WO2015133716A1

WO2015133716A1 - A pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient

Info

Publication number: WO2015133716A1
Application number: PCT/KR2015/000025
Authority: WO
Inventors: Sang Hyun Park; Beom Su Jang; Jung Ae KANG
Original assignee: Korea Atomic Energy Research Institute
Priority date: 2014-03-07
Filing date: 2015-01-02
Publication date: 2015-09-11
Also published as: KR20150104950A

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient. Particularly, in the mouse group having tissue damage induced by gamma-ray irradiation, body weight, blood cell number, and antioxidant material/antioxidant enzyme in the liver tissue were all significantly increased by the administration of hesperetin before or after gamma-ray irradiation, compared with those of the group irradiated with gamma-ray but not administered with hesperetin. In addition, ALT and AST in blood plasma, and lipid peroxidation and XO in the liver tissue of the group treated with hesperetin were all significantly decreased, compared with those of the gamma-ray irradiation group, indicating that oxidative stress was relieved. Therefore, the hesperetin of the present invention can be effectively used as a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation.

Description

[DESCRIPTION]

[invention Title]

A pharmaceutical composition for the. prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient

[Technical Field]

The present invention relates to a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient .

[Background Art]

The recent radiation exposure state is getting intense because of Fukushima nuclear power plant accident caused by the great Japan earthquake. In addition, the increase of medical use of radiation such as radio-therapy and clinical use of radio- isotope along with the increasing natural radiation exposure on cosmic radiation cause defect or disability in human (Meister 2005). Therefore, the people are highly concerned about ^άί3^οη-θ3 εθά human defect and accordingly interested in human defense system as well. in Korea, anxiety about radiation exposure is growing, and "Safety Control of Radiation Around Living Environment" has been enforced to prevent unnecessary radiation exposure in daily life. Radiation has been used to eliminate cancer cells or to inhibit cancer cell growth in order to treat various cancers and to extend cancer patient's life thereby. However, it also destroys normal tissues in long-term cancer patients, causing a secondary cancer (Dormand et al . 2005). Radiation can directly damage tissues and can also damage tissues indirectly by inducing ionization of biological materials. The reactive oxygen species (ROS) generated by radiation induce indirectly cell, damage including the damages of DNA, protein, and cell membrane, and even induce apoptosis (Halliwell and Gutterige 1999) . Studies have been conducted to develop a radioprotective agent for the prevention of organism damage and for the protection of cells with less side effects and with protecting normal cells under radiation exposure (Phillips 1981).

Studies on the radioprotective agent have been mainly focused on synthetic materials containing thiol group such as WR-2721 (amifostine) , cytokines, and immunomodulators . However, the synthetic material displayed a strong toxicity, and the immunomodulator accompanied many side effects and was very expensive, so that their use has been limited. So, it is still requested to develop a radioprotective agent which is safe in living body and less expensive but effective.

Flavonoid which is abundant in leaves, flowers, and fruits of a plant is a polyphenol compound. According to the structure and location of its substituent, flavonoid is classified into anthocyanidin, flavone, flavonol, flavanone, flavanolol, chalcone, catechin, and isoflavone (Rauha et al . 2001) . It has been known that flavonoid has various biological effects such as anti -oxidative activity, antiviral activity, anti-bacterial activity, cancer growth inhibitory effect, and immuno-stimulating activity etc (Korkina and Afanas ' ev 1997) .

Hesperidin having the molecular formula of Ci₈H₃₄.Oi5 and the molecular weight of 610.57 is a disaccharide rutinose conjugated glycoside, which is found in citrus fruits such as orange and lemon. Hesperidin is hydrolyzed in vivo by glycosidase into aglycone type hesperetin (Figure 1) . The molecular formula of hesperetin is Ci₆Hi₄0₆ and the molecular weight thereof is 302.27 (Garg et al . 2001) . Hesperetin is absorbed in the stomach and intestines and shows the physiological activity in vivo. So, it is detected as hesperetin in blood and liver tissues (Ameer et al . 1996) . The present inventors tried to find out a natural radioprotective agent without toxicity in order to prevent damage by radiation in living body. In the course of our study, the present inventors confirmed that the antioxidant material and antioxidant enzyme had been significantly increased in liver tissues along with the body weight and blood cell count in the group treated with hesperetin before or after gamma-ray- irradiation, compared with the group irradiated with gamma-ray but not treated with hesperetin. It was also confirmed that the plasma AST (aspartate aminotransferase) , ALT (alanine aminotransferase) , lipid peroxidation in liver tissue, and XO (xanthine oxidase) were significantly reduced in the experimental group. Thereafter, the present inventors proved that hesperetin could be effectively used as a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation, leading to the completion of this invention.

[Disclosure]

[Technical Problem]

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient. [Technical Solution]

To achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cell or tissue damage by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

The present invention also provides a method for the prevention and treatment of cell or tissue damage by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

The present invention further provides a use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition for the prevention and treatment of cell or tissue damage by radiation.

The present invention also provides a pharmaceutical composition for the prevention and treatment of oxidative stress caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient . The present invention also provides a method for the prevention and treatment of oxidative stress caused by- radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

The present invention further provides a use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition for the prevention and treatment of oxidative stress caused by radiation.

The present invention also provides a radioprotective agent comprising hesperetin or the . pharmaceutically acceptable salts thereof as an active ingredient.

The present invention also provides a radioprotective method containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

The present invention also provides a use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a radioprotective agent.

The present invention also provides a health food for the prevention and improvement of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient . The present invention also provides a health food for the prevention and improvement of oxidative stress caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

In addition, the present invention provides a health food for radioprotection comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient . [Advantageous Effect]

Hesperetin of the present invention displays the effect of preventing and treating liver cell damage and hematopoietic -immune system damage caused by radiation in the mouse model irradiated with gamma-ray and also shows the effect of preventing and recovering tissue damage caused by oxidative stress. Thus, hesperetin of the present invention can be effectively used as a radioprotective agent in the incidence of radiation exposure .

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a diagram illustrating the chemical structures of hesperetin and hesperidin.

Figure 2 is a diagram illustrating the DPPH (1,1- diphenyl-2-picryl-hydrazyl) radical scavenging activities of hesperetin and hesperidin:

*: p<0.05; and

**: p<0.01.

Figure 2 is a diagram illustrating the ABTS [(2, 2'- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activities of hesperetin and hesperidin.

Figure 4 is a diagram illustrating the result of FRAP (ferric reducing antioxidant power) analysis of hesperetin and hesperidin.

