preparation method of seabuckthorn pomace flavonoid extract freeze-dried powder and application thereof, belonging to the technical field of natural product extraction. 2. Background Technology

[0002] Seabuckthorn is a deciduous shrub of the genus Hippophae rhamnoides of the family Elaeagnaceae. It is planted all over the world and is one of the early medicinal and edible plants in my country. Seabuckthorn is rich in a variety of bioactive substances such as vitamins, flavonoids and phenols, unsaturated fatty acids, triterpenes, 5-hydroxytryptamine, etc. It has a variety of pharmacological and health-care effects such as anti-oxidation, anti-aging, anti-tumor, hypoglycemic, immune enhancement, anti-inflammatory and myogenic effects, and can effectively relieve cardiovascular diseases, inhibit bacteria, improve intestinal flora and other disease prevention functions. Seabuckthorn seeds, seabuckthorn fruits and seabuckthorn leaves are good sources of precious nutrients. There are many existing applications of seabuckthorn. For example, patent CN202411393767.5 discloses a seabuckthorn flash-release direct drinking powder with antioxidant function and its preparation method. The product has high nutritional content and significant antioxidant effect; patent CN202411434926.1 discloses ultrasonic extraction using ethanol to obtain a crude extract of total flavonoids from seabuckthorn branches and trunks, and experiments have proved that the prepared extract has good antioxidant and anti-inflammatory effects. The invention relates to the extraction of flavonoids from pomace, a byproduct of seabuckthorn processing. The obtained lyophilized powder of flavonoids extract has good α-glucosidase and α-amylase inhibitory activity and has potential blood sugar lowering effect.

[0003] Seabuckthorn pomace is a byproduct of seabuckthorn processing, accounting for 20% of the fresh weight of seabuckthorn. At present, seabuckthorn fruit in my country is mainly used for the production of juice. Every year, tens of thousands of tons of pomace produced by seabuckthorn processing are discarded as waste, with low utilization efficiency, loss of a large amount of nutrients, waste of resources and environmental pollution. Therefore, the utilization of seabuckthorn pomace has become a new research hotspot. Seabuckthorn pomace is rich in nutrients, including protein, dietary fiber, fatty acids, carotenoids, 17 kinds of amino acids, vitamins, minerals, etc. Seabuckthorn pomace is rich in bioactive ingredients such as flavonoids and polyphenols. Studies have shown that seabuckthorn flavonoids have the functions of anti-oxidation, lowering blood sugar, promoting wound healing, enhancing immunity, lowering blood lipids, and regulating oxidative stress damage. Therefore, extracting flavonoids from seabuckthorn pomace and deep processing can promote resource utilization and economic development. Traditional extraction methods of flavonoids include solvent extraction and heat reflux extraction. There are also many methods for extracting flavonoids in the prior art, such as the ultrasonic-assisted extraction method for extracting flavonoids mentioned in patent CN202411845396.X; patent CN202410842155.3 discloses a method for separating and purifying seabuckthorn flavonoids from seabuckthorn pomace, but the extraction is divided into three steps, the steps are cumbersome, and multiple organic solvents are involved. This project adopts the ultrasonic-assisted bioenzymatic extraction method, which is more efficient and effective. Effectively developing seabuckthorn pomace-related products and improving the comprehensive utilization value of seabuckthorn have important research significance and application prospects. III. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing freeze-dried powder of flavonoid extract of seabuckthorn pomace and its application, which adopts ultrafine grinding, ultrasonic synergistic bioenzymatic method for extraction, macroporous resin purification, and freeze drying to obtain the product. The mechanical effect, cavitation effect, emulsification, diffusion, crushing, chemical effect and thermal effect of ultrasonic waves cause the plant cell wall and the whole organism to break, and the whole breaking process is completed in an instant, which is conducive to the release and dissolution of effective components in plants, and can ensure that the structure and biological activity of effective components remain unchanged. The action of enzymes can fully destroy the cell wall structure of plants and the pectin connected between cells, so that pectin is completely decomposed into small molecules, reducing the mass transfer resistance of extraction, and promoting the full release of flavonoids from plants. In the extraction process, taking the flavonoid extraction amount as an index, a single factor experiment is carried out by controlling variables, and the optimal process parameters for extracting seabuckthorn flavonoids are found. Under the parameters, the total flavonoid extraction amount in seabuckthorn pomace is high, and the product obtained after purification and freeze drying has good α-glucosidase and α-amylase inhibitory activity, which can be applied to the development of functional foods such as hypoglycemic products.

