A flavonoid extract based on shiya tea and its preparation method and application

Abstract

The invention relates to the technical field of effective component extraction process of yarrow tea, and specifically discloses a flavonoid extract based on yarrow tea, a preparation method and application thereof. The flavonoid components in the raw material are extracted by crushing, crushing after freezing, and cellulase-assisted pure water reflux extraction, providing a more efficient and high-yield extraction method of yarrow tea flavonoid extract, laying a foundation for the industrial production of high value-added products of yarrow tea; solving the problem that the extraction of flavonoid components of yarrow tea mainly depends on finished tea, resulting in high cost; solving the problem that flavonoid components of old tea leaves at different periods cannot be effectively utilized; the extracted flavonoid extract of yarrow tea has a more excellent in vitro antibacterial effect, and also has a stronger effect of enhancing immunity function, and the extracted flavonoid extract of yarrow tea has the effect of preventing and treating poultry H9 subtype avian influenza.

Description

Technical Field

[0001] The invention belongs to the technical field of effective component extraction technology of shiya tea, and particularly relates to a flavonoid extract based on shiya tea, and a preparation method and application thereof. Background Art

[0002] Stone cliff tea, also known as: stone rock tea, stone mountain tea. The scientific name is bright yellow Ruimu, genus Yangtong, tree. It is named because it grows on the cliff. Its buds and leaves are thick, and the tea soup is bright. The taste is pure and sweet, and it is resistant to brewing. According to the test of relevant departments, because stone cliff tea does not contain caffeine, it does not affect sleep after drinking, and it is rich in flavonoids and has good health care effects, so it is deeply loved by consumers. "Wild stone cliff tea" is green like emerald, fragrant and refreshing, and contains low theophylline. After tasting, it has an endless aftertaste, does not affect sleep, and long-term drinking in the morning and evening has the effects of clearing away heat and detoxification, protecting the liver and improving eyesight, anti-inflammatory and moisturizing the lungs, nourishing the skin, lowering blood pressure, reducing fat, and strengthening the stomach and digestion. It has obvious effects on pharyngitis, obesity, diabetes, hypertension, and hyperlipidemia. Wild cliff tea is a treasure of the Dayao Mountains. It grows in various counties and cities in Guangxi, on stone cliffs or by mountain streams in the southern mountainous area where the sunshine is short, the temperature difference is large, and it is cool and humid. Because the tea grows on cliffs, it can be said to absorb the spiritual energy of heaven and earth and the essence of the sun and the moon. The unique ecological environment has given birth to a pure natural beverage with a unique style.

[0003] Modern pharmacological studies have shown that the extract of Shiya tea has multiple pharmacological activities such as anti-tumor, antioxidant, and antibacterial. Phytochemical studies have found that the chemical composition of Shiya tea is complex, containing flavonoids, tea polyphenols, alkaloids and other components, of which flavonoids account for about 28%, which is one of the main active ingredients of Shiya tea and also one of the plants with the highest flavonoid content in natural plants. The extraction of natural flavonoids is usually carried out by solvent extraction, such as acetone, ethanol, water, etc., through immersion, leakage, heating and reflux. In recent years, due to the application of new technologies such as microwaves, ultrasound, and bio-enzymes, the extraction technology of flavonoid components has been further sublimated. Regarding the extraction of flavonoid components of Shiya tea, the literature reports mainly include heating reflux extraction, ultrasonic extraction, microwave extraction, and carbon dioxide supercritical extraction. Each extraction method has its own advantages and disadvantages. For example, the heating reflux extraction method has a high yield of the target component, but the solvent consumption is large and the extraction time is long. The carbon dioxide supercritical extraction method has a high product purity, but the equipment requirements are high and the production cost is high. This study used a combined extraction method, namely bio-enzyme + reflux extraction, to extract flavonoids from Shiya tea, aiming to explore a more efficient and high-yield extraction method and lay the foundation for the industrial production of high value-added Shiya tea products.

[0004] In addition, nowadays, the extraction of flavonoids from Shiya tea mainly relies on finished tea, which leads to high costs. In fact, old tea leaves that have not been made into finished tea also contain a large amount of flavonoids. In addition, the composition and content of flavonoids in old tea leaves at different periods also change. Such components also need to be effectively utilized. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of flavonoid extract based on cliff tea, aiming to explore a more efficient and high-yield extraction method, laying a foundation for the industrial production of high value-added products of cliff tea; it also solves the problem that the extraction of flavonoid components of cliff tea mainly relies on finished tea, resulting in high costs; at the same time, it solves the problem that flavonoid components of old tea leaves at different periods cannot be effectively utilized.

[0006] To achieve the above object, the present invention provides a method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0007] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 15-18 parts of Shiya tea, 40-45 parts of dried leaves of 6-month-old tea leaves, and 30-35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying Shiya tea leaves picked in October;

[0008] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0009] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly and crushing them to obtain old tea powder;

[0010] (3) freezing the old tea powder at -15 to -25°C for 8 to 10 hours, taking it out, vacuum drying it, and then crushing it into 300 to 500 meshes to obtain old tea fine powder;

[0011] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0012] (5) The flavonoid components in the mixed raw material are extracted by cellulase-assisted pure water reflux extraction method to obtain the flavonoid extract based on Shiya tea.

[0013] Furthermore, the raw materials of the flavonoid extract based on Shiya tea in the step (1) include, by weight: 16-18 parts of Shiya tea, 42-45 parts of dried leaves of 6-month-old tea leaves, and 32-35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying Shiya tea leaves picked in October.

[0014] Furthermore, the raw materials of the flavonoid extract based on Shiya tea in the step (1) include, by weight: 17-18 parts of Shiya tea, 44-45 parts of dried leaves of 6-month-old tea leaves, and 33-35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying Shiya tea leaves picked in October.

[0015] Furthermore, in the step (2), the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in the step (1) are mixed evenly and crushed to 50-100 meshes to obtain old tea powder.

[0016] Furthermore, in the step (3), the old tea powder is frozen in three stages at -15 to -25°C for a total of 8 to 10 hours, taken out, vacuum dried, and then crushed to 300 to 500 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -20 to -25°C for 4 hours, then freezing at -15 to -18°C for 2 hours, and the remaining time freezing at -22 to -24°C.

