A puerarin microcapsule for intestinal targeted release and its preparation method and application

Abstract

The present invention discloses a puerarin microcapsule for intestinal targeted release and its preparation method and application in the field of puerarin microcapsule embedding technology. The preparation method is as follows: using yeast β-glucan as an embedding carrier, in a solution environment, puerarin is embedded in yeast β-glucan under ultrasound to form a core material to obtain a first mixed solution; then sodium alginate powder is added to the first mixed solution under stirring to coat the surface of the core material with sodium alginate to form a layer of wall material to obtain a second mixed solution; using a sharp hole method, the second mixed solution is added dropwise to a coagulation bath for solidification, washed, and freeze-dried after solid-liquid separation to obtain puerarin microcapsules. The preparation method of the present invention is simple and easy to operate, selects medicine and food homologous substances as raw materials, does not introduce toxic and harmful chemical preparations such as cross-linking agents, avoids chemical pollution and potential health risks, and the puerarin microcapsules of the present invention have good intestinal sustained release and antioxidant properties.

Description

Technical Field

[0001] The invention relates to the technical field of puerarin microcapsule embedding, and in particular to an intestinal targeted release puerarin microcapsule and a preparation method and application thereof. Background Art

[0002] Pueraria root is a tuberous dried root of the leguminous plant Pueraria lobata, and is one of the commonly used Chinese herbal medicines. Puerarin is mainly extracted from the dried root of Pueraria lobata, and has multiple pharmacological effects such as anti-oxidation, improving cardiovascular and cerebrovascular diseases, and alleviating alcoholic hepatitis.

[0003] At present, the clinical application of puerarin mainly includes three dosage forms: injection, tablets and capsules. However, the chemical structure characteristics of puerarin lead to its poor water solubility and fat solubility, low oral absorption and bioavailability, and limited clinical application. In addition, due to its short half-life, frequent or large-dose injections or infusions are required. Therefore, it is particularly important to develop an effective drug delivery system to prolong the drug's duration of action and improve the bioavailability of puerarin. Finding alternative crystal forms, preparing amorphous solid dispersions or encapsulating them into structured carriers are the most commonly used methods to solve this problem.

[0004] In order to solve the above technical problems, those skilled in the art have proposed that puerarin can be made into puerarin microcapsules by an encapsulation method to prolong the drug action time and improve the bioavailability of puerarin. However, the current encapsulation method usually requires the introduction of a cross-linking agent, and the long-term use of the cross-linking agent will lead to potential health risks and the risk of chemical pollution; and the puerarin microcapsules prepared by this method have weak antioxidant capacity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a puerarin microcapsule for intestinal targeted release and its preparation method and application. The present invention uses yeast-β-glucan as an embedding carrier, firstly embeds puerarin in yeast β-glucan by ultrasound to form a core material, and then forms a capsule shell on the surface of the core material by sodium alginate-calcium chloride method, thereby preparing puerarin microcapsules. The puerarin microcapsules prepared by the present invention have good sustained release effect and solubility, improve bioavailability, and also have good antioxidant properties, providing theory and basis for the development of puerarin preparations.

[0006] The intestinal targeted release puerarin microcapsules and the preparation method and application thereof of the present invention are achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing puerarin microcapsules with intestinal targeted release, comprising the following steps:

[0008] Step 1: using yeast β-glucan as an embedding carrier, dispersing yeast β-glucan and puerarin in acetic acid solution in sequence to obtain a suspension.

[0009] Step 2, subjecting the suspension to ultrasonic treatment, so that the puerarin in the suspension is embedded in the yeast β-glucan under the action of ultrasound to form a core material, thereby obtaining a first mixed solution.

[0010] Step 3, adding sodium alginate powder to the first mixed solution, followed by stirring, so that the sodium alginate powder is coated on the surface of the core material under stirring and forms a layer of wall material, thereby obtaining a second mixed solution.

[0011] Step 4, dispersing the calcium salt in water to obtain a coagulation bath.

[0012] Step 5, using the sharp hole method, dropwise adding the second mixed solution into the coagulation bath for solidification, followed by washing, solid-liquid separation, freeze-drying after obtaining the solid component, and obtaining the puerarin microcapsules.

[0013] It should be noted that the sequence numbers of the above steps of the present invention are only for the convenience of description and do not represent the preparation order. The order of the above steps can be adjusted according to actual conditions. And since the preparation of puerarin microcapsules by the sharp hole method is a very common technical means in the field, those skilled in the art should know it, so the present invention will not repeat it.

[0014] In view of the potential health risks and weak antioxidant capacity of puerarin microcapsules prepared by existing encapsulation technology, the present invention attempts to improve the antioxidant capacity of puerarin microcapsules on the basis of reducing health risks. To this end, the present invention uses yeast β-glucan with strong antioxidant capacity as an encapsulation carrier. Yeast β-glucan is a polysaccharide extracted from yeast cells, which can remove free radicals in the body and improve the activity of antioxidant enzymes. Among them, yeast β-glucan has a hollow porous spherical structure, which is conducive to the encapsulation of puerarin. Moreover, yeast β-glucan has good dispersibility in water, and the surface of yeast β-glucan has a negative charge, so that yeast β-glucan can be combined with the positive charge on the surface of puerarin powder through electrostatic adsorption through the negative charge on its surface, and then on the basis of improving the encapsulation effect of yeast β-glucan on puerarin, the dispersibility of puerarin powder in water is further improved by the combination of yeast β-glucan. In addition, the main components of yeast β-glucan are not digested in the gastrointestinal tract, which can protect puerarin from passing through the gastrointestinal tract. Yeast β-glucan also has good targeting and biodegradability, so that after puerarin is embedded in yeast β-glucan, it can achieve intestinal targeted release, thereby prolonging the drug action time of puerarin microcapsules.

