Polymethoxylated flavone component enriched from citrus chachiensis hortorum as well as enrichment method and application of polymethoxylated flavone component

Abstract

The invention discloses a polymethoxylated flavone component enriched from citrus chachiensis hortorum as well as an enrichment method and application of the polymethoxylated flavone component, and relates to the technical field of extraction, separation and purification of effective components in natural plants. The polymethoxylated flavone component enriched from citrus chachiensis hortorum disclosed by the invention comprises seven polymethoxylated flavone components, namely 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxylated flavone, nobiletin, hesperetin, 5, 6, 7, 3 ', 4'-pentamethoxylated flavone, 5, 7, 8, 3 ', 4'-pentamethoxylated flavone, 3 ', 4', 5 ', 6, 7, 8-hexamethoxy-5-hydroxylated flavone and 5-hydroxy-4', 6, 7, 8-tetramethoxylated flavone. Meanwhile, various polymethoxylated flavonoids in citrus chachiensis hortorum are contained, so that the blood fat level of high-fat diet mice can be reduced, the accumulation of lipid plaques and the oxidative stress level are reduced, and the effect of preventing and treating atherosclerosis is shown.

Description

Technical Field

[0001] The invention relates to a polymethoxyflavonoid component enriched from dried orange peel and an enrichment method and application thereof, and relates to the technical field of extraction, separation and purification of effective components in natural plants. Background Art

[0002] AS (Atherosclerosis) is a cardiovascular disease that is prone to cause a variety of complications such as myocardial infarction, cerebral ischemia, stroke and other high-risk diseases, and has a high morbidity and mortality rate. Atherosclerosis is considered to be the result of the interaction between various mechanisms, including lipid disorders, inflammatory cell infiltration, oxidative stress, immune dysfunction and vascular endothelial cell damage. Although the pathogenesis of AS has not been fully clarified, it is currently believed that lipid metabolism disorders and chronic inflammation are key factors in the formation of AS. The main manifestations of lipid metabolism disorders are the imbalance of cholesterol uptake, esterification and efflux metabolism in the body, which leads to the formation and accumulation of foam cells in blood vessels, which is also considered to be a sign of early atherosclerosis. Therefore, regulating lipid metabolism disorders in the body to inhibit the formation of foam cells helps to reduce the risk of AS. At present, the main treatment for AS is lipid-lowering. Statins are the most common drugs for the treatment of AS, but AS is a long-term process. Long-term use of statins will produce inevitable side effects, such as allergies, muscle pain, myopathy, rhabdomyolysis, hepatotoxicity and new-onset diabetes. Therefore, more effective, safer and healthier means should be sought for the treatment of AS.

[0003] Guang Chenpi is a local medicinal material of tangerine peel. It is mainly derived from the mature peel of citrus fruits aged for more than 3 years in Xinhui or Sihui District, Guangdong Province, China. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm. PMFs (polymethoxy flavones) are the main active and characteristic flavonoid components in Guang Chenpi. It contains a variety of PMFs monomers with different numbers of methoxyl groups or hydroxyl groups and their locations. It has good antioxidant and anti-inflammatory activities, and has a good lipid-lowering effect. It can improve lipid metabolism and prevent the occurrence of atherosclerosis. However, most of the current research focuses on the extraction of crude PMFs or some PMFs components in Guang Chenpi, and few studies focus on how to extract multiple PMFs components in Guang Chenpi at the same time. But in fact, the content of these monomer components is low and the development value is not high; by enriching PMF components, the multi-component and multi-target pharmacodynamic mechanism of PMF can be fully utilized, which has a high practical application value. Summary of the invention

[0004] According to one aspect of the present invention, a polymethoxyflavonoid component enriched from dried orange peel is provided, comprising 20-25 mg / g of 3,5,6,7,8,3',4'-heptamethoxyflavone, 102-115 mg / g of nobiletin, 65-75 mg / g of tangerine peelin, 8-11 mg / g of 5,6,7,3',4'-pentamethoxyflavone, 6-9 mg / g of 5,7,8,3',4'-pentamethoxyflavone, 0.45-0.52 mg / g of 3',4',5',6,7,8-hexamethoxy-5-hydroxyflavone and 0.18-0.21 mg / g of 5-hydroxy-4',6,7,8-tetramethoxyflavone.

[0005] The polymethoxyflavonoids component enriched from the peel of dried oranges provided by the present invention includes a variety of PMFs monomer components, which can improve lipid metabolism and prevent atherosclerosis through a variety of pharmacodynamic mechanisms. The content of the above monomer components is respectively within the range of 20-25 mg / g, 102-115 mg / g, 65-75 mg / g, 8-11 mg / g, 6-9 mg / g, 0.45-0.52 mg / g, and 0.18-0.21 mg / g, which can achieve full recovery of the PMFs component in the peel of dried oranges, with a recovery rate of more than 90%.

[0006] In some embodiments, the purity of the polymethoxylated flavonoids component enriched from dried orange peel is 20-23%.

