Preparation method of tea tree flower extract, product and application thereof

Abstract

The application relates to a preparation method of a tea flower extract and products and applications thereof, and the preparation method comprises the following steps: mixing tea flowers with water to perform micro-jet flow extraction, and then performing negative pressure cavitation treatment to obtain an extraction liquid; filtering the extraction liquid through a ceramic membrane to obtain a filtrate; and performing concentration sterilization on the filtrate, and then performing freeze-drying, so that the tea flower extract is obtained.The dynamic high-pressure micro-jet flow technology, the negative pressure cavitation extraction and the membrane filtration technology are adopted, the content of active substances in the tea flower extract is improved, the contents of total polyphenols and total flavonoids of the functional components are high, and the anti-allergy, anti-inflammatory and anti-aging effects are good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of substance extraction, and relates to a preparation method of tea flower extract, a product thereof and application. BACKGROUND

[0002] The tea flower contains components with the effects of detoxification, bacteriostasis, sugar reduction, anti-aging, cancer prevention and enhancement of immunity, and the contents of protein, tea polysaccharide, tea polyphenol and active antioxidant substances in the tea flower are higher than those in tea leaves, and the contents of pesticide residues and heavy metals are lower than the European Union standard. The tea flower is a high-quality protein nutrient source and can be comparable to rosemary, a world-recognized antioxidant plant.

[0003] In the extraction and preparation process of the tea flower extract, general reflux extraction and macroporous resin adsorption are adopted, but these methods have the defects of high energy consumption and great loss of effective components in retaining effective components and removing ineffective components. CN 117100811A adopts ultrasonic extraction of tea flowers, and the content of tea flower polyphenol is 5.10-6.31%. CN 116574563 A adopts cellulase, metal ions and ethylene glycol as extraction solvents to extract tea flowers to obtain tea flower extract liquid.

[0004] Traditional column chromatography and organic reagent extraction are generally used for separation and purification of the tea flower extract, which cannot be truly applied in the food field, and has the problems of low yield of active substances, high industrial cost, loss of flavor characteristics, complex industrial production and the like. On the other hand, the tea flower contains various bioactive components, and the high-retention industrial production has been a difficult problem to be solved in the field. SUMMARY

[0005] In view of the defects of the prior art, the purpose of the present application is to provide a preparation method of tea flower extract, a product thereof and application.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a preparation method of tea flower extract, which comprises the following steps:

[0008] (1) mixing tea flowers with water for microfluidization extraction, and then performing negative pressure cavitation treatment to obtain an extract liquid;

[0009] (2) filtering the extract liquid through a ceramic membrane to obtain a filtrate;

[0010] (3) concentrating and sterilizing the filtrate, and then freeze-drying to obtain the tea flower extract.

[0011] The present research uses RO water as the extraction solvent, combines dynamic high-pressure microfluidization technology, negative pressure cavitation extraction and step-by-step membrane filtration technology, improves the content of active substances in tea flower extract, and has high content of functional components such as total polyphenols and total flavonoids, and good anti-allergy, anti-inflammatory and anti-aging effects; using RO water as the solvent, the safety is high, the production is intensive, and the operation is easy.

[0012] Preferably, the mass ratio of the tea flower to water is 1:(10-30), and the extraction times are 1-6 times.

[0013] Specific point values in (10-30) can be selected as 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc., and the extraction times can be selected as 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0014] Preferably, the temperature of the microfluidization extraction is 40-70℃.

[0015] The temperature of the extraction can be selected as 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0016] Preferably, the pressure of the microfluidization extraction is 100-250MPa, for example, 100MPa, 110MPa, 120MPa, 130MPa, 140MPa, 150MPa, 160MPa, 170MPa, 180MPa, 190MPa, 200MPa, 210MPa, 220MPa, 230MPa, 240MPa, 250MPa, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the pressure of the negative pressure cavitation extraction is 0.04-0.08MPa, and the extraction times are 1-4 times.

[0018] The pressure can be selected as 0.04MPa, 0.05MPa, 0.06MPa, 0.07MPa, 0.08MPa, etc., and the extraction times can be selected as 1 time, 2 times, 3 times, 4 times, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0019] Preferably, the temperature of the negative pressure cavitation extraction is 20-70℃, and the time is 30-120min.

[0020] The temperature can be selected from 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc., and the time can be selected from 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min, 100min, 105min, 110min, 115min, 120min, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0021] Preferably, the pore size of the ceramic membrane is 200-400nm, for example, 200nm, 220nm, 240nm, 260nm, 280nm, 300nm, 320nm, 340nm, 360nm, 380nm, 400nm, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0022] Preferably, the sterilization temperature is 110-150℃, and the time is 15-30s.

