Uric acid-reducing apigenin cyclodextrin inclusion compound liposome and preparation method thereof

Abstract

The invention discloses a uric acid reducing apigenin cyclodextrin inclusion compound liposome and a preparation method thereof. The uric acid reducing apigenin cyclodextrin inclusion compound liposome is prepared from the following components in parts by weight: 1 part of apigenin, 2 to 50 parts of beta-cyclodextrin, 0.8 to 30 parts of phospholipid and 0.1 to 10 parts of surfactant. According to the invention, apigenin is included in beta-cyclodextrin to obtain the apigenin cyclodextrin inclusion compound, and the water solubility of apigenin can be improved and the molecular volume can be increased through a cyclodextrin inclusion method to reduce the ability of apigenin to penetrate through a phospholipid bilayer; and the apigenin cyclodextrin inclusion compound is encapsulated in the liposome to obtain the apigenin cyclodextrin inclusion compound liposome, so that the stability of the liposome is fundamentally improved, and the problem of liposome drug leakage in a liquid state is solved. The whole water solubility of the apigenin cyclodextrin inclusion compound liposome provided by the invention is improved, the apigenin cyclodextrin inclusion compound liposome has good stability and slow release effect, the bioavailability of apigenin is improved, and the uric acid reducing effect is obviously superior to that of untreated apigenin.

Description

Technical Field

[0001] The invention belongs to the technical field of food and medicine, and particularly relates to a uric acid-reducing apigenin cyclodextrin inclusion compound liposome and a preparation method thereof. Background Art

[0002] Hyperuricemia is a metabolic disease that causes elevated uric acid levels in the blood due to purine metabolism disorders and / or uric acid excretion disorders. Studies have shown that hyperuricemia is the most dangerous factor inducing gout, and is also associated with hypertension, chronic kidney disease, metabolic syndrome, hyperlipidemia, diabetes, islet resistance and other diseases. In recent years, as people's living standards have continued to improve, their dietary structure has also changed. Excessive intake of purine and protein has caused the incidence of gout to rise year by year.

[0003] Xanthine oxidase (XO) is a key enzyme in the production of uric acid. Its substrate hypoxanthine is converted into xanthine under the action of XO, and finally into uric acid, while also generating superoxide radicals and hydrogen peroxide. When XO activity is abnormally high, it will lead to an increase in uric acid levels in the body, clinically manifested as hyperuricemia, and with the deposition of urate, gout attacks will occur.

[0004] Apigenin (API), also known as apigenin or apigenin, is a natural flavonoid compound. It can be extracted from many common fruits, vegetables, beans and tea leaves, especially in celery. Naturally derived apigenin is non-toxic and non-genotoxic, and has very rich biological activities, such as anti-tumor, anti-oxidation, anti-inflammatory, anti-viral, uric acid-lowering, blood sugar-lowering, and prevention and treatment of cardiovascular diseases.

[0005] It has been reported that apigenin can achieve the effect of lowering uric acid by inhibiting the activity of xanthine oxidase, and can significantly enhance the body's total antioxidant and oxygen free radical scavenging capabilities, reducing lipid peroxidation. However, due to the poor water solubility of apigenin, its bioavailability is extremely low, which will greatly affect its uric acid-lowering effect, which greatly limits the application of apigenin.

[0006] Patent CN201811563044.X "A responsive sustained-release aggregate of apigenin and preparation method" uses Tween 80, soybean lecithin, water-soluble chitosan, ethyl oleate and water to prepare a responsive sustained-release aggregate of apigenin to improve the solubility and sustained-release effect of apigenin. Patent CN201310593357.0 "A apigenin polylactic acid sustained-release microsphere and preparation method thereof" uses polylactic acid as a matrix material to prepare apigenin polylactic acid sustained-release microspheres to improve bioavailability. Patent CN201711121219.7 "A drug carrier of apigenin and preparation method" is prepared by mixing sodium deoxycholate, sucrose stearate, isopropanol, ethyl acetate and sodium chloride water to form a microemulsion to improve the solubility and antioxidant activity of apigenin. However, these methods currently have problems such as instability in water systems, easy precipitation, low drug loading, and easy leakage, and the dosage forms used are limited.

[0007] Cyclodextrin inclusion liposome technology combines the advantages of cyclodextrin and liposome into one system. Compared with traditional liposome technology and cyclodextrin inclusion technology, it can increase the solubility of poorly soluble drugs and make them have sustained release in the human body, prolonging the drug action time. At the same time, it can avoid the problem of hydrophobic substances interfering with the lipid bilayer structure, leading to instability of the liposome system and drug leakage during storage.

