WO2008035925A1

WO2008035925A1 - A feed composition comprising a leaf extract of pinus koraiensis showing antioxidative activity

Info

Publication number: WO2008035925A1
Application number: PCT/KR2007/004576
Authority: WO
Inventors: Sang Kyum Kim; Chang-Sik Park
Original assignee: Sang Kyum Kim; Chang-Sik Park
Priority date: 2006-09-22
Filing date: 2007-09-20
Publication date: 2008-03-27

Abstract

The extract of Pinus Koraneisis shows potent antioxidative activity through experimental experiment such as TOSC assay, therefore, it can be useful as a feed composition for preventing and alleviating chronic animal diseases caused by increased oxygen radicals or for enhancing the immunity of animal. The animal bred by feeding with the extract of Pinus Koraiensis and the processed animal product using thereby shows various advantages such as, meat improvement, odor reduction, prevention against adult disease, improved digestion, shortened breeding period, reduced feeding, improved immunity against various diseases etc.

Description

A FEED COMPOSITION COMPRISING A LEAF EXTRACT OF PINUS KORAIENSIS SHOWING ANTIOXIDATIVE ACTIVITY

Technical Field

[1] The present invention relates to a feed composition comprising a leaf extract of Pinus

Koraiensis with potent antioxidative activity, as well as a method for breeding animal using thereby.

[2]

Background Art

[3] The oxidative substances involved in oxygen radical in human body can be classified into two species, i.e., reactive oxygen species (ROS) and reactive nitrogen species (RNS), for example, free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl, hy- droperoxyl radicals etc. and non-free radicals such as hydrogen peroxide, hypochlorous acid, ozone, singlet oxygen, peroxynitrite etc.. Among them, hydroxyl radicals, peroxyl radicals, and peroxinitrite are the most reactive, which causes to direct toxicity (Bayir H. et al., Crit Care Med., 33(12 Suppl), ppS498-501, 2005; Niles et al., Nitric Oxide, 14, pplO9-121, 2006).

[4] A peroxidation reaction caused by the free radicals not only stimulates the permeability of cell membrane, but also causes total cell toxicity, resulting in aging, various pathological syndromes and carcinogenesis. There have been reported that the radical reaction takes an effect on the progress of lots of chronic diseases correlated with oxidative stress, for example cancer, hypertension, myocardial infarction, atherosclerosis, rheumatoid etc. or functions as a factor for decreasing immunity system (De Souza LC. et al., Bioorg. Med. Chem. Lett., 14, pp5859-5861, 2004; Pike J. et al., Int. J. Vitam. Nutr. Res., 65, ppl 17-120, 1995).

[5] Regoli and Winston developed an assay for determining the antioxidative activity of a living organism and natural product using α-keto-γ-methiolbutyric acid (KMBA), which oxidizes to ethylene when reacting with internal oxidative substances (Winston et al., Free Radical Biol. Med., Feb., 24G). pp480-493, 1998; Regoli and Winston, Toxicol. Appl. Pharmacol., Apr., 15, 156(2). pp96-105, 1999). The amount of ethylene in a sealed reaction container can be quantified by using gas chromatography. The advantage of this method is useful in various applications. Since KMBA reacts with variable substances, such as hydroxyl radical, peroxyl radical, peroxynitrite, hypochlorous acid etc., resulting in producing ethylene gas, the occurrence of the ethylene gas in high antioxidative blood sample or tissues are very low. The concentration of ethylene gas formed by the reaction KMBA with hydroxyl radical, peroxyl radical, peroxynitrite etc. is determined according to the change of reaction time and the value of TOSC, transformed by the ratio of the calculated value of AUC (area under the curve) and those of control group and treatment group. Therefore, the assay shows highly reproducible and accurate since the error occurring in the other assay measuring only a single reaction point may be compensable. Since the determined value of TOSC from the samples is compared with that of the control group, the value is not affected by other factors, such as the sensitivity of measurement instrument, reagents, and other reaction conditions, theologically. Furthermore, the method shows broad scope of applicability by evaluating the antioxidative activity for the intracellular reactive oxygen species in human body (Ghiselli A. et al., Free Radical Biol. Med., 18, pp29-36, 1995).