[0015] In the above step 1, in order to fully mix the yeast β-glucan and puerarin, the present invention preferably first disperses the yeast β-glucan powder in a solvent, and then adds the puerarin powder and disperses it evenly, so that the yeast β-glucan powder and the puerarin powder are fully mixed by gradually mixing.

[0016] The present invention uses acetic acid solution as a solvent, which can not only improve the dispersibility and solubility of yeast β-glucan, but also is more user-friendly. In addition, the acidity of acetic acid solution is not strong, and will not damage the efficacy of puerarin. In some preferred embodiments of the present invention, the volume concentration of the acetic acid solution used is 5% to 10%, so as to achieve the effect of dissolving yeast β-glucan with the lowest acetic acid concentration without damaging the functional activity of puerarin.

[0017] In some preferred embodiments of the present invention, in the suspension, the concentration of yeast β-glucan is 1.0 mg / mL to 1.5 mg / mL, and the concentration of puerarin is 2.0 mg / mL to 2.5 mg / mL, so as to avoid the situation that the amount of puerarin encapsulated is small and the encapsulation rate is low due to the concentration of the added yeast β-glucan being too low relative to the puerarin, or the situation that the drug loading rate is low due to the concentration of the added yeast β-glucan being too high relative to the puerarin, so that the added puerarin can be fully encapsulated in the yeast β-glucan, so that the encapsulation rate and drug loading rate of the obtained puerarin microcapsules are optimal.

[0018] In some preferred embodiments of the present invention, the yeast β-glucan used in the present invention is prepared by the following steps:

[0019] 1) Yeast Extract Peptone Dextrose Medium, abbreviated as YEPD liquid medium, was used as the culture medium. The selected yeast strain was inoculated into the YEPD liquid medium for activation, cultured at 28°C at 120r / min~150r / min for 48h~72h, centrifuged to obtain the precipitate, and washed with sterile water for several times to obtain bacterial mud. In the YEPD liquid medium, there were 10.0g / L yeast extract, 20.0g / L peptone, and 20.0g / L D-glucose.

[0020] 2) Using a NaCl solution with a concentration of 3% to 3.5% w / v as a self-solvent, the bacterial mud is dispersed in the self-solvent to prepare a bacterial solution with a bacterial concentration of 10% to 15% w / v.

[0021] 3) The bacterial solution is shaken at 100 r / min to 120 r / min for autolysis at 55° C. to 60° C. for 20 h to 24 h, the precipitate is collected by centrifugation, and washed with sterile water for multiple times to obtain autolyzed yeast cells.

[0022] 4) Using 0.02M phosphate buffer solution with a pH of 7.2-7.5 as a solvent, the solvent is mixed with the yeast cells after the autolysis in step 3) to prepare a first bacterial suspension with a concentration of 10%-15% w / v.

[0023] 5) extracting the first bacterial suspension at 100°C to 110°C for 3h to 4h, centrifuging to obtain a precipitate, and washing it with sterile water for multiple times to obtain a clean precipitate. Subsequently, dispersing the obtained clean precipitate in a NaOH solution with a concentration of 1% to 3% w / v to prepare a second bacterial suspension with a concentration of 10% to 15% w / v.

[0024] 6) The second bacterial suspension was ultrasonically treated at an ultrasonic power of 400 W for 30 to 45 min, and the precipitate was collected by centrifugation. The precipitate was then dispersed in a 0.02 M phosphate buffer having a pH of 7.2 to 7.5 to prepare a third bacterial suspension having a concentration of 15% w / v.

[0025] 7) adding papain to the third bacterial suspension in an amount of 1.5% w / v, followed by enzymolysis at 55° C. to 60° C. for 4 h, boiling water bath for 10 min to 15 min after enzymolysis, centrifuging at 4000 r / min to 5000 r / min and 4° C. for 15 min, washing the precipitate with distilled water for multiple times, and freeze-drying to obtain yeast β-glucan for later use.

[0026] In the above steps 2 and 3, the present invention first mixes the yeast β-glucan suspension and the puerarin solution, so that the puerarin can be fully loaded in the hollow porous spherical structure of the yeast β-glucan, and is combined with the yeast β-glucan by electrostatic adsorption to form the yeast β-glucan modified puerarin. Since the yeast β-glucan has good dispersibility in water, the yeast β-glucan modified puerarin can be evenly dispersed in the solution environment, so that the added wall material sodium alginate powder can evenly coat the yeast β-glucan modified puerarin in the solution environment under stirring, so that in the subsequent sharp hole method-coagulation bath treatment process, the sodium alginate is combined with Ca 2+ After the ion gel reaction occurs, a capsule shell is formed on the surface of the core material to obtain puerarin microcapsules. In the puerarin microcapsules, due to the physical interaction between puerarin and the microcapsule shell, the lattice characteristics of puerarin are reduced, the water solubility of puerarin microcapsules is improved, and the bioavailability of puerarin is further improved.

[0027] In the above step 2, in order to ensure that the yeast β-glucan and puerarin are fully mixed, the present invention adopts stirring and ultrasonic mixing. In some preferred embodiments of the present invention, ultrasonic stirring is performed for 10 min to 15 min at a stirring rate of 8000 r / min to 8500 r / min, so that puerarin can be fully loaded in the hollow porous spherical structure of yeast β-glucan to form yeast β-glucan-modified puerarin, so that the yeast β-glucan-modified puerarin is used as the core material.