Figure 5 is a diagram illustrating the changes of blood cell component in the mouse with tissue damage induced by gamma-ray irradiation, examined after the administration of hesperetin:

Normal control group: the group of mice supplied with CMC (carboxy methyl cellulose) for 7 days;

6 Gy irradiation: the group of mice irradiated with 6 Gy of gamma-ray after provided with CMC for 7 days;

25 mg/kg HT + 6 Gy irradiation: the group of mice irradiated with 6 Gy of gamma-ray after orally administered with 25 mg/kg of hesperetin for 7 days;

50 mg/kg HT + 6 Gy irradiation: the group of mice irradiated with 6 Gy of gamma-ray after orally administered with 50 mg/kg of hesperetin for 7 days;

100 mg/kg HT + 6 Gy irradiation: the group of mice irradiated with 6 Gy of gamma-ray after orally administered with 100 mg/kg of hesperetin for 7 days;

#: p<0.05 and ## : p<0.01, compared with the normal control group; and

*: p<0.05 and **: p<0.01, compared with the 6 Gy irradiated group.

Figure 6 is a diagram illustrating the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in blood plasma of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin.

Figure 7 is a diagram illustrating the changes of lipid peroxidation in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin.

Figure 8 is a diagram illustrating the changes of XO in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin.

Figure 9 is a diagram illustrating the changes of GSH (glutathione) in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin. Figure 10 is a diagram illustrating the changes of SOD (superoxide dismutase) , catalase, and GPx (glutathione peroxidase) in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin.

Figure 11 is a diagram illustrating the morphological changes in sternum tissue of the mouse with tissue damage induced by gamma-ray irradiation, measured after the administration of hesperetin.

Figure 12 is a diagram illustrating the weight changes of the mouse with tissue damage induced by gamma- ray irradiation before the administration of: hesperetin:

Normal control group: the group of mice provided with CMC for 7 days;

50 mg/kg HT: the group of mice provided with 50 mg/kg of hesperetin for 7 days;

6 Gy irradiation: the group of mice provided with CMC after irradiated with 6 Gy of gamma-ray;

6 Gy irradiation + 25 mg/kg HT: the group of mice provided with 25 mg/kg of hesperetin for 7 days after irradiated with 6 Gy of gamma-ray;

6 Gy irradiation + 50 mg/kg HT: the group of mice provided with 50 mg/kg of hesperetin for 7 days after irradiated with 6 Gy of gamma-ray;

6 Gy irradiation + 100 mg/kg HT : the group of mice provided with 100 mg/kg of hesperetin for 7 days after irradiated with 6 Gy of gamma-ray;

#: p<0.05 and ## : p<0.01, compared with the normal control group; and

*: p<0.05 and **: p<0.01, compared with the 6 Gy irradiated group.

Figure 13 is a diagram illustrating the changes of spleen index of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 14 is a diagram illustrating the activities of AST and ALT in blood plasma of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 15 is a diagram illustrating the changes of lipid peroxidation in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 16 is a diagram illustrating the changes of XO in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 17 is a diagram illustrating the changes of GSH in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 18 is a diagram illustrating the changes of SOD, catalase, and GPx in liver tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Figure 19 is a diagram illustrating the morphological changes in sternum tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

[Best Mode]

Hereinafter, the present invention is described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient .

The present invention also provides a method for the prevention and treatment of cell or tissue damage caused by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

The present invention further provides a use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by- radiation.

The said hesperetin is the compound represented by the following [Formula 1] :

[Formula 1]

The radiation herein is selected from the group consisting of UV, X-ray, a-ray, β-ray, γ-ray, electron beam, and neutron beam, but not always limited thereto.

The cell or tissue herein is hematopoietic system or liver cell or tissue, but not always limited thereto.

The composition herein is preferably, supposed to recover the weight reduced by radiation exposure, but not always limited thereto.

The composition herein preferably reduces the activity of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) , the serum marker enzyme which has been increased by radiation exposure, but not always limited thereto.

The composition of the present invention preferably reduces the lipid peroxidation and XO activity increased by radiation exposure, but not always limited thereto.

The composition herein preferably recovers the activity of SOD (superoxide dismutase) , catalase, or GPx (glutathione peroxidase) , the enzymatic antioxidant which has been reduced by radiation exposure, but not always limited thereto .

The composition of the present invention preferably recovers the activity of GSH, the antioxidant agent which has been reduced by radiation exposure, but not always limited thereto.

The composition of the present invention preferably recovers the number of blood cells which has been reduced by radiation exposure, but not always limited thereto.

In a preferred embodiment of the present invention, the antioxidant activities of hesperetin and hesperidin were compared. As a result, it was confirmed that hesperetin has significantly higher DPPH scavenging activity, ABTS scavenging activity, and FRAP, than those of hesperidin, indicating that hesperetin in the form of aglycone has more excellent antioxidant activity than hesperidin in the form of glycoside (see Figure 2, Figure 3, and Figure 4 ) .

To examine the activity of hesperetin to prevent damage from irradiation, the present inventors had BALB/c mice irradiated with gamma-ray, to which hesperetin was orally administered for 7 days, resulting in the construction of the animal model with cell or tissue damage caused by gamma-ray irradiation. Then, any change in weight, spleen, and thymus index was observed. As a result, in the group treated with hesperetin a day after gamma-ray irradiation, the weight was significantly increased, compared with the group irradiated with gamma-ray but not treated with hesperetin. In the investigation of spleen and thymus index, the group irradiated with gamma-ray after being treated with hesperetin displayed significant increase in both indexes, compared with the group irradiated with gamma-ray but not treated with hesperetin, suggesting that hesperetin had the preventive effect on the damage in hematopoietic-immune system (see Table 1) . Blood cell component of the mouse with cell or tissue damage caused by gamma-ray irradiation after being treated with hesperetin was analyzed. As a result, the number of white blood cell in the group irradiated with gamma-ray after being treated with hesperetin was increased, suggesting that hesperetin had the activity of protecting hematoblasts from irradiation and the activity of regenerating the same (see Figure 5) .

To investigate the activity of hesperetin to protect cell or tissue from being damaged by irradiation, the present inventors analyzed blood AST and ALT of the mouse with cell or tissue damage caused by gamma-ray irradiation after the administration of hesperetin. As a result, blood AST and ALT in the group irradiated with gamma-ray after being treated with hesperetin were significantly reduced, compared with those in the group irradiated with gamma-ray but not treated with hesperetin, suggesting hesperetin had the preventive effect on liver cell damage (see Figure 6) .

To investigate the effect of hesperetin on preventing damage and oxidative stress caused by irradiation, the present inventors measured lipid peroxidation and XO in the liver tissue of the mouse with damage induced by gamma-ray irradiation after the administration of hesperetin. As a result, the level of lipid peroxidation in the group irradiated with gamma-ray after being treated with hesperetin was significantly lower than that of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that the damage caused by radiation was minimized. In the meantime, XO in the group irradiated with gamma-ray after being treated with hesperetin was significantly reduced, compared with that of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that the administration of hesperetin could prevent and protect the cell from being damaged by irradiation (see Figure 7 and Figure 8) .

