Preparation of a nanometer particle of cortex moutan and application thereof

Abstract

The application discloses a preparation method of nanometer particles of cortex moutan, and the content of polyphenol components with bactericidal and antiviral properties is effectively improved through a multi-step purification method of hydrothermal treatment, dialysis and extraction, so that the nanometer particles have better broad-spectrum bactericidal and antiviral properties than traditional cortex moutan preparations and crude extracts without purification. In addition, the nanometer particles of cortex moutan prepared by the method have a size of nanometer level through the preparation method of hydrothermal reaction and dialysis purification, and the size effect of the ultra-small size can provide better tissue penetration and better drug delivery capacity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical material engineering, and particularly relates to a preparation method of cortex moutan nanoparticles and application thereof. BACKGROUND

[0002] Fatal viral infection reduces the immunity of patients, increases the susceptibility to bacterial infection, including Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, etc., among which children and the elderly with low immunity are high-risk groups. The treatment of bacterial and viral infection pneumonia is greatly increased in difficulty because the diagnosed infection state is complex and the treatment method is complex. In addition to antibiotics, antiviral drugs also need to be taken for treatment. However, the drugs used not only reduce the immunity of patients, but also interact with each other, resulting in a decrease in drug effect. Therefore, compared with single bacterial or viral infection, complex pneumonia leads to a higher mortality rate of patients. Although it is generally believed that epithelial cell damage caused by viruses leads to bacterial invasion, recent studies have shown that the immune defense dysfunction of the body after influenza is the main reason for increasing the susceptibility to secondary bacterial infection. Therefore, how to design a broad-spectrum antibacterial agent with antibacterial, antiviral and immunomodulatory functions becomes particularly important.

[0003] Natural products have been used for the treatment of bacterial and viral infections, such as wound healing, pneumonia and enteritis. On the one hand, natural products with mixed ingredients contain a large number of antibacterial and antiviral monomers. However, the complex ingredients and large size limit their application. On the one hand, the complex ingredients cannot maximize the effect of the effective components therein, and the harmful substances contained therein also have toxic side effects on the body. On the other hand, the large size is also not conducive to its penetration into tissues and organs, thus limiting its delivery capacity in the body. Therefore, how to reduce the dose of natural products without affecting the therapeutic effect of natural products is very important. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art, and provides a preparation method of cortex moutan nanoparticles. The cortex moutan nanoparticles prepared by the method have good biocompatibility, strong antibacterial performance and high safety.

[0005] The present application is realized by the following technical solutions:

[0006] A preparation method of cortex moutan nanoparticles comprises the following steps:

[0007] Step 1, preparation of the crude extract of the nanometer particles of the cortex moutan: the cortex moutan is crushed to less than 80 mesh (preferably 80-140 mesh), and the crushed cortex moutan is mixed with deionized water to obtain a first mixture, after stirring for 1-3 h, a hydrothermal reaction is carried out, the temperature of the hydrothermal reaction is 100-140℃, and the reaction time is 16-20 h, to obtain a hydrothermal reaction product;

[0008] The hydrothermal reaction product is subjected to centrifugal solid-liquid separation to obtain a solid, which is the crude extract of the nanometer particles of the cortex moutan;

[0009] The mass ratio of the crushed cortex moutan to deionized water in the first mixture is 1:(15-80);

[0010] Step 2, purification of the nanometer particles of the cortex moutan: the crude extract of the nanometer particles of the cortex moutan obtained in step 1 is mixed with deionized water to obtain a second mixture, the second mixture is placed in a dialysis bag with a molecular weight of 800-1200 for dialysis for 6-8 days; the contents of the dialysis bag are freeze-dried to obtain a dried powder;

[0011] The dried powder is mixed with deionized water to obtain a third mixture, and the third mixture is extracted with ethyl acetate, and after extraction, a lower clear liquid is obtained, and the lower clear liquid is subjected to centrifugal separation to obtain a solid, which is the nanometer particles of the cortex moutan; the volume ratio of the third mixture to ethyl acetate in the extraction process is 1:(2-16);

[0012] The mass ratio of the crude extract of the nanometer particles of the cortex moutan to deionized water in the second mixture is 1:(40-60);

[0013] The concentration of the third mixture is the mass ratio of the dried powder to deionized water, which is 1:(50-100).

[0014] In the above technical solution, the reaction temperature of the hydrothermal reaction in step 1 is 120-130℃, and the reaction time is 17-19 h.

[0015] In the above technical solution, the centrifugal solid-liquid separation process in step 1 is carried out at a speed of 500-2000 rpm, and the centrifugal separation time is 6-14 min.