[0028] In the above step 3, in some preferred embodiments of the present invention, the dosage ratio of the sodium alginate powder to the first mixed solution is 17.5 mg to 18.0 mg / mL, so as to avoid the situation that the added sodium alginate concentration is too low and cannot react completely with the calcium salt, thereby resulting in poor molding of the obtained puerarin microcapsules; it also avoids the situation that the solution is viscous due to the high concentration of the added sodium alginate, thereby resulting in poor shape of the obtained puerarin microcapsules.

[0029] In order to promote the mixing of sodium alginate powder and the first mixed solution to form the second mixed solution, the present invention adopts a stirring method for mixing. In some preferred embodiments of the present invention, the stirring temperature is 40°C to 45°C, and the stirring time is 30min to 45min, so as to achieve that the biological activity of puerarin is not reduced while the sodium alginate powder is fully stirred and dissolved in the first mixed solution.

[0030] In the above step 4, the present invention preferably uses an aqueous solution of calcium salt as a coagulation bath to solidify the wall material to form the puerarin microcapsule shell. In some preferred embodiments of the present invention, the concentration of calcium salt in the coagulation bath is 40 mg / mL to 60 mg / mL, so that the obtained puerarin microcapsules have a higher embedding rate and a regular shape. Among them, in some preferred embodiments of the present invention, the calcium salt used is CaCl2.

[0031] In the above step 5, in some preferred embodiments of the present invention, the second mixed liquid is added dropwise into the coagulation bath, and the second mixed liquid is solidified in the coagulation bath at room temperature, and the solidification time is 30 min to 45 min, so that the sodium alginate and calcium chloride react fully to form a regular capsule shell, thereby obtaining regularly formed puerarin microcapsules.

[0032] It is understandable that the solidification process is carried out in a solution environment, so the solidified product exists in the solidification bath. In order to obtain solid puerarin microcapsules, the present invention also performs post-treatment on them, removes excess solidification bath and other impurities by washing, and then separates and removes liquid components to obtain wet capsules, and then dries the wet capsules to obtain the final product puerarin microcapsules. In some preferred embodiments of the present invention, the drying method used is freeze drying, so as to achieve drying on the basis of retaining the original structure of the capsule, that is, the wet capsules are frozen in a -80°C refrigerator for 8h to 12h, and then freeze-dried for 48 to 72h.

[0033] The second object of the present invention is to provide puerarin microcapsules prepared by the above preparation method.

[0034] The third object of the present invention is to provide an application of puerarin microcapsules in the preparation of puerarin pharmaceutical preparations.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The invention uses yeast beta-glucan as an embedding carrier, mixes yeast beta-glucan and puerarin in an acetic acid solution, so that both yeast beta-glucan and puerarin are fully dissolved without damaging the functional activity of puerarin; then ultrasonic action is performed to enable puerarin to be fully loaded in the hollow porous spherical structure of yeast beta-glucan, and the negative charge on the surface of yeast beta-glucan is combined with the positive charge on the surface of puerarin powder through electrostatic adsorption to obtain yeast beta-glucan-modified puerarin, and on the basis of improving the embedding effect of yeast beta-glucan on puerarin, the dispersibility of puerarin powder in water is further improved. Puerarin modified with yeast β-glucan is used as the core material. Since yeast β-glucan has good dispersibility in water, the core material can be evenly dispersed in an acetic acid solution environment, so that the added wall material sodium alginate powder can be coated on the surface of the core material under stirring to form a layer of wall material, and then fully react with the wall material through the sharp hole method-coagulation bath method, and freeze-dry to form a regular capsule shell to obtain puerarin microcapsules. In the obtained puerarin microcapsules, due to the physical interaction between puerarin and the microcapsule shell, the lattice characteristics of puerarin are reduced, the water solubility of puerarin microcapsules is improved, and the bioavailability of puerarin is further improved. In addition, the yeast β-glucan used in the present invention is a polysaccharide extracted from yeast cells, which can remove free radicals in the body and improve the activity of antioxidant enzymes; and its main components are not digested in the gastrointestinal tract, which can protect puerarin from passing through the gastrointestinal tract, and the yeast β-glucan also has good targeting and biodegradability, so that after puerarin is embedded in the yeast β-glucan, it can achieve intestinal targeted release, thereby prolonging the drug action time of the puerarin microcapsule.

[0037] The preparation method of the invention is simple and easy to operate, selects medicine and food homologous substances as raw materials, does not use traditional cross-linking agents, and does not introduce toxic and harmful chemical agents, thereby avoiding chemical pollution and potential health risks. The prepared puerarin microcapsules have good intestinal sustained-release effect and antioxidant performance, and provide a theory and basis for the development of puerarin preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the standard curve of puerarin.

[0039] Figure 2 This is a scanning electron microscope image of the yeast β-glucan of Comparative Example 2.

[0040] Figure 3 This is a scanning electron microscope image of the puerarin microcapsules of Example 1.

[0041] Figure 4 The infrared spectra of the puerarin microcapsules of Example 1, the physical mixture of Comparative Example 1, the yeast β-glucan of Comparative Example 2, the sodium alginate of Comparative Example 3 and the puerarin of Comparative Example 4 are shown.

[0042] Figure 5 Schematic diagram of thermogravimetric analysis of puerarin microcapsules of Example 1 and puerarin of Comparative Example 4.

[0043] Figure 6 The XRD diagrams are of the puerarin microcapsules of Example 1, the physical mixture of Comparative Example 1, the yeast β-glucan of Comparative Example 2, the sodium alginate of Comparative Example 3 and the puerarin of Comparative Example 4.

[0044] Figure 7 These are the in vitro simulated release test results of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4. pH 1.2 is the gastric juice stage, pH 6.8 is the small intestine stage, and pH 7.4 is the colon stage.

[0045] Figure 8 The test results of the DPPH free radical scavenging ability of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 after digestion are shown.