[0007] According to another aspect of the present invention, there is provided a method for enriching polymethoxylated flavonoid components in dried orange peel, comprising the following steps:

[0008] S1. After crushing the tangerine peel, soak it in an alcohol solution for 60 to 120 minutes, wherein the solid-liquid ratio of the tangerine peel to the alcohol solution is 1: (10 to 20), and after the soaking is completed, heating and alcohol extraction is performed, the heating and alcohol extraction temperature is 65 to 75° C., and the total alcohol extraction time is 180 to 300 minutes. After the alcohol extraction is completed, a crude extract of tangerine peel is obtained;

[0009] S2. Use HP20 resin to perform column chromatography on the crude extract of dried orange peel obtained in step S1, and use water, 25-35 vol% and 80-95 vol% alcohol solution as mobile phases for elution. After the elution is completed, the PMFs enriched product can be obtained.

[0010] The applicant was surprised to find that HP20 resin has excellent adsorption and desorption rates for PMFs components. After the crude extract of dried tangerine peel is loaded onto HP20 resin, various components in PMFs can be firmly adsorbed and cannot be eluted by water and 25-35 vol% alcohol solution, but can be eluted by 80-95 vol% alcohol solution. Therefore, pure water can be used to elute impurities such as polysaccharides and water-soluble pigments in dried orange peel first, and then a 25-35 vol% alcohol solution can be used to elute interfering components such as simple phenolic acids and flavonoid glycosides (such as hesperidin) in dried orange peel. Finally, an 80-95 vol% alcohol solution is used as the mobile phase to enrich the PMFs components in dried orange peel into the eluent, thereby achieving simultaneous extraction of seven PMFs components, namely, 3,5,6,7,8,3',4'-heptamethoxyflavone, nobiletin, hesperidin, 5,6,7,3',4'-pentamethoxyflavone, 5,7,8,3',4'-pentamethoxyflavone, 3',4',5',6,7,8-hexamethoxy-5-hydroxyflavone, and 5-hydroxy-4',6,7,8-tetramethoxyflavone. Among them, in the last step of elution, when the ethanol concentration in the alcohol solution is 80 vol%, the desorption rate of the resin to the PMFs component can reach the maximum.

[0011] In some embodiments, the alcohol solutions used in steps S1 to S2 are all aqueous solutions of ethanol.

[0012] In some embodiments, after the alcohol extraction is completed, the step of filtering out large particles in the alcohol extract using gauze is also included, and after filtering out the large particles, a crude extract of dried orange peel can be obtained, and the crude extract is in the form of a solution. The purpose of the above operation is to prevent the crude extract of dried orange peel from clogging the resin pores after loading, resulting in a decrease in elution efficiency.

[0013] In some embodiments, before using HP20 resin for column chromatography, the steps of resin pretreatment, column packing and sample loading are also included.

[0014] In some embodiments, the pretreatment method of the HP20 resin in step S2 of the present invention is: add 95 vol% ethanol to the HP20 resin and soak it for 4 hours, then elute the resin with 95 vol% ethanol until a small amount of eluate is taken in a test tube and an equal amount of water is added without turbidity, and finally wash it with pure water until the ethanol content is less than 1%.

[0015] In some embodiments, the column packing method in step S2 of the present invention is wet column packing.

[0016] In some embodiments, after the column is loaded, an appropriate amount of water may be added to rinse the resin.

[0017] In some embodiments, the sample loading method in step S2 of the present invention is wet sample loading.

[0018] Preferably, when water is used for elution in step S2, the volume of the mobile phase is 3 to 4 BV.

[0019] More preferably, when water is used for elution in step S2, the elution flow rate is 3 to 4 BV / h.

[0020] The purpose of pure water elution is to remove impurities such as polysaccharides and water-soluble pigments in the peel of dried orange peel, but at the same time, it is necessary to ensure that the mobile phase will not affect the PMFs components. In the experiment, about 3-4BV of pure water was used for elution. It was found that the color of the eluent in the early stage was turbid light yellow, and the eluent was close to colorless at the end of the elution, indicating that impurities such as polysaccharides and water-soluble pigments were basically removed. Since most of the impurities eluted with pure water are sugars, the eluent has a certain viscosity. During the elution process, it was found that when the pure water elution flow rate is less than 3BV / h, the eluent is not easy to flow out, and the elution efficiency is low. Therefore, 3-4BV of pure water is used for elution at a flow rate of 3-4BV / h, and this process can remove a large number of impurities.

[0021] Preferably, when 25-35 vol% alcohol solution is used for elution in step S2, the volume of the mobile phase is 5-7 BV.

[0022] More preferably, when 25-35 vol% alcohol solution is used for elution in step S2, the elution flow rate is 1-3 BV / h.

[0023] When eluting with 25-35 vol% alcohol solution, the mobile phase volume is 5-7 BV, and the flow rate is 1-3 BV / h, which is the best, and can remove a large number of irrelevant impurities and fully retain the PMFs components.

[0024] Preferably, when elution is performed using an alcohol solution of 80-95 vol% in step S2, the elution flow rate is 1-3 BV / h.

[0025] More preferably, when elution is performed using an alcohol solution of 80 to 95 vol% in step S2, the elution time is 130 to 200 min.