[6] Antioxidants have been used to minimize the loss of vitamins and essential amino acids, and to delay or prevent lipid peroxidation by reacting with free radicals, not by absorbing or eliminating the oxygen. Synthesized antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), propyl galate (PG), teri- tiarybutyl hydroquinone (TBHQ) etc. are conventionally used in various food or medicine. However, it has been known to induce carcinogenesis or liver hypertrophy in case of being administrated at high concentration into experimental animals. Especially, BHT has been known to increase the microsomal enzyme activity in liver, and those phenolic synthetic antioxidants cause to various problems in safety resulting that the using amount thereof has been under regulation (Brannen et al., J. Amer. Oil Chem. Soc, 52, pp59-63, 1975 ; Ito N. et al., J. Natl. Cancer Inst., 70, p343, 1983 ; Chan KM. et al., J. Food ScL, 58, ppl-4, 1993).

[7] Several natural antioxidants such as tocopherol, flavonoids, gossypol, sesamol, oryzanol, vitamin C etc. and L-ascorbic acid has been reported till now however, tocopherol, a representative antioxidant, shows little locking ability of oxidation reaction and high cost in spite of its good safety (Halliwell B. et al., FASEB J., 2, pp2867-2870, 1988).

[8] Pinus Koraiensis belonging to Pinaceae, has been known to contain essential oil, pinnotol. pinnosylvin, pinnosylvin-methylester, flavoniod cresin, pinocembrin, pinobancesin, creptostrobin, amino acid such as arginine, histidine, lysine, tyrosine, leucine, glutamic acid, tannin and etc..

[9] However, it has been not reported or disclosed on the antioxidative activity of Pinus

Koraiensis in any of the above cited literatures, the disclosures of which are incorporated herein by reference.

[10] Accordingly, the present inventors have confirmed that the extract of Pinus

Koraiensis shows potent antioxidative activity through experimental experiment such as TOSC assay, therefore, it can be useful as a feed composition. [11] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter.

[12]

Disclosure of Invention

Technical Problem

[13] It is an object of the present invention to provide a feed composition for promoting the growth of animals, which shows potent antioxidative activity as an active ingredient for preventing and alleviating chronic animal diseases caused by increased oxygen radicals.

[14] Another object of the present invention is to provide a method of breeding comprising feeding the animals with feed composition showing potent antioxidative activity.

[15]

Technical Solution

[16] To achieve the above objects, the present invention provides a feed composition comprising a leaf extract of Pinus Koraiensis showing potent antioxidative activity as an active ingredient for preventing and alleviating chronic animal diseases caused by increased oxygen radicals.

[17] The present invention provides a feed additive comprising a leaf extract of Pinus

Koraiensis showing potent antioxidative activity as an active ingredient.

[18] The present invention provides a method of treating or preventing chronic animal diseases caused by increased oxygen radicals by administering an effective amount of a leaf extract of Pinus Koraiensis showing potent antioxidative activity to said animal, together with an acceptable additive or carrier thereof.

[19] The present invention provides a method for improving the antioxidative activity, wherein said method comprises administering to said animal an effective amount of a composition comprising a leaf extract of Pinus Koraiensis showing potent antioxidative activity.

[20] The present invention also provides a method of using a composition comprising a leaf extract of Pinus Koraiensis for the preparation of a veterinary therapeutics for stimulating growth and enhancing the immune system in animals in need thereof.

[21] The present invention also provides a method of breeding comprising feeding the animals with feed composition comprising a leaf extract of Pinus Koraiensis as an active ingredient showing potent antioxidative activity.

