Preparation method and application of heracleum moellendorffii microemulsion

Abstract

The invention relates to a preparation method and application of a heracleum moellendorffii microemulsion. The preparation method comprises the following steps: (1) extracting heracleum moellendorffii hance by using a PEG800-ammonium sulfate aqueous two-phase system to obtain a heracleum moellendorffii hance flavone crude extract and a heracleum moellendorffii hance polysaccharide crude extract; (2) purifying the heracleum moellendorffii flavone crude extract by adopting macroporous resin to obtain purified flavone; treating the heracleum moellendorffii polysaccharide crude extract by adopting alcohol extraction desalination to obtain purified polysaccharide; and (3) preparing microemulsion: preparing the purified flavone into a grease type solution, preparing the purified polysaccharide into a heracleum moellendorffii polysaccharide aqueous solution, adding the grease type solution into the heracleum moellendorffii polysaccharide aqueous solution in a stirring process, and emulsifying. The heracleum moellendorffii microemulsion prepared by the invention has the effects of reducing blood sugar, reducing blood fat, resisting oxidation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and application of effective components of natural plants, and in particular relates to a preparation method of a celery microemulsion and application thereof. Background Art

[0002] Heracleum dissectum is a perennial herbaceous plant of the genus Angelica in the Apiaceae family. It is commonly known as large-leaf celery, old mulberry celery, wild hollyhock, and Northeastern cow-wind-proof. It is primarily distributed in Northeast, North, and Southwest my country. Its crispy roots, rich aroma, delicious flavor, and high nutritional value make it a popular choice among local people, earning it the nickname "green gold" of mountain vegetables. Heracleum dissectum is commonly used in folk medicine to treat rheumatoid arthritis, muscle and bone pain, and indigestion. Its roots and stems are pungent, bitter, and warm in nature, and have the effects of dispelling wind and dampness, promoting blood circulation and relieving pain, and strengthening the spleen and stomach.

[0003] Traditional extraction methods (maceration, reflux, sublimation) and modern extraction methods (supercritical fluid extraction, membrane extraction and separation) require the addition of large amounts of organic solvents, which are flammable, explosive, and environmentally polluting. Furthermore, natural products from celery (total flavonoids, total phenols, and polysaccharides) suffer from poor stability and storage difficulties. Therefore, it is crucial to develop a celery-related product with superior performance and combined antioxidant, blood sugar-lowering, and lipid-lowering properties. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a preparation method of a celery microemulsion and application thereof.

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

[0006] The present invention provides a method for preparing a celery microemulsion, comprising the following steps:

[0007] (1) Using PEG800-ammonium sulfate aqueous two-phase system to extract celery, crude flavonoid extracts and crude polysaccharide extracts of celery were obtained;

[0008] (2) Purifying the crude extract of flavonoids from the celery vine using macroporous resin to obtain purified flavonoids; treating the crude extract of polysaccharides from the celery vine using alcohol extraction and desalination to obtain purified polysaccharides;

[0009] (3) Preparation of microemulsion: preparing the purified flavonoids into an oily solution; preparing the purified polysaccharide into an aqueous solution of celery polysaccharide; and adding the oily solution into the aqueous solution of celery polysaccharide during stirring for emulsification.

[0010] The beneficial effects of adopting the above technical solution include:

[0011] The microemulsion prepared by the present invention has the advantages of uniform size, simple preparation method, low cost, and thermodynamic stability. The present invention prepares the effective components of celery (crude polysaccharide, total flavonoids) into an oil-in-water microemulsion in which the flavonoids are encapsulated by celery polysaccharide. The dispersion of the celery microemulsion is determined by testing the zeta potential, the thermal stability of the celery microemulsion is tested by TG and DSC experiments, and the encapsulation integrity of the celery microemulsion is tested by infrared spectroscopy. The stability of the product is also confirmed. This lays a foundation for the microemulsion preparation to effectively enhance the medicinal value of celery and further increase its bioavailability.

[0012] The present invention uses PEG-800 as an organic solvent and ammonium sulfate as a salt solvent, which has the advantages of high efficiency, convenience, and environmental protection. At the same time, the use of a PEG800-ammonium sulfate two-phase aqueous system can increase the yield of effective ingredients. The microemulsion prepared by the present invention has the advantages of good stability, small particle size, good dispersibility, and is conducive to body absorption. It can improve the medicinal value and market acceptance of celery, and effectively solve the problems of instability and difficulty in preservation of natural active ingredients. The use of macroporous adsorption resin for the purification of celery flavonoids has the advantages of good adsorption effect, easy operation, and regeneration.

[0013] Furthermore, in step (1), the liquid-to-solid ratio is (20-40) mL:1 g; preferably, (20-35) mL:1 g; more preferably, (34-35) mL:1 g.

[0014] Furthermore, in step (1), the extraction temperature is 40-60°C; preferably, 45-55°C; more preferably, the extraction temperature is 50-51°C.

[0015] Furthermore, in step (1), the ultrasonic time is 10-50 min; preferably, the ultrasonic time is 30-50 min; more preferably, the ultrasonic time is 40-41 min.

[0016] Furthermore, in step (1), the ultrasonic power is 160-320W; preferably, the ultrasonic power is 240W.

[0017] Furthermore, in step (1), in the PEG800-ammonium sulfate two-phase aqueous system, the volume ratio of PEG800 to the ammonium sulfate aqueous solution is 1:1, and the ammonium sulfate concentration in the ammonium sulfate aqueous solution is 40%.

[0018] The beneficial effects of adopting the above technical solution include: adopting the above conditions is conducive to increasing the yield of total flavonoids, total phenols and crude polysaccharides.

[0019] Furthermore, in step (2), D-101 resin is used for purification.

[0020] The beneficial effects of adopting the above technical solution include: there are currently many types of macroporous resins, and different resins have a great influence on the yield of the target product. The present invention screened six macroporous adsorption resins, S-8, AB-8, HPD-100, D-101, DM-130, and D-406, and found that the use of D-101 resin can significantly increase the concentration of the target product.

[0021] Furthermore, in step (2), a dialysis bag is used to desalinate the celery polysaccharide.

[0022] The beneficial effects of adopting the above technical solution include: a large amount of inorganic salts exist in the lower phase of the extract of celery by the two-phase aqueous method, so the celery polysaccharide is desalted by using a dialysis bag, which is beneficial to the purification of the effective components of celery.

[0023] Furthermore, in step (2), static adsorption and desorption are adopted.

[0024] Furthermore, the loading temperature is 30-50°C; preferably, 40°C.

[0025] Furthermore, the loading concentration is 50-250 mg / mL, preferably 100 mg / mL.

[0026] Furthermore, the pH of the loading solution is 5-9, preferably, the pH is 7.

[0027] Furthermore, the eluent is a 60-100% ethanol solution, preferably anhydrous ethanol.

[0028] Furthermore, in step (2), dynamic adsorption and desorption are adopted.

[0029] Furthermore, the sample loading flow rate is 0.5-2.5 mL / min, preferably 1.5 mL / min.

[0030] Furthermore, the volume of the eluent is 70-110 mL, preferably 100 mL.

[0031] Furthermore, the eluent flow rate is 0.5-2.5 mL / min, preferably 1.5 mL / min.

[0032] The beneficial effects of adopting the above technical solution include: being conducive to improving the effects of adsorption and desorption, thereby significantly improving the purity of the purified product.

[0033] Furthermore, in step (3), HLB=12.

[0034] Furthermore, in step (3), the purified flavonoids, medium-chain fatty acid edible oil, surfactant and co-surfactant are mixed to obtain an oil-type solution.

[0035] Furthermore, the mass ratio of surfactant to co-surfactant is (2-2.1):1.

[0036] Furthermore, the ratio of the purified flavonoids, the medium-chain fatty acid edible oil, the surfactant and the purified polysaccharide is 10 mg:10 mL:10.5 g:5 mg.

[0037] Furthermore, the surfactant includes Tween 80 and Span 80.

[0038] Furthermore, the cosurfactant is anhydrous ethanol.

[0039] The invention provides a genus celery microemulsion, which is prepared by adopting the method.

[0040] The use of the above-mentioned celery microemulsion in the preparation of any one or several products (1) to (3).

[0041] (1) Lowering blood sugar;

[0042] (2) Lowering blood lipids;

[0043] (3) Antioxidant.

[0044] The products include but are not limited to food, health products, medicines, nutritional products, antioxidants, daily chemical products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The purified flavonoid powder and polysaccharide powder are described, wherein A is flavonoid powder and B is polysaccharide powder.

[0046] Figure 2 For the preparation of celery microemulsion.

[0047] Figure 3 In the figure, A is the phase diagram of the two-phase aqueous system; B is the effect of ATPS on the yield of total flavonoids, total phenols, and crude polysaccharides; C is the single-factor test result of liquid-to-solid ratio; D is the single-factor test result of ultrasonic time; E is the single-factor test result of ultrasonic power; F is the single-factor test result of extraction temperature.

[0048] Figure 4 This is the response surface 3D diagram of crude polysaccharide.

[0049] Figure 5 This is the response surface 3D diagram of flavonoids.

[0050] Figure 6In the figure, A is the effect of loading temperature on the adsorption rate; B is the effect of loading concentration on the adsorption rate of flavonoids; C is the effect of loading solution pH on the adsorption rate of flavonoids; D is the effect of eluent concentration on the adsorption rate of flavonoids; E is the effect of loading flow rate on the adsorption effect of D-101 macroporous resin; F is the effect of eluent volume on the desorption effect; G is the effect of eluent flow rate on the desorption effect.

[0051] Figure 7 This is the HPLC chromatogram of the polysaccharide derivatives of Rhizoma Coptidis.

[0052] Figure 8 HPLC chromatogram of standard monosaccharides.

[0053] Figure 9 Transmission electron microscope photos of celery microemulsion at different magnifications.

[0054] Figure 10 This is the Zeta potential spectrum of Celery microemulsion.

[0055] Figure 11 This is the particle size spectrum of the old celery microemulsion.

[0056] Figure 12 It is an infrared spectrum.

[0057] Figure 13 This is the DSC detection spectrum, where a is the celery microemulsion and b is the celery original powder.

[0058] Figure 14 TG spectrum, where a is celery powder and b is celery microemulsion.

[0059] Figure 15 In the figure, A is the binding rate of celery powder, celery extract and microemulsion on sodium cholate; B is the binding rate of celery powder, celery extract and microemulsion on sodium glycocholate; C is the binding rate of celery powder, celery extract and microemulsion on sodium taurocholate; D is the inhibitory effect of celery powder, celery extract and microemulsion on pancreatic lipase activity; E is the inhibitory effect of acarbose, celery powder, celery extract and microemulsion on α-glucosidase; F is the inhibitory effect of acarbose, celery powder, celery extract and microemulsion on α-amylase; G is the scavenging effect of Vc control solution, celery powder, celery extract and celery microemulsion on DPPH free radicals; H is the inhibitory effect of Vc control solution, celery powder, celery extract and microemulsion on ABTS + Free radical scavenging effect. DETAILED DESCRIPTION

[0060] The principles and features of the present invention are described below with reference to 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.