[0017] Furthermore, the step (5) specifically includes the following steps:

[0018] 5.1) adding the mixed raw material to a cellulase solution having a cellulase content of 0.18-0.22%, adjusting the pH value of the solution to 3.9-4.1, and performing enzymolysis at 48-52° C. for 55-65 min to obtain a cellulase hydrolyzate;

[0019] 5.2) extracting the cellulase hydrolyzate with pure water reflux for 2 to 3 times at 60 to 80° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0020] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0021] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding ethanol to the extract, stirring sufficiently, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Camellia sinensis.

[0022] The present invention also provides a flavonoid extract based on Camellia oleifera, which is prepared by the aforementioned preparation method.

[0023] The present invention also provides the use of the flavonoid extract based on Camellia oleifera in preparing an in vitro antibacterial composition.

[0024] The present invention also provides the use of the flavonoid extract based on the Herba Lysimachiae in preparing a composition with the function of enhancing immunity.

[0025] The present invention also provides the use of the flavonoid extract based on the shiyala tea in preparing a composition for preventing and treating poultry H9 subtype avian influenza.

[0026] The present invention discloses a flavonoid extract based on yam tea, and a preparation method and application thereof. Compared with the prior art, the present invention has the following beneficial effects: (1) a more efficient and high-yield method for extracting flavonoid extract from yam tea is provided, laying a foundation for the industrial production of high value-added products of yam tea; (2) the problem that the extraction of flavonoid components of yam tea mainly relies on finished tea, resulting in high costs is solved; (3) the problem that flavonoid components of old tea leaves at different periods cannot be effectively utilized is solved; (3) the extracted flavonoid extract from yam tea has a more excellent in vitro antibacterial effect, and also has a stronger effect of enhancing immunity, and the extracted flavonoid extract from yam tea has the effect of preventing and treating poultry H9 subtype avian influenza.

[0027] The invention discloses a flavonoid extract based on Camellia sinensis, and a preparation method and application thereof. The old tea powder is subjected to three-stage freezing at -15 to -25 DEG C, so that the cell wall of the old tea is more broken, the subsequent cellulase hydrolysis effect is better, the pure water reflux extraction effect is better, and the yield of the flavonoid extract is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a high performance liquid chromatogram of the present invention, in which: A: camellia glycoside A reference substance; B: apigenin reference substance; C: test substance; 1 camellia glycoside A; 2 apigenin.

[0029] Figure 2 This is a graph showing the effect of different enzymatic hydrolysate concentrations on the yield of flavonoid components in the present invention.

[0030] Figure 3 This is a graph showing the effect of the pH value of the enzymatic hydrolyzate on the yield of flavonoid components.

[0031] Figure 4 This is a graph showing the effect of the enzymatic hydrolysis temperature on the yield of flavonoid components in the present invention.

[0032] Figure 5This is a graph showing the effect of the enzymatic hydrolysis time on the yield of flavonoid components in the present invention.

[0033] Figure 6 This is a graph showing the effect of the material-liquid ratio on the yield of flavonoid components in the present invention.

[0034] Figure 7 This is a graph showing the effect of the extraction time on the yield of flavonoid components in the present invention. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0036] The present invention discloses a flavonoid extract based on shiya tea and a preparation method and application thereof;

[0037] Test equipment:

[0038] VARIAN 210 high performance liquid chromatograph (Varian, USA); BSA124S 1 / 10,000 electronic analytical balance (Sartorius); SB-5200D ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); ZF-8 dark box four-purpose UV analyzer (Shanghai Jiapeng Technology Co., Ltd.); BIOCEN 22R centrifuge (WIGGENS, Germany); RE-2000 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); conventional reflux extraction device, etc.

[0039] Experimental materials and test drugs:

[0040] Shiya tea: purchased from Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region, identified as the dried leaves of Adinandra nitida Merr. ex HLLi, Theaceae, by Lan Mingsheng, chief pharmacist of Guangxi Medicinal Botanical Garden; apigenin reference substance (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: B1911231, content is 98%); camellia glycoside A reference substance (Chengdu Purifa Technology Development Co., Ltd., number: PRF22112244, content is 98%); cellulase (10000U / g, Shanghai Aladdin Biochemical Technology Co., Ltd.); KBT-ZTC Chinese medicine clarifier (Shanghai Kelier Biotechnology Co., Ltd.); methanol was chromatographic grade, and the other reagents were all analytical grade.

[0041] Front-end experimental methods and results:

[0042] Selection of extraction method:

[0043] At present, the main extraction methods of flavonoids in Shiya tea include heating reflux extraction, ultrasonic extraction, microwave extraction, carbon dioxide supercritical extraction, etc. By referring to the literature method and conducting verification experiments, the total flavonoid yield of different extraction methods is investigated as shown in Table 1.

[0044] Table 1 Comparison of total flavonoids yields from different extraction methods of Shiya tea

[0045] Serial number Extraction method Total flavonoid yield (%) 1 Ethanol reflux extraction 34.35 2 Pure water reflux extraction method 31.62 3 Ultrasonic extraction 28.32 4 Microwave extraction 29.64 5 Continuous countercurrent extraction 30.25 6 Cellulase method 20.98 7 Supercritical Carbon Dioxide Extraction 32.54

[0046] According to the experimental results in Table 1, combined with the actual situation, existing equipment and experimental costs and other factors, it was decided to use cellulase-assisted pure water reflux extraction to extract flavonoids from Shiya tea.

[0047] Preparation of cellulase solution:

[0048] Cellulase was dissolved in a pure water solution of a certain pH (prepared with dilute hydrochloric acid) to prepare 450 ml of solutions of a certain concentration.

[0049] Shiya tea sample processing:

[0050] Weigh 10g of Shiya tea accurately, wash it quickly with pure water in a suitable container for 3 times, stir for 3 minutes after adding pure water each time, filter out the washing liquid with a single-layer filter cloth (filter out fine impurities thoroughly), and filter the medicinal materials dry for later use.

