CN114515271A

CN114515271A - Costunolide nanoemulsion with acid stability and preparation method and application thereof

Info

Publication number: CN114515271A
Application number: CN202210085235.XA
Authority: CN
Inventors: 牛霞; 李桂玲; 何红伟; 牛冰羽; 王玉成; 蒙亚楠; 王晓梅
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-05-20

Abstract

The invention discloses costunolide nanoemulsion with acid stability and a preparation method and application thereof. The invention provides a costunolide-loaded nanoemulsion preparation which is prepared from the following raw materials: costunolide, oil phase, emulsifier, auxiliary emulsifier and water. The invention also provides a method for preparing the costunolide-loaded nanoemulsion preparation, which comprises the following steps: and uniformly mixing the costunolide, the oil phase, the emulsifier and the co-emulsifier, and then dropwise adding into water to obtain the costunolide-loaded nanoemulsion preparation. The costunolide-loaded nanoemulsion preparation prepared by the invention obviously increases the solubility of costunolide and improves the stability of costunolide to acid, light and heat. The costunolide-loaded nanoemulsion preparation provided by the invention is suitable for oral administration (more stable in a gastric acid environment), so that the absorption efficiency of an organism can be improved, and the effect of treating hepatic fibrosis is further improved.

Description

Costunolide nanoemulsion with acid stability and preparation method and application thereof

Technical Field

The invention belongs to the field of nano-drugs, and relates to costunolide nanoemulsion with acid stability and a preparation method and application thereof, in particular to application of costunolide nanoemulsion as an anti-hepatic fibrosis drug.

Background

In recent years, chronic liver disease has become an important issue threatening global human health, and the long-term persistence of chronic liver injury and malignant transformation lead to the occurrence of liver fibrosis, cirrhosis and even liver cancer. 2015 surveys showed that 81 million people die worldwide from liver cancer. The severe social situation warns us that liver diseases become the urgent health problem for people.

Liver fibrosis is an important transitional stage for the development of liver cirrhosis and even liver cancer from liver injury, while early liver fibrosis is reversible, so that early diagnosis and reversal of liver fibrosis through drug treatment become important breakthrough for treating various chronic liver injuries. But the anti-hepatic fibrosis drugs are seriously deficient in the world. The FDA has not yet approved drugs for the treatment of liver fibrosis and cirrhosis. Currently, only 2 kinds of Chinese medicines approved in China, namely compound turtle shell liver softening tablets and capsules for strengthening body resistance and removing blood stasis, are used for treating hepatic fibrosis and cirrhosis. To expedite drug development in the field of liver fibrosis, the FDA has given a number of drug fast-check channels for the treatment of liver fibrosis. In this regard, the development of anti-hepatic fibrosis drugs has become one of the hot spots of current research.

Studies show that Costunolide (COS) has an anti-fibrosis effect. However, COS, as a sesquiterpene lactone, has poor water solubility, low bioavailability and very poor stability under the acidic conditions of gastric juice, which greatly limits its oral application.

Disclosure of Invention

The invention aims to provide costunolide nanoemulsion with acid stability and a preparation method and application thereof.

The invention provides a costunolide-loaded nanoemulsion preparation which is prepared from the following raw materials: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

Specifically, the preparation raw materials are as follows: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

The invention also provides a method for preparing the costunolide-loaded nanoemulsion preparation, which comprises the following steps: preparing a costunolide-loaded nanoemulsion preparation from raw materials; the raw materials comprise: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

Specifically, the raw materials are as follows: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

Any one of the oil phases is medium carbon chain triglyceride.

The oil phase described above may also be ethyl oleate, isopropyl myristate or isopropyl palmitate.

Any emulsifier is one of the following (i), (ii) or (iii): polyoxyethylene castor oil; ② polyoxyethylene hydrogenated castor oil; ③ polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil.

Specifically, the polyoxyethylene castor oil and the polyoxyethylene hydrogenated castor oil may be equal mass of the polyoxyethylene castor oil and the polyoxyethylene hydrogenated castor oil (i.e., 1 part by mass of the polyoxyethylene castor oil and 1 part by mass of the polyoxyethylene hydrogenated castor oil).

Any of the above co-emulsifiers is ethanol (i.e., absolute ethanol).

Any of the above co-emulsifiers may also be propylene glycol.

The proportion of any one of the raw materials is as follows: 16-70mg costunolide: 0.4g oil phase: 0.2-0.4g emulsifier: 1-2ml co-emulsifier: 10ml of water.

The proportion of any one of the raw materials is as follows: 16-70mg costunolide: 0.4g oil phase: 0.4g of emulsifier: 2ml of co-emulsifier: 10ml of water.

The preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: and (3) uniformly mixing costunolide, the oil phase, the emulsifier and the co-emulsifier, and then dropwise adding into water to obtain the costunolide-loaded nanoemulsion preparation.

The preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: the costunolide, the oil phase, the emulsifier and the co-emulsifier are mixed uniformly, and then are added into water drop by drop to form a uniform dispersion system spontaneously, namely the costunolide-loaded nanoemulsion preparation.

The preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: the costunolide, the oil phase, the emulsifier and the co-emulsifier are uniformly mixed, and then are dropwise added into water (the system is kept in a stirring state in the dropwise adding process), and a uniform dispersion system is spontaneously formed, namely the costunolide-loaded nanoemulsion preparation.

The preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: the costunolide, the oil phase, the emulsifier and the co-emulsifier are uniformly mixed, and then are dropwise added into water (the system is kept in a stirring state at the rotating speed of 100-1000rpm in the dropwise adding process), and a uniform dispersion system, namely the costunolide-loaded nano-emulsion preparation, is spontaneously formed.

The preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: the costunolide, the oil phase, the emulsifier and the co-emulsifier are uniformly mixed, and then are dropwise added into water (the system is kept in a stirring state at the rotating speed of 800rpm in the dropwise adding process), and a uniform dispersion system is spontaneously formed, namely the costunolide-loaded nanoemulsion preparation.

The preparation of the costunolide-loaded nanoemulsion preparation is carried out at room temperature.

The costunolide-loaded nanoemulsion preparation prepared by any one of the methods also belongs to the protection scope of the invention.

In any of the costunolide-loaded nanoemulsion preparation, the content of costunolide is 1.0-5.3 mg/ml.

In any one of the costunolide-loaded nanoemulsion preparations, the content of costunolide is 1.2-5.3 mg/ml.

In any one of the costunolide-loaded nanoemulsion preparations, the content of costunolide is 1.3-5.3 mg/ml.

Any of the above carbon chain triglycerides may be MIGLYOL 812N.

Any of the above carbon chain triglycerides may specifically be caprylic/capric triglyceride.

Any one of the costunolide with molecular formula of C₁₅H₂₀O₂CAS number 553-21-9.

The above polyoxyethylene castor oil (also called castor oil polyoxyethylene ether) has CAS number of 61791-12-6.

Any one of the isopropyl myristate of formula C₁₇H₃₄O₂CAS number 110-27-0.

Any one of the above ethyl oleate having a molecular formula of C₂₀H₃₈O₂CAS number 111-62-6.

Any one of the isopropyl palmitate having the molecular formula C₁₉H₃₈O₂CAS number 142-91-8.

Any of the above polyoxyethylene hydrogenated castor oils having a CAS number of 61788-85-0.

The invention also protects the application of any costunolide-loaded nanoemulsion preparation in preparing anti-hepatic fibrosis medicines. In the application, the costunolide-loaded nanoemulsion preparation can be used as the only active ingredient of a medicament and also can be used as one of the active ingredients of the medicament.

The invention also provides an anti-hepatic fibrosis medicament, which comprises any one of the costunolide-loaded nanoemulsion preparation. In the medicine, the costunolide-loaded nanoemulsion preparation can be used as the only active ingredient and also can be used as one of the active ingredients.

In the above application, carrier material can be added during preparation of the medicine.

The medicine can also comprise a carrier material.

Costunolide is unstable to acid, light and heat, and has very low water solubility (saturated solubility in water is only 26 μ g/ml). The costunolide-loaded nanoemulsion preparation prepared by the invention obviously increases the solubility of costunolide and improves the stability of costunolide to acid, light and heat. The costunolide-loaded nanoemulsion preparation provided by the invention is suitable for oral administration (more stable in a gastric acid environment), so that the absorption efficiency of an organism can be improved, and the effect of treating hepatic fibrosis is further improved.

The costunolide-loaded nanoemulsion preparation prepared by the invention has the following advantages: (1) the preparation method is simple and can be operated at room temperature; (2) the water solubility is good, and the concentration can reach 5.3 mg/ml; (3) the stability is good under the acidic condition; (4) after the administration dosage is reduced, the anti-hepatic fibrosis effect is good.

Drawings

FIG. 1 is a chromatogram of example 2.

Fig. 2 shows the results of the blood concentration-time curve and pharmacokinetic parameters of costunolide raw material and nanoemulsion in rats.

FIG. 3 is the pictures of HE staining and sirius red staining of rat liver treated with costunolide bulk drug and nanoemulsion.

FIG. 4 shows the effect of costunolide bulk drug and nanoemulsion on the level of liver fibrosis-related factors of BDL rats.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Room temperature: 25 ℃ plus or minus 2 ℃. PDI: polydispersity index (Polydispersity index), which represents the degree of uniform particle size; the smaller the PDI, the more uniform the particle size.

Unless otherwise stated, the quantitative tests in the following examples were carried out in triplicate, and the results were averaged.

The quantitative tests in the following examples, data were processed using SPSS19.0 statistical software and the results were expressed as mean ± standard deviation, with One-way ANOVA test, P < 0.05 (x) indicating significant difference between the two groups and P < 0.01 (x) indicating very significant difference between the two groups.

Costunolide with molecular formula of C₁₅H₂₀O₂CAS number 553-21-9; purchased from Nanjing Biotech Ltd. Medium carbon chain triglyceride (MIGLYOL 812N): the manufacturer is SASOL company, and the seller is Jiya North chemical industry Co., Ltd, Beijing; MIGLYOL 812N is caprylic/capric triglyceride. Polyoxyethylene castor oil (also known as castor oil polyoxyethylene ether) with CAS number 61791-12-6; available from mclin, catalog No. C804845. Isopropyl myristate of formula C₁₇H₃₄O₂CAS number 110-27-0; available from mclin, inc, catalog No. I811858. Ethyl oleate with molecular formula C₂₀H₃₈O₂CAS number 111-62-6; available from mclin, catalog No. E828279. Isopropyl palmitate with the molecular formula C₁₉H₃₈O₂CAS number 142-91-8; available from mclin, inc, catalog number I811770. Ethyl acetate of the formula C₄H₈O₂CAS number 141-78-6; purchased from national chemical group, ltd. Soybean oil: purchased from Jiangxi Yipu crude drug industry Co. Polyoxyethylene hydrogenated castor oil, CAS number 61788-85-0; purchased from chemical company Limited, Waverrucke, Beijing, under the number HWG 55665.

