Camellia pollen total flavone extract as well as preparation method and application thereof

Abstract

The invention discloses a camellia pollen total flavone extract as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the method, the optimal process for ultrasonically extracting the total flavonoids of the camellia pollen is explored, the influence of three single factors, namely the ethanol concentration, the ultrasonic time and the liquid-solid ratio, on the extraction rate of the total flavonoids of the camellia pollen is explored, and finally the three optimal single factors are selected. On the basis, through design of Design-Expert software, extraction conditions are optimized through a response surface method, and the optimal process is obtained as follows: the ethanol concentration is 73%, the ultrasonic time is 31 min, and the liquid-solid ratio is 31: 1 (mL.g <-1 >). Meanwhile, the extract is used for blood fat reduction research, and experiments prove that the camellia pollen total flavonoids have the drug effects of reducing the fat rate and improving hyperlipidemia. The method lays a foundation for extraction, separation and pharmacological action research of the camellia pollen total flavonoids.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a total flavone extract of tea pollen, a preparation method and application thereof. BACKGROUND

[0002] Hyperlipidemia is a pathological state caused by fat metabolism disorder, and its incidence rate increases year by year. It is a main inducing factor of atherosclerosis, fatty liver and other cardiovascular diseases, and seriously affects the quality of life of patients. Hyperlipidemia is mainly manifested by high content of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and / or low content of high-density lipoprotein cholesterol (HDL-C) in blood. The main methods for reducing blood lipids are statins, fibrates, niacin drugs and cholesterol absorption inhibitors, but they can cause adverse reactions such as abnormal liver and kidney function, muscle pain, nerve damage, diabetes and digestive tract reactions.

[0003] Plant extracts have obvious hypolipidemic activity, and screening of highly effective and low-toxicity anti-lipid drugs from traditional Chinese medicines has gradually become a research hotspot. Tea flower bee pollen is a yellow to brownish yellow flat round mass with a diameter of about 2-4 mm, which is gathered by worker bees of Apis cerana Fabricius and other bees and added with a small amount of glandular secretions. It has a slight odor and a slightly sweet taste. It has the effects of benefiting qi and consolidating the body and is used for treating fatigue, insomnia, post-illness weakness, and chronic liver disease with the above symptoms.

[0004] As a major tea planting country, tea flower bee pollen has a large output, high nutritional value, and activities of antioxidant, immune enhancement, and blood sugar reduction, and is favored by consumers, and has a broad market prospect. Tea flower bee pollen is rich in flavonoids, polyphenols, coumarins, triterpene alcohols, vitamins, trace elements and other substances, and is called a "concentrated nutrient bank" and a "complete nutritional food". The flavonoids and their biological activities are the research hotspots.

[0005] Bee pollen has a certain hypolipidemic effect, but the hypolipidemic effect of tea pollen has not been studied. Ma Fumin's research shows that ultrasonic wall breaking treatment is beneficial to the release of nutritional ingredients in tea pollen, but the dissolution amount of total flavones is low after only wall breaking treatment. SUMMARY

[0006] The purpose of the present application is to provide a total flavone extract of tea pollen, a preparation method and application thereof, so as to solve the problems existing in the prior art. The total flavone extract of tea pollen prepared by the present application has high total flavone content and certain hypolipidemic effect.

[0007] To achieve the above purpose, the present application provides the following solutions.

[0008] In a first aspect, the present application provides a preparation method of Camellia nitidissima H.T.Chang total flavonoids extract, which comprises: taking Camellia nitidissima H.T.Chang powder, and then ultrasonic treating the powder under the conditions of 60-80% ethanol concentration, 20-40 mL:0.5-1.5 g liquid-solid ratio, and 50-150 W ultrasonic power for 10-50 min to obtain Camellia nitidissima H.T.Chang total flavonoids extract.

[0009] Preferably, the ethanol concentration is 73%.

[0010] Preferably, the liquid-solid ratio is 31 mL:1 g.

[0011] Preferably, the ultrasonic power is 100 W.

[0012] Preferably, the ultrasonic time is 31 min.

[0013] In a second aspect, the present application also provides Camellia nitidissima H.T.Chang total flavonoids extract prepared according to the preparation method.

[0014] In a third aspect, the present application also provides a hypolipidemic composition comprising the Camellia nitidissima H.T.Chang total flavonoids extract and a pharmaceutically acceptable carrier or excipient.