[0061] The present invention uses celery as a raw material, screens for the optimal aqueous two-phase system, optimizes the extraction conditions of total flavonoids, total phenols, and crude polysaccharides from celery using the aqueous two-phase system through response surface design, and then purifies the system. A polysaccharide-encapsulated flavonoid oil-in-water microemulsion is prepared and tested for its physicochemical indices. Based on these in vitro hypoglycemic activity, hypolipidemic activity, and antioxidant activity are then verified. The specific details are as follows:

[0062] (1) Using a PEG800-ammonium sulfate aqueous two-phase system, the extract of celery was studied. The effects of liquid-to-solid ratio, ultrasonic time, extraction temperature, and ultrasonic power on the yield of total flavonoids, total phenols, and crude polysaccharides in celery were investigated by single-factor experiments. Plackett-Burman experiments identified liquid-to-solid ratio, ultrasonic time, and extraction temperature as significant factors. On this basis, a response surface optimization experiment was conducted, and the optimal extraction process conditions were obtained as follows: liquid-to-solid ratio of 35:1, extraction temperature of 51°C, and ultrasonic time of 41 min. Three parallel validation experiments were conducted according to these extraction process conditions, and the actual yield of crude polysaccharides was 10.03% ± 2.28%, the actual yield of total flavonoids was 0.53% ± 0.05%, and the actual yield of total phenols was 2.57% ± 0.72%.

[0063] (2) The total flavonoids extracted from the old celery were purified using macroporous resins. The static adsorption and desorption capacities of the macroporous resins S-8, AB-8, D-101, HPD-100, DM-130, and D-406 for the total flavonoids were compared, and D-101 resin was determined to be the best choice for purifying the total flavonoids. The optimal purification parameters for static adsorption and desorption were: sample temperature 40°C, sample concentration 100 mg / mL, pH 7 of the sample flavonoid solution, and anhydrous ethanol as the eluent. The optimal purification parameters for dynamic adsorption and desorption were: sample flow rate 1.5 mL / min, eluent volume 100 mL, and elution flow rate 1.5 mL / min. The purity of the flavonoids in the old celery extract could be increased to 62.8%. At the same time, the extracted crude polysaccharide of celery was subjected to alcohol extraction and desalting treatment, and analysis determined that there were 7 monosaccharide components in the polysaccharide of celery, namely mannose, ribose, rhamnose, xylose, arabinose, glucuronic acid, and galacturonic acid. Among them, the content of glucuronic acid and galacturonic acid was relatively high, indicating that the polysaccharide of celery has certain antioxidant capacity.

[0064] (3) The separated and purified flavonoids and polysaccharides of celery were made into water-in-oil microemulsions with polysaccharide-encapsulated flavonoids. The average particle size was 140.8 nm, the Zeta potential was -19.36 mV, and the curve fitting was good, indicating that the celery microemulsion had good dispersibility. The TG and DSC analysis results showed that the melting point of the celery powder was T max The melting point of the old celery microemulsion is 179.15℃ maxThe temperature of the celery microemulsion was 225.01°C, indicating that the celery microemulsion has good thermal stability. Infrared spectroscopy showed no significant changes in the absorption peak of the celery microemulsion, indicating its relative stability. The polysaccharide drug loading was 2.03%, and the flavonoid drug loading was 1.72%. The polysaccharide encapsulation efficiency was 87.55%, and the flavonoid encapsulation efficiency was 85.31%. These results demonstrate that the celery microemulsion has a small particle size, good dispersibility, and excellent thermal stability.

[0065] (4) Through the α-glucosidase activity inhibition test, IC 50 The IC value calculator calculates the IC value of the old celery powder. 50 The value is 17.5mg / mL, and the IC 50 The value is 15.95mg / mL, and the IC 50 The value is 13.61mg / mL. In the α-amylase activity inhibition experiment, the IC 50 The value is 16.6mg / mL, and the IC 50 The value is 14.77mg / mL, and the IC 50 The value was 12.69 mg / mL. The order of in vitro hypoglycemic activity was: celery microemulsion > celery extract > celery powder, indicating that celery microemulsion can inhibit the activity of α-glucosidase and α-amylase, and has a hypoglycemic effect.

[0066] (5) In vitro lipid-lowering studies were conducted by measuring the binding rates of sodium cholate, sodium glycocholate, and sodium taurocholate, as well as the inhibition rate of pancreatic lipase activity. The sodium cholate binding rate of celery powder was 22.45% ± 2.21%, the sodium cholate binding rate of celery extract was 65.83% ± 1.97%, and the sodium cholate binding rate of celery microemulsion was 70.15% ± 2.32%. The sodium glycocholate binding rate of celery powder was 16.42% ± 1.37%, the sodium glycocholate binding rate of celery extract was 47.51% ± 2.3%, and the sodium glycocholate binding rate of celery microemulsion was 59.92% ± 2.4%. The binding rate of sodium taurocholate of celery powder was 22.2%±1.78%, the binding rate of sodium taurocholate of celery extract was 29.1%±2.33%, and the binding rate of sodium taurocholate of celery microemulsion was 56.9%±2.19%. The results of pancreatic lipase activity inhibition experiment showed that the IC 50 The value is 41.23 mg / mL, and the IC 50 The value is 30.83 mg / mL, and the IC 50 The value was 24.06 mg / mL. The order of in vitro lipid-lowering activity was: celery microemulsion > celery extract > celery powder, indicating that celery microemulsion can increase the binding rate of bile salts, inhibit pancreatic lipase activity, and have lipid-lowering effects.

[0067] (6) Verify the removal of DPPH and ABTS + The results of DPPH in vitro antioxidant test showed that the IC 50 The value is 0.081mg / mL, and the IC of celery powder is 50 The value is 49.06mg / mL, and the IC 50 The value is 38.54 mg / mL, and the IC 50 The value is 30.66 mg / mL. + The results of in vitro antioxidant experiments showed that the IC 50 The value is 0.057mg / mL, and the IC value of celery powder is 0.057mg / mL. 50 The value is 24.11mg / mL, and the IC 50 The value is 15.86 mg / mL, and the IC 50 The value was 10.72 mg / mL. The order of in vitro antioxidant capacity was: celery microemulsion > celery extract > celery powder, indicating that celery microemulsion has good antioxidant capacity.

[0068] The in vitro hypoglycemic activity, in vitro hypolipidemic activity and in vitro antioxidant activity of celery powder, celery extract and celery microemulsion were studied. The results showed that the celery microemulsion preparation had better hypoglycemic, hypolipidemic and antioxidant effects than the original powder.

[0069] The present invention determines the optimal extraction process of celery in a PEG800-ammonium sulfate aqueous two-phase system, and prepares an oil-in-water microemulsion with flavonoids encapsulated by celery polysaccharide. In vitro activity experiments prove that the celery microemulsion has hypoglycemic activity, hypolipidemic activity, and antioxidant capacity, which is conducive to the deep processing of celery and the development of celery food and medicine.

[0070] In the examples, rutin standard, gallic acid standard, and anhydrous glucose were all analytically pure and purchased from Shanghai Yuanye Biotechnology Co., Ltd. HPD-100 macroporous adsorption resin, D-101 macroporous adsorption resin, AB-8 macroporous adsorption resin, DM-130 macroporous adsorption resin, S-8 macroporous adsorption resin, and D-406 macroporous adsorption resin were all purchased from New Era Chemical Co., Ltd. Medium- and long-chain fatty acid edible oil (chemically pure) was purchased from Qingdao Haizhiyuan Life Science Technology Co., Ltd. Tween 80 and Span 80 were both analytically pure and purchased from Tianjin Guangfu Fine Chemical Research Institute. Phosphate buffer solution of pH 6.8, phosphate buffer solution of pH 7.4 (i.e., PBS buffer solution), ABTS +and DPPH, all analytically pure, were purchased from Sinopharm Chemical Reagent Co., Ltd. Anhydrous ethanol as the cosurfactant was purchased from Xi'an Sanpu Chemical Reagent Co., Ltd. p-NPG was purchased from Sigma-Aldrich, catalog number 292710. DNS reagent was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R30592.

[0071] Unless otherwise specified, all solutions were prepared using water. Room temperature in this invention is 20 ± 2°C.

[0072] In the embodiment, the method for detecting the total flavonoid content comprises the following steps:

[0073] ① Create a standard curve: Weigh 10 mg of rutin standard into a 100 mL volumetric flask. Prepare rutin standard solutions at concentrations of 0.01 mg / mL, 0.015 mg / mL, 0.02 mg / mL, 0.025 mg / mL, 0.03 mg / mL, and 0.035 mg / mL. Take 1 mL of the standard solution and add 60% ethanol solution to a total volume of 5 mL. Then add 0.3 mL of 5% sodium nitrite solution, shake well, and let stand in the dark for 10 minutes. Add 0.3 mL of 10% aluminum chloride solution, shake well, and let stand in the dark for 8 minutes. Add 4 mL of 4% sodium hydroxide solution, dilute to the mark with 60% ethanol solution, shake well, and let stand in the dark for 15 minutes. Measure the absorbance of the flavonoids at 510 nm. Draw a flavonoid standard curve with the rutin solution concentration as the horizontal axis and the absorbance as the vertical axis. The standard curve equation of total flavonoids content obtained by the above method is Y=6.424x-0.0024, R 2 =0.9934, where x is the rutin concentration (mg / mL) and Y is the absorbance.

[0074] ② Detect the total flavonoid content in the sample: Take 1.0 mL of the total flavonoid sample solution to be tested, measure the absorbance value, and calculate the total flavonoid content according to formula 1:

[0075]

[0076] Where: W1-total flavonoids content, (%); C1-total flavonoids mass concentration, (g / mL); V1-total flavonoids volume, (mL); D1-solution dilution multiple; M1-sampling volume, (mg).

[0077] In the embodiment, the total phenol content is detected, comprising the following steps:

[0078] ① Make a standard curve: Weigh 50 mg of gallic acid standard and place it in a 100 mL volumetric flask, and prepare standard solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL and 0.5 mg / mL respectively. Take 1 mL of the standard solution in a 10 mL volumetric flask, add 60% ethanol, and make up to volume. Take 1 mL of the fixed solution and place it in a 10 mL stoppered colorimetric tube, add 2.5 mL of 0.1 mol / mL forin phenol, shake well, react for 5 minutes, add 2 mL of 7.5% sodium carbonate solution, shake well, react in the dark for 30 minutes, and measure the absorbance at 765 nm. Use the concentration of gallic acid solution as the horizontal axis and the absorbance as the vertical axis to draw a standard curve for total phenol content. The standard curve equation for total phenol content is Y=0.512x-0.0094, R 2 =0.9951, where x is the gallic acid concentration (mg / mL) and Y is the absorbance.

[0079] ② Detect the total phenol content in the sample: Take 1.0 mL of the total phenol sample solution to be tested, measure the absorbance value, and calculate the total phenol content according to Formula 2.

[0080]

[0081] Where: W2-total phenol content, (%); C2-total phenol mass concentration, (g / mL); V2-total phenol volume, (mL); D2-solution dilution multiple; M2-sample volume, (mg).

[0082] In the embodiment, the detection of polysaccharide content comprises the following steps:

[0083] ① Make a standard curve: Weigh 10 mg of anhydrous glucose and place it in a 100 mL volumetric flask, and prepare standard solutions with concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL and 0.06 mg / mL respectively. Take 1 mL of the standard solution and place it in a 10 mL stoppered colorimetric tube, shake it well, add 1 mL of 5% phenol solution and 3 mL of sulfuric acid, shake and mix well, place it in a 100 ° C water bath and heat for 10 minutes, take it out and cool to room temperature, measure the absorbance at 490 nm, and draw a glucose standard curve with the glucose solution concentration as the horizontal axis and the absorbance as the vertical axis. The standard curve equation for glucose content is Y = 5.978x + 0.0076, R 2 =0.9993, where x is the glucose concentration (mg / mL) and Y is the absorbance.

[0084] ② Detection of polysaccharide content in the sample: Take 1.0 mL of the test solution, measure the absorbance value, and calculate the polysaccharide content according to formula (3).

[0085]

[0086] Where: W3-polysaccharide content, (%); C3-mass concentration of polysaccharide, (g / mL); V3-volume of polysaccharide, (mL); D3-solution dilution factor; M3-sample mass, (mg).

[0087] The following is an introduction through specific embodiments.