[0051] Preparation of total flavonoids extract from Camellia oleifera:

[0052] Take the washed Shiya tea sample, add a certain amount of cellulase solution of a certain concentration under a certain pH value, stir and mix, place at a certain temperature (monitored and adjusted with a thermometer in an oven) for a certain time, transfer to a round-bottom flask, reflux extract for a certain time, filter while hot on a Buchner funnel with a single-layer filter cloth, and store the filtrate in another container for later use. Add the same amount of pure water to the residue and continue to reflux extract 1 to 2 times, filter while hot in the same way as above, combine the filtrate, add a certain amount of KBT-ZTC Chinese medicine clarifier, let stand for 20 to 30 minutes, concentrate by rotary evaporation, add an appropriate amount of 80% ethanol to the extract, stir thoroughly, let stand and precipitate, collect the precipitate by centrifugation, and dry to obtain the crude extract of total flavonoids.

[0053] Preparation of test solution:

[0054] Take 1 mg of the crude total flavonoids prepared above, transfer it to a 25 ml volumetric flask after accurate weighing, add 20 ml of methanol, seal it, weigh it, ultrasonicate it at 40°C until it is completely dissolved, cool it, weigh it, make up the loss with methanol, shake it well, filter it, filter it with a microporous membrane, and take the filtrate to obtain it.

[0055] Preparation of reference solution:

[0056] Apigenin reference solution: Weigh 1 mg of apigenin reference, accurately weigh, transfer to a 10 mL volumetric flask, dissolve with appropriate amount of methanol by ultrasonication, make up to volume with methanol, shake well, and obtain (concentration is 0.102 mg / ml).

[0057] Camellia glycoside A reference solution: Weigh 0.5 mg of Camellia glycoside A reference, accurately weigh, transfer to a 10 mL volumetric flask, dissolve with an appropriate amount of 70% methanol by ultrasonication, dilute to the mark with 70% methanol, shake well, and the solution is obtained (concentration is 0.058 mg / ml).

[0058] Determination of flavonoid content:

[0059] Chromatographic conditions:

[0060] Chromatographic column: Kromasil 100-5-C18 (250×4.6mm, 5μm); mobile phase: acetonitrile-water (35:65); flow rate: 1.0mL / min; detection wavelength: 270nm; injection volume: 10μL.

[0061] System adaptability:

[0062] Under the above chromatographic conditions, 10 μl of the above camellia glycoside A reference solution, apigenin reference solution, and test solution were accurately pipetted into the HPLC instrument and the spectra were recorded. Figure 1 As shown in the figure, the chromatographic peaks of camelliaside A and apigenin were separated well, indicating that the system has good applicability and good specificity.

[0063] Investigation of linear relationship:

[0064] Accurately measure 1, 2, 3, 4, 5, 6, 7, and 8 mL of the camellia glycoside A and apigenin reference solution, respectively, and place them in different 10 mL volumetric flasks, dilute to the scale with 50% methanol, filter with a microporous filter membrane, and determine according to the above chromatographic conditions. Draw a standard curve with the peak area (Y) as the ordinate and the injection volume (X, μg) as the abscissa, and get the regression equation of camellia glycoside A: 1854693x+38022, (R 2 =0.9998); Apigenin regression equation: Y = Y = 365396X + 112.49 (R 2 =0.9996); indicating that the linear relationship is good when the injection amount of camellia glycoside A is in the range of 0.058μg to 0.464μg and the injection amount of apigenin is in the range of 0.102μg to 0.816μg.

[0065] Precision test:

[0066] The camellia glycoside A and apigenin reference solution were accurately measured, and the samples were continuously injected into the chromatograph for 6 times according to the chromatographic conditions for determination, the chromatogram was recorded, the peak area was determined, and the change of the peak area of ​​baicalin reference was investigated. The results showed that the RSD value of the peak area of ​​camellia glycoside A was 1.02% (n=6), and the RSD value of the peak area of ​​apigenin was 0.97% (n=6), indicating that the precision of the content determination method was good.

[0067] Stability test:

[0068] Another 5g of the same batch of Shiya tea samples were taken, accurately weighed, and prepared into a test stock solution using the test solution preparation method. The samples were injected at 0, 2, 4, 8, 16, and 24 hours under the same chromatographic conditions, and the peak areas were recorded. The results showed that the RSD value of the peak area of ​​camellia glycoside A was 1.65% (n=6), and the RSD value of the peak area of ​​apigenin was 1.17% (n=6), indicating that the test solution had good stability within 24 hours.

[0069] Repeatability test:

[0070] Take another 6 samples of the same batch of cliff tea, 5g each, accurately weighed, and prepared the test sample stock solution according to the above test sample solution preparation method, and determine according to the chromatographic conditions according to the law, inject samples according to the above chromatographic conditions, record the peak area, and calculate the content of camellia glycoside A and apigenin. The RSD values ​​of camellia glycoside A and apigenin content were 0.35% and 0.72% (n=6), respectively, indicating that the repeatability of the content determination method is good and meets the requirements.

[0071] Sample recovery test:

[0072] Take 9 samples of the same batch of cliff tea with known camellia glycoside A and apigenin content (245.6534 mg / g, 9.6552 mg / g), each about 5g, accurately weighed, add camellia glycoside A and apigenin reference substances in a certain proportion, prepare the test solution according to the above method, determine the content of camellia glycoside A and apigenin under the same chromatographic conditions, and calculate the recovery rate. Recovery rate (%) = [measured value (mg) - sample content (mg)] / reference substance addition amount (mg) × 100%. Results The average recovery rate of camellia glycoside A was 99.6%, and the RSD value was 1.11% (n = 9); the average recovery rate of apigenin was 99.7%, and the RSD value was 0.67% (n = 9), indicating that the recovery rate was good. See Table 2-Table 3 for details.

[0073] Table 2 Camellia glycoside A recovery test results (n = 9)

[0074]

[0075] Table 3 Apigenin sample recovery test results (n = 9)

[0076]

[0077]

[0078] Single factor test:

[0079] The two main links that affect the yield of flavonoid components are enzymatic hydrolysis and water extraction. The influencing factors mainly include enzymatic hydrolysis solution concentration, enzymatic hydrolysis solution pH value, enzymatic hydrolysis temperature, enzymatic hydrolysis time, water extraction solid-liquid ratio, and extraction time. A single-factor experiment was designed to examine the influence of each factor.

