A cinnamaldehyde-based nanoemulsion and uses thereof

Abstract

The application discloses cinnamyl aldehyde-based nanoemulsion and application thereof, and relates to cinnamyl aldehyde-based preparation of nanoemulsion, which can be used for resisting liver fibrosis. Based on the feature of cinnamyl aldehyde as an oily liquid, cinnamyl aldehyde is used as an oil phase to prepare nanoemulsion and drug-loaded nanoemulsion, the solubility and stability of cinnamyl aldehyde in water are increased, and the drug efficacy is enhanced; experimental data show that the nanoemulsion and the drug-loaded nanoemulsion both have the effect of resisting liver fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a nanoemulsion based on cinnamyl aldehyde and application thereof. BACKGROUND

[0002] Liver fibrosis is a pathological process of abnormal proliferation of connective tissue in the liver caused by acute and chronic liver damage. Viral hepatitis, excessive alcohol intake, fatty liver disease, cholestasis and other primary diseases can cause repeated damage and repair of liver cells, eventually leading to fibrosis-like pathological changes. Early liver fibrosis is reversible, and can be reversed and subsided through drug treatment and other interventions, so the diagnosis and treatment of early liver fibrosis is very important, otherwise it can progress to cirrhosis, and even further develop into hepatocellular carcinoma, which seriously endangers the life and health of patients. Liver fibrosis cannot heal itself and needs to be treated simultaneously for the primary disease and liver fibrosis. However, there is currently no specific effective drug treatment method for liver fibrosis, which makes it difficult to reverse liver fibrosis. Therefore, anti-liver fibrosis drugs are still a hot spot in the research and development of hepatobiliary disease drugs, and drug treatment to block or even reverse the progression of liver fibrosis will have a profound impact on human health.

[0003] Cinnamyl aldehyde is a natural aldehyde organic compound extracted from a traditional Chinese medicine and food homologous plant (cinnamon), which is a light yellow oily liquid at room temperature. The structural formula of cinnamyl aldehyde is as follows:

[0004]

[0005] Cinnamyl aldehyde has anti-inflammatory, antioxidant and other pharmacological activities, but there is no literature report on the association of cinnamyl aldehyde with liver disease treatment. SUMMARY

[0006] In view of the above technical problems existing in the prior art, the present application provides a nanoemulsion based on cinnamyl aldehyde and application thereof, which is prepared based on cinnamyl aldehyde and can be used for anti-liver fibrosis.

[0007] The present application discloses a nanoemulsion based on cinnamyl aldehyde, which comprises cinnamyl aldehyde, an emulsifier and water.

[0008] Preferably, the nanoemulsion further comprises a fat-soluble drug.

[0009] Preferably, the fat-soluble drug comprises valsartan or vitamin A.

[0010] Preferably, the mass ratio of cinnamyl aldehyde, emulsifier, vitamin A and water is 12:6-18:1-4:100.

[0011] The mass ratio of cinnamyl aldehyde, emulsifier, valsartan and water is 60:60:6:500.

[0012] Preferably, the nanoemulsion further comprises a co-emulsifier.

[0013] Preferably, the emulsifier comprises any one of the following components or a combination thereof: polyoxyethylene castor oil (EL) and polyoxyethylene hydrogenated castor oil (EH); and the co-emulsifier comprises ethanol, propylene glycol, polyethylene glycol 400 or n-butanol.

[0014] Preferably, the preparation method of the nanoemulsion comprises: uniformly mixing cinnamaldehyde and an emulsifier to obtain a first oil phase system; adding water dropwise into the first oil phase system and stirring to obtain the nanoemulsion.

[0015] Preferably, the preparation method of the nanoemulsion comprises: uniformly mixing cinnamaldehyde, an emulsifier and a fat-soluble drug to obtain a second oil phase system; adding water dropwise into the second oil phase system and stirring to obtain the drug-loaded nanoemulsion.

[0016] Preferably, the nanoemulsion is used for preparing a drug or food for resisting liver fibrosis.

[0017] Preferably, the nanoemulsion is used for prolonging the residence time of cinnamaldehyde and a fat-soluble drug in the intestinal tract, enhancing the intestinal absorption of the drug, and thus enhancing the anti-liver fibrosis effect of the drug.

