Nano-emulsion for relieving chronic obstructive pulmonary disease as well as preparation method and application of nano-emulsion

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a nano-emulsion for relieving chronic obstructive pulmonary disease as well as a preparation method and application of the nano-emulsion. The nano-emulsion provided by the invention is prepared by taking fructus hippophae as a raw material, taking a fructus hippophae extract obtained by enzymolysis, mould mixed liquor fermentation and ethanol extraction as a main active ingredient, and performing high-pressure homogenization on the fructus hippophae extract, an emulsifier and other auxiliary materials. The nano-emulsion obtained by the invention is administrated in the form of atomized liquid in a mode of oral and nasal inhalation, and experimental results show that the nano-emulsion can significantly reduce mucus secretion, improve respiratory tract airflow obstruction, relieve lung inflammation and improve lung functions, and can effectively relieve chronic obstructive pulmonary diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a nanoemulsion for alleviating chronic obstructive pulmonary disease, and a preparation method and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease, which is mainly manifested by persistent airflow limitation and dyspnea. At present, it is mostly relieved and improved by taking oral chemical drugs.

[0003] However, oral medications are easily affected by gastrointestinal function and contents during absorption, resulting in low bioavailability and reduced efficacy. Long-term use may also cause the body to develop drug resistance. Therefore, the cycle of oral drug treatment is long and the effect is not ideal.

[0004] To address the drawbacks of oral medications, existing research has begun to formulate drugs that relieve COPD into sprays or nasal drops, which are then delivered to the body through the nasal cavity. However, due to persistent airflow limitation, the efficacy of inhaled drugs (including nanomedicines) is also affected. In these patients, mucus secretion is aggravated, producing a thicker and more difficult-to-clear mucus layer, which traps inhaled particles. In addition, the chronic inflammation of COPD increases the activity of alveolar macrophages, thereby enhancing phagocytosis and reducing the dose reaching the lungs.

[0005] Plant extracts have shown great potential in the treatment of COPD due to their multi-target, multi-pathway characteristics and low toxic side effects.

[0006] Sea buckthorn fruit is the dried mature fruit of the sea buckthorn plant of the Elaeagnaceae family. "Jingzhu Materia Medica" records: "The fruits of both large and small sea buckthorns are boiled into a paste, which is beneficial to the lungs and throat." In addition, "Chinese Pharmacopoeia" also records: Sea buckthorn "is sour, astringent, and warm. It enters the spleen, stomach, lung, and heart meridians. It strengthens the spleen and aids digestion, relieves cough and expectoration, and promotes blood circulation and relieves blood stasis."

[0007] However, the traditional method of extracting active ingredients from seabuckthorn fruit results in limited active ingredients in the obtained seabuckthorn fruit extract and low bioavailability, which restricts its clinical application. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a nanoemulsion for alleviating chronic obstructive pulmonary disease, and a preparation method and application thereof. The seabuckthorn fruit nanoemulsion product prepared by this method has higher bioavailability.

[0009] The nanoemulsion provided by the present invention comprises seabuckthorn fruit extract, an emulsifier accounting for 1% to 5% by volume of the seabuckthorn fruit extract, and other excipients accounting for 0.1% to 0.5% by volume of the seabuckthorn fruit extract; The emulsifier is selected from at least one of soy protein isolate, sodium caseinate, whey protein isolate, sorbitan monooleate, and sorbitan monooleate; The other auxiliary materials are selected from any one of sodium alginate and sodium carboxymethyl cellulose.