[0046] Fig. 9 The test results of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 for the iron ion reducing ability after digestion are shown in FIG.

[0047] Fig.10 The test results of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 for superoxide anion free radicals after digestion are shown in FIG.

[0048] Fig.11The test results of the ABTS free radical scavenging ability of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 after digestion are shown.

[0049] Fig.12 The cytotoxicity test results of puerarin microcapsules in Example 1 are shown in the figure. The lowercase letters in the figure are used to mark the significant differences between different groups. a and b indicate P<0.05, which is significant. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention will be described clearly and completely below. It should be noted that in the following embodiments of the present invention, the yeast strain used is Kluyveromyces marxianus LSY9-3, with a deposit number of CCTCCNO: M2024566. And it is obtained by screening according to the method of CN202410354113.5 in the prior art. And in the following embodiments of the present invention, the yeast β-glucan used is prepared by the following steps:

[0051] 1) Kluyveromyces marxianus LSY9-3 was inoculated into YEPD liquid medium for activation, cultured at 28°C and 120 r / min for 48 h, centrifuged to obtain the precipitate, and washed several times with sterile water to obtain bacterial sludge. The YEPD liquid medium contained 10.0 g / L yeast extract, 20.0 g / L peptone, and 20.0 g / L D-glucose.

[0052] 2) Using a 3% w / v NaCl solution as a self-solvent, the bacterial sludge is dispersed in the self-solvent to prepare a bacterial solution with a bacterial concentration of 15% w / v.

[0053] 3) The bacterial cell solution was autolyzed at 55° C. and 120 rpm for 24 h, the precipitate was collected by centrifugation, and the precipitate was washed with sterile water for multiple times to obtain autolyzed yeast cells.

[0054] 4) Using 0.02M phosphate buffer solution at pH 7.5 as solvent, the solvent was mixed with the autolyzed yeast cells to prepare a first bacterial suspension with a concentration of 10% w / v.

[0055] 5) The first bacterial suspension was extracted at 100°C for 4 hours, centrifuged to obtain a precipitate, and washed with sterile water for several times to obtain a clean precipitate. Subsequently, the clean precipitate was dispersed in a 1% w / v NaOH solution to prepare a second bacterial suspension with a concentration of 15% w / v.

[0056] 6) The second bacterial suspension was ultrasonically treated at an ultrasonic power of 400 W for 30 min, and the precipitate was collected by centrifugation. The precipitate was then dispersed in a 0.02 M phosphate buffer solution with a pH of 7.2 to 7.5 to prepare a third bacterial suspension with a concentration of 15% w / v.

[0057] 7) adding papain to the third bacterial suspension in an amount of 1.5% w / v, followed by enzymolysis at 55° C. for 4 h, boiling water bath for 10 min after enzymolysis, centrifuging at 4500 r / min, 4° C. for 15 min, washing the precipitate with distilled water for multiple times, freeze-drying, and obtaining yeast β-glucan for standby use. The yeast β-glucan extracted by the present invention is light yellow granules.

[0058] It should also be noted that the THLE-2 cells used in the present invention were purchased from Wuhan Pronocell Life Science Co., Ltd. with the product number CL-0833.

[0059] Example 1

[0060] This embodiment provides a puerarin microcapsule for intestinal targeted release, which is prepared by the following steps:

[0061] Step 1: Using acetic acid solution with a volume concentration of 10% as a solvent, yeast-β-glucan powder and puerarin powder are dispersed in the acetic acid solution in sequence to prepare a suspension. In the suspension of this embodiment, the concentration of yeast β-glucan powder is 1.0 mg / mL, and the concentration of puerarin powder is 2.0 mg / mL.

[0062] Step 2, subjecting the suspension obtained above to ultrasonic treatment at a stirring rate of 8000 r / min for 10 min to obtain a second mixed solution.

[0063] Step 3, adding a corresponding amount of sodium alginate powder to the first mixed solution obtained in the above step 2 according to the dosage ratio of sodium alginate to the first mixed solution of 18.0 mg:1 mL, and then stirring at 40° C. for 45 min to obtain a second mixed solution.

[0064] Step 4, disperse CaCl2 in sterile water to prepare a coagulation bath with a concentration of 50 mg / mL.

[0065] Step 5, using the sharp hole method, the second mixed solution obtained in step 3 is added dropwise to the coagulation bath, solidified at room temperature for 30 minutes, then washed, solid-liquid separated, and the separated liquid component and solid component are collected respectively. The solid component is frozen in a -80°C refrigerator for 8 hours, and then freeze-dried for 48 hours to obtain puerarin microcapsules, and the puerarin microcapsules obtained in this example are white spherical solids.

[0066] And the liquid component of this embodiment is used for the determination of embedding rate.

[0067] Example 2

[0068] This embodiment provides a puerarin microcapsule for intestinal targeted release, which is prepared by the following steps:

[0069] Step 1: Using acetic acid solution with a volume concentration of 5% as a solvent, yeast-β-glucan powder and puerarin powder are dispersed in the acetic acid solution in sequence to prepare a suspension. In the suspension of this embodiment, the concentration of yeast β-glucan powder is 1.2 mg / mL, and the concentration of puerarin powder is 2.3 mg / mL.

[0070] Step 2, subjecting the suspension obtained above to ultrasonic treatment at a stirring rate of 8300 r / min for 13 min to obtain a second mixed solution.

[0071] Step 3, adding a corresponding amount of sodium alginate powder to the first mixed solution obtained in the above step 2 according to the dosage ratio of sodium alginate to the first mixed solution of 17.5 mg:1 mL, and then stirring at 42° C. for 30 min to obtain a second mixed solution.