[0080] Effect of enzyme hydrolysate concentration on the yield of flavonoids:

[0081] According to the above test sample preparation method, 40 times the amount of pure water solution with pH value of 4.0 (prepared with dilute hydrochloric acid) of the medicinal material was used to prepare different concentrations of cellulase solutions (0.05%, 0.1%, 0.2%, 0.3%, g / v). The enzyme was hydrolyzed at 50°C for 1 hour and refluxed for 1 hour to investigate the effect of different enzyme hydrolyzate concentrations on the yield of flavonoid components. The results are as follows Figure 2 shown.

[0082] Effect of pH value of enzymatic hydrolysate on the yield of flavonoid components:

[0083] A 0.2% (g / v) cellulase solution was prepared with 40 times the amount of pure water of the medicinal material with different pH values ​​(3.0, 3.5, 4.0, 4.5). The solution was hydrolyzed at 50°C for 1 hour and refluxed for 1 hour to investigate the effect of different pH values ​​of the hydrolyzate on the yield of flavonoid components. The results are as follows: Figure 3 shown.

[0084] Effect of enzymatic hydrolysis temperature on the yield of flavonoids:

[0085] A 0.2% (g / v) cellulase solution was prepared with 40 times the amount of pure water solution with a pH value of 4.0 of the medicinal material. The solution was enzymatically hydrolyzed for 1 hour at a certain temperature (40°C, 45°C, 50°C, 55°C) and refluxed for 1 hour to investigate the effect of different enzymatic hydrolysis temperatures on the yield of flavonoid components. The results are as follows: Figure 4 shown.

[0086] Effect of enzymatic hydrolysis time on the yield of flavonoids:

[0087] A 0.2% (g / v) cellulase solution was prepared with 40 times the amount of pure water solution with a pH value of 4.0. The solution was hydrolyzed at 50°C for a certain time (0.5h, 1h, 1.5h, 2h), and refluxed for 1h to investigate the effect of different hydrolysis times on the yield of flavonoid components. The results are as follows: Figure 5 shown.

[0088] Effect of solid-liquid ratio on the yield of flavonoids:

[0089] A 0.2% (g / v) cellulase solution was prepared with a certain amount (20, 40, 60, 80 times) of pure water with a pH value of 4.0 using the medicinal materials. The solution was enzymatically hydrolyzed at 50°C for 1 hour and refluxed for 1 hour to investigate the effect of different material-liquid ratios on the yield of flavonoid components. The results are as follows: Figure 6 shown.

[0090] Effect of extraction time on the yield of flavonoids:

[0091] A 0.2% (g / v) cellulase solution was prepared with 40 times the amount of pure water solution with a pH value of 4.0. The solution was hydrolyzed at 50°C for 1 hour and refluxed for a certain period of time (0.5h, 1h, 1.5h, 2h). The effect of different extraction times on the yield of flavonoid components was investigated. The results are as follows: Figure 7 shown.

[0092] Orthogonal test:

[0093] According to the results of the single factor test, combined with the influence of each factor on the flavonoid yield and the magnitude of the inflection point fluctuation, four factors were selected, including solid-liquid ratio (A), enzyme concentration (B), enzymatic hydrolysis temperature (C) and extraction time (D), and three levels were selected for each factor. L9 (3 4 ) Orthogonal table was used for experimental design. The yield of total flavonoids and representative components camelliaside A and apigenin were used as the evaluation indexes to select the best extraction process of flavonoid components. The results are shown in Tables 4 to 7.

[0094] Table 4 Orthogonal test factor level table

[0095] level A B(％) C(℃) D(h) 1 1:40 0.1 40 1 2 1:60 0.2 45 1.5 3 1:80 0.3 50 2

[0096] Table 5L9(3 4 )Orthogonal test plan and results

[0097]

[0098] Table 6 Total flavonoids yield variance analysis table

[0099] Sum of Squares of Deviations Degrees of Freedom Mean Square F A 51.831 2 25.916 19.395* B(error) 2.672 2 1.336 —— C 18.195 2 9.097 6.808 D 3.555 2 1.777 1.330

[0100] Note: F 0.05 (2,2)=19.00, *significant difference

[0101] Table 7 Analysis of variance of the yield of camelliaside A + apigenin

[0102] Sum of Squares of Deviations Degrees of Freedom Mean Square F A 31.929 2 15.964 50.092* B(error) 0.637 2 0.319 —— C 6.382 2 3.191 10.012 D 2.263 2 1.131 3.550

[0103] Note: F 0.05 (2,2)=19.00, *significant difference

[0104] From the range analysis in Table 5, it can be seen that the order of influence of each factor on the flavonoid yield is: A>C>D>B, and the extraction liquid ratio has the greatest influence on the flavonoid yield, and from the variance analysis in Tables 6 and 7, it can be seen that the extraction liquid ratio has a significant effect on the flavonoid yield. Comprehensive analysis shows that the optimal process is A2B2C3D2, that is, using 60 times the amount of medicinal materials and pure water with a pH value of 4.0 to prepare a 0.2% (g / v) cellulase solution, enzymolysis at 50°C for 1 hour, and reflux extraction for 1.5 hours.

[0105] Verification test:

[0106] Weigh 10g of Shiya tea and weigh it accurately. Test according to the optimal process, and conduct parallel tests 3 times. The results show that the yield of total flavonoids and representative components is stable, which further verifies the reliability of the process. See Table 8 for details.

[0107] Table 8 Optimal process verification experiment

[0108]

[0109] About the preparation of crude flavonoid extracts:

[0110] Through research and practical experience, the extraction method of crude flavonoid extract is determined. The present invention adopts enzymatic hydrolysis combined with water extraction and alcohol precipitation for extraction, which is mainly divided into three steps: enzymatic hydrolysis-water extraction-alcohol precipitation. The water extraction method is simple to operate, but the extract has more impurities. The water extract can be preliminarily purified by using a clarifier. According to preliminary experiments, the optimal amount of clarifier is 10 mg per milliliter of extract, and the optimal ethanol concentration for alcohol precipitation is 80%. When performing alcohol precipitation, pay attention to stirring while adding alcohol, and ensure that the precipitation is allowed to stand for enough time before centrifugation, not less than 20 minutes.

[0111] Regarding the choice of enzyme type:

[0112] The enzymes that can hydrolyze plant cell walls mainly include cellulase and pectinase, which are composed of cellulose and pectin respectively. Under normal circumstances, the application of compound enzymes is more destructive to plant cell walls, but because the product obtained after pectinase hydrolyzes pectin has an adsorption effect on flavonoids, it reduces the yield of flavonoids to a certain extent. Therefore, cellulase is the preferred enzyme used.