Example 1 screening and optimization of Components

First, screening of oil phase and emulsifier

The oil phase respectively adopts: isopropyl myristate (IPM), Medium Chain Triglycerides (MCT), ethyl oleate, isopropyl palmitate, ethyl acetate or soybean oil. The emulsifying agents respectively adopt: polyoxyethylene hydrogenated castor oil (RH), polyoxyethylene castor oil (EL), tween 80 or an RH-EL mixture (RH-EL mixture consists of equal masses of RH and EL). Auxiliary emulsifier: anhydrous ethanol.

The preparation method of the nano-emulsion (under the room temperature condition) comprises the following steps: 0.4g of oil phase, a plurality of emulsifiers and 2ml of auxiliary emulsifier are taken and uniformly mixed, and then the mixture is dropwise added into 10ml of water (the system is kept in a stirring state at the rotating speed of 800rpm in the dropwise adding process), and a uniform dispersion system, namely the nano-emulsion, is spontaneously formed.

The particle size and PDI of the freshly prepared nanoemulsion were measured. Then, the mixture was left at room temperature for a certain period of time, and the particle size and PDI were measured again.

The effect of 0.4g of emulsifier is firstly detected, and if the formed nano-emulsion is stable, the effect of 0.2g of corresponding emulsifier is detected. The smaller the PDI value, the better, and the field generally considers that the effect of the PDI value less than 0.3 is good. And (3) monitoring that the appearance is changed or PDI or the particle size is greatly changed at any time in the placing process, and abandoning the corresponding scheme.

The results are shown in Table 1. When the oil phase is medium-carbon chain triglyceride, stable nanoemulsion can be formed by using the emulsifier EL (0.4 g), RH (0.2 g or 0.4g), Tween 80 (0.4 g) or RH-EL mixture (0.2 g or 0.4g in total). When the oil phase is ethyl oleate, stable nanoemulsion can be formed by using the emulsifier EL (the dosage is 0.4g) or RH-EL mixture (the total dosage is 0.2 g). When the oil phase is isopropyl myristate, stable nanoemulsion can be formed by using EL (0.2 g or 0.4g) or RH-EL mixture (0.2 g or 0.4g in total) as emulsifier. When the oil phase is isopropyl palmitate, the emulsifier is RH-EL mixture (the total dosage is 0.4g) and can form stable nanoemulsion. When the oil phase is ethyl acetate or soybean oil, stable nano-emulsion cannot be formed. The nano-emulsion formed when the medium carbon chain triglyceride is in an oil phase has smaller particle size, and correspondingly, more types of emulsifiers can form stable nano-emulsion. In summary, in the present invention, it is preferable that the oil phase is medium-chain triglyceride.

TABLE 1 screening of emulsifier types and amounts

Screening of coemulsifier

Oil phase: medium carbon chain triglycerides. Emulsifier: RH-EL mixtures (RH-EL mixtures consist of equal masses of RH and EL). The auxiliary emulsifying agent respectively adopts: ethanol (anhydrous ethanol), propylene glycol, n-butanol or glycerol.

The preparation method of the nano-emulsion (under the room temperature condition) comprises the following steps: 0.4g of oil phase, 0.4g of emulsifier and a plurality of coemulsifiers are taken and evenly mixed, and then the mixture is dropwise added into 10ml of water (the system is kept in a stirring state at the rotating speed of 800rpm in the dropping process), and a uniform dispersion system, namely the nano-emulsion, is spontaneously formed.

The results are shown in Table 2. The oil phase is medium carbon chain triglyceride, the emulsifier is RH-EL mixture, and the auxiliary emulsifier is ethanol (with the addition amount of 1ml) and propylene glycol (with the addition amount of 2ml) which can form stable nanoemulsion. Ethanol is preferred as a co-emulsifier in the present invention because of its wider use.

TABLE 2 selection results of the kind and amount of co-emulsifier

Third, screening the highest drug-loading rate

Oil phase: medium carbon chain triglycerides. Emulsifier: RH-EL mixtures (RH-EL mixtures consist of equal masses of RH and EL). Auxiliary emulsifier: anhydrous ethanol.

The preparation method of the drug-loaded nanoemulsion (under the room temperature condition) comprises the following steps: a plurality of costunolide, 0.4g of oil phase, 0.4g of emulsifier (namely 0.2g of RH +0.2g of EL) and 2ml of auxiliary emulsifier are taken, uniformly mixed and then dropwise added into 10ml of water (the system is kept in a stirring state at the rotating speed of 800rpm in the dropping process) to spontaneously form a uniform dispersion system, namely the drug-loaded nanoemulsion.

The newly prepared drug-loaded nano-emulsion is divided into three parts, one part is directly used for detecting the particle size, the PDI and the costunolide content, the other part is placed at room temperature for one week and then is used for detecting the particle size, the PDI and the costunolide content, and the other part is placed at 4 ℃ for one week and then is used for detecting the particle size, the PDI and the costunolide content.