[0008] There is no report on using cyclodextrin inclusion liposomes to improve the water solubility and bioavailability of apigenin to enhance the uric acid-lowering effect. Summary of the invention

[0009] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a cyclodextrin inclusion complex liposome of uric acid-lowering apigenin with high solubility and stability and capable of achieving sustained release.

[0010] Another object of the present invention is to provide a method for preparing apigenin cyclodextrin inclusion liposomes with uric acid lowering effect. The present invention adopts a cyclodextrin inclusion method combined with a nanoliposome method to prepare the cyclodextrin inclusion liposomes.

[0011] Another object of the present invention is to provide an application of apigenin cyclodextrin inclusion compound liposome as a health food for relieving gout, which can reduce the level of uric acid in serum and has a protective effect on the kidneys.

[0012] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0013] The invention firstly discloses a uric acid-reducing apigenin cyclodextrin inclusion compound liposome, which comprises the following components in parts by weight: 1 part of apigenin, 2-50 parts of beta-cyclodextrin (beta-cyclodextrin abbreviated as beta-CD), 0.8-30 parts of phospholipids and 0.1-10 parts of surfactant.

[0014] Furthermore, the surfactant is one of Tween 20, Tween 60 or Tween 80.

[0015] Furthermore, the phospholipid is one of soybean lecithin and egg yolk lecithin.

[0016] A method for preparing uric acid-lowering apigenin cyclodextrin inclusion complex liposomes comprises the following steps:

[0017] S1, weigh 2-50 parts of β-cyclodextrin (β-CD), add water at a certain temperature to dissolve it to prepare a β-cyclodextrin aqueous solution, take 1 part of apigenin, add anhydrous ethanol to dissolve it, add it to the prepared β-cyclodextrin aqueous solution under a certain temperature and stirring conditions, continue stirring for a certain time after adding, filter, and prepare an apigenin β-CD aqueous solution, which is stored at a certain temperature for later use;

[0018] S2. Weigh 0.8-30 parts of phospholipids and 0.1-10 parts of surfactant, add anhydrous ethanol to dissolve, and inject into the above-mentioned apigenin β-CD aqueous solution under stirring. After the injection is completed, keep the conditions unchanged and continue to stir for a certain period of time, then take out and remove the anhydrous ethanol. The resulting solution is homogenized by a high-pressure homogenizer to obtain apigenin β-cyclodextrin inclusion liposomes, that is, uric acid-lowering apigenin cyclodextrin inclusion liposomes.

[0019] Furthermore, in the step S1, the water temperature for dissolving β-CD is 30-100°C.

[0020] Furthermore, in step S1, the source of apigenin is the rhizomes and seeds of celery, western celery and parsley.

[0021] Furthermore, in step S1, the method of dissolving apigenin in the solvent is one of heating, stirring, ultrasound or grinding.

[0022] Furthermore, in step S1, the stirring speed is 600-1200 r / min, the temperature is 40-60° C., and the time is 1 h-24 h.

[0023] Furthermore, in step S1, the filtering method may be one of membrane filtration, paperboard filtration or plate and frame filtration, and the pore size of the membrane, paperboard or filter cloth is 0.1-15 μm.

[0024] Furthermore, in step S1, the storage temperature of the apigenin β-CD aqueous solution is 4°C-30°C.

[0025] Furthermore, in step S2, the phospholipid used is one of soybean lecithin and egg yolk lecithin.

[0026] Furthermore, in step S2, the surfactant used may be one of Tween 20, Tween 60 or Tween 80.

[0027] Furthermore, in step S2, the stirring speed is 100-800 r / min, and stirring is continued for 0.5-8 h after all the apigenin β-cyclodextrin inclusion complex is injected.

[0028] Furthermore, in step S2, the method for removing anhydrous ethanol can be one of reduced pressure rotary evaporation, freeze drying, and vacuum drying.

[0029] Furthermore, in step S2, the homogenization pressure is 500-1000 bar, and the homogenization cycle is 1-5 times.

[0030] By adopting the above technical solution, the present invention has the following beneficial effects:

[0031] 1. The preparation method of the uric acid-lowering apigenin cyclodextrin inclusion complex liposome provided by the present invention is simple to operate and has mild reaction conditions. Apigenin is included in β-cyclodextrin to obtain an apigenin cyclodextrin inclusion complex. The cyclodextrin inclusion method can improve the water solubility of apigenin and increase the molecular volume to reduce its ability to pass through the phospholipid bilayer. The apigenin cyclodextrin inclusion complex is then encapsulated in liposomes to obtain uric acid-lowering apigenin cyclodextrin inclusion complex liposomes, which can fundamentally improve the stability of the liposomes and solve the problem of drug leakage in the liposomes in the liquid state, thereby reducing the solubility.

