KR102356581B1 - Composition Comprising Liposome and Lactobacillus spp. for Preventing or Treating Porcine Infectious Disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a porcine infectious disease containing liposomes and lactic acid bacteria, and more particularly, to a porcine reproductive and respiratory syndrome by administering together a liposome containing a malotus philippensis extract and a Lactobacillus genus lactic acid bacteria. It relates to a composition for preventing or treating porcine infectious diseases, including. The liposome of the present invention and the lactic acid bacteria of the genus Lactobacillus inhibit the proliferation of viruses and enhance the immunity of animals, and thus can be usefully used to treat porcine infectious diseases, including porcine reproductive and respiratory syndrome.

Description

Composition for the prevention or treatment of swine infectious diseases, including liposomes and lactic acid bacteria {Composition Comprising Liposome and Lactobacillus spp. for Preventing or Treating Porcine Infectious Disease}

The present invention relates to a composition and feed additive for the prevention or treatment of porcine infectious diseases, including liposomes and lactic acid bacteria, and more specifically, to the porcine reproductive system by administering together liposomes containing Malotus filifensis extract and Lactobacillus lactic acid bacteria. It relates to a composition for preventing or treating swine infectious diseases, including respiratory syndrome.

Porcine Reproductive and Respiratory Syndrome (PRRS) was first discovered in the United States in 1986 and in Europe in 1990. Then, in 1991, Dr. Wensvoort at the Central Veterinary Research Institute in the Netherlands isolated the causative virus for the first time using pig alveolar macrophages and revealed that it was a disease caused by a virus (PRRSV), and was named Lelystad virus after the place where the institute was located. . After that, Lelystad virus was called by names such as swine infertility and respiratory syndrome (SIRS) virus, porcine epidemic abortions and respiratory syndrome (PEARS) virus, blue-eared pig disease virus, etc. I decided to call it a virus. When piglets or growing pigs contract porcine reproductive and respiratory syndrome due to the above virus, respiratory symptoms such as cough, shortness of breath, and pneumonia are shown. It is known to cause abnormalities, and as the name suggests, it is characterized by growth retardation due to reproductive disorders and respiratory symptoms. Attenuated vaccines and dead poison vaccines are known as vaccines for preventing porcine reproductive and respiratory syndrome. In addition, since the PRRS virus has a characteristic of replicating and propagating in cells, an appropriate therapeutic agent for porcine reproductive and respiratory syndrome is not known.

Nico Van Rooijen, Annemarie Sanders. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods 174 (1994) 83-93

The present inventors made intensive research efforts to develop a therapeutic agent for porcine reproductive and respiratory syndrome caused by a virus having the characteristic of intracellular replication. As a result, liposome extract containing Phosphatidylcholine, Sterol Compound, Phosphatidyl Ethanolamine, Polyethyleneglycolated Phosphatidyl Ethanolamine and Mallotus Philippensis. (liposome) has excellent stability and was effective in the treatment of porcine reproductive and respiratory syndrome, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a liposome comprising the Malotus philippensis extract.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating porcine infectious disease comprising the liposome as an active ingredient.

Another object of the present invention is to provide an animal feed additive for preventing or improving porcine infectious diseases comprising the liposome.

Another object of the present invention is to provide a method for treating a swine infectious disease comprising inoculating a pig with a pharmaceutical composition comprising the liposome as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating porcine infectious diseases, including liposomes and Lactobacillus genus lactic acid bacteria consisting of phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, and polyethylene glycolized phosphatidyl ethanolamine. will be.

Another object of the present invention is to provide an animal feed additive for the prevention or improvement of porcine infectious diseases, including liposomes and Lactobacillus genus lactic acid bacteria composed of phosphatidylcholine, sterol-based compounds, phosphatidyl ethanolamine, and polyethylene glycolated phosphatidyl ethanolamine. will be.

According to one aspect of the present invention, the present invention provides a liposome comprising phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, polyethyleneglycolized phosphatidyl ethanolamine, and an extract of malotus philippensis.

As used herein, the term liposome refers to a phospholipid bilayer capable of encapsulating an active drug.

As used herein, the term “polyethylene glycolized (PEGylated)” refers to chemically bonding polyethylene glycol (PEG).

Malotus philippensis of the present invention is a plant belonging to the genus oleifera that grows in India, the Philippines, Southeast Asia, and the like, and is also called a kamala tree. The extract of Malotus philippensis may include Rottlerin (Rottlerin).

The extraction site of the Malotus philippensis is selected from the group consisting of leaves, branches, wood parts, bark, roots, etc., but is not limited thereto. More specifically, the extraction site of the Malotus philippensis is a tree bark.

As used herein, the term "extract" has a meaning commonly used as a crude extract in the art, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. That is, the Malotus philippensis extract includes not only those obtained by using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the extract of the present invention.

The Malotus philippensis extract used in the present invention can be obtained by extracting Malotus philippensis, or it can be purchased and used. In the case of obtaining Malotus philippensis by treatment with an extraction solvent, various extraction solvents may be used. More specifically, a polar solvent or a non-polar solvent may be used. Suitable as polar solvents are: (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol), (iii) acetic acid, (iv) DMFO (dimethyl-formamide) and (v) DMSO (dimethyl sulfoxide).

Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.

In the present invention, when extracting Malotus philippensis, hot water extraction method, cold extraction method, reflux extraction method, room temperature extraction method, or ultrasonic extraction method may be used, but is not limited thereto.

In one embodiment of the present invention, the Malotus philippensis extract is extracted with any one extraction solvent selected from the group consisting of water, an organic solvent, and a mixed solvent thereof, and the organic solvent is alcohol, petroleum ether, It is selected from the group consisting of hexane, benzene, chloroform, methylene chloride, ether, ethyl acetate, and acetone.

In one embodiment of the present invention, the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, and a mixed solvent thereof.

In one embodiment of the present invention, the concentration of the ethanol solution is 30 to 100% by volume.

More specifically, the concentration of the ethanol solution is 40 to 90% by volume, 40 to 80% by volume, 40 to 70% by volume, 50 to 70% by volume.

In one embodiment of the present invention, the liposome contains O-palmitoylgalactose (OPG) on the surface.

The OPG is used to prepare galactosylated liposomes, and the galactosylated liposomes may target the lectin receptor of macrophages. The OPG uses the electrostatic attraction induced by the positive charge of the liposome caused by phosphatidyl ethanolamine and the negative charge of the OPG to be included on the surface of the liposome.

In one embodiment of the present invention, the phosphatidylcholine, sterol-based compound, phosphatidyl ethanolamine, polyethylene glycolated phosphatidyl ethanolamine and malotus philippensis extract have a weight ratio of 4-12:1-3:0.5-2: It is 1-5:1.

More specifically, the weight ratio is 6-10:1.25-2.5:0.5-2:1-5:1, 4-12:1-3:0.8-1.2:1-5:1, 4-12:1-3: 0.5-2:2-4:1, 7-9:1.75-2.25:0.5-2:1-5:1, but not limited thereto. Most specifically, the weight ratio is 7-9:1.75-2.25:0.8-1.2:2-4:1.

In one embodiment of the present invention, the phosphatidylcholine is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, DMPC), 1 ,2-dihexanoyl-sn-glycero-3-phosphocholine (1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC), 1,2-diheptanoyl-sn-glycero-3- Phosphocholine (1,2-diheptanoyl-sn-glycero-3-phosphocholine), 1,2-Dioctanoyl-sn-glycero-3-phosphocholine (1,2-dioctanoyl-sn-glycero-3- phosphocholine, 1,2-dinonanoyl-sn-glycero-3-phosphocholine), 1,2-didecanoyl-sn-glycero-3-phosphocholine (1,2-didecanoyl-sn-glycero-3-phosphocholine) , 1,2-diundecanoyl-sn-glycero-3-phosphocholine (1,2-diundecanoyl-sn-glycero-3-phosphocholine), 1,2-dilauroyl-sn-glycero- 3-Phosphocholine (1,2-dilauroyl-snglycero-3-phosphocholine, DLPC), 1,2-Ditridecanoyl-sn-glycero-3-phosphocholine (1,2-ditridecanoyl-snglycero-3 -phosphocholine), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (1,2-dipentadecanoyl-sn-glycero-3-phosphocholine), 1,2-dipalmitoyl-sn-glycer Rho-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (1,2-diheptadecanoyl) -sn-glycero-3-phosphocholine), from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC) what is chosen I am not limited to this.

More specifically, the phosphatidylcholine is 2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC).

In one embodiment of the present invention, the sterol-based compound is cholesterol, 3b-[N-(N',N'-dimethylaminoethane)-carbamyl}cholesterol (3b-[N-(N',N'-) dimethylaminoethane)-cabamyl]cholesterol, DC-Chol), stigmasterol, campesterol, sitosterol, ergosterol, lanosterol, dinosterol, gorgosterol ( gogosterol), avenasterol, saringosterol, fucosterol, cholesteryl hemisuccinate, cholesteryl benzoate, cholesteryl ol cholesteryl oleate, cholesteryl oleyl carbonate, cholesteryl isostearate, cholesteryl linoleate, cholesteryl acetate, cholester consisting of cholesteryl palmitate, cholesteryl stearate, cholesteryl chloride, cholesteryl nonanoate and cholesteryl arachidonate It is selected from the group, but is not limited thereto.

More specifically, the sterol-based compound is cholesterol.

In one embodiment of the present invention, the phosphatidyl ethanolamine is a positively charged phosphatidyl ethanolamine.

The surface of the liposome is positively charged by the phosphatidyl ethanolamine, and can be connected to the negatively charged OPG through electrostatic attraction.

In one embodiment of the present invention, the phosphatidyl ethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2-Distearoyl-sn-Glycero-3-phosphoethanolamine, DSPE ), 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine), 1,2-dioctanoyl-sn-glycero- 3-phosphoethanolamine (1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine), 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (1,2-didecanoyl-sn- glycero-3-phosphoethanolamine), 1,2-diauroyl-sn-glycero-3-phosphoethanolamine (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, DLPE), 1,2-dimyri Stoyl-sn-glycero-3-phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), 1,2-dipentadecanoyl-sn-glycero-3-phospho Ethanolamine (1,2-dipentadecanoyl-snglycero-3-phosphoethanolamine), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl-snglycero-3-phosphoethanolamine, DPPE) And phosphatidylethanolamine (Phosphatidylethanolamine, PE) is selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the phosphatidyl ethanolamine of the polyethyleneglycolized phosphatidyl ethanolamine is the same as defined above.

More specifically, the phosphatidyl ethanolamine of the polyethylene glycolated phosphatidyl ethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2-Distearoyl-sn-Glycero-3-phosphoethanolamine , DSPE).

In one embodiment of the present invention, the polyethylene glycolated phosphatidyl ethanolamine is DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol) -2000]).

According to an embodiment of the present invention, the liposome of the present invention is 2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, phosphatidyl ethanolamine, 1,2-distearoyl-sn-glycero -3-Phosphoethanolamine-N-[amino(polyethylene glycol)-2000] and Malotus pilifensis extract, the weight ratio of which is 7-9:1.75-2.25:0.8-1.2:2-4: 1 is

In addition, according to an embodiment of the present invention, the liposome of the present invention may include OPG on the surface.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a swine infectious disease comprising the liposome as an active ingredient.

In one embodiment of the present invention, the swine infectious disease is foot-and-mouth disease, African swine fever, swine fever, swine blister disease, swine Ozeski disease, swine Japanese encephalitis, swine Tessen's disease, swine influenza, swine infectious gastroenteritis, swine alone, swine Genital respiratory syndrome, swine epidemic diarrhea, swine atrophic rhinitis, swine glasser disease, swine proliferative enteropathy, porcine circovirus infection is selected from the group consisting of, but is not limited thereto. More specifically, the porcine infectious disease is porcine reproductive and respiratory syndrome (PRRS).

As used herein, the term “porcine reproductive and respiratory syndrome” refers to a disease caused by a porcine reproductive and respiratory syndrome virus, which is a virus belonging to Arteriviridae as a positive-strand RNA virus. The porcine reproductive and respiratory syndrome virus has an RNA genome of about 15 kb and encodes 9 open reading frames (ORFs). There are GP5, M, and N encoded by ORFs 5, 6, and 7 as major structural proteins. GP5 protein is a 25 kDa envelope protein and induces strong neutralizing ability. M protein is an 18 kDa matrix protein that induces a cellular immune response.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the composition as an active ingredient.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration , intracerebral administration, intracranial administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the subject, An ordinarily skilled veterinarian can readily determine and prescribe an effective dosage (a pharmaceutically effective amount) for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.

As used herein, the term “prevention” refers to the prevention or protective treatment of a disease or disease state. As used herein, the term “treatment” refers to reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. At this time, the dosage form can be prepared in various ways such as oral medicine, injection, etc., in the form of a solution, suspension or emulsion in oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, dispersant or stable Additional topics may be included.

According to another aspect of the present invention, the present invention provides an animal feed additive for preventing or improving porcine infectious disease comprising the liposome.

The animal of the present invention is selected from the group consisting of dog, horse, cow, pig, sheep, goat, chicken, duck, goose and turkey, but is not limited thereto. More specifically, the animal is a pig.

As used herein, the term “feed additive” refers to a substance added to feed. The feed additive may be used for the purpose of improving the productivity and health of the target animal, but is not limited thereto.

As used herein, the term “improvement” refers to any action that reduces the symptoms of porcine reproductive and respiratory syndrome (PRRS), which is the target disease of the present invention.

According to another aspect of the present invention, the present invention provides a method for treating a swine infectious disease comprising inoculating a pig with a pharmaceutical composition comprising the liposome as an active ingredient.

In one embodiment of the present invention, the treatment method is to administer the pharmaceutical composition and the lactic acid bacteria of the genus Lactobacillus in combination.

As used herein, the term "administration" or "administering" refers to the administration of a therapeutically or prophylactically effective amount of the composition of the present invention directly to a pig suffering from, or likely to suffer from, the target disease in the body of a pig. This means that the same amount is formed.

The “therapeutically effective amount” of the composition means an amount of the composition sufficient to provide a therapeutic or prophylactic effect to a pig to which the composition is administered, and includes a “prophylactically effective amount”.

According to another aspect of the present invention, the present invention provides a pharmaceutical for preventing or treating porcine infectious diseases, including liposomes and Lactobacillus spp. consisting of phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, and polyethylene glycolized phosphatidyl ethanolamine. to provide a medical composition.

In one embodiment of the present invention, the lactic acid bacteria is Lactobacillus animalis SWLA-1 (Accession No.: KCCM11968P).

According to another aspect of the present invention, the present invention provides a liposome composed of phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, and polyethylene glycolated phosphatidyl ethanolamine, and an animal for the prevention or improvement of porcine infectious diseases, including lactic acid bacteria of the genus Lactobacillus Feed additives are provided.

Since the contents of phosphatidylcholine, sterol-based compounds, phosphatidyl ethanolamine, and polyethylene glycolated phosphatidyl ethanolamine, which are the components of the liposome of the present invention, are the same as those of the liposome containing the Malotus filifensis extract, In order to avoid excessive complexity of the present specification, descriptions of common content between the two are omitted.

The liposome of the present invention has excellent stability because it contains DSPE-PEG as a component, and can target macrophages because it contains OPG on the surface. Therefore, it is possible to effectively treat porcine reproductive and respiratory syndrome using the liposome of the present invention. In addition, when the liposome of the present invention is administered together with Lactobacillus animalis SWLA-1, it exhibits excellent PRRS therapeutic effect.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides liposomes comprising phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, polyethyleneglycolized phosphatidyl ethanolamine, and an extract of malotus philippensis.

(b) The present invention provides a pharmaceutical composition for preventing or treating porcine infectious disease comprising the liposome as an active ingredient.

(c) The present invention provides an animal feed additive for preventing or improving porcine infectious diseases comprising the liposome.

(d) provides a method for treating a swine infectious disease comprising the step of inoculating a pig with the pharmaceutical composition comprising the liposome of the present invention as an active ingredient.

(E) The present invention provides a pharmaceutical composition for the prevention or treatment of porcine infectious diseases comprising liposomes and Lactobacillus spp. lactic acid bacteria consisting of phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, and polyethylene glycolated phosphatidyl ethanolamine.

(f) The present invention provides an animal feed additive for preventing or improving porcine infectious diseases, including liposomes and Lactobacillus genus lactic acid bacteria composed of phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, and polyethylene glycolized phosphatidyl ethanolamine.

The liposome of the present invention and the lactic acid bacteria of the genus Lactobacillus inhibit the proliferation of viruses and enhance the immunity of animals, and thus can be usefully used to treat porcine infectious diseases, including porcine reproductive and respiratory syndrome.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Materials and Methods

DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), cholesterol (cholesterol) and PE (phosphatidyl ethanolamine) described herein were purchased from Sigma-Aldrich (Missouri, USA), and DSPE-PEG (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) was purchased from Avanti polar lipids (Alabama, USA), and Mallotus Philippensis extract was obtained from Maruzen ( Hiroshima, Japan). The Malotus philippensis extract is a Malantus philippensis extract obtained by treating the bark of a Malantus philippensis tree with ethanol.

Pig alveolar macrophage described herein was prepared by the following method.

Three-week-old piglets were purchased from a PRRS-negative farm, the lungs were removed after euthanasia, and 30 ml of Roswell Park Memorial Institute medium (RPMI) medium was injected through the airway. Elution was attempted. After repeating the medium injection and massage 3 times, centrifuge the collected RPMI medium at 3,000 rpm for 10 minutes to obtain 10 ⁷ cells/ml and mix 1:1 with Cell Banker 2 (Amsbio, USA) solution at -70 ℃ stored frozen in

Preparation Example 1: Preparation of liposomes containing malotus philippensis extract

Liposomes containing an extract of Malotus philippensis were prepared by thin-layer hydration.

In order to enhance the stability of the liposomes containing the Malotus philippensis extract, DSPE-PEG was used in the preparation of the liposomes.

8 mg of DPPC (dipalmito phosphatidylcholine), 2 mg of CH (cholesterol), 1 mg of PE (phosphatidyl ethanolamine), 3 mg of DSPE-PEG (1,2-distearoyl-sn-glycero- 3-Phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), and 1 mg of Malotus philipensis extract were quantified and DPPC:CH:PE:DSPE-PEG:Malotus philipensis extract was quantified. It was added to a round flask in a weight ratio of 8:2:1:3:1. 4 ml of an organic solvent (chloroform) was added to the round flask, and the mixed lipids were well dissolved. Thereafter, the mixed lipids were vacuum dried with a rotary concentrator to remove the organic solvent, and a lipid film containing the Malotus philippensis extract was formed.

1 mL of CEP (Cremophor EL:ethanol:phosphate buffered saline (PBS)=20:15:65 [volume ratio]) was added to the lipid film thus produced, and suspended while warming to 60° C. to form liposomes. In order to control the particle size, a sonication treatment was performed at 60°C. Then, the liposome was extruded through a polycarbonate membrane having a 200 nm pole size.

Finally, the unencapsulated Malotus philippensis extract was removed by ultrafiltration using a membrane filter.

In addition, in order to compare the properties of the liposome according to the weight ratio, liposomes were prepared at the weight ratio shown in Table 1 using the above-described method.

division Weight ratio of DPPC:CH:PE:DSPE-PEG:Malotus philippensis extract Preparation 1-1 8:2:1:3:1 Preparation 1-2 8:2:1:0:1 Preparation 1-3 12:3:1:3:1 Preparation Example 1-4 4:1:1:3:1

Preparation Example 2: Preparation of liposomes containing O-palmitoyl galactose (OPG, O-palmitoyl galactose) on the surface

To prepare liposomes targeting the lectin receptor of macrophages, OPG was used.

The following method was used to prepare OPG.

2 g of galactose was dissolved in 100 ml dry dimethylformamide at 70°C. To the resulting solution was added 0.5 ml dry dimethylformamide in which 2 ml of dry pyridine and 0.2 g of palmitoyl chloride were dissolved. The mixture was stirred at 60 °C for 3 hours and then at room temperature for 2 hours. Then, the mixture was slowly added to 100 ml of absolute ethanol with stirring. The precipitate formed in the same manner as above was collected and washed three times with 120 ml of absolute ethanol and 80 ml of dry diethyl ether. The obtained white solid material (OPG) was vacuum-dried at 50 °C for 2 hours to prepare OPG.

The liposome produced in Preparation Example 1-1 was mixed with the OPG solution (OPG dissolved in PBS, pH 7.4) prepared by the above method, and stirred at room temperature. After stirring, the mixture was rotated through a Sephadex G-50 column at 2000 rpm for 10 minutes to remove unbound OPG.

Finally, liposomes containing OPG on the surface were prepared through the above process.

Example 1: Evaluation of the physical properties of the prepared liposome

Example 1-1. Measurement of the size of the prepared liposome

In order to measure the size of the liposome prepared in Preparation Example 1, a certain amount of the liposome suspension was taken and diluted so that the cps (count per second) was in the range of 8,000 to 12,000, and the liposome using a laser particle analyzer (OPARIII, Ostuka Electronics, Japan) was measured.

The particle size of the liposome was variously measured from 315 nm to 340 nm before extrusion, but was uniformly measured to be from 128 to 134 nm after extrusion (Table 2).

division Liposome size (nm) before extrusion after extrusion Preparation 1-1 325 nm 134 nm Preparation 1-2 315 nm 128 nm Preparation 1-3 340 nm 134 nm Preparation Example 1-4 321 nm 133 nm

Example 1-2. Measurement of the encapsulation rate of the prepared liposome

In order to measure the encapsulation rate of the liposomes prepared in Preparation Examples 1 and 2, an HPLC method was used. More specifically, 1 ml of the prepared liposome suspension was added to the gel bed of a Sephadex G-25 column, and then the free drug and the drug encapsulated in the liposome were separated using a pH 4,0 phosphate buffer as a mobile phase. Thereafter, the concentration of the drug encapsulated in the liposome was measured using HPLC. The HPLC system used the LC-10Ai (Shimadzu, Tokyo, Japan) model, the column was Develosil C30-UG-5 (3.0 x 150 mm, Nomura Chemical Co, Seto, Japan), the mobile phase was 80% CH ₃ CN-0.05% TFA, the flow rate was 0.4 mL/min, and the detection wavelength was 350 nm.

From the obtained drug content, the encapsulation rate was measured using Equation 1 below.

[Equation 1]

Encapsulation efficiency (EE: %) = drug content/initial drug amount × 100

The encapsulation rates of the liposomes prepared in Preparation Example 1 were 47.3%, 35.6%, 42.3%, and 32.8%, respectively, and the liposome prepared in Preparation Example 1-1 showed the highest encapsulation rate (Table 3). Since the liposome prepared in Preparation Example 1-1 exhibited the highest inclusion rate, Preparation Example 1-1 was selected as the liposome to be used in Preparation Example 2.

division nesting rate Preparation 1-1 47.3% Preparation 1-2 39.6% Preparation 1-3 42.3% Preparation Example 1-4 32.8%

Examples 1-3. Measurement of drug release ability of the prepared liposome

A drug release experiment was performed to measure the drug release ability of the prepared liposome. After fixing one end of the pre-activated dialysis membrane (Molecular weight cut-off 12,000) with a closure, 1.5 mL of PBS as a release solvent is added, and 0.5 mL of liposome suspension containing drug is added to remove air bubbles. After fixing the other end of the bag with a closure and mixing it well, put the bag in 98 mL of PBS, which is the release medium, and stir at 37 °C and 100 rpm, taking 200 µL of the eluate for each hour, and quantifying the amount of drug released by HPLC. . The 100% release amount was defined as the amount of Malotus philippensis extract treated with Triton X-100 (final concentration 5%).

Additionally, in order to compare the stability of liposomes to plasma, a release experiment was performed by adding pig plasma to a bag instead of PBS.

Although the drug release ability of the liposome was significantly reduced compared to the actual drug, it was highest in Preparation Example 1-2, and there was no significant difference between Preparation Example 1-1, Preparation Example 1-3, Preparation Example 1-4, Preparation Example 2 (Table 4). In addition, in the experiment in which plasma was added instead of PBS, the drug release ability of the liposome was increased compared to that in the experiment with PBS, but it was highest in Preparation Example 1-1, which is a liposome to which DSPE-PEG was added (Table 5).

The liposome to which DSPE-PEG was added showed slower drug release than the liposome to which DSPE-PEG was not added (Preparation Example 1-2). This is because the drug is more stabilized and the leakage of the drug due to the destruction of the liposome membrane is reduced. In addition, the reason for the increase in the release of liposomes from plasma is judged to be due to the interaction of liposomes with proteins present in plasma, and in the case of liposomes containing DSPE-PEG, stability is increased, so that it can be maintained for a long time in plasma, and the target You can easily reach the target.

division drug release capacity (PBS) 1 h 2 h 3 h 6 h 9 h 12 h 15 h Preparation 1-1 6.2% 11.9% 16.8% 20.5% 27.9% 30.6% 35.4% Preparation 1-2 9.6% 13.2% 19.2% 26.7% 32.9% 36.4% 43.9% Preparation 1-3 6.1% 10.8% 17.9% 25.6% 26.7% 30.2% 37.1% Preparation Example 1-4 7.8% 12.2% 14.8% 21.8% 28.9% 31.2% 36.2% Preparation 2 8.9% 11.8% 13.9% 20.9% 26.8% 30.2% 35.9%

division drug release capacity (plasma) 1 h 2 h 3 h 6 h 9 h 12 h 15 h Preparation 1-1 6.9% 13.4% 17.3% 23.9% 28.1% 32.6% 37.1% Preparation 1-2 10.3% 18.6% 24.7% 30.8% 39.7% 48.8% 62.7% Preparation 1-3 6.2% 11.9% 20.2% 28.9% 30.2% 35.8% 42.7% Preparation Example 1-4 7.6% 16.3% 21.3% 27.8% 36.7% 39.8% 40.2% Preparation 2 7.8% 15.6% 19.7% 25.9% 31.8% 38.9% 41.3%

Example 2: Evaluation of intracellular drug transport capacity of liposomes containing extracts of Malotus philippensis

Pig alveolar macrophages were used to evaluate the intracellular drug-carrying ability of liposomes containing Malotus filifensis extract. A 24-well plate with 4 columns was seeded with 100 μL of the cell suspension so ^{that the cells were 1 x 10 4 cells/well.} Thereafter, 10 μL of the malotus philippensis extract was added to the first column, the liposome of Preparation Example 1-1 to the second column, and the liposome of Preparation Example 2 to the third column, respectively. Then, it was cultured for 48 hours and incubated at room temperature. Cell suspensions from each well were transferred to polycarbonate filters at 0, 1, 4, 8, 20 and 48 hours during incubation. The filter was centrifuged at 4,000 rpm for 15 minutes to separate the cells from the medium in the form of pellets. To rupture the cells, about 0.5 ml of Triton X-100 was added to the pellet, and incubated at 25° C. for 5 to 6 hours. Drug intake was assessed by HPLC method.

Experimental results confirmed that the liposome of Preparation Example 2 (liposome containing OPG on the surface) had the highest drug transport ability into cells (Table 6). These results indicate that in the case of liposomes containing OPG on the surface, the drug transport capacity in macrophages is excellent.

division Evaluation of intracellular drug-carrying capacity of liposomes (unit: μg/10) ⁶ cell) 0 h 2 h 4 h 6 h 8 h 10 h 12 h 24 h 48 h not nested
malotus philippensis extract 0 0.01 0.02 0.02 0.05 0.09 0.11 0.26 0.32 Preparation 1-1 0 0.14 0.26 0.41 0.56 0.76 0.87 1.21 1.35 Preparation 2 0 0.32 0.48 0.69 0.95 1.10 1.43 1.95 2.30

Example 3: Evaluation of cytotoxicity of liposomes containing extracts of Malotus philippensis

The cytotoxicity of the liposomes containing the malotus philippensis extract was evaluated by the MTT assay method.

Pig alveolar macrophages were used as test cells. Macrophages were seeded in 96-well plates at 1×10 ³ cells/well.

After culturing for 24 hours, 10 μL of the liposomes of Preparation Examples 1 and 2 were added to each well, and PBS was used as a control. After 72 hours of exposure, MTT solution was added to a final concentration of 0.5 mg/mL and incubated for 4 hours. Thereafter, the resulting formazan was dissolved with a formazan solution (10% SDS + 0.02 N HCl) to evaluate the cytotoxicity of the liposome. Cell viability was evaluated relative to the PBS-treated group.

Through the cytotoxicity evaluation, it was confirmed that there is no cytotoxicity in the case of the liposomes of Preparation Examples 1 and 2 (Table 7).

division Cell viability (relative value) Control (PBS) One Preparation 1-1 0.98 Preparation 2 1.07

Example 4: Evaluation of the therapeutic effect on PRRS of liposomes containing Malotus philippensis extract

The therapeutic effect on PRRS of the liposome containing the Malotus philippensis extract was evaluated by a virus titration analysis method.

To isolate PRRS, serum samples from farm A where North American (NA) PRRS virus that is 95% similar to the sequencing analysis of North American (NA) PRRS virus ORF were collected.

^{After thawing 10 7} cells/ml alveolar macrophages frozen in Cell Banker 2 solution at -70 ° C., inoculated with the serum collected from farm A, _{and incubated at 37 ° C., 5% CO 2} 3 to 4 days later, CPE was confirmed. The supernatant of alveolar macrophages in which CPE occurred was collected ^{and re-inoculated into 10 5} /ml MARC-145 cells (ATCC, Virginia, USA) prepared in advance, and cultured at _{37° C., 5% CO 2 .} The PRRS virus was finally isolated by repeating subculture and inoculation until the MARC-145 cells showed CPE. The final supernatant was collected for use in the experiment.

Pig alveolar macrophages were used as test cells. Macrophages were seeded in 96-well plates at 1×10 ³ cells/well. After 24 hours, 100 μL of the collected supernatant diluted 10-fold was inoculated, and 10 μL of the experimental group was treated in each well. TCID ₅₀ was calculated by the Reed-Muench method 72 hours after inoculation. As an experimental group, a control group (PBS), a malotus philippensis extract, the liposome of Preparation Example 1-1, and the liposome of Preparation Example 2 were used.

The experimental results showed that the liposome of Preparation Example 2 (liposome containing OPG on the surface) inhibited virus proliferation (Table 8). These results indicate that in the case of liposomes containing OPG on the surface, they effectively inhibit the proliferation of PRRS because they have excellent drug transport capacity in macrophages.

division Log ₁₀ TCID ₅₀ value Control (PBS) 7.1 Malotus philippensis extract not encapsulated in liposomes 6.3 Preparation 1-1 5.4 Preparation 2 5.1

Example 5: Evaluation of therapeutic effect on PRRS of liposomes and lactic acid bacteria containing Malotus philippensis extract

In order to confirm the therapeutic effect on PRRS of liposomes and lactic acid bacteria containing the Malotus philippensis extract, the virus titration analysis method described in Example 4 was evaluated.

Lactobacillus animalis SWLA-1, a lactic acid bacterium co-administered with liposomes, was deposited with the Korea Microbial Conservation Center under the accession number KCCM11968P.

Lactobacillus animalis SWLA-1 was inoculated into the nasal passages of pigs, and alveolar macrophages were obtained 3 days later using the above-described alveolar macrophage collection method, and then used in the experiment.

As a result of the experiment, when liposomes were inoculated with lactic acid bacteria into the nasal passages of pigs, the Log ₁₀ TCID ₅₀ value was 4.2. These results indicate the fact that when the lactic acid bacteria and the liposome are treated together, the growth of PRRS is more effectively inhibited than when the liposome is treated alone.

Korea Microorganism Conservation Center (Overseas) KCCM11968P 20170203

Claims (14)

In a liposome comprising phosphatidylcholine, a sterol-based compound, phosphatidyl ethanolamine, polyethylene glycolized phosphatidyl ethanolamine, and malotus philippensis extract in a weight ratio of 4-12:1-3:0.5-2:1-5:1 ,
The phosphatidylcholine is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, DMPC), 1,2-dihexanoyl-sn- Glycero-3-phosphocholine (1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (1,2-diheptanoyl -sn-glycero-3-phosphocholine), 1,2-dioctanoyl-sn-glycero-3-phosphocholine (1,2-dioctanoyl-sn-glycero-3-phosphocholine, 1,2-dinonanoyl-sn -glycero-3-phosphocholine), 1,2-didecanoyl-sn-glycero-3-phosphocholine (1,2-didecanoyl-sn-glycero-3-phosphocholine), 1,2-diundecanoyl -sn-glycero-3-phosphocholine (1,2-diundecanoyl-sn-glycero-3-phosphocholine), 1,2-dilauroyl-sn-glycero-3-phosphocholine (1,2-diundecanoyl-sn-glycero-3-phosphocholine) -dilauroyl-snglycero-3-phosphocholine, DLPC), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (1,2-ditridecanoyl-snglycero-3-phosphocholine), 1,2-di pentadecanoyl-sn-glycero-3-phosphocholine (1,2-dipentadecanoyl-sn-glycero-3-phosphocholine), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (1,2-diheptadecanoyl-sn-glycero-3-phosphocholine) , 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, ) is selected from the group consisting of;
The sterol-based compound is cholesterol, 3b-[N-(N',N'-dimethylaminoethane)-carbamoyl}cholesterol (3b-[N-(N',N'-dimethylaminoethane)-cabamoyl]cholesterol, DC- Chol), stigmasterol, campesterol, sitosterol, ergosterol, lanosterol, dinosterol, gorgosterol, avenasterol , saringosterol (saringosterol), and fucosterol (fucosterol) will be selected from the group consisting of, liposomes.
The liposome according to claim 1, wherein the liposome comprises O-palmitoyl galactose (OPG) on its surface.
delete delete delete The liposome according to claim 1, wherein the phosphatidyl ethanolamine is a positively charged phosphatidyl ethanolamine.
According to claim 1, wherein the polyethylene glycolated phosphatidyl ethanolamine is DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000) ]), which is a liposome.
A pharmaceutical composition for preventing or treating a swine infectious disease comprising the liposome of claim 1 as an active ingredient.
An animal feed additive for the prevention or improvement of swine infectious disease comprising the liposome of claim 1.
A method of treating a swine infectious disease comprising inoculating a pig with a pharmaceutical composition comprising the liposome of claim 1 as an active ingredient.
The method of claim 10, wherein the treatment method is to administer the pharmaceutical composition and the lactic acid bacteria of the genus Lactobacillus in combination.
A pharmaceutical composition for preventing or treating porcine infectious disease comprising the liposome of claim 1 and Lactobacillus genus lactic acid bacteria.
The pharmaceutical composition of claim 12, wherein the lactic acid bacteria is Lactobacillus animalis SWLA-1 (accession number: KCCM11968P).
An animal feed additive for preventing or improving porcine infectious diseases comprising the liposome of claim 1 and Lactobacillus genus lactic acid bacteria.