[0023] The temperature can be selected from 110℃, 115℃, 120℃, 125℃, 130℃, 135℃, 140℃, 145℃, 150℃, etc., and the time can be selected from 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s, 30s, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0024] In a second aspect, the present application provides a tea flower extract prepared by the preparation method of the tea flower extract according to the first aspect.

[0025] In a third aspect, the present application provides an application of the tea flower extract according to the first aspect in the preparation of a product with anti-aging, anti-inflammatory and anti-allergic effects.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] In this study, RO water is used as the extraction solvent, combined with dynamic high-pressure microfluidization technology, negative pressure cavitation extraction and step-by-step membrane filtration technology, to improve the active content of the tea flower extract, and the total polyphenol and total flavonoid contents of the active ingredients are high, and the anti-allergic, anti-inflammatory and anti-aging effects are good. Using RO water as the solvent, the safety is high, the production is intensive, and the operation is easy.

[0028] High efficiency: the extraction method used in the present application can complete the extraction process in a short time, improving the yield of active substances and production efficiency.

[0029] Environmental protection: the solvent used in the extraction process is RO water, no organic solvent is involved, which improves the safety of the product and reduces the pollution to the environment.

[0030] Improved biological activity: the present application effectively improves the biological activity of tea flower extract by specific extraction conditions, and improves the application effect.

[0031] Wide application field: the present application is not only suitable for food and health care product field, but also can be applied to cosmetic field, which widens the application range of tea flower extract.

[0032] Simple operation: the preparation method of the present application is simple and easy to operate, which is beneficial to industrial production BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is the neutrophil quantity result graph.

[0034] Figure 2 is the normal group zebra fish phenotype graph.

[0035] Figure 3 is the model group zebra fish phenotype graph.

[0036] Figure 4 is the positive control group zebra fish phenotype graph.

[0037] Figure 5 is the zebra fish phenotype graph of tea flower extract obtained in example 1.

[0038] Figure 6 is the trypsin content result graph.

[0039] Figure 7 is the zebra fish collagen gene expression level result graph. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be further described through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitation to the present application.

[0041] Example 1

[0042] The present embodiment provides a preparation method of tea flower extract, which comprises:

[0043] (1) The tea flower powder is crushed and passed through a 100-mesh screen. The tea flower powder is mixed with RO water at a solid-liquid ratio of 1:30, and subjected to microfluidization extraction (the temperature of the microfluidization extraction is 60°C, the pressure is 200 MPa, and the number of times is 3), and then subjected to negative pressure cavitation treatment (the pressure of the negative pressure cavitation treatment is 0.06 MPa, the temperature is 60°C, the time is 45 min, and the number of times is 1), to obtain an extract;

[0044] (2) The extract is filtered through 1 μm and 200 nm ceramic membranes in sequence, to obtain a filtrate;

[0045] (3) After the filtrate is concentrated and sterilized, freeze-drying is performed, to obtain the tea flower extract.

[0046] Example 2

[0047] The present example provides a preparation method of a tea flower extract, which comprises:

[0048] (1) The tea flower powder is crushed and passed through a 100-mesh screen. The tea flower powder is mixed with RO water at a solid-liquid ratio of 1:10, and subjected to microfluidization extraction (the temperature of the microfluidization extraction is 50°C, the pressure is 150 MPa, and the number of times is 5), and then subjected to negative pressure cavitation treatment (the pressure of the negative pressure cavitation treatment is 0.08 MPa, the temperature is 50°C, the time is 120 min, and the number of times is 4), to obtain an extract;

[0049] (2) The extract is filtered through 1 μm and 400 nm ceramic membranes in sequence, to obtain a filtrate;

[0050] (3) After the filtrate is concentrated and sterilized, freeze-drying is performed, to obtain the tea flower extract.

[0051] Example 3

[0052] The present example provides a preparation method of a tea flower extract, which comprises:

[0053] (1) The tea flower powder is crushed and passed through a 100-mesh screen. The tea flower powder is mixed with RO water at a solid-liquid ratio of 1:20, and subjected to microfluidization extraction (the temperature of the microfluidization extraction is 70°C, the pressure is 250 MPa, and the number of times is 2), and then subjected to negative pressure cavitation treatment (the pressure of the negative pressure cavitation treatment is 0.04 MPa, the temperature is 70°C, the time is 30 min, and the number of times is 2), to obtain an extract;

[0054] (2) The extract is filtered through 1 μm and 300 nm ceramic membranes in sequence, to obtain a filtrate;

[0055] (3) After the filtrate is concentrated and sterilized, freeze-drying is performed, to obtain the tea flower extract.