[22] The term "extract" disclosed herein includes a crude extract which can be extracted with polar solvent selected from water, C -C lower alcohol or the mixture thereof,

1 4 preferably a mixture solvent with water and methanol, more preferably 50% to 80% mixture ratio of water and methanol.

[23]

[24] Hereinafter, the preparation of Pinus Koraiensis extract is described in detail.

[25]

[26] For the present invention, the dried Pinus Koraiensis leaf is cut into small pieces and the pieces were mixed with approximately 1 to 20-fold per weight (kg), more preferably 3 to 10-fold volume of water, C -C lower alcohol such as methanol, ethanol, butanol or the mixtures thereof, more preferably 50 to 80% mixture ratio of water and methanol; and is heated at the temperature ranging from 20 to 100⁰C, more preferably 50 to 100⁰C, for the period ranging 1 hour to 10 days, more preferably 1 to 5 hour, by reflux extraction with cold water, hot water extraction, ultra-sonication or conventional extraction; the residue was filtered and then the filtrate is dried by vacuum freeze-drying to obtain the crude extract of the present invention.

[27] It has confirmed that the extract of Pinus Koraiensis, showed potent antioxidative activity through experimental experiment such as TOSC assay, therefore, it can be useful as a feed composition for preventing and alleviating chronic animal diseases caused by increased oxygen radicals or for enhancing the immunity of animal.

[28] The term "chronic animal diseases caused by increased oxygen

[29] radicals" disclosed herein includes various animal diseases correlated with oxidative stress, for example cancer, hypertension, myocardial infarction, atherosclerosis or rheumatoid.

[30] An animal feed composition as used herein may be in the form of tablets, pills, powder, elixirs, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsules, sterilized injection and sterilized powder. The feed composition may additionally comprise fillers, anti- aggregating agents, lubricants, wetting agents, perfumes, emulsifiers, preservatives and the like. The inventive composition may be formulated by a method well known in the art, such that it can provide the rapid, sustained or delayed release of the active ingredients after administration. A particularly advantageous feed composition comprises a foodstuff selected from the group consisting of dry forages and roughages, energy feeds, protein feeds, mineral feeds, vitamin feeds, yeast products, normal premix, cornmeal, cotton seed wheat gluten, maize silage rutabaga, sugar beet pulp, apple pulp, ryegrass, fescue grass, alfalfa feed concentrate and feed supplement.

[31] The invention therefore comprises a method for preparation of animal feed comprising the steps of providing an animal feed stuff and mixing said feed stuff with an effective dose of the leaf extract of Pinus Koraiensis.

[32] The leaf extract of Pinus Koraiensis may be mixed with any suitable base feed material, such as rape seed, cotton seed, soybean, fish meal, wheat bran, wheat feed meal, minerals, vitamins and binders or prepared as a premix with, for example amino acids, salts, phosphorous or cornmeal.

[33] To develop feed composition for animal, at least one of the other ingredients selected from organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid and the like; phosphate salt such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate and the like; and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further be added to the active ingredient of the present invention.

[34] For example, the inventive feed composition of the present invention could be prepared by the procedure comprising the steps of: mixing inventive extract of present invention with the combination of various aid component such as amino acid, inorganic salt, vitamin, antibiotic, antibacterial agent, antioxidant, anti-fungal agent, live microbial preparation and the like; and grain such as macerated or pulverized wheat, oat, barley, corn or rice; vegetable protein feed such as bean or sunflower seed; animal protein feed such as blood powder, meat powder, bone powder or fish powder; sugar powder or milk product such as various powdered milk or powdered whey and the like together; heating to obtain remaining fluid component, for example, lipid component such as fluidized animal fat, vegetable lipid and the like; mixing the lipid component as a main component with other component such as nutrient supplement, digestive improving agent, growth stimulator, disease preventing agent and the like to prepare purposed inventive composition.

[35] The above-described animal feed composition can be administrated into animal as a sole or the combinations with other feed additive.