[0005] To achieve the above object, the present invention discloses a method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract and its application, comprising the following steps:

[0006] Step 1, pretreatment of seabuckthorn pomace: the seabuckthorn pomace raw material is the seabuckthorn pomace obtained as a byproduct of squeezing seabuckthorn fruit juice during the processing of seabuckthorn fruit, which is placed in an oven for drying to remove impurities such as seabuckthorn seeds, and then crushed to obtain seabuckthorn pomace powder;

[0007] Step 2, preparation of crude extract of seabuckthorn pomace flavonoids: extracting the seabuckthorn pomace powder mentioned in step 1 by using ultrasound-assisted bio-enzymatic hydrolysis to obtain crude extract of seabuckthorn pomace flavonoids;

[0008] Step 3, purification of the seabuckthorn pomace flavonoids extract: removing impurities from the crude seabuckthorn pomace flavonoids extract mentioned in step 2, and purifying it with a macroporous resin to obtain a purified seabuckthorn pomace flavonoids eluate;

[0009] Step 4, drying the seabuckthorn pomace flavonoids extract: removing ethanol from the seabuckthorn pomace flavonoids purified eluate mentioned in step 3 by rotary evaporation, and freeze-drying to obtain a seabuckthorn pomace flavonoids extract freeze-dried powder;

[0010] Further: the drying temperature in step 1 is 55-65° C. and the drying time is 22-24 hours; the sea buckthorn seeds are removed through a 20-mesh sieve, and the pomace is crushed into fine powder using a vibrating superfine grinder, and then passed through an 80-mesh sieve for later use;

[0011] Further: in step 2, the ultrasonic synergistic bioenzymatic extraction is mentioned, the ultrasonic power is 300W, pectinase is selected for bioenzymatic hydrolysis, the amount of bioenzyme added is 1500U / g to 3000U / g, the extraction solvent is 40% to 70% ethanol, the extraction temperature is 30°C to 60°C, and the extraction time is 20 to 40min; the solid-liquid ratio is 1:15 to 1:35;

[0012] Further: In step 3, the seabuckthorn pomace flavonoid extract is purified by first removing protein by sevage method, then removing sugar by alcohol precipitation method, and finally purifying by AB-8 macroporous adsorption resin, and adsorbing for 3 to 5 hours. When eluting, first use 2 times the volume of distilled water to wash away water-soluble impurities, and then use 2 times the volume of ethanol with a concentration of 55% to 70% for elution, the elution rate is 1.8 mL / min, and the elution time is 1 to 3 hours.

[0013] Further: in step 4, the seabuckthorn pomace flavonoid extract is freeze-dried by first pre-freezing the extract in a -80°C refrigerator for 12 hours, and then drying it in a vacuum freeze dryer for 24 to 26 hours;

[0014] Furthermore: freeze-dried powder of seabuckthorn pomace flavonoid extract was obtained, with a total flavonoid extraction amount of 17.79-23.05 mg / g seabuckthorn pomace dry weight and a purity of 50-65%. It has good antioxidant, α-glucosidase and α-amylase inhibitory activities and can be used in the development of functional foods such as hypoglycemic foods.

[0015] Beneficial effects:

[0016] (1) The method for preparing the freeze-dried powder of the seabuckthorn pomace flavonoid extract of the present invention adopts ultrafine grinding, ultrasonic synergistic bioenzymatic extraction, and no harmful or complex organic solvents are used in the extraction process, which is green and environmentally friendly. The seabuckthorn flavonoid extraction efficiency is high, the total flavonoid extraction amount is large, energy saving and time saving, the preparation process is simple, the operability is strong, and large-scale production is easy to achieve.

[0017] (2) The freeze-dried powder of seabuckthorn pomace flavonoid extract obtained by the present invention has a high flavonoid content, good antioxidant activity, and the potential to enhance the body's immunity; it has a significant inhibitory effect on α-glucosidase and α-amylase, can inhibit the decomposition of sugars into glucose and thus inhibit the increase in blood sugar, and can be used for the development of functional products such as lowering blood sugar.

[0018] (3) The seabuckthorn pomace flavonoid extract freeze-dried powder of the present invention adopts seabuckthorn pomace, which is a by-product in the seabuckthorn processing process, as its raw material. The seabuckthorn pomace can be rationally utilized to turn waste into treasure, protect the ecological environment, improve the comprehensive utilization of seabuckthorn, and be applied in the development of functional foods, thereby increasing the added value of products, increasing farmers' income, and thus driving the development of the agricultural economy. IV. Description of the drawings

[0019] Figure 1 Response surface optimization of extraction of flavonoids from seabuckthorn pomace by ultrasound-assisted bio-enzymatic hydrolysis, where A is the interaction between ethanol concentration and extraction time, B is the interaction between solid-liquid ratio and extraction time, and C is the interaction between solid-liquid ratio and ethanol concentration;