[0056] Example 4

[0057] The embodiment provides a preparation method of tea flower extract, which is only different from the embodiment 1 in that the step (2) is "filtering the extraction solution through 1 mu m and 500 nm ceramic membranes in sequence to obtain a filtrate".

[0058] Other operations remain unchanged.

[0059] Example 5

[0060] The embodiment provides a preparation method of tea flower extract, which is only different from the embodiment 1 in that the step (2) is "filtering the extraction solution through 1 mu m and 100 nm ceramic membranes in sequence to obtain a filtrate".

[0061] Other operations remain unchanged.

[0062] Comparative Example 1

[0063] The comparative example provides a preparation method of tea flower extract, which is only different from the embodiment 1 in that the step (1) is "mixing tea flowers and RO water in a solid-liquid ratio of 1:30, performing ultrasonic extraction (ultrasonic power 400 W, time 60 min, temperature 60 DEG C), and then performing negative pressure cavitation treatment (the pressure of the negative pressure cavitation treatment is 0.06 MPa, the temperature is 60 DEG C, the time is 45 min, and the number of times is 1), to obtain an extraction solution".

[0064] Other operations remain unchanged.

[0065] Comparative Example 2

[0066] The comparative example provides a preparation method of tea flower extract, which is only different from the embodiment 1 in that the step (1) is "mixing tea flowers and RO water in a solid-liquid ratio of 1:30, performing microjet extraction (the temperature of the microjet extraction is 60 DEG C, the pressure is 200 MPa, and the number of times is 3), and then performing normal pressure extraction (the pressure of the normal pressure extraction is indoor pressure, the temperature is 60 DEG C, the time is 45 min, and the number of times is 1), to obtain an extraction solution".

[0067] Other operations remain unchanged.

[0068] Comparative Example 3

[0069] The comparative example provides a preparation method of tea flower extract, which is only different from the embodiment 1 in that the step (1) is "mixing tea flowers and RO water in a solid-liquid ratio of 1:30, performing microjet extraction (the temperature of the microjet extraction is 60 DEG C, the pressure is 200 MPa, and the number of times is 3), and then performing high pressure extraction treatment (the pressure of the high pressure extraction treatment is 100 MPa, the pressure maintaining time is 5 min, and the number of times is 1), to obtain an extraction solution".

[0070] Other operations remain unchanged.

[0071] Comparative Example 4

[0072] The present embodiment provides a preparation method of tea flower extract, which comprises:

[0073] (1) Tea flower is pulverized and passed through a 100-mesh screen. Tea flower powder is mixed with RO water at a solid-liquid ratio of 1:20, heated and refluxed for 2 times, each time for 1 h, to obtain an extract;

[0074] (2) The extract is filtered through a 1-μm and a 200-nm ceramic membrane in sequence to obtain a filtrate;

[0075] (3) The filtrate is sterilized after concentration, and then freeze-dried to obtain the tea flower extract.

[0076] Test Example 1

[0077] Flavonoid and polyphenol content test

[0078] Total polyphenol content determination:

[0079] 1. Reagent preparation

[0080] 1.1 10% Folin phenol reagent (freshly prepared): 20 mL of Folin phenol reagent (1 mol / L) is transferred to a 200-mL volumetric flask, diluted to the mark with water, and shaken well.

[0081] 1.2 7.5% sodium carbonate solution: 37.50 g ± 0.01 g of sodium carbonate (Na2CO3) is dissolved in water, transferred to a 500-mL volumetric flask, diluted to the mark, and shaken well (can be stored for 1 month at room temperature).

[0082] 2. Sample preparation

[0083] 2.1 Test sample: Examples 1-5, Comparative Examples 1-4

[0084] 2.2 Test sample mother liquor: 0.2 g of the test sample is accurately weighed into a 10-mL centrifuge tube, 5 mL of 70% methanol water solution preheated at 70°C is added, and the mixture is thoroughly stirred and moistened with a glass rod. The mixture is immediately transferred to a 70°C water bath, extracted for 10 min (stir every 5 min), and then cooled to room temperature. The mixture is centrifuged at 3500 r / min for 10 min, the supernatant is transferred to a 10-mL volumetric flask, the residue is extracted once more with 5 mL of 70% methanol water solution, and the above operation is repeated. The combined extract is diluted to 10 mL, shaken well, filtered with a 0.45-μm membrane, and used as needed (the extract can be stored for up to 24 h at 4°C).