The present inventors also investigated the effect of hesperetin on preventing damage and oxidative stress caused by irradiation. To do so, the inventors analyzed the changes of antioxidant material and antioxidant enzyme in the liver tissue of the mouse with cell or tissue damage caused by gamma-ray irradiation after the administration of hesperetin. As a result, the activity of each GSH, SOD, catalase, and GPx in the group irradiated with gamma-ray after being treated with hesperetin was all significantly increased, compared with that of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that hesperetin was able to normalize the in vivo antioxidant defense system (see Figure 9 and Figure 10) .

To investigate the effect of hesperetin on preventing damage caused ,by irradiation, the inventors analyzed the morphological changes in sternum breast bone tissue of the mouse group with tissue damage induced by gamma-ray irradiation after the administration of hesperetin. As a result, the loss of myelocytes in the group administered with hesperetin was less, suggesting that the tissue damage was minimized. That is, the administration of hesperetin before the gamma-ray irradiation was effective in preventing tissue damage from gamma-ray irradiation (see Figure 11) . To investigate the treatment effect of hesperetin on the damaged cell/tissue induced by irradiation, the present inventors, had BALB/c mice irradiated with gamma-ray, to which hesperetin was orally administered for 7 days, resulting in the construction of an animal model having cell/tissue damage caused by gamma-ray irradiation. Then, weight change and spleen index were observed. As a result, the weight that had been reduced 7 days after gamma-ray irradiation was peculiarly recovered in the group treated with hesperetin after gamma-ray irradiation, compared with that of the group irradiated with gamma-ray but not treated with hesperetin. Spleen index was increased in the group treated with hesperetin, compared with that of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that hesperetin had the therapeutic effect on the damage in hematopoietic- immune system (see Figure 12 and Figure 13) . Blood cell component of the mouse with cell or tissue damage caused by gamma-ray irradiation before the administration of hesperetin was analyzed. As a result, the numbers of leucocytes, neutrophils, lymphocytes, and monocytes in the group irradiated with gamma-ray before the administration of hesperetin were all increased, suggesting that hesperetin had the activity of protecting hematoblasts from irradiation and the activity of regenerating the same (see Table 2) . To investigate the therapeutic effect of hesperetin on the cell/tissue damage caused by irradiation, the inventors analyzed blood AST and ALT of the mouse with cell/tissue damage induced by gamma-ray irradiation before the administration of hesperetin. The levels of AST and ALT of the group treated with 50 mg/kg of hesperetin after gamma-ray irradiation were significantly reduced, compared with those of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that hesperetin had the therapeutic effect on the liver damage (see Figure 14).

To investigate the preventive effect of hesperetin on cell/tissue damage and oxidative stress induced by irradiation, the present inventors analyzed lipid peroxidation and XO in the liver tissue of the mouse having cell/tissue damage induced by gamma-ray irradiation before the administration of hesperetin. As a result, the level of lipid peroxidation in the group treated with hesperetin after being irradiated with gamma-ray was significantly lower than that of the group irradiated with gamma-ray but not treated with hesperetin. In the meantime, XO in the group treated with 50 mg/kg of hesperetin after gamma-ray irradiation was significantly reduced, compared with that of the group irradiated with gamma-ray but not administered with hesperetin. Therefore, it was confirmed that hesperetin had the therapeutic effect on cell damage caused by radiation and the activity of protecting cells from radiation (see Figure 15 and Figure 16) .

To investigate the therapeutic effect of hesperetin on cell/tissue damage and oxidative stress caused by irradiation, the present inventors analyzed the changes of antioxidant material and antioxidant enzyme in the liver tissue of the mouse having cell or tissue damage caused by gamma-ray irradiation before the administration of hesperetin. As a result, the activity of GSH in the group treated with hesperetin after being irradiated with gamma- ray was significantly increased, compared with that of the group irradiated with gamma-ray but not treated with hesperetin. The antioxidant enzyme activity in the group treated with 25 mg/kg of hesperetin after being irradiated with gamma-ray was significantly increased, compared with that of the group irradiated with gamma-ray but not treated with hesperetin, suggesting that the defense system was recovered (see Figure 17 and Figure 18) .

To investigate the therapeutic effect of hesperetin on damage caused by irradiation, the present inventors analyzed the morphological changes in breast bone tissue of the mouse group with tissue damage induced by gamma-ray irradiation before the administration of hesperetin. As a result, the loss of myelocytes in the group administered with hesperetin was less, suggesting that the administration of hesperetin was effective in recovering the damage induced by irradiation (see Figure 19) .

Therefore, hesperetin of the present invention displayed the preventive and therapeutic effect on the liver cell and hematopoietic-immune system damage caused by radiation in the mouse model having damage induced by gamma-ray irradiation, and it was also confirmed to be effective in preventing and recovering tissue damage caused by oxidative stress, indicating that hesperetin of the invention could be effectively used as a radioprotective agent against radiation exposure.

The hesperetin of the present invention can be used as the form of a pharmaceutically acceptable salt, in which the salt is preferably acid addition salt formed by pharmaceutically acceptable free acids. The acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid and phosphorous acid, or non-toxic organic acids such as aliphatic mono/dicarboxylate , phenyl-substituted alkanoate, hydroxy alkanoate, alkandioate, aromatic acids and aliphatic/aromatic sulfonic acids. The pharmaceutically non-toxic salts are exemplified by sulfate, pyrosulfate, bisulfate, sulphite, bisulphite, nitrate, phosphate, monohydrogen phosphate _> dihydrogen phosphate, metaphosphate , pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutylate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, cabacate, fumarate, maliate, butyne-1, 4-dioate, hexane-1, 6-dioate, benzoate, chlorobenzoate , methylbenzoate , dinitrobenzoate , hydroxybenzoate , methoxybenzoate , phthalate, terephthalate , benzenesulfonate, toluenesulfonate , chlorobenzenesulfonate ,- xylenesulfonate , phenylacetate , phenylpropionate , phenylbutylate , citrate, lactate, hydroxybutylate , glycolate, malate, tartrate, methanesulfonate , propanesulfonate, naphthalene- 1-sulfonate, naphthalene-2- sulfonate and mandelate.

The acid addition salt of the present invention can be prepared by the conventional method. For example, the said hesperetin is dissolved in excessive acid aqueous solution and then the salt can be prepared by the precipitation using a water-miscible organic solvent which is exemplified by methanol, ethanol, acetone, or acetonitrile . Then, the solvent or the excessive acid is evaporated from the mixture, followed by drying the mixture to give addition salt or suction- filtering the precipitated salt to give the same .

A pharmaceutically acceptable metal salt can be prepared by using base. For example, alkali metal or alkali earth metal salt can be prepared by the following steps: dissolving the compound in excessive alkali metal hydroxide or alkali earth metal hydroxide solution, filtering the non-soluble compound salt, evaporating the remaining solution, and drying thereof. At this time, the metal salt is preferably sodium salt, potassium salt, or calcium salt for the pharmaceutical purpose. And the counter silver salt can be obtained by the reaction between alkali metal or alkali earth metal salt and a proper silver salt (ex, silver nitrate) .

