Dendrobium officinale flavone nano-liposome with uric acid reducing activity as well as preparation method and application of dendrobium officinale flavone nano-liposome

Abstract

The invention discloses a dendrobium officinale flavone nano-liposome with uric acid reducing activity as well as a preparation method and application thereof. The nano-liposome is prepared based on a sterol substance, a phospholipid substance and a cationic lipid. Wherein the sterol substance comprises phytosterol ester and cholesterol, the phospholipid substance is dipalmitoyl phosphatidylcholine (DPPC), and the cationic lipid is (2, 3-dioleoyl-propyl)-trimethylamine (DOTAP); and a functional active substance dendrobium officinale flavone is encapsulated. The dendrobium officinale flavone nano-liposome is prepared by adopting an ethanol injection method, and the nano-liposome can reduce the content of uric acid and xanthine oxidase, inhibit the level of inflammatory factors and relieve kidney and liver injury, so that hyperuricemia is relieved.

Description

Technical Field

[0001] The invention relates to the technical field of nano liposome preparation, and in particular to a dendrobium officinale flavonoid nano liposome with uric acid lowering activity, and a preparation method and application thereof. Background Art

[0002] Uric acid (UA) is the end product of purine metabolism in the human body. In a normal body, the production and excretion of uric acid are in a dynamic balance. When the balance is broken and the concentration of uric acid in the body is too high, it is easy to cause hyperuricemia (HUA). Hyperuricemia is a major risk factor for gout and chronic kidney disease. At present, the prevention and treatment of hyperuricemia mainly include drug therapy and reducing the intake of purine-containing compounds. However, these drugs can easily cause kidney and liver damage. Therefore, finding methods to improve hyperuricemia has received increasing attention.

[0003] Dendrobium officinale flavonoids are natural flavonoid compounds with multiple physiological activities, such as antioxidant, antibacterial and anti-inflammatory activities. Existing studies have reported that Dendrobium officinale flavonoids can inhibit the activities of xanthine oxidase and adenosine deaminase. In addition, Dendrobium officinale flavonoids protect the kidneys of hyperuricemia mice from damage by inhibiting the increase in the levels of proinflammatory cytokines. However, the low solubility of Dendrobium officinale flavonoids hinders its transport across biological membranes, thereby reducing its availability. This has largely limited the application of Dendrobium officinale flavonoids in the pharmaceutical field.

[0004] Nanoliposomes are spherical closed vesicles containing a lipid bilayer structure formed by the hydration of amphiphilic lipid molecules such as phospholipids dispersed in an aqueous phase. Nanoliposomes can encapsulate both water-soluble and fat-soluble drugs. As a drug carrier, nanoliposomes have attracted attention and been widely used due to their high bioavailability, long drug action time, and ability to control drug release. Summary of the invention

[0005] The purpose of the present invention is to provide a Dendrobium officinale flavonoids nanoliposome with uric acid lowering activity and a preparation method thereof, wherein the preparation process of the method is short in time consumption and high in efficiency, and the obtained Dendrobium officinale flavonoids nanoliposomes have high encapsulation rate and good stability. The embedding of the nanoliposomes improves the uric acid lowering ability of Dendrobium officinale flavonoids, and provides an important theoretical basis for expanding the application of Dendrobium officinale flavonoids in the food field.

[0006] The specific technical solutions adopted by the present invention are as follows:

[0007] A dendrobium officinale flavonoid nanoliposome with uric acid lowering activity is prepared based on sterols, phospholipids and cationic lipids, and encapsulates dendrobium officinale flavonoids, wherein the sterols include one or more of phytosterol esters or cholesterol.

[0008] Furthermore, the phospholipid substance is dipalmitoylphosphatidylcholine (DPPC), and the cationic lipid is (2,3-dioleoyl-propyl)-trimethylamine (DOTAP).

[0009] The preparation method of the Dendrobium officinale flavonoid nanoliposome comprises the following steps:

[0010] (1) Weigh sterols, phospholipids and DOTAP and add them to anhydrous ethanol-chloroform; add Dendrobium officinale flavonoids to anhydrous ethanol-chloroform; mix and completely dissolve by ultrasonication.

[0011] (2) Use a syringe to absorb the organic phase obtained in (1) and slowly inject it into a glucose aqueous solution containing Tween 80 to obtain a suspension.