[0010] Preferably, the preparation method of the seabuckthorn fruit extract is as follows: S1 Seabuckthorn fruit pretreatment: fresh and ripe seabuckthorn fruits are selected, washed, removed, and crushed into pulp to obtain seabuckthorn pulp; S2 uses a mixed enzyme preparation to enzymatically hydrolyze the sea buckthorn fruit pulp obtained in S1, inactivates the enzyme and sterilizes the fruit to obtain a sea buckthorn fruit enzymatic hydrolyzate; The mixed enzyme preparation comprises at least two of cellulase, pectinase and hemicellulase; S3: adding the mold mixture to the sea buckthorn fruit hydrolyzate obtained in S2, mixing and fermenting, and centrifuging to obtain a sea buckthorn fruit fermentation liquid, wherein the mold mixture is a mixture of species of Mucor racemosus and Aspergillus oryzae; S4 adds ethanol to the sea buckthorn fruit fermentation liquid obtained in S3 for extraction, and concentrates the extract to obtain the sea buckthorn fruit extract.

[0011] In the above-mentioned method for preparing sea buckthorn fruit extract, preferably, the mixed enzyme preparation described in S2 includes: cellulase, pectinase, and hemicellulase, based on the volume of sea buckthorn pulp, 0.1%-0.5% cellulase, 0.1%-0.5% pectinase, and 0.05%-0.3% hemicellulase; the enzymatic hydrolysis temperature is 45-55°C, the pH is 4.0-6.0, and the enzymatic hydrolysis is 1-5 h.

[0012] Preferably, the inoculation volume of the mold mixture in S3 accounts for 3%-15% of the volume of the sea buckthorn fruit hydrolysate, the inoculation volume ratio of Mucor racemosus to Aspergillus oryzae in the mold mixture is 1:1-2, the fermentation temperature is 20-32°C, and the fermentation time is 24-96h.

[0013] Preferably, the volume fraction of ethanol in S4 is 55%-95%, and the volume ratio of seabuckthorn fruit fermentation liquid to ethanol is 1:1-3.

[0014] Furthermore, the method for preparing the nanoemulsion is as follows: Mix sea buckthorn fruit extract, emulsifier and water accounting for 70%-90% of the total volume, heat to 30-60℃, homogenize at 80-120 MPa for 5-15 minutes, then homogenize at 30-60 MPa for 3-10 minutes, then add 0.1%-0.5% of other excipients to the homogenized emulsion and adjust the pH value to 6.5-7.5.

[0015] The present invention also provides a nanoemulsion for alleviating chronic obstructive pulmonary disease. The nanoemulsion is prepared by the method described above.

[0016] In addition, the present invention also provides an atomized liquid, wherein the atomized liquid contains the nanoemulsion prepared by the above method.

[0017] Furthermore, the method for using the atomized liquid is as follows: The atomized liquid is atomized by electronic heating atomization, the heating temperature is 200-220 ° C, the single heating time is 2-4 s, the cooling time is 27-57 s, and the cycle works. Each time the atomization is started, it lasts 30-60 minutes, 1-3 times a day, and is inhaled through the mouth and nose.

[0018] In addition, the use of the nanoemulsion or the atomized liquid containing the nanoemulsion in the preparation of a drug for alleviating chronic obstructive pulmonary disease is also the focus of protection of the present invention.