[0016] In the above technical solution, the hydrothermal reaction in step 1 is carried out in a stainless steel hydrothermal reaction kettle.

[0017] In the above technical solution, the dialysis process in step 2 is carried out in a regenerated cellulose membrane dialysis bag.

[0018] In the above technical solution, the extraction process in step 2 is divided into 2-4 times, each time the water phase is mixed with ethyl acetate, the volume ratio of the water phase to ethyl acetate is 1:1-4; after standing and layering, the lower clear liquid is collected, and the lower water phase is mixed with ethyl acetate again to start the next extraction process; when all the extraction processes are completed, the lower clear liquid is collected and enters the centrifugal separation process.

[0019] In the above technical solution, the centrifugal solid-liquid separation process in step 2 has a rotation speed of 8000-12000 rpm, and the centrifugal separation time is 15-25 min.

[0020] A nano-particle of cortex moutan prepared by the above technical solution, the nano-particle of cortex moutan is a nano-particle formed by self-assembly of small molecules through intermolecular hydrogen bonding force, and the small molecules at least include one or more of paeonol and paeonoside. Compared with traditional cortex moutan preparations and unpurified crude extracts, the nano-particle of cortex moutan has a smaller size, and thus can better penetrate tissues to achieve efficient drug delivery; the preparation method of dialysis and extraction removes small molecules (sugars, oils, pigments, volatile oils, etc.) without antibacterial and antiviral properties, improves the content of components with antibacterial and antiviral properties, and thus improves the treatment effect of the nano-particle of cortex moutan.

[0021] The application of a nano-particle of cortex moutan prepared by the above technical solution in preparing a material for killing bacteria and / or viruses.

[0022] In the above technical solution, the application of the nano-particle of cortex moutan in preparing a material for killing Staphylococcus aureus and / or influenza virus.

[0023] The application of a nano-particle of cortex moutan prepared by the above technical solution in preparing a medicine for treating influenza.

[0024] The advantages and beneficial effects of the present application are as follows:

[0025] 1. The nano-particle of cortex moutan prepared by the present application reduces the size of the nano-particle to nanoscale through the preparation method of hydrothermal reaction and dialysis purification, and the size effect of the ultra-small size can have better tissue penetration and better drug delivery capacity.

[0026] 2. The nano-particle of cortex moutan prepared by the present application is effectively improved in the content of polyphenolic components with bactericidal and antiviral properties through the multi-step purification of the preparation method of hydrothermal reaction, dialysis and extraction, so that it has better broad-spectrum bactericidal and antiviral properties than traditional cortex moutan preparations and unpurified crude extracts.

[0027] 3、The prepared Paeonia Nanoparticle has the immune regulation characteristics of traditional Chinese medicine, and can significantly regulate the expression level of inflammatory factors in the infected microenvironment after being atomized or intravenously injected, thereby realizing immunotherapy. The large batch preparation can be realized by a simple method, the product cost is low, the implementation difficulty is small, the preparation means is simple, and the Paeonia Nanoparticle has good medical application prospect.

[0028] 4、The Paeonia Nanoparticle is a traditional herbal extract, has good biocompatibility, and can be used as a drug to treat bacterial and viral infectious pneumonia. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a TEM diagram of the Paeonia Nanoparticle of Example 1.

[0030] Figure 2 is a liquid chromatogram of the Paeonia Nanoparticle of Example 1.

[0031] Figure 3 is a molecular dynamics simulation diagram of the Paeonia Nanoparticle of Example 1.

[0032] Figure 4 is a tissue penetration diagram of the Paeonia Nanoparticle of Example 1.

[0033] Figure 5 is an antibacterial performance diagram of the Paeonia Nanoparticle of Example 1 on Staphylococcus aureus.

[0034] Figure 6 is a killing performance diagram of the Paeonia Nanoparticle of Example 1 on influenza virus.

[0035] Figure 7 is an inflammatory factor regulation diagram of the Paeonia Nanoparticle of Example 1.

[0036] For those skilled in the art, other related drawings can be obtained according to the above drawings without creative labor. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical scheme of the present application, the technical scheme of the present application will be further described below in combination with specific embodiments.