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

[0019] Based on the characteristics of cinnamaldehyde as an oily liquid, the nanoemulsion and the drug-loaded nanoemulsion prepared by taking cinnamaldehyde as the oil phase increase the solubility and stability of cinnamaldehyde in water and enhance the drug efficacy; experimental data show that the nanoemulsion and the drug-loaded nanoemulsion both have an anti-liver fibrosis effect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a flow chart of the preparation method of the nanoemulsion of the present application;

[0021] Figure 2 is a result image of fluorescence imaging;

[0022] Figure 3 is a result image of liver tissue sample staining. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0024] The application will be further described in detail below with reference to the drawings:

[0025] A cinnamaldehyde-based nanoemulsion, comprising cinnamaldehyde, an emulsifier and water. The cinnamaldehyde serves as an oil phase, and the nanoemulsion increases the solubility and stability of cinnamaldehyde in water, thereby enhancing the drug efficacy. In specific tests, the nanoemulsion has been shown to have an anti-liver fibrosis effect.

[0026] The emulsifier comprises any one of the following components or a combination thereof: Tween 80, polyoxyethylene castor oil (EL) and polyoxyethylene hydrogenated castor oil (EH).

[0027] As shown in Figure 1 The preparation method of the nanoemulsion comprises the following steps:

[0028] Step S1: uniformly mixing cinnamaldehyde and an emulsifier to obtain a first oil phase system.

[0029] Step S2: adding water dropwise into the first oil phase system and stirring to obtain a nanoemulsion.

[0030] A fat-soluble drug can be added to the oil phase to form a drug-loaded nanoemulsion. The fat-soluble drug includes valsartan or vitamin A (Va), but is not limited thereto. The preparation method of the drug-loaded nanoemulsion comprises the following steps:

[0031] Step 101: uniformly mixing cinnamaldehyde, an emulsifier and a fat-soluble drug to obtain a second oil phase system.

[0032] Step 102: adding water dropwise into the second oil phase system and stirring to obtain a drug-loaded nanoemulsion.

[0033] Emulsifier screening

[0034] The preparation method of the nanoemulsion comprises the following steps:

[0035] Step 201: under room temperature conditions, weighing 600 mg of cinnamaldehyde and 300-900 mg of an emulsifier, and uniformly mixing to obtain a first oil phase system.

[0036] Step 202: adding 5 ml of water dropwise into the first oil phase system, and maintaining a rotation speed of 600 rpm during the dropwise adding process, so that a uniform dispersion system, i.e., a cinnamaldehyde nanoemulsion, is spontaneously formed.

[0037] Step 203: investigating the type and amount of the emulsifier, with the particle size, PDI (polydispersity index) and appearance of the cinnamaldehyde nanoemulsion as the investigation indexes; after being placed at 4℃ for 7 days, the particle size, PDI and appearance of the cinnamaldehyde nanoemulsion are determined again. The determination results are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041] In Table 1, EL is polyoxyethylene castor oil, and RH is polyoxyethylene hydrogenated castor oil. From the results in Table 1, the preferred combination of the type and amount of emulsifier can be obtained: when cinnamaldehyde is used as the oil phase and Tween 80 is used as the emulsifier, it is difficult to form emulsion with uniform particle size, the PDI is large, and stratification occurs after standing; when cinnamaldehyde is used as the oil phase and polyoxyethylene castor oil EL is used as the emulsifier, the mass ratio of emulsifier to cinnamaldehyde is > 0.5, especially ≥ 1, which can form nanoemulsion with uniform particle size; when the emulsifier is polyoxyethylene hydrogenated castor oil RH, nanoemulsion with relatively uniform particle size can be formed, but when the mass ratio of emulsifier to cinnamaldehyde is ≥ 1, the PDI is large, the uniformity of particle size is poor, and when the amount of emulsifier is increased, stratification occurs after standing.

[0042] Screening of the composition of drug-loaded nanoemulsion

[0043] Drug-loaded nanoemulsion is prepared with cinnamaldehyde as the oil phase, polyoxyethylene castor oil EL or polyoxyethylene hydrogenated castor oil RH as the emulsifier, and fat-soluble drugs such as vitamin A or valsartan. The preparation method includes:

[0044] Step 301: Under room temperature conditions, 600 mg of cinnamaldehyde, 300-900 mg of emulsifier, and 50-200 mg of Va are mixed uniformly to obtain a third oil phase system. Alternatively, under room temperature conditions, 600 mg of cinnamaldehyde and 600 mg of emulsifier EL are mixed uniformly, and then 60 mg of valsartan is added in portions to obtain a fourth oil phase system.

[0045] Step 302: 5 ml of water is added dropwise to the third oil phase system or the fourth oil phase system, and the rotation speed is maintained at 600 rpm during the dropwise addition process to form a uniform dispersion system, i.e., drug-loaded nanoemulsion / drug-loaded emulsion, spontaneously.