[0032] 2. The uric acid-lowering apigenin cyclodextrin inclusion complex liposomes prepared by the present invention have improved overall water solubility, good stability and sustained-release effect, and increased the bioavailability of apigenin. The uric acid-lowering effect is significantly better than that of untreated apigenin.

[0033] 3. The apigenin β-cyclodextrin inclusion complex liposome provided by the present invention expands the application scope of apigenin in the food field. Its good water solubility and stability enable it to be applied to various dosage forms such as solid beverages, liquid beverages and jellies, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other design solutions and drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is a comparison chart of the solubility of apigenin and apigenin β-cyclodextrin inclusion liposomes;

[0036] Figure 2 This is a comparison chart of the cumulative release of apigenin and apigenin β-cyclodextrin inclusion liposomes;

[0037] Figure 3 This is a comparison chart of the bioavailability of apigenin and apigenin β-cyclodextrin inclusion liposomes.

[0038] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. DETAILED DESCRIPTION

[0039] The content of the present invention can be further understood by combining the following detailed description of the preferred implementation method of the present invention and the embodiments included. Unless otherwise stated, all technical and scientific terms used in this article have the same meaning as those of ordinary skill in the art to which the present invention belongs. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in the present invention, the definition of the term provided in the present invention shall prevail.

[0040] It should be noted that the implementation conditions used in the examples can be further adjusted according to the specific experimental environment, and the implementation conditions not specified are usually the conditions in conventional experiments. The preparation methods mentioned in the present invention are all conventional methods unless otherwise specified; the various chemical reagents and chemicals mentioned in the following examples are all commercially available unless otherwise specified.

[0041] The invention firstly discloses a uric acid-reducing apigenin cyclodextrin inclusion compound liposome, which comprises the following components in parts by weight: 1 part of apigenin, 2-50 parts of beta-cyclodextrin (beta-cyclodextrin abbreviated as beta-CD), 0.8-30 parts of phospholipids and 0.1-10 parts of surfactant.

[0042] As a further preferred embodiment of the present invention, the surfactant is one of Tween 20, Tween 60 or Tween 80.

[0043] In some specific embodiments, the phospholipid is one of soybean lecithin and egg yolk lecithin.

[0044] The present invention further discloses a method for preparing apigenin cyclodextrin inclusion compound liposomes having uric acid lowering effect, comprising the following steps:

[0045] S1, weigh 2-50 parts of β-CD, add water at a certain temperature to dissolve it to prepare a β-CD solution, take 1 part of apigenin, add anhydrous ethanol to dissolve it, add it to the prepared β-CD solution under a certain temperature and stirring conditions, continue stirring for a certain time after adding, filter, prepare an apigenin β-CD aqueous solution, and store it at a certain temperature for later use;

[0046] S2. Weigh 0.8-30 parts of phospholipids and 0.1-10 parts of surfactant, add anhydrous ethanol to dissolve, and inject into the above-mentioned apigenin β-CD aqueous solution under stirring. After the injection is completed, keep the conditions unchanged and continue to stir for a certain period of time, then take out and remove the anhydrous ethanol. The resulting solution is homogenized by a high-pressure homogenizer to obtain apigenin β-cyclodextrin inclusion complex liposomes, that is, uric acid-lowering apigenin cyclodextrin inclusion complex liposomes.

[0047] As a further illustration of the technical solution of the present invention, in step S1, the water temperature for dissolving β-CD is 30-100°C.

[0048] In a specific embodiment, in step S1, the source of apigenin is the rhizomes and seeds of celery, western celery and parsley.

[0049] As a further preferred embodiment of the present invention, in step S1, the method of dissolving apigenin in the solvent is one of heating, stirring, ultrasound or grinding.

[0050] In a preferred embodiment, in step S1, the stirring speed is 600-1200 r / min, the temperature is 40-60° C., and the time is 1 h-24 h.

[0051] As a further preferred embodiment of the present invention, in step S1, the filtering method is one of membrane filtration, paperboard filtration or plate and frame filtration, and the pore size of the membrane, paperboard or filter cloth is 0.1-15 μm.

[0052] During specific implementation, in step S1, the storage temperature of the apigenin β-CD aqueous solution is 4°C-30°C.