[0085] Test sample test solution: 1 mL of the above mother liquor is transferred to a 100-mL volumetric flask, diluted to the mark with water, shaken well, and used for testing.

[0086] Preparation of standard solution: Gallic acid standard stock solution (1 000 μg / mL): 0.110 g ± 0.001 g of gallic acid (CAS: 149-91-7) standard was weighed into a 100 mL volumetric flask, dissolved and diluted to the mark, and shaken (freshly prepared).

[0087] Gallic acid working solution: 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL of gallic acid standard stock solution was respectively transferred into a 100 mL volumetric flask, diluted to the mark with water, and shaken, with concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

[0088] 3Detection

[0089] 1.0 mL of gallic acid working solution, water (as a blank control), and test solution was respectively transferred into a calibrated test tube, 5 mL of 10% Folin phenol reagent (1.1) was added to each, and shaken. After 5 min of reaction, 4 mL of 7.5% sodium carbonate solution (1.2) was added to each, diluted to the mark with water, and shaken. After 60 min of room temperature standing, the absorbance was measured at 765 nm with the reagent blank solution as the reference. The absorbance of the working curve solution was plotted against the corresponding gallic acid concentration to obtain a standard curve. The concentration of total polyphenols in the test solution was read from the standard curve, and the content of total polyphenols in the sample was calculated, with the results shown in Table 1.

[0090]

[0091] Total flavone content determination:

[0092] 1Flavone compounds refer to a series of compounds with a 2-phenyl chromone basic core structure in the molecular structure of the substance. The flavone core contains basic oxygen atoms and more than half of them have phenolic hydroxyl groups. In the presence of neutral or weakly basic and sodium nitrite, flavonoids form chelates with aluminum salts. After adjusting the alkalinity with sodium hydroxide, a red-orange color is shown, with a maximum absorption at about 500 nm, and the absorption value is proportional to the content of flavonoids. Rutin is used as a control, aluminum nitrate is used as a flavonoid developing agent, and the absorbance is linearly related to the concentration of rutin.

[0093] 2Test samples: Examples 1-5, Comparative Examples 1-4

[0094] 3. Test Method: Accurately measure 1.0 mL each of tea tree flower extract solution (2 mg / mL) and reference solutions (1, 0.8, 0.6, 0.4, 0.2 mg / mL) into 10 mL volumetric flasks. Accurately add 0.5 mL of 5% sodium nitrite solution to each flask, mix well, and let stand for 6 min. Then, accurately add 0.5 mL of 10% aluminum nitrate solution to each flask, mix well, and let stand for 6 min. Next, add 4.0 mL of 4% sodium hydroxide solution to each flask, mix well, and dilute to the mark with 95% ethanol. Mix well and let stand at room temperature for 15 min. After the reaction, pipette 200 μL of each sample into a 96-well plate. Using the reagent as a blank, measure the absorbance at a wavelength of 500 ± 2 nm. Plot a standard curve based on the absorbance values ​​and the concentration of rutin reference solution. Calculate the total flavonoid content based on the standard curve. The results are shown in Table 1.

[0095]

[0096] Table 1

[0097] Group Polyphenol (%) Flavonoid (%) Example 1 16.41 12.32 Example 2 16.17 12.27 Example 3 15.93 12.03 Example 4 12.80 13.12 Example 5 11.75 6.65 Comparative Example 1 14.22 13.74 Comparative Example 2 12.98 12.71 Comparative Example 3 12.27 12.92 Comparative Example 4 12.83 14.51

[0098] Test Example 2

[0099] Anti-inflammatory effect test

[0100] 1. Test Method: RAW264.7 cells in logarithmic growth phase with good morphology were selected and prepared into 5×10⁶ cells / years using complete culture medium. 4 A cell suspension of 1000 μL / mL was seeded into 24-well plates at a rate of 5 × 10⁶ cells / mL. 4 Each sample was incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0101] Drug administration and LPS: A blank control group, an induction stimulation group, a positive control group, and a sample group were set up.

[0102] In the blank control group, complete culture medium was added to each well.

[0103] Positive control group: 5 μM dexamethasone acetate (DEX) diluted with complete culture medium was added to each well.