[0015] In a fourth aspect, the present application also provides use of the Camellia nitidissima H.T.Chang flavonoids extract or the hypolipidemic composition in the preparation of a hypolipidemic product.

[0016] Preferably, the hypolipidemic product is a hypolipidemic drug.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0018] The present application discloses the following technical effects:

[0019] The Camellia nitidissima H.T.Chang total flavonoids extract product prepared by the present application has high total flavonoids content and certain hypolipidemic effect. Experiments prove that Camellia nitidissima H.T.Chang total flavonoids has the pharmacodynamic effect of reducing fat rate and improving hyperlipidemia. The present application lays a foundation for the extraction, separation and pharmacological effect research of Camellia nitidissima H.T.Chang total flavonoids. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 Rutin standard curve;

[0022] Figure 2 The effect of ethanol concentration on the extraction rate of total flavonoids from camellia pollen;

[0023] Figure 3 The effect of ultrasonic time on the extraction rate of total flavonoids from camellia pollen;

[0024] Figure 4 The effect of liquid-solid ratio on the extraction rate of total flavonoids from camellia pollen;

[0025] Figure 5 The effects of ethanol concentration and ultrasonic time on the extraction rate of total flavonoids from camellia pollen;

[0026] Figure 6 The effect of the interaction between ethanol concentration and liquid-solid ratio on the extraction rate of total flavonoids from camellia pollen;

[0027] Figure 7 The effect of the interaction between ultrasonic time and liquid-solid ratio on the extraction rate of total flavonoids from camellia pollen;

[0028] Figure 8 is the effect of CPFs on the body weight of mice; n = 6;

[0029] Figure 9 Effects of CPFs on fat percentage in mice; n = 3, *p < 0.05, **p < 0.01;

[0030] Figure 10 The effects of CPFs on serum biochemical indicators of mice; A is the effect of CPFs on serum TG content of mice, B is the effect of CPFs on serum TC content of mice, C is the effect of CPFs on serum HDL-C content of mice, and D is the effect of CPFs on serum LDL-C content of mice; ABCD: n = 6, compared with the blank group, ### p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001;

[0031] Figure 11 Effects of CPFs on aortic plaques in mice; A is the result of macroscopic oil red staining, B is the aortic plaque area in different treatment groups; AB: n=3, **p<0.01, ***p<0.001;

[0032] Figure 12 Effects of CPFs on HE staining and Masson staining of mouse aorta (10×); A: HE staining; B: Masson staining; C: collagen volume fraction; n=3, **p<0.01. DETAILED DESCRIPTION

[0033] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The term "exemplary" used throughout this description means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other examples.

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "including," "comprising," "having," "containing," and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless otherwise indicated, the use of the terms "a" and "an" are meant to encompass the plural as well as the singular. The use of the term "comprising" is meant to encompass the terms "including," "containing," "having," and the like. The use of the term "about" is meant to encompass the term "comprising" and variations thereof. The use of the term "comprising" is meant to encompass the terms "consisting essentially of" and "consisting of." The use of the term "consisting essentially of" is meant to encompass the term "consisting of."

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0036] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative only.

[0037] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0038] Example 1 Preparation of Camellia sinensis L. powder flavonoid extract

[0039] The instruments and equipment are shown in Table 1 and the materials and reagents are shown in Table 2.

[0040] Table 1 Instruments and equipment

[0041]

[0042]

[0043] Table 2 Materials and reagents

[0044]

[0045] Camellia sinensis L. powder pretreatment: The Camellia sinensis L. powder was crushed and passed through an 80 mesh sieve, dried at 60°C and stored for use.

[0046] 1. Preparation of standard curve

[0047] 1.1 Preparation of rutin standard solution

[0048] Accurately weigh 0.0101 g of rutin standard, dissolve in 65% ethanol solution, then transfer to a 50 mL volumetric flask, and dilute to the mark with 65% ethanol solution. The concentration of the rutin standard solution is 0.2020 mg·mL -1 .