[0113] Example 1

[0114] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0115] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 15 parts of Shiya tea, 40 parts of dried leaves of 6-month-old tea leaves, and 30 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0116] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0117] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 50 meshes to obtain old tea powder;

[0118] (3) freezing the old tea powder in three stages at -15 to -25°C for a total of 8 hours, taking it out, vacuum drying it, and then crushing it into 300 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -20°C for 4 hours, then freezing at -15°C for 2 hours, and freezing at -22°C for the remaining time;

[0119] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0120] (5) extracting the flavonoid components in the mixed raw material by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on the tea of ​​the morning glory;

[0121] The specific steps include:

[0122] 5.1) adding the mixed raw material into a cellulase solution with a cellulase content of 0.18%, adjusting the pH value of the solution to 3.9, and performing enzymolysis at 48° C. for 55 minutes to obtain a cellulase hydrolyzate;

[0123] 5.2) extracting the cellulase hydrolyzate twice with pure water reflux at 60° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0124] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0125] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0126] Example 2

[0127] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0128] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 18 parts of Shiya tea, 45 parts of dried leaves of 6-month-old tea leaves, and 35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0129] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0130] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 100 meshes to obtain old tea powder;

[0131] (3) freezing the old tea powder in three stages at -15 to -25°C for a total of 10 hours, taking it out, vacuum drying it, and then crushing it into 500 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -25°C for 4 hours, then freezing at -18°C for 2 hours, and freezing at -24°C for the remaining time;

[0132] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0133] (5) extracting the flavonoid components in the mixed raw material by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on the tea of ​​the morning glory;

[0134] The specific steps include:

[0135] 5.1) adding the mixed raw material into a cellulase solution with a cellulase content of 0.22%, adjusting the pH value of the solution to 4.1, and performing enzymolysis at 52° C. for 65 minutes to obtain a cellulase hydrolyzate;

[0136] 5.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 80° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0137] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0138] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0139] Example 3

[0140] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0141] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 16 parts of Shiya tea, 42 parts of dried leaves of 6-month-old tea leaves, and 32 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0142] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0143] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 80 meshes to obtain old tea powder;

[0144] (3) freezing the old tea powder in three stages at -15 to -25°C for a total of 9 hours, taking it out, vacuum drying it, and then crushing it into 400 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -22°C for 4 hours, then freezing at -16°C for 2 hours, and freezing at -23°C for the remaining time;

[0145] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0146] (5) extracting the flavonoid components in the mixed raw material by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on the tea of ​​the morning glory;

[0147] The specific steps include:

[0148] 5.1) adding the mixed raw material into a cellulase solution with a cellulase content of 0.2%, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 minutes to obtain a cellulase hydrolyzate;

[0149] 5.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0150] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0151] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0152] Example 4

[0153] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0154] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 17 parts of Shiya tea, 44 parts of dried leaves of 6-month-old tea leaves, and 33 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0155] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0156] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 80 meshes to obtain old tea powder;

[0157] (3) freezing the old tea powder in three stages at -15 to -25°C for a total of 9 hours, taking it out, vacuum drying it, and then crushing it into 400 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -22°C for 4 hours, then freezing at -16°C for 2 hours, and freezing at -23°C for the remaining time;

[0158] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0159] (5) extracting the flavonoid components in the mixed raw material by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on the tea of ​​the morning glory;

[0160] The specific steps include:

[0161] 5.1) adding the mixed raw material into a cellulase solution with a cellulase content of 0.2%, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 minutes to obtain a cellulase hydrolyzate;

[0162] 5.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0163] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0164] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0165] Example 5

[0166] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0167] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 17 parts of Shiya tea, 44 parts of dried leaves of 6-month-old tea leaves, and 33 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0168] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0169] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 80 meshes to obtain old tea powder;

[0170] (3) freezing the old tea powder at -25°C for 9 hours, taking it out, vacuum drying it, and then grinding it into 400 meshes to obtain old tea fine powder;

[0171] (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material;

[0172] (5) extracting the flavonoid components in the mixed raw material by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on the tea of ​​the morning glory;

[0173] The specific steps include:

[0174] 5.1) adding the mixed raw material into a cellulase solution with a cellulase content of 0.2%, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 minutes to obtain a cellulase hydrolyzate;

[0175] 5.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0176] 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0177] 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0178] Comparative Example 1

[0179] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0180] (1) The raw material of the flavonoid extract based on Shiya tea is Shiya tea;

[0181] (2) extracting the flavonoid components in the Shiya tea by using a cellulase-assisted pure water reflux extraction method to obtain the Shiya tea-based flavonoid extract;

[0182] The specific steps include:

[0183] 2.1) adding the Shiya tea to a cellulase solution with a cellulase content of 0.2%, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 minutes to obtain a cellulase hydrolyzate;

[0184] 2.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0185] 2.3) adding KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, standing and clarifying to obtain a clarified solution;

[0186] 2.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0187] Comparative Example 2

[0188] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0189] (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 44 parts of dried leaves of 6-month-old tea leaves and 33 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying old Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0190] Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio;

[0191] (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly, and crushing them into 80 meshes to obtain old tea powder;

[0192] (3) freezing the old tea powder at -25°C for 9 hours, taking it out, vacuum drying it, and then grinding it into 400 meshes to obtain old tea fine powder;

[0193] (4) extracting the flavonoid components in the old tea powder by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on Shiya tea;

[0194] The specific steps include:

[0195] 4.1) adding the old tea powder to a cellulase solution containing 0.2% cellulase, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 min to obtain a cellulase hydrolyzate;

[0196] 4.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0197] 4.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0198] 4.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0199] Comparative Example 3

[0200] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0201] (1) The raw material of the flavonoid extract based on Shiya tea is the dried leaves of six-month-old tea leaves; the dried leaves of six-month-old tea leaves are obtained by drying the old Shiya tea leaves picked in June;

[0202] (2) crushing the 6-month-old dry tea leaves obtained in step (1) into 80 meshes to obtain old tea powder;

[0203] (3) freezing the old tea powder at -25°C for 9 hours, taking it out, vacuum drying it, and then grinding it into 400 meshes to obtain old tea fine powder;