[0019] The beneficial effects of the present invention are: (1) Provided is a nanoemulsion that can alleviate chronic obstructive pulmonary disease. The nanoemulsion contains sea buckthorn extract as the main functional ingredient and is administered in the form of a nebulized liquid by inhalation through the mouth and nose. Experimental results show that the nebulized liquid can significantly reduce mucus secretion, improve airway airflow obstruction, reduce lung inflammation, improve lung function, and effectively alleviate chronic obstructive pulmonary disease. (2) The present invention uses a mold mixture to ferment the sea buckthorn fruit hydrolysate. Compared with traditional yeast fermentation, the enzyme system of the present invention is richer and more complementary, has a stronger ability to resist miscellaneous bacteria, and can improve the quality of the fermented product. The obtained fermentation liquid is extracted and concentrated with ethanol to prepare a nanoemulsion, which further improves the physicochemical stability and atomization characteristics of the sea buckthorn fruit extract, and the bioavailability of the sea buckthorn fruit is higher; (3) Experiments on mice showed that the content of inflammatory factors in the alveolar lavage fluid of mice decreased significantly after treatment with nanoemulsion, and the effect of the fermentation group was significantly better than that of the alcohol extraction group. This shows that the seabuckthorn fruit extract nanoemulsion prepared by the method of the present invention has a reducing effect on the increase of inflammatory factors in the lungs of mice and can effectively alleviate COPD symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results of the determination of inflammatory factors in the alveolar lavage fluid of mice after aerosol inhalation of nanoemulsions prepared by different methods of the present invention; Figure 2 The results of measuring the lung function indicators FEV20 / FVC (%) and FEV50 / FVC (%) of mice after aerosol inhalation of nanoemulsions prepared by different methods of the present invention; Figure 3 The figures are HE staining results of mouse lung tissue sections after aerosol inhalation of nanoemulsions prepared by different methods of the present invention. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0022] Example 1 A nanoemulsion, the preparation method is as follows: The sea buckthorn fruit extract, sorbitan monooleate accounting for 3% of the volume of the sea buckthorn fruit extract, and water accounting for 75% of the total volume were mixed and homogenized in two stages using a high-pressure homogenizer. The homogenization pressure of the first stage was 90 MPa and the homogenization time was 10 min. The homogenization pressure of the second stage was 50 MPa and the homogenization time was 5 min. The flow rate of the high-pressure homogenizer was 30 L / h. An emulsion was prepared at a temperature of 55°C. Then, 0.5% sodium carboxymethyl cellulose was added to the homogenized emulsion, and the pH value was adjusted to 7.0 with an acid / alkali solution to obtain the nanoemulsion, which was then refrigerated and stored at 4°C.

[0023] The method for preparing the above-mentioned seabuckthorn fruit extract comprises the following steps: S1 Seabuckthorn fruit pretreatment: fresh and ripe seabuckthorn fruits are selected, washed, removed, and crushed into pulp to obtain seabuckthorn pulp; S2 used a mixed enzyme preparation to enzymatically hydrolyze the sea buckthorn fruit pulp obtained in S1 at 45°C and pH=5.0 for 3 h. After the enzymatic hydrolysis, an appropriate amount of sterile water was added to the hydrolyzate, and the enzyme was inactivated and sterilized at 85°C for 10 min to obtain the sea buckthorn fruit hydrolyzate. The mixed enzyme preparation includes cellulase, pectinase and hemicellulase, and based on the volume of sea buckthorn pulp, the added volume of each enzyme is: 0.2% cellulase, 0.2% pectinase and 0.1% hemicellulase; S3: adding the mold mixture to the sea buckthorn fruit hydrolysate obtained in S2, and fermenting them at 30° C. for 72 h. After the fermentation is completed, centrifuging at 4000 rpm for 10 min to remove bacteria to obtain sea buckthorn fruit fermentation liquid, wherein the mold mixture is a mixture of species of Mucor racemosus and Aspergillus oryzae, the mold mixture accounts for 8% of the mass of the sea buckthorn hydrolysate, and the ratio of species of Mucor racemosus to Aspergillus oryzae in the mold mixture is 1:1; S4 added 70% ethanol by volume to the sea buckthorn fruit fermentation liquid obtained in S3 at a volume ratio of 1:1, extracted for 40 min, stirred and allowed to stand, collected the ethanol phase, concentrated the extract at 60°C, removed the ethanol, and obtained the sea buckthorn fruit extract.

[0024] Example 2 The difference from Example 1 is that, when preparing the seabuckthorn fruit extract, the inoculation ratio of Mucor racemosus to Aspergillus oryzae in the mold mixture described in S3 is 1:2, and the rest is the same as Example 1.

[0025] Example 3 The difference from Example 1 is that when preparing the seabuckthorn fruit extract, in S3, the inoculation amount of the mold mixture accounts for 4% and 12% of the volume of the seabuckthorn fruit enzymatic hydrolyzate, and the rest is the same as Example 1.

