Efficient green naringin nano system and preparation method thereof

Abstract

The invention relates to the technical field of nano-drug preparation, in particular to an efficient green naringin nano-system and a preparation method thereof.The preparation method comprises the following steps that 1, naringin and citrus pectin in a certain proportion are evenly dispersed in deionized water, and a uniform suspension is obtained; step 2, heating and stirring the suspension at the temperature of 75 DEG C to 90 DEG C, so as to obtain a naringin nano suspension; 3, the cooled naringin nano suspension is subjected to multi-step centrifugal treatment, precipitates are taken and dispersed again with deionized water, and finally the naringin nano system is obtained.According to the efficient and green naringin nano system and the preparation method thereof, the preparation process is simple, and the oral absorption efficiency can be promoted under the condition that the anti-inflammatory drug effect is reserved.

Description

Technical Field

[0001] The invention relates to the technical field of nano drug preparation, and in particular to a high-efficiency green naringin nano system and a preparation method thereof. Background Art

[0002] Naringin is an important active ingredient in many traditional Chinese medicines and has significant biological activities, such as antitussive, anti-inflammatory and antioxidant. However, due to the low solubility and slow dissolution rate of naringin, its oral bioavailability is poor and its efficacy cannot be fully exerted in the body, thus limiting its extensive clinical application. At present, the solubility improvement methods for poorly soluble drugs mainly include salt formation, co-solvent, micronization, solid dispersion and prodrug preparation, but each of these methods has certain limitations.

[0003] Nanotechnology involves the technology of manufacturing single atoms or molecules, or processing macromolecules into nanometer-sized particles, including drug-embedded nanocapsules made of polymer materials. Drug preparations prepared by nanotechnology have many excellent properties, including sustained release, extended half-life of drugs in the body, extended duration of drug action, targeted delivery to specific organs, reduced dosage, reduced or eliminated toxic side effects, improved drug stability, increased membrane permeability and improved drug solubility.

[0004] There are many forms of naringin nanosystems. For example, Chinese patent CN114344268A discloses naringin nanoparticles with poly(hydroxyethyl methacrylate) as a carrier and a preparation method thereof, which can increase the drug loading capacity of naringin, enhance the sustained-release effect, and effectively improve its anti-inflammatory effect, while suppressing cough and reducing the toxic and side effects of the drug; Chinese patent CN101543476B discloses a naringin solid dispersion and a preparation method thereof, which improves its solubility in water and in vitro dissolution rate, and can also improve its bioavailability.

[0005] However, the above-mentioned preparations and preparation methods have problems such as difficulty in obtaining materials, complex preparation processes, and limited promotion and application. Therefore, how to develop an oral nanosystem with simple preparation conditions, easy-to-obtain materials, simple process, and high efficiency and greenness has become a technical problem that needs to be solved urgently by those skilled in the art. Therefore, in view of the above status quo, it is urgent to develop an efficient and green naringin nanosystem and its preparation method to overcome the shortcomings in current practical applications. Summary of the invention

[0006] The object of the present invention is to provide a highly efficient green naringin nanosystem and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A highly efficient green naringin nanosystem, wherein the system uses citrus pectin as a carrier, and the mass ratio of naringin to citrus pectin is 1:8 to 1:15.

[0009] As a further solution of the present invention: the mass ratio of naringin to citrus pectin is 1:10.

[0010] A method for preparing a high-efficiency green naringin nanosystem, using the high-efficiency green naringin nanosystem as described above, comprises the following steps:

[0011] Step 1, evenly dispersing a certain proportion of naringin and citrus pectin in deionized water to obtain a uniform suspension;

[0012] Step 2, heating and stirring the suspension at a temperature of 75° C. to 90° C. to obtain a naringin nanosuspension;

[0013] Step 3: subjecting the cooled naringin nanosuspension to multi-step centrifugation treatment, taking the precipitate and re-dispersing it with deionized water to finally obtain a naringin nanosystem.