The present invention includes not only hesperetin or the pharmaceutically acceptable salts thereof, but also solvates and hydrates possibly produced from the same.

The addition salt in this invention can be prepared by the conventional method known to those in the art. For example, hesperetin is dissolved in water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, to which excessive organic acid or acid aqueous solution of inorganic acid is added to induce precipitation or crystallization. Then, the solvent or the excessive acid is evaporated from the mixture, followed by drying the mixture to give addition salt or suction- filtering the precipitated salt to give the same.

The pharmaceutical composition of the present invention comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient can be administered orally or parenterally and be used in general forms of pharmaceutical formulation, but not always limited thereto.

The formulations for oral administration are exemplified by tablets, pills, hard/soft capsules, solutions, suspensions, emulsions, syrups, granules, and elixirs, etc. These formulations can include diluents (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine) and lubricants (for example, silica, talc, stearate and its magnesium or calcium salt, and/or polyethylene glycol) in addition to the active ingredient. Tablets can include binding agents such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, , sodium carboxymethylcellulose and/or polyvinylpyrolidone, and if necessary disintegrating agents such as starch, agarose, alginic acid or its sodium salt or azeotropic mixtures and/or absorbents, coloring agents, flavors, and sweeteners can be additionally included thereto .

The pharmaceutical composition of the present invention comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient can be administered orally or parenterally. The parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection and .. intrathoracic injection. To prepare the composition as a formulation for parenteral administration, hesperetin or the pharmaceutically acceptable salts thereof are mixed with a stabilizer or a buffering agent to produce a solution or suspension, which is then formulated as ampoules or vials. The composition can be sterilized and can additionally include preservatives, resolvents, stabilizers, wetting agents, emulsifiers, osmosis controlling salts, buffering agents, and other therapeutically valuable additives.

The effective dosage of the hesperetin of the present invention can be determined according to age, weight, gender, administration method, health condition, and severity of disease. For example, the dose for an adult in 60 kg of body weight is 0.001 ~ 1,000 nig/day and preferably 0.01 ~ 500 nig/day. This administration can be performed once a day ~ several times a day according to the decision of a doctor or a pharmacist.

The present invention also provides a pharmaceutical composition for the prevention and treatment of oxidative stress caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient . The present invention also provides a method for the prevention and treatment of oxidative stress caused by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

The said hesperetin is the compound represented by the following [Formula 1] :

The radiation herein is selected from the group consisting of UV, X-ray, -ray, β-ray, γ-ray, electron beam, and neutron beam, but not always limited thereto.

Hesperetin of the present invention has the activity of preventing and treating liver cell damage and hematopoietic- immune system damage caused by radiation in the mouse model having damage induced by gamma-ray irradiation along with the effect of preventing and recovering tissue damage caused by oxidative stress, so that it can be effectively used as a radioprotective agent against radiation exposure.

The present invention also provides a radioprotective agent comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

The said hesperetin is the compound represented by the following [Formula 1] :

[Formula 1]

Hesperetin of the present invention has the activity of preventing and treating liver cell damage and hematopoietic-immune system damage caused by radiation in the mouse model having damage induced by gamma-ray irradiation along with the effect of preventing and recovering tissue damage caused by oxidative stress, so that it can be effectively used as a radioprotective agent against radiation exposure.

The present invention also provides a health food for the prevention and improvement of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient .

The present invention also provides a health food for the prevention and improvement of oxidative stress caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

In addition, the present invention provides a health food for radioprotection comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient .

The said hesperetin is the compound represented by the following [Formula 1] : Formula 1]

Hesperetin of the present invention has the activity of preventing and treating liver cell damage and hematopoietic- immune system damage caused by radiation in the mouse model having damage induced by gamma-ray irradiation along with the effect of preventing and recovering tissue damage caused by oxidative stress, so that it can be effectively used as a radioprotective agent against radiation exposure. The food herein is not limited. For example, the composition of the present invention can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included.

The hesperetin or the pharmaceutically acceptable salts thereof of the present invention can be used as food additives. In that case, the hesperetin or the pharmaceutically acceptable salts thereof can be added as they are or as mixed with other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use (prevention or health enhancement) . In general, to produce health food or beverages, the hesperetin or the pharmaceutically acceptable salts thereof are added preferably by 0.1 ~ 90 weight part. However, if long term administration is required for health and hygiene or for regulating health condition, the content can be lower than the above but higher content can be accepted as well since the compound has been proved to be very safe .

The composition for health beverages of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages in addition to the extract of crude drug complex. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. Besides, natural sweetening agents (thaumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic sweetening agents (saccharin, aspartame, etc . ) can be included as a sweetening agent . The content of the natural carbohydrate is preferably 1 ~ 20 g and more preferably 5 ~ 12 g in 100 ml of the composition.

In addition to the ingredients mentioned above, the hesperetin or the pharmaceutically acceptable salts thereof of the present invention can include in variety of nutrients, vitamins, minerals (electrolytes) , flavors including natural flavors and synthetic flavors, coloring agents and extenders (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The hesperetin or the pharmaceutically acceptable salts thereof of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 0.1 ~ 20 weight part per 100 weight part of the hesperetin or the pharmaceutically acceptable salts thereof of the invention. [Mode for Invention] Practical and presently preferred embodiments of the present invention : are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Construction of the animal model having tissue damage induced by irradiation

<!-!> Construction of the animal model having tissue damage induced by gamma-ray irradiation after the administration of hesperetin

The animal model having tissue damage induced by gamma-ray irradiation after being administered with hesperetin was constructed, with which the preventive effect of hesperetin on the damage induced by gamma-ray irradiation was investigated.

Particularly, female BALB/c mice at 5 weeks of age (19.4+1 g) were purchased from Orientbio, Co. Ltd. The animals were adapted in a raising cage at the temperature of 23±2°C, humidity of 55±5%, with 12 hour dark/light cycle for a week, during which solid feeds and drinking water were given freely. Then, the mice were grouped into 5 groups, 7 mice per group; the normal control group provided with CMC (carboxy methyl cellulose) for 7 days, the gamma- ray irradiation group which was irradiated with 6 Gy of gamma-ray after given with CMC for 7 days, the gamma-ray irradiation group which was irradiated with 6 Gy of gamma- ray after given with 25 rag/kg of hesperetin (HT, Sigma- Aldrich, USA) for 7 days, the gamma-ray irradiation group which was irradiated with 6 Gy of gamma-ray after given with 100 mg/kg of hesperetin for 7 days, and the gamma-ray irradiation group which was irradiated with 6 Gy of gamma- ray after given with 100 mg/kg of hesperidin (HD, Sigma- Aldrich, USA) for 7 days. The test sample was orally administered once a day for 7 days. After the last administration, the animals were irradiated with gamma-ray. 24 hours later, the animals were sacrificed. Gamma-ray irradiation was performed at the dose rate of 1.1 Gy/min by using Gammacell 40 exactor (MDS Nordion, Canada) at Advanced Radiation Technology Institute (ARTI) , Korea Atomic Energy Research Institute (KAERI) . Except the normal control group, the BALB/c mice of other groups were placed in an acrylic box, which were irradiated with ¹³⁷CS - Y ray at the absorbed dose of 6 Gy over the whole body of the animal

<l-2> Construction of the animal model having tissue damage induced by gamma-ray irradiation before the administration of hesperetin To investigate whether or not the administration of hesperetin to the animal model having tissue damage induced by gamma-ray irradiation was effective in treating the already made damage, the animal model having tissue damage induced by gamma-ray irradiation was first constructed -before the administration of hesperetin.