[0072] Step 4, disperse CaCl2 in sterile water to prepare a coagulation bath with a concentration of 40 mg / mL.

[0073] Step 5, using the sharp hole method, the second mixed solution obtained in step 3 is added dropwise to the coagulation bath, solidified at room temperature for 40 minutes, then washed, solid-liquid separated, and the separated liquid component and solid component are collected respectively. The solid component is frozen in a -80°C refrigerator for 10 hours, and then freeze-dried for 60 hours to obtain puerarin microcapsules, and the puerarin microcapsules obtained in this example are white spherical solids.

[0074] And the liquid component of this embodiment is used for the determination of embedding rate.

[0075] Example 3

[0076] This embodiment provides a puerarin microcapsule for intestinal targeted release, which is prepared by the following steps:

[0077] Step 1: Using acetic acid solution with a volume concentration of 8% as a solvent, yeast-β-glucan powder and puerarin powder are dispersed in the acetic acid solution in sequence to prepare a suspension. In the suspension of this embodiment, the concentration of yeast β-glucan powder is 1.5 mg / mL, and the concentration of puerarin powder is 2.5 mg / mL.

[0078] Step 2, subjecting the suspension obtained above to ultrasonic treatment at a stirring rate of 8500 r / min for 15 min to obtain a second mixed solution.

[0079] Step 3, adding a corresponding amount of sodium alginate powder to the first mixed solution obtained in the above step 2 according to the dosage ratio of sodium alginate to the first mixed solution of 17.8 mg:1 mL, and then stirring at 45° C. for 43 min to obtain a second mixed solution.

[0080] Step 4, disperse CaCl2 in sterile water to prepare a coagulation bath with a concentration of 60 mg / mL.

[0081] Step 5, using the sharp hole method, the second mixed solution obtained in step 3 is added dropwise to the coagulation bath, solidified at room temperature for 45 minutes, then washed, solid-liquid separated, and the separated liquid component and solid component are collected respectively. The solid component is frozen in a -80°C refrigerator for 12 hours, and then freeze-dried for 72 hours to obtain puerarin microcapsules, and the puerarin microcapsules obtained in this example are white spherical solids.

[0082] And the liquid component of this embodiment is used for the determination of embedding rate.

[0083] Comparative Example 1

[0084] This comparative example provides a physical mixture of puerarin, yeast β-glucan and sodium alginate, and the mixture is prepared by the following steps:

[0085] The yeast-β-glucan powder and puerarin powder were added to the acetic acid solution with a volume concentration of 10% as solvent to obtain a third mixed solution. In the third mixed solution of this comparative example, the concentration of the yeast β-glucan powder was 1.0 mg / mL, and the concentration of the puerarin powder was 2.0 mg / mL.

[0086] According to the dosage ratio of sodium alginate to the third mixed solution of 18.0 mg:1 mL, a corresponding amount of sodium alginate powder was added to the third mixed solution obtained in the above step 2 and mixed to obtain a fourth mixed solution.

[0087] That is, the difference between this comparative example and Example 1 is only that:

[0088] In this comparative example, no ultrasonic treatment and stirring treatment were performed, and the fourth mixed solution obtained was a physically mixed solution of puerarin, yeast β-glucan and sodium alginate.

[0089] Comparative Example 2

[0090] This comparative example uses the yeast β-glucan prepared by the present invention as a comparison.

[0091] Comparative Example 3

[0092] This comparative example uses untreated sodium alginate as a comparison.

[0093] Comparative Example 4

[0094] This comparative example uses untreated puerarin as a comparison.

[0095] Experimental Section

[0096] (I) Determination of embedding efficiency and drug loading rate

[0097] Take the liquid component obtained in step 5 of each embodiment, filter it through a 0.2 μm microporous membrane to obtain a test solution, take 1 mL of the test solution in a 10 mL volumetric flask, and add pure water to the scale. After shaking, measure the absorbance at a wavelength of 250 nm, and then calculate the puerarin content in the liquid component from the puerarin standard curve.

[0098] Wherein, the puerarin standard curve is prepared by the following steps:

[0099] Accurately weigh 5 mg of puerarin standard solution in a 25 mL volumetric flask, dilute to volume with 95% ethanol, shake well for later use, take 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.7 mL, 0.8 mL, 0.9 mL and 1 mL of the above puerarin standard solution respectively and place them in a 10 mL volumetric flask, and add ethanol to make the total sample volume 1 mL, then add deionized water to dilute to volume, shake well, and obtain the standard solution to be tested. Take another 1.0 mL of ethanol, add water to dilute to 10 mL as a blank control. At a wavelength of 250 nm, test the absorbance values ​​of the above standard solution to be tested and the blank control respectively, use the measured absorbance value as the horizontal coordinate x, and the corresponding puerarin concentration as the vertical coordinate y to draw a graph, and obtain the following: Figure 1 The puerarin standard curve was obtained by the regression equation y=0.0134x.

[0100] According to the above puerarin standard curve, the puerarin concentration in the liquid component obtained in step 5 of each embodiment was measured, and then the corresponding embedding rate and drug loading rate were calculated according to the following formula 1 and formula 2.

[0101]

[0102]

[0103] In formula 1 and formula 2, C is the concentration of puerarin in the supernatant, in mg / mL; V is the total volume of the solution; M1 is the puerarin content of the original solution, in mg; and M2 is the mass of puerarin microcapsules, in mg.

[0104] According to the above method, the embedding rate of Example 1 was 88.5%, and the drug loading rate was 11.3%. The embedding rate of Example 2 was 85.7%, and the drug loading rate was 9.1%. The embedding rate of Example 3 was 84.6%, and the drug loading rate was 10.6%. The above shows that the puerarin microcapsules prepared by the present invention have excellent embedding rate and drug loading rate.