[0026] Example 4 The difference from Example 1 is that, when preparing the seabuckthorn fruit extract, the volume fraction of ethanol in S4 is 90%, and the rest is the same as Example 1.

[0027] Example 5 The difference from Example 1 is that the emulsifier used in the preparation of the seabuckthorn fruit extract nanoemulsion is soy protein isolate, and the rest is the same as Example 1.

[0028] Comparative Example 1 The difference from Example 1 is that the preparation method of seabuckthorn fruit extract is different. Based on Example 1, the enzymatic hydrolysis and fermentation operations in S2-S3 are omitted and the effective components in the seabuckthorn pulp are directly extracted with ethanol to obtain the seabuckthorn fruit extract.

[0029] Comparative Example 2 The difference from Example 1 is that the preparation method of sea buckthorn fruit extract is different. In this comparative example, Aspergillus niger is used instead of Mucor racemosus in Example 1. The addition ratio of Aspergillus oryzae to Aspergillus niger is 1:1. The inoculation amount of the mold mixture and other operations are the same as in Example 1.

[0030] Comparative Example 3 The difference from Example 1 is that the preparation method of the seabuckthorn fruit extract is different, the inoculation ratios of Mucor racemosus and Aspergillus oryzae are 2:1, 0:1, and 1:0, and the rest are the same as Example 1.

[0031] Comparative Example 4 The difference from Example 1 is that the preparation method of the seabuckthorn fruit extract is different, and Mucor rouxii is used to replace Mucor racemosus in Example 1. The rest is the same as Example 1.

[0032] Comparative Example 5 The difference from Example 1 is that the preparation method of the seabuckthorn fruit extract is different, step S4 is omitted, and the seabuckthorn fruit fermentation liquid obtained in S3 is directly concentrated to obtain the seabuckthorn fruit extract.

[0033] Comparative Example 6 The difference from Example 1 is that sodium carboxymethyl cellulose is not added during the preparation of the nanoemulsion. The rest is the same as Example 1.

[0034] Test Example 1 The sea buckthorn fruit extract nanoemulsions obtained in each embodiment and comparative example were used as atomized liquids, and the mouse model was atomized by electronic heating atomization. The atomization heating temperature was 220°C, the single heating time was 3 s, the cooling time was 57 s, and the cycle worked.

[0035] Using C57BL / 6J model mice, different types of sea buckthorn fruit extract nanoemulsions were designed for aerosol inhalation, once a day for 1 hour each time. After 80 days of inhalation, the inflammatory factors in the alveolar lavage fluid of each group of mice were examined. The measurement results are shown in Table 1 below.

[0036] Table 1 Effects of different types of sea buckthorn fruit extract nanoemulsions TNF-α (pg / mL) IL-6 (pg / mL) IL-1β (pg / mL) Example 1 8 11 16 Example 2 10 13 19 Example 3 11 12 17 Example 4 10 11 15 Example 5 9 13 19 Comparative Example 1 18 30 29 Comparative Example 2 20 28 31 Comparative Example 3 20 31 35 Comparative Example 4 22 30 32 Comparative Example 5 21 32 30 Comparative Example 6 22 34 33

[0037] Test Example 2 The sea buckthorn fruit extract nanoemulsions obtained in Example 1 and Comparative Example 1 were used as atomized liquids, and the mouse model was atomized by electronic heating atomization. The atomization heating temperature was 220°C, the single heating time was 3 s, the cooling time was 57 s, and the cycle worked.

[0038] Using C57BL / 6J model mice, we designed a nebulized inhalation of different types of sea buckthorn fruit extract nanoemulsions (fermentation group - Example 1, alcohol extraction group - Comparative Example 1), once a day for 1 hour each time. The mice were divided into four groups: blank group (NOR), modeling group (CS), fermentation group (FJ), and alcohol extraction group (CT). After 80 days of inhalation, the inflammatory factors in the alveolar lavage fluid of each group of mice were examined. The results are shown in the attached figure. Figure 1 shown.