[0104] Negative control group: LPS at a final concentration of 1 μg / mL

[0105] Add the test sample (concentration of 250 μg / mL) diluted with complete culture medium to each well of the sample group.

[0106] Each group was set up with 3 replicates and incubated in a 37℃, 5% CO2 incubator for 2 hours. Except for the blank control group, LPS was added to each well to a final concentration of 1 μg / mL and incubated in a 37℃, 5% CO2 incubator for 24 hours.

[0107] 2Cell supernatant inflammatory factor detection: the cell supernatant was centrifuged at 4°C, 1000g, 10min, and collected according to the steps of the nitric oxide detection kit instructions, and the content of NO in the collected cell supernatant was detected. The experimental results were analyzed by data processing software, and the results are shown in Table 2.

[0108] Table 2

[0109] Group NO production (μmol / L) Example 1 7.45 Example 2 7.62 Example 3 7.78 Example 4 8.10 Example 5 14.01 Comparative Example 1 8.00 Comparative Example 2 9.26 Comparative Example 3 9.62 Comparative Example 4 7.94 Blank control group 6.72 DEX 17.25 LPS 21.01

[0110] Test Example 3

[0111] Antioxidant effect test

[0112] DPPH free radical scavenging experiment

[0113] DPPH (1,1-diphenyl-2-picrylhydrazyl) is a very stable nitrogen-centered free radical with a single electron. When a free radical scavenger is present, the single electron of DPPH is captured, causing the color to become lighter, and the absorbance value at the maximum light absorption wavelength decreases. The decrease in absorbance level indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample.

[0114] Test method: DPPH solution was dissolved in anhydrous ethanol with a concentration of 0.1 mg / mL, and a 0.5 mg / mL vitamin C standard solution was prepared. The tea flower extract prepared by dissolving Examples 1-5 and Comparative Examples 1-4 in different proportions of aqueous solution (1-0.03125 mg / mL) was prepared. 150 μL of DPPH ethanol solution and 150 μL of tea flower extract solution were mixed uniformly, and a blank control group was set. The reaction was carried out at room temperature for 30 min in the dark, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate of the sample was calculated according to the following formula:

[0115]

[0116] In this experiment, vitamin C was used as a positive control. The concentration of the sample to be tested was taken as the X axis, and the absorbance was taken as the Y axis to produce a scatter plot with a smooth line and data markers. Select the upper, middle and lower three points with a clearance rate of about 50% to draw a straight line, and obtain the equation of the straight line. The DPPH free radical scavenging IC50 value and clearance rate of the sample and vitamin C were calculated. The DPPH free radical scavenging IC50 value results are shown in Table 3,

[0117] Table 3

[0118] Group Inhibition IC50 value / μg / mL Example 1 22.47 Example 2 23.07 Example 3 25.51 Example 4 31.70 Example 5 60.84 Comparative Example 1 29.71 Comparative Example 2 44.40 Comparative Example 3 30.05 Comparative Example 4 41.92

[0119] Test Example 4

[0120] Relieving efficacy test:

[0121] Hyaluronidase (HAase) can hydrolyze HA to generate β-N-acetylglucosamine, which can be condensed with p-dimethylaminobenzaldehyde to generate a chromogenic 2-methyl-3- diacetylpyrrole derivative (purple color) under alkaline conditions. Hyaluronidase is strongly related to inflammation and allergy, and is a participant of type I allergic reaction. Therefore, the in vitro inhibition experiment of hyaluronidase can be used as a quick screening method for soothing efficacy raw materials.

[0122] Test method:

[0123] 2.5-10 mg was weighed, dissolved in 10 mL of 0.1 mM acetic acid buffer and vortexed to prepare a 0.25-1 mg / mL HAS solution. 10 mg was weighed, dissolved in 10 mL of 0.1 mM acetic acid buffer and vortexed to prepare a 1 mg / mL HA solution. A 1 mg / mL tannic acid solution was prepared with water as a solvent, and tea flower extracts prepared in Examples 1-5 and Comparative Examples 1-4 (test concentration: 1 mg / mL). The operation is shown in Table 4:

[0124] Table 4

[0125]

[0126]

[0127] A - absorbance value of sample solution

[0128] B - absorbance value of sample blank

[0129] C - absorbance value of control solution

[0130] D - absorbance value of control blank

[0131] Tannic acid was used as a positive control in this experiment. A scatter plot with a smooth line and data markers was made with the test sample concentration as the X axis and the absorbance as the Y axis. Three points with an inhibition rate of about 50% were selected to draw a straight line, and the straight line equation was obtained to calculate the hyaluronidase inhibition IC50 value and inhibition rate of the sample and tannic acid. The hyaluronidase inhibition rate value of the tea flower extract is shown in Table 5.