[0088] Example 1

[0089] The preparation of celery microemulsion comprises the following steps:

[0090] (1) Pretreatment of celery: freeze-dry the whole plant of celery, crush it, and pass it through an 80-mesh sieve to prepare celery raw powder for later use.

[0091] (2) Extraction of celery powder using PEG800-ammonium sulfate aqueous two-phase system:

[0092] An ATPS solution was prepared, which included PEG800 and an ammonium sulfate aqueous solution. The volume ratio of PEG800 to the ammonium sulfate aqueous solution was 1:1, the ammonium sulfate concentration in the ammonium sulfate aqueous solution was 40% (mass percentage), and celery powder was added at a liquid-to-material ratio of 35 mL:1 g (i.e., the volume of the ATPS solution and the mass ratio of the celery powder was 35 mL:1 g). The extraction temperature was 51°C, the ultrasonic power was 240 W, the ultrasonic time was 41 min, and the celery extract was obtained by filtration. The extract was allowed to stand for 20 min, the upper phase was brownish yellow, and the lower phase was brown. Detection by ultraviolet spectrophotometer showed that the flavonoid content in the upper phase was 25.7%, and the polysaccharide content in the lower phase was 20.9%.

[0093] The upper phase is dried to obtain a crude extract of flavonoids from celery (also known as total flavonoids from celery), and the lower phase is dried to obtain a crude extract of polysaccharides from celery (also known as crude polysaccharides from celery).

[0094] (3) Purification of flavonoids using macroporous adsorption resin

[0095] Activate the macroporous adsorption resin: Rinse the macroporous resin (D-101) with deionized water and then filter it. Then add anhydrous ethanol, soak it for 12 hours, filter it again, and then rinse it with deionized water until the ethanol smell disappears. Then add 5% hydrochloric acid solution, soak it again for 12 hours, and rinse it with deionized water until the liquid pH is 7. Finally, add 5% NaOH solution, soak it for 12 hours, and rinse it with deionized water until the pH is 7. Then filter it and set it aside.

[0096] Regeneration of macroporous adsorption resin: Add a large amount of deionized water to the macroporous resin and filter it. Then, add anhydrous ethanol to completely submerge it. Soak for 12 hours and filter it again. Rinse with deionized water until the ethanol smell disappears and filter it again. Next, add 5% hydrochloric acid solution, soak for 12 hours, then rinse with deionized water to a pH of 7. Then add 5% NaOH solution, soak for 12 hours, then rinse with deionized water to a pH of 7. Finally, filter it and reuse it.

[0097] When purifying flavonoids, either static adsorption-desorption or dynamic adsorption-desorption can be used.

[0098] The static adsorption-desorption of macroporous resin includes the following steps: weigh 5g of activated macroporous resin (D-101), put it into a 50mL conical flask, add 20mL of 100mg / mL crude extract of flavonoids from old celery, adjust the pH to 7, the sample temperature to 40℃, and oscillate and adsorb at a rate of 100 revolutions per minute in a shaking box for 240min. After shaking and filtering, the adsorbed macroporous resin is obtained, 20mL of anhydrous ethanol is added to a 50mL conical flask, and the mixture is fully shaken and desorbed for 4h. The purified flavonoid powder is obtained by the above method, such as Figure 1 As shown, the flavonoid content in the powder was detected by ultraviolet spectrophotometer, and the results showed that the purity of flavonoids in the flavonoid powder was 62.8%, indicating that this method can effectively improve the purity of the effective components of celery. The obtained flavonoid powder was used to prepare celery microemulsion.

[0099] Dynamic adsorption-desorption using a macroporous resin involves the following steps: 5 g of activated macroporous resin (D-101) is weighed and loaded into a glass column. A 50 mL solution of a 100 mg / mL crude extract of flavonoids from Cephalotaxus chinensis (pH 7, 40°C) is then pumped into the column via a peristaltic pump at a rate of 1.5 mL / min. After adsorption, the column is eluted with 100 mL of anhydrous ethanol at a flow rate of 1.5 mL / min.

[0100] (4) The crude polysaccharide extract of the old celery was added to anhydrous ethanol for extraction for 12 hours, centrifuged at 8000rpm for 15 minutes, and the precipitate was freeze-dried at -55℃ for 48 hours. The dried crude polysaccharide of the old celery was prepared into a solution and loaded into a dialysis bag with a molecular weight of 3500. The loading volume should not exceed 1 / 3 of the volume of the dialysis bag, and the dialysis bag was sealed after removing the bubbles in the bag. It was placed in a beaker filled with distilled water for 12 hours. The product after dialysis and desalination was freeze-dried to remove moisture to obtain purified polysaccharide powder. The crude polysaccharide extract of the old celery was purified by the above method, as shown in FIG. Figure 1 As shown, the content of polysaccharide in the powder was detected by ultraviolet spectrophotometer, and the results showed that the purity of polysaccharide in the polysaccharide powder was 59.2%, indicating that this method can effectively improve the purity of the effective components of old celery.

[0101] (5) Preparation of Celery Microemulsion

[0102] At a rotation speed of 1 rpm, 10 mg of flavonoid powder was dissolved in 10 mL of medium-chain fatty acid edible oil, heated at 80°C for 40 min, and 7.5 g of surfactant Tween 80, 3 g of Span 80 and 5 g of co-surfactant (anhydrous ethanol) were added to make the microemulsion KM value (mass ratio of surfactant to co-surfactant) (2-2.1):1, HLB=12, and mixed into an oil-type solution.

[0103] 5 mg of polysaccharide powder was dissolved in 20 mL of water and stirred to dissolve completely to obtain an aqueous solution of celery polysaccharide. The temperature of the aqueous phase was 20°C.

[0104] The oil-in-water rotation titration method was used to add the oil-type solution dropwise into the aqueous solution of celery polysaccharide in a high-speed stirrer (stirring speed was 100r / min) for emulsification to obtain an oil-in-water microemulsion in which the polysaccharide aqueous solution encapsulated the flavonoid oil, which was named celery microemulsion.

[0105] Old celery microemulsion Figure 2 As shown, the celery microemulsion is a yellow clear and transparent liquid, and under the irradiation of laser, the Tyndall phenomenon occurs.

[0106] The present invention uses a high-speed stirring method to prepare an oil-in-water microemulsion in which flavonoids are encapsulated in a polysaccharide aqueous solution. It is found that the microemulsion exhibits the Tyndall phenomenon, and its physical and chemical properties are characterized: under a transmission electron microscope, the old celery microemulsion is spherical, with the inner core being a mixed solution of flavonoid oil and surfactant, and the outer ring being a polysaccharide water film, proving that this microemulsion is an oil-in-water preparation. The average particle size of the microemulsion is 140.8nm, the Zeta potential value is -19.36mV, and the microemulsion particle size is small and has good dispersibility; the polysaccharide loading in the old celery microemulsion is 2.03%, and the flavonoid loading in the old celery microemulsion is 1.72%; the polysaccharide encapsulation rate in the old celery microemulsion is 87.55%, and the flavonoid encapsulation rate in the old celery microemulsion is 85.31%. The results of TG experiments, DSC experiments, and infrared spectroscopy tests show that the first endothermic peak T max The first endothermic peak T of the old celery powder is 225.01℃. max The melting point of the microemulsion is 179.15℃, and the higher melting point of the original powder indicates that the microemulsion preparation has a higher thermal stability for the effective components of celery (polysaccharides and flavonoids), providing an effective basis for the future product development of celery.

[0107] Example 2 Screening of two-phase aqueous system

[0108] (1) Draw the phase diagram of the ATPS system: Add 25 mL of organic solution to a test tube, slowly add salt solutions of different concentrations to the test tube, constantly shaking and mixing while adding. The solution is transparent at first. As the salt solution is continuously added, the clear liquid will become turbid. Calculate the mass fraction of the organic solvent at the cloud point. Repeat several times and draw the phase diagram curve.

[0109] Figure 3 A is a phase diagram of a two-phase aqueous system. The curve marks the boundary between a single phase and two phases. The region above the curve represents the two-phase region, and the region below the curve represents the single-phase region. Using the information in the phase diagram, we formed a two-phase aqueous system by combining organic solvents with salt ion solutions in varying mass ratios. The optimal combination was selected by combining the systems that yielded the highest yield of the active ingredients of the old celery.

[0110] (2) Extraction of the active ingredients of Agave arborescens: Prepare ATPS solution in a centrifuge tube according to the phase diagram curve.

[0111] The ATPS solutions were PEG800-ammonium sulfate, PEG400-ammonium sulfate, dipotassium hydrogen phosphate-ethanol, PEG4000-ammonium sulfate, and sodium carbonate-ethanol.

[0112] In the PEG800-ammonium sulfate, the volume ratio of PEG800 to the ammonium sulfate solution is 1:1, and the concentration of ammonium sulfate in the ammonium sulfate solution is 40% (mass percentage).

[0113] In PEG400-ammonium sulfate, the volume ratio of PEG400 to ammonium sulfate solution is 1:1, and the concentration of ammonium sulfate in the ammonium sulfate solution is 40% (mass percentage).

[0114] In the dipotassium hydrogen phosphate-ethanol, the volume ratio of the dipotassium hydrogen phosphate solution to the anhydrous ethanol is 1:1, and the concentration of the dipotassium hydrogen phosphate in the dipotassium hydrogen phosphate solution is 40% (mass percentage).

[0115] In the PEG4000-ammonium sulfate, the volume ratio of PEG4000 to the ammonium sulfate solution is 1:1, and the concentration of ammonium sulfate in the ammonium sulfate solution is 40% (mass percentage).

[0116] In the sodium carbonate-ethanol, the volume ratio of the sodium carbonate solution to the anhydrous ethanol is 1:1, and the concentration of the sodium carbonate in the sodium carbonate solution is 40% (mass percentage).

[0117] Add celery powder at a liquid-to-solid ratio of 40 mL:1 g (i.e., the volume of ATPS solution to the mass of celery powder is 40 mL:1 g). The extraction temperature is room temperature, the ultrasonic power is 240 W, and the ultrasonication is carried out for 30 min. The celery extract is filtered to obtain the celery extract. The upper and lower phases of the celery extract are separated, and the volumes of the upper and lower phases are calculated to calculate the yield:

[0118]

[0119] Wherein: V1 and V2 are the volumes of the upper and lower phases of the celery extract (mL), respectively; C1 and C2 are the concentrations of the celery active ingredients in the upper and lower phases (mg / mL), respectively; D1 is the dilution multiple of the solution; Y1 and Y2 are the yields of the celery active ingredients in the upper and lower phases, respectively.

[0120] Total flavonoids and total phenols of celery are mainly concentrated in the upper phase of the solution, while crude polysaccharides of celery are concentrated in the lower phase of the solution. Figure 3 As shown in Figure B, the maximum yield of total flavonoids from celery was 0.39% ± 0.08% in the PEG800-ammonium sulfate system, the maximum yield of crude celery polysaccharides was 11.35% ± 0.7% in the dipotassium hydrogen phosphate-ethanol system, and the maximum yield of total phenols from celery was 2.42% ± 0.2% in the PEG800-ammonium sulfate system. In summary, PEG800-ammonium sulfate was selected as the aqueous two-phase system for use in the subsequent examples.

[0121] Example 3

[0122] Referring to the method of Example 1, a single factor experiment was conducted using PEG800-ammonium sulfate as ATPS to investigate the effects of extraction temperature, ultrasonic power, ultrasonic time and liquid-to-solid ratio on the yields of total flavonoids, crude polysaccharides and total phenols from celery.