[0053] As a further preferred embodiment of the present invention, in step S2, the phospholipid used is one of soybean lecithin and egg yolk lecithin.

[0054] In some preferred embodiments, in step S2, the surfactant used may be one of Tween 20, Tween 60 or Tween 80.

[0055] Preferably, in step S2, the stirring speed is 100-800 r / min, and stirring is continued for 0.5-8 h after all the apigenin β-cyclodextrin inclusion complex is injected.

[0056] As a further preferred embodiment of the present invention, in step S2, the method for removing anhydrous ethanol can be one of the methods of reduced pressure rotary evaporation, freeze drying, and vacuum drying.

[0057] In specific implementation, in step S2, the homogenization conditions are: homogenization pressure 500-1000 bar, and homogenization cycle 1-5 times.

[0058] The present invention will be further described below in conjunction with embodiments:

[0059] Example 1

[0060] This embodiment provides a method for preparing apigenin cyclodextrin inclusion complex liposomes with uric acid lowering effect, comprising the following steps:

[0061] S1. Weigh 50g of β-CD, add 80℃ water to dissolve it to prepare β-CD solution, take another 1g of apigenin, add anhydrous ethanol ultrasonic treatment (200w, 80kHZ) to dissolve it, then add it to the above prepared β-CD solution under stirring conditions of 40℃ and 800r / min, continue stirring for 6h, filter with 0.45μm membrane to obtain apigenin β-CD aqueous solution, and store it at 4℃ for later use.

[0062] S2. Weigh 30g of soybean lecithin and 10g of Tween 80 in a beaker, add anhydrous ethanol to dissolve and set aside. Slowly inject into the above-mentioned apigenin β-CD aqueous solution under stirring at 160r / min, continue stirring for 1h, take out, remove anhydrous ethanol by rotary evaporation under reduced pressure, and circulate and homogenize the obtained solution in a high-pressure homogenizer set at a pressure of 800bar for 3 times to obtain apigenin β-cyclodextrin inclusion liposomes.

[0063] Example 2

[0064] This embodiment provides a method for preparing apigenin cyclodextrin inclusion complex liposomes with uric acid lowering effect, comprising the following steps:

[0065] S1. Weigh 8g of β-CD and dissolve it in 45℃ water to prepare a β-CD solution. Take another 1g of apigenin and dissolve it in anhydrous ethanol. Then add it to the prepared β-CD solution under stirring conditions of 45℃ and 1000r / min. Continue stirring for 10h, filter it with a 0.8μm membrane to obtain an apigenin β-CD aqueous solution, and store it at 4℃ for later use.

[0066] S2. Weigh 2g of egg yolk lecithin and 0.5g of Tween 20 into a beaker, add anhydrous ethanol to dissolve, inject into the above-mentioned apigenin β-CD aqueous solution under stirring at 200r / min, continue stirring for 0.5h, then take out, remove the anhydrous ethanol by rotary evaporation under reduced pressure, and circulate and homogenize the obtained solution in a high-pressure homogenizer (pressure 400bar) for 5 times to obtain apigenin β-cyclodextrin inclusion liposomes.

[0067] Example 3

[0068] This embodiment provides a method for preparing apigenin cyclodextrin inclusion complex liposomes with uric acid lowering effect, comprising the following steps:

[0069] S1. Weigh 9.6g of β-CD and dissolve it in 55℃ water to prepare a β-CD solution. Take another 1g of apigenin and stir it in anhydrous ethanol at 600r / min to dissolve it. Then add it to the prepared β-CD solution at 55℃ and 600r / min, and continue stirring for 8h. Filter it through 0.45μm paperboard to obtain an apigenin β-CD aqueous solution, and store it at 4℃ for later use.

[0070] S2. Weigh 6.9g soybean lecithin and 2.4g Tween 40 in a beaker, add anhydrous ethanol to dissolve and set aside. Inject into the above apigenin β-CD aqueous solution under stirring at 300r / min, continue stirring for 2h, take out, freeze-dry to remove anhydrous ethanol, homogenize the obtained solution once in a high-pressure homogenizer at a pressure of 1000bar, and obtain apigenin β-cyclodextrin inclusion liposomes.