[0012] (3) The suspension is transferred to another container, connected to a rotary evaporator, and the anhydrous ethanol-chloroform solution is completely removed in a water bath under vacuum to obtain a nanoliposome suspension. The water bath temperature is preferably 45°C.

[0013] (4) The nanoliposome suspension is passed through a micro extruder with a polycarbonate filter having a pore size of 100 nm for 5 to 10 times, thereby obtaining Dendrobium officinale flavonoid nanoliposomes having uric acid-lowering activity.

[0014] Furthermore, the sterol substance in step (1) is phytosterol ester or cholesterol, and the phospholipid substance is DPPC. The mass ratio of the sterol substance, DPPC and DOTAP is 1-4.5:3.5-10:1.

[0015] Furthermore, the concentration of Dendrobium officinale flavonoids in the mixed solution in step (1) is 2.5 to 9 mg / mL;

[0016] Furthermore, in the mixed solution in step (1), the volume ratio of anhydrous ethanol-chloroform to the nanoliposome material, namely, sterols, DPPC and DOTAP, is 7-20:1.

[0017] Furthermore, in the glucose aqueous solution described in step (2), the mass fraction of glucose is 5%, the pH value of the solution is 5.4, the content of Tween 80 is 0.2 mg / mL, and the amount of glucose aqueous solution added is equivalent to the volume of anhydrous ethanol-chloroform in step (1).

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The present invention aims to prepare Dendrobium officinale flavonoid nanoliposomes using different nanoliposome wall materials. Among them, in particular, plant sterol esters are used instead of cholesterol as the membrane material of nanoliposomes, which overcomes the adverse effects of traditional cholesterol nanoliposomes and gives the nanoliposomes the beneficial physiological activity of plant sterol esters, while having considerable physicochemical properties and encapsulation efficiency. The protection and sustained release of the nanoliposomes protect the embedded Dendrobium officinale flavonoids from the destruction of the digestion process, so that its biological activity can be better exerted.

[0020] (2) The Dendrobium officinale flavonoids nanoliposome prepared by the present invention not only solves the problem of low oral bioavailability of Dendrobium officinale flavonoids, but also gives it a special uric acid-lowering ability due to its nano-scale size. Dendrobium officinale flavonoids nanoliposomes can inhibit inflammatory factors, significantly improve kidney and liver damage, and relieve hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 H&E staining of mouse kidney tissue sections of the animal experiment control group, model group, comparative example group (positive drug benzbromarone and Dendrobium officinale flavonoids treatment) and example group (Dendrobium officinale flavonoids nanoliposome treatment) provided by the present invention. The scale is 100 μm, and different letters represent significant differences (p<0.05).

[0022] Figure 2 The statistical results of ALT and AST in the serum and liver of mice in the animal experiment control group, model group, comparative example group (treated with positive drug benzbromarone and Dendrobium officinale flavonoids) and embodiment group (treated with Dendrobium officinale flavonoids nanoliposomes) provided by the present invention. Different letters represent significant differences (p<0.05).

[0023] Figure 3 The statistical results of the animal experiment control group, model group, comparative example group (positive drug benzbromarone and Dendrobium officinale flavonoids treatment) and embodiment group (Dendrobium officinale flavonoids nanoliposome treatment) on mouse serum and kidney inflammatory factors (IL-1β, IL-18) provided by the present invention. Different letters represent significant differences (p<0.05).

[0024] Figure 4 The immunohistochemical staining diagram of IL-1β in the kidney of mice in the animal experiment control group, model group, comparative example group (treated with positive drug benzbromarone and Dendrobium officinale flavonoids) and embodiment group (treated with Dendrobium officinale flavonoids nanoliposomes) provided by the present invention. The scale is 50 μm, and different letters represent significant differences (p<0.05). DETAILED DESCRIPTION

[0025] The present invention is further explained below in conjunction with specific examples. The present invention is implemented according to the technical solution of the invention, and detailed implementation methods and operating steps are given, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0026] Example 1. Preparation and characterization of phytosterol ester Dendrobium officinale flavonoid nanoliposomes

[0027] First, a Dendrobium officinale flavonoid nanoliposome is prepared, and the specific steps are as follows:

[0028] (1) The total lipid and flavonoids of Dendrobium officinale were dissolved in 5 mL of anhydrous ethanol:chloroform (volume ratio 1:1) solution respectively and mixed; the concentration of flavonoids of Dendrobium officinale was 0.6 mg / mL, and the components of the total lipid were phytosterol esters, DPPC, and DOTAP, and the mass ratio of the three was 2.5:6.5:1. Ultrasonic frequency was 50 Hz, and ultrasonication was performed for 10-15 min to completely dissolve them.