[0014] As a further solution of the present invention: in step 1, the mass ratio of naringin to deionized water is 1:1000.

[0015] As a further solution of the present invention: in step 2, the heating stirring speed is 500 rpm or the ultrasonic frequency is 200 kHz, and the duration is 30 to 40 minutes.

[0016] As a further solution of the present invention: In step 3, the multi-step centrifugation treatment includes: first, centrifuging at 8000 rpm for 10 minutes, taking the supernatant and placing it in a new centrifuge tube, and then centrifuging at 16000 rpm for 10 minutes and discarding the supernatant.

[0017] As a further solution of the present invention: the preparation method further comprises optimizing the preparation conditions by response surface methodology to obtain a naringin nanosystem with small particle size, low polydispersity index and high drug loading.

[0018] As a further solution of the present invention: in step 2, the heating temperature is 78° C. and the stirring time is 32 minutes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention provides a green and safe process for preparing naringin nanosystems, using deionized water as a solvent to avoid the use of chemical solvents. This feature not only ensures the safety and simplicity of operation, but also significantly reduces the production cost, meeting the environmental protection requirements of modern pharmaceuticals.

[0021] (2) The prepared naringin nanosystem exhibits good stability and can be quickly re-dissolved after drying, ensuring its effectiveness in clinical applications. This feature enables it to have greater flexibility during storage and transportation.

[0022] (3) The experimental results show that the prepared naringin nanosystem retains the anti-inflammatory effect of naringin while improving its bioavailability by 124%. These improvements provide patients with a more effective treatment plan, which may reduce side effects during treatment and improve medication compliance.

[0023] (4) Naringin, as an important bioactive ingredient, has significant antitussive, anti-inflammatory and antioxidant effects. The present application provides a highly efficient green naringin nanosystem with citrus pectin as a carrier, which not only has a simple preparation process but also can promote oral absorption efficiency while retaining anti-inflammatory efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a response surface diagram for optimizing the levels of various factors in the preparation method of the high-efficiency green naringin nanosystem in the embodiment of the present invention.

[0025] Figure 2 Schematic diagram of the effects of different drug delivery systems on the relative expression of IL-6 mRNA and TNF-α mRNA and NO release in RAW264.7 cells in the examples of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0028] See also Figure 1 and Figure 2 , an embodiment of the present invention provides an efficient green naringin nanosystem, wherein the system uses citrus pectin as a carrier, and the mass ratio of naringin to citrus pectin is 1:8 to 1:15.

[0029] The mass ratio of naringin to citrus pectin is 1:10.

[0030] A method for preparing a high-efficiency green naringin nanosystem, using the high-efficiency green naringin nanosystem as described above, comprises the following steps:

[0031] Step 1, evenly dispersing a certain proportion of naringin and citrus pectin in deionized water to obtain a uniform suspension;

[0032] Step 2, heating and stirring the suspension at a temperature of 75° C. to 90° C. to obtain a naringin nanosuspension;

[0033] Step 3: subjecting the cooled naringin nanosuspension to multi-step centrifugation treatment, taking the precipitate and re-dispersing it with deionized water to finally obtain a naringin nanosystem.

[0034] In step 1, the mass ratio of naringin to deionized water is 1:1000.

[0035] In step 2, the heating stirring speed is 500 rpm or the ultrasonic frequency is 200 kHz, and the duration is 30 to 40 minutes.

[0036] In step 3, the multi-step centrifugation treatment includes: first, centrifuging at 8000 rpm for 10 minutes, taking the supernatant and placing it in a new centrifuge tube, and then centrifuging at 16000 rpm for 10 minutes and discarding the supernatant.

[0037] The preparation method further comprises: optimizing the preparation conditions by response surface methodology to obtain a naringin nanosystem with small particle size, low polydispersity index and high drug loading.

[0038] In step 2, the heating temperature is 78° C. and the stirring time is 32 minutes.