[0204] (4) extracting the flavonoid components in the old tea powder by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on Shiya tea;

[0205] The specific steps include:

[0206] 4.1) adding the old tea powder to a cellulase solution containing 0.2% cellulase, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 min to obtain a cellulase hydrolyzate;

[0207] 4.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0208] 4.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0209] 4.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0210] Comparative Example 4

[0211] A method for preparing a flavonoid extract based on Camellia sinensis, comprising the following steps:

[0212] (1) The raw material of the flavonoid extract based on Shiya tea is 10-month-old dried tea leaves; the 10-month-old dried tea leaves are obtained by drying old Shiya tea leaves picked in October;

[0213] (2) crushing the 10-month-old dry tea leaves obtained in step (1) into 80 meshes to obtain old tea powder;

[0214] (3) freezing the old tea powder at -25°C for 9 hours, taking it out, vacuum drying it, and then grinding it into 400 meshes to obtain old tea fine powder;

[0215] (4) extracting the flavonoid components in the old tea powder by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on Shiya tea;

[0216] The specific steps include:

[0217] 4.1) adding the old tea powder to a cellulase solution containing 0.2% cellulase, adjusting the pH value of the solution to 4, and performing enzymolysis at 50° C. for 60 min to obtain a cellulase hydrolyzate;

[0218] 4.2) extracting the cellulase hydrolyzate with pure water reflux for 3 times at 70° C., filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining a total filtrate of pure water reflux extraction;

[0219] 4.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution;

[0220] 4.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding alcohol to the extract with an alcohol volume fraction of 80%, stirring thoroughly, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Shiya tea.

[0221] The performance experiment was carried out using the flavonoid extract based on the tea of ​​Shiya prepared in Comparative Example 1. The specific experiment was as follows: (I) Studying the effect of the flavonoid components in the tea of ​​Shiya on the immune function of mice

[0222] 1. Principle of macrophage phagocytosis experiment:

[0223] Phagocytosis is the main way for single-celled animals to absorb nutrients, and it is also a non-specific immune protection mechanism for higher animals. This study used macrophages in the peritoneal cavity of mice to conduct an experiment on the phagocytosis of chicken red blood cells. By observing the phagocytosis of chicken red blood cells by cells, the phagocytic rate and phagocytic index were calculated according to the formula to analyze and judge the body's immune level.

[0224] 2. Experimental instruments and test drugs:

[0225] IFM-880C microscope (Shanghai Tuming Optical Instrument Co., Ltd.), 1% chicken red blood cells (CRBO suspension, Hank's working solution, physiological saline, Giemsa staining solution; sterile syringes, needles, scissors, dissection trays, slides, etc.

[0226] 3. Experimental methods:

[0227] In order to explore and analyze the new uses of flavonoids in Shiya tea, this paper took 60 km mice provided by Guangxi Experimental Animal Center (animal license number: SCXK Gui 2014-0002) for the experiment, half of which were male and half were female, and randomly divided into control group, model group, low-dose, medium-dose, high-dose and treatment group, with 10 mice in each group. Among them, the control group was given 40 mg / kg of cyclophosphamide for injection, and the model group was gavaged with an equal amount of normal saline. The experimental group was gavaged with Shiya tea crude flavonoid solution; the low-dose group was 50 mg / kg.bw.d; the low-medium dose group was 1500 mg / kg.bw.d; the high-dose group was 450 mg / kg.bw.d; and the treatment group was 450 mg / kg.bw.d. Each experimental group was gavaged with Shiya tea flavonoid solution for 15 consecutive days. The grouping and dosing regimen are as shown in the following table.

[0228] Table 9 Grouping and dosing regimen

[0229] Group n Drug dosage by oral gavage Normal control group 10 Normal saline Model Group 10 Normal saline Positive control group 10 <![CDATA[Cyclophosphamide for injection 40mg·kg -1 > High dose group 10 Shiya tea flavonoids solution 50mg / kg.bw.d Medium dose group 10 Shiya tea flavonoids solution 150mg / kg.bw.d Low dose group 10 Shiya tea flavonoids solution 450mg / kg.bw.d

[0230] After the last administration of the test substance, all experimental mice were immunized with 0.5mL to 1mL of 5% chicken red blood cell saline suspension in the peritoneal cavity of each mouse, and the abdomen of the mouse was gently rubbed with hands after immunization. Between 2h and 10h after immunization, the mouse was killed, and 2.5mL of Hank's working solution was injected into the peritoneal cavity of the mouse. When the abdomen of the mouse was gently rubbed, the injected Hank's working solution would fully rub the macrophages in the peritoneal cavity of the mouse.

[0231] A small incision was made in the center of the mouse's abdomen. The mouse's peritoneal lavage fluid was sucked with a pipette and then dropped onto a glass slide. Each experimental mouse was dripped with about 2 to 3 glass slides. A glass slide was placed in a white disk that was pre-covered with multiple layers of gauze and soaked and flattened with saline, and then the white disk was placed in an incubator at 37°C for 30 minutes of incubation. After the incubation time, the unphagocytic chicken red blood cells and suspended white membranes in the white slide were rinsed off with saline, and then the white disk was dried. The dried white disk was placed in a staining jar filled with methanol for 5 minutes to fix it, and then the white disk was taken out and blown dry with cold air. Then the Giemsa staining solution was used for 5 minutes to 10 minutes of staining. After the staining time was reached, the staining solution was rinsed with distilled water and the white disk was blown dry. The number of chicken red blood cells phagocytosed and engulfed by 100 to 200 macrophages was counted and recorded by observing with an oil immersion lens under a microscope. At the same time, the phagocytic rate and phagocytic index were calculated according to the following formula.

[0232] Phagocytic rate (%) = number of macrophages engulfing chicken red blood cells / 100 macrophages × 100.0%; phagocytic index = number of macrophages engulfing chicken red blood cells / 100 macrophages × 100.0%.

[0233] 4. Effects on phagocyte function

[0234] The effect of flavonoids in Shiya tea on phagocytic function is shown in Table 9. After using different doses of Shiya tea flavonoid extracts, the phagocytic percentage and phagocytic index of mouse peritoneal macrophages were significantly enhanced compared with the control group; and the phagocytic percentage and phagocytic index of the treatment group were higher than those of the control group. It can be seen that after Shiya tea flavonoid extracts were continuously administered at doses of 150 mg / kg and 450 mg / kg for 15 days, the phagocytic function of mouse macrophages was also enhanced, which was conducive to the simultaneous improvement of the nonspecific immune function of mice.