[0029] (2) Using a 1 ml sterile syringe, the organic phase was slowly injected into 10 ml of a Tween 80-containing glucose aqueous solution (glucose mass fraction 5%, pH = 5.3; Tween 80 content 0.2 mg / mL) to obtain a suspension.

[0030] (3) The suspension was transferred to a 250 mL round-bottom flask, and the flask was connected to a rotary evaporator. The anhydrous ethanol-chloroform solution was completely removed in a 45° C. water bath under vacuum to obtain a nanoliposome suspension.

[0031] (4) The nanoliposome suspension is passed through a micro extruder with a polycarbonate filter having a pore size of 100 nm for 5 to 10 times to obtain Dendrobium officinale flavonoid nanoliposomes.

[0032] The above-mentioned Dendrobium officinale flavonoids nanoliposomes were put into a sample bottle to obtain the potential, particle size and encapsulation efficiency; and animal experiments were carried out.

[0033] Example 2: Preparation and characterization of phytosterol ester Dendrobium officinale flavonoid nanoliposomes

[0034] In this example, the mass ratio of total fat added is adjusted to phytosterol ester: DPPC: DOTAP = 1.5: 6.5: 1. The rest of the contents are exactly the same as those in Example 1. The results are shown in Table 1.

[0035] Example 3. Preparation and characterization of phytosterol ester Dendrobium officinale flavonoid nanoliposomes

[0036] In this example, the mass ratio of total fat added was adjusted to phytosterol ester: DPPC: DOTAP = 3.5: 6.5: 1. The rest of the contents are exactly the same as those in Example 1. The results are shown in Table 1.

[0037] Example 4. Preparation and characterization of cholesterol-dendrobe flavonoids nanoliposomes

[0038] Except that the added sterol is cholesterol, the rest of the contents of this example are exactly the same as those of Example 1. The results are shown in Table 1.

[0039] Comparative Example 1, Benzbromarone solution

[0040] Benzbromarone was dissolved in a 0.5% sodium carboxymethyl cellulose solution to prepare a 2 mg / mL benzbromarone solution. In this comparative example, only the positive drug benzbromarone 20 mg / kg / d was used to treat hyperuricemia mice to evaluate its inhibitory effect on hyperuricemia. The results are as follows Figures 1 to 4 shown.

[0041] Comparative Example 2, Dendrobium officinale flavonoids solution

[0042] Dissolve the flavonoids of Dendrobium officinale in a 0.5% sodium carboxymethyl cellulose solution to prepare a 5 mg / mL solution of flavonoids of Dendrobium officinale. This comparative example only uses 50 mg / kg / d of the flavonoids of Dendrobium officinale solution to treat hyperuricemia mice to evaluate its inhibitory effect on hyperuricemia. The results are as follows: Figures 1 to 4 shown.

[0043] Experiment on hyperuricemia mice

[0044] (1) Experimental animals

[0045] A total of 42 healthy male Kunming mice (18-22 g) were selected and purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. (Hangzhou, China) (SCXK (Zhe) 2022-0005). The animals were adapted for 1 week before the experiment. All animals were allowed to eat and drink normally. Animal breeding environment: temperature 22±2℃, humidity 60±5% specific pathogen-free (SPF) conditions, fixed 12h artificial light. This study was guided and approved by the Animal Ethics Committee of the School of Medicine, Zhejiang University of Technology (MGS20230711097).

[0046] (2) Establishment of hyperuricemia mouse model and drug administration

[0047] All mice were randomly divided into 6 groups (n=7). Except for the control group, the rest of the mice were given potassium oxonate (200 mg / kg / d) and hypoxanthine (100 mg / kg / d) intraperitoneal injection to induce hyperuricemia. One hour after the administration of the model, the control group and the model group were gavaged with normal saline, and the other groups were gavaged with the corresponding drugs. The dose of the Dendrobium officinale flavonoids nanoliposome group was 40 mg / kg / d for 14 days. The whole experiment lasted 21 days. One hour after the treatment on the 14th day, the mice were killed, and blood samples and liver and kidney tissues were collected and stored at -80°C for later use.