[0039] Embodiment 1:

[0040] 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 80°C, and maintained at this temperature for 30 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0041] After drying, the yield of the prepared naringin nanosystem was 93.23%. The average particle size of the nanosystem measured by Malvern particle size analyzer was 393.56 nm, and the polydispersity index (PDI) was 0.123.

[0042] Embodiment 2:

[0043] 200 mg of naringin was mixed with 1.6 g of citrus pectin and added to 200 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 75°C, and maintained at this temperature for 30 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0044] After drying, the yield of the prepared naringin nanosystem was 90.78%. The average particle size of the nanosystem measured by Malvern particle size analyzer was 433.65 nm, and the polydispersity index (PDI) was 0.743.

[0045] Embodiment three:

[0046] 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 78°C, and maintained at this temperature for 32 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0047] After drying, the yield of the prepared naringin nanosystem was 97.80%. The average particle size of the nanosystem measured by Malvern particle size analyzer was 350.47 nm, and the polydispersity index (PDI) was 0.415.

[0048] Embodiment 4:

[0049] 1 g of naringin was mixed with 10 g of citrus pectin and added to 1000 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 85°C, and maintained at this temperature for 35 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0050] After drying, the yield of the prepared naringin nanosystem was 96.22%. The average particle size of the nanosystem measured by Malvern particle size analyzer was 423.13 nm, and the polydispersity index (PDI) was 0.279.

[0051] Example 5: Preparation method of the optimal high-efficiency green naringin nanosystem;

[0052] Weigh 5 mg of naringin and a certain amount of citrus pectin, add them to 5 ml of deionized water, and mix them in proportion. Heat them to a set temperature (such as 70°C) in a water bath, and stir for a certain period of time to ensure full dissolution. The naringin-pectin nanosystem is obtained by multi-step centrifugation and redispersion.

[0053] Response surface methodology design: Selecting the heating temperature (X 1 ), pectin dosage (X 2 )、Stirring time (X 3 ) as the independent variable. Three levels were selected for each factor for experimental design, as shown in the following table:

[0054] Table 1 Factors and levels

[0055]

[0056]

[0057] According to the Box-Behnken design, 15 groups of experiments were conducted, and the average particle size (Y 1 )、polydispersity index (PDI) (Y 2 ) and drug loading (Y 3 ).

[0058] Performance characterization: ① Particle size and PDI: Malvern particle size analyzer was used to measure the average particle size and polydispersity index (PDI) of the nanosystem. ② Drug loading: The content of the active ingredient was determined by high performance liquid chromatography.

[0059] The collected data were subjected to variance analysis, and the design-expert software was used for quadratic regression analysis to establish a mathematical regression model. The specific model is as follows:

[0060] When the particle size is used as the response value, Y 1 =2981.4925-51.60163X 1 -1.4727X 2 -33.99575X 3 -0.04582X 1 X 2 -0.051375X 1 X 3 -0.0239X 2 X 3 +0.341263X 1 2 +0.078008X 2 2 +0.6127X 3 2 .

[0061] When PDI is the response value, Y 1 =2.92772-0.60431X 1 -0.007085X 2 -30.024112X 3 -0.000043X 1 X 2 -0.000062X 1 X 3 -0.000083X 2 X 3 +0.000407X 1 2 +0.000196X 2 2 +0.000456X 3 2 .

[0062] When the drug loading is taken as the response value, Y 1 =-38.20531+0.901562X 1 +2.15095X 2 +1.72788X 3 -0.00076X 1 X 2 +0.0102X 1 X 3 +0.02303X 2 X 3 -0.007091X 1 2 -0.022814X 2 2 -0.054638X 3 2 .

[0063] Based on the model results, the interaction between the factors was analyzed by response surface plots and contour plots to determine the optimal preparation conditions (such as Figure 1 The optimal preparation conditions were finally determined to be: heating temperature of 78°C, ratio of naringin to pectin of 1:10 (i.e., 50 mg of pectin), and stirring time of 32 minutes.