[0123] ①The influence of liquid-to-material ratio on yield

[0124] The experimental results are as follows: under the conditions of material-liquid ratio of 20mL:1g, 25mL:1g, 30mL:1g, 35mL:1g, 40mL:1g, extraction temperature of 40℃, ultrasonic power of 240W, and ultrasonic time of 50min, respectively. Figure 3 As shown in Figure C, when the ATPS solution volume to celery powder mass ratio ranged from 25:1 to 35:1, the yields of total flavonoids and total phenolics significantly increased (P < 0.05). When the solution ratio reached 35:1, the yields of total flavonoids, total phenolics, and crude polysaccharides reached their maximum values of 0.503%, 2.058%, and 11.818%, respectively. This may be because the increasing solution ratio enhanced the diffusivity of celery cells, allowing for the release of active ingredients. The yield decreased with increasing liquid-to-solid ratio, due to a relatively lower ammonium sulfate concentration and a decrease in crude polysaccharide yield. Furthermore, a relatively lower PEG800 concentration led to a gradual decrease in the yield of fat-soluble total flavonoids, while the yield of less fat-soluble total phenolics continued to increase within this concentration range.

[0125] ②The effect of ultrasonic time on yield

[0126] The experimental results are shown in the following table: the material-liquid ratio was 35mL:1g, the extraction temperature was 40℃, the ultrasonic power was 240W, and the ultrasonic time was 10min, 20min, 30min, 40min, and 50min respectively. Figure 3 As shown in Figure D, as the ultrasonic treatment time increased from 10 min to 40 min, the yield of crude polysaccharides from celery increased significantly, with a significance level of (P<0.05). When the ultrasonic time reached 40 min, the yield of crude polysaccharides from celery was 11.21%. When the ultrasonic time was 50 min, the yield of crude polysaccharides from celery decreased. This may be due to the long ultrasonic time, which led to the decomposition of flavonoids and the inability to increase the total flavonoid yield. At the same time, the polysaccharides and total phenols were at 10-40 min. This is because as the ultrasonic time increases, the cell tissue of celery is destroyed, accelerating the release of polysaccharides and total phenols from celery.

[0127] ③The influence of ultrasonic power on yield

[0128] The experimental results are shown in the following table under the experimental conditions of material-liquid ratio of 25mL:1q, extraction temperature of 40℃, ultrasonic power of 160W, 200W, 240W, 280W, 320W, and ultrasonic time of 50min. Figure 3 As shown in Figure E, when the ultrasonic power varied between 160-320W, the yields of total flavonoids and crude polysaccharides in the old celery increased significantly with the increase in power (P<0.05). When the power reached 240W, the yields of total flavonoids and crude polysaccharides reached their maximum values, at 0.39% and 9.26%, respectively. This is because as the power increased, the thermal effect within the old celery cells increased, causing the cells to vibrate and break, promoting the dissolution of components. When the power was further increased, the yields of total flavonoids and crude polysaccharides began to decrease, because excessive power caused the structures of flavonoids and polysaccharides to change and degrade. The yield of total phenols did not increase significantly, but excessive power would also cause changes in the structure of total phenols, so it is necessary to avoid setting too high an ultrasonic power.

[0129] ④The effect of extraction temperature on yield

[0130] The experimental results are shown in the following table under the experimental conditions of material-liquid ratio of 35mL:1g, extraction temperatures of 40℃, 45℃, 50℃, 55℃, and 60℃, ultrasonic power of 240W, and ultrasonic time of 40min. Figure 3As shown in Figure F, when the extraction temperature was between 40 and 50°C, the extraction yields of total flavonoids and crude polysaccharides from jacaranda increased significantly with increasing extraction temperature (P < 0.05). When the extraction temperature reached 50°C, the extraction yields of total flavonoids and crude polysaccharides reached their highest values, at 0.58% and 11.36%, respectively. This phenomenon is attributed to the release of total flavonoids and crude polysaccharides as the extraction temperature increased. However, when the extraction temperature was further increased, the extraction yields of total flavonoids and crude polysaccharides from jacaranda decreased. This is because the excessively high extraction temperature caused the structural degradation of the flavonoid and polysaccharide molecules.

[0131] Example 4 Plackett-Burman test

[0132] On the basis of single factor experiment, the total flavonoids, total phenols and crude polysaccharide yields were used as indicators, and the factors of liquid-to-solid ratio, ultrasonic power, extraction temperature and ultrasonic time were selected as independent variables. The Plackett-Burman test was designed and the factors with significant influencing factors were screened out through analysis of the results.

[0133] According to the results of the single-factor experiment, the Plackett-Burman experiment was designed with the total flavonoids, crude polysaccharides and total phenol yields as the experimental response values. The experimental design method and results are shown in Table 1.

[0134] Table 1 Factor levels and results of Plackett-Burman experimental design

[0135]

[0136] According to the experimental results, the extraction temperature (D) was a significant factor (P<0.05) affecting the crude polysaccharide yield of celery, and the ultrasonic time (B) and liquid-to-solid ratio (A) were the main factors. The liquid-to-solid ratio (A) was an extremely significant factor (P<0.01) affecting the total flavonoid yield of celery, and the extraction temperature (D) and ultrasonic time (B) were significant factors (P<0.05). The liquid-to-solid ratio (A), extraction temperature (D), and ultrasonic power (C) were all extremely significant factors affecting the total phenol yield of celery (P<0.01). In summary, the liquid-to-solid ratio (A), extraction temperature (D), and ultrasonic time (B) were significant factors and were selected as the three factors for the next step of the response surface experiment design.

[0137] A Plackett-Burman test identified three significant factors: liquid-to-solid ratio, ultrasonication time, and extraction temperature. A three-factor, three-level response surface experiment was designed based on these factors. The optimized conditions were: liquid-to-solid ratio of 34.51:1, ultrasonication time of 41.03 min, and extraction temperature of 50.92°C. Under these optimal extraction conditions, the software predicted a crude polysaccharide yield of 8.62%, a total flavonoid yield of 0.518%, and a total phenol yield of 2.43%. Three parallel validation experiments were conducted using these extraction conditions, and the results were generally consistent with the model's predictions, demonstrating that the equation closely matched the actual experimental results and that the model was effective and feasible. Compared with traditional extraction methods, this method achieved higher yields of crude polysaccharides and total flavonoids from celery. Furthermore, this experiment demonstrated environmental friendliness, high yields, and ease of operation.

[0138] Example 5 Corresponding surface design

[0139] Based on the Plackett-Burman experiment, a three-factor, three-level design was used to investigate the effects of ultrasonic power, ultrasonic time, and liquid-to-solid ratio on the yields of total flavonoids, crude polysaccharides, and total phenolics from celery. These three factors were represented by A, B, and C, respectively, with -1, 0, and 1 representing low, medium, and high levels, respectively. The factor coding table is shown in Table 2, and the detailed scheme is shown in Table 3.

[0140] Table 2 Experimental factors and levels

[0141]

[0142] Table 3 Response surface experimental design scheme

[0143]

[0144]

[0145] ① Response surface Box-Behnken experimental design

[0146] Based on the results of the Plackett-Burman test, crude polysaccharides (Y1), total flavonoid yield (Y2) and total phenol yield (Y3) were used as response values. The factors liquid-to-solid ratio (A), ultrasonic time (B) and extraction temperature (C) were selected as independent variables. A three-factor three-level response surface experimental design was carried out using Design-Expert11. The results of the Box-Behnken test are shown in Table 4.

[0147] Table 4 Response surface experimental design and results

[0148]

[0149] ②Response surface interaction analysis

[0150] The crude polysaccharide yield (Y1) was used as the response value, and the equation obtained by quadratic polynomial regression fitting was: crude polysaccharide yield (Y1) = 8.652-0.04625A+0.14125B-0.465C-0.4625AB-0.065AC+0.43BC-0.43725A 2 -0.22725B 2 -0.40475C 2 .

[0151] As shown in Table 5, the model is significant (P<0.05), and the lack of fit term is not significant (P>0.05), which shows that the model is credible and can correctly reflect the variation of crude polysaccharide yield and various factors. In addition, as shown in Table 5, the determination coefficient R 2 =0.9238, indicating that the regression equation simulates the test well, the test error is relatively small, and the correction determination coefficient R 2 acj =0.8257, which shows that the predicted value of the software is correlated with the actual value of the experiment, and the correlation is very high. In addition, the coefficient of variation CV is 3.1%, indicating that the variability of the experiment is 3.1%. This model can be used to analyze and predict the extraction process of old celery. From the P value, we can get that the linear terms AB and BC of the equation and the quadratic term C 2 The effect on the crude polysaccharide binding rate was significant (P<0.05), indicating that there was an interaction between the liquid-to-solid ratio (A) and the ultrasonic time (B), the ultrasonic time (B) and the extraction temperature (C). 2 The effect was extremely significant (P<0.01), while other factors had no significant effect (P>0.05). The F value showed that the order of influence of each factor on the crude polysaccharide yield was: extraction temperature (C) > ultrasonic time (B) > liquid-to-solid ratio (A). This regression equation can be used to predict the crude polysaccharide yield under different extraction conditions.

[0152] Table 5 Variance analysis of regression model of crude polysaccharides

[0153]

[0154]

[0155] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); CV = 3.1%, R 2 =0.9238, R 2 adj=0.8257

[0156] Taking the total flavonoid yield (Y2) as the response value, the equation obtained by quadratic polynomial regression fitting was: Y2 = 0.5172 + -0.011A + 0.00175B + 0.00575C + -0.0275AB + 0.007AC + 0.0075BC + -0.0681A 2 +-0.0246B 2 +-0.0321C 2 .

[0157] As shown in Table 6, the model is significant (P < 0.05), and the lack of fit term is not significant (P > 0.05), which shows that the model is credible and can correctly reflect the variation of total flavonoid yield and various factors. 2 =0.9452, which shows that the linear regression equation simulates the test well, the error of the test results is small, and the correction determination coefficient R 2 adj = 0.8748, from which we can see that the predicted value of the software is correlated with the actual value of the experiment, and the correlation is very high. In addition, the coefficient of variation CV is 3.63%, indicating that the variability of the experiment is 3.63%, which can be used to analyze and predict the extraction process of old celery with this model. From the P value, we can see that the interaction term AB in the equation indicates that there is an interaction between the liquid-to-solid ratio (A) and the ultrasonic time (B). The quadratic term B in the equation 2 The quadratic term A of the equation with significant effect (P<0.05) 2 、C 2 The effect on the total flavonoid yield was extremely significant (P<0.01), while the other factors had no significant effect (P>0.05). The F value shows that the order of influence of each factor on the total flavonoid yield is: liquid-to-solid ratio (A) > extraction temperature (C) > ultrasonic time (B). This regression equation can be used to predict the total flavonoid yield under different extraction conditions.

[0158] Table 6 Analysis of variance of regression model of total flavonoids

[0159]

[0160]

[0161] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); Coefficient of variation CV=3.63%, R 2 =0.9452, R 2 adj=0.8748

[0162] See Figure 4As shown, the elliptical contour map shows that the ultrasonic time and liquid-to-material ratio have a significant interaction, which is consistent with the analysis results in Table 5. When the ultrasonic time and extraction temperature are kept constant, the yield of crude polysaccharides shows a trend of first increasing and then decreasing with the increase of the liquid-to-material ratio. This is because with the increase of the liquid-to-material ratio, the fluidity weakens, which is not conducive to the dissolution of crude polysaccharides. Therefore, it is beneficial to extract crude polysaccharides under conditions of a lower liquid-to-material ratio. Under the condition of keeping the extraction temperature and liquid-to-material ratio unchanged, the yield of crude polysaccharides also shows a trend of first increasing and then decreasing with the increase of the ultrasonic time. This is because the structure of the crude polysaccharides of old mountain celery is unstable, and too long an ultrasonic extraction time will lead to the decomposition of the crude polysaccharides, thereby reducing the yield of crude polysaccharides.