[0049] 1.2 Determination of maximum absorption wavelength

[0050] Accurately take 5.00 mL of rutin standard solution and place it in a 10 mL volumetric flask. Accurately add 0.3 mL of 5% sodium nitrite solution, shake well, and let stand at room temperature for 6 min. After the timing is complete, add 0.3 mL of 10% aluminum nitrate solution, shake well, and let stand at room temperature for 6 min. After the timing is complete, add 4.0 mL of 4% sodium hydroxide solution, shake well, and let stand at room temperature for 15 min. After the timing is complete, add 65% ethanol solution to the mark. Take another 10 mL volumetric flask and add the above color reagents in order, then add 65% ethanol solution to the mark as a blank. Scan the UV wavelength range from 200 to 600 nm to obtain the required maximum wavelength:

[0051] 1.3 Drawing of standard curve

[0052] Refer to the standard curve drawing method of total flavonoids in the reference "Study on the Hypolipidemic and Hepatoprotective Effects of Bupleurum and Camellia Powder on Hyperlipidemic Model Rats" (Wang Zhe, Liang Lin, He Hongyuan, et al. Journal of Shandong University of Traditional Chinese Medicine, 2015, 39(06): 557.) and make modifications.

[0053] Use NaNO2-Al(NO3)3-NaOH colorimetric method to determine the absorbance. Use a pipette to accurately transfer 0.00, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, and 4.00 mL of rutin standard solution into 10 10 mL volumetric flasks. Add NaNO2 solution, Al(NO3)3 solution, and NaOH solution as color reagents. After color development using the above method, measure the absorbance at the maximum wavelength of 507 nm.

[0054] Take the concentration of rutin (mg·mL -1 ) as the abscissa and the measured absorbance value (Abs) as the ordinate to draw the standard curve as Figure 1 , and the standard curve equation is: Y = 13.003x - 0.002 (R 2= 0.9992) with a linear range of 0.00505-0.0808 (mg·mL -1 ).

[0055] 1.4 Determination of the extraction rate of total flavonoids from tea flower powder

[0056] Accurately weigh about 0.5 g of tea flower powder, extract with a fixed ethanol concentration, ultrasonic time and liquid-solid ratio to prepare the test solution. Accurately transfer 2.0 mL of the test solution into a 10 mL volumetric flask, add 0.3 mL of 5% sodium nitrite solution, shake well and stand at room temperature for 6 min; add 0.3 mL of 10% aluminum nitrate solution, shake well and stand at room temperature for 6 min; add 4.0 mL of 4% sodium hydroxide solution, shake well and stand at room temperature for 15 min. Replace the test solution with 70% ethanol solution, add the color developing agent in the same way as the blank control. Measure the absorbance value at 507 nm. Substitute the absorbance value into the rutin standard curve equation to calculate the concentration of total flavonoids, and then calculate the extraction rate using equation (1) as follows.

[0057] W = (C x V x D) / M Equation (1)

[0058] Appendix: In the above equation (1), W is the extraction rate of total flavonoids from tea flower powder; C is the concentration of total flavonoids, mg·mL -1 ; V is the volume of the extraction liquid, mL; D is the dilution factor; and M is the amount of tea flower powder, g.

[0059] 2. Single factor experiment

[0060] 2.1 Effect of ethanol concentration on the extraction rate of total flavonoids from tea flower powder

[0061] Weigh 0.5 g of tea flower powder and add it to a 25 mL volumetric flask. The liquid-solid ratio is 30:1 (mL·g -1 ), the ultrasonic time is 30 min, the ultrasonic power is 100 W, and the extraction is carried out using ethanol solutions with a volume fraction of 40%, 50%, 60%, 70% and 80%, respectively. After the extraction is completed, place the sample in cold water (4 min) to reduce the temperature to room temperature. Dilute with the corresponding ethanol, take 2 mL, and place it in a 10 mL volumetric flask. Add the color developing agent, develop the color, dilute to 10 mL, and measure the absorbance.

[0062] As can be seen from Figure 2 , as the ethanol concentration increases, the extraction rate of total flavonoids from tea flower powder also increases, and when the ethanol concentration reaches 70%, the extraction rate reaches the highest value. Therefore, an ethanol concentration of 70% is selected for investigation. When the ethanol concentration reaches 70% or above, the extraction rate of flavonoids from tea flower powder decreases, which may be due to the fact that as the ethanol concentration increases, the extraction rate of flavonoids decreases, and the extraction rate of other lipophilic compounds increases, thereby reducing the total flavonoid extraction rate.