[0046] Step 303: The particle size, PDI, and appearance of the prepared drug-loaded nanoemulsion (cinnamaldehyde-Va) are detected; after being placed at 4°C for 7 days, the particle size, PDI, and appearance of the drug-loaded nanoemulsion are detected again. The detection results are shown in Table 2:

[0047] Table 2

[0048]

[0049]

[0050] The drug-loaded nanoemulsion (cinnamaldehyde-valartan) prepared based on the fourth oil phase system has a particle size of 141.2 nm, a PDI of 0.27, and a good appearance, which indicates that the cinnamaldehyde-valartan drug-loaded nanoemulsion with uniform particle size is prepared; and the particle size, PDI and appearance do not change obviously after being placed for 3 days.

[0051] It can be known from Examples 10-23 that: when the oil phase is cinnamaldehyde, the drug for encapsulation is Va, and the emulsifier is polyoxyethylene hydrogenated castor oil RH, it is difficult to form cinnamaldehyde-Va drug-loaded emulsion with uniform particle size, and the PDI is large. When the oil phase is cinnamaldehyde, the drug for encapsulation is Va, and the emulsifier is polyoxyethylene castor oil EL, cinnamaldehyde-Va drug-loaded nanoemulsion with uniform particle size can be formed. And when the amount of EL is 600 mg, the maximum drug loading amount of Va is 100 mg. In the present application, PDI≤0.3 is used as the index for measuring the uniform particle size.

[0052] Screening of co-emulsifiers:

[0053] Cinnamaldehyde-Va drug-loaded nanoemulsion is prepared by taking cinnamaldehyde as the oil phase, polyoxyethylene castor oil EL as the emulsifier, and Va as the fat-soluble drug, and taking anhydrous ethanol, propylene glycol, PEG400 and n-butanol as the co-emulsifiers, respectively.

[0054] Preparation method:

[0055] Step 401: 600 mg of cinnamaldehyde, 600 mg of EL, 50-150 mg of Va and 1 ml of co-emulsifier are weighed and uniformly mixed at room temperature to obtain a third oil phase system.

[0056] Step 402: 5 ml of water is added dropwise into the third oil phase system, and the self-formation of a uniform dispersion system, i.e., cinnamaldehyde-Va drug-loaded nanoemulsion, is realized by maintaining a rotation speed of 600 rpm during the dropwise addition process.

[0057] Step 403: The types of co-emulsifiers are investigated, and the particle size, PDI and appearance of the newly prepared cinnamaldehyde-Va drug-loaded nanoemulsion under different conditions are detected; after being placed at 4°C for 7 days, the particle size, PDI and appearance of the drug-loaded nanoemulsion are detected again. The detection results are shown in Table 3.

[0058] Table 3

[0059]

[0060] From the results of Table 3, it can be seen that when the oil phase is cinnamaldehyde, the drug loaded is Va, the emulsifier is polyoxyethylene castor oil EL, and the co-emulsifier is anhydrous ethanol, propylene glycol, and n-butanol, it is difficult to form drug-loaded nanoemulsion with uniform particle size and stable storage. When polyethylene glycol 400 (PEG400) is added, stable nanoemulsion can be formed, but the PDI is large (>0.3). Referring to the results of Table 2 and Table 3, it can be seen that the co-emulsifier is not an essential component for forming nanoemulsion / drug-loaded nanoemulsion, and the co-emulsifier is not added in the subsequent research of the present application.

[0061] Content stability test:

[0062] The cinnamaldehyde raw material (dispersed in water), the cinnamaldehyde nanoemulsion prepared in Example 8, and the cinnamaldehyde-Va drug-loaded nanoemulsion prepared in Example 18 were respectively placed in a Schlenk flask, sealed, and stored in the dark at 25°C and 4°C. The cinnamaldehyde content was determined at different days, and the results are shown in Table 4:

[0063] Table 4

[0064]

[0065] From the results of Table 4, it can be seen that the nanoemulsion significantly improves the stability of the cinnamaldehyde content, and the cinnamaldehyde-Va drug-loaded nanoemulsion has better stability than the cinnamaldehyde nanoemulsion.

[0066] In vivo distribution test:

[0067] Experimental animals: KM male mice, body weight 25-35g.

[0068] Free fluorescein group (18): 18 KM mice were randomly selected as the free fluorescein group (referred to as "free group"). After fasting for 12h, they were given 1 dose of sample liquid containing fluorescein D I R (D I R is a fat-soluble cell membrane fluorescent probe; D I R was diluted with olive oil to form a uniform oily liquid). The D I R dosage was 1mg / kg.