[0020] Figure 2 This is the HPLC-MS spectrum (360nm) of the flavonoids extract from seabuckthorn pomace. V. Specific implementation methods

[0021] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0022] The present invention first pre-treats the seabuckthorn pomace, and then prepares a crude extract of seabuckthorn pomace flavonoids. On the one hand, the extraction methods are explored as ultrasound only, enzymatic hydrolysis only, ultrasound-assisted biological enzymatic hydrolysis, and different enzymes required, including cellulase, pectinase, and cellulase and pectinase complex enzymes, and it is found that selecting pectinase for ultrasonic-assisted enzymatic hydrolysis is a better process technology; on the other hand, the effects of the amount of biological enzyme added, enzymatic hydrolysis temperature, enzymatic hydrolysis time, ultrasonic treatment time, and ultrasonic power on the extraction yield are explored to obtain the process preparation parameters of the crude extract of seabuckthorn flavonoids. Then, a response surface experiment of ultrasonic-assisted enzymatic extraction of seabuckthorn flavonoids is carried out to obtain the optimal process parameters, and then the crude extract of seabuckthorn pomace flavonoids is purified, and finally a freeze-dried powder of seabuckthorn pomace flavonoid extract is obtained, which specifically includes the following steps:

[0023] Step 1: Pretreatment of seabuckthorn pomace: Dry the seabuckthorn pomace, a byproduct obtained during the processing of seabuckthorn fruit juice, in an oven at 55-65°C for 22-24 hours, remove the seabuckthorn seeds through a 20-mesh sieve, crush the pomace into fine powder using a vibrating superfine grinder, and pass through an 80-mesh sieve for later use;

[0024] Step 2: Preparation of crude extract of flavonoids from seabuckthorn pomace: extract the seabuckthorn pomace powder mentioned in step 1 by ultrasonic synergistic bioenzymatic hydrolysis, with an ultrasonic power of 300W, pectinase is selected for bioenzymatic hydrolysis, the amount of bioenzyme added is 1500U / g to 3000U / g, the extraction solvent is 40% to 70% ethanol, the extraction temperature is 30°C to 60°C, and the extraction time is 20 to 40min; the solid-liquid ratio is 1:15 to 1:35;

[0025] Step 3: Purification of seabuckthorn pomace flavonoid extract: Remove impurities from the crude seabuckthorn pomace flavonoid extract mentioned in step 2, first remove protein by sevage method, then remove sugar by alcohol precipitation method, and finally purify by AB-8 macroporous adsorption resin, and let stand for 2 to 5 hours. When eluting, first use 2 times the volume of distilled water to wash away water-soluble impurities, and then use 2 times the volume of ethanol with a concentration of 50% to 70% for elution, the elution rate is 1.8mL / min, and the elution time is 1 to 2 hours.

[0026] Step 4: Freeze-drying of the seabuckthorn pomace flavonoid extract: remove ethanol from the purified seabuckthorn pomace flavonoid eluate mentioned in step 3 by rotary evaporation, pre-freeze the extract in a -80°C refrigerator for 12 hours, and then dry it in a vacuum freeze dryer for 24 to 26 hours;

[0027] The present invention also includes the seabuckthorn pomace flavonoid extract prepared by any of the above methods.

[0028] Example 1

[0029] Step 1: Pretreatment of seabuckthorn pomace: Pretreatment of seabuckthorn pomace: Dry the seabuckthorn pomace, a byproduct obtained during the juicing process of seabuckthorn fruit, in a 60°C oven for 24 hours, remove the seabuckthorn seeds through a 20-mesh sieve, and crush the pomace into fine powder using a vibrating superfine grinder, and pass through an 80-mesh sieve for later use;

[0030] Step 2: Preparation of crude extract of flavonoids from seabuckthorn pomace: The seabuckthorn pomace powder mentioned in step 1 was extracted by ultrasonic synergistic bioenzymatic hydrolysis, the ultrasonic power was 300W, pectinase was selected for bioenzymatic hydrolysis, the amount of bioenzyme added was 1500U / g, the extraction solvent was 60% ethanol, the extraction temperature was 50°C, the extraction time was 30min, and the solid-liquid ratio was 1:30;

[0031] Step 3: Purification of seabuckthorn pomace flavonoid extract: The crude seabuckthorn pomace flavonoid extract mentioned in step 2 is removed from impurities, firstly deproteinized by sevage method, then desugared by alcohol precipitation method, and finally purified by AB-8 macroporous adsorption resin, and adsorbed for 5 hours. When eluting, first use 2 times the volume of distilled water to wash away water-soluble impurities, and then use 2 times the volume of 60% ethanol for elution, the elution rate is 1.8mL / min, and the elution time is 3h.