[0163] See Figure 5 As shown in the figure, the elliptical contour map shows that the interaction between ultrasonic time and liquid-to-solid ratio is significant, which is consistent with the analysis results in Table 6. When the ultrasonic time increases with the increase of liquid-to-solid ratio, the yield of total flavonoids shows a trend of first increasing and then decreasing. It may be because too long ultrasonic time will cause the internal structure of the total flavonoids in the old celery to change, so it is helpful to extract the total flavonoids under the condition of shorter ultrasonic time. When the extraction temperature and ultrasonic time remain unchanged, with the increase of liquid-to-solid ratio, the yield of total flavonoids shows a trend of first increasing and then decreasing. This is because the total flavonoids in the old celery are relatively fully extracted within a certain liquid-to-solid ratio range. Continuing to increase the liquid will not change the content significantly, but will increase the concentration time and the loss amount will also increase relatively.

[0164] Because total flavonoids in celery have a higher medicinal value, the total flavonoid yield was used as the primary response. Using Design-Expert 13 software, the optimal extraction conditions were predicted to be: a liquid-to-solid ratio of 34.51:1, an ultrasonication time of 41.03 minutes, and an extraction temperature of 50.92°C. Under these conditions, the software predicted a crude polysaccharide yield of 8.62%, a total flavonoid yield of 0.518%, and a total phenol yield of 2.43%. Based on actual results, the optimal extraction conditions were: a liquid-to-solid ratio of 34.5:1, an ultrasonication time of 41 minutes, and an extraction temperature of 51°C. Three parallel validation experiments were conducted using these extraction conditions, resulting in actual values of 10.03% ± 2.28% for crude polysaccharide yield, 0.53% ± 0.35% for total flavonoid yield, and 2.57% ± 0.72% for total phenol yield. The crude polysaccharide yield was within 5% of the model prediction value, and the total flavonoid yield was within 1% of the model prediction value, which was basically consistent with the model prediction, indicating that the equation had a good fit with the actual experimental results. The optimal parameters obtained through the experiment had good stability and reliability, indicating that the model was effective and feasible.

[0165] Example 6 Screening conditions for flavonoid purification

[0166] Calculation of adsorption and desorption

[0167] 1 mL of the test solution was taken and the adsorption capacity, adsorption rate, desorption capacity, desorption rate and purity of the macroporous resin were calculated based on the flavonoid content.

[0168] Adsorption amount Q t The formula is as follows:

[0169]

[0170] Where Q t : adsorption amount at the moment (mg / g); C0: flavonoid content of the solution before adsorption (mg / mL); C t : flavonoid content of the solution after adsorption (mg / mL); V1: volume of adsorption solution (mL); M1: mass of adsorption resin (g).

[0171] Adsorption rate A t The formula is as follows:

[0172]

[0173] Wherein, C0: flavonoid content of solution before adsorption (mg / mL); Ct: flavonoid content of solution after adsorption (mg / mL).

[0174] Desorption amount Q t The formula is as follows:

[0175]

[0176] Where: C t ′: flavonoid content of the solution after desorption (mg / mL); V2: volume of desorption solution (mL); M2: mass of desorption resin (g).

[0177] Desorption rate A t The formula is as follows:

[0178]

[0179] Where, C0: flavonoid content of solution before adsorption (mg / mL); C t : flavonoid content of solution after adsorption (mg / mL); C t ': flavonoid content of the solution after desorption (mg / mL); V1: volume of adsorption solution (mL); V2: volume of desorption solution (mL).

[0180] The purity formula is as follows:

[0181]

[0182] (1) Screening of macroporous adsorption resins

[0183] The crude extract of flavonoids from celery was prepared into a 100 mg / mL crude extract solution of flavonoids from celery. 10 g of the six treated macroporous resins (S-8, AB-8, HPD100, D-101, DM-130, and D-406) were taken out and placed in a conical flask. Then 40 mL of the crude extract solution of flavonoids from celery was weighed and poured into the conical flask. The flask was placed in a shaker with the shaking parameter set to 100 times per minute for 12 hours. After adsorption equilibrium, 1 mL of sample was taken to detect the flavonoid content in the sample. The adsorption rate and adsorption amount were calculated using the above formula.

[0184] Weigh 5 g of the adsorbed macroporous resin, add 20 mL of 60% ethanol and shake for 12 h to reach desorption equilibrium. Take 1 mL of the desorbed sample and detect the flavonoid content in the sample. Calculate the desorption rate and desorption amount using the above formula.

[0185] Six macroporous adsorption resins were used to adsorb the same mass concentration of flavonoids from C. chinensis. The adsorption and desorption effects are shown in Table 7. In the static adsorption experiment, HPD-100, AB-8, and D-101 had higher adsorption rates. In the desorption experiment, D-101 had the highest desorption rate. Therefore, D-101 was selected as the optimal macroporous resin for purifying flavonoids from C. chinensis.

[0186] Table 7 Comparison of purification effects of macroporous adsorption resins

[0187]

[0188] (2) Static adsorption and desorption of macroporous resin

[0189] ① Detect the effect of sample loading temperature on static adsorption: Weigh 5g of the optimal macroporous resin and add 20mL of a 100mg / mL crude flavonoid extract from celery (pH 6.0). Then, set the temperature to 30°C, 35°C, 40°C, 45°C, and 50°C, respectively, and adjust the speed to 100 rpm. Oscillate and adsorb in a constant temperature oscillating chamber for 240 minutes. Afterwards, aspirate 1mL of the solution, detect the flavonoid content in the sample, and calculate the adsorption rate to determine the optimal purification temperature.

[0190] like Figure 6 Figure A shows the adsorption rate of flavonoids by D-101 macroporous adsorption resin at different temperatures. The resin exhibited different trends in flavonoid adsorption with varying temperatures. When the loading temperature was 40°C, the adsorption rate reached a maximum of 78.31%, a significant increase (P < 0.05). Above 40°C, the adsorption rate decreased significantly (P < 0.05). The structure of flavonoid compounds in celery undergoes structural changes at elevated temperatures. Setting the loading temperature at 40°C was considered the optimal adsorption temperature.

[0191] ② Detect the effect of sample concentration on static adsorption: Prepare 20 mL of crude extract of flavonoids from celery of different concentrations, where the total flavonoid concentrations of celery of celery are 50 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, and 250 mg / mL, respectively. The pH of the crude extract of flavonoids from celery of celery is 6.0. Weigh 5 g of the best macroporous resin and place it in a 50 mL conical flask. Subsequently, add crude extract of flavonoids from celery of different concentrations to the conical flask, place it in a shaking box, and shake at 30°C at a speed of 100 revolutions per minute for 240 minutes for adsorption. Then, take 1 mL of solution samples, detect the flavonoid content in the samples, and calculate the adsorption rate to determine the optimal sample concentration.

[0192] like Figure 6 As shown in Figure B, when the sample concentration was between 50 and 100 mg / mL, the adsorption rate increased significantly (P < 0.05), reaching a maximum of 59.5%. As the sample concentration exceeded 100 mg / mL, the adsorption rate decreased significantly (P < 0.05). At too low a concentration, the macroporous adsorption resin did not fully contact the flavonoids, resulting in a lower adsorption rate. At too high a concentration, the macroporous adsorption resin reached saturation prematurely, resulting in loss of flavonoid compounds. Therefore, the optimal sample concentration should be set at 100 mg / mL.

[0193] ③ Detect the effect of sample solution pH on static adsorption: Use 0.1% sodium hydroxide solution and 0.1% hydrochloric acid solution by mass to adjust the pH of the crude extract of flavonoids from celery to 5, 6, 7, 8, and 9 respectively. Weigh 5g of the best macroporous resin and place it in a 50mL conical flask. Add 20mL of 100mg / mL crude extract of flavonoids from celery to each flask. Oscillate and adsorb at 40℃ in a shaking box at 100 rpm for 240min. Then, take 1mL of solution samples, detect the flavonoid content in the samples, and calculate the adsorption rate to determine the optimal sample pH.

[0194] like Figure 6 As shown in C, when the pH of the sample solution is in the range of 5-7, the adsorption rate will first increase significantly (P<0.05). When the pH of the sample solution is 7, the adsorption rate of flavonoids reaches the highest, which is 82.24%. When the pH of the sample solution is higher than 7, the adsorption rate of flavonoids will decrease significantly with the increase of pH (P<0.05). When the pH is too high, the flavonoid molecules of the old celery will form salts or the H on the hydroxyl group of the flavonoid molecules will be + Therefore, the sample solution is relatively stable under neutral conditions with a pH of 7.

[0195] ④ Screening for the optimal eluent: Weigh 5g of the optimal macroporous resin and place it in a 50mL Erlenmeyer flask. Add 20mL of a 100mg / mL crude flavonoid extract from Celery of Oldenlandia chinensis. Adjust the pH to 7 and adsorb for 240min at 40°C in a shaker at 100 rpm. After shaking and filtering, obtain the adsorbed macroporous resin. Add 20mL of ethanol solutions of varying mass fractions (60%, 70%, 80%, 90%, and 100%) to the 50mL Erlenmeyer flask. After vigorous shaking, desorb for 4h. Measure the flavonoid concentration and calculate the resolution to determine the optimal eluent concentration.

[0196] like Figure 6 As shown in Figure D, the ethanol concentration significantly affects the desorption rate of D-101 macroporous adsorption resin (P < 0.05). Since higher ethanol concentrations reduce the polarity of the eluent, and D-101 resin is non-polar, it dissolves flavonoids with relatively low polarity better. Therefore, anhydrous ethanol was selected as the optimal eluent concentration, achieving a maximum desorption rate of 88.53%.

[0197] (3) Dynamic adsorption and desorption of macroporous resin

[0198] ① To test the effect of sample flow rate on dynamic adsorption: Weigh 5 g of the optimal macroporous resin after activation and load it into a glass column. Using a peristaltic pump, pump 50 mL of a crude flavonoid extract of C. chinensis (pH 7, 40°C) at the optimal concentration into the column at rates of 0.5 mL / min, 1.0 mL / min, 1.5 mL / min, 2.0 mL / min, and 2.5 mL / min, respectively. Determine the flavonoid content and calculate the adsorption rate.

[0199] like Figure 6 As shown in Figure E, dynamic adsorption experiments were conducted by pumping the solution through a glass-packed column at varying speeds. Adsorption efficiency significantly increased (P < 0.05) when the sample flow rate ranged from 0.5 mL / min to 1.5 mL / min. When the sample flow rate was too high, the flavonoids were not fully dissolved in the eluent, resulting in incomplete adsorption. When the sample flow rate was too low, the elution time was prolonged, slowing the experimental progress. Therefore, the optimal sample flow rate was 1.5 mL / min, achieving a maximum adsorption efficiency of 82.36%.

[0200] ② Detect the effect of eluent volume on dynamic desorption: Weigh 5 g of the activated optimal macroporous resin and load it into a glass-packed column. Pump 50 mL of a 100 mg / mL crude flavonoid extract of C. chinensis (pH 7, 40°C) into the glass-packed column using a peristaltic pump at a rate of 1.5 mL / min. Pump the eluent (anhydrous ethanol) into the glass-packed column at a flow rate of 2 mL / min using a peristaltic pump. The eluent volumes were 70 mL, 80 mL, 90 mL, 100 mL, and 110 mL, respectively. Determine the flavonoid content and calculate the desorption rate.

[0201] like Figure 6 Figure F shows the effect of different eluent volumes on resolution. When the eluent volume was between 70 mL and 100 mL, the desorption rate significantly increased (P < 0.05). Because using too much eluent would result in waste, 100 mL was selected as the optimal eluent volume for subsequent experiments.