[0235] Table 10 Effects of flavonoids in Shiya tea on cellular phagocytic function

[0236]

[0237] Note: # P < 0.05; ## P < 0.01 vs control group; ** P<0.01VS model group.

[0238] 5. Effect on the weight of immune organs

[0239] As the body ages, its immune organs, such as the thymus and spleen, gradually degenerate and atrophy. The use of more immunosuppressants may have a certain impact on the immune organs of humans or animals, resulting in a decrease in organ weight or decreased function. As shown in Table 10, the use of flavonoid extracts from Shiya tea helps to improve the spleen index of mice. Compared with the control group, there is a significant gap between the medium-dose and high-dose groups, and the effect is proportional to the dosage. The spleen index of mice in the treatment group is also significantly higher than that in the model group. Combined with the above, it can be seen that the application of flavonoid extracts from Shiya tea has played a positive role in delaying the decline of immune organs, and the ability to prevent and resist diseases will also be strengthened.

[0240] Table 11 Effects of flavonoids in Shiya tea on the weight of immune organs

[0241]

[0242] Note: # P < 0.05; ## P < 0.01 vs control group; * P < 0.05; ** P<0.01VS model group.

[0243] (II) Study on the in vitro antibacterial effect of flavonoids of Shiya tea on common clinical bacteria

[0244] 1. Principle of in vitro antibacterial experiment of bacteria:

[0245] Based on the effect of compounds or substances on the growth and reproduction process of bacteria, by preparing solutions of compounds or substances of different concentrations, and observing the effect of solutions of compounds or substances of different concentrations on bacterial growth on culture medium, the antibacterial effect can be determined.

[0246] 2. Experimental instruments and test drugs:

[0247] Experimental instruments: JBQ-ZD8 type gold temperature oscillator (Jintan Jingbo Experimental Instrument Factory), YXQ-LS-100SⅡ type vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), SW-CJ-2FD type clean workbench (Suzhou Antai Air Technology Co., Ltd.), BSA124S type 1 / 10,000 electronic analytical balance (Sartorius Company), MX-S type mixer (Shanghai Keya Biotechnology Co., Ltd.), Tiangen 05E type 100℃ constant temperature metal bath (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0248] The tested strains: Staphylococcus aureus [CMCC(B)26003], Pseudomonas aeruginosa [CMCC(B)10104], Escherichia coli [CMCC(B)44102], and Candida albicans [CMCC(F)98001] were all purchased from China Food and Drug Inspection Institutes.

[0249] Drugs and reagents: Shiya tea (Guangxi Jinxiu Yao Autonomous County Dayaoshan Natural Plant Development Co., Ltd.), agar powder (Sangon Biotech), Sabouraud dextrose agar medium SDA (Beijing Luqiao Technology Co., Ltd.), tryptic soy peptone agar medium TSA (GCP168B, Beijing Luqiao Technology Co., Ltd.), compound Huangsong wash (201607062, Guangxi Yuanantang Pharmaceutical Co., Ltd.)

[0250] 3. Experimental methods:

[0251] (1) Preparation of drug test sample: Weigh 150 g of Shiya tea and prepare the total flavonoid extract according to the Shiya tea total flavonoid extraction process. Add appropriate amount of water to the extract, heat in a water bath to suspend and disperse, grind the concentrate with 3 mL of Tween 80 and 20 g of calamine, add pure water to make 150 ml, seal, and place in a vertical pressure steam sterilizer at 121°C for 15 min for use.

[0252] (2) Sterilization of culture medium: Weigh 4 g of TSA and 6.5 g of SDA respectively and dissolve them in 100 ml of distilled water. Then sterilize them in a vertical pressure steam sterilizer at 121°C for 15 min.

[0253] The following operations are all carried out in a clean workbench, and a sterile environment is guaranteed at all times during the operation.

[0254] (3) Preparation of test bacterial solution: Take 20 ml of each of overnight grown Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, add them to 980 ml of pure water, mix well, then take 20 ml of the solution and add it to 1980 ml of pure water, mix well, and dilute to 1×10 6 cfu / ml, for future use.

[0255] (4) Plate preparation: Using the pouring method, first add 1 ml of the four bacterial solutions into eight plates, then pour 50°C SDA culture medium into the plate containing Candida albicans, and add 50°C TSA culture medium to the rest. Gently shake to mix and wait for the plate to solidify.

[0256] (5) Punching: Punch small holes at equal distances on the plate and inject 50 μl of the test solution into each well. Repeat for one plate and transfer the plate to an incubator and culture at 36 ± 1°C overnight.

[0257] (6) Measurement record: Observe whether a transparent “inhibition zone” appears around the punch hole, measure the diameter of the inhibition zone with a vernier caliper and make a record.

[0258] 4. Experimental results

[0259] The experimental results are shown in Table 12:

[0260] Table 12 Comparison of the antibacterial effects of Shiya Tea Skin Treasure on different bacteria

[0261]

[0262] According to the pharmacological method, the diameter of the inhibition zone d < 10 mm indicates that the bacteria are resistant to the drug and have no antibacterial effect; d = 10 mm indicates mild sensitivity; 11 mm ≤ d ≤ 15 mm indicates moderate sensitivity; d ≥ 16 mm indicates high sensitivity;

[0263] 5. Experimental conclusion

[0264] Through experiments, it was found that the total flavonoids of the stone cliff tea (Dayaoshan Natural Plant Development Co., Ltd., Jinxiu Yao Autonomous County, Guangxi) in comparative example 1 were highly sensitive to Staphylococcus aureus and Pseudomonas aeruginosa; moderately sensitive to Escherichia coli; and had no inhibitory effect on white myxococci.