[0071] Example 4

[0072] This embodiment provides a method for preparing apigenin cyclodextrin inclusion complex liposomes with uric acid lowering effect, comprising the following steps:

[0073] S1. Weigh 2g of β-CD and dissolve it in 60℃ water to prepare a β-CD solution. Take another 1g of apigenin, dissolve it in anhydrous ethanol ultrasonic treatment (200w, 80kHZ), then add it to the prepared β-CD solution under stirring conditions of 60℃ and 1000r / min, and continue stirring for 10h. Filter through a 0.45μm membrane to obtain an apigenin β-CD aqueous solution, and store it at 4℃ for later use.

[0074] S2. Weigh 0.8g of egg yolk lecithin and 0.1g of Tween 40 in a beaker, add anhydrous ethanol to dissolve and set aside. Inject into the above-mentioned apigenin β-CD aqueous solution under stirring at 400r / min, continue stirring for 8h, take out, freeze-dry to remove anhydrous ethanol, and homogenize the obtained solution twice in a high-pressure homogenizer at a pressure of 700bar to obtain apigenin β-cyclodextrin inclusion liposomes.

[0075] Example 5

[0076] This embodiment provides a method for preparing apigenin cyclodextrin inclusion complex liposomes with uric acid lowering effect, comprising the following steps:

[0077] S1. Weigh 3.5g of β-CD and dissolve it in 80℃ water to prepare a β-CD solution. Take another 1g of apigenin and stir it in anhydrous ethanol at 600r / min to dissolve it. Then add it to the prepared β-CD solution at 80℃ and 600r / min, and continue stirring for 24h. Filter it through a 0.8μm membrane to obtain an apigenin β-CD aqueous solution, and store it at 4℃ for later use.

[0078] S2. Weigh 1.5g of phospholipid and 0.3g of Tween 40 in a beaker, add anhydrous ethanol to dissolve and set aside. Inject into the above-mentioned apigenin β-CD aqueous solution under stirring at 500r / min, continue stirring for 5h, take out, remove anhydrous ethanol by rotary evaporation under reduced pressure, homogenize the obtained solution 3 times in a high-pressure homogenizer at a pressure of 600bar, and obtain apigenin β-cyclodextrin inclusion liposomes.

[0079] Experiment 1: Solubility test and in vitro release test of liposomes of apigenin cyclodextrin inclusion complex of the present invention

[0080] Sample preparation: Refer to Example 1 to prepare apigenin β-cyclodextrin inclusion liposome 1. Refer to Example 3 to prepare apigenin β-cyclodextrin inclusion liposome 2. Refer to Example 4 to prepare apigenin β-cyclodextrin inclusion liposome 3.

[0081] Solubility experiment: Add excess untreated apigenin and apigenin β-cyclodextrin inclusion liposome 1, apigenin β-cyclodextrin inclusion liposome 2 and apigenin β-cyclodextrin inclusion liposome 3 into deionized water respectively, place in a constant temperature water bath oscillator, shake at a constant temperature of 30°C for 24 hours, centrifuge at 6000r / min in a high-speed centrifuge for 15 minutes, take the supernatant and dilute it appropriately with methanol and pass through a 0.45μm filter membrane, and detect the apigenin concentration therein by HPLC.

[0082] In vitro release experiment: Take untreated apigenin, apigenin β-cyclodextrin inclusion liposome 1, apigenin β-cyclodextrin inclusion liposome 2 and apigenin β-cyclodextrin inclusion liposome 3 containing 1 mg of apigenin, add 1 mL of methanol and 1 mL of PBS solution to dissolve them respectively, put them in 4 dialysis bags, tie them tightly at both sides to prevent leakage, put the dialysis bags in 80 mL of PBS solution (make sure the liquid levels inside and outside the dialysis bag are consistent), keep them at 37°C with constant temperature magnetic stirring, and release them at constant temperature for 24 hours. After the experiment started, 1 mL of sample was taken at 4.8 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 240 min, 360 min, 480 min and 720 min, and then 1 mL of PBS solution was added back to ensure that the total volume of the system remained unchanged. The samples were centrifuged and passed through the membrane, and the concentration of apigenin in the samples was detected by HPLC. The proportion of the cumulative release amount at each sampling moment to the total dosage was calculated, which was the cumulative release rate.

[0083] The content of apigenin was determined by high performance liquid chromatography. Chromatographic conditions: liquid chromatography column C18 column (250 mm x 4.6 mm, 5 μm); column temperature: room temperature; mobile phase composition: methanol and water ratio of 7:3 (containing 0.2% phosphoric acid); flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 350 nm.