[0038] Example One

[0039] A Paeonia Nanoparticle preparation and application, comprising the following steps:

[0040] 1) Preparation of crude extract of nanometer particles of Radix Paeoniae: First, the medicinal material of Radix Paeoniae was ground into 120 mesh powder with a grinding rod for 30 min, then 2 g of Radix Paeoniae powder was weighed and added to 60 mL of deionized water, stirred for 2 h, and then added to a 100 mL stainless steel reaction kettle for hydrothermal reaction, the reaction temperature was 125°C, and the reaction lasted for 18 h. After the reaction was completed, the hydrothermal reaction product was centrifuged at a speed of 1000 rpm for 10 min, and the obtained solid was the crude extract of nanometer particles of Radix Paeoniae.

[0041] 2) Preparation of nanometer particles of Radix Paeoniae: 0.4 g of crude extract of nanometer particles of Radix Paeoniae was dissolved in 20 mL of deionized water, and then dialyzed in a 1000 molecular weight dialysis bag for 1 week. The contents of the dialysis bag were freeze-dried. Then the obtained dried powder was prepared into a solution with a concentration of 15 mg / mL, and extracted with ethyl acetate for 3 times. In each extraction process, the water phase was mixed with ethyl acetate, and the volume ratio of the water phase to ethyl acetate was 1:2. After standing and layering, the lower clear liquid was collected, and the lower water phase was mixed with ethyl acetate again to start the next extraction process. When all the extraction processes were completed, the final lower clear liquid was collected and subjected to centrifugal separation. The lower clear liquid was centrifuged at a speed of 10000 rpm for 20 min, and the solid part was the nanometer particles of Radix Paeoniae.

[0042] 1) TEM experiment of nanometer particles of Radix Paeoniae

[0043] The nanometer particles of Radix Paeoniae prepared in Example 1 were prepared into a solution with a concentration of 1 mg / mL, and then 20 μL of the solution was taken out and dropped on a ultra-thin copper mesh after ultrasonic treatment for 10 min with a cell pulverizer. Then the copper mesh was placed on the sample stage of a TEM electron microscope for observation, and the results are shown in Figure 1 According to Figure 1 it can be seen that the size distribution of the nanometer particles of Radix Paeoniae is uniform, and the size is about 2.5 nm, which meets the size of nanometer level.

[0044] 2) Liquid chromatogram of nanometer particles of Radix Paeoniae

[0045] The nanometer particles of Radix Paeoniae and the crude extract of nanometer particles of Radix Paeoniae prepared in Example 1 were prepared into solutions with a concentration of 0.05 mg / mL, and their liquid chromatograms were detected by a liquid chromatograph, and the results are shown in Figure 2 According to Figure 2 it can be seen that the nanometer particles of Radix Paeoniae are at least composed of small molecular components such as paeonol and paeonoside. Compared with the crude extract of nanometer particles of Radix Paeoniae, the impurities such as sugars, oils, pigments, and volatile oils are removed by dialysis purification and extraction process.

[0046] 3) Molecular dynamics simulation of nanometer particles of Radix Paeoniae

[0047] The components of the nanometer particles of cortex radicis paeoniae were placed in the receptor pocket respectively, and possible binding conformations were obtained by adjusting the ligand conformation; each binding conformation was scored and evaluated to screen the best scoring conformation; the results are shown in Figure 3 . According to Figure 3 It can be seen that the main force between different molecules is intermolecular hydrogen bond.

[0048] 4) Tissue penetration experiment of the nanometer particles of cortex radicis paeoniae

[0049] The nanometer particles of cortex radicis paeoniae and the crude extract of the nanometer particles of cortex radicis paeoniae prepared in Example 1 were respectively blended with fluorescein sodium and stirred for 1 h, and then centrifuged and configured into a solution with a concentration of 1 mg / mL. Then different tissues (lungs, stomachs, intestines and skins) of mice in vitro were completely soaked for 20 min, and then sliced and placed on a fluorescence microscope to observe the staining of the tissues, and the results are shown in Figure 4 . According to Figure 4 It can be seen that compared with the crude extract of the nanometer particles of cortex radicis paeoniae, more fluorescently labeled nanometer particles of cortex radicis paeoniae are distributed in the tissues, thus representing that the nanometer particles of cortex radicis paeoniae have better tissue penetration ability.

[0050] 5) Antibacterial experiment of the nanometer particles of cortex radicis paeoniae

[0051] The nanometer particles of cortex radicis paeoniae and the crude extract of the nanometer particles of cortex radicis paeoniae prepared in Example 1 were configured into a solution with a concentration of 1 mg / mL, and were incubated with Staphylococcus aureus with an initial absorbance of 0.06 nm for 6 h. The absorbance value was measured every 2 h using a microplate reader, and the results are shown in Figure 5 . According to Figure 5 It can be seen that compared with the crude extract of the nanometer particles of cortex radicis paeoniae, the nanometer particles of cortex radicis paeoniae have better antibacterial effect, because the smaller size makes the nanometer particles of cortex radicis paeoniae have more contact area with bacteria and the ability to penetrate the bacterial membrane, thus better killing bacteria.