[0032] Step 4: freeze-drying of seabuckthorn pomace flavonoid extract: remove ethanol from the purified seabuckthorn pomace flavonoid eluate mentioned in step 3 by rotary evaporation, pre-freeze the extract in a -80°C refrigerator for 12 hours, and then dry it in a vacuum freeze dryer for 24 hours to obtain freeze-dried seabuckthorn pomace flavonoid extract powder, with a total flavonoid extraction amount of 23.05 mg / g pomace dry weight and a purity of 65%.

[0033] Comparative Example 1

[0034] The difference from the embodiment is that the biological enzymes are cellulase and pectinase complex enzymes in a ratio of 1:1, and the rest of the processing methods are the same to prepare the seabuckthorn pomace flavonoid crude extract.

[0035] Comparative Example 2

[0036] The difference from the embodiment is that the biological enzymes are cellulase and pectinase complex enzymes in a ratio of 1:2, and the rest of the processing methods are the same to prepare the seabuckthorn pomace flavonoid crude extract.

[0037] Comparative Example 3

[0038] The difference from the embodiment is that the biological enzymes are cellulase and pectinase complex enzymes in a ratio of 2:1, and the rest of the processing methods are the same to prepare the seabuckthorn pomace flavonoids crude extract.

[0039] Comparative Example 4

[0040] The difference from the embodiment is that the biological enzyme is cellulase, and the rest of the processing methods are the same to prepare the crude extract of seabuckthorn pomace flavonoids.

[0041] Table 1 Effects of different bio-enzyme selection on the extraction yield of flavonoids from seabuckthorn pomace

[0042]

[0043] As shown in Table 1, single pectinase has higher extraction yield than compound enzyme, lower cost and simplified steps, so it is the best choice.

[0044] Comparative Example 5

[0045] The difference from the embodiment is that the treatment method is only ultrasonic extraction, and the other treatment methods are the same to prepare the sea buckthorn pomace flavonoids crude extract.

[0046] Comparative Example 6

[0047] The difference from the embodiment is that the treatment methods are respectively only enzymatic extraction, and the other treatment methods are the same to prepare the sea buckthorn pomace flavonoids crude extract.

[0048] Table 2 Effects of different treatment methods on the extraction yield of flavonoids from seabuckthorn pomace

[0049]

[0050] Conclusion: By comparing the flavonoid extraction yields of the embodiments and comparative examples 1-6, it can be seen that the ultrasonic-assisted enzymatic hydrolysis process can effectively improve the flavonoid extraction yield of seabuckthorn pomace, so the ultrasonic-assisted enzymatic hydrolysis extraction method was selected.

[0051] At the same time, the active function of the seabuckthorn pomace flavonoid extract of the present invention was further detected.

[0052] Comparative Example 7

[0053] The difference from the embodiment is that the treatment method of step 3 is omitted, and the crude extract of seabuckthorn pomace flavonoids is directly freeze-dried, and the remaining treatment methods are the same to prepare the freeze-dried powder of the crude extract of seabuckthorn pomace flavonoids.

[0054] Detection of Antioxidant Activity of Examples

[0055] The antioxidant activities of crude and purified seabuckthorn pomace flavonoids extracts were analyzed by measuring the iron ion reducing ability (FRAP method), DPPH free radical scavenging ability and ABTS free radical scavenging ability. The results showed that the purified seabuckthorn pomace flavonoids extract had better antioxidant activity, and the ability to reduce iron ions and scavenge DPPH and ABTS free radicals was significantly improved.

[0056] Table 3 Antioxidant activity determination of flavonoid extracts from seabuckthorn pomace

[0057]

[0058] Detection of enzyme inhibitory activity in Example

[0059] Table 4 Enzyme inhibition activity of seabuckthorn flavonoid extract

[0060] α-amylase inhibition rate α-glucosidase inhibition rate Example (Purified product) 50.8±0.1%(0.1mg / mL) 79.1±0.6%(0.1mg / mL) <![CDATA[IC 50 (μg / mL)]]> 97.81±0.421 52.89±0.087 Comparative Example 7 (crude extract) 15.9±0.1%(0.3mg / mL) 49.4±0.1%(0.1mg / mL) <![CDATA[IC 50 (μg / mL)]]> 316.70±1.430 131.04±0.413 Acarbose (positive control drug) 67.8±1.2%(0.03mg / mL) 85.7±0.6%(0.1mg / mL) <![CDATA[IC 50 (μg / mL)]]> 18.44±0.082 8.00±0.002