And a particle size analyzer is adopted for detecting the particle size and the PDI.

The method for detecting the costunolide content comprises the following steps: taking 1ml of a drug-loaded nano-emulsion sample, placing the sample in a 10ml measuring flask, adding methanol (used as a demulsifier) to a scale mark, shaking to fully demulsify, filtering, taking filtrate as a sample solution to be detected, carrying out high performance liquid chromatography, recording a chromatogram, and measuring the area of a main peak (a target peak) of costunolide.

The conditions of the high performance liquid chromatography are as follows:

chromatograph: ultra-high pressure liquid chromatograph, model: LC 20403D; the manufacturer: shimadzu corporation, Japan;

a chromatographic column: shimadzu Shim-pack GIST C18, 2 μm, 2.1 × 50mm (Shimadzu corporation, japan);

mobile phase: the pH value of the mixture is adjusted to 2.0 by phosphoric acid solution, wherein the mixture consists of 70 parts by volume of methanol and 30 parts by volume of water;

column temperature: 25 ℃;

flow rate of mobile phase: 0.2 ml/min;

sample introduction volume: 5 mu L of the solution;

detection wavelength: 225 nm.

In addition, dissolving costunolide in methanol to prepare solutions with different costunolide concentrations as a series of standard solutions, performing high performance liquid chromatography under the same chromatographic conditions, recording chromatogram, and measuring target peak area; and (4) regressing the concentration of the costunolide by using the target peak area, and drawing a standard curve.

Substituting a target peak area obtained by performing high performance liquid chromatography analysis on the sample solution to be detected into a standard curve to obtain the costunolide concentration, and further calculating to obtain the costunolide content in the drug-loaded nanoemulsion.

The results of the maximum drug loading screening are shown in Table 3. The results show that when the costunolide is added in an amount of 100mg, part of the drug is precipitated. When the addition amount of the costunolide is less than or equal to 70mg, the drug-loaded nanoemulsion can be formed, and the changes of the particle size, PDI and the costunolide content are very small after the costunolide is placed at room temperature or 4 ℃ for one week, so that the drug-loaded nanoemulsion with the drug content can exist stably. In conclusion, the highest loading of the drug-loaded nanoemulsion on costunolide is 5.3 mg/ml.

TABLE 3 screening results for maximum drug loading

Example 2 preparation and Performance testing of Costunolide-loaded nanoemulsion formulations

Preparation method of nanoemulsion preparation loaded with costunolide

Preparation method of costunolide-loaded nanoemulsion preparation (at room temperature): taking a proper amount of costunolide, 0.4g of oil phase, 0.4g of emulsifier (namely 0.2g of RH +0.2g of EL) and 2ml of co-emulsifier, uniformly mixing, and then dropwise adding into 10ml of water (keeping the system in a stirring state at the rotating speed of 800rpm in the dropwise adding process) to spontaneously form a uniform dispersion system, namely the costunolide-loaded nanoemulsion preparation.

Second, stability test

1. Acid stability

(1) The sample (low concentration) was mixed with an acidic medium (aqueous hydrochloric acid solution pH 1.0) in a ratio of 1:2

Costunolide group: accurately weighing 1.5mg of costunolide, placing in a 25ml measuring flask, adding methanol to scale, and shaking to dissolve costunolide completely to obtain costunolide methanol solution (60 μ g/ml). 3.33ml of costunolide methanol solution was weighed into a 10ml measuring flask and diluted to the mark with aqueous hydrochloric acid solution of pH 1.0. The solution was stored at 37 ℃ for 2 hours in the dark. The samples were taken at 0 hour after being kept in the dark, and then at 30-minute intervals, and the costunolide content in the sample liquid was measured by high performance liquid chromatography (the same method as in step three of example 1).

The costunolide-loaded nanoemulsion preparation group comprises the following components: preparing costunolide-loaded nanoemulsion preparation (with costunolide content of 3mg/ml) according to the method of the first step, and diluting with water to 50 times volume (with costunolide content of 60 μ g/ml); then, 3.33ml was measured and placed in a 10ml measuring flask, and diluted to the mark with an aqueous hydrochloric acid solution of pH 1.0. The solution was stored at 37 ℃ for 2 hours in the dark. Samples were taken at time 0 by keeping them protected from light, and then at 30 minute intervals (1ml for each sample). Putting 1ml of sample solution taken each time into a 10ml measuring flask, diluting the sample solution to the scale with methanol, shaking to fully demulsify, and filtering to obtain filtrate as sample solution to be detected. The costunolide content was determined by HPLC (same as in step three of example 1).

The chromatogram is shown in FIG. 1 (arrows indicate peaks of interest). In fig. 1, a is a chromatogram of a sample at the time of 0-hour stability of the costunolide acid, B is a chromatogram of a sample at the time of 2-hour (120-minute) stability of the costunolide acid, C is a chromatogram of a sample at the time of 0-hour stability of the costunolide-loaded nanoemulsion preparation acid, and D is a chromatogram of a sample at the time of 2-hour (120-minute) stability of the costunolide-loaded nanoemulsion preparation acid.

The results of the costunolide content in each sample are shown in Table 4.