[0105] (B) Scanning electron microscope test

[0106] The present invention takes the yeast β-glucan of comparative example 2 and the puerarin microcapsules of example 1 as examples, and performs scanning electron microscope tests on them respectively, and the test results are as follows: Figure 2 and Figure 3 As shown, Figure 2 and Figure 3 The scale bars in the graphs are all 50 μm.

[0107] Figure 2 This is a scanning electron microscope image of the yeast β-glucan of Comparative Example 2. It can be seen that the yeast β-glucan extracted by the present invention has obvious wrinkles and a rough appearance, is broken into pieces, and has a porous structure.

[0108] Figure 3 This is a scanning electron microscope image of the puerarin microcapsule of Example 1. It can be seen that the surface of the puerarin microcapsule of Example 1 is smooth, and traces of yeast β-glucan encapsulated by sodium alginate layer by layer can be seen from the side, indicating that the puerarin microcapsule of the present invention is successfully prepared.

[0109] (III) Fourier infrared spectroscopy test

[0110] The present invention takes the puerarin microcapsules of Example 1, the physical mixture of Comparative Example 1, the yeast β-glucan of Comparative Example 2, the sodium alginate of Comparative Example 3 and the puerarin of Comparative Example 4 as examples, and performs Fourier transform infrared spectroscopy tests on them respectively, and the test results are as follows: Figure 4 shown.

[0111] Depend on Figure 4 It can be seen that the physical mixture of puerarin, yeast β-glucan and sodium alginate in Comparative Example 1 presents a different peak shape from the puerarin microcapsules in Example 1, and the characteristic peak of puerarin does not appear in the puerarin microcapsule group. The yeast-β-glucan in Comparative Example 2 and the sodium alginate in Comparative Example 3 are compared with the puerarin microcapsules in Example 1. In the puerarin microcapsules, the characteristic peaks of yeast-β-glucan and sodium alginate appear respectively. It can be concluded that puerarin is successfully encapsulated by yeast β-glucan and sodium alginate as the outer shell through the method of the present invention.

[0112] (IV) Thermogravimetric analysis test

[0113] The present invention takes the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 as examples, and conducts thermogravimetric analysis tests on them respectively, and the test results are as follows: Figure 5 shown.

[0114] Depend on Figure 5 It can be seen that compared with the untreated puerarin of Comparative Example 4, the thermal stability of the puerarin microcapsules formed after embedding in Example 1 is significantly improved.

[0115] (V) XRD test

[0116] The present invention takes the puerarin microcapsules of Example 1, the physical mixture of Comparative Example 1, the yeast β-glucan of Comparative Example 2, the sodium alginate of Comparative Example 3 and the puerarin of Comparative Example 4 as examples, and performs XRD tests on them respectively, and the test results are as follows: Figure 6 shown.

[0117] Depend on Figure 6 It can be seen that no characteristic diffraction peak of puerarin is detected in the XRD pattern of Example 1, proving that puerarin is completely embedded. This may be due to the physical interaction between puerarin and the microcapsule shell, which interferes with the purity of puerarin, resulting in a decrease in its lattice characteristics and an increase in water solubility.

[0118] (VI) In vitro simulated release test

[0119] The present invention takes the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 as examples, and conducts in vitro simulated release tests on them respectively, and the test results are as follows: Figure 7 shown.

[0120] The test method for the simulated release test is:

[0121] Accurately weigh 50 mg of puerarin, dissolve it in artificial gastric juice, small intestinal juice, and colonic juice, make up to 100 mL, mix well and dilute the concentration to 0.004-0.02 mg / mL, and detect the OD value at 250 nm.

[0122] Appropriate amounts of puerarin microcapsules and puerarin of Example 1 were weighed, and in vitro release tests were performed using artificial gastric juice, artificial small intestinal juice, and artificial colonic juice as release media, respectively. Artificial gastric juice was denoted as SGF, pH=1.2; artificial small intestinal juice was denoted as SIF, pH=6.8; and artificial colonic juice was denoted as SCF, pH=7.4.

[0123] The artificial gastric juice SGF, artificial small intestinal juice SIF and artificial colonic juice SCF used in the present invention are all prepared according to the method in the Chinese Pharmacopoeia.

[0124] Among them, artificial gastric juice SGF: take 16.4mL of dilute hydrochloric acid, add 800mL of water and 10g of pepsin, shake well, and dilute with water to 1000mL; wherein, the dilute hydrochloric acid is 234mL of concentrated hydrochloric acid diluted with water to 1000mL to obtain 9.5% to 10.5% dilute hydrochloric acid.

[0125] Artificial small intestinal fluid: pH is 6.8, take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve it, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution, take 10g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two liquids, and dilute with water to 1000mL.

[0126] Artificial colon fluid: pH 7.8, 5.59g of dipotassium hydrogen phosphate and 0.41g of potassium dihydrogen phosphate, add water to dissolve into 1000mL.

[0127] Test conditions for simulated release test:

[0128] The temperature was 37 ± 0.5 ° C and the stirring speed was 55 rpm. Samples were taken at predetermined time intervals, filtered with a 0.45 μm micromembrane, and the OD value was measured at a wavelength of 250 nm after dilution. The puerarin content in the release medium was obtained by calibration curve, and an equal volume of fresh release medium was added at the same time. The cumulative release percentage of the drug at different time points Q n Calculate according to the following formula 3:

[0129]

[0130] Among them, Q n It is the cumulative percentage of drug released at the preset time point.

[0131] C n is the concentration of puerarin in the release solution at a predetermined time.

[0132] V is the total volume of the release medium.

[0133] C i The puerarin concentration of the solution measured for each sampling.