[0048] (3) Detection

[0049] The kit detects serum UA, urea nitrogen, creatinine and xanthine oxidase levels;

[0050] The kit detects the ALT and AST levels in serum and liver.

[0051] ELISA kits were used to detect the levels of IL-1β and IL-18 in mouse serum and kidney.

[0052] H&E staining: kidney tissue was fixed in 4% paraformaldehyde solution, dehydrated with gradient alcohol, routinely embedded in paraffin, sliced ​​at 5 μm, and read under a light microscope after H&E staining.

[0053] IHC staining: paraffin-embedded kidney sections were dewaxed with xylene and dehydrated with graded concentrations of alcohol. Antigen retrieval was performed by autoclaving at 120°C for 15 min in citrate buffer (pH 6.0), and then the sections were incubated with 3% hydrogen peroxide to inactivate endogenous peroxidase. Blocked and incubated with IL-1β antibody at 4°C overnight. Secondary antibodies were then incubated at room temperature, color was developed, and cell nuclei were counterstained. Finally, the sections were dehydrated and mounted, and the results were obtained by microscopy.

[0054] Table 1 Particle size, potential and encapsulation efficiency of different Dendrobium officinale flavonoid nanoliposomes

[0055] Group Particle size (nm) Potential (mV) Encapsulation rate (%) Example 1 <![CDATA[179.50±3.96 c ]]> <![CDATA[40.87±0.97 a ]]> <![CDATA[79.93±2.74 a ]]> Example 2 <![CDATA[204.78±2.61 b ]]> <![CDATA[37.40±0.92 b ]]> <![CDATA[55.51±8.89 b ]]> Example 3 <![CDATA[226.79±3.43 a ]]> <![CDATA[37.47±1.00 b ]]> <![CDATA[52.56±5.53 b ]]> Example 4 <![CDATA[184.77±1.46 bc ]]> <![CDATA[35.16±0.02 c ]]> <![CDATA[77.33±4.55 a ]]>

[0056] As can be seen from Table 1, the particle size of the Dendrobium officinale flavonoids nanoliposomes prepared in Example 1 and Example 4 is relatively small, and there is no significant difference, and the particle size is within the range of 179.50 to 184.77 nm. The Zeta potential of all the Dendrobium officinale flavonoids nanoliposomes prepared in the experiment is positively charged, and the Zeta potential is between 35.16 and 40.87 mV, indicating that all the nanoliposomes are in a stable stage. In addition, there are differences in the encapsulation efficiency of Dendrobium officinale flavonoids provided by the nanoliposomes in Examples 1 to 4. Among them, the encapsulation efficiency of Dendrobium officinale flavonoids in Example 1 and Example 4 is the largest.

[0057] Table 2 Physiological parameters of hyperuricemia mice

[0058]

[0059] Table 2 shows the serum uric acid and uric acid index content of each treatment group mouse after treatment, and multiple comparisons and significant difference marks are performed on the data therein. As shown in Table 2, the model group uric acid, urea nitrogen, creatinine content and xanthine oxidase activity are the highest, and the uric acid content is reduced to about half of it in Comparative Example 1 (positive drug), and the xanthine oxidase activity is also reduced, indicating that the positive drug can reduce the uric acid content and the ability to inhibit the activity of xanthine oxidase. And in Examples 1 and 4, nanoliposomes intervene in the synthesis of xanthine oxidase and uric acid in hyperuricemia mice, causing serum uric acid physicochemical indexes and xanthine oxidase activity to present various degrees of reduction. Wherein, Example 1 (phytosterol ester Dendrobium officinale flavonoids nanoliposomes) is the most significant.

[0060] like Figure 1 As shown in A, the renal tissue of the control group mice was normal in morphology and showed no signs of inflammation. The renal tissue of the model group showed severe renal histopathological damage, characterized by atrophic glomeruli and unusual tubular structures. However, Examples 1 and 4 showed relatively intact glomeruli and regular tubules. Compared with the model group (3.94), the histopathological scores of Examples 1 and 4 were significantly reduced to 1.06-1.33 (p<0.05; Figure 1 B). This indicates that Dendrobium officinale flavonoids can effectively reduce kidney damage caused by hyperuricemia.