The costunolide content of the sample is multiplied by the volume of the sample (1ml), divided by the amount of costunolide (μ g) added as the raw material in the sample, and then multiplied by 100 percent to obtain the costunolide relative percentage (%) of the sample. The results of the relative percentage of costunolide content of each sample are shown in Table 4.

TABLE 4

(2) The sample (high concentration) was mixed with an acidic medium (aqueous hydrochloric acid solution pH 1.0) in a ratio of 1:2

The costunolide-loaded nanoemulsion preparation group comprises the following components: preparing costunolide-loaded nanoemulsion formulation (containing costunolide about 3mg/ml) according to the method of step one, measuring 3.33ml, placing in a 10ml measuring flask, and diluting to scale with hydrochloric acid aqueous solution with pH 1.0. The solution was stored at 37 ℃ for 2 hours in the dark. Samples were taken at 0 o' clock with protection from light, and then at 30min intervals (1ml per sample). Putting 1ml of sample solution taken each time into a 10ml measuring flask, diluting the sample solution to the scale with methanol, shaking to fully demulsify, and filtering to obtain filtrate as sample solution to be detected. The costunolide content was determined by HPLC (same as in step three of example 1).

Because the costunolide raw material medicine is difficult to dissolve in water, the solubility of the final concentration of the nanoemulsion of 1.0mg/ml in an acidic medium (pH1.0 hydrochloric acid aqueous solution) can not be achieved by far, and the solubility can not be achieved even if a solubilizer is added into the medium; and the addition of the solubilizer changes the medium composition, and the measured stability result is influenced by the solubilizer. Thus, the high concentration sample: under the condition that the acidic medium is 1:2, the stability of the costunolide-loaded nanoemulsion preparation is only examined, but the stability of the raw material drug cannot be examined.

The results of the costunolide content of each sample are shown in Table 5.

The results of the relative percentage of costunolide content of each sample are shown in Table 5.

TABLE 5

Sampling sample	Costunolide content (mg/ml)	Relative percentage of costunolide (%)
			Time 0	0.92±0.02	98.9±2.34
30 minutes	0.92±0.02	98.1±2.22
			60 minutes	0.89±0.02	94.9±1.80
90 minutes	0.87±0.02	93.2±1.71
			120 minutes	0.85±0.03	90.8±2.71

(3) The sample was mixed with an acidic medium (aqueous hydrochloric acid solution pH 1.0) in a ratio of 1:100

Costunolide group: accurately weighing costunolide 50.5mg, placing in 25ml measuring flask, adding methanol to scale, and shaking to dissolve costunolide completely to obtain costunolide methanol solution (2.02 mg/ml). 1ml of costunolide methanol solution and 100ml of aqueous hydrochloric acid solution with pH of 1.0 were weighed and mixed well. The solution was stored at 37 ℃ for 2 hours in the dark. Sampling is carried out at the time 0, then sampling is carried out at intervals of 30 minutes, and the costunolide content in the sample liquid is detected by adopting a high performance liquid chromatography (the method is the same as the method in the third step of the example 1).

The costunolide-loaded nanoemulsion preparation group comprises the following components: preparing costunolide-loaded nanoemulsion formulation according to the method of step one (wherein costunolide content is 2.02 mg/ml). 1ml of the costunolide-loaded nanoemulsion preparation and 100ml of hydrochloric acid aqueous solution with the pH value of 1.0 are weighed and fully mixed. The solution was stored at 37 ℃ for 2 hours in the dark. Samples were taken at time 0, followed by 30 minute intervals (1ml each). Putting 1ml of sample solution taken each time into a 10ml measuring flask, diluting the sample solution to the scale with methanol, shaking to fully demulsify, and filtering to obtain filtrate as sample solution to be detected. The costunolide content was determined by HPLC (same as in step three of example 1).

The results of the costunolide content in each sample are shown in Table 6.

The results of the relative percentage of costunolide content of each sample are shown in Table 6.

TABLE 6

The costunolide-loaded nanoemulsion preparation has good stability when placed in an acidic medium (mixed according to the ratio of a low-concentration sample to the acidic medium being 1:2 and mixed according to the ratio of a high-concentration sample to the acidic medium being 1: 2), and the loss ratio of the medicine is only 5.4% and 9.2% after 2 hours; the costunolide (bulk drug) is unstable when placed in an acidic medium (mixed according to the ratio of a low-concentration sample to the acidic medium of 1: 2), and the loss ratio of the drug is up to 67.3 percent after 2 hours of mixing. After the costunolide-loaded nanoemulsion preparation is placed in an acidic medium (mixed according to the ratio of the sample to the acidic medium of 1: 100), the stability is reduced, and the relative percentage content of the residual medicine is 36.6 percent after 2 hours; and the residual medicine content of the costunolide (bulk drug) is only 2.36 percent after the costunolide is placed for 2 hours under the same condition. The results show that, compared with costunolide bulk drug, the costunolide-loaded nanoemulsion preparation has a protection effect on costunolide under acidic conditions.

In the sample: after the nano-emulsion preparation carrying the costunolide is placed for 2 hours under the condition of mixing the acidic medium with the ratio of 1:100, the relative percentage content of the costunolide in the nano-emulsion preparation carrying the costunolide is 15.5 times of the relative percentage content of the costunolide in the costunolide bulk drug. The suggestion is that after the costunolide-loaded nanoemulsion preparation is orally taken, the costunolide-loaded nanoemulsion preparation can be kept in a gastric acid environment and is not damaged, so that the amount of the medicine entering the small intestine is obviously higher than that of the costunolide bulk drug.