[0134] V i is the sampling volume for each time.

[0135] M is the mass of original puerarin.

[0136] Figure 7 The test results of the in vitro simulated release test of puerarin microcapsules and puerarin in Example 1 are shown in the figure. In the figure, pH 1.2 is the gastric juice stage, pH 6.8 is the small intestine stage, and pH 7.4 is the colon stage.

[0137] And by Figure 7 It can be seen that in the simulated release test, the puerarin microcapsules of Example 1 have a lower release amount in the gastric juice stage, a release rate of more than half in the small intestine stage, and a continuous release in the colon stage. The final release rate is as high as 90%, and the sustained release effect is better. The puerarin microcapsules prepared using sodium alginate and β-glucan as wall materials have a good sustained release effect, are pH responsive, release less at low pH, and release a lot at high pH.

[0138] (VII) Antioxidant performance test

[0139] The present invention also tests the antioxidant properties of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 after digestion obtained after the above in vitro simulated release test, and the test results are as follows: Figure 8-Figure 11 shown.

[0140] The test method is:

[0141] Preparation of puerarin extract and puerarin microcapsule extract:

[0142] The digestion solution of puerarin of Comparative Example 4 and the digestion solution of puerarin microcapsules of Example 1 obtained after the previous digestion were respectively taken, filtered through 0.22 μm, and gradiently diluted to concentrations of 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL and 0.1 mg / mL as test solutions for later use.

[0143] 1) DPPH scavenging ability determination:

[0144] DPPH free radicals and anhydrous ethanol were prepared to form a 0.4 mol / L DPPH free radical solution, and 0.2 mL of DPPH free radical solution and 2 mL of deionized water were added to 1.0 mL of the above-mentioned test solution of different concentrations in turn to mix and obtain sample solutions of different concentrations. The sample solutions of different concentrations were placed in the dark at room temperature for 30 minutes, that is, after being protected from light for 30 minutes, the absorbance at a wavelength of 517 nm was detected by ultraviolet absorption method, and the DPPH free radical scavenging rate R1 of the test solution was calculated according to formula 4. The DPPH free radical solution was replaced by the same volume of deionized water as a blank group, and the absorbance was measured at a wavelength of 517 nm. The above-mentioned test solutions of different concentrations were replaced by the same volume of deionized water as a control group, and the absorbance was measured at a wavelength of 517 nm.

[0145]

[0146] Wherein, R1 is DPPH free radical scavenging rate, %.

[0147] A i1 It is the absorbance of the sample solution of different concentrations after reacting in the dark at room temperature for 30 minutes.

[0148] A j1 is the absorbance of the control group.

[0149] A c1 is the absorbance of the blank group.

[0150] And the test results are as follows Figure 8 As shown, Figure 8The following are the test results of the DPPH free radical scavenging ability of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 after digestion. Figure 8 It can be seen that the DPPH free radical scavenging ability of the puerarin microcapsules after digestion in Example 1 is better than that of puerarin alone, and shows a certain concentration dependence.

[0151] 2)Fe 3+ Reducing power determination:

[0152] Take 1mL of the leaching solution and place it in a centrifuge tube, add 1mL of 0.2mol / L, pH=6.6 phosphate buffer, add 1mL of 1% potassium ferricyanide solution, heat in a 50℃ water bath for 20min, then quickly put in an ice bucket for 5min, add 1mL of 10% trichloroacetic acid solution, centrifuge at 4000r / min for 10min, take 1mL of the supernatant, add 1mL of distilled water, 1mL of 0.5% ferric chloride solution, shake well, let stand at room temperature for 10min, and detect the OD value at a wavelength of 70nm, where OD value is the abbreviation of optical density, also known as absorbance. In general, the higher the concentration of the substance being tested, the higher the absorbance value. Use 1mL of distilled water sample as the control group, and the calculation formula is shown in Formula 5.

[0153] R c =OD i -OD0 formula 5.

[0154] Among them, R c Fe 3+ Restore capability.

[0155] OD i is the absorbance of the sample solution with different concentrations.

[0156] OD0 is the absorbance of the control group.

[0157] And the test results are as follows Fig. 9 As shown, Fig. 9 Fe of puerarin microcapsules after digestion 3+ Resilience test results.

[0158] Depend on Fig. 9 It can be seen that the Fe 3+ The reducing ability is better than that of puerarin alone and shows a certain concentration dependence.

[0159] 3) Determination of superoxide anion free radical scavenging ability:

[0160] Add 2.8mL of Tris-HCl buffer solution with a concentration of 0.05mol / L and pH=8.2, and 0.1mL of the above-mentioned test solutions of different concentrations to the tubes respectively, and keep them in a 25℃ water bath for 10min. Add 0.1mL of 0.03mol / L pyrogallol solution that has been preheated in a 25℃ water bath for 10min, react at 25℃ for 4min, add 0.1mL of 10mmol / LHCl to terminate the reaction, and obtain sample solutions of different concentrations. Finally, quickly measure the absorbance of the sample solutions of different concentrations at 320nm. Replace the different test solutions with the same volume of distilled water as the blank group. Replace the pyrogallol solution with the same volume of distilled water as the control group. The clearance calculation formula is shown in Formula 6.

[0161] R2(%)=1-(Ai2-Aj2) / Ac2×100%Formula 6.

[0162] Wherein, R2 is the superoxide anion free radical scavenging rate, %.

[0163] A i2 is the absorbance of the sample solution with different concentrations.

[0164] A j2 is the absorbance of the blank group.

[0165] A c2 is the absorbance of the control group.

[0166] And the test results are as follows Fig.10 As shown, Fig.10 The test results of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 for the superoxide anion radical scavenging ability after digestion are shown in FIG.