[0061] like Figure 2 As shown in AD, in order to evaluate the potential hepatotoxicity of the experimental group to hyperuricemia mice, serum AST, ALT and liver ALT and AST levels were measured. Compared with the model group, Comparative Example 1 (benzbromarone) had no significant difference in serum ALT and AST levels in hyperuricemia mice, showing certain liver damage side effects. While Examples 1 and 4 showed lower levels, indicating that the nanoliposomes did not damage the liver.

[0062] In order to evaluate the degree of renal inflammation, the levels of inflammatory cytokines in serum and renal tissue of hyperuricemia mice were further measured. Compared with the model group, Comparative Example 2 (Dendrobium officinale flavonoids) did not show a significant inhibitory effect on inflammatory factors in the kidney. The embedding of nanoliposomes better exerts the physiological activity of Dendrobium officinale flavonoids. The nanoliposomes provided in Example 1 significantly inhibited the expression of inflammatory cytokines, including serum IL-1β, IL-18 and renal IL-18 ( Figure 3 AD; p<0.05). In addition, immunohistochemical examination of renal IL-1β also showed similar results ( Figure 4AB). The results show that after encapsulation by nanoliposomes, Example 1 and Example 4 can significantly reduce the level of renal inflammatory factor IL-1β, thereby reducing renal damage and achieving the effect of relieving HUA.

Claims

1. A Dendrobium officinale flavonoid nanoliposome with uric acid-lowering activity, characterized in that: The invention is prepared based on sterol substances, phospholipid substances and cationic lipids and is embedded with Dendrobium officinale flavonoids; the sterol substances include one or more of cholesterol and plant sterol esters.

2. The Dendrobium officinale flavonoid nanoliposome with uric acid lowering activity according to claim 1, characterized in that: The phospholipid substance is dipalmitoylphosphatidylcholine (DPPC), and the cationic lipid is (2,3-dioleoyl-propyl)-trimethylamine (DOTAP).

3. The Dendrobium officinale flavonoid nanoliposome with uric acid lowering activity according to claim 1, characterized in that: The sterol substances are phytosterol esters.

4. The method for preparing the Dendrobium officinale flavonoid nanoliposomes having uric acid lowering activity according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Weigh sterols, phospholipids, and DOTAP into a container, add anhydrous ethanol-chloroform, and dissolve Dendrobium officinale flavonoids in anhydrous ethanol-chloroform; mix and sonicate to completely dissolve; (2) using a syringe to absorb the organic phase obtained in (1), and injecting it into a glucose aqueous solution containing Tween 80 to obtain a suspension; (3) transferring the suspension into another container and connecting it to a rotary evaporator to completely remove the anhydrous ethanol-chloroform solution in a water bath under vacuum to obtain a nanoliposome suspension; (4) The nanoliposome suspension is passed through a micro extruder with a polycarbonate filter having a pore size of 100 nm for 5 to 10 times, thereby obtaining Dendrobium officinale flavonoid nanoliposomes having uric acid-lowering activity.

5. The method for preparing the Dendrobium officinale flavonoid nanoliposomes having uric acid lowering activity according to claim 4, characterized in that: The sterol substance in step (1) is plant sterol ester or cholesterol, and the phospholipid substance is DPPC; the mass ratio of sterol substance, DPPC and DOTAP is 1.5-4:4.5-10:

1.

6. The method for preparing the Dendrobium officinale flavonoid nanoliposomes having uric acid lowering activity according to claim 4, characterized in that: The concentration of Dendrobium officinale flavonoids in the mixed solution is 2.5-9 mg / mL.

7. The method for preparing the Dendrobium officinale flavonoid nanoliposomes having uric acid lowering activity according to claim 4, characterized in that: After mixing, the volume ratio of anhydrous ethanol-chloroform to nanoliposome materials, namely sterol substances, DPPC and DOTAP, in the solution is 7-20:

1.

8. The method for preparing the Dendrobium officinale flavonoid nanoliposomes having uric acid lowering activity according to claim 4, characterized in that: In the glucose aqueous solution described in step (2), the mass fraction of glucose is 5%, the pH value of the solution is 5.4, the content of Tween 80 is 0.2 mg / mL, and the amount of glucose aqueous solution added is 1 times the volume of the anhydrous ethanol-chloroform used in step (1).

9. Use of the Dendrobium officinale flavonoid nanoliposomes with uric acid-lowering activity as claimed in any one of claims 1 to 3 in the preparation of uric acid-lowering drugs.

Images