[0063] 2.2 Effect of ultrasonic time on the extraction rate of total flavonoids in tea flower powder

[0064] Take tea flower powder 0.5 g, add 25 mL volumetric flask, ethanol concentration is 70%, liquid-solid ratio is 30:1 (mL·g -1 ), ultrasonic power is 100 W, investigate the difference of tea flower powder total flavonoids extraction rate under 10 min, 20 min, 30 min, 40 min, 50 min ultrasonic time. After extraction, put into cold water (4 min) to room temperature. After cooling, use 70% ethanol solution to constant volume to the calibration line, remove 2 mL, put into 10 mL volumetric flask, add color developing agent, constant volume to 10 mL after color development, measure absorbance.

[0065] As Figure 3 can be seen, the ultrasonic time from 10 min to 30 min, the tea flower powder total flavonoids extraction rate shows an upward trend, and the ultrasonic time from 30 min to 40 min, the tea flower powder total flavonoids extraction rate shows a downward trend. Therefore, choose ultrasonic time 30 min for investigation. When the ultrasonic time exceeds 30 min, the tea flower powder total flavonoids extraction rate begins to decrease, which may be because the ultrasonic time is too long, which destroys the structure of flavonoids, resulting in the decrease of tea flower powder total flavonoids extraction rate.

[0066] 2.3 Effect of liquid-solid ratio on the extraction rate of total flavonoids in tea flower powder

[0067] Take tea flower powder 0.5 g, add 25 mL volumetric flask, ultrasonic power is 100 W, ethanol concentration is 70%, ultrasonic time is 30 min, liquid-solid ratio is 10:1, 20:1, 30:1, 40:1, 50:1 (mL·g -1 ) extraction. After extraction, put into cold water (4 min) to room temperature. After cooling, use 70% ethanol solution to constant volume to the calibration line, remove 2 mL, put into 10 mL volumetric flask, add color developing agent, constant volume to 10 mL after color development, measure absorbance.

[0068] From Figure 4 can be seen, the liquid-solid ratio is 40:1, the tea flower powder total flavonoids extraction rate is the largest, the tea flower powder total flavonoids extraction rate under 10:1, 20:1, 30:1 and 50:1 conditions is low, therefore choose 40:1 liquid-solid ratio as the investigation condition. When the liquid-solid ratio is less than 40:1, the tea flower powder total flavonoids extraction rate is low, which may be because when the liquid-solid ratio is 10:1, 20:1, 30:1, the contact area of ethanol and tea flower powder is small, and flavonoids cannot be fully extracted; when the liquid-solid ratio increases, flavonoids extraction tends to be saturated, and other alcohol compounds are extracted, resulting in the decrease of tea flower powder total flavonoids extraction rate.

[0069] 3. Response surface optimization of extraction process

[0070] 3.1 Response surface experiment results and analysis

[0071] Based on the single factor experiment results, with three independent variables: ethanol concentration (A), ultrasonic time (B), liquid-solid ratio (C), and the response value: total flavonoids extraction rate of tea flower powder (Y), the Design-Expert software was used to perform a three-factor and three-level Box-Behnken design to optimize the extraction process of total flavonoids of tea flower powder. The factor levels are shown in Table 3, and the experimental scheme and results are shown in Table 4:

[0072] Table 3 Factor levels

[0073]

[0074] Table 4 Response surface experiment scheme and results

[0075]

[0076] 3.2 Regression equation fitting and variance analysis

[0077] Analyzing the data in Table 4, the regression equation between the total flavonoids extraction rate of tea flower powder (Y) and the three independent variables: ethanol concentration (A), ultrasonic time (B), and liquid-solid ratio (C) is obtained as follows:

[0078] Y = 9.64 + 0.5895xA - 0.1034xB - 0.2261xC + 0.3322xAB + 0.2888xAC + 0.2310xBC - 1.09xA 2 - 1.38xB 2 + 0.0604xC 2 , R 2 = 0.9963 Equation (2);

[0079] The results are shown in Table 5. As can be seen from the table, in this model, the P value is less than 0.0001, indicating that the model is significant, and the lack of fit value is 0.4141, which is greater than 0.05, indicating that the model is not significant, which means that this method is reliable and the equation fitting is good, and can be used to optimize the extraction process of total flavonoids of tea flower powder. The P values of the first-order term A and the quadratic term A 2 and B 2 are less than 0.0001, indicating that these three terms have a very significant effect on the extraction rate of total flavonoids of tea flower powder. The P values of the first-order terms B and C and the interaction terms AB, AC, and BC are less than 0.05, indicating that these five terms have a significant effect on the results, while the quadratic term C 2 has no significant effect on the results. According to the F value, the influence of the three factors on the extraction rate of total flavonoids of tea flower powder is as follows: ethanol concentration (%) > liquid-solid ratio (mL·g -1 ) > ultrasonic time (min).