[0202] ③ Detect the effect of eluent flow rate on dynamic desorption: Weigh 5 g of the optimal activated macroporous resin and load it into a glass-packed column. Pump 50 mL of a 100 mg / mL crude flavonoid extract of Cercidiphyllum sibiricum (pH 7, 40°C) into the glass-packed column using a peristaltic pump at a rate of 1.5 mL / min. Pump the eluent into the glass-packed column using the above-mentioned optimal eluent volume using a peristaltic pump. Set the eluent flow rate to 0.5 mL / min, 1.0 mL / min, 1.5 mL / min, 2.0 mL / min, and 2.5 mL / min, respectively. Determine the flavonoid content and calculate the desorption rate.

[0203] like Figure 6 As shown in Figure G, the desorption rate significantly increased when the eluent flow rate ranged from 0.5 mL / min to 1.5 mL / min (P < 0.05). When the eluent flow rate was too high, the ethanol could not effectively contact the flavonoids, resulting in incomplete elution from the resin. However, when the eluent flow rate was too low, the experimental progress was affected. Therefore, the optimal eluent flow rate was 1.5 mL / min, achieving a desorption rate of 86.61%.

[0204] Example 7 Product Testing

[0205] (1) Analysis of monosaccharide components of celery polysaccharide: The monosaccharide composition of celery polysaccharide (prepared by the method of Example 1) was analyzed by PMP pre-column derivatization-high performance liquid chromatography. The experimental method was referred to the literature:

[79] Zhang Weijia, Wang Qian, Dou Xin, et al. Ultrasonic-assisted extraction of polysaccharide from Phellinus igniarius fruiting body and analysis of its monosaccharide components [J]. Grain and Oils, 2023, 36(06): 139-162.

[0206] like Figure 7 and Figure 8As shown in the figure, by comparing the HPLC chromatogram with the standard monosaccharide, it was determined that the polysaccharide of jasmine is mainly composed of 7 monosaccharides, namely mannose, ribose, rhamnose, xylose, arabinose, glucuronic acid, and galacturonic acid. Among them, the content of glucuronic acid and galacturonic acid is relatively high, indicating that the polysaccharide of jasmine has antioxidant capacity.

[0207] (2) Determination of encapsulation efficiency and drug loading of celery microemulsion

[0208] ① Determination of drug loading of celery microemulsion: Accurately weigh 5g of celery microemulsion (prepared using the method of Example 1) into a 50mL volumetric flask, add 25mL of 60% ethanol, place in an ultrasonic cleaner, sonicate for 30 minutes, and then adjust the volume to 50mL with 60% ethanol. Centrifuge, take the supernatant, measure the flavonoid absorbance of the sample solution at 500nm, calculate the flavonoid concentration, and substitute it into Formula 11 to calculate the flavonoid drug loading in the celery microemulsion. After centrifugation, reconstitute the precipitate with distilled water, measure the polysaccharide absorbance of the sample solution at 490nm, calculate the polysaccharide concentration, and substitute it into Formula 12 to calculate the drug loading of the crude polysaccharide in the celery microemulsion.

[0209]

[0210] The above method was used for detection, and the drug loading of polysaccharide in the celery microemulsion was 2.03%, and the drug loading of flavonoids was 1.72%.

[0211] ② Determination of the encapsulation efficiency of the celery microemulsion: Accurately weigh 5g of the celery microemulsion (prepared using the method of Example 1) into a 50mL volumetric flask, add 25mL of 60% ethanol by mass, sonicate for 30min, and then add 60% ethanol by mass to make the volume 50mL. Centrifuge, take the supernatant solution, measure the flavonoid absorbance of the sample solution at 500nm, calculate the flavonoid concentration, and substitute it into Formula 13 to calculate the encapsulation efficiency of the flavonoids in the celery microemulsion. The precipitate after centrifugation was re-dissolved in distilled water, and the polysaccharide absorbance of the sample solution was measured at 490nm to calculate the polysaccharide concentration. Substitute it into Formula 14 to calculate the encapsulation efficiency of the crude polysaccharide in the celery microemulsion.

[0212]

[0213] The above method was used for detection, and the flavonoid encapsulation efficiency in the celery microemulsion was 85.31%, and the polysaccharide encapsulation efficiency was 87.55%.

[0214] (3) Characterization of the physicochemical properties of microemulsions

[0215] ① Observation of the morphological characteristics of the microemulsion under a transmission electron microscope: 1 g of the celery microemulsion (prepared by the method of Example 1) was diluted 10 times and lightly applied to a copper grid with a carbon film. Tungstic acid solution was added to stain the celery microemulsion. After the copper grid was dried, the surface morphology of the microemulsion was observed under an electron microscope.

[0216] The morphological characteristics of microemulsion under transmission electron microscopy are as follows Figure 9 As shown in the figure, the morphology of the microemulsion under transmission electron microscopy is spherical, with the inner core being oil droplets loaded with flavonoids and the outer ring being a water film loaded with crude polysaccharides, indicating that the microemulsion is an oil-in-water type microemulsion.

[0217] ② Detecting the potential of the microemulsion: dilute the celery microemulsion (prepared by the method of Example 1) 40 times, take 1 mL of the diluted microemulsion solution, transfer it to a polymer plastic cuvette, insert an electrode, and place it in a laser particle size analyzer to detect the potential of the celery microemulsion.

[0218] The results were measured by laser particle size analyzer potential mode. Figure 10 As shown in the figure, the Zeta potential value of the celery microemulsion is -19.36mV, which is consistent with the curve, indicating that the celery microemulsion colloidal solution has good stability.

[0219] ③ Detection of particle size: dilute the celery microemulsion (prepared by the method of Example 1) 40 times, take 1 mL of the diluted microemulsion solution, transfer it to a polymer plastic color dish, and detect its particle size using a laser particle size analyzer.

[0220] The results of microemulsion particle size test are as follows Figure 11 The average particle size of the old celery microemulsion is 140.8nm, indicating that the microemulsion particle size is small and evenly distributed.

[0221] ④ Infrared spectroscopy (FT-IR) detection: Fourier transform infrared spectrometer was used to perform infrared spectroscopy detection on flavonoid polysaccharide microemulsion (i.e., celery microemulsion), flavonoid microemulsion, polysaccharide microemulsion, and blank microemulsion, and the differences in the functional group fingerprint areas in the spectra of the four were compared and analyzed.

[0222] The flavonoid polysaccharide microemulsion (i.e., celery microemulsion) was prepared by referring to the method of Example 1. Referring to the method of Example 1, the flavonoid microemulsion used flavonoids in water, the polysaccharide microemulsion used corn oil in polysaccharide, and the blank microemulsion used corn oil in water.

[0223] Infrared spectroscopy (FT-IR) test results analysis Figure 12 As shown, infrared spectroscopy was performed on blank microemulsion, flavonoid microemulsion, polysaccharide microemulsion and flavonoid polysaccharide microemulsion, and the results showed no significant changes, indicating that the flavonoid polysaccharide microemulsion (i.e., celery microemulsion) was relatively stable.

[0224] ⑤ Differential scanning calorimetry (DSC) detection: A differential scanner was used to detect the melting point of the celery powder and celery microemulsion. The DSC detection conditions were: temperature range 20-300°C, heating rate 10°C / min. This experiment needed to be carried out under nitrogen protection.

[0225] Differential scanning calorimetry (DSC) test results analysis Figure 13 As shown, the first endothermic peak T max The first endothermic peak T of the old celery microemulsion is 179.15℃. max It is 225.01℃, indicating that the old celery microemulsion has higher thermal stability than the original powder.

[0226] ⑥ Thermogravimetric (TG) analysis: The thermal stability and composition of celery powder and celery microemulsion were determined using a thermogravimetric analyzer. TG testing conditions: temperature range 25-600°C, heating rate 10°C / min. Thermogravimetric curves are plotted with time on the horizontal axis and mass on the vertical axis, representing the cumulative weight loss of the sample during heating, with mass expressed in mg and time expressed in minutes.

[0227] Thermogravimetric analysis (TG) test results are as follows Figure 14 As shown in the figure, the mass of the old celery raw powder began to decline at about 28 minutes after the temperature rise procedure, while the old celery microemulsion began to decline at about 24 minutes, indicating that the old celery microemulsion began to decompose when the melting point of the microemulsion was reached at about 24 minutes. Compared with the old celery raw powder, the old celery microemulsion had a higher weight loss rate when reaching thermal stability, indicating that the old celery microemulsion had better thermal stability than the old celery raw powder.

[0228] Example 8 Detection of in vitro lipid-lowering activity

[0229] The celery powder and celery microemulsion were prepared by referring to the method of Example 1. The celery extract was prepared by the optimal two-phase aqueous extraction method of Example 1. The celery extract includes an upper phase and a lower phase, and the upper phase was used in this example.

[0230] (1) Detection of bile salt binding rate

[0231] To prepare a standard curve: Prepare sodium cholate, sodium glycocholate, and sodium taurocholate standard solutions at varying concentrations (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, and 0.45 mmol / L, respectively). Place 2 mL of each standard solution into a volumetric flask. Add 6 mL of 60% sulfuric acid. Incubate in a 70°C water bath for 20 minutes, then cool on ice for 5 minutes. Measure the absorbance at 387 nm using a UV spectrophotometer. Plot a standard curve with the bile salt content as the horizontal axis and the absorbance as the vertical axis.

[0232] The standard curve equation of sodium cholate is Y=0.8407x+0.0878(R 2 =0.9942), where x is the sodium cholate concentration (mg / mL) and Y is the absorbance; the standard curve equation for sodium glycocholate is Y=1.2695x-0.0096(R 2 =0.9945), where x is the concentration of sodium glycocholate (mg / mL) and Y is the absorbance; the standard curve equation of sodium taurocholate is Y=1.8793+0.1544(R 2 =0.9914), where x is the sodium taurocholate content (mg / mL) and Y is the absorbance.

[0233] Afterwards, 5 g of the sample to be tested was taken and 0.2 mol / mL sodium cholate, sodium glycocholate, and sodium taurocholate were added respectively. The sample was detected by ultraviolet spectrophotometry. The bile salt content was calculated according to the standard curve, and the bile salt binding rate was calculated according to Formula 15.

[0234]

[0235] like Figure 15 As shown in A, the sodium cholate binding rate of celery powder was 22.45%±2.21%, the sodium cholate binding rate of celery extract was 65.83%±1.97%, and the sodium cholate binding rate of microemulsion was 70.15%±2.32%.

[0236] See Figure 15 As shown in B, the binding rate of sodium glycocholate of celery powder is 16.42%±1.37%, the binding rate of sodium glycocholate of celery extract is 47.51%±2.3%, and the binding rate of sodium glycocholate of microemulsion is 59.92%±2.4%.

[0237] See Figure 15 As shown in Figure C, the binding rate of sodium taurocholate of celery powder is 22.2%±1.78%, the binding rate of sodium taurocholate of celery extract is 29.1%±2.33%, and the binding rate of sodium taurocholate of microemulsion is 56.9%±2.19%.

[0238] Therefore, celery microemulsion can significantly increase (P<0.05) the binding rate of bile salt, which has the effect of lowering blood lipids.

[0239] (2) Detection of pancreatic lipase activity inhibition rate

[0240] Prepare 0.08% (w / v) 4-nitrophenyl laurate (p-PNL) solution: weigh 0.4 g p-PNL and add it to 5 mmol / L 1% Triton X-100 sodium acetate solution. Boil in water for 1 min to dissolve it. Cool to room temperature, dilute to 50 mL with 5 mmol / L sodium acetate solution, and store in a refrigerator at 4°C.