[0265] The statistical results of various parameters of the flavonoid extracts based on Camellia sinensis prepared in the above Examples 1-5 and Comparative Examples 1-4 are shown in Table 13 below;

[0266] Table 13 Statistical results of various parameters of flavonoid extracts from Shiya tea

[0267]

[0268] Comparative Example 1 The total flavonoids of stone cliff tea are highly sensitive to Staphylococcus aureus and Pseudomonas aeruginosa; moderately sensitive to Escherichia coli; and have no inhibitory effect on white myxococcus; The flavonoid extracts based on stone cliff tea prepared in Examples 1-5 of the present invention are highly sensitive to Staphylococcus aureus and Pseudomonas aeruginosa; highly sensitive to Escherichia coli; and moderately sensitive to white myxococcus; It can be seen that the stone cliff tea flavonoid extracts extracted in Examples 1-5 of the present invention have better in vitro antibacterial effects.

[0269] Comparative Example 1 High-dose group of total flavonoids from Shiya tea (sample dose 450 mg / kg): Cell phagocytosis rate (%) was 74.7±4.5 ## , phagocytic index (%) was 80.4±3.2 ## , spleen index (mg / 10g) was 46.7±3.5 ## ; When the sample dosage of the flavonoid extract based on Shiya tea prepared in Examples 1-5 of the present invention is 350 mg / kg, its effect is equivalent to that of the high-dose group of total flavonoids from Shiya tea in Comparative Example 1; when the sample dosage of the flavonoid extract based on Shiya tea prepared in Examples 1-5 of the present invention is 450 mg / kg, its effect is better; it can be seen that the flavonoid extract of Shiya tea extracted in Examples 1-5 of the present invention has a more excellent function of enhancing immunity.

[0270] It can be seen from this that the flavonoid extract based on Shiya tea and its preparation method and application of the present invention provide a more efficient and high-yield extraction method of Shiya tea flavonoid extract, laying the foundation for the industrial production of high value-added products of Shiya tea; solve the problem that the extraction of flavonoid components of Shiya tea mainly relies on finished tea, resulting in high costs; solve the problem that the flavonoid components of old tea leaves at different periods cannot be effectively utilized; the extracted Shiya tea flavonoid extract has a more excellent in vitro antibacterial effect, and also has a stronger effect of enhancing immunity, and the extracted Shiya tea flavonoid extract has the effect of preventing and treating poultry H9 subtype avian influenza. The old tea powder is frozen in three stages at -15 to -25°C, so that the cell wall of the old tea leaves is more broken, the subsequent cellulase enzymolysis effect is better, the pure water reflux extraction effect is better, and the yield of flavonoid extract is higher.

[0271] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for preparing a flavonoid extract based on Camellia sinensis, characterized in that: The following steps are involved: (1) The raw materials of the flavonoid extract based on Shiya tea include, by weight: 15-18 parts of Shiya tea, 40-45 parts of 6-month-old dried tea leaves, and 30-35 parts of 10-month-old dried tea leaves; The 6-month old tea leaves are obtained by drying old tea leaves of Shiya tea picked in June; the 10-month old tea leaves are obtained by drying old tea leaves of Shiya tea picked in October; Weigh the raw materials for preparing the flavonoid extract based on Camellia sinensis according to the weight ratio; (2) mixing the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in step (1) evenly and crushing them to obtain old tea powder; (3) freezing the old tea powder in three stages at -15 to -25°C for a total of 8 to 10 hours, taking it out, vacuum drying it, and then crushing it into 300 to 500 meshes to obtain old tea fine powder; the three-stage freezing is specifically: first freezing at -20 to -25°C for 4 hours, then freezing at -15 to -18°C for 2 hours, and freezing at -22 to -24°C for the remaining time; (4) mixing the Shiya tea and the old tea powder obtained in step (1) evenly to obtain a mixed raw material; (5) The flavonoid components in the mixed raw material are extracted by cellulase-assisted pure water reflux extraction to obtain the flavonoid extract based on Shiya tea.

2. The method for preparing a flavonoid extract based on Camellia sinensis according to claim 1, characterized in that: The raw materials of the flavonoid extract based on Shiya tea in step (1) include, by weight: 16-18 parts of Shiya tea, 42-45 parts of dried leaves of 6-month-old tea leaves, and 32-35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying Shiya tea leaves picked in October.

3. The method for preparing a flavonoid extract based on Camellia sinensis according to claim 1, characterized in that: The raw materials of the flavonoid extract based on Shiya tea in the step (1) include, by weight: 17-18 parts of Shiya tea, 44-45 parts of dried leaves of 6-month-old tea leaves, and 33-35 parts of dried leaves of 10-month-old tea leaves; the dried leaves of 6-month-old tea leaves are obtained by drying Shiya tea leaves picked in June; the dried leaves of 10-month-old tea leaves are obtained by drying Shiya tea leaves picked in October.

4. The method for preparing a flavonoid extract based on Camellia sinensis according to claim 1, characterized in that: In the step (2), the 6-month-old dry tea leaves and the 10-month-old dry tea leaves obtained in the step (1) are mixed evenly and crushed into 50-100 meshes to obtain old tea powder.

5. The method for preparing a flavonoid extract based on Camellia sinensis according to claim 1, characterized in that: The step (5) specifically includes the following steps: 5.1) adding the mixed raw material to a cellulase solution having a cellulase content of 0.18-0.22%, adjusting the pH value of the solution to 3.9-4.1, performing enzymolysis at 48-52°C for 55-65 minutes, and obtaining a cellulase hydrolyzate; 5.2) extracting the cellulase hydrolyzate with pure water reflux for 2-3 times at 60-80°C, filtering after each extraction with pure water reflux, taking the filtrate, combining the filtrates, and obtaining the total filtrate of pure water reflux extraction; 5.3) Add KBT-ZTC Chinese medicine clarifier to the total filtrate of the pure water reflux extraction, let it stand for clarification, and obtain a clarified solution; 5.4) Concentrating the clarified liquid by rotary evaporation to obtain an extract; adding ethanol to the extract, stirring sufficiently, standing to precipitate, collecting the precipitate by centrifugation, and drying to obtain the flavonoid extract based on Camellia sinensis.

6. A flavonoid extract based on Shiya tea, characterized in that: The flavonoid extract based on Camellia oleifera is prepared by the preparation method described in any one of claims 1 to 5.

7. Use of the flavonoid extract based on Camellia oleifera according to claim 6 in the preparation of a composition having the function of enhancing immunity.

8. Use of the flavonoid extract based on Camellia oleifera according to claim 1 in the preparation of an in vitro antibacterial composition.

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