[0080] Table 5 Analysis of variance of regression model

[0081]

[0082] Note: **P value < 0.01 means extremely significant, *P value < 0.05 means significant.

[0083] 3.3 Interaction results of each factor

[0084] Design-Expert software can draw the interaction between ethanol concentration, ultrasonic time and liquid-solid ratio as Figure 5 , Figure 6 , Figure 7 3D graph can more intuitively reflect the influence of the interaction of three single factors on the total flavonoids extraction rate of tea flower powder. Through the analysis of the surface graph and contour graph of the response surface, it can be known that the 3D graph of A (ethanol concentration) and B (ultrasonic time) response surface is steep, and the contour is elliptical, which shows that the interaction of ethanol concentration and ultrasonic time is obvious, and has a great influence on the total flavonoids extraction rate of tea flower powder. In the response surface graph of A (ethanol concentration) and C (liquid-solid ratio) and the response surface graph of B (ultrasonic time) and C (liquid-solid ratio), the slope of the surface along the C (liquid-solid ratio) direction is gentle, which shows that the influence of C (liquid-solid ratio) on the total flavonoids extraction rate of tea flower powder is not as significant as that of ethanol concentration and ultrasonic time on the total flavonoids extraction rate of tea flower powder, which is consistent with the analysis in Table 5.

[0085] 3.4 Optimal extraction process and verification experiment

[0086] The optimal extraction process predicted by Design-Expert software is: ethanol concentration is 73.316%, ultrasonic time is 30.826 min, liquid-solid ratio is 30.906:1 (mL·g -1 ). According to the actual situation, the predicted process conditions are modified: ethanol concentration is 73%, ultrasonic time is 31 min, liquid-solid ratio is 31:1 (mL·g -1 ). And under this optimal condition, three groups of parallel verification experiments are repeated, and the average extraction rate of total flavonoids of tea flower powder is 9.666%, which is close to the predicted value 9.810%, and the deviation from the response surface experiment prediction result is 0.144%, which shows that the extraction process can be used for the extraction of total flavonoids of tea flower powder.

[0087] Example 2 Study on hypolipidemic effect of tea flower powder flavonoids extract

[0088] 1. Materials

[0089] 1.1 Animals

[0090] SPF C57BL / 6J mice 6 (as Control group), ApoE- / - Mice 18 (divided into 3 groups), 6 weeks old, purchased from Beijing Vito Leehua Experimental Animal Technology Co., Ltd., production license number SYXK(E)2019-0031. Indoor environment: 23±2℃, relative humidity: 55±5%, 12h alternating daylight illumination. The animal experiment was approved by the Experimental Animal Ethics Committee of Hubei Medical College.

[0091] 1.2 Instruments and reagents

[0092] Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) detection kit, glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST) detection kit, Nanjing Jiancheng Biological Engineering Institute; modified Masson three-color staining kit, Beijing Solaybao; modified oil red O staining kit, hematoxylin-eosin (HE) staining kit and Trizol reagent, Shanghai Biyun Tian Biotechnology Co., Ltd.; chloroform, National Pharmaceutical Group Chemical Reagent Co., Ltd.; Plus All-in-one 1st Strand cDNA Synthesis SuperMix kit, SYBR qPCR SuperMix Plus kit, Suzhou Jinan Protein Technology Co., Ltd.; primers were synthesized by Chengke Biological Technology Co., Ltd. Centrifuge, Eppendorf; gradient PCR instrument and fluorescent quantitative PCR instrument, BIO RAD.

[0093] 2. Animal grouping and administration

[0094] 18 ApoE - / - The mice were randomly divided into model group (Model), CPFs low-dose group, CPFs high-dose group (CPFs are total flavonoids extract of camellia japonica), and were adaptively fed for 1 week, and were fed with high-fat feed. Another 6 C57BL / 6J mice were used as normal control group, and were fed with ordinary feed. According to the conversion of human and animal body surface area, the daily administration dose of mice in low-dose and high-dose groups was 0.33g / kg and 1.32g / kg, and the normal control group and model group were given equal amount of 0.5% CMC-Na solution by gavage, for 12 weeks. After the last administration, the eyeball blood of each group of mice was taken, centrifuged at 3000rpm for 10min, and the serum was taken; part of the aorta was placed in paraformaldehyde solution for preservation, and the abdominal fat was weighed and the remaining aorta tissue was stored in a-80℃ refrigerator.