Particularly, the BALB/c mice adopted by the same manner as described in Example <1-1> were grouped into 6 groups, 6 mice per each group; the normal control group provided with CMC for 7 days, the group treated with 50 mg/kg of hesperetin (HT) for 7 days, the group irradiated with 6 Gy of gamma-ray and thereafter administered orally with CMC for 7 days , the group irradiated with 6 Gy of gamma-ray and thereafter administered orally with 25 mg/kg of hesperetin for 7 days, the group irradiated with 6 Gy of gamma-ray and thereafter administered orally with 50 mg/kg of hesperetin for 7 days, the group irradiated with 6 Gy of gamma-ray and thereafter administered orally with 100 mg/kg of hesperidin (HD) for 7 days. The test sample was orally administered 1 hour after gamma-ray irradiation, once a day for 7 days. 24 hours after the last administration, the animals were sacrificed. Gamma-ray irradiation was performed by the same manner as described in Example <1-1>. Except the normal control group and the group treated with hesperetin for 7 days, the BALB/c mice of other groups were placed in an acrylic box, which were irradiated with ¹³⁷Cs-y ray at the absorbed dose of 6 Gy over the whole body of the animal .

Comparative Example 1: Comparison of antioxidant activities of hesperetin and hesperidin

<!-!> Comparison of DPPH radical scavenging activities of hesperetin and hesperidin

The investigation of DPPH radical scavenging activity, that is the action to eliminate free radicals directly, is one way to evaluate the antioxidant activity. When DPPH is reduced in the course of the reaction, it means free radical extinction is going on, by which the suppression rate of early lipid peroxidation can be predicted (Hatano et al. 1989) . Thus, in order to compare the antioxidant activities of hesperetin and hesperidin, DPPH radical scavenging activity of each of them was measured.

Particularly, DPPH radical scavenging activity was measured by the method of Dietz et al . (2005) . 900 μΐ of 0.1 mM DPPH solution (DPPH was dissolved in 80% methanol) was added to 100 μί of hesperetin or hesperidin solution at different concentrations, which stood at room temperature for 30 minutes. Then, 0D₅i₇ was measured by using Multiskan FC microplate photometer (Thermo, Finland) . DPPH radical scavenging activity was calculated by the below mathematical formula 1. The test result was presented as mean + standard deviation after performing the measurement in triplicate. The significance in the difference between the two materials was verified by using student's t-test.

[Mathematical Formula l]

EDA (electro donating ability, %) =

(control OD - sample OD / control OD) x 100

As a result, as shown in Figure 2, EDA of hesperetin was significantly higher than that of hesperidin dose- dependently from the concentration of 100 μΜ, suggesting that the DPPH radical scavenging activity of hesperetin was greater than that of hesperidin (Figure 2) .

<l-2> Comparison of ABTS radical scavenging activities of hesperetin and hesperidin

The investigation of ABTS radical scavenging activity is another way to measure the antioxidant activity, which is similar to the method to examine DPPH radical scavenging activity. Precisely, ABTS and potassium persulfate are left in a dark place to induce chemical reaction between them. The resultant ABTS⁺ · radical solution is used for the measurement of antioxidant activity. When ABTS⁺ · is extinct by an antioxidant material, the typical radical color blue- green is discolored (Re et al . 1999). Based on that, ABTS radical scavenging activities of hesperetin and hesperidin were measured in order to compare the antioxidant activities of hesperetin and hesperidin.

Particularly, ABTS radical scavenging activity was measured by the method of Re et al. (1999). 7.4 mM ABTS [2, 2¹ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] (Sigma-Aldrich, USA) and 2.45 mM of potassium persulfate (Sigma-Aldrich, USA) were left in a cold/dark place for 16 hours, resulting in the generation of ABTS⁺, which was then diluted with phosphate buffer (pH 7.4) until OD reached 1.0. 900 μί of ABTS solution was added to 100 μί of hesperetin or hesperidin solution at different concentrations. 6 minutes later, OD₇₃₄ was measured by using Multiskan FC microplate photometer. ABTS radical scavenging activity was calculated by mathematical formula 1. The test result was presented as mean ± standard deviation after performing the measurement in triplicate. The significance in the difference between the two materials was verified by using student's t-test.

As a result, as shown in Figure 3, EDA of hesperetin was significantly higher than that of hesperidin dose- dependently from the concentration of 100 μΜ, suggesting that the ABTS radical scavenging activity of hesperetin was greater than that of hesperidin (Figure 3) . <l-2> Comparison of antioxidant activities of hesperetin and hesperidin

FRAP (ferric reducing antioxidant power) analysis is the method to measure the total antioxidant activity of a sample. This method is the way to evaluate the antioxidant activity by measuring OD of Fe(2) when Fe(3) is reduced into Fe(2) by a reducing agent at low pH condition (Benzie and Strain 1996) . Herein, in order to compare the antioxidant activities of hesperetin and hesperidin, FRAP analysis was performed with hesperetin and hesperidin.