[36] Providing that the inventive extract of the composition is administrated into the animal independently on other animal feed, the extract may be combined with pharmaceutically acceptable non-toxic edible carrier to make imprompt releasing or sustained releasing preparation. Solid type or fluid type edible carrier, for example corn starch, lactose, sucrose, bean flake, bean oil, olive oil, sesame oil and propylene glycol may be used as edible carrier of the present invention. In case of solid carrier, the dosage form may contain the other aid component such as preservative, stabilizer, humidifier, emulsifier and solubilizer.

[37] The inventive feed composition of the present invention has no toxicity and adverse effect therefore can be used with safe.

[38] The desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and the period of administration, and may be chosen by those skilled in art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.01 mg/kg to 10 g/kg per day, more preferably 1 mg/kg to 1 g/kg of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day.

[39] The inventive composition of the present invention could be used by the procedure comprising the steps of: pulverizing the dried leaves of Pinus Koraiensis or the extract thereof; mixing with other feed components with the mix ratio ranging from about 5 to 10% (w/w) ratio of the total feed; administrating the mixture to animal to provide functional animal meat products.

[40] The animal feed or feed additive of the present invention can be applied to various animals including mammals, poultry and fish, preferably commercially important mammals, for example horse, pig, cow, sheep, goat, raising fish such as red snapper, bastard halibut, flatfish, yellowtail, trout, swellfish, catfish and the like, shellfish such as abalone, scallop, oyster, Crustacea such as lack tiger shrimp, blue crab, poultry such as chicken, turkey, duck, goose, quail and the like and pet animals. The administration may be by any of the procedures well known in art, for example oral, rectal, intravenous, intramuscular injections and the like.

[41] The animal feed additive of the present invention can be added to animal feed for use as an appetizer, for example conventionally available optional protein-comprising organic grain such as corn powder, bean powder or the mix therewith.

[42] The above-described feed additive can be prepared by any mixing means known to one skilled in the art such as mechanical blending, extrusion, palletizing, and spray drying.

[43] The animal feed additive of the present invention can be mixed with animal feed in the amount ranging from about 1 to 100 g per lkg of animal feed.

[44] For example, the inventive feed composition of the present invention could be prepared by the procedure comprising the steps of: mixing inventive extract of present invention with other feed component to obtain cohesive granule type to be used directly or other type to purpose further processing and packaging steps, for example adding water to said feed to perform further conventionally necessary procedure such as pellet, expansion or compression etc.

[45] Accordingly, the present invention also provides a processed animal product of which animal has been bred by the above-described method of the present invention.

[46] The term "processed animal product" disclosed herein comprises the meat of pheasant, chicken, pig, horse, cow, goat, sheep, duck, artificial, turkey, rabbit, and egg, powder egg, liver and the like. Also the pate, paste made from liver, meat liquid, meat jelly, rennet, bacon, bouillon, broth, black pudding (blood sausage), beef steak, charcuterie, sausage, cow liver, edible jelly, beef jerky, hamburger pellet and the like.

[47] The inventive additive is believed to have a considerable effect of the live-stock's health, production efficiency, feed intake, daily weight gain and feed utilization. It is further believed that the inventive additive reduces the usage of other antioxidants such as vitamin E and selenuim in production animals and pets, has antimicrobial and antiviral characteristics, improves nitrogen utilization, and improves the nutrient substance utilization generally. The inventive additive may be used by animal production including animal breeding and production of animal products such as meat, eggs and milk.

[48] It will be apparent to those skilled in the art that a various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[49]

Advantageous Effects

[50] The extract of Pinus Koraiensis, showed potent antioxidative activity through experimental experiment such as TOSC assay, therefore, it can be useful as a feed composition for preventing and alleviating chronic animal diseases caused by increased oxygen radicals or for enhancing the immunity of animal, as well as a method for breeding animal using thereby. Thus, the feed composition is useful for the preparation of highly functional feed.