2. Stability in standing away from light at room temperature

Costunolide group: 4 parts of costunolide (each about 5mg, see table 7 for specific mass) were weighed in parallel, placed in 4 100ml measuring bottles, respectively, placed in the dark at room temperature (1 st part was placed for 0 days, 2 nd part was placed for 10 days, 3 rd part was placed for 20 days, and 4 th part was placed for 30 days), and then dissolved and diluted to the scale with methanol, respectively, to obtain a costunolide methanol solution. The costunolide content was determined by HPLC (same as in step three of example 1). And calculating according to the costunolide content to obtain a costunolide detection value. The remaining percentage of costunolide is detected as ÷ measured mass × 100%.

The costunolide-loaded nanoemulsion preparation group comprises the following components: weighing 13mg of costunolide, and preparing the costunolide-loaded nanoemulsion preparation according to the method in the step one. Placing in dark at room temperature (setting time of 0, 10, 20 or 30 days), sampling 1ml, placing in a 10ml measuring flask, diluting with methanol to scale, shaking to break emulsion, filtering, collecting filtrate, and detecting costunolide content by high performance liquid chromatography (the method is the same as in step three of example 1). Taking the detection value of the costunolide content in the nanoemulsion preparation at the time 0 as 100%, and calculating the relative value of the costunolide content in the nanoemulsion preparation after standing as the residual percentage of costunolide.

The results are shown in Table 7. The results show that after being placed in dark at room temperature for 30 days, the residual percentage of costunolide in the costunolide bulk drug is 87.0%, and the residual percentage of costunolide in the costunolide-loaded nanoemulsion preparation is 95.0%. The results show that compared with costunolide bulk drugs, the costunolide-loaded nanoemulsion preparation has a protection effect on costunolide under the conditions of room temperature and light shielding.

Table 7 costunolide and costunolide-loaded nanoemulsion stability results at room temperature in the dark

3. Light stability

Costunolide group: 4 parts of costunolide (about 5mg each, specific mass shown in Table 8) were weighed in parallel, placed in 4 100ml measuring bottles, respectively, placed at room temperature and continuously illuminated (1 st part was placed for 0 day, 2 nd part was placed for 10 days, 3 rd part was placed for 20 days, 4 th part was placed for 30 days; illumination intensity was 1600lx), and then dissolved and diluted to scale with methanol to obtain a costunolide methanol solution. The costunolide content was determined by HPLC (same as in step three of example 1). And calculating according to the costunolide content to obtain a costunolide detection value. The remaining percentage of costunolide is detected as ÷ measured mass × 100%.

The costunolide-loaded nanoemulsion preparation group comprises the following components: 13mg of costunolide is weighed and prepared into the nanoemulsion preparation carrying the costunolide according to the method in the first step. Standing at room temperature and continuously illuminating (the standing time is respectively set as 0 time, 10 days, 20 days or 30 days; the illumination intensity is 1600lx), then sampling 1ml, placing in a 10ml measuring flask, diluting to the scale with methanol, shaking to fully demulsify, filtering, taking the filtrate, and detecting the costunolide content by high performance liquid chromatography (the method is the same as the method in step three of example 1). Taking the detection value of the costunolide content in the nanoemulsion preparation at the time 0 as 100%, and calculating the relative value of the costunolide content in the nanoemulsion preparation after standing as the residual percentage of costunolide.

The results are shown in Table 8. The results show that after being placed under the condition of room temperature and illumination for 30 days, the residual percentage of the costunolide in the costunolide bulk drug is 79.7 percent, and the residual percentage of the costunolide in the costunolide-loaded nanoemulsion preparation is 89.2 percent. The stability of both costunolide and costunolide-loaded nanoemulsion is reduced under the illumination condition, but compared with the two, the stability of the nanoemulsion is better.

Table 8 costunolide and costunolide-loaded nanoemulsion stability results under ambient light conditions

Example 3 in vivo efficacy test in animals

Preparation of nanoemulsion preparation loaded with costunolide

Preparation method of costunolide-loaded nanoemulsion preparation (at room temperature): 52mg of costunolide, 0.4g of oil phase, 0.4g of emulsifier (namely 0.2g of RH +0.2g of EL) and 2ml of auxiliary emulsifier are taken, uniformly mixed and then added into 10ml of water dropwise (the system is kept in a stirring state at the rotating speed of 800rpm in the dropwise adding process), and a uniform dispersion system, namely the costunolide-loaded nanoemulsion preparation, is spontaneously formed.

Second, in vivo absorption effect

Test animals: SD rats, body weight 200 + -20 g.

Costunolide group: orally intragastrically administering costunolide (2 ml per rat intragastrically suspended with 0.5% sodium carboxymethylcellulose water solution) at a dose of 40 mg/kg.

The costunolide-loaded nanoemulsion preparation group comprises the following components: and (3) orally administering the costunolide-loaded nanoemulsion preparation prepared in the step one through intragastric gavage, wherein the administration dose is 40mg/kg calculated by costunolide.