[0132] Table 5

[0133] Group Hyaluronidase inhibition rate (%) Positive control 100.00％ Example 1 76.18％ Example 2 73.53％ Example 3 70.64％ Example 4 39.28％ Example 5 5.08％ Comparative Example 1 49.84％ Comparative Example 2 28.37％ Comparative Example 3 21.90％ Comparative Example 4 58.91％

[0134] Test Example 5

[0135] Collagenase inhibition efficacy test

[0136] Test method:

[0137] 1 Reagent preparation: preparation of tea flower extract (500, 250, 125, 62.5 μg / mL); preparation of Tris-HCl buffer solution (37°C, pH = 7.5 ± 0.2, containing 5 mmol / mL CaCl2), tetracycline hydrochloride solution (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL); preparation of 0.5 mg / mL substrate FALGPA buffer salt solution (the absorbance at 330 nm should be less than 0.9); preparation of 0.2 mg / mL collagenase type I Tris-HCl solution (the absorbance at 330 nm should be less than 0.3).

[0138] 2 Sample testing: select the optimal enzyme activity concentration, and determine the positive drug and sample. Add 140 μL of 0.2 mg / mL collagenase solution, 60 μL of tetracycline hydrochloride solution of different concentrations and sample solution, and set up a 100% enzyme activity control group, i.e. replace the tetracycline solution with 60 μL of PBS, and replace the same volume of buffer salt for the blank control and sample blank group. After incubation at 37°C for 20 min, add 40 μL of substrate FALGPA buffer salt solution, and determine the absorbance change at 330 nm for 0-20 min. Calculate the collagenase inhibition rate according to the following formula:

[0139]

[0140] ΔA330B = the difference between the absorbance values at the two time points (0 min and 20 min) of the control group.

[0141] ΔA330A = the difference between the absorbance values at the two time points (0 min and 20 min) of the blank group.

[0142] ΔA330D = the difference between the absorbance values at the two time points (0 min and 20 min) of the sample group.

[0143] ΔA330C = the difference between the absorbance values at the two time points (0 min and 20 min) of the sample blank group.

[0144] This experiment uses tetracycline as a positive control. With the concentration of the sample to be tested as the X axis and the absorbance as the Y axis, a scatter plot with a smooth line and data markers is prepared. Select the upper, middle and lower three points with an inhibition rate of about 50% to draw a straight line, and obtain the equation of the straight line to calculate the collagenase activity inhibition rate IC50 value of the test sample and tetracycline. The collagenase enzyme inhibition rate IC50 value of tea flower extract is shown in Table 6.

[0145] Table 6

[0146] Group Inhibition IC50 value / μg / mL Positive control 552.52 Example 1 143.05 Example 2 149.87 Example 3 167.63 Example 4 305.48 Example 5 470.55 Comparative Example 1 284.51 Comparative Example 2 205.00 Comparative Example 3 318.00 Comparative Example 4 161.80

[0147] Test Example 6

[0148] Zebrafish skin inflammation test

[0149] Test method:

[0150] 1 Zebrafish type: neutrophil red fluorescent labeled zebrafish Tg(mpX; dSRED).

[0151] 2 Zebrafish embryo collection

[0152] The zebrafish breeding method is carried out according to The. zebrafish book. The breeding water temperature is maintained at about 28.5°C, the light is on for 14h and off for 10h per day, and the fish is fed twice a day. The night before collecting the embryos, 1 female fish and 2 male fish are placed in the spawning tank and separated by a partition, and placed in a dark environment. The next morning, after the light is turned on, the partition is removed, and the embryos are collected after spawning is completed. The embryos are placed in egg water and cultured in a light incubator at 28.5°C.

[0153] 3 Anti-inflammatory active substance efficacy evaluation method

[0154] 3.1 On the day of embryo collection, about 7-8hpf, the embryos were thoroughly cleaned and the poor embryos were removed, and fresh egg water was replaced, and the culture was continued in a 28.5°C incubator to 3dpf (during the culture period, the poor embryos were picked out in time, and fresh egg water was replaced every day).

[0155] 3.2 Test grouping

[0156] The 3dpf zebrafish were randomly divided into groups, 15 zebrafish per group

[0157] a) Normal control group: containing zebrafish and standard dilution water.