[51]

Brief Description of the Drawings

[52] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[53]

[54] Fig. 1 shows the antioxidative activity against peroxyl radical after the treatment with an extract of Pinus Koraiensis;

[55] Fig. 2 shows the antioxidative activity against peroxyl radical after the treatment with an extract of Allium Tuberosum;

[56] Fig. 3 shows the antioxidative activity against peroxyl radical after the treatment with glutathione;

[57] Fig. 4 shows the antioxidative activity against hydroxyl radical after the treatment with an extract of Pinus Koraiensis;

[58] Fig. 5 shows the antioxidative activity against hydroxyl radical after the treatment with an extract of Allium Tuberosum;

[59] Fig. 6 shows the antioxidative activity against hydroxyl radical after the treatment with glutathione;

[60] Fig. 7 shows the antioxidative activity against peroxynitrite after the treatment with an extract of Pinus Koraiensis;

[61] Fig. 8 shows the antioxidative activity against peroxynitrite after the treatment with an extract of Allium Tuberosum;

[62] Fig. 9 shows the antioxidative activity against peroxynitrite after the treatment with glutathione.

[63]

Best Mode for Carrying Out the Invention

[64] The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

[65]

Mode for the Invention

[66] Comparative Example 1. Preparation of the extract of Allium Tuberosum

[67] 80.45 g of leaf of Allium Tuberosum purchased from Gongju-city (Korea) was cut into small pieces with the size of about 2.0 to 4.0 cm, mixed with 500 ml of 80% methanol thoroughly and the mixture was subjected to sonication extraction at room temperature 3 times per hour to obtain the leaf extract of Allium Tuberosum.

[68] The extract was concentrated in vaccuo for 2 hours at 45⁰C using by rotary evaporator and the concentrates were lyophilized for 3 days to obtain 5.24 g of a powder of Allium Tuberosum to be used as a test sample in following experiments (designated as 'AT' hereinafter).

[69]

[70] Example 1. Preparation of the extract of Pinus Koraiensis

[71] 80.45 g of leaf of Pinus Koraiensis purchased from Gongju-city (Korea) was cut into small pieces with the size of about 2.0 to 4.0 cm, mixed with 500 ml of 80% methanol thoroughly and the mixture was subjected to sonication extraction at room temperature 3 times per hour to obtain the leaf extract of Pinus Koraiensis.

[72] The extract was concentrated in vaccuo for 2 hours at 45⁰C using by rotary evaporator and the concentrates were lyophilized for 3 days to obtain 5.31 g of a powdered extract of Pinus Koraiensis to be used as a test sample in following experiments (designated as 'PK' hereinafter).

[73]

[74] Reference Example 1. Preparation of experimental animal and caring conditions

[75] The Pinus Koraneisis were collected in Korea, dried and pulverized into powder, and mixed with commercial animal feed by 5% ratio (w/w) and administrated freely to 5 pigs consisting of 7 weeks old male and female Duroc, Landrace and Large white pigs, for 4 weeks. The size of breeding room was 3x2.5 m and water was freely accessible using by an automatic water supplier.

[76] The blood samples collected from previous animals were used as test samples in following experiments.

[77]

[78] Experimental Example 1. Antioxidative activity

[79] 1-1 Animal blood sample

[80] The blood sample was collected from the jugular vein of the pigs, centrifuged at

5,000xg for 1 minute in room temperature, and kept in -7O⁰C until used for further experiments. To test the antioxidative activities, the blood samples were diluted with 200 to 250-fold volume of distilled water for injection.

[81] The blood sample collected from the 4 week fed animals was analyzed for the antioxidative activities in Table 1. As shown, the results show no difference in hydroxyl radical, but scavenging activity 1.5 and 1.3-fold respectively of the peroxy radical and peroxynitrite.