[0064] Experimental verification:

[0065] The naringin-pectin nanosystem was prepared again under the optimal conditions, and the particle size was measured to be 350.4 nm, the PDI was 0.057, and the drug loading was 97.8%, which were close to the predicted values, verifying the reliability of the model.

[0066] Example 6: In vitro anti-inflammatory effect of naringin nanosystem;

[0067] Naringin nanosystem 1: 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 80°C, and maintained at this temperature for 30 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0068] Naringin nanosystem 2: 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 78°C, and maintained at this temperature for 32 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with deionized water and redispersed to obtain a naringin nanosystem.

[0069] RAW264.7 cells were pre-protected for 2 hours before administration, and then treated with different groups of administration solutions (nanosystem 1, nanosystem 2, naringin monomer, naringin-pectin physical mixture; each system contained naringin 100 μg / mL), and then stimulated with LPS for 19 hours. The supernatant of each group was collected, and the expression of TNF-α, IL-6 and the amount of NO were detected by ELISA.

[0070] The experimental results show (such as Figure 2 As shown in Figure 2, compared with the control group, the levels of IL-6 and TNF-α in the model group were significantly increased, indicating that the inflammatory model was successfully established. After administration of naringin nanosystem 1, naringin nanosystem 2, and naringin monomer, the levels of IL-6 and TNF-α and the release of NO in each group were significantly reduced (P<0.01), showing a good anti-inflammatory effect.

[0071] Example 7: Transmembrane transport of naringin nanosystem;

[0072] Naringin nanosystem: 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 80°C, and maintained at this temperature for 30 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, the precipitate was washed with deionized water and redispersed to obtain the naringin nanosystem.

[0073] Caco-2 cells were seeded on Transwell plates at a seeding density of 2×10^5 cells / well, and 0.5 mL of culture medium was added to the inner layer and 1.5 mL was added to the outer layer. The cells were grown to form a tight monolayer for 21 days. Bidirectional transport experiments were performed to evaluate the transmembrane permeability of naringin and naringin nanosystems. Transport experiments were performed from the top (AP) to the base (BL) of the Caco-2 cell monolayer and in the reverse direction under pH conditions of 7.2-7.4.

[0074] Calculate the apparent permeability coefficient (P APP ) to evaluate the transport of drugs. The formula is as follows:

[0075]

[0076] in:

[0077] dQ / dt is the amount of drug transported per unit time (μg / min);

[0078] C 0 is the initial drug concentration in the dosing solution (μg / mL);

[0079] A is the surface area of ​​the monolayer cells (cm 2 ).

[0080] Table 2 P of naringin and naringin nanosystem in Caco-2 monolayer cells APP Value (n = 3, )

[0081]

[0082] The experimental results showed that the transport properties of naringin and naringin nanosystem in the Caco-2 cell model were significantly different. Specifically, the apparent permeability coefficient (P APP ) in the transport from the apical end (AP) to the basal end (BL) was significantly higher than that from the basal end to the apical end, indicating that the nanosystem can effectively improve the intestinal absorption efficiency of naringin. Through comparative analysis, the P APP The value was significantly higher than that of unencapsulated naringin, which indicated that nano-processing significantly enhanced the bioavailability of the drug, thereby improving the absorption rate of the drug.

[0083] Example 8: Study on the concentration and pharmacokinetics of naringin in rat plasma;

[0084] Naringin nanosystem: 100 mg of naringin was mixed with 1 g of citrus pectin and added to 100 ml of deionized water. The suspension was transferred to a heated stirrer, heated to 80°C, and maintained at this temperature for 30 minutes with continuous stirring. The heated suspension was centrifuged at 8000 rpm for 10 minutes and the supernatant was taken. The suspension was centrifuged again at 16000 rpm for 10 minutes, the supernatant was discarded, the precipitate was washed with deionized water and redispersed to obtain the naringin nanosystem.