[0052] 6) Antiviral experiment of the nanometer particles of cortex radicis paeoniae

[0053] First, 0.1 mg / mL H1N1 influenza virus was co-cultured with lung cancer human alveolar basal epithelial cells A549 for 1 h, and then the culture solution was taken out and replaced with cell maintenance solution, and 1 mg / mL of the crude extract of the nanometer particles of cortex radicis paeoniae and the nanometer particles of cortex radicis paeoniae were added respectively and continued to be cultured for 1 d. Then the culture medium was aspirated, and an equal volume of tetramethyl azo salt solution (2.5 mg·mL -1 ) was added. After continuing to culture for 4 h, the tetramethyl azo salt solution was aspirated, and dimethyl sulfoxide solution was added to terminate the reaction, and the cell activity at 490 nm was detected using a microplate reader, and the results are shown in Figure 6 . According to Figure 6It can be seen that the activity of the cells cultured by the nanometer particles of Danpi is slightly lower than that of the blank, but higher than that of the nanometer particle extract of Danpi, so it has a better antiviral effect than the nanometer particle extract of Danpi.

[0054] 7) Inflammation factor regulation experiment of nanometer particles of Danpi

[0055] 30 four to six weeks old male C57BL / 6J mice were evenly divided into four groups, namely a blank group (without Staphylococcus aureus infection), a Staphylococcus aureus infected PBS treatment control group, a Staphylococcus aureus infected nanometer particle of Danpi treatment group and a Staphylococcus aureus infected nanometer particle extract of Danpi treatment group. After the modeling with Staphylococcus aureus infection was completed, PBS (50 μL), 1 mg / mL nanometer particles of Danpi (50 μL) and 1 mg / mL nanometer particle extract of Danpi (50 μL) were injected respectively. Peripheral blood was taken at the end of the treatment to analyze the expression amount of inflammation factor IL-6, and the results are shown in Figure 7 . According to Figure 7 It can be seen that the nanometer particles of Danpi have a lower expression content of inflammation factor than the nanometer particle extract of Danpi, so it has a better anti-inflammatory effect than the nanometer particle extract of Danpi.

[0056] Example Two

[0057] A preparation and application of nanometer particles of Danpi, comprising the following steps:

[0058] 1) Preparation of nanometer particle extract of Danpi: first, grind the medicinal material of Danpi into 80 mesh powder with a grinding rod for 30 min, then weigh 1 g of Danpi powder and add it to 40 mL of deionized water, stir for 1 h, then add it to a 50 mL stainless steel reaction kettle for hydrothermal reaction, the reaction temperature is 120°C, and the reaction lasts for 17 h. After the reaction is completed, the hydrothermal reaction product is centrifuged at a speed of 500 rpm for 8 min to obtain the solid nanometer particle extract of Danpi.

[0059] 2) Preparation of nanometer particles of Danpi: 0.2 g of nanometer particle extract of Danpi is added to 8 mL of deionized water, and dialysis is performed for 6 days using a dialysis bag with a molecular weight of 800. Then the contents in the dialysis bag are freeze-dried. Then the obtained dried powder is configured to a concentration of 10 mg / mL, and extracted twice with ethyl acetate. In each extraction process, the water phase and ethyl acetate are mixed, and the volume ratio of the water phase to ethyl acetate is 1:1; after standing and layering, the lower clear liquid is collected, and the lower water phase is mixed with ethyl acetate again to start the next extraction process; when all the extraction processes are completed, the final lower clear liquid is collected into a centrifugal separation process. The lower clear liquid is centrifuged at a speed of 8000 rpm for 15 min, and the solid part is the nanometer particles of Danpi.

[0060] Example Three

[0061] A preparation and application of nanometer particles of cortex moutan, comprising the following steps:

[0062] 1) Preparation of crude extract of nanometer particles of cortex moutan: first, grind the medicinal material of cortex moutan into 140 meshes with a grinding rod for 30 min, then take 3 g of cortex moutan and dissolve it in 80 mL of deionized water, stir for 3 h, then add it into a 200 mL stainless steel reaction kettle for hydrothermal reaction, the reaction temperature is 130 ℃, and the reaction time is 19 h. After the reaction is completed, centrifuge the hydrothermal reaction product at a speed of 2000 rpm for 12 min to obtain the solid crude extract of nanometer particles of cortex moutan.