[0167] Depend on Fig.10 It can be seen that the superoxide anion free radical scavenging ability of the puerarin microcapsules after digestion of Example 1 is better than that of puerarin alone, and shows a certain concentration dependence.

[0168] 4) Determination of ABTS free radical scavenging ability:

[0169] The ABTS working solution was prepared by mixing 5 mL of 7 mM ABTS free radical solution and 88 μL of 140 mM potassium persulfate solution, and standing at room temperature in the dark for 12 to 16 hours.

[0170] Before the measurement, the ABTS working solution was diluted with anhydrous ethanol so that its absorbance at 734 nm was in the range of 0.7±0.05 to obtain the ABTS dilution solution.

[0171] Mix 100 μL of the above-mentioned test solution of different concentrations with 900 μL of the above-mentioned ABTS dilution to obtain sample solutions of different concentrations. After mixing, keep it at room temperature and away from light for 6 minutes, and then measure its absorbance value at a wavelength of 734 nm. Repeat three times and take the average value as the final test result. Replace the test solution of different concentrations with an equal volume of deionized water as the control group. The clearance calculation formula is shown in Formula 7.

[0172] R3(%)=(A0-Am) / A0×100%Formula 7.

[0173] Wherein, R3 is the ABTS free radical scavenging rate, %.

[0174] A m is the absorbance of the sample solution with different concentrations.

[0175] A0 is the absorbance of the control group.

[0176] And the test results are as follows Fig.11 As shown, Fig.11 The test results of the ABTS free radical scavenging ability of the puerarin microcapsules of Example 1 and the puerarin of Comparative Example 4 after digestion are shown.

[0177] And by Fig.11 It can be seen that the ABTS free radical scavenging ability of the puerarin microcapsules after digestion in Example 1 is better than that of puerarin alone, and shows a certain concentration dependence.

[0178] (8) Cytotoxicity test

[0179] Transformed human liver epithelial-2 cells, referred to as THLE-2 cells, are one of the few human normal liver cells that are currently mature and can be selected for experimental research. Therefore, the present invention uses THLE-2 cells as test cells and takes the puerarin microcapsules prepared in Example 1 as an example to explore the cytotoxic effect of puerarin microcapsules on THLE-2 cells. The test method is as follows:

[0180] After recovery, THLE-2 cells were transferred to DMEM complete medium, and the cell culture flask was placed in an incubator at 37°C and 5% CO2. The cell adhesion was observed every 12 hours. When the adhesion rate reached more than 80%, the cells were used for passage operation or experiment.

[0181] THLE-2 cells were expressed at 1.2×10 4A cell suspension of 100 μg / mL was inoculated in a 96-well plate. After the cells adhered to the wall, it was replaced with 100 μL of 30 μg / mL to 180 μg / mL puerarin microcapsule solution. In the blank group, DMEM high-glucose medium was used instead of cell suspension, 100 μL per well, cultured for 24 hours, and 5 parallel wells were set up in each group. After the culture, the cell viability was determined by CCK-8 method. The OD value of the well containing cells but not treated with samples was set as A1, the OD value of the well containing cells and treated with samples was set as A2, and the OD value of the well containing only culture medium was set as A0. The cell survival rate R4 was calculated by formula 8, and the results calculated at different concentrations were summarized as follows. Fig.12 shown.

[0182] R4(%)=(A2-A0) / (A1-A0)×100%Formula 8.

[0183] Depend on Fig.12 It can be seen that the cell viability of the experimental groups exceeded 100%, indicating that at these concentrations, cell viability was not affected by puerarin microcapsules, and puerarin microcapsules could also promote cell growth.

[0184] Obviously, the above embodiments are only some embodiments of the present invention, not all 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.

Claims

1. A method for preparing puerarin microcapsules for intestinal targeted release, characterized in that: The following steps are involved: Using yeast β-glucan as an embedding carrier, yeast β-glucan and puerarin are dispersed in acetic acid solution in sequence to obtain a suspension; subjecting the suspension to ultrasonic treatment, so that puerarin in the suspension is embedded in yeast β-glucan under the action of ultrasound to form a core material, thereby obtaining a first mixed solution; Adding sodium alginate powder to the first mixed solution, followed by stirring, so that the sodium alginate powder is coated on the surface of the core material under stirring and forms a layer of wall material, thereby obtaining a second mixed solution; Disperse calcium salt in water to obtain a coagulation bath; The second mixed solution is added dropwise to the coagulation bath to solidify by using a sharp hole method, followed by washing, solid-liquid separation, and freeze-drying to obtain the solid component to obtain the puerarin microcapsules; In the suspension, the concentration of yeast β-glucan is 1.0 mg / mL to 1.5 mg / mL, and the concentration of puerarin is 2.0 mg / mL to 2.5 mg / mL; The dosage ratio of the sodium alginate powder to the first mixed solution is 17.5 mg-18.0 mg:1 mL.

2. The preparation method according to claim 1, characterized in that The volume concentration of the acetic acid solution is 5% to 10%.

3. The preparation method according to claim 1, characterized in that: The concentration of calcium salt in the coagulation bath is 40 mg / mL to 60 mg / mL; The calcium salt is CaCl2.

4. The preparation method according to claim 1, characterized in that: The ultrasonic time of the ultrasonic treatment is 10 min to 15 min.

5. The preparation method according to claim 1, characterized in that: The stirring temperature is 40°C to 45°C, and the stirring time is 30min to 45min.

6. The preparation method according to claim 1, characterized in that: The curing time is 30min~45min.

7. A puerarin microcapsule prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the puerarin microcapsules according to claim 7 in the preparation of puerarin pharmaceutical preparations.

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