[0158] b) Model control group: containing zebrafish and 1 μM copper sulfate solution

[0159] c) Positive control group: containing zebrafish, 1 μM copper sulfate solution and positive drug (50.0 μM dexamethasone acetate is used as the positive drug in this experiment)

[0160] d) Test substance test group: containing zebrafish, 1 μM copper sulfate solution and test substance, the test substance is 8 μg / mL tea flower extract obtained in Example 1

[0161] 3.3 Test substance treatment

[0162] According to the experimental requirements, a sufficient number of 72pf zebrafish with uniform development were pre-selected and randomly assigned to six-well plates, 15 zebrafish per well. The standard dilution water was removed from the six-well plates without harming the embryos, and then 3 mL of the test solution was quickly added to each well. After thorough mixing, the plate was covered and wrapped with aluminum foil, and incubated in a 28°C biochemical incubator in the dark for 2 hours.

[0163] 3.4 Observation and photography

[0164] Neutrophils labeled with green fluorescence (T8(pX:dSRED)) in zebrafish emit green fluorescence, allowing direct observation of neutrophil migration and other behaviors in live zebrafish under a stereofluorescence microscope. After incubation, zebrafish with normal phenotype and behavior were randomly selected, fixed with 3% methylcellulose, and observed and photographed under a stereofluorescence microscope. All zebrafish photographs were taken under identical instrument and environmental conditions, and the zebrafish positions were kept consistent.

[0165] Neutrophil count results as follows Figure 1 As shown, the zebrafish phenotypic diagram is as follows: Figures 2-5 As shown in the figure, tea tree flower extract significantly reduced the number of neutrophils in zebrafish, showing a significant difference compared to the model group (P < 0.001), indicating its anti-inflammatory effect.

[0166] Test Example 7

[0167] Anti-allergy test

[0168] 1. Zebrafish type

[0169] Wild-type AB strain zebrafish juveniles.

[0170] 2. Zebrafish embryo collection

[0171] Zebrafish husbandry was conducted according to "The Zebrafish Book". Maintain a water temperature of approximately 28.5℃, providing 14 hours of light and 10 hours of darkness daily, feeding twice a day, morning and evening. The night before collecting embryos, place one female and two males in the spawning tank, separating them with a partition and placing them in a dark environment. The following morning, after turning on the lights, remove the partition and collect the embryos after spawning. Place the embryos in egg water and incubate them in a 28.5℃ lit incubator.

[0172] 3. Type I sensitization assessment method

[0173] 3.1 Collect embryos on the day of the experiment, about 7-8hpf, thoroughly clean the embryos and remove the poor embryos, replace fresh egg water, and continue to cultivate in a 28.5°C incubator to 2dpf (during the cultivation period, remove the poor embryos in time, and replace fresh egg water every day).

[0174] 3.2 Test grouping

[0175] Randomly group 2dpf zebrafish, 10 per group:

[0176] a) Normal control group: contains zebrafish larvae and standard dilution water (100 μL) + 100 μg / mL BAPNA (100 μL).

[0177] b) Model control group: contains 3 μg / mL dinitrochlorobenzene prepared with standard dilution water (100 μL) + 100 μg / mL BAPNA (100 μL).

[0178] c) Positive control test group: contains 3 μg / mL dinitrochlorobenzene and 1000 μg / mL cromolyn sodium prepared with standard dilution water (100 μL) + 100 μg / mL BAPNA (100 μL).

[0179] d) Test substance test group: contains 3 μg / mL dinitrochlorobenzene and 8 μg / mL tea flower extract obtained in Example 1 prepared with standard dilution water (100 μL) + 100 μg / mL BAPNA (100 μL).

[0180] 3.3 Test substance concentration setting

[0181] According to the test needs, the tea flower extract concentration for this experiment is set to 8 μg / mL.

[0182] 3.4 Test substance treatment

[0183] Select 2dpf zebrafish larvae with normal development, place them in a 12-well cell culture plate, 10 per well, remove the embryo culture solution in the 12-well plate without harming the larvae, quickly add 1 mL of the corresponding concentration of the test substance diluent to each well. Wrap the 12-well cell culture plate with aluminum foil, and incubate in a (28.5°C ± 1.0°C) biochemical incubator for 22h in the dark. After incubation, remove the 12-well plate, shake well, and then aspirate 200 μL of liquid from each well into a 96-well plate, and read the absorbance at 405 nm.