[0063] 2) Preparation of nanometer particles of cortex moutan: add 0.6 g of crude extract of nanometer particles of cortex moutan into 36 mL of deionized water, dialyze it for 8 days using a dialysis bag with a molecular weight of 1200, then freeze-dry the contents in the dialysis bag. Then configure the obtained dried powder into a concentration of 20 mg / mL, and extract it with ethyl acetate for 4 times. In each extraction process, mix the water phase with ethyl acetate, the volume ratio of the water phase to ethyl acetate is 1:4; after standing and layering, collect the lower clear liquid, and mix the lower water phase with ethyl acetate again to start the next extraction process; when all the extraction processes are completed, collect the final lower clear liquid into a centrifugal separation process. Centrifuge the lower clear liquid at a speed of 12000 rpm for 10 min, and the solid part is the nanometer particles of cortex moutan.

[0064] The relational terms such as "first" and "second" are only used to distinguish one from another of the same-named components, and do not necessarily require or imply any such actual relationship or order between the components.

[0065] The above has exemplarily described the present application, it should be explained that, without departing from the core of the present application, any simple modification, modification or other equivalent replacement which can not cost creative labor of the person skilled in the art falls into the protection scope of the present application.

Claims

1. A method for preparing peony bark nanoparticles, characterized in that, Includes the following steps: Step 1, Preparation of crude extract of peony bark nanoparticles: Peony bark is pulverized to less than 80 mesh, and the pulverized peony bark is mixed with deionized water to obtain a first mixture. After stirring for 1 to 3 hours, a hydrothermal reaction is carried out. The hydrothermal reaction temperature is 100 to 140°C and the reaction time is 16 to 20 hours to obtain the hydrothermal reaction product. The hydrothermal reaction product was centrifuged to separate solid and liquid phases, and the solid obtained was crude extract of peony root nanoparticles. The mass ratio of pulverized peony bark to deionized water in the first mixture is 1:(15-80); Step 2, purification of peony bark nanoparticles: The crude extract of peony bark nanoparticles obtained in Step 1 is mixed with deionized water to obtain a second mixture. The second mixture is placed in a dialysis bag with a molecular weight of 800-1200 and dialyzed for 6-8 days. The contents of the dialysis bag are then freeze-dried to obtain a dried powder. The dried powder was mixed with deionized water to obtain a third mixture. The third mixture was extracted with ethyl acetate to obtain a lower clear liquid. The lower clear liquid was centrifuged to obtain solid peony bark nanoparticles. The volume ratio of the third mixture to ethyl acetate during the extraction process was 1:(1-16). The mass ratio of the crude extract of peony bark nanoparticles to deionized water in the second mixture is 1:(40-60); The concentration of the third mixture is such that the mass ratio of the dried powder to deionized water is 1:(50-100).

2. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, The hydrothermal reaction in step 1 is carried out at a temperature of 120–130°C for 17–19 hours.

3. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, In step 1, the centrifugal solid-liquid separation process is carried out at a speed of 500–2000 rpm for 6–14 min.

4. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, The hydrothermal reaction in step 1 is carried out in a stainless steel hydrothermal reactor.

5. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, In step 2, the dialysis process is carried out in a regenerated cellulose membrane dialysis bag.

6. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, The extraction process in step 2 is carried out in 2 to 4 times. In each extraction process, the aqueous phase is mixed with ethyl acetate, and the volume ratio of the aqueous phase to ethyl acetate is 1:1 to 4. After standing and separating, the lower clear liquid is collected, and the lower aqueous phase is mixed with ethyl acetate again to start the next extraction process. When all extraction processes are completed, the lower clear liquid is collected and put into the centrifugation process.

7. The method for preparing peony bark nanoparticles according to claim 1, characterized in that, In step 2, the centrifugal solid-liquid separation process is carried out at a speed of 8000-12000 rpm for 15-25 min.

8. A method for preparing peony bark nanoparticles according to any one of claims 1 to 7, characterized in that, The peony bark nanoparticles are nanoparticles formed by the self-assembly of small molecules through intermolecular hydrogen bonding forces, and the small molecules include at least one or more of paeonol and paeonol.

9. The application of peony bark nanoparticles prepared by the preparation method according to any one of claims 1 to 7 in the preparation of materials for killing bacteria and / or viruses, specifically in the preparation of materials for killing Staphylococcus aureus and / or influenza virus.

10. The application of peony bark nanoparticles prepared by the preparation method according to any one of claims 1 to 7 in the preparation of drugs for treating influenza.

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