Particularly, FRAP analysis was performed by the method of Benzie and Strain (1996) . For FRAP analysis, 0.3 M sodium acetate (pH 3.6) buffer, 10 mM TPTZ (2,4,6- tripyridyl-S-triazine) dissolved in 40 mM HC1, and 20 mM ferric chloride solution were prepared. Then, the prepared sodium acetate buffer, TPTZ solution, and ferric chloride solution were mixed at the ratio of 10:1:1 (v/v/v) , which stood at 37^°C, resulting in the preparation of FRAP solution. 100 βί of FRAP solution was added to 30 μΐ of hesperetin or hesperidin solution at different concentrations, which stood at room temperature for 10 minutes. Then, OD₅₉₃ was measured by using Multiskan FC microplate photometer. The test result was presented as mean ± standard deviation after performing the measurement in triplicate. The significance in the difference between the two materials was verified by using student's t-test. As a result, as shown in Figure 4 , FRAP was significantly increased by hesperetin dose-dependently, compared with that increased by hesperidin, suggesting that hesperetin displayed higher reducing activity. Therefore, it was confirmed that hesperetin had stronger antioxidant activity than hesperidin (Figure 4 ) . The above results of •cComparative Example 1> confirmed that hesperetin had greater antioxidant activity than hesperidin. Example 2: Confirmation of the preventive effect of hesperetin on tissue damage induced by gamma-ray irradiation and oxidative stress

<2-l> Investigation of the changes of weight, spleen index, and thymus index of the mouse having tissue damage induced by gamma-ray irradiation after the administration of hesperetin

Weight and dietary intake were reduced in the animal model by irradiation (Linn et al . 1996; Pradeep et al . 2007) . So, in order to investigate whether or not the administration of hesperetin before the gamma-ray irradiation was effective in preventing tissue damage caused by gamma-ray irradiation, weight, spleen index, and thymus index of the mouse having tissue damage induced by gamma-ray irradiation were measured after the administration of hesperetin. Particularly, the mice having tissue damage induced by gamma-ray irradiation after the administration of hesperetin, selected from the 5 experimental groups prepared in Example <1-1>, were anesthetized with a inhalation anesthetic a day after gamma-ray irradiation. The abdomen was opened and blood was collected from the postcaval vein. Then, the liver, the sternum, the spleen, and the thymus were extracted and washed with cold saline, whose dried weights were ^" measured respectively. Spleen index and thymus index were calculated by the formula: (spleen and thymus weight/body weight) x 100. The test result was examined by one-way A OVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean + standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Table 1, the body weight of the normal control group mouse, measured a day after gamma- ray irradiation, was 19.243±0.353 , while the body weight of the mouse of the group irradiated with gamma-ray was 18.171+0.289, indicating that the weight of the irradiation group was reduced compared with that of the normal group. In the group irradiated with gamma-ray after administered with 25 mg/kg of hesperetin, the body weight of the mouse was 19.614±0.295 , suggesting that the body weight was significantly increased, compared with that of the gamma- ray irradiation group.

When a living body was irradiated at 6.5 Gy or higher, the spleen and the thymus, which are the hematopoietic- immune system organs sensitive to radiation, were getting defect by radiation, and accordingly numbers of lymphocytes became dead, resulting in the weight and volume loss of the spleen and the thymus (Gough et al . 1977) . The spleen index of the gamma-ray irradiation group was 0.197+0.004 a day after gamma-ray irradiation, which was significantly lower than that of the normal control. In the meantime, in the group irradiated with gamma-ray after administered with 25 mg/kg of hesperetin, the spleen index was 0.234±0.006, indicating that the spleen index was significantly increased, compared with that of the gamma-ray irradiation group.

A day after gamma-ray irradiation, the thymus index of the gamma-ray irradiation group was 0.131+0.010, which was significantly lower than that of the normal control. In the grout irradiated with gamma-ray after administered with 25 mg/kg of hesperetin, the thymus index was 0.200+0.012, which was significantly increased compared with that of the gamma-ray irradiation group. The above results indicate that the administration of hesperetin before the irradiation was effective in preventing the hematopoietic- immune system damage caused by radiation (Table 1) .

[Table l]

p<0.05 and p<0.01: significant difference from the normal control .

p<0.05 and p<0.01: significant difference from the 6 Gy irradiation group.

<2-2> Investigation of blood cell component of the mouse having tissue damage induced by gamma-ray irradiation after the administration of hesperetin

After irradiation, the number of immune cells in peripheral blood is rapidly reduced. In the recovery, the recovery of cell number is a key factor, according to the previous study (Patchen et al . 1991). So, in order to investigate whether or not the administration of hesperetin before the gamma-ray irradiation could be effective in preventing damage induced by gamma-ray irradiation, the present inventors investigated blood cell component in blood of the mouse having tissue damage induced by gamma- ray irradiation after the administration of hesperetin.

Particularly, blood was collected from the mice of the 5 experimental groups treated by the same manner as described in Example <2-l>, which were administered with hesperetin and then irradiated with gamma-ray. The collected blood was loaded in the tube treated with heparin, the anticoagulant, followed by the measurement of the number of white blood cell (WBC) using HEMAVET HV950 (Drew Scientific, Inc., USA). The test result was examined by one-way A OVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 5, the number of white blood cell (WBC) in the group irradiated with gamma-ray was significantly reduced a day after gamma-ray irradiation, compared with that of the normal control group. In the group administered with 50 mg/kg of hesperetin before being irradiated with gamma-ray, the number of WBC was significantly increased, compared with that of the gamma- ray irradiation group. Therefore, it was confirmed that the administration of hesperetin before the gamma-ray irradiation was effective in protecting hematoblasts from irradiation and in regenerating the same (Figure 5).

<2-3> Investigation of biochemical changes in blood plasma of the mouse having tissue damage induced by gamma-ray irradiation after the administration of hesperetin

AST and ALT are the aminotransferases existing in liver cells. When the liver cells are damaged, these enzymes are released into blood and become more active, making them good testing materials for hepatotoxicity test. AST and ALT are also found in the kidney, muscle, and heart, in addition to the liver tissue. So, they might not directly be a reason of liver injury but particularly since ALT is mainly distributed in the liver tissue, the up- regulation of ALT seems to be directly involved in liver injury (Reckhagel et al . 1989) . When a rat is irradiated with gamma-ray, the activities of blood AST and ALT are rapidly increased (Pradeep et al. 2008). To investigate whether or not the administration of hesperetin before the gamma-ray irradiation could be effective in preventing tissue damage induced by gamma-ray irradiation, blood AST and ALT in the mouse having tissue damage induced by gamma- ray irradiation after being administered with hesperetin were measured.

Particularly, blood was collected from the mice of the 5 experimental groups having tissue damage induced by gamma-ray irradiation by the same manner as described in Example <2-l>. Each tube containing the blood was centrifuged at 3000 rpm for 20 minutes to separate blood plasma, which was then stored at 4^°C. AST and ALT in the blood plasma were measured by the method of Bergmeye et al . (1978). 1.0 mi of AST and ALT substrate was added to each test tube, which stood at 37^°C for 5 minutes. 0.2 mi .of blood plasma was added thereto and mixed well therein. Reaction was induced at 37°C, and then^' 1 mi of DNPH was added and well mixed. The mixture stood at room temperature for 20 minutes, and then the reaction was terminated. Lastly, 0.4 M sodium hydroxide was added thereto. After well mixed, the reaction mixture stood at room temperature for 10 minutes. Then, OD_{5 0} was measured by using Multiskan FC microplate photometer. The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean + standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result_s as shown in Figure 6, AST and ALT in blood plasma of the gamma-ray irradiation group were significantly increased a day after the irradiation, compared with those of the normal control, indicating hepatotoxicity was induced. In the meantime, in the group treated with hesperetin or hesperidin before being irradiated with gamma-ray, ALT and AST were significantly decreased. In particular, hesperetin was more effective in reducing significantly the levels of ALT and AST than hesperidin. Therefore, it was confirmed that the administration of hesperetin before irradiation was effective in preventing cell damage from irradiation and protecting thereby the cells by significantly inhibiting the over-activation of the liver injury marker enzyme in blood plasma (Figure 6) .