[0095] 3. Mouse weight detection

[0096] From the beginning of the mouse adaptation for a week, weigh the body weight at the same time every week, draw the growth curve.

[0097] 4. Detection of serum biochemical indicators in mice

[0098] After feeding, 24 mice were fasted overnight without water, anesthetized with 10% chloral hydrate, and the eyeball blood of the mice was taken. The collected blood was placed at 4°C for 30 min, then centrifuged at 3000 rpm for 15 min, and the serum was aspirated and divided into 1.5 mL EP tubes. Then immediately stored in a -80°C refrigerator. According to the kit instructions, the contents of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in mouse serum were detected.

[0099] 5. Gross oil red staining of mouse aorta

[0100] Under the dissecting microscope, the excess fat around the aorta was stripped, and after stripping, the aorta was cut along the back. Next, oil red O staining was performed, and Image-Pro Plus 6.0 software was used for quantitative analysis of lesion area. The ratio of aortic plaque area to total vascular area was calculated to assess the degree of lesion.

[0101] 6. HE staining of mouse aorta

[0102] After fixing the aorta in 4% paraformaldehyde, HE staining was performed, and the prepared tissue sections were photographed to analyze the aortic AS lesion.

[0103] 7. Masson staining of mouse aorta

[0104] After fixing the aorta in 4% paraformaldehyde at room temperature, Masson staining was performed, and the prepared tissue sections were photographed and analyzed for collagen volume fraction using Image J software.

[0105] 8. Statistical analysis

[0106] The experimental data were input into GraphPad Prism 8.0 software for analysis, and the results were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to compare the differences between multiple groups, and p<0.05 was considered statistically significant.

[0107] 9. Results

[0108] 9.1 Effect of CPFs on mouse body weight

[0109] During the experiment, the body weight of the mice was monitored every week to evaluate the effect of CPFs intervention on body weight. The results are shown in Figure 8, After 12 weeks of feeding, the body weight of mice in each group showed a stable growth trend, and there was no significant difference in body weight between the high-dose CPFs group and the Model group (p>0.05). At 12 weeks, compared with the Model group, the body weight of mice in the high-dose CPFs group showed a downward trend, but there was no significant difference (p>0.05), indicating that CPFs intervention had no significant effect on ApoE - / - The body weight of mice was not significantly affected.

[0110] 9.2 Effect of CPFs on fat rate of mice

[0111] At the end of the experiment, after the mice were sacrificed, the entire abdominal fat was taken out and weighed to calculate the fat rate (abdominal fat weight / body weight). The results are shown in Figure 9 The fat rate of mice in the Model group increased, indicating that the modeling was successful (p<0.01), and after gavage with CPFs, the fat rate of mice in the blank group was significantly lower than that in the Model group (p<0.05) in a dose-dependent manner, indicating that CPFs had a lipid-lowering effect and could significantly reduce the increase in fat rate caused by high-fat diet feeding.

[0112] 9.3 Effect of CPFs on serum biochemical indicators of mice

[0113] AS is often accompanied by the occurrence of hypercholesterolemia and other lipid metabolism abnormalities. The results are shown in Figure 10 Compared with Control, the TG, TC, and LDL-C levels in the Model group were significantly increased, and the HDL-C level was significantly decreased. After administration, compared with the Model group, the TG level in the low-dose CPFs group was significantly reduced, and the HDL-C level was significantly increased. The TG and LDL-C levels in the high-dose group were significantly reduced, and the HDL-C level was significantly increased. The results showed that CPFs could significantly reduce the TG and LDL-C levels of ApoE - / - mice, significantly increase the HDL-C level of ApoE - / - mice, and have a certain improvement effect on the lipid abnormalities of ApoE - / - mice, and the high-dose group had the best effect.

[0114] 9.4 Effect of CPFs on gross oil red staining of mouse aorta

[0115] The results are shown in Figure 11 The staining results showed that compared with the Model group, the lesion plaques gradually decreased in the administration groups. By calculating the plaque area, it can be seen that with the increase of the dosage of tea flower powder extract, the plaque lesion area showed a decreasing trend, and the plaque reduction in the high-dose CPFs group was the most obvious (p<0.001).