[82]

[83] 1-2 Total Oxyradical Scavenging Capacity (TOSC) assay

[84] To determine the antioxidative activity of the extracts prepared in Comparative

Example and Examples, the radical scavenging assay using TOSC method was performed according to the procedure disclosed in the literature as follows (Regoli and Winston, Toxicol. Appl. Pharmacol., Apr 15, 156(2). pp96-105, 1999).

[85] Peroxyl radical induces to form 2,2'-azobisamidinopropane (ABAP) through thermal homolysis at 35⁰C (Winston et al., Free Radic. Biol Med., Feb, 24G). pp480-493, 1998) and hydroxyl radical induces through fenton reaction using Fe and ascorbate (Winston and Cederbaum, Alcholol Clin. Exp. Res., 9(2), pp95-102, 1985). Peroxynitrite induces to form 2,2'-azobisamidinopropane (ABAP) through spontaneous decomposition of SIN-I. The produced reactive oxygen species react with α- keto-γ-methiolbutyric acid (KMBA) to produce ethylene gas, of which temperature within certain range does not affect on any difference in the reaction (Winston et al., Free Radic. Biol. Med., 24G). pp480-493, 1998; Regoli and Winston, Toxicol. Appl. Pharmacol., 156(2). pp96-105, 1999).

[86] The reaction was performed in 10 ml volume of sealed container with rubber septum with 1 ml of total volume of reactants. The amount of ethylene gas was determined by using gas chromatography (GC) equipped with Poropack N column, Flame ionization detector (FID) running with He gas as a mobile phase at the speed of 30 ml/min, of which temperature had been adjusted to 6O⁰C in oven and to 18O⁰C in injector and detector. For quantitative determination of the amount of ethylene gas, 150 D of inner gas in the vial was sampled with gas-tight injector using by head-space technique method and injected to injector to determine the amount of ethylene gas.

[87] At the result, the area under the kinetic curve (AUC) was measured from the integration of determined graph through the instrument, and the value of TOSC was calculated by following Math Figure 1.

[88]

[89] MathFigure 1

[Math.l]

TOSC = 100 - (JSA/JCA x 100)

IS A = integrated are a from the curve of the sample re action

J CA = integrated area from the curve of the control reaction

[90] The sample with no oxyradical scavenging capacity will obtain JSA/JCA = 1, TOSC

= 0. In other words, JSA → 0, the value of TOSC is close to 100. The value of TOSC is compared with that of the control group, of which value is regarded as being not interfered by the sensitivity of the instrument, reagent, and other reaction conditions.

[91]

[92] As shown in following Table 1 and Table 2, the scavenging activity of the group treated with PK extract expressed 1.7 to 1.9-fold potent radical- scavenging effect comparing with that treated with AT extract. Furthermore, the values of TOSC against peroxy radical in the group treated with PK extract and AT extract showed about 2-fold and 3 to 4-fold more effective, respectively, comparing with that that of the group treated with glutathione (940 TOSC/mg), a representative antioxidant ( See Figs. 1, 2 and 3).

[93]

[94] The hydroxyl radical scavenging effect of the group treated with PK extract and AT extract showed about 1.6 to 1.7-fold more effective comparing with that of the group treated with glutathione (260 TOSC/mg), a representative antioxidant ( See Figs. 4, 5 and 6).

[95] The peroxynitrite radical scavenging effect of the group treated with PK extract and

AT extract showed about 2.4 to 3.1-fold more effective comparing with that of the group treated with glutathione, a representative antioxidant ( See Figs. 7, 8 and 9).

[96]

[97] Table 1 [Table 1] [Table ]

[98] Table 2 [Table 2] [Table ]

[99] [100] All the results are designated as mean+standard deviation, and statistical significance were tested by Student's t-test.

[101] [102] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

[103] [104] Preparation of feed composition [105] PK extract 100 mg [106] PK powder 16 mg [107] β-glucan containing extract 0.7 mg [108] Yeast culture 24 mg [109] Vitamin E 0.7 mg [HO] L-carnitine 0.7 mg [111] Feed composition preparation was prepared by mixing with the above components altogether.