[0039] By the attached Figure 1 It can be seen that by measuring the inflammatory factors TNF-α, IL-6, and IL-1β in the alveolar lavage fluid of mice, the results showed that compared with the blank group, the content of inflammatory factors in the alveolar lavage fluid of the mice in the modeling group was significantly increased, and the content of inflammatory factors in the alveolar lavage fluid of the mice was significantly decreased after treatment with sea buckthorn fruit extract nanoemulsion, and the effect of the fermentation group was significantly better than that of the alcohol extraction group, indicating that the sea buckthorn fruit extract nanoemulsion prepared by the method of the present invention has a reducing effect on the increase of inflammatory factors in the lungs of mice and can effectively alleviate COPD symptoms.

[0040] Test Example 3 Using C57BL / 6J model mice, different types of sea buckthorn fruit extract nanoemulsions were designed for aerosol inhalation (fermentation group - Example 1, alcohol extraction group - Comparative Example 1), once a day for 1 hour each time. The mice were divided into four groups: blank group (NOR), modeling group (CS), fermentation group (FJ), and alcohol extraction group (CT). After 80 days of inhalation, the lung function indicators FEV20 / FVC (%) and FEV50 / FVC (%) of the mice in each group were observed. The results are shown in the attached figure. Figure 2 shown.

[0041] Depend on Figure 2 It can be seen that by measuring the lung function indicators FEV20 / FVC and FEV50 / FVC of mice, the results showed that the lung function of the mice in the modeling group was significantly reduced compared with the blank group, and the primary indicator for evaluating COPD disease, FEV20 / FVC, was improved after treatment with the sea buckthorn fruit extract nanoemulsion prepared in Example 1 or Comparative Example 1. However, the therapeutic effect of the sea buckthorn fruit extract nanoemulsion obtained by fermentation in Example 1 was better than that of the alcohol extraction group in Comparative Example 1. The above results can be inferred that the reason why the nanoemulsion in Example 1 is better than the alcohol extraction group is that the active ingredients therein may be richer in variety and have a high content of active ingredients, and it is these active ingredients that play a significant role in showing the above results; FEV50 / FVC recovered lung function after treatment with the sea buckthorn fruit extract nanoemulsion, which shows that the sea buckthorn fruit extract nanoemulsion has a certain improvement effect on the lung function treatment of mice and can alleviate COPD symptoms.

[0042] Test Example 4 C57BL / 6J model mice were used to design aerosol inhalation of different types of sea buckthorn fruit extract nanoemulsions (fermentation group - Example 1, alcohol extraction group - Comparative Example 1), once a day for 1 hour each time. The mice were divided into four groups, namely blank group (NOR), modeling group (CS), fermentation group (FJ) and alcohol extraction group (CT). After 80 days of inhalation, the lung tissue pathological section staining of the mice in each group was investigated. The results are shown in the attached figure. Figure 3 shown.

[0043] Depend on Figure 3 It can be seen that the results of lung tissue pathological section staining showed that compared with the blank group, the alveolar cavity structure of the modeling group was disordered, the alveolar wall was significantly thickened, and there was a large amount of inflammatory infiltration; after treatment with sea buckthorn fruit extract nanoemulsion, the area of ​​inflammatory infiltration was reduced to varying degrees, the alveolar wall became thinner, and the morphology of the alveolar cavity gradually recovered. In addition, the therapeutic effect of the fermentation group was better than that of the alcohol extraction group. Sea buckthorn fruit extract nanoemulsion has a significant improvement effect on alleviating chronic obstructive pulmonary disease.