[0085] Plasma collection of drug-administered animals: 8 SD rats in each group, blank whole blood was first collected (recorded as 0min), followed by oral administration (naringin group: 100mg / kg; naringin nanosystem group: 110mg / kg). Whole blood was collected from the fundus venous plexus after 15min, 30min, 1h, 1.5h, 2h, 4h, and 6h, about 0.6mL per point, and placed in a centrifuge tube containing heparin. Centrifuge at 3000rpm for 15 minutes, and the supernatant plasma was separated and stored at -20℃.

[0086] Preparation of test samples for drug-administered animals: accurately pipette 200 μL of plasma sample, add 200 μL of ethyl acetate for extraction, collect the supernatant after centrifugation and blow dry with nitrogen, and reconstitute with 200 μL of methanol for analysis.

[0087] Blood drug concentration determination and analysis: HPLC was used to test the treated samples. The blood drug concentration was calculated and the blood drug concentration-time curve was drawn. All data were expressed as mean ± standard deviation (mean ± SD) and statistically analyzed using analysis of variance (ANOVA). The blood drug concentration-time data were processed using PKsolution software.

[0088] Table 3 Pharmacokinetic parameters of SD rats after oral administration of different drugs (n=6)

[0089]

[0090] The results show that:

[0091] The peak time of naringin nanosystem and naringin monomer was 1 hour, but the peak concentration of the nanosystem was 2.234μg / mL, which was higher than 1.043μg / mL of the monomer; the concentration of naringin in rat plasma before the peak of the nanosystem was significantly higher than that of the monomer group, indicating that the nanosystem effectively improved the absorption efficiency and rate of naringin; the elimination half-life of the nanosystem was 1.167 hours, while that of the monomer was 1.896 hours, which showed that the nanosystem helped the metabolism of naringin in the body. The bioavailability of the naringin nanosystem (with AUC as an indicator) was increased by about 124% compared with the naringin monomer, and the peak concentration (Cmax) was increased by about 114%. This shows that the nanosystem significantly enhanced the oral absorption efficiency of naringin.

[0092] It should be noted that in the present invention, it should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that those skilled in the art can understand.

Claims

1. A highly efficient green naringin nanosystem, characterized in that: The system uses citrus pectin as a carrier, and the mass ratio of naringin to citrus pectin is 1:8 to 1:

15.

2. The high-efficiency green naringin nanosystem according to claim 1, characterized in that: The mass ratio of naringin to citrus pectin is 1:

10.

3. A method for preparing a high-efficiency green naringin nanosystem, using the high-efficiency green naringin nanosystem as claimed in claim 1 or 2, characterized in that: The following steps are involved: Step 1, evenly dispersing a certain proportion of naringin and citrus pectin in deionized water to obtain a uniform suspension; Step 2, heating and stirring the suspension at a temperature of 75° C. to 90° C. to obtain a naringin nanosuspension; Step 3: subjecting the cooled naringin nanosuspension to multi-step centrifugation treatment, taking the precipitate and re-dispersing it with deionized water to finally obtain a naringin nanosystem.

4. The method for preparing the high-efficiency green naringin nanosystem according to claim 3, characterized in that: In step 1, the mass ratio of naringin to deionized water is 1:1000.

5. The method for preparing the high-efficiency green naringin nanosystem according to claim 4, characterized in that: In step 2, the heating stirring speed is 500 rpm or the ultrasonic frequency is 200 kHz, and the duration is 30 to 40 minutes.

6. The method for preparing the high-efficiency green naringin nanosystem according to claim 5, characterized in that: In step 3, the multi-step centrifugation treatment includes: first, centrifuging at 8000 rpm for 10 minutes, taking the supernatant and placing it in a new centrifuge tube, and then centrifuging at 16000 rpm for 10 minutes and discarding the supernatant.

7. The method for preparing the high-efficiency green naringin nanosystem according to claim 3, characterized in that: The preparation method also includes optimizing the preparation conditions by response surface methodology to obtain a naringin nanosystem with small particle size, low polydispersity index and high drug loading.

8. The method for preparing the high-efficiency green naringin nanosystem according to claim 5, characterized in that: In step 2, the heating temperature is 78° C. and the stirring time is 32 minutes.

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