[0116] 9.5 Effect of CPFs on pathological features of mouse aorta

[0117] Results are shown in Figure 12 Figure 2A shows that HE staining results show that the arterial blood vessel wall of the Control group has no obvious lipid deposition, no obvious stenosis is observed in the lumen, the endothelial cells and smooth muscle cells have normal morphology and complete structure, and no obvious increase in inflammatory cells and foam cells is observed. The aortic valve of the Model group forms atherosclerotic plaques, the deep part is a large amount of lipid-rich necrotic material and needle-shaped cholesterol crystal voids; the bottom of the plaque has a thin tunica media, atrophic smooth muscle and damaged elastic fibers; a large amount of lymphocyte infiltration is observed in the plaque and adipose tissue. The pathological and morphological changes of the aorta of the mice in the low-dose and high-dose CPFs groups are improved to some extent compared with the Model group, and the improvement of the degree of the lesion shows an upward trend with the increase of the dosage. Meanwhile, the Masson staining results show that, compared with the Model group, the collagen deposition area of the aorta of the mice in the CPFs groups is significantly reduced, and the fibrosis lesion shows a decreasing trend with the increase of the dosage (see Figure 12 Figure 2B-C). The above results show that CPFs can reduce the pathological changes of the aorta of ApoE - / - mice, and the effect of the high dose is better than that of the low dose.

[0118] When the tea flower powder is crushed, it should be sieved more than three times to make the tea flower powder uniform, the same batch of crushed tea flower powder should be used for each extraction, and the tea flower powder that is bonded into a group should not be weighed to reduce the errors caused by the uneven particle size, inconsistent storage time and powder grouping, and to interfere with the experimental results. When the ultrasonic extraction is performed, the extraction temperature should be ensured to be consistent to avoid affecting the total flavonoids leaching of the tea flower powder. When the color development is performed, the cap of the volumetric flask should be covered to reduce the evaporation of ethanol and affect the extraction rate.

[0119] In summary, the optimal process for ultrasonic extraction of total flavonoids from tea flower powder is explored, the effects of three single factors of ethanol concentration, ultrasonic time and liquid-solid ratio on the extraction rate of total flavonoids from tea flower powder are explored, and finally the optimal three single factors are selected. On this basis, the extraction conditions are optimized by the Design-Expert software through the response surface method, and the optimal process is obtained as follows: the ethanol concentration is 73%, the ultrasonic time is 31 min, the liquid-solid ratio is 31:1 (mL·g -1 ), and the extraction rate of total flavonoids from tea flower powder is 9.666%. Meanwhile, it is proved by experiments that the total flavonoids from tea flower powder have the pharmacological effects of reducing the fat rate and improving hyperlipidemia. The present application lays a foundation for the extraction and separation of total flavonoids from tea flower powder and the research on the pharmacological effects.

[0120] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a total flavonoid extract from camellia pollen, characterized in that: The preparation method comprises the following steps: taking camellia pollen powder, and ultrasonically treating it for 10-50 minutes under the conditions of 60-80% ethanol concentration, 20-40 mL:0.5-1.5 g liquid-solid ratio, and 50-150 W ultrasonic power to obtain a camellia pollen total flavonoid extract.

2. The preparation method according to claim 1, characterized in that The ethanol concentration is 73%.

3. The preparation method according to claim 1, characterized in that The liquid-to-solid ratio is 31 mL:1 g.

4. The preparation method according to claim 1, characterized in that The ultrasonic power is 100W.

5. The preparation method according to claim 1, characterized in that The ultrasonic time is 31 minutes.

6. The total flavonoids extract of camellia pollen obtained by the preparation method according to any one of claims 1 to 5.

7. A blood lipid lowering composition comprising the camellia pollen total flavonoids extract according to claim 6 and a pharmaceutically acceptable carrier or excipient.

8. Use of the camellia pollen flavonoid extract according to claim 6 or the lipid-lowering composition according to claim 7 in the preparation of a product for treating lipid-lowering.

9. The use according to claim 8, characterized in that The lipid-lowering product is a lipid-lowering drug.

10. The use according to claim 9, characterized in that The medicine further comprises a pharmaceutically acceptable carrier or excipient.

Images