[0069] Cinnamaldehyde nanoemulsion group (18): 18 KM mice were randomly selected as the cinnamaldehyde nanoemulsion group (referred to as "nanoemulsion group"). After fasting for 12h, they were given 1 dose of cinnamaldehyde nanoemulsion prepared in Example 8 containing D I R. The D I R dosage was 1mg / kg, and the cinnamaldehyde dosage was 360mg / kg.

[0070] Cinnamaldehyde-Va drug-loaded nanoemulsion group (18): 18 KM mice were randomly selected as the cinnamaldehyde-Va drug-loaded nanoemulsion group (referred to as "drug-loaded group"). After fasting for 12h, they were given 1 dose of cinnamaldehyde-Va drug-loaded nanoemulsion prepared in Example 18 containing D I R. The D I R dosage was 1mg / kg, and the cinnamaldehyde dosage was 360mg / kg.

[0071] After administration, the mice in each group were sacrificed by over-anesthesia in batches according to the preset time (4-72 hours), and then their livers, intestines, spleens, and kidneys were collected for imaging. Since both fluorescein DIR and cinnamaldehyde are fat-soluble and are simultaneously encapsulated in the nanoemulsion, the DIR content is positively correlated with the cinnamaldehyde content in this research system. Figure 2 Fluorescence imaging results showed that compared with the free and nanoemulsion groups, the drug-loaded emulsion significantly prolonged the drug's intestinal retention time and enhanced intestinal absorption. Furthermore, compared with the free group, the nanoemulsion and drug-loaded emulsions significantly increased the DIR content (i.e., cinnamaldehyde content) in the liver. In particular, the drug-loaded group showed the highest liver accumulation of cinnamaldehyde, which persisted 72 hours after administration. Compared with the nanoemulsion group, the Va in the cinnamaldehyde-Va-loaded nanoemulsion exhibited an intestinal retention effect, prolonging drug absorption and exhibiting liver-targeting properties, resulting in long-term drug storage in the liver.

[0072] Efficacy test:

[0073] Experimental animals: SD male rats, weighing 180-200 g.

[0074] Animal model of cholestatic liver fibrosis (BDL): 30 experimental animals were fasted for 12 hours before surgery. Surgery: After anesthesia with isoflurane, the abdomen was opened under sterile conditions, the liver margin was elevated, the duodenum was pulled open, and the common bile duct was separated by 2-3 cm. Two ligatures were made near the duodenum and near the liver hilum with No. 000 silk thread. The common bile duct was cut between the two ligatures. The liver was restored to its original position and the incision was sutured. After the animals woke up from anesthesia, they were given a normal diet and free access to water.

[0075] BDL model group (6 animals): 6 of the above BDL model animals were selected as the BDL model group (abbreviated as "model group"), and physiological saline was administered by gavage once a day starting from the second day after surgery.

[0076] API control group (6 animals): 6 of the above-mentioned BDL model animals were selected as the API control group (referred to as the "control group"). Starting from the second day after surgery, cinnamaldehyde API sample solution (cinnamaldehyde was diluted with olive oil to form a uniform oily liquid) was administered by gavage once a day, with a single dose of 120 mg / kg / day.

[0077] Cinnamaldehyde nanoemulsion group (6 animals): 6 of the BDL model animals were selected as the cinnamaldehyde nanoemulsion group (referred to as the "nanoemulsion group"), and starting from the second day after surgery, the cinnamaldehyde nanoemulsion of Example 8 was administered by gavage once a day at a dose of 120 mg / kg / day.

[0078] Cinnamaldehyde-Va-loaded nanoemulsion group (6): 6 of the above BDL model animals were taken as the cinnamaldehyde-Va-loaded nanoemulsion group (referred to as "drug-loaded group") and were given the cinnamaldehyde-Va-loaded nanoemulsion of Example 18 by gavage once a day at a dose of 120 mg / kg / day from the second day after the operation.

[0079] Sham group (6): The experimental animals were fasted for 12 h before the operation; operation: after being anesthetized with isoflurane, the abdomen was opened under aseptic operation conditions, and then the incision was sutured; after the animal was anesthetized and awakened, the animal was given normal diet and free water; normal saline was given by gavage once a day from the second day after the operation.

[0080] After the above groups were given the drug (or normal saline) for 14 days, the blood, bile, liver tissue and other samples were collected after the animals were fasted for 12 h. The serum was taken for serum biochemical index detection, and the detection results are shown in Table 5. The serum biochemical index detection results show that, compared with the BDL model group, the nanoemulsion group and the drug-loaded group can significantly reduce the levels of ALT, AST and TBA in the serum, indicating that the cinnamaldehyde nanoemulsion and the cinnamaldehyde-Va-loaded nanoemulsion can both significantly improve the liver function level of the BDL rats, and the cinnamaldehyde-Va-loaded nanoemulsion has a better anti-liver fibrosis effect in vivo; and the raw material drug comparison group has no significant improvement in the serum biochemical index. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bile acid (TBA) are used to measure liver function.