[0061] By measuring the inhibition rates of α-amylase and α-glucosidase, the ability of seabuckthorn pomace flavonoid extract to regulate glucose and lipid metabolism was analyzed. As shown in Table 4, seabuckthorn flavonoid extract has an inhibitory effect on α-glucosidase. As the purity of the sample increases, the inhibition rate of seabuckthorn flavonoid extract on α-glucosidase increases accordingly. When the concentration is 0.1 mg / mL, the inhibition rate of the embodiment on α-glucosidase reaches 79.1±0.6%, which is close to the positive control drug acarbose (85.7±0.6%). Seabuckthorn flavonoid extract also has a certain inhibitory effect on α-amylase, inhibiting 50.8±0.1% at a concentration of 0.1 mg / mL, but the inhibitory activity is weaker than acarbose. From the above two sets of data, it can be concluded that seabuckthorn flavonoids can inhibit the decomposition of carbohydrates to form glucose and inhibit the increase of blood sugar by inhibiting the activity of α-glucosidase and α-amylase, so it is concluded that seabuckthorn flavonoids have a certain effect on lowering blood sugar.

[0062] HPLC-MS qualitative analysis of detection examples

[0063] like Figure 1 As shown in the figure, the flavonoid components in the flavonoid extract of seabuckthorn pomace were analyzed by HPLC-MS. The results showed that this variety of seabuckthorn mainly contained 12 flavonoid compounds. Combined with the literature, the 12 substances were judged as follows through retention time comparison, accurate molecular weight matching and MS / MS fragment information analysis: 1. Gallicatechin; 2. Proanthocyanidin B2; 3. (+)-catechin; 4. Rutin; 5. Isoquercetin; Quercetin-3-O-glucoside; 6. Kaempferol-3-O-rutinoside; 7. Isorhamnetin-3-O-neohesperidin; 8. Astragaloside; 9. Quercetin; 10. Naringenin; 11. Kaempferol; 12. Isorhamnetin. Among them, quercetin has the function of enhancing capillary resistance, kaempferol has the ability to resist infection and inflammation, and has strong nutritional and health functions.

[0064] The following is a selection experiment of the method for extracting flavonoids from seabuckthorn pomace

[0065] Method for detecting total flavonoids

[0066] Take 3 mL of seabuckthorn pomace flavonoids extract and follow the same method for making a standard curve (take 2 mL of appropriately diluted and filtered sample solution and mix with 0.2 mL of 5% NaNO2 solution for 6 minutes, then add 0.2 mL of 10% AlCl3·6H2O solution and shake well. After 5 minutes, add 2 mL of 1M NaOH solution, wait for the reaction solution to be fully mixed, and measure the absorbance at a wavelength of 510 nm after 15 minutes). Calculate the total flavonoids content based on the standard curve.

[0067] Method for detecting antioxidant activity—FRAP assay

[0068] Freshly prepared 180 μL FRAP working solution was mixed and placed in a water bath at 37 degrees Celsius for 30 minutes, mixed with 5 μL of each concentration of sea buckthorn pomace flavonoid extract sample solution in a 96-well ELISA plate, and double distilled water was used as a blank control. After shaking for 1 minute in an ELISA reader, the reaction was incubated at 37°C for 10 minutes. The absorbance was measured at 593 nm and a standard curve was drawn. The standard curve was obtained from the prepared FeSO4·7H2O standard solutions of different concentrations: 10mMFeSO4 solution was diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM respectively; FeSO4·7H2O was diluted with pure water to adjust the concentration. The antioxidant activity was calculated according to the standard curve, and the results were expressed as FeSO4 equivalent concentration (μmolFeSO4 / mL).

[0069] Method for detecting antioxidant activity - DPPH free radical scavenging ability assay

[0070] Take a certain amount of DPPH and 80% methanol to prepare a 120μmol / L DPPH solution, and measure the absorbance at 515nm to be between 0.6 and 1.0; take a certain amount of Trolox and dilute it with 50% ethanol to prepare 0, 50, 100, 200, 400, and 600μM / L Trolox solutions. Add 100μL Trolox solution or seabuckthorn pomace flavonoid extract sample solution and 1.9mL DPPH dilution solution to the test tube, mix well and place in the dark to react for 2h. After the reaction, measure the absorbance of the reaction solution at 515nm with an enzyme marker to prepare a standard curve. Calculate the in vitro antioxidant capacity of the sample by the linear equation of the standard curve, and its unit is μmolTrolox / L.