[0026] The elution time of the last step is controlled to be 130-200 min, which is also to control the volume of the eluent. When the elution flow rate is 1-3 BV / h, the elution time is 130-200 min, and the volume of the eluent is 2.2-10 BV. Using 2.2-10 BV 80-95 vol% alcohol solution for elution and controlling the flow rate to 1-3 BV / h, most of the PMFs components in the dried orange peel can be eluted, thereby improving the enrichment efficiency and the content and purity of PMFs in the final enrichment product.

[0027] Preferably, in step S2, when the crude extract of Citrus aurantium obtained in step S1 is subjected to column chromatography using HP20 resin, the loading speed is 2 to 6 BV / h.

[0028] More preferably, in step S2, when the crude extract of Citrus aurantium obtained in step S1 is subjected to column chromatography using HP20 resin, the sample is loaded in a multiple loading manner, and the number of loadings is 2 to 8 times.

[0029] When the loading speed of the crude extract of Guang Chenpi is less than 2BV / h, the flow rate is too slow, the adsorption efficiency is low, and the chromatographic column is easily blocked. However, when the loading speed is greater than 6BV / h, the flow rate is too fast, which will lead to incomplete adsorption of the PMFs component in Guang Chenpi. For this reason, it is necessary to control the loading flow rate to 2-6BV / h. Under the premise of controlling the loading flow rate, the sample is loaded multiple times. It can be observed that the color of the Guang Chenpi crude extract gradually becomes lighter to light yellow, which can be judged that the PMFs component in Guang Chenpi has been basically completely adsorbed on the HP20 resin.

[0030] Preferably, the heating and alcohol extraction in step S1 is performed multiple times, and the number of heating and alcohol extraction is 2 to 4 times.

[0031] In some embodiments, during multiple heating and alcohol extractions, the alcohol extract is separated after each heating and alcohol extraction, and then the next heating and alcohol extraction is performed.

[0032] More preferably, the alcohol extraction is performed multiple times, and the time for each heating and alcohol extraction is 90 to 150 minutes.

[0033] More preferably, the volume fraction of the alcohol solution in step S1 is 60-80 vol%.

[0034] The orthogonal experimental method was used to optimize the process conditions for heating and reflux alcohol extraction of effective components in dried orange peel. The results showed that when the solid-liquid ratio was 1:(10-20), the number of heating and alcohol extraction was 2-4 times, the time of each alcohol extraction was 90-150 min, and the volume fraction of the alcohol solution was 60-80 vol%, which can improve the extraction rate of PMFs components in dried orange peel.

[0035] According to another aspect of the present invention, there is provided a use of a polymethoxylated flavonoid component enriched from dried orange peel in the preparation of a drug for preventing atherosclerosis.

[0036] According to another aspect of the present invention, there is provided a use of a polymethoxylated flavonoid component enriched from dried orange peel in the preparation of a lipid-lowering drug.

[0037] The polymethoxylated flavonoids component enriched from dried orange peel provided by the present invention can reduce the blood lipid level of mice fed a high-fat diet, and exhibits the effect of preventing and treating atherosclerosis.

[0038] According to another aspect of the present invention, there is provided a use of a polymethoxylated flavonoid component enriched from Pericarpium Citri Reticulatae in the preparation of a drug for reducing lipid plaque accumulation and lowering oxidative stress levels.

[0039] The polymethoxylated flavonoids component enriched from dried orange peel provided by the present invention can reduce the accumulation of lipid plaques and the level of oxidative stress in mice fed a high-fat diet, and exhibits the effect of preventing and treating atherosclerosis.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The polymethoxyflavonoids component enriched from the peel of dried orange peel provided by the present invention comprises 7 PMFs components, namely 3,5,6,7,8,3',4'-heptamethoxyflavone, nobiletin, hesperidin, 5,6,7,3',4'-pentamethoxyflavone, 5,7,8,3',4'-pentamethoxyflavone, 3',4',5',6,7,8-hexamethoxy-5-hydroxyflavone and 5-hydroxy-4',6,7,8-tetramethoxyflavone, and achieves full recovery of the PMFs component in the peel of dried orange peel, with a recovery rate of more than 90% and a purity of more than 20.62%. The polymethoxyflavonoids component enriched from the peel of dried orange peel provided by the present invention can effectively reduce the blood lipid level of mice fed a high-fat diet, reduce the accumulation of lipid plaques and reduce the level of oxidative stress, thereby showing the effect of preventing and treating atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the adsorption and desorption rates of PMFs by four different types of macroporous resins, HP20, HPD450, AB-8, and D101, in the investigation of column chromatography resin types of the present invention.

[0043] Figure 2 This is a graph showing the desorption rate of PMFs at different alcohol solution concentrations, elution volumes and elution flow rates obtained by the single factor experimental method in the column chromatography parameter investigation of the present invention, wherein A corresponds to the alcohol solution concentration, B corresponds to the elution volume, and C corresponds to the elution flow rate.

[0044] Figure 3 This is a graph showing the desorption rate of PMFs at different alcohol solution concentrations, elution volumes and elution flow rates obtained by the response surface methodology in the investigation of column chromatography parameters of the present invention, wherein A corresponds to the alcohol solution concentration, B corresponds to the elution volume, and C corresponds to the elution flow rate.