[0241] 3mL of pH 7.4 phosphate buffer solution, 3mL of 0.08% lauric acid 4-nitrophenyl ester solution, and 3mL of 1.0mg / mL pancreatic lipase solution were respectively taken and mixed together to obtain a mixed solution. The mixed solution was mixed evenly with the samples, and the samples were respectively celery powder, celery extract or celery microemulsion. The sample solutions with concentrations of 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 50mg / mL and 60mg / mL were prepared using the above method. The sample solutions were placed in a constant temperature water bath at 37°C for 30min, and then the absorbance value was measured at 410nm and recorded as A. At the same time, the absorbance at 410nm of the sample replaced by water was A1, and the absorbance at 410nm of the sample without adding pancreatic lipase solution (phosphate buffer was used instead of an equal amount of pancreatic lipase solution) was A0. The formula for calculating the pancreatic lipase inhibition rate is as follows:

[0242]

[0243] Wherein, C is the inhibition rate of pancreatic lipase (%); A is the absorbance of the sample group; A1 is the absorbance of the blank group; A0 is the absorbance of the control group.

[0244] Depend on Figure 15 As can be seen, the inhibitory effects of celery powder, celery extract, and celery microemulsion on pancreatic lipase were concentration-dependent and increased with increasing concentration. When the celery powder solution concentration was less than 40 mg / mL, its inhibitory effect on pancreatic lipase activity increased rapidly with increasing concentration. After 40 mg / mL, the concentration dependence of the inhibitory effect weakened, reaching an inhibition rate of 64.71% ± 1.4% at 60 mg / mL. The celery extract showed significantly stronger inhibitory effects on pancreatic lipase, also exhibiting a dose-dependent effect, with an inhibition rate of 75.21% ± 2.1% at 60 mg / mL. The celery microemulsion had the greatest inhibitory effect on pancreatic lipase activity, reaching an inhibition rate of 78.8% ± 1.8% at a concentration of 60 mg / mL, demonstrating that the celery microemulsion indeed enhanced the inhibitory effect on pancreatic lipase activity. The order of inhibitory effect on pancreatic lipase activity was: celery microemulsion > celery extract > celery powder.

[0245] Example 9 Evaluation of hypoglycemic activity in vitro

[0246] The celery powder and celery microemulsion were prepared by referring to the method of Example 1. The celery extract was prepared by the optimal two-phase aqueous extraction method of Example 1. The celery extract includes an upper phase and a lower phase, and the upper phase was used in this example.

[0247] (1) Determination of α-glucosidase inhibition activity

[0248] Sample group: Samples (Ceratitis chinensis powder, Ceratitis chinensis extract, and Ceratitis chinensis microemulsion) were prepared into solutions of varying concentrations (5, 10, 15, 20, 25, and 50 mg / mL). 1 mL of each sample solution was uniformly mixed with 0.5 mL of 0.1 U / mL α-glucosidase solution (dissolved in 25 mmol / L phosphate buffer, pH 6.8). The mixture was reacted in a 37°C water bath for 10 min. Then, 0.5 mL of 5 mmol / L p-NPG solution (dissolved in 0.1 mol / L phosphate buffer, pH 6.8) was added. The mixture was reacted in a 37°C water bath for 15 min. The reaction was terminated by the addition of 1 mL of 0.1 mol / L sodium carbonate solution. The absorbance was measured at 405 nm using a UV spectrophotometer.

[0249] Positive group: The samples were replaced by equal volumes of acarbose solutions of different concentrations, and the rest were the same as those in the sample group.

[0250] Control group: an equal volume of 0.1 mol / L pH 6.8 phosphate buffer was used instead of α-glucosidase, and the rest were the same as those in the sample group.

[0251] The inhibition rate formula is as follows:

[0252]

[0253] Wherein: A-absorbance of the sample group; A0-absorbance of the control group; A1-absorbance of the mixed reaction of PBS buffer, p-NPG solution and enzyme solution, the concentration and volume of the added components are all referred to the sample group; A2-absorbance of the mixed reaction of PBS buffer and p-NPG solution, the concentration and volume of the added components are all referred to the sample group.

[0254] Depend on Figure 15 E It can be seen that the three samples of celery powder, celery extract, and celery microemulsion have different inhibitory effects on α-glucosidase, and are dose-dependent. As the mass concentration of the solution increases, the inhibition rate of acarbose, celery powder, celery extract, and celery microemulsion on α-glucosidase continues to increase. At different concentrations of 5mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, and 50mg / mL:

[0255] The inhibition rates of celery powder on α-glucosidase were 12.22%±2.24%, 37.26%±2.11%, 40.23%±2.31%, 56.32%±2.14%, 64.43%±1.82%, and 80.54%±1.93% respectively;

[0256] The inhibition rates of the extracts of Rhizoma Cibotii on α-glucosidase were 14.42%±2.04%, 30.51%±2.22%, 47.75%±2.44%, 57.72%±2.09%, 72.1%±1.81%, and 83.67%±2.4% respectively;

[0257] The inhibition rates of celery microemulsion on α-glucosidase were 15.76%±2.02%, 39.42%±2.33%, 51.17%±2.19%, 65.4%±2.12%, 75.92%±2.2%, and 88.41%±2.15% respectively;

[0258] The inhibition rates of α-glucosidase by acarbose at concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1.0 mg / mL were 40.74%±2.16%, 56.84%±2.53%, 67.81%±2.12%, 84.52%±1.67%, and 88.08%±3.14%, respectively.

[0259] Therefore, in the experiment on the inhibition of α-glucosidase activity, the order of inhibition rate is: acarbose > celery microemulsion > celery extract > celery original powder.

[0260] (2) Determination of α-amylase inhibition activity: Samples (Ceratitis chinensis powder, Ceratitis chinensis active ingredient solution, and Ceratitis chinensis microemulsion) were prepared into sample solutions of different mass concentrations (5, 10, 15, 20, 25, and 50 mg / mL). 1 mL of sample solution was mixed with 2 mL of 1 U / mL α-amylase solution (dissolved in pH 6.8 phosphate buffer) in a 37°C water bath for 10 min. 1 mL of 1% soluble starch solution was added and mixed in a 37°C water bath for 10 min. 1 mL of DNS reagent was then added and the mixture was heated in a boiling water bath for 5 min to terminate the reaction. The mixture was cooled and the volume was adjusted to 10 mL. The absorbance was measured at 540 nm.

[0261] Positive group: The samples were replaced by equal volumes of acarbose solutions of different concentrations, and the rest were the same as those in the sample group.

[0262] Control group: an equal volume of 0.1 mol / L pH 6.8 phosphate buffer was used instead of α-amylase, and the rest were the same as the sample group.

[0263] The inhibition rate formula is as follows:

[0264]

[0265] Where: A-absorbance of sample group; A0-absorbance of control group.

[0266] Depend on Figure 15 As can be seen from the results, the four solutions all have different inhibitory effects on α-amylase, and are dose-dependent. As the concentration of the solution increases, the inhibition rates of celery powder, celery extract, celery microemulsion, and acarbose on α-amylase continue to rise. At different concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, and 50 mg / mL:

[0267] The inhibition rates of celery powder on α-amylase were 26.28%±2.22%, 32.53%±2.13%, 41.52%±2.3%, 56.1%±2.12%, 67.38%±1.8%, and 75.28%±1.92% respectively;

[0268] The inhibition rates of celery extract on α-amylase were 25.4%±2.01%, 35.31%±2.23%, 47.24%±2.41%, 57.75%±2.11%, 68.69%±1.81%, and 79.61%±2.39% respectively;

[0269] The inhibition rates of celery microemulsion on α-amylase were 26.35%±2.01%, 38.78%±2.31%, 58.89%±2.21%, 64.5%±2.14%, 72.62%±2.2% and 80.38%±2.13% respectively;

[0270] When acarbose is at a concentration of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, the inhibition rates of α-amylase are 35.24%±2.35%, 45.52%±2.28%, 58.27%±2.45%, 72.7%±2.31%, 88.51%±1.88%, and 92.39%±1.88%, respectively.

[0271] Therefore, in the experiment on the inhibition effect of α-amylase activity, the order of inhibition rate is: acarbose > celery microemulsion > celery extract > celery original powder.

[0272] Example 10 Antioxidation

[0273] The celery powder and celery microemulsion were prepared by referring to the method of Example 1. The celery extract was prepared by the optimal two-phase aqueous extraction method of Example 1. The celery extract includes an upper phase and a lower phase, and the upper phase was used in this example.

[0274] (1) Evaluation of the antioxidant capacity of DPPH in vitro

[0275] Prepare DPPH solution: Accurately weigh 3.94 mg of DPPH into a 100 mL volumetric flask and add anhydrous ethanol to make up to volume to obtain a 0.1 mmol / L DPPH ethanol solution.

[0276] Vc control solution: Weigh 25 mg of Vc into a 10 mL volumetric flask and add deionized water to the volume to prepare a 2.5 mg / mL Vc reference solution; then dilute with deionized water to 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL reference solutions.

[0277] Preparation of sample supernatant: Take samples (Celeriaceae powder, Celeriaceae extract and Celeriaceae microemulsion), make the samples into sample solutions of 0.025 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL and 100 mg / mL respectively, add 70% ethanol solution to dissolve, the volume ratio of sample solution to 70% ethanol solution is 1:25, ultrasonic extraction is carried out at 280W and 45℃ for 1h, and then centrifuged at 8000r / min for 20min, and the supernatant is taken for use.

[0278] Determination of scavenging ability: 2 mL of each concentration of Vc control solution and each sample supernatant were respectively aspirated, 2 mL of DPPH solution was added, and after thorough mixing, the mixture was sealed and placed in the dark for 30 minutes. The absorbance of each sample was measured at 517 nm, and the obtained results were substituted into the DPPH scavenging rate formula to calculate the scavenging rate of each concentration of Vc control solution, celery powder, celery extract and microemulsion. The scavenging rate calculation formula is as follows:

[0279]

[0280] Where: A0-absorbance of 2 mL DPPH solution and 2 mL anhydrous ethanol; A1-absorbance of 2 mL sample supernatant and 2 mL anhydrous ethanol; A2-absorbance of 2 mL DPPH solution and 2 mL sample supernatant.

[0281] The experimental results are as follows Figure 15 As shown in Figure G, the scavenging rates of Vc solutions at various concentrations were 5.15% ± 2.2%, 24.81% ± 2.1%, 59.57% ± 2.1%, 95.72% ± 2.3%, and 98.44% ± 2.2%. It can be seen that with the increase in the concentration of Vc solution, the scavenging ability of DPPH free radicals continued to increase;

[0282] The scavenging rates of different concentrations of celery powder were 0.12% ± 0.06%, 23.1% ± 0.21%, 39.17% ± 1.18%, 50.65% ± 2.30%, 69.05% ± 2.32%, and 72.78% ± 2.40%, respectively. It can be seen that the scavenging ability of DPPH free radicals was relatively weak.

[0283] The scavenging rates of the various concentrations of the celery extract were 0.17% ± 0.04%, 27.2% ± 2.2%, 43.34% ± 2.4%, 55.19% ± 2.1%, 72.02% ± 1.8%, and 78.86% ± 2.4%. When the concentration reached 80 mg / mL, the scavenging rate exceeded 70%, and the scavenging rate of DPPH free radicals was significantly higher than that of the celery powder.

[0284] The scavenging rates of the celery microemulsion at each concentration were 9.67% ± 0.6%, 29.18% ± 1.01%, 46.24% ± 1.40%, 61.32% ± 1.98%, 75.14% ± 2.11%, and 84.27% ± 1.77%. At low concentrations, the scavenging ability of the microemulsion was lower than that of the vitamin C solution. However, as the concentration increased, the microemulsion had a scavenging ability similar to that of the vitamin C solution, indicating that the celery microemulsion had a relatively strong scavenging ability against DPPH. Therefore, the order of DPPH free radical scavenging ability was: vitamin C > celery microemulsion > celery extract > celery powder.