[0071] Method for detecting antioxidant activity - ABTS free radical scavenging ability assay

[0072] Take a certain amount of ABTS to prepare a 7mM / L ABTS solution, take a certain amount of potassium persulfate to prepare a 2.45mM / L K2S2O8 solution, mix the ABTS solution and the K2S2O8 solution in equal proportions, and store them in the dark for more than 16 hours to obtain an ABTS mixed solution; take a certain amount of Trolox and dilute it with 50% ethanol to prepare 0, 50, 100, 200, 400, and 600μM / L Trolox solutions. Dilute the ABTS mixed solution 20 times with 80% methanol, and measure the absorbance at about 0.7 at 734nm; add 200μL of Trolox solution or sea buckthorn pomace flavonoid extract sample solution and 1.8mL ABTS dilution solution to the test tube, mix well, and place in the dark to react for 6 minutes. After the reaction, the absorbance of the reaction solution is measured at 734nm with an enzyme marker to prepare a standard curve. The in vitro antioxidant capacity of the sample is calculated by the linear equation of the standard curve, and its unit is μmol Trolox / L.

[0073] Method for detecting α-amylase inhibitory activity

[0074] The sea buckthorn pomace flavonoid extract samples and acarbose were respectively prepared with PBS into standard solutions with gradient mass concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, and then α-amylase solution and 1% soluble starch solution were prepared with a concentration of 1 mg / mL. 50 μL of α-amylase solution and sample solution were added to the experimental group; 50 μL of α-amylase solution and PBS were added to the sample control group; 50 μL of PBS solution and sample solution were added to the blank control group; and 100 μL of PBS buffer was added to the solvent control group. The above four groups of solutions were placed in a 37°C water bath for 10 minutes, and then 100 μL of 1% starch solution was added to each of them, and then kept at 37°C for 10 minutes again. Finally, 400 μL of DNS (3.5-dinitrosalicylic acid) solution was added, and the solution was placed in a 100°C water bath for 10 minutes. After cooling, the absorbance was measured at 540nm. Acarbose, a hypoglycemic drug, was used as a positive control group to calculate the inhibition rate (%) and half inhibitory concentration (IC 50 ).

[0075] Method for detecting α-glycosidase inhibitory activity

[0076] The sea buckthorn pomace flavonoid extract samples and acarbose were prepared with PBS into standard solutions with gradient mass concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively, and then α-glucosidase solution with a concentration of 1 mg / mL and 1% pNPG p-nitrophenyl-α-D-pyranoglucoside (6 mM / L) solution were prepared. 50 μL of α-glucosidase solution and sample solution were added to the experimental group; 50 μL of α-glucosidase solution and PBS were added to the sample control group; 50 μL of PBS solution and sample solution were added to the blank control group; and 100 μL of PBS buffer was added to the solvent control group. The above four groups of solutions were placed in a 37°C water bath for 10 minutes, and then 100 μL of 1% pNPG was added to each of them, and then kept at 37°C for 30 minutes. Finally, 500 μL of Na2CO3 solution was added to measure the absorbance at 405 nm, and the inhibition rate (%) and half inhibition concentration (IC) were calculated. 50 ).

[0077] The following is the process selection of flavonoids extraction method of seabuckthorn pomace

[0078] (1) Accurately weigh 1.0 g of seabuckthorn pomace, the ethanol concentration of the extract was 50%, the solid-liquid ratio was 25:1, the ultrasonic power was 300 w, the extraction time was 30 min, the extraction temperature was 50 ° C, and the total flavonoids extraction amount of seabuckthorn pomace under different enzyme addition amounts was determined when the pectinase addition amount was 1000 U / g, 1500 U / g, 2000 U / g, 2500 U / g, and 3000 U / g.

[0079] (2) Accurately weigh 1.0 g of seabuckthorn pomace, the ethanol concentration of the extract was 50%, the solid-liquid ratio was 25:1, the ultrasonic power was 300 w, the extraction time was 30 min, the pectinase addition amount was 1500 U / g, and the total flavonoids extraction amount of seabuckthorn pomace at the enzymatic hydrolysis temperature was determined at 20°C, 30°C, 40°C, 50°C, and 60°C.

[0080] (3) Accurately weigh 1.0 g of seabuckthorn pomace, set the ethanol concentration of the extract to 50%, the solid-liquid ratio to 25:1, the ultrasonic power to 300 w, the pectinase addition to 1500 U / g, the enzymatic hydrolysis temperature to 50 °C, and determine the total flavonoids extraction amount of seabuckthorn pomace at different enzymatic hydrolysis times and ultrasonic times.

[0081] (4) Accurately weigh 1.0 g of seabuckthorn pomace, the ethanol concentration of the extract was 50%, the solid-liquid ratio was 25:1, the extraction time was 30 min, the amount of pectinase added was 1500 U / g, the extraction temperature was 50°C, and the total flavonoids extraction amount of seabuckthorn pomace was determined when the ultrasonic power was 0, 100 w, 150 w, 200 w, 250 w, and 300 w, respectively.