[112] [113] Preparation of powder

[114] PK extract 20 mg

[115] Lactose 100 mg

[116] Talc 10 mg

[117] Powder preparation was prepared by mixing above components and filling sealed package. [118]

[119] Preparation of tablet

[120] PK extract 10 mg

[121] Corn Starch 100 mg

[122] Lactose 100 mg

[123] Magnesium Stearate 2 mg

[124] Tablet preparation was prepared by mixing above components and entabletting. [125]

[126] Preparation of capsule

[127] PK extract 10 mg

[128] Crystalline cellulose 3 mg

[129] Lactose 14.8 mg

[130] Magnesium Stearate 0.2 mg

[131] Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. [132]

[133] Preparation of injection

[134] PK extract 10 mg

[135] Mannitol 180 mg

[136] Distilled water for injection 2974 mg

[137] Na HPO , 12H O 26 mg

[138] Injection preparation was prepared by dissolving the components in 2D ample and sterilizing by conventional injection preparation method. [139]

[140] Preparation of liquid medicine

[141] PK extract 20 mg

[142] Isomerized sugar 1O g

[143] Mannitol 5 g

[144] Distilled water optimal amount

[145] Liquid medicine was prepared by dissolving the components to distilled water with a proper dose of lemon scent, mixing, adjusting to 100 ml with distilled water in brown bottle and sterilizing by conventional liquid medicine preparation method. [146]

[147] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[148]

Industrial Applicability

[149] As described in the above, the extract of Pinus Koraneisis shows potent antioxidative activity through experimental experiment such as TOSC assay, therefore, it can be useful as a feed composition for preventing and alleviating chronic animal diseases caused by increased oxygen radicals or for enhancing the immunity of animal. The animal bred by feeding the extract of Pinus Koraiensis and the processed animal product using thereby shows various advantages such as, meat improvement, odor reduction, prevention against adult disease, improved digestion, shortened breeding period, reduced feeding, improved immunity against various diseases etc.

Claims

[1] A feed composition comprising a leaf extract of Pinus Koraiensis showing potent antioxidative activity as an active ingredientfor preventing and alleviating chronic animal diseases caused by increased oxygen radicals or enhancing the immunity of animal.

[2] The feed composition according to the claim 1, wherein the feed compostion is in the form of tablets, pills, powder, elixirs, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsules, sterilized injection or sterilized powder.

[3] The feed composition according to the claim 1 wherein said extract is extracted with a polar solvent selected from water, C -C lower alcohol or the mixture

1 4 thereof. [4] The feed composition according to the claim 1 wherein said animal diseases caused by increased oxygen radicals are various animal diseases correlated with oxidative stress, which is selected from cancer, hypertension, myocardial infarction, atherosclerosis or rheumatoid. [5] A method for improving the antioxidative activity, wherein said method comprises administering to said animal an effective amount of feed composition as set forth in claim 1. [6] A method of using a composition comprising a leaf extract of Pinus Koraiensis for the preparation of a veterinary therapeutics for stimulating growth and enhancing the immune system in animals in need thereof. [7] A method for preparation of animal feed comprising the steps of providing an animal feed stuff and mixing said feed stuff with an effective dose of the leaf extract of Pinus Koraiensis. [8] A method of breeding comprising feeding the animals with feed composition as set forth in claim 1. [9] A processed animal product of which animal has been bred by the method of claim 8. [10] The animal product according to the claim 9, wherein said product is selected from the meat of pheasant, chicken, pig, horse, cow, goat, sheep, duck, artificial, turkey, rabbit, egg, powder egg, a pate, a paste made from liver, meat liquid, meat jelly, rennet, bacon, bouillon, broth, black pudding (blood sausage), beef steak, charcuterie, sausage, cow liver, edible jelly, beef jerky or hamburger pellet.