<2-4> Investigation of lipid peroxidation and XO in the liver tissue of the mouse having tissue damage induced by gamma-ray irradiation after the administration of hesperetin

When lipid, the major component of biomembrane, is oxidized by ionizing radiation such as X-ray and γ-ray, it produces MDA showing lipid peroxidation which is frequently used as the oxidative stress index (Emerit et al . 2003). In vivo XO generates free radicals, which may damage cells to produce lipid peroxides (Mccord 1985). To investigate whether or not the administration of hesperetin before the gamma-ray irradiation could be effective in preventing cell damage induced by gamma-ray irradiation and be effective in treating oxidative stress, lipid peroxidation and XO (xanthine oxidase) were measured in the liver tissue of the mouse having tissue damage induced by gamma- ray irradiation after the administration of hesperetin.

Particularly, to measure the lipid peroxidation of liver tissue by irradiation, MDA (malondialdehyde) was measured by the method of Ohakawa, et al. (1979) . Liver tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation after being treated with hesperetin selected from each of the 5 experimental groups, according to the method described in Example <2-l>. The extracted liver tissues were mixed with phosphate buffer (0.1 M, pH 7.4) by using Precellys 24-dual (Bertin, France), resulting in 10% homogenized solution. The solution was centrifuged at 4^°G at lOOOOxg for 10 minutes to separate supernatant. 100 id of the supernatant was mixed with 100 id of 8.1% SDS lysis buffer, 150 id of 20% TCA solution, and 150 fd of 0.8% TBA solution, which was reacted at 100 ^°C for 1 hour. Upon completion of the reaction, centrifugation was performed at 4^°C at 3000xg for 10 minutes. 300 id of the supernatant was mixed with 300 id of n-butanol, followed by centrifugation at lOOOOxg for 5 minutes. Then, 200 id of the supernatant was obtained, and OD₅₄₀ was measured by using Multiskan FC microplate photometer (Figure 7) .

To investigate the changes of XO in the liver tissue induced by irradiation, liver tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation after being treated with hesperetin selected from each of the 5 experimental groups, according to the method described in Example <2-l>. XO in the extracted liver tissues was measured by using a measuring kit reagent (Biovision, USA) according to the manufacturer's instruction (Figure 8) . Protein content in the liver tissue was measured according to the method of Bradford (1976) using BSA (bovine serum albumin) as the standard. The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 7, MDA of the gamma- ray irradiation group was significantly increased a day after the irradiation, compared with that of the normal control group. In the meantime, MDA of the group treated with hesperetin or hesperidin before irradiation was significantly reduced, compared with that of the group irradiated with gamma-ray. It was also confirmed that hesperetin reduced MDA more significantly than hesperidin did (Figure 7) .

As shown in Figure 8 , XO of the group irradiated with gamma-ray was significantly increased a day after the irradiation, compared with that of the normal control group In the meantime, in the group treated with 50 mg/kg of hesperetin before being irradiated with gamma-ray, XO was significantly reduced (Figure 8). From the above results, it was confirmed that the administration of hesperetin before irradiation was effective in preventing cell damage and protecting the cells by inhibiting significantly the unnecessary increase of XO and lipid peroxidation in liver tissues.

<2-5> Investigation of the changes of antioxidant material and antioxidant enzyme in the liver tissue of the mouse with tissue damage induced by gamma-ray irradiation after the administration of hesperetin

Excessive free radical generated by radiation is eliminated by in vivo antioxidant material or antioxidant enzyme or the generation thereof itself is suppressed by the same. The in vivo antioxidant material includes reduced GSH (glutathione) which is acting as a substrate of GPx (glutathione peroxidase) capable of scavenging free radical and metabolizing H₂0₂ and lipid peroxidation. The in vivo antioxidant enzyme is exemplified by SOD (superoxide dismutase) , catalase, and GPx. SOD converts 0₂ ^~ into H₂0₂. Catalase and GPx decompose H₂0₂ into H₂0 to eliminate intracellular radicals (Fridovich I. 1986). When an animal is irradiated with gamma-ray, the antioxidant material GSH and the antioxidant enzymes SOD, catalase, and GPx are less activated in order to protect cells from oxidative damage (Pratheeshkumar and Kuttan 2010; Pradeep 2012) . To investigate whether or not the administration of hesperetin could be effective in preventing oxidative damage induced by gamma-ray irradiation, the antioxidant material GSH and the antioxidant enzymes SOD, catalase, and GPx in the liver tissue of the mouse with tissue damage induced by gamma-ray irradiation after the administration of hesperetin were measured.

Particularly, liver tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation after being treated with hesperetin selected from each of the 5 experimental groups, according to the method described in Example <2-l>. GSH in the extracted liver tissues was measured by using a measuring kit reagent (Biovision, USA) according to the manufacturer's instruction (Figure 9) , and so were SOD, catalase, and GPx (Figure 10) . Protein content in the liver tissue was measured according to the method of Bradford (1976) using BSA (bovine serum albumin) as the standard. The test result was examined by one-way A OVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean + standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 9, GSH of the group irradiated with gamma-ray was significantly reduced a day after the irradiation, compared with that of the normal control group. In the meantime, GSH of the group irradiated with gamma-ray after being administered with 50 mg/kg of hesperetin was significantly increased. These results suggest that the administration of hesperetin before the irradiation was effective in suppressing significantly the decrease of in vivo antioxidant material in the liver tissue, resulting in the prevention of cell damage and protection of cells thereby (Figure 9) .

As shown in Figure 10, SOD, catalase, and GPx of the group irradiated with gamma-ray were all decreased significantly a day after the irradiation, compared with those of the normal control group. In the meantime, in the group treated with hesperetin and hesperidin before being irradiated with gamma-ray, the enzymes were significantly increased, compared with those of the gamma-ray irradiation group. In particular, the increase of SOD and catalase was more peculiar when hesperetin was treated than when hesperidin was treated. Therefore, it was confirmed that the administration of hesperetin was effective in preventing cell damage and thereby protecting cells by suppressing the decrease of in vivo antioxidant enzymes in the liver tissue caused by irradiation (Figure 10) .

<2-6> Investigation of the morphological changes of the mouse with tissue damage induced by gamma-ray irradiation after the administration of hesperetin

To investigate the effect of hesperetin on preventing damage caused by gamma-ray irradiation, sternum tissue of the mouse with tissue damage induced by gamma-ray irradiation after the administration of hesperetin was analyzed.