[0082] (5) Accurately weigh 1.0 g of seabuckthorn pomace, set the ethanol concentration of the extract to 50%, the ultrasonic power to 300 w, the extraction time to 30 min, the amount of pectinase added to 1500 U / g, the extraction temperature to 50 °C, and determine the total flavonoids extraction amount of seabuckthorn pomace at a solid-liquid ratio of 1:10, 1:15, 1:20, 1:25, 1:30, and 1:35.

[0083] (6) Accurately weigh 1.0 g of seabuckthorn pomace, with a solid-liquid ratio of 1:30, an ultrasonic power of 300 w, an extraction time of 30 min, a pectinase addition of 1500 U / g, and an extraction temperature of 50°C. Determine the total flavonoids extraction amount of seabuckthorn pomace when the ethanol concentration of the extract is 40%, 50%, 60%, 70%, 80%, and 90%.

[0084] Table 5 Effect of different bio-enzyme addition amounts on the total flavonoids extraction from seabuckthorn pomace

[0085]

[0086]

[0087] Table 6 Effect of different enzymatic hydrolysis temperatures on the extraction of total flavonoids from seabuckthorn pomace

[0088]

[0089] Table 7 Effect of different solid-liquid ratios on the extraction of total flavonoids from seabuckthorn pomace

[0090]

[0091] Table 8 Effect of different ultrasonic powers on the extraction of total flavonoids from seabuckthorn pomace

[0092]

[0093] Table 9 Effect of different ethanol concentrations on the extraction of total flavonoids from seabuckthorn pomace

[0094]

[0095]

[0096] Through the above single factor optimization experiment, the optimal extraction process of seabuckthorn pomace flavonoids was obtained as follows: solid-liquid ratio 1:30, ethanol concentration 60%, ultrasonic power 300w, extraction time 30min, pectinase addition 1500U / g, and extraction temperature 50℃.

[0097] Further optimization of the extraction process

[0098] According to the optimal parameters, three parameters, extraction time, ethanol concentration and liquid-to-solid ratio, were selected to design a response surface experiment and conduct the measurement. The results are as follows Figure 1As shown in the figure, the optimal conditions for ultrasonic-assisted enzyme extraction of flavonoids from seabuckthorn pomace obtained by regression model are: solid-liquid ratio 1:33.47, ethanol concentration 47.97%, ultrasonic power 300w, extraction time 28.01min, pectinase addition 1500U / g, extraction temperature 50℃, and the predicted flavonoids can reach up to 21.72mg / g. Considering the feasibility of actual operation, the ultrasonic time, ethanol concentration, and liquid-solid ratio were adjusted to 28min, 48%, and 1:34 respectively. Under these conditions, the actual content of flavonoids obtained by three repeated experiments was 21.57mg / g, which is consistent with the predicted value, indicating that the optimal process parameters obtained by the response surface are reliable.

[0099] Further optimization of the macroporous adsorption resin purification process

[0100] First, the protein was removed by sevage method, then the sugar was removed by alcohol precipitation method, and finally, the AB-8 macroporous adsorption resin was used for purification, and the adsorption was allowed to stand for 3 to 5 hours. When eluting, water-soluble impurities were first washed away with 2 times the volume of distilled water, and then eluted with 2 times the volume of 55% to 70% ethanol, the elution rate was 1.8 mL / min, and the elution time was 1 to 3 hours.

[0101] (1) Accurately weigh 1 g of the pretreated resin, add 50 ml of a 2 mg / ml seabuckthorn flavonoids crude extract solution, mix well and seal, place in an oscillator for 6 hours at 25 °C and 120 rpm, and absorb 1 mL of the filtrate every hour, taking care to avoid the resin. After centrifugation, determine the residual total flavonoids content in the solution and calculate the adsorption rate.

[0102] (2) Filter the saturated resin by suction, rinse with distilled water to remove the residual solution on the surface of the resin, put the resin that has reached adsorption equilibrium into a 150 ml conical flask, add 30 ml of 85% ethanol solution, place the conical flask in a 25°C water bath shaker at a frequency of 100 r / min for desorption, and absorb 1 ml of supernatant every hour, determine the total flavonoids content in the supernatant, and calculate the desorption rate.

[0103] (3) Adsorption was carried out according to the preferred adsorption conditions. After saturated adsorption and washing with water, 50 ml of eluent was added and placed in a constant temperature incubator for oscillation for 4 h. The effect of the ethanol volume fraction of the eluent (40%, 50%, 60%, 70%, 80%) on the elution effect was studied. The first 10 ml of the mobile phone outflow liquid was discarded, the total flavonoid concentration was determined, and the flavonoid resolution rate was calculated.