[0084] The experimental results and conclusions are detailed in Figure 1 and Figure 2 .Depend on Figure 1 It can be seen that the solubility of untreated apigenin in deionized water is extremely low, the solubility of apigenin β-cyclodextrin inclusion liposome 1 and apigenin β-cyclodextrin inclusion liposome 2 is not much different, and the solubility of apigenin β-cyclodextrin inclusion liposome 3 is slightly lower. The solubility of the three apigenin β-cyclodextrin inclusion liposomes in deionized water is significantly improved compared with that of untreated apigenin, which are 640 times, 619 times and 407 times that of untreated apigenin respectively.

[0085] Depend on Figure 2It can be seen that the cumulative release of the three apigenin β-cyclodextrin inclusion liposomes is higher than that of untreated apigenin at all times. Untreated apigenin is slowly released from 0 to 60 minutes, and the release amount remains almost unchanged thereafter, reaching dissolution equilibrium at 60 minutes, and the cumulative release rate within 720 minutes is 24.61%. The three apigenin β-cyclodextrin inclusion liposomes are continuously slowly released from 0 to 120 minutes. As the release progresses, the release rate gradually slows down after 120 minutes. Apigenin β-cyclodextrin inclusion liposome 1 and apigenin β-cyclodextrin inclusion liposome 3 finally reach release equilibrium at 360 minutes, and the cumulative release rates at 720 minutes are 96.58% and 91.5%, respectively, which are 3.92 times and 3.72 times that of untreated apigenin. The release rate of apigenin β-cyclodextrin inclusion liposomes gradually slows down after 120 minutes, and the dissolution rate is close to equilibrium at 480 minutes, and the cumulative release rate at 720 minutes is 98.7%. It is 4.01 times that of untreated apigenin. It can be seen that the three apigenin β-cyclodextrin inclusion liposomes have effectively prolonged the release time, achieved the effect of sustained release, and greatly improved the cumulative release rate. Therefore, apigenin β-cyclodextrin inclusion liposomes may be rapidly absorbed by the body after oral administration, delaying release and producing better clinical application effects. Among them, apigenin β-cyclodextrin inclusion liposome 2 reaches the release equilibrium time longer and has a higher cumulative release rate. Therefore, Example 3 was selected to prepare apigenin β-cyclodextrin inclusion liposomes for subsequent animal tests.

[0086] Experiment 2: Experiment on reducing uric acid in hyperuricemia rats by using apigenin β-cyclodextrin inclusion complex liposomes of the present invention

[0087] (1) Animal husbandry:

[0088] 32 SPF male SD rats, 7-8 weeks old, weighing 180-200 g, were housed in a room with a temperature of 20-25°C and a humidity of 50-60%, and water and feed were changed regularly every day.

[0089] (2) Sample preparation:

[0090] Referring to Example 3, apigenin β-cyclodextrin inclusion liposomes were prepared.

[0091] (3) Experimental methods

[0092] The animals were purchased and observed for 3 days and then randomly divided into 4 groups: negative control group, model control group, untreated apigenin group, and apigenin β-cyclodextrin inclusion liposome group. There were 8 animals in each group. The model was established by potassium oxonate-yeast extract combined method. At 8 am every day, the untreated apigenin group and apigenin β-cyclodextrin inclusion liposome group were given a dose of 300 mg / kg (calculated based on apigenin content), and the negative control group and model control group were given an equal amount of 0.5% CMC-Na solution. This lasted for 14 consecutive days, once a day. At 4 pm every day, except for the negative control group which was given 0.5% CMC-Na solution, the mice were given yeast extract by gavage at 10 g / (kg·d) for 14 consecutive days, once a day. During the experiment, each group had free access to water and fasted at night (19:00-7:00 the next day). At 8:00 a.m. on the 14th day, each group was given the corresponding test article. One hour later, except for the normal group which was injected with an equal amount of 0.5% CMC-Na solution, each group was intraperitoneally injected with 300 mg / kg of potassium oxonate. 30 minutes after administration, 1.5 mL of blood was collected from the retro-orbital venous plexus of the mice. The blood was naturally coagulated at room temperature for 1 hour and centrifuged at 7000 r / min for 10 minutes. The supernatant was taken to determine serum uric acid, creatinine and urea nitrogen.