Respectively carrying out eye socket blood collection on rats at different time points (0.25, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24h) after administration, collecting serum after 3000rpm centrifugation, precipitating protein by 4 times of volume of methanol, then carrying out centrifugation at 12000rpm for 30min, taking supernatant, detecting costunolide concentration in the supernatant at different times by adopting a liquid chromatography-mass spectrometry (LC-MS) method, drawing a blood concentration-time curve graph of costunolide and nanoemulsion as raw material medicines in rats, and calculating related pharmacokinetic parameters.

LC-MS was performed using a XEVO-TQS # WAC1607 ultra high performance liquid chromatography-tandem mass spectrometer (Waters, USA).

A chromatographic column: ACQUITY

BEH C18 1.7μm；

Column temperature: at 40 ℃; sample introduction amount: 10 mu l of the mixture;

flow rate of mobile phase: 0.4 ml/min;

mobile phase A: acetonitrile; mobile phase B: 0.1% (volume ratio) formic acid aqueous solution;

elution procedure for chromatography: at 0-0.5min, the mobile phase B accounts for 44% of the mobile phase by volume, and the corresponding mobile phase A accounts for 56% of the mobile phase by volume; in 0.5-1min, the volume percentage of the mobile phase B in the mobile phase is linearly reduced to 2%, and the volume percentage of the corresponding mobile phase A in the mobile phase is linearly increased to 98%; in 1-2min, the volume percentage of the mobile phase B in the mobile phase is linearly reduced to 0%, and the volume percentage of the corresponding mobile phase A in the mobile phase is linearly increased to 100%; the mobile phase is the mobile phase A in the 2-3 min; at 3-4min, the volume percentage of the mobile phase B in the mobile phase linearly increases to 44%, and the volume percentage of the corresponding mobile phase A in the mobile phase linearly decreases to 56%.

The mass spectrometry system was a triple quadrupole equipped with electrospray ionization source (ESI), and the analysis was performed in positive ion mode, operating conditions were as follows: capillary voltage was 2800V, desolventizing gas temperature was 550 ℃, ion source temperature: at 150 ℃. Costunolide quantitative ion pair: m/z 233.1/187.1, internal standard quantitation ion pair: m/z 231.1/185.1.

The results of the blood concentration-time curve and pharmacokinetic parameters of costunolide bulk drugs and nanoemulsion in rats are shown in figure 2 and table 9. Compared with costunolide bulk drug, the bioavailability of the costunolide-loaded nanoemulsion preparation is improved to 4.95 times.

TABLE 9 pharmacokinetic parameters of costunolide bulk drug and costunolide-loaded nanoemulsion in rats (n ═ 5)

Parameter(s)	Costunolide	Costus lactone-loaded nanoemulsion preparation
			C_max(μg/L)	3.8	44.6
T_max(h)	10	5.3
			AUC_0～24h(μg/L h)	39.6	196
MRT_0～24h	12.7	5.2

Third, evaluation of drug efficacy

Test animals: SD male rats weighing 180-.

1. Preparation of cholestatic liver fibrosis (BDL) animal model.

The test animals were fasted for 12h before surgery.

And (3) operation: after anesthesia with isoflurane, the abdomen is opened under aseptic conditions, the hepatic margin is raised, the duodenum is pulled open, the common bile duct is separated by 2-3cm, two ligatures are respectively tied at the position near the duodenum and the position near the hepatic portal by using a number 000 silk thread, the common bile duct is cut off from the middle of the two ligature positions, and the liver is recovered to the original position and then is sutured.

After the animals are anesthetized and conscious, the animals eat the normal diet and drink water freely.

2. Group administration

BDL model group (labeled BDL): taking 7 of the BDL modeling animals as a BDL model group, beginning on the 2 nd day after the operation, and feeding 1 time of saline solution by intragastric administration every day for 14 continuous days;

costunolide bulk drug (marked as COS): taking 9 of the BDL modeling animals as costunolide raw material medicines, and performing intragastric administration for 1 time every day on the 2 nd day after operation (the medicine is fully suspended by 0.5% sodium carboxymethylcellulose aqueous solution to obtain suspension), wherein the single administration dosage of costunolide is 80mg/kg for 14 days;

costunolide-loaded nanoemulsion formulation high dose group (labeled OH): taking 9 of the BDL modeling animals as a costunolide-loaded nanoemulsion preparation high-dose group, beginning on the 2 nd day after the operation, performing intragastric gavage on the costunolide-loaded nanoemulsion preparation prepared in the first step 1 time every day, wherein the single administration dose is 24mg/kg calculated by costunolide, and continuously administering for 14 days;

nanoemulsion formulation low dose group with costunolide (labeled OL): taking 9 of the BDL modeling animals as a costunolide-loaded nanoemulsion preparation low-dose group, and performing gavage administration on the costunolide-loaded nanoemulsion preparation prepared in the first step 1 time every day on the 2 nd day after the operation, wherein the single administration dose is 12mg/kg calculated by costunolide and is continuously 14 days;

sham group (marked Sham) (7): the test animals are fasted for 12h before operation; and (3) operation: after anesthesia with isoflurane, opening abdomen under aseptic condition, then suturing incision, after the animal is anesthetized and conscious, eating normally, drinking freely; beginning on postoperative day 2, the stomach was perfused with 1 time saline daily for 14 consecutive days.

After 14 days of administration, fasting is carried out for 12 hours, and then samples of blood, bile, liver and the like are collected.