[0081] Table 5

[0082] Index Model group Sham operation group Control group Nanoemulsion group Drug-loaded group ALT 113.57±15.27*** 46.50±3.39 105.57±22.79 81.80±11.56## 85.17±16.85## AST 577.43±115.23*** 106.50±15.64 529.29±153.85 437.60±91.13# 400.67±120.19# TBA 278.96±65.22*** 48.52±41.38 254.69±64.37 243.38±47.36 204.15±54.70#

[0083] Among them, *** means that the difference is significant compared with the sham operation group, P < 0.001; # means that the difference is significant compared with the model group, P < 0.05; ## means that the difference is significant compared with the model group, P < 0.01.

[0084] The liver tissue samples of each group were made into paraffin sections and were subjected to hematoxylin-eosin staining (H&E staining), Sirius red staining and Masson staining, respectively, and the staining results are shown in Table 6. Figure 3 The results show that, compared with the sham operation group, the bile duct proliferation and necrosis of the animals in the model group increased significantly, indicating that the modeling was successful; compared with the model group, the bile duct proliferation and necrosis of the comparison group did not decrease significantly; the nanoemulsion group and the drug-loaded group can improve the bile duct proliferation and necrosis of the liver after being given the drug, and the improvement of the drug-loaded group is more obvious.

[0085] The Sirius red staining can show collagen deposition in the liver, and the more collagen deposition, the more serious the degree of liver fibrosis. The model group can show serious collagen deposition, the contrast group does not significantly reduce collagen deposition compared with the model group, while the nanoemulsion group and the drug-loaded group significantly reduce collagen deposition after administration, and the drug-loaded group is better.

[0086] The Masson staining can show fibrous connective tissue in the liver, and the more fibrous connective tissue proliferation, the higher the degree of liver fibrosis. The model group can show serious fibrous connective tissue proliferation, the contrast group does not significantly inhibit fibrous connective tissue proliferation compared with the model group, while the nanoemulsion group and the drug-loaded group significantly inhibit fibrous connective tissue proliferation after administration, and the drug-loaded group is better.

[0087] The above slice staining results show that the cinnamyl aldehyde nanoemulsion and the cinnamyl aldehyde-Va drug-loaded nanoemulsion can improve the degree of liver fibrosis of the BDL model rats, and the drug-loaded nanoemulsion has better in-vivo anti-liver fibrosis efficacy. Among them, vitamin A is stored in the liver and has a certain liver targeting. It is speculated that fat-soluble vitamins such as vitamin D, vitamin E and vitamin K and drugs stored or metabolized in the liver can also be used to prepare the drug-loaded nanoemulsion in the application.

[0088] The nanoemulsion and the drug-loaded nanoemulsion of the application can be used as food or medicine, and can also be used to prepare anti-liver fibrosis drugs or food. It can prolong the retention time of cinnamyl aldehyde and fat-soluble drugs in the intestinal tract, and enhance the intestinal absorption of the drugs, thereby enhancing the anti-liver fibrosis efficacy.

[0089] The above is only a preferred embodiment of the application and is not used to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A nanoemulsion based on cinnamaldehyde, characterized in that The invention comprises cinnamaldehyde, an emulsifier, water and a fat-soluble drug, wherein the fat-soluble drug comprises valsartan or vitamin A; the mass ratio of the cinnamaldehyde, the emulsifier, the vitamin A and the water is 12:6-18:1-4:100, wherein the emulsifier is polyoxyethylene castor oil; the mass ratio of the cinnamaldehyde, the emulsifier, the valsartan and the water is 60:60:6:500, wherein the emulsifier is polyoxyethylene hydrogenated castor oil or polyoxyethylene castor oil.

2. The nanoemulsion according to claim 1, wherein Also included are co-emulsifiers.

3. The nanoemulsion according to claim 2, characterized in that The auxiliary emulsifier includes: ethanol, propylene glycol, polyethylene glycol 400 or n-butanol.

4. The nanoemulsion according to claim 1, wherein The preparation method of the nanoemulsion comprises: Mixing cinnamaldehyde, emulsifier, and fat-soluble drug uniformly to obtain a second oil phase system; Water is added dropwise into the second oil phase system and stirred to obtain the drug-loaded nanoemulsion.

5. A use of the nanoemulsion according to any one of claims 1 to 4, characterized in that: Used for preparing drugs for treating liver fibrosis.

Images