Claims

1. A nanoemulsion, characterized in that The nanoemulsion comprises seabuckthorn fruit extract, an emulsifier accounting for 1% to 5% of the volume of the seabuckthorn fruit extract, and other auxiliary materials accounting for 0.1% to 0.5%; The emulsifier is selected from at least one of soy protein isolate, sodium caseinate, whey protein isolate, sorbitan monooleate, and sorbitan monooleate; The other auxiliary materials are selected from any one of sodium alginate and sodium carboxymethyl cellulose.

2. A nanoemulsion as claimed in claim 1, characterized in that, The seabuckthorn fruit extract is prepared as follows: S1 Seabuckthorn fruit pretreatment: fresh and ripe seabuckthorn fruits are selected, washed, removed, and crushed into pulp to obtain seabuckthorn pulp; S2 uses a mixed enzyme preparation to enzymatically hydrolyze the sea buckthorn fruit pulp obtained in S1, inactivates the enzyme and sterilizes the fruit to obtain a sea buckthorn fruit enzymatic hydrolyzate; The mixed enzyme preparation comprises at least two of cellulase, pectinase and hemicellulase; S3: adding the mold mixture to the sea buckthorn fruit hydrolyzate obtained in S2, mixing and fermenting, and centrifuging to obtain a sea buckthorn fruit fermentation liquid, wherein the mold mixture is a mixture of species of Mucor racemosus and Aspergillus oryzae; S4 adds ethanol to the sea buckthorn fruit fermentation liquid obtained in S3 for extraction, and concentrates the extract to obtain the sea buckthorn fruit extract.

3. A nanoemulsion as claimed in claim 2, characterized in that, The mixed enzyme preparation described in S2 includes: cellulase, pectinase, and hemicellulase. Based on the volume of sea buckthorn pulp, the cellulase is 0.1%-0.5%, the pectinase is 0.1%-0.5%, and the hemicellulase is 0.05%-0.3%; the enzymatic hydrolysis temperature is 45-55°C, the pH is 4.0-6.0, and the enzymatic hydrolysis time is 1-5 hours.

4. The nanoemulsion according to claim 2, wherein The inoculation volume of the mold mixture in S3 accounts for 3%-15% of the volume of the sea buckthorn fruit hydrolysate, the inoculation volume ratio of Mucor racemosus to Aspergillus oryzae in the mold mixture is 1:1-2, the fermentation temperature is 20-32°C, and the fermentation time is 24-96 h.

5. The nanoemulsion for alleviating chronic obstructive pulmonary disease according to claim 2, characterized in that: The volume fraction of ethanol in S4 is 55%-95%, and the volume ratio of seabuckthorn fruit fermentation liquid to ethanol is 1:1-3.

6. A method for preparing the nanoemulsion according to any one of claims 1 to 5, characterized in that The following steps are involved: Mix sea buckthorn fruit extract, emulsifier and water, with water accounting for 70%-90% of the total volume of sea buckthorn fruit extract and emulsifier, heat to 30-60°C, homogenize at 80-120 MPa for 5-15 minutes, then homogenize at 30-60 MPa for 3-10 minutes, then add 0.1%-0.5% of other excipients to the homogenized emulsion, and adjust the pH value to 6.5-7.

5.

7. A nanoemulsion for alleviating chronic obstructive pulmonary disease, characterized in that: The nanoemulsion is prepared by the method according to claim 6.

8. An atomized liquid, characterized in that: The atomized liquid contains the nanoemulsion according to claim 1.

9. The atomized liquid according to claim 8, wherein The method of using the atomized liquid is as follows: The atomized liquid is atomized by electronic heating atomization, the heating temperature is 200-220 ° C, the single heating time is 2-4 s, the cooling time is 27-57 s, and the cycle works. Each time the atomization is started, it lasts 30-60 minutes, 1-3 times a day, and is inhaled through the mouth and nose.

10. Use of the nebulized liquid according to claim 8 in the preparation of a medicament for alleviating chronic obstructive pulmonary disease.

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