[0184] 3.5 Observation and photography

[0185] After incubation, observe whether the zebrafish has a toxic reaction, 200 μL of liquid is taken from each well in a 96-well plate, and the absorbance value is read at a wavelength of 405 nm, and the results are shown in Table 2. Figure 6 Dinitrochlorobenzene induces a significant increase in serum trypsin levels, and trypsin is complexed with the BAPNA substrate, which has a maximum absorption peak at 405 nm, and the degree of allergy can be detected. Figure 6 It can be seen that the tea flower extract can significantly reduce the content of zebrafish trypsin, and has a significant difference from the model group (P<0.001), and has an anti-allergic effect.

[0186] Test Example 8

[0187] Zebrafish collagen expression

[0188] 1. Zebrafish strain: wild type AB strain.

[0189] 2. Test method: 180 randomly selected wild type AB strain zebrafish of 4 dpf normal growth and development morphology in a 6-well plate, 30 in each well. Add 125 μg / mL tea flower extract solution to the 6-well plate, and each well is 3 mL, and set up a normal control group without adding tea flower extract and 3 biological repeats.

[0190] After incubation in a 28°C incubator in the dark for 24 h, the total RNA of each group of zebrafish was extracted using an RNA rapid extraction kit, and the total RNA concentration and purity were determined using a UV-visible spectrophotometer. According to the operation of the cDNA first strand synthesis kit, 2 μg of zebrafish sample total RNA was synthesized into 20 μL of cDNA, and the gene expression of β-actin and collagen was detected by q-PCR. Taking β-actin as the internal reference gene, the RNA relative expression amount of collagen type I (zebrafish genes col1a1a, col1a1b) gene was calculated.

[0191] The RNA relative expression amount of collagen gene is calculated according to the following formula:

[0192] RNA relative expression amount = 2^ΔΔC(t)

[0193] ΔΔC(t) = ΔC(t) normal control group - ΔC(t) sample group

[0194] ΔC(t) = ΔC(t) target gene - ΔC(t) β-actin

[0195] C(t) target gene: collagen gene expression level detected by q-PCR

[0196] C(t) (β-actin): β-actin gene expression level detected by q-PCR

[0197] AC(t) normal control: average value of AC(t) of three repeated tests of normal control group

[0198] AC(t): difference between collagen gene expression level and β-actin gene expression level detected by q-PCR

[0199] C(t) target gene: collagen gene expression level detected by q-PCR

[0200] C(t) (β-actin): β-actin gene expression level detected by q-PCR

[0201] AC(t) normal control: average value of AC(t) of three repeated tests of normal control group

[0202] AC(t): difference between collagen gene expression level and β-actin gene expression level detected by q-PCR

[0203] The results, as shown in Table 1, show that the tea flower extract significantly improves the collagen type I gene expression level, and has a significant difference (P<0.001) compared with the normal group, and has an anti-aging effect. Figure 7

[0204] The applicant states that the present application is illustrated by the above-mentioned embodiments, and the product and application of a tea flower extract preparation method of the present application, but the present application is not limited to the above-mentioned embodiments, i.e. it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0205] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications of the technical solutions of the present application can be made, and these simple modifications all belong to the protection scope of the present application.

[0206] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination methods.​

Claims

1. A method of preparing an extract of tea tree flowers, characterised in that, The preparation method comprises: (1) mixing tea flowers with water for micro-jet extraction, and then performing negative pressure cavitation treatment to obtain an extraction solution; (2) filtering the extraction solution through a ceramic membrane to obtain a filtrate; (3) concentrating and sterilizing the filtrate, and then freeze-drying to obtain the tea flower extract; The temperature of the micro-jet extraction is 40-70 DEG C, and the pressure is 100-250 MPa; The pressure of the negative pressure cavitation treatment is 0.04-0.08 MPa, the extraction times is 1-4 times, the temperature is 20-70 DEG C, and the time is 30-120 min.

2. The method of claim 1, wherein the tea tree flower extract is prepared by the steps of: The mass ratio of the tea flowers to water is 1:(10-30), and the extraction times is 1-6 times.

3. The method of claim 1, wherein the tea tree flower extract is prepared by the steps of: The pore size of the ceramic membrane is 200-400 nm.

4. The method of claim 1, wherein the tea tree flower extract is prepared by the steps of: The sterilization temperature is 110-150 DEG C, and the time is 15-30 s. 5.A tea flower extract prepared by the preparation method of any one of claims 1-4. 6.Use of the tea flower extract of claim 5 in the preparation of a product having anti-aging, anti-inflammatory and anti-allergic effects.

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