[0104] Table 10 Effect of adsorption time on adsorption rate of seabuckthorn flavonoids

[0105]

[0106] It can be seen from the above table that between 0 and 5 hours of adsorption, the adsorption rate increases with the extension of the adsorption time. After 3 hours, the adsorption rate reaches more than 80%. After 5 hours, the adsorption rate remains at around 84% and no longer increases. Therefore, 3-5 hours is selected for process optimization.

[0107] Table 11 Effect of desorption time on adsorption rate of seabuckthorn flavonoids

[0108]

[0109] It can be seen from the above table that within 1 hour of analysis, the analysis rate increases with the extension of the analysis time. After 1 hour, the analysis rate reaches more than 80%. After 3 hours, the analysis rate remains at around 88% and no longer increases. Therefore, 1-3 hours is selected for process optimization.

[0110] Table 12 Effect of ethanol concentration on the resolution rate of seabuckthorn flavonoids

[0111]

[0112]

[0113] It can be seen from the above table that the resolution rate first increases and then decreases with the ethanol concentration, reaching a maximum value at 60%. The seabuckthorn flavonoids purification process selects an eluent with an ethanol concentration of 55-70%.

[0114] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0115] In addition, it should be understood that although the present specification is described in accordance with implementation modes, not every implementation mode includes only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to make various possible equivalent changes or substitutions to form other implementation modes that those skilled in the art can understand, but all these changes or substitutions should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract and its application, characterized in that: The following steps are involved: Step 1: Seabuckthorn pomace pretreatment: The seabuckthorn pomace raw material is the seabuckthorn pomace obtained as a byproduct of the seabuckthorn fruit juice extraction process, which is placed in an oven for drying to remove impurities such as seabuckthorn seeds, and then crushed to obtain seabuckthorn pomace powder; Step 2: Preparation of crude extract of flavonoids from seabuckthorn pomace: extracting the seabuckthorn pomace powder mentioned in step 1 by using ultrasound-assisted bio-enzymatic hydrolysis to obtain crude extract of flavonoids from seabuckthorn pomace; Step 3: Purification of seabuckthorn pomace flavonoids extract: removing impurities from the crude seabuckthorn pomace flavonoids extract mentioned in step 2, and purifying it with a macroporous resin to obtain a purified seabuckthorn pomace flavonoids eluate; Step 4: Drying the seabuckthorn pomace flavonoids extract: subjecting the seabuckthorn pomace flavonoids purification eluate mentioned in step 3 to rotary evaporation to remove ethanol, and freeze-drying to obtain a seabuckthorn pomace flavonoids extract freeze-dried powder; 2. The method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract according to claim 1, characterized in that: The drying temperature is 55-65°C for 22-24 hours. The sea buckthorn seeds are removed through a 20-mesh sieve. The pomace is crushed into fine powder using a vibrating ultrafine grinder and passed through an 80-mesh sieve for later use.

3. The method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract according to claim 1, characterized in that: The biological enzyme selected is pectinase, the biological enzyme addition amount is 1500U / g-3000U / g, the enzymatic hydrolysis temperature is 30℃-60℃, the extraction solvent is 40%-70% ethanol, the ultrasonic power is 300W, the extraction time is 20-40min, and the solid-liquid ratio is 1:15-1:

35.

4. The method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract according to claim 1, characterized in that: The purification process uses AB-8 macroporous adsorption resin, the extract freeze-dried powder is first deproteinized by the sevage method, then desugared by the alcohol precipitation method, and finally the crude flavonoid extract after preliminary treatment is added to the resin column at a rate of 2 mL / min using a constant flow pump and allowed to stand for adsorption for 3 to 5 hours. When eluting, first use 2 times the volume of distilled water to wash away water-soluble impurities, then use 2 times the volume of ethanol with a concentration of 55% to 70% for elution, the elution rate is 1.8 mL / min, and the elution time is 1 to 3 hours.

5. The method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract according to claim 1, characterized in that: The freeze-drying process is to first pre-freeze the extract in a -80°C refrigerator for 12 hours, and then put it into a vacuum freeze dryer for 24 to 26 hours.

6. The method for preparing a freeze-dried powder of seabuckthorn pomace flavonoid extract according to claim 1, characterized in that: A freeze-dried powder of seabuckthorn pomace flavonoid extract is prepared by the method described in any of the above claims, with a total flavonoid extraction amount of 17.79-23.05 mg / g seabuckthorn pomace dry weight and a purity of 50-65%, and has good α-glucosidase and α-amylase inhibitory activity, and can be used in the development of functional foods such as hypoglycemic foods.

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