3. Evaluation of drug efficacy

And (3) centrifuging the collected blood sample to obtain a serum sample, and carrying out serum biochemical index detection. The results are shown in Table 10. The results show that the costunolide-loaded nanoemulsion low dose group (OL) was able to significantly reduce the levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), TBA (bile acid), TG (triglyceride), TBili (total bilirubin) compared to the BDL model group; the costunolide raw material drug can only reduce the TBA level. Shows that the costunolide-loaded nanoemulsion preparation can remarkably improve the liver function level of BDL rats.

TABLE 10 Effect of Costunolide bulk drug and Costunolide-loaded nanoemulsion on serum biochemical indices of BDL model rats

	Sham	BDL	COS	OH	OL
						ALT(U/L)	41.0±3.83	97.29±23.32^***	90.71±27.13	69.00±18.38^#	63.86±20.18^#
AST(U/L)	127.86±8.40	487.57±94.12^***	473.71±143.87	452.14±183.07	320.71±107.42^##
						ALP(U/L)	172.57±15.46	357.29±41.23^***	323.86±45.13	281.43±39.84^##	278.86±78.92^#
TBA((μmol/L))	21.26±11.09	209.11±23.04^***	180.73±24.58^#	184.84±19.66	149.53±40.86^##
						TG(mmol/L)	0.85±0.41	1.37±0.17^***	1.25±0.22	1.21±0.20	1.03±0.24^#
TBiLi(μmol/L)	0.66±0.54	159.23±24.28^***	145.67±45.17	154.44±28.11	121.09±38.30^#

Note:^***P<0.001vs Sham group;^#P<0.05,^##P<0.01vs BDL group.

Taking the collected liver sample, making paraffin sections and carrying out HE and sirius red staining. The staining picture is shown in FIG. 3. HE staining results showed: bile duct proliferation and necrosis were significantly increased in the BDL model group (BDL) compared to the Sham group (Sham); after the costunolide bulk drug (COS) is administrated, the hyperplasia and necrosis of the bile duct of the liver are improved to a certain extent compared with a BDL model group; after the costunolide-loaded nanoemulsion preparation is administered to the high-dose group (OH) and the low-dose group (OL), the hyperplasia and necrosis of the bile duct of the liver can be obviously improved compared with the BDL model group, and the effect is better than that of the COS group. Collagen deposition in the liver can be seen in the sirius red staining picture, and the more the collagen deposition, the more serious the liver fibrosis degree is proved; severe collagen deposition was seen in the stained pictures of the BDL model group compared to the Sham group (Sham); compared with the BDL group, the costunolide-loaded nanoemulsion preparation has the advantages that the collagen deposition phenomenon is obviously reduced in the high-dose group (OH) and the low-dose group (OL); compared with the BDL group, the costunolide bulk drug group (COS) has a certain improvement on the collagen deposition phenomenon, but the effect is not as good as that of the nanoemulsion group. The result shows that the costunolide-loaded nanoemulsion preparation can obviously improve the hepatic fibrosis degree of BDL rats.

Liver tissues were taken, total protein was extracted and Western Blot analysis was performed. The results are shown in FIG. 4. As can be seen from the results, the levels of fibrosis-associated proteins MMP2 and TGF β 1 were significantly increased in liver tissue of the animals of the BDL model group compared to the Sham group (Sham); compared with BDL group, the water level of MMP2 and TGF beta 1 of the costunolide-loaded nanoemulsion preparation in low dose group (OL), high dose group (OH) and costunolide bulk drug group (COS) is obviously reduced, wherein MMP2 has a reduction effect, and OL group is superior to OH group and COS group. The results suggest that costunolide-loaded nanoemulsion formulations may inhibit liver fibrosis levels by inhibiting the TGF β 1 pathway.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. A costunolide-loaded nanoemulsion preparation is prepared from the following raw materials: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

2. A method for preparing costunolide-loaded nanoemulsion preparation comprises the following steps: preparing a costunolide-loaded nanoemulsion preparation from raw materials; the raw materials comprise: costunolide, oil phase, emulsifier, auxiliary emulsifier and water.

3. The formulation of claim 1 or the method of claim 2, wherein: the oil phase is medium-carbon chain triglyceride.

4. A formulation or method as claimed in any one of claims 1 to 3, wherein: the emulsifier is (i), (ii) or (iii): polyoxyethylene castor oil; ② polyoxyethylene hydrogenated castor oil; ③ polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil.

5. A formulation or method as claimed in any one of claims 1 to 4, wherein: the auxiliary emulsifier is ethanol.

6. A formulation or method as claimed in any one of claims 1 to 5, wherein: the raw materials are as follows: 16-70mg costunolide: 0.4g oil phase: 0.2-0.4g emulsifier: 1-2ml co-emulsifier: 10ml of water.

7. A formulation or method as claimed in any one of claims 1 to 6, wherein: the preparation method of the costunolide-loaded nanoemulsion preparation comprises the following steps: and uniformly mixing the costunolide, the oil phase, the emulsifier and the co-emulsifier, and then dropwise adding into water to obtain the costunolide-loaded nanoemulsion preparation.

8. A costunolide-loaded nanoemulsion formulation prepared by the method of any one of claims 2 to 7.

9. Use of the costunolide-loaded nanoemulsion formulation of claim 8 in the preparation of anti-hepatic fibrosis drugs.

10. An anti-hepatic fibrosis drug, which comprises the costunolide-loaded nanoemulsion formulation of claim 8.