[0285] (2)ABTS + In vitro antioxidant assay

[0286] Preparation of ABTS + Test solution: Accurately weigh 6.62 mg of potassium persulfate and ABTS + 38.4 mg was dissolved in a 10 mL volumetric flask with distilled water and made up to volume to prepare ABTS. + After 16 h of reaction at room temperature in the dark, dilute ABTS with 1 mmol / L PBS buffer at pH 7.4. + The absorbance of the mother solution is about 0.70±0.02nm at a wavelength of 734nm, which means ABTS is obtained. + Test solution.

[0287] Vc reference solution: Accurately weigh 25 mg of Vc into a 10 mL volumetric flask and dilute to volume with deionized water to prepare a 2.5 mg / mL Vc reference solution; then dilute with deionized water to 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1.0 mg / mL reference solutions.

[0288] Preparation of sample supernatant: Weigh the samples (Celeriaceae powder, Celeriaceae extract and Celeriaceae microemulsion) respectively, and make the samples into sample solutions of 0.025 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL and 80 mg / mL respectively. Add 70% ethanol solution to the sample solution to dissolve it. The volume ratio of the sample solution to 70% ethanol solution is 1:25. Ultrasonic extraction is carried out at 280W and 45℃ for 1h, followed by centrifugation at 8000r / min for 20min. The supernatant is taken for later use.

[0289] Determination of ABTS + Clearing capacity: Pipette 4.8mL ABTS + To the test solution, 0.2 mL of each concentration of Vc control solution and each sample supernatant were added respectively. Then all the liquids were ultrasonically vibrated for 30 seconds, sealed and protected from light. After reacting for 6 minutes, the absorbance of each sample was measured at a wavelength of 734 nm using an ultraviolet spectrophotometer. The absorbance value was recorded as A. The blank was zeroed with 1 mmol / L phosphate buffer with a pH of 7.4, and the result was substituted into ABTS. + The clearance formula is used to calculate the clearance of each concentration of Vc control solution, celery powder, celery extract and celery microemulsion solution. The clearance calculation formula is as follows:

[0290]

[0291] Where: A-sample absorbance.

[0292] The experimental results are as follows Figure 15 As shown in Figure H, the clearance rates of Vc solution were 15.49% ± 1.83%, 37.96% ± 2.21%, 68.97% ± 2.15%, 87.2% ± 2.1%, and 98.81% ± 2.3%, respectively. This shows that Vc solution has a strong effect on ABTS. + Free radicals have strong scavenging power;

[0293] The clearance rates of different concentrations of celery powder were 0.12% ± 0.02%, 22.12% ± 0.21%, 28.40% ± 1.18%, 40.50% ± 2.30%, 61.20% ± 2.32%, 71.12% ± 2.40%, and 80.35% ± 1.69%, respectively. The clearance rates increased with the increase of sample concentration, indicating that it has a strong effect on ABTS. + Free radicals have scavenging capabilities;

[0294] The clearance rates of each concentration of the celery extract were 0.17% ± 0.02%, 25.72% ± 2.2%, 36.69% ± 2.4%, 57.41% ± 2.1%, 65.22% ± 1.8%, 79.9% ± 2.4%, and 86.2% ± 2.1% respectively;

[0295] The clearance rates of different concentrations of celery microemulsion were 0.36%±0.07%, 29.36%±2.31%, 41.53%±2.27%, 58.19%±2.1%, 71.26%±2.22%, 83.33%±2.18% and 93.58%±2.32% respectively.

[0296] In summary, the effect of celery microemulsion on ABTS + The scavenging ability of celery microemulsion was significantly improved, and it had a scavenging ability similar to that of Vc solution, indicating that the scavenging ability of celery microemulsion on ABTS + The scavenging ability of free radicals is relatively strong. Therefore, the order of scavenging free radicals is: Vc>Ceratum microemulsion>Ceratum extract>Ceratum powder.

[0297] Example 11 Calculation of IC 50 value

[0298] IC 50 The value refers to the concentration at which the sample can scavenge half of the free radicals. The DPPH and ABTS of the celery powder, celery extract and microemulsion were + The experimental results obtained from the scavenging ability assay and the experimental results obtained from the different concentrations of each sample, pancreatic lipase, α-amylase and α-glucosidase assays, were input into IC 50 The DPPH and ABTS of celery powder, celery extract and celery microemulsion were calculated in the calculator software. + IC of scavenging ability and in vitro lipid-lowering ability 50 IC values and in vitro hypoglycemic activity 50 .

[0299] Through IC 50 IC value calculator for calculating the IC value of celery powder in the pancreatic lipase activity inhibition test 50 The value is 41.23 mg / mL, and the IC 50 The value is 30.83 mg / mL, and the IC 50 The value was 24.06 mg / mL. Therefore, the inhibitory activity was ranked as follows: celery microemulsion > celery extract > celery powder.

[0300] See Table 8, through the α-glucosidase activity inhibition test, after IC 50 IC value calculator to calculate acarbose 50 The value is 0.208mg / mL, and the IC value of celery powder is 0.208mg / mL. 50 The value is 17.5mg / mL, and the IC 50 The value is 15.95mg / mL, and the IC 50The value is 13.61mg / mL. In the α-amylase activity inhibition experiment, the IC 50 The value is 0.158mg / mL, and the IC value of celery powder is 0.158mg / mL. 50 The value is 16.6mg / mL, and the IC 50 The value is 14.77mg / mL, and the IC 50 The value was 12.69 mg / mL. Therefore, the order of inhibitory ability of hypoglycemic activity was: acarbose > celery microemulsion > celery extract > celery powder.

[0301] See Table 9, through DPPH clearance test, after IC 50 Value calculator to calculate and get Vc IC 50 The value is 0.081mg / mL, and the IC of celery powder is 50 The value is 49.06mg / mL, and the IC 50 The value is 38.54 mg / mL, and the IC 50 The value was 30.66 mg / mL. Therefore, the order of DPPH scavenging ability is: Vc>microemulsion>Ceratum chinense extract>Ceratum chinense powder.

[0302] See Table 9, ABTS + In the in vitro antioxidant test, the IC 50 The value is 0.057mg / mL, and the IC 50 The value is 24.11mg / mL, and the IC 50 The value is 15.86 mg / mL, and the IC 50 The value was 10.72 mg / mL. Therefore, the order of free radical scavenging ability is: Vc>microemulsion>Ceratum extract>Ceratum powder.

[0303] Table 8 IC for inhibition of α-glucosidase and α-amylase activities 50 value

[0304]

[0305] Table 9 DPPH, ABTS + Clearance IC 50 value

[0306]

[0307] The present invention respectively studies the in vitro blood sugar lowering activity, in vitro blood lipid lowering activity and in vitro antioxidant capacity of celery powder, celery extract and celery microemulsion.

[0308] In the bile salt binding rate experiment, the sodium cholate binding rate of old celery raw powder was 22.45% ± 2.21%, the sodium cholate binding rate of old celery extract was 65.83% ± 1.97%, and the sodium cholate binding rate of old celery microemulsion was 70.15% ± 2.32%. The sodium glycocholate binding rate of old celery raw powder was 16.42% ± 1.37%, the sodium glycocholate binding rate of old celery extract was 47.51% ± 2.3%, and the sodium glycocholate binding rate of old celery microemulsion was 59.92% ± 2.4%. The sodium taurocholate binding rate of old celery raw powder was 22.2% ± 1.78%, the sodium taurocholate binding rate of old celery extract was 29.1% ± 2.33%, and the sodium taurocholate binding rate of old celery microemulsion was 56.9% ± 2.19%. In the pancreatic lipase activity inhibition activity experiment, the IC of old celery raw powder was 2. 50 The value is 41.23 mg / mL, and the IC 50 The value is 30.83 mg / mL, and the IC 50 The value is 24.06 mg / mL. By analyzing the bile salt binding rate and pancreatic lipase inhibition activity experiments of celery microemulsion, the results showed that the order of in vitro lipid-lowering activity is: celery microemulsion > celery extract > celery powder, indicating that celery microemulsion has good lipid-lowering activity.

[0309] In vitro hypoglycemic studies showed that the inhibitory effects on α-glucosidase and α-amylase activity were ranked as follows: acarbose > celery microemulsion > celery extract > celery powder. The celery microemulsion exhibited the highest inhibition rates for α-glucosidase, reaching 88.41% ± 2.20%, and α-amylase, reaching 80.38% ± 2.12%, demonstrating its robust hypoglycemic activity.

[0310] Finally, DPPH and ABTS + Comparison of scavenging ability: Vc> microemulsion> celery extract> celery powder. The scavenging rate of celery microemulsion on DPPH reached 84.27%±1.77%, and on ABTS + The clearance rate can reach 93.58%±2.32%, indicating that the celery microemulsion has good antioxidant activity.

[0311] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a celery microemulsion, characterized in that: The following steps are involved: (1) Using PEG800-ammonium sulfate aqueous two-phase system to extract celery, crude flavonoid extracts and crude polysaccharide extracts of celery were obtained; (2) Purifying the crude extract of flavonoids from the celery vine using macroporous resin to obtain purified flavonoids; treating the crude extract of polysaccharides from the celery vine using alcohol extraction and desalination to obtain purified polysaccharides; (3) Preparation of microemulsion: preparing the purified flavonoids into an oily solution; preparing the purified polysaccharide into an aqueous solution of celery polysaccharide; and adding the oily solution into the aqueous solution of celery polysaccharide during stirring for emulsification.

2. The preparation method according to claim 1, characterized in that In step (1), the liquid-to-solid ratio is (20-40) mL:1 g, preferably, (20-35) mL:1 g, and more preferably, (34-35) mL:1 g.

3. The preparation method according to claim 1, characterized in that: In step (1), the extraction temperature is 40-60°C, preferably, the extraction temperature is 45-55°C, and more preferably, the extraction temperature is 50-51°C.

4. The preparation method according to claim 1, characterized in that In step (1), the ultrasonic time is 10-50 min, preferably, the ultrasonic time is 30-50 min, and more preferably, the ultrasonic time is 40-41 min.

5. The preparation method according to any one of claims 1 to 4, characterized in that In step (2), D-101 resin is used for purification.

6. The preparation method according to any one of claims 1 to 4, characterized in that In step (2), static adsorption and desorption are adopted, the loading temperature is 30-50°C, preferably 40°C; the loading concentration is 50-250 mg / mL, preferably 100 mg / mL; the loading solution pH is 5-9, preferably pH 7; the eluent is 60-100% ethanol solution, preferably anhydrous ethanol.

7. The preparation method according to any one of claims 1 to 4, characterized in that In step (2), dynamic adsorption and desorption are adopted, the sample flow rate is 0.5-2.5 mL / min, preferably, 1.5 mL / min; the eluent volume is 70-110 mL, preferably, 100 mL; the eluent flow rate is 0.5-2.5 mL / min, preferably, 1.5 mL / min.

8. The preparation method according to any one of claims 1 to 4, characterized in that: In step (3), the purified flavonoids, medium-chain fatty acid edible oil, surfactant and co-surfactant are mixed to obtain an oil-type solution; the ratio of the purified flavonoids, medium-chain fatty acid edible oil, surfactant and purified polysaccharide is 10 mg:10 mL:10.5 g:5 mg.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the surfactant to the cosurfactant is (2-2.1):1; the surfactant comprises Tween 80 and Span 80; the cosurfactant is anhydrous ethanol; and HLB=12.

10. Use of celery microemulsion in preparing any one or more of the products (1) to (3), characterized in that: The celery microemulsion is prepared by the method according to any one of claims 1 to 9; (1) Lowering blood sugar; (2) Lowering blood lipids; (3) Antioxidant.

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