Particularly, sternum tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation after being treated with hesperetin selected from each of the 5 experimental groups , according to the method described in Example <2-l>. The extracted sternum tissues were fixed in 10% formalin for histopathological analysis. The fixed sternum tissue was washed with ethanol and xylene stepwise, and then embedded in paraffin wax. The paraffin block was sliced . in 5 πι sections by using a microtome (Leica Microsystems, Germany) . Then, the wax was eliminated, followed by staining with hematoxylin and eosin The sample was observed under microscope.

As a result, as shown in Figure 11, the cellularity of the normal control was excellent, but the myelocytes of the group irradiated with gamma-ray were almost lost, indicating myelocyte damage. In the meantime, the loss of myelocytes in the group administered with hesperetin before being irradiated with gamma-ray was less than that in the group irradiated with gamma-ray without being treated with hesperetin. Therefore, it was confirmed that the administration of hesperetin before the gamma-ray irradiation was effective in preventing tissue damage from gamma-ray irradiation (Figure 11) .

Example 3: Confirmation of the therapeutic effect of hesperetin on tissue damage induced by gamma-ray irradiation and oxidative stress

<3-l> Investigation of the changes of weight, spleen index, and thoracic index of the mouse having tissue damage induced by gamma-ray irradiation before the administration of hesperetin

To investigate whether or not the administration of hesperetin after gamma-ray irradiation was effective in treating tissue damage induced by gamma-ray irradiation, weight, spleen index, and thymus index of the mouse with tissue damage induced by gamma-ray irradiation before being administered with hesperetin were measured.

Particularly, blood was collected from the mice of the

6 experimental groups with tissue damage induced by gamma- ray irradiation before being treated with hesperetin, prepared by the same manner as . described in Example <l-2>,

7 days after the irradiation. Blood collection was performed by the same manner as described in Example <2-l>. The liver, the breast bone, and the spleen were extracted and weighed (Figure 12) . Spleen index was calculated (Figure 13) . The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 6 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 12, 7 days after gamma-ray irradiation, the weight of the normal control group was 102.24% and the weight of the gamma-ray irradiation group was 89.51%, which was decreased, compared with that of the normal control group. In the meantime, the weight of the group administered with 25 mg/kg of hesperetin after the irradiation was 98.95% and the weight of the group administered with 50 mg/kg of hesperetin after the irradiation was 96.84%, indicating the weight was recovered, compared with that of the gamma-ray irradiation group (Figure 12) .

As shown in Figure 13, spleen index of the group irradiated with gamma-ray was significantly decreased 7 days after the irradiation, compared with that of the normal control group. On the other hand, in the group treated with hesperetin after gamma-ray irradiation, spleen index was significantly increased hesperetin dose- dependently, indicating that the administration of hespereti after the irradiation was effective in recovering spleen damage caused by radiation. Therefore, it was confirmed that the administration of hesperetin after the irradiation was effective in protecting hematoblasts from radiation exposure and in regenerating the same (Figure 13) .

<3-2> Investigation of blood cell component of the mouse having tissue damage induced by gamma-ray irradiation before the administration of hesperetin

To investigate whether or not the administration of hesperetin after gamma-ray irradiation could be effective in treating damage induced by gamma-ray irradiation, the present inventors analyzed blood cell component in blood of the mouse having tissue damage induced by gamma-ray irradiation before the administration of hesperetin.

Particularly, blood was collected from the mice of the 5 experimental groups treated by the same manner as described in Example <2-l>, which were administered with hesperetin and then irradiated with gamma-ray. The collected blood was loaded in the tube treated with heparin, the anticoagulant, followed by the measurement of the number of white blood cell (WBC) using HEMAVET HV950 (Drew Scientific, Inc., USA). The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 5, the number of white blood cell (WBC) in the group irradiated with gamma-ray was significantly reduced a day after gamma-ray irradiation, compared with that of the normal control, group. In the group administered with 50 mg/kg of hesperetin before being irradiated with gamma-ray, the number of WBC was significantly increased, compared with that of the gamma- ray irradiation group. Therefore, it was confirmed that the administration of hesperetin before the gamma-ray irradiation was effective in protecting hematoblasts from irradiation and in regenerating the same (Figure 5) .

Particularly, blood was collected from the mice of the 6 experimental groups by the same manner as described in Example <3-l>. The numbers of white blood cells (WBC), neutrophils, and lymphocytes in blood of the mouse having tissue damage induced by gamma-ray irradiation before the administration of hesperetin were measured by the method described in Example <2-2> using a hematocytometer . which were administered with hesperetin and then irradiated with gamma-ray. The test result was examined by one-way A OVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 6 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Table 2, the numbers of WBC, neutrophils, and lymphocytes of the group irradiated with gamma-ray were significantly decreased 7 days after the irradiation, compared with those of the normal control group: On the other hand, the numbers of WBC, neutrophils, and lymphocytes of the group administered with hesperetin after gamma-ray irradiation were significantly increased, compared with those of the group irradiated with gamma-ray alone. In the group administered with 50 mg/kg of hesperetin, the significance in the difference from the group irradiated with gamma-ray was not peculiar but the numbers of WBC, neutrophils, and lymphocytes were still increased. The above results indicate that the administration of hesperetin after the irradiation was effective in regenerating hematoblasts damaged by irradiation (Table 2) .

[Table 2]

p<0.05 and ^#p<0.01: significant difference from the normal control .

^*p<0.05 and ^**p<0.01: significant difference from the 6 Gy irradiation group.

<3 -3 > Investigation of biochemical changes in blood plasma of the mouse having tissue damage induced by gamma-ray irradiation before the administration of hesperetin

To investigate whether or not the administration of hesperetin after gamma-ray irradiation was effective in treating damage induced by gamma-ray irradiation, AST and ALT in blood of the mouse with tissue damage induced by gamma-ray irradiation before being administered with hesperetin were measured.

, Particularly, blood was collected from the mice of the 6 experimental groups having tissue damage induced by gamma-ray irradiation before the administration of hesperetin by the same manner as described in Example <3-l> Blood plasma was separated therefrom by the same manner as described in Example <2-3>, then AST and ALT in blood plasma were measured. The test result was examined by one- way A OVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 6 mice of each group were presented as mean + standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 14, AST and ALT in blood plasma of the gamma-ray irradiation group were significantly increased 7 days after the irradiation, compared with those of the normal control group, indicating hepatotoxicity was induced. In the meantime, in the group administered with hesperetin after gamma-ray irradiation, ALT and AST were significantly decreased. In particular, hesperetin was more effective in reducing significantly the levels of ALT and AST than hesperidin. Therefore, it was confirmed that the administration of hesperetin after irradiation was effective in treating cell damage caused by irradiation and in protecting cells by significantly suppressing the over-activation of the liver injury marker enzymes in blood plasma induced by irradiation (Figure 14) . <3-4> Investigation of lipid peroxidation and XO in the