[0045] Figure 4 LC-MS chart of various products obtained in steps S1 to S2 in Example 1 of the present invention.

[0046] Figure 5 It is a schematic diagram of the serum blood lipid test results in the pharmacological action investigation experiment of the present invention.

[0047] Figure 6 This is a schematic diagram of the detection results of serum TNF-α, IL-1β, and IL-6 in the pharmacological action investigation experiment of the present invention.

[0048] Figure 7-8 This is a diagram showing the results of gross Oil Red O staining and plaque quantitative detection of the aorta in the pharmacological action investigation experiment of the present invention.

[0049] Figure 9-10 This is a diagram showing the results of Oil Red O staining and plaque quantitative detection of the aortic root in the pharmacological action investigation experiment of the present invention.

[0050] Fig.11 The pharmacological effect of PMFs of dried orange peel on ApoE - / - The effect results of HE staining pathological sections of mouse liver.

[0051] Fig.12 The pharmacological effect of PMFs of dried orange peel on ApoE - / - The results of the effect on TG and TC content in mouse liver.

[0052] Fig.13 The pharmacological effect of PMFs of dried orange peel on ApoE - / - The results of the effects on the levels of SOD, MDA, and GSH-Px in mouse liver.

[0053] Fig.14 The pharmacological effect of PMFs of dried orange peel on ApoE - / - The results of the effect on the expression of CD36, SRB1, ABCG5, and ABCG1 proteins in mouse liver. DETAILED DESCRIPTION

[0054] Investigation of column chromatography resin types

[0055] Pre-treat four macroporous resins: HP20, HPD450, AB-8, and D101: add 95 vol% ethanol aqueous solution to the resin and soak for 4 hours, then elute the resin with 95 vol% ethanol aqueous solution until a small amount of eluate is taken in a test tube and an equal amount of water is added without turbidity, finally wash with pure water until the ethanol content is less than 1%, and store in pure water for later use, to obtain four hydrated resins. Take a small amount of each hydrated resin, filter to remove the solution, and weigh; weigh the same mass of resins respectively and place them in a 60°C oven to dry until the mass no longer changes, and the water content of the four resins can be measured respectively, which can be converted into dry weight according to the proportion.

[0056] Take 1g (dry weight) of each type of macroporous resin in a 100mL stoppered conical flask, add 50mL of crude extract of dried orange peel (PMFs content is C0), place it in a shaking table and vibrate (150rpm) for 12h, collect the filtrate, and measure the PMFs content (C1) in the filtrate. Then wash the resin with pure water, absorb the moisture on the surface of the resin with filter paper, add 60mL 95vol% ethanol to desorb the resin in adsorption equilibrium, place it in a shaking table and vibrate (150rpm) for 12h, collect the filtrate afterwards, and measure the final concentration (C2) of PMFs. Calculate according to the following formula to compare the adsorption rate and desorption rate of different resins.

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] Wherein, C0 is the concentration of PMFs in the crude extract of Dangpi (mg / mL); C1 is the concentration of PMFs in the filtrate collected after shaking the crude extract of Dangpi (mg / mL); C2 is the concentration of PMFs in the eluent (mg / mL); V0 is the volume of the crude extract of Dangpi (mL); V1 is the volume of the filtrate collected after shaking the crude extract of Dangpi (mL); V2 is the volume of the eluent (mL); M is the mass of the macroporous resin.

[0063] The experimental results are shown in Table 1 and Figure 1 shown.

[0064] Table 1. Comparison of static adsorption and elution performance of different types of resins

[0065]

[0066]

[0067] As shown in Table 1 and Figure 1 As shown in the figure, HP20 resin has obvious advantages in both retention (adsorption) and elution (desorption) capabilities for PMFs components.

[0068] Investigation of column chromatography parameters

[0069] Response surface methodology was used to investigate the parameters of the last elution step in column chromatography.

[0070] First, a single factor test method was used to select three factors that have a greater impact on the desorption rate of the PMFs component (alcohol solution concentration, elution flow rate and elution volume), and 5 levels were set for each, where the alcohol solution concentration was 50vol%, 60vol%, 70vol%, 80vol%, 90vol%, the elution flow rate was 1.0, 2.0, 3.0, 4.0, 5.0BV / h, and the elution volume was 2, 3, 4, 5, 6, 7, 8BV, to examine the effects of different factors and their different levels on the desorption rate of the PMFs component. The results of the single factor test are shown in Figure 2. Figure 2 As shown in the figure, the alcohol solution concentration and elution volume corresponding to the maximum desorption rate of the PMFs group are 80 vol% and 2 BV / h respectively; the elution flow rate corresponding to the maximum change in the desorption rate of the PMFs group is 5 BV. Taking these three levels as the midpoints, 70 vol%, 80 vol%, and 90 vol% were selected in the alcohol solution concentration, 4, 5, and 6 BV were selected in the elution volume, and 1, 2, and 3 BV / h were selected in the elution flow rate for subsequent response surface experiments.

[0071] The Box-Behnken Design experimental principle (BBD) in Design-Expert 8.06 software was used to carry out response surface experimental design. A total of 17 experiments were carried out to obtain the desorption rate at different factor levels. Finally, the desorption rate of each group of experiments was input, and the software was used to analyze and obtain the optimal process conditions.

[0072] The response surface experimental design scheme and corresponding data are shown in Table 2 and Figure 3 As shown:

[0073] Table 2. Response surface experimental design and its corresponding data

[0074]

[0075]

[0076] As shown in Table 2 and Figure 3 As shown in the figure, the best process conditions obtained by response surface methodology are: alcohol solution concentration is 85.409 vol%, ethanol elution volume is 5.140 BV, and elution flow rate is 2.036 BV / h. Under the above parameter conditions, the maximum desorption rate prediction value is 96.242%.

[0077] According to the actual situation and the convenience and operability of the adsorption-desorption process, the optimal process conditions obtained by the response surface experiment were modified, and finally the parameters of the last elution step in this application were obtained, which are: the concentration of the alcohol solution is 86 vol%, the elution flow rate is 2 BV / h, the elution volume is 5.5 BV, that is, the elution time is 165 min.

[0078] Example 1

[0079] A method for enriching polymethoxylated flavonoid components in dried orange peel comprises the following steps:

[0080] S1. alcohol extraction: After crushing the tangerine peel, soak it in a 70 vol% ethanol aqueous solution for 90 minutes, wherein the solid-liquid ratio of the tangerine peel to the ethanol aqueous solution is 1:14, and after the soaking, it is heated and extracted twice, each time at a temperature of 70°C and a time of 120 minutes, that is, the total extraction time is 240 minutes, and the alcohol extract is separated after each heating and extraction, and then the next heating and extraction is carried out; after the two heating and extractions are completed, the alcohol extracts of the two heating and extractions are combined, and the large particles in the alcohol extract are filtered out with gauze to obtain a crude extract of tangerine peel;

[0081] S2. Enrichment of PMFs components: Using HP20 resin to perform column chromatography on the crude extract of dried orange peel obtained in step S1, first pre-treat the HP20 resin, add 95 vol% ethanol aqueous solution to the HP20 resin and soak it for 4 hours, then elute the resin with 95 vol% ethanol aqueous solution until a small amount of eluate is taken in a test tube and an equal amount of water is added without turbidity, and finally wash with pure water until the ethanol content is less than 1%; after the pre-treatment, the column is loaded by wet column loading, and then an appropriate amount of water is added to rinse the resin, and wet loading is performed by multiple loading, and the number of loadings is 5 times; after the loading is completed, first use 3BV of water for elution, and the elution flow rate is 4BV / h; then use 6BV, 30vol% ethanol aqueous solution for elution, and the elution flow rate is 2BV / h; finally, use 89vol% ethanol aqueous solution for elution, the elution flow rate is 2BV / h, and the elution time is 165min. After the elution is completed, the PMFs enriched product can be obtained, the PMFs enriched product is concentrated, and after removing the alcohol solution, it is placed at -80°C for solidification and then freeze-dried to obtain the PMFs enriched product freeze-dried powder for standby use.

[0082] Three parallel experiments were performed to characterize the products obtained in steps S1 to S2. The experimental results are shown in Tables 3 to 4. Figure 4 As shown:

[0083] Table 3. Mass and recovery of PMFs components in the freeze-dried powder of PMFs-enriched product obtained in step S2

[0084]

[0085]

[0086] Note: P1, P2, and P3 in the above table are the PMFs enrichment products obtained in step S2 in three parallel experiments.

[0087] Table 4. Content and purity of PMFs components in the freeze-dried powder of PMFs enriched product obtained in step S2

[0088]

[0089] Note: P1, P2, and P3 in the above table are the PMFs enrichment products obtained in step S2 in three parallel experiments.

[0090] As shown in Tables 3 and 4, the polymethoxyflavonoid components enriched from the peel of dried orange of the present invention include 20 to 25 mg / g of 3,5,6,7,8,3',4'-heptamethoxyflavones, 102 to 115 mg / g of nobiletin, 65 to 75 mg / g of tangerine peel, 8 to 11 mg / g of 5,6,7,3',4'-pentamethoxyflavones, 6 to 9 mg / g of 5,7,8,3',4'-pentamethoxyflavone, 3',4',5',6,7,8-hexamethoxy-5-hydroxyflavone with a content of 0.45-0.52 mg / g and 5-hydroxy-4',6,7,8-tetramethoxyflavone with a content of 0.18-0.21 mg / g achieved the full recovery of PMFs components in Guang Chenpi, with a recovery rate of more than 90% and a purity of more than 20.62%.

[0091] Figure 4 LC-MS images of various products obtained in steps S1 to S2 of Example 1 of the present invention, specifically, LC-MS images of the crude extract of dried orange peel obtained in step S1 of Example 1 of the present invention, the eluate after elution with 30 vol% alcohol solution in step S2, and the eluate after elution with 86% alcohol solution. Figure 4 It can be seen that the method provided by the present invention can successfully extract the PMFs components in 7 kinds of dried orange peels at the same time.

[0092] Experimental study on the pharmacological effects of PMFs enrichment products

[0093] Next, the PMFs enriched product P1 obtained by the first parallel experiment in Example 1 was used for animal experiments to explore the pharmacological effects of the PMFs enriched product. This experiment provides a series of experiments on the use of the PMFs enriched product obtained in Example 1 for treating early atherosclerosis in mice. - / - Mice (apolipoprotein E knockout mice) were used as an early atherosclerosis model and were given the drug by gavage. The lipid-lowering and anti-atherosclerotic effects of the PMFs enriched product prepared by the present invention on mice were explored by detecting the blood lipid content in the mouse serum, oil red staining of the aorta and the root, HE staining of the liver, as well as related experiments such as liver TG, TC and oxidative stress indicators.

[0094] 1. Animal grouping and modeling

[0095] All mice were weighed for the first time after one week of adaptive feeding in the same feeding environment and randomly divided into 6 groups, with 10 mice in each group, and free access to food and water. When administering, the powdered drug (freeze-dried powder of PMFs enrichment product, Atorvastatin) was dissolved in a blank solvent and administered by gavage. The specific dosage was calculated according to the real-time weight of the mice at 0.1 mL / 10 g, and the dosage was as follows:

[0096] Normal control group (control group): C57BL / 6J mice, normal feed + daily administration of corresponding amount of normal saline

[0097] Model group: ApoE - / - Mice, high-fat diet + daily administration of corresponding amount of normal saline

[0098] Atorvastatin positive drug group (Ator group): ApoE - / - Mice, high-fat diet + Atorvastatin 5 mg / kg / day

[0099] PMFs low-dose group (PMFs-L group): ApoE - / - Mice, high-fat diet + PMFs enrichment product obtained in Example 1 75 mg / kg / day

[0100] PMFs medium dose group (PMFs-M group): ApoE - / - Mice, high-fat diet + PMFs enrichment product obtained in Example 1 150 mg / kg / day

[0101] PMFs high-dose group (PMFs-H group): ApoE - / - Mice, high-fat diet + PMFs enrichment product obtained in Example 1 300 mg / kg / day

[0102] The high-fat feed ratio is: sucrose 20%, lard 15%, cholesterol 1.2%, sodium cholate 0.2%, casein 10%, calcium hydrogen phosphate 0.6%, stone powder 0.4%, premix 0.4% and basic feed 52.2%.

[0103] The mass ratio of components in high-fat feed is: protein 19%, fat 18%, carbohydrate 50%; calorie ratio: protein 17%, fat 37%, carbohydrate 46%.

[0104] In the first week of the experiment, except for the normal control group, the mice in other groups were fed with high-fat diet. The drug-treated group was given adaptive oral administration on the third and fifth days, and the control group and the model group were given corresponding amounts of blank solvent.

[0105] From the 2nd to the 12th week of the experiment, the normal control group was fed with ordinary feed, and the mice in the other groups continued to be fed with high-fat feed. According to the above grouping and dosage, the blank group and the model group were gavaged with blank solvent, and the drug-treated group was gavaged with three different doses of PMFs solution: low, medium and high. The mice were gavaged once a day and weighed once a week. The dosage was adjusted according to the real-time weight of the mice. During the experiment, the mice were free to eat and the daily food intake was recorded. In the middle of the experiment, blood was taken from the orbits once to measure TG and TC to observe the modeling. The results showed that the TG and TC of the mice in the model group were increased, and there was a significant difference compared with the blank group, indicating that the early atherosclerosis model was successfully established.

[0106] 2. Animal tissue sampling

[0107] On the last night of the 12th week of the experiment, the mice were fasted for 12 hours. The next day, the eyeballs were removed and blood was collected. After the whole blood was placed at room temperature for 30-60 minutes, it was centrifuged to separate the serum (4°C, 3000rpm, 15 minutes). The upper serum was divided and stored in a -80°C refrigerator. After the mice were killed, their abdominal and chest cavities were opened, the auricles were cut off, and ice PBS buffer was used to perfuse from the left ventricle. The entire aorta of the mouse was dissected from the proximal root of the aorta to the iliac artery branch, and the heart was taken out together. The connective tissue around the blood vessels was peeled off as much as possible, and the heart was immediately separated and placed in dry ice. The aorta was roughly placed in 4% paraformaldehyde. The liver was removed, the surface blood stains were washed with PBS buffer, and then weighed and photographed. The liver of all mice was cut from the same position of one lobule and placed in 4% paraformaldehyde. The remaining part was first placed in liquid nitrogen and then stored in a -80°C refrigerator.

[0108] 3. Determination of relevant indicators

[0109] 1) Serum lipid test

[0110] The treated serum was taken out from the -80℃ freezer and thawed on ice. The single reagent GPO-PAP method was used to determine the levels of triglyceride TG, total cholesterol TC, low-density lipoprotein LDL-C, and high-density lipoprotein HDL-C in serum. All steps were carried out according to the kit instructions.

[0111] The experimental results are as follows Figure 5 As shown in Figure 2, the PMFs fraction of dried orange peel can reduce the ApoE level in early AS induced by high-fat diet. - / - The levels of serum TG, TC, and LDL-C in mice were decreased, and the level of serum HDL-C was increased.

[0112] 2) ELISA method to measure serum TNF-α, IL-1β, IL-6 levels

[0113] The treated serum was taken out from the -80°C refrigerator, thawed on ice, and centrifuged (4°C, 3000 rpm, 10 min). The ELISA method was used to detect the levels of TNF-α, IL-1β, and IL-6 in the serum. All steps were carried out strictly in accordance with the instructions of the corresponding kits.

[0114] The experimental results are as follows Figure 6 As shown in Figure 2, the PMFs fraction of dried orange peel can reduce the ApoE level in early AS induced by high-fat diet. - / - The levels of TNF-α and IL-1β in mouse serum were significantly different in the low-dose group.

[0115] 3) Aorta gross oil red O staining and plaque quantification

[0116] The aorta fixed with 4% paraformaldehyde for 24 hours was removed, washed with PBS buffer solution, and the peripheral fat adhered to the aorta was carefully peeled off with anatomical forceps. The blood vessels were gently dissected along the opposite sides of the three arteries of the aortic arch, and the entire aorta was longitudinally dissected along the direction of the common iliac artery. After immersing in 60% isopropanol for 3-5 seconds, it was immersed in the oil red working solution prepared in a ratio of saturated oil red dye: pure water = 3:2 and filtered, and stained at room temperature for 10-60 minutes. After staining, the blood vessels were transferred to 60% isopropanol for differentiation and then washed with water until the fat plaques in the wall of the tube were bright red and clearly visible, and the background was white or almost colorless. The stained blood vessels were spread flat in a culture dish, dried with filter paper, placed on a table with a black background, and photographed with an ordinary SLR camera. Image-pro Plus6.0 software was used for analysis.

[0117] The experimental results are as follows Figure 7-8 As shown in Figure 2, the PMFs fraction of dried orange peel can reduce ApoE in the early AS induced by high-fat diet. - / - Generation of macroscopic atherosclerotic plaques in mouse aorta.

[0118] 4) Oil Red O staining and plaque quantification of aortic root

[0119] The mouse heart tissue placed on dry ice was removed, embedded in OCT, and then frozen and serially sliced ​​using a freezing microtome to obtain aortic root slices. The slices for Oil Red O staining were rinsed in 60% isopropanol for 20 seconds, 6 mL of saturated Oil Red dye solution was taken, 4 mL of distilled water was added, mixed and allowed to stand for 10 minutes, and then filtered for staining. The Oil Red O staining time was 5 minutes, and the excess dye solution was slightly washed away with 60% isopropanol and washed with distilled water. The slices were observed under a microscope and analyzed using Image-pro Plus 6.0 software.

[0120] The experimental results are as follows Figure 9-10As shown in Figure 2, the PMFs fraction of dried orange peel can reduce ApoE in early AS induced by high-fat and high-cholesterol diet. - / - Atherosclerotic plaque formation in the aortic root of mice.

[0121] 5) HE staining of liver pathological sections

[0122] The mouse liver tissue fixed in 4% paraformaldehyde for 24 hours was dehydrated, embedded in paraffin, and then sliced ​​to a thickness of 5 μm. The liver slices were placed at room temperature to dry before HE staining. Finally, the liver tissue pathological sections were observed under an optical microscope.

[0123] The specific steps of HE staining are as follows: before staining, place the paraffin sections in a 60°C oven for 1 hour to melt the paraffin, dewax in xylene I and xylene II solutions for 10 minutes each, and then immerse in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol I, 95% ethanol II, 90% ethanol, and 80% ethanol for 5 minutes each for hydration, and then immerse in ultrapure water for 3 minutes to remove residual ethanol. After the sections are immersed in hematoxylin for 20 minutes, take out the sections and rinse them gently with tap water until they are colorless, immediately immerse them in a color separation solution (75% hydrochloric acid ethanol) for differentiation for 1 second, then immerse them in 80% ethanol for 1 minute, and then immerse the sections in eosin for staining for 3 minutes. After hematoxylin and eosin staining, the sections were immersed in 80% ethanol, 90% ethanol for 20 seconds each, 95% ethanol for 1 minute, and anhydrous ethanol for 2 minutes for dehydration, and then immersed in xylene for 2 minutes to make it transparent. Finally, the sections were taken out and immediately sealed with neutral gum. After leaving them at room temperature overnight, the sections were observed and images were collected using an upright microscope.

[0124] The experimental results are as follows Fig.11 As shown, ApoE - / - PMFs administration to mice with atherosclerosis reduced lipid vacuoles and inflammatory infiltration in the liver.

[0125] 6) Determination of liver TG and TC content

[0126] Preparation of liver homogenate: Take out the mouse liver tissue from the -80℃ refrigerator, place it on ice to thaw, and then accurately weigh 20mg of liver tissue (accurately record the liver mass W weighed) according to the ratio of weight (g): volume (mL) = 1:9, and add the corresponding amount of anhydrous ethanol. Use a homogenizer to homogenize twice, each time for 30s. Then centrifuge (4℃, 2500rpm, 10min), and take the supernatant for testing. The single-reagent GPO-PAP method was used to determine TG and TC in the liver. All steps were carried out according to the instructions of the kit.

[0127] The experimental results are as follows Fig.12As shown in the results, the PMFs fraction of dried orange peel can reduce the ApoE level in early AS induced by high-fat and high-cholesterol diet. - / - The TG and TC content in mouse liver has a certain improvement effect on liver lipid accumulation.

[0128] 7) Determination of liver SOD, MDA, GSH-Px content

[0129] The protein concentration of liver homogenate was detected by BCA method, and the liver homogenate was detected according to the instructions of the SOD, MDA, and GSH-Px kits.

[0130] The experimental results are as follows Fig.13 As shown in the results, intervention with PMFs components of Dangpi can improve oxidative stress, which is mainly manifested by reducing the content of MDA in the liver of mice and increasing the content of SOD and GSH-Px.

[0131] 8) Western blot analysis of the effect of PMFs on the expression of cholesterol transport-related proteins

[0132] 30 mg of liver tissue was placed in a homogenizer tube containing 1 magnetic bead, and 600 μL of RIPA cell lysis buffer containing protease inhibitors and phosphatase inhibitors was added. After full lysis, centrifugation was performed (4°C, 12000 rpm, 15 min). The supernatant was taken to detect the protein concentration according to the BCA method, and 50 μg of protein was separated on an 8% SDS-PAGE gel, and then the protein was transferred to a PVDF membrane (280 mA, 90 min), blocked with 5% skim milk powder for 1.5 h, and then incubated with the corresponding antibody at 4°C overnight. The bands were then incubated with secondary antibodies for 1 h, and the bands were visualized using the ECL detection system, and the band density and calculation were analyzed using Image J software. The expression of CD36, SRB1, ABCG5 and ABCG1 in each group of cells was detected.

[0133] The experimental results are as follows Fig.14 As shown in the results, PMFs intervention can upregulate the expression of SRB1, ABCG5 and ABCG1 proteins in the liver of early AS mice and downregulate the expression of CD36 protein. Therefore, by inhibiting CD36 expression and increasing the expression of ABCG1, ABCG5 and SRB1, PMFs can maintain cholesterol homeostasis in foam cells and reduce lipid accumulation in foam cells.

[0134] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A polymethoxyflavonoid component enriched from dried orange peel, characterized in that: The invention comprises 20-25 mg / g of 3,5,6,7,8,3',4'-heptamethoxyflavone, 102-115 mg / g of nobiletin, 65-75 mg / g of cisperetin, 8-11 mg / g of 5,6,7,3',4'-pentamethoxyflavone, 6-9 mg / g of 5,7,8,3',4'-pentamethoxyflavone, 0.45-0.52 mg / g of 3',4',5',6,7,8-hexamethoxy-5-hydroxyflavone and 0.18-0.21 mg / g of 5-hydroxy-4',6,7,8-tetramethoxyflavone.

2. The polymethoxyflavonoid component enriched from dried orange peel according to claim 1, characterized in that: The purity of the polymethoxy flavonoids component enriched from dried orange peel is 20-23%.

3. The method for enriching polymethoxylated flavonoid components in dried orange peel according to claim 1, characterized in that: The steps include: S1. After crushing the tangerine peel, soak it in an alcohol solution for 60 to 120 minutes, wherein the solid-liquid ratio of the tangerine peel to the alcohol solution is 1: (10 to 20), and then heated for alcohol extraction at a temperature of 65 to 75° C. The total alcohol extraction time is 180 to 300 minutes, and after the alcohol extraction is completed, a crude extract of tangerine peel is obtained; S2. Use HP20 resin to perform column chromatography on the crude extract of dried orange peel obtained in step S1, and use water, 25-35 vol% and 80-95 vol% alcohol solution as mobile phases for elution. After the elution is completed, the PMFs enriched product can be obtained.

4. The method according to claim 3, characterized in that When 25-35 vol% alcohol solution is used for elution in step S2, the volume of the mobile phase is 5-7 BV.

5. The method according to claim 4, characterized in that When 25-35 vol% alcohol solution is used for elution in step S2, the elution flow rate is 1-3 BV / h.

6. The method according to claim 3, characterized in that When 80-95 vol% alcohol solution is used for elution in step S2, the elution flow rate is 1-3 BV / h.

7. The method according to claim 6, characterized in that When 80-95 vol% alcohol solution is used for elution in step S2, the elution time is 130-200 min.

8. Use of the polymethoxylated flavonoids component enriched from dried orange peel according to claim 1 or 2 in the preparation of drugs for preventing atherosclerosis.

9. Use of the polymethoxylated flavonoids component enriched from dried orange peel according to claim 1 or 2 in the preparation of lipid-lowering drugs.

10. Use of the polymethoxylated flavonoids component enriched from dried orange peel according to claim 1 or 2 in the preparation of drugs for reducing lipid plaque accumulation and lowering oxidative stress levels.

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