[0093] (4) Determination of liver xanthine oxidase activity

[0094] After the last administration, 0.4 g of liver was taken and added to 4 ml of normal saline to prepare liver homogenate. Centrifuge at 10000 r / min for 15 min, take 500 μL of supernatant and place it in a 1.5 ml centrifuge tube, add 500 μL Tris-HCl buffer (pH 8.4), 30 μl xanthine solution (24 mmol / L), and 50 μl xanthine oxidase enzyme solution (3200 μ / L) in sequence. Mix well and heat in a 37°C constant temperature water tank for 20 min, and add 30 μl HCL (7 mol / L) to terminate the reaction. The reaction solution was filtered with a 0.45 μm filter membrane and analyzed by HPLC. The amount of enzyme required to convert 1 μmol xanthine into uric acid per minute is defined as one xanthine oxidase activity unit (U), and the unit is μmol / min. The enzyme activity calculation formula is: enzyme activity = (C-C0) × V0 × n÷t;

[0095] Wherein, C is the amount of uric acid produced (mmol / L); C0 is the blank control (mmol / L); V0 is the total volume of the system (ml); n is the dilution multiple, and t is the reaction time (20 min).

[0096] Table 1 Effects of different treatment groups on urea, creatinine, uric acid and xanthine oxidase activity in rats

[0097]

[0098] Note: Compared with the negative control group #p<0.05; ##p<0.01.

[0099] Compared with the model control group *p<0.05; **p<0.01.

[0100] Since uric acid has low solubility, excessive deposition in the body can cause serious harm such as urinary tract stones or gout. Therefore, uric acid value is the most direct indicator of whether the model is successful or not and the effect of lowering uric acid. As can be seen from the table, the uric acid (SUA) and xanthine oxidase (XO) values ​​in the serum of the model control group were significantly different from those in the negative control group (p<0.01), indicating that the rat hyperuricemia model was successfully established. The untreated apigenin group showed a decreasing trend in SUA and XO values ​​compared with the model control group, but there was no significant difference. Compared with the model control group, the apigenin β-cyclodextrin inclusion complex liposome group can reduce the uric acid content in the serum of hyperuricemia rats, which is extremely significant (p<0.01), and can also significantly (p<0.05) reduce the activity of xanthine oxidase.

[0101] Since hyperuricemia can cause a certain degree of damage to the kidneys, the renal function indicators urea nitrogen (BUN) and creatinine (CRE) were detected to determine the health of the renal function of mice after administration. As can be seen from the table, the BUN and CRE values ​​in the serum of the model control group were significantly (p<0.01) higher than those in the negative control group, indicating that the kidneys were severely damaged. The untreated apigenin group had lower BUN and CRE values ​​than the model control group, but the difference was not statistically significant. Compared with the model control group, the apigenin β-cyclodextrin inclusion liposome group can significantly (p<0.01) reduce the level of BUN, and the level of CRE also has a significant (p<0.05) difference. This shows that apigenin β-cyclodextrin inclusion liposomes have a protective effect on the renal function of animals while lowering uric acid.

[0102] Experiment 3: Determination of bioavailability of apigenin β-cyclodextrin inclusion complex liposomes of the present invention.

[0103] (1) Animal husbandry: Ten healthy female SD rats (230 ± 17 g) were selected and fed normally in the animal laboratory for one week before the experiment to allow them to adapt to the current environment.

[0104] (2) Sample preparation: Prepare apigenin β-cyclodextrin inclusion complex liposomes according to Example 3.

[0105] (3) Experimental method: The night before the experiment, rats were fasted but not watered. At the beginning of the experiment, SD rats were randomly divided into two groups, 5 rats in each group, and parallel experiments were carried out. The two groups of rats were gavaged with untreated apigenin group and apigenin β-cyclodextrin inclusion liposome group, respectively, and a dose of 60 mg / kg (in terms of apigenin content) was given according to the body weight of the rats. At 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h, 8h, 12h, and 24h after administration, the rats were stunned and an appropriate amount of blood was quickly collected from the fundus venous plexus of the rats using a capillary glass tube. After the blood was fully mixed with the sodium heparin aqueous solution in a centrifuge tube, the lower layer of red blood cells was removed by centrifugation, and 200μL of the upper layer of serum was collected and stored in a -40℃ environment.

[0106] (4) Serum treatment method: First, the serum was thawed at room temperature, and then 600uL of methanol was added to each centrifuge tube containing serum. The tubes were shaken and vortexed for 3 minutes. Then, centrifugation was performed at 10,000rpm for 10 minutes. After centrifugation, the supernatant was removed and placed in another 2mL centrifuge tube. 600μL of methanol was added to the remaining precipitate. The tubes were shaken and vortexed for 3 minutes. The tubes were centrifuged at 10,000rpm for 10 minutes. The supernatant was removed and combined with the first supernatant. The combined total supernatant was placed in a nitrogen blower. The methanol and water in the supernatant were blown dry by a gentle stream of nitrogen. The apigenin residue in the serum adhered to the wall of the centrifuge tube. Before testing, accurately take 100 μL of methanol and place it in a centrifuge tube containing apigenin residues, and ultrasonically treat it for 3 minutes to fully dissolve the apigenin adhering to the centrifuge tube in methanol. Then, centrifuge it at 10,000 rpm for 10 minutes, take out the supernatant, and perform HPLC detection.

[0107] Figure 3 is the bioavailability of apigenin and apigenin β-cyclodextrin inclusion liposomes. The apigenin concentration in the apigenin β-cyclodextrin inclusion liposomes continued to rise from 0 to 1.5h, reaching a maximum at 1.5h. From 1.5h to 24h, the apigenin concentration in the apigenin β-cyclodextrin inclusion liposomes continued to decrease. For untreated apigenin, it reached the maximum blood concentration at 2.5h. It can be seen that the time (T) for apigenin β-cyclodextrin inclusion liposomes to reach the maximum blood concentration in rats max ) is significantly shorter than that of untreated apigenin. By calculation, the maximum blood drug concentration value (C maxThe value) is 4.67 times that of untreated apigenin. And by calculating the area under the plasma concentration-time curve (AUC), it can be concluded that apigenin β-cyclodextrin inclusion liposomes are 6.82 times that of untreated apigenin. Therefore, apigenin β-cyclodextrin inclusion liposomes have higher oral bioavailability than untreated apigenin.

[0108] The above description is only a preferred embodiment of the present invention, which is merely illustrative of the present invention and does not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A cyclodextrin inclusion compound liposome of apigenin for lowering uric acid, characterized in that: The uric acid-lowering apigenin cyclodextrin inclusion compound liposome comprises the following components in parts by weight: 1 part of apigenin, 2-50 parts of beta-cyclodextrin, 0.8-30 parts of phospholipids, and 0.1-10 parts of surfactants.

2. The cyclodextrin inclusion compound liposome of apigenin for lowering uric acid as claimed in claim 1, characterized in that: The surfactant is one of Tween 20, Tween 60 or Tween 80.

3. The cyclodextrin inclusion compound liposome of apigenin for lowering uric acid as claimed in claim 1, characterized in that: The phospholipid is one of soybean lecithin and egg yolk lecithin.

4. A method for preparing uric acid-lowering apigenin cyclodextrin inclusion compound liposomes, characterized in that: The following steps are involved: S1. Preparation of apigenin β-cyclodextrin aqueous solution Weigh 2-50 parts of β-cyclodextrin, add water at a certain temperature to dissolve it and prepare a β-cyclodextrin aqueous solution; take 1 part of apigenin, add anhydrous ethanol to dissolve it, add it to the prepared β-cyclodextrin aqueous solution under a certain temperature and stirring conditions, continue stirring for a certain time after adding, filter, and prepare the apigenin β-cyclodextrin aqueous solution, which is stored at a certain temperature for later use; S2. Preparation of apigenin β-cyclodextrin inclusion liposomes Weigh 0.8-30 parts of phospholipids and 0.1-10 parts of surfactants, add anhydrous ethanol to dissolve, and inject them into the above-mentioned apigenin β-cyclodextrin aqueous solution under stirring. After the injection is completed, continue to stir for a certain period of time and then take them out, remove the anhydrous ethanol, and homogenize the obtained solution through a homogenizer to obtain apigenin β-cyclodextrin inclusion liposomes.

5. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S1, the filtering method is membrane filtration, paperboard filtration or plate and frame filtration.

6. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S1, the method of dissolving apigenin in the solvent is one of heating, stirring, ultrasound or grinding.

7. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S1, the water temperature for dissolving the β-cyclodextrin is 30-100°C; the storage temperature of the apigenin β-cyclodextrin aqueous solution is 4°C-30°C.

8. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S1, the stirring speed is 600-1200 r / min, the temperature is 40-60° C., and the stirring time is 1 h-24 h; In step S2, the stirring speed is 100-800 r / min, and stirring is continued for 0.5-8 h after all the apigenin β-cyclodextrin aqueous solution is injected.

9. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S2, the method for removing anhydrous ethanol can be one of the methods of reduced pressure rotary evaporation, freeze drying, and vacuum drying.

10. The method for preparing the uric acid-lowering apigenin cyclodextrin inclusion compound liposome according to claim 4, characterized in that: In step S2, the homogenization conditions are: homogenization pressure 500-1000 bar, and homogenization cycle 1-5 times.

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