Composition for preventing or treating stroke or degenerative brain disease

Abstract

The present invention relates to a composition for preventing and/or treating a stroke or a degenerative brain disease comprising: at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil; olive oil; and soybean oil. The composition of the present invention has an excellent neuroprotective effect but no toxicity or side effects, and thus can be effectively and safely used for preventing, treating or ameliorating a stroke or a degenerative brain disease.

Claims

1. A method for ameliorating or treating stroke or neuronal cell damage caused by cerebral ischemia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition consisting essentially of olive oil, soybean oil, and: (a) egg yolk lecithin, glycerol, and sodium oleate or (b) medium chain triglyceride and refined fish oil.

2. The method of claim 1, wherein the composition consists essentially of 0.1-20 wt % of the olive oil, 0.1-20 wt % of the soybean oil, 0.1-20 wt % of the medium chain triglyceride, and 0.1-10 wt % of the refined fish oil.

3. The method of claim 1, wherein the composition consists essentially of 1.0-50 wt % of the olive oil, 0.1-20 wt % of the soybean oil, 0.01-5 wt % of the egg yolk lecithin, 2.25 wt % of the glycerol and 0.01-1 wt % of the sodium oleate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an image showing a result of an observation via TTC staining on brain slices of a control group wherein a saline solution is administered to middle cerebral artery occlusion model rats, and experimental groups wherein oleic acid, a sodium oleate solution, the Clinoleic20% or the SMOFlipid20% is administered to middle cerebral artery occlusion model rats.

(2) FIG. 2 is a graph illustrating comparison in damage rates of brain tissue (infarction area) in the control group, and the experimental groups of the middle cerebral artery occlusion model animals wherein the oleic acid, the sodium oleate solution, the Clinoleic20% or the SMOFlipid20% is administered.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(3) The inventors confirmed that the pharmaceutical composition for preventing or treating stroke or degenerative brain disease comprising at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil; olive oil; and soybean oil has superior neuroprotective effect for neuronal cell damages caused by cerebral ischemia, and that the pharmaceutical composition has less toxicity and side effects upon result of low death rate after administration in the middle cerebral artery occlusion model (MCAo), and succeedingly completed the present invention.

(4) The present invention will be described more in detail hereinafter.

(5) Pharmaceutical Composition

(6) The pharmaceutical composition of the present invention includes at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil; olive oil; and soybean oil.

(7) The composition of the present invention can be administered orally or parenterally, more preferably be administered parenterally, and most preferably be administered intravenously. In case of formulation, it can be prepared by using conventional diluents or excipients such as a filler, an extender, a binder, a humectants, a disintegrant and a surfactant.

(8) Formulation for parenteral administration includes a sterile aqueous solution, a nonaqueous solution, a suspension, an emulsifiable concentrate, a lyophilized formulation and a suppository. Propylene glycol, polyethylene glycol, vegetable oil and an injectable ester such as ethyl oleate, etc. can be used as a nonaqueous solvent and a solvent for suspension. Witepsol, macrogol, tween61, cacao butter, laurinum, glycerol, gelatin, etc. can be used as a base of the suppository. The pharmaceutical composition of the present invention can be administered hypodermically, intravenously and intramuscularly when administering parenterally.

(9) Dosage of the pharmaceutical composition of the present invention can vary depending on the patient's condition, weight, degree of disease, drug form, administration passage and period, and it can be selected properly by those skilled in the art. However, it is preferred to administer 0.1-1000 mg/kg of the composition of the present invention per a day to achieve desirable effect. Administration frequency can be once or several times per a day. However, the said dosage does not limit the scope of the present invention.

(10) The composition of the present invention can be used for preventing or treating stroke or degenerative brain disease solely or jointly with an operation, hormone therapy, chemotherapy and methods using biological response regulators.

(11) Health Functional Food

(12) The present invention provides the health functional food for preventing or ameliorating stroke or degenerative brain disease comprising at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil; olive oil; and soybean oil.

(13) The health functional food of the present invention can be used by adding at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil, olive oil, and soybean oil intactly, or with other food or food ingredients, and can be used according to conventional methods. Content of the active ingredients can be adequately adjusted according to a purpose of use (e.g. for prevention, health or therapeutic purpose). In general, components of the present invention can be added in an amount of 0.01-50 wt % of total food weight when preparing food or beverage.

(14) Additionally, types of the food are not specifically limited. Examples of foods that the pharmaceutical composition of the present invention can be added thereto could be beverages, gums, vitamin complexes, drink preparations among others and furthermore, the food includes all conventional health functional foods.

(15) Like other common beverages, beverage composition of the present invention can include various flavoring agents or natural carbohydrates, etc. as additional ingredients. The natural carbohydrates can be general sugars such as monosaccharides (e.g. glucose and fructose among others); disaccharides (e.g. maltose and sucrose among others); and polysaccharides (e.g. dextrin and cyclodextrin among others); and sugar alcohols such as xylitol, sorbitol and erythritol among others. Furthermore, other flavoring agents which are not mentioned before can also be used (e.g. saccharin and aspartame among others).

(16) The health functional food of the present invention can include various nutritional supplements, vitamin, minerals (e.g. electrolyte), synthetic flavoring agents, coloring agents and enhancers (e.g. cheese and chocolate among others), pectic acid and salt thereof, alginic acid and salt thereof, organic acid, protective colloid thickener, pH modifier, stabilizing agent, preservative, glycerin, alcohol and carbonator which is used for carbonated drinks among others.

(17) Preparation Method

(18) The pharmaceutical composition and the health functional food of the present invention can be prepared by conventional methods. The pharmaceutical composition and the health functional food can be prepared by mixing at least two substances selected from the group consisting of egg yolk lecithin, glycerol, sodium oleate, medium chain triglyceride and refined fish oil; olive oil; and soybean oil, and by selectively mixing pharmaceutically or nutritionally acceptable excipients, diluents or other additives.

(19) For an example, the composition was prepared by mixing olive oil, soybean oil, medium chain triglyceride, refined fish oil and distilled water. The ingredients were mixed by using a homogenizer, and were mixed several more times by using a high pressure homogenizer, resulting in the composition of the present invention.

(20) As an another example, the composition was prepared by mixing olive oil, soybean oil, egg yolk lecithin, glycerol, sodium oleate and distilled water. The ingredients were mixed by using a homogenizer, and were mixed, several more times by using a high pressure homogenizer, resulting in the composition of the present invention.

(21) Practical and presently preferred embodiments of the present invention are illustrative as shown in following Example, Experimental Examples and Comparative Example. However, it will be appreciated that those skilled in the art, on consideration of this invention, may make modifications and improvements within the spirit and scope of the present invention.

EXAMPLE

(22) The SMOFlipid 20% Injection that is manufactured and sold by the Fresenius-Kabi Korea.sup.©, and the Clinoleic 20% Injection that is manufactured and sold by the Baxter International Inc. were used as the compositions of the present invention comprising at least two substances selected from the group consisting of the egg yolk lecithin, the glycerol, the sodium oleate, the medium chain triglyceride and the refined fish oil; the olive oil; and the soybean oil. 100 ml of the SMOFlipid20% Injection comprises 6 g of the purified soybean oil, 6 g of the purified olive oil, 6 g of the medium chain triglyceride and 3 g of the refined fish oil. 100 ml of the Clinoleic20% Injection includes 16 g of the purified olive oil, 4 g of the purified soybean oil, 1.2 g of the egg yolk lecithin, 2.25 g of the glycerol and 0.03 g of the sodium oleate.

Comparative Example

(23) The oleic acid and the sodium oleate solution were used as compositions for comparison with the effects of the Clinoleic20% Injection and the SMOFlipid20% Injection of the present invention.

(24) The oleic acid was purchased from Sigma Aldrich Korea (Catalog No. 01008). The sodium oleate is the sodium salt of the oleic acid and is more soluble to water because it is in form of powder unlike the oleic acid. The sodium oleate was purchased from Sigma Aldrich Korea (Catalog No. 07501). The sodium oleate solution was prepared by adding 0.25 ml of the glycerol into the 10 mg of the sodium oleate and vortexing; mixed with triple distilled water (Millipore DQ-3) to have total 10 ml of volume; and comminuted for three hours by using a sonicator.

Experimental Example 1: Neuroprotective Effect of the Composition of the Present Invention—Middle Cerebral Artery Occlusion Model (MCAo)

(25) 1.1. Preparation of Laboratory Animals

(26) 8 week-old adult male Sprague-Dawley (SD) rats, obtained from Samtako (Korea), weighing 300 g were kept in an environment to adapt to the experimental environment while providing enough feed and water. After one week of adaptation period, the animals were used for the following experiments.

(27) 1.2. Preparation of Middle Cerebral Artery Occlusion Model

(28) Intraluminal suture method was used to measure neuroprotective effect of the oleic acid, the sodium oleate solution, the SMOFlipid20% and the Clinoleic20% on neuronal cell damage caused by local cerebral ischemia (Zea Longa, et al., Stroke, 20:84-91, 1989).

(29) In detail, a nylon suture (4-0, 22 mm) was prepared by silicone coating about 5-8 mm of the end of the suture to have 0.36 mm of diameter, and was used for experiment. The rats prepared in Experimental Example 1.1 were anesthetized with 5% of isoflurane in a mixture of gas (i.e. 70% N.sub.2O and 30% O.sub.2). A syringe was inserted in one side for blood collection and a probe was inserted in the other side to measure blood pressure. Then the blood glucose level and the blood gas level in the collected blood were measured.

(30) Through midline neck incision, right carotid artery and external carotid artery (ECA), which are branches of external carotid artery, were separated. Superior thyroid artery and laryngeal artery were cauterized by using an electrocauter and a probe was introduced from external carotid artery to internal carotid artery by inserting it about 18-19 mm from the common carotid artery branch and fixing it with a suture. After suturing the skin incision and the rats were allowed to recover from anesthesia. Same number of animals were induced in all experimental groups in the very day and observed the operation while maintaining 37 g 0.5° C. of body temperature. The rats were anesthetized again in the same way as the above after 90 minutes of the operation and the probe was withdrawn to allow reperfusion.

(31) 24 hours after the reperfusion, the animals were sacrificed by cervical spine dislocation. The brains were removed within two minutes and six 2 mm thick slices were obtained. Then the tissue slices were immersed in a 12-well plate containing 2% TTC (triphenyltertrazolium chloride) solution for 30 minutes at 37° C., and the tissue slices were fixed with 4% paraformaldehyde for observation. All operation procedures were conducted beneath an operating microscope. The body temperature was controlled not to drop below 37° C. by using a heat lamp and isofluran was maintained at 2% in the course of anesthesia. Each stained brain tissue slice was photographed one by one by using a digital camera. The files were stored in a computer, and the infarct area (%) in each tissue slice was calculated according to the following equation 1 by using an analysis program (i.e. Optimas 6.5, Bioscan). The adjusted volume of the infarct area was calculated through following Equation 2.
Infarct area(%)=(A−B/A)s 100  [Equation 1]

(32) A: volume of normal left hemisphere

(33) B: adjusted infarct volume (mm.sup.3)
Adjusted infarct volume (mm.sup.3)=(volume of normal left hemisphere)−(normal area in damaged hemisphere)  [Equation 2]
1.3. Administration of Clinoleic 20%, SMOFlipid20%, Oleic Acid and Sodium Oleate Solution

(34) Right after ischemia induction, to Example groups, 200 mg/kg of the Clinoleic20% (based on fatty acid) or 500 mg/kg of the SMOFlipid20% (based on fatty acid) was administered intravenously in a volume of 0.3 ml per 100 g of rat weight; to Comparative Example groups, 100 mg/kg of the oleic acid or 3 mg/kg of the sodium oleate solution was administered intravenously in a volume of 0.3 ml per 100 g of rat weight; and to control group, same volume of saline solution was administered.

(35) 1.4. Neuroprotective Effect

(36) Upon measurement of brain tissue and neuroprotective effect of each Clinoleic20%, SMOFlipid20%, oleic acid and sodium oleate solution, groups in which Clinoleic20%, SMOFlipid20%, oleic acid or sodium oleate solution was administered respectively had less TTC-unstained area due to neuronal cell death, and TTC-stained dark-red colored area was found to be large because neuronal cells were not damaged compared to the control group. For reference, when brain tissue is stained with TTC, area where neuronal cell death incurred due to damage would not be stained and be shown as white color, and normal area would be stained and be shown as dark-red color (see FIG. 1).

(37) Upon evaluation of protective effect on neuronal cell death induced by middle cerebral artery occlusion, significant efficacy was shown in every group, especially 47% of high neuroprotective effect was shown in Clinoleic20% group (p<0001) (see FIG. 2).

Experimental Example 2: Safety Evaluation—the Death Rate of Experimental Animals

(38) Right after degenerative brain disease induction, death rate was calculated by counting number of dead animals in every group from the time of administration until they were sacrificed.

(39) Following Table 1 shows death rate of the animals of the control group and the experimental groups in the middle cerebral artery occlusion model.

(40) TABLE-US-00001 TABLE 1 Number of Administered Number of Death Rate Group Animals Dead Animals (%) Oleic Acid 116 21 18.1 Sodium 37 8 21.6 Oleate Clinoleic20% 114 5 4.3 SMOFlipid20% 21 0 0

(41) As shown in Table 1, in case of the experimental group in which oleic acid was administered to brain infarction induced rats, 21 rats died out of 116 rats and death rate was 181%, and the death rate was highest as 21.6% in sodium oleate solution administered group where 8 rats died out of 37 rats. On the other hand, death rate of Clinoleic20% administered, group was 4.3% with 2 deaths out of 88 rats, and no rats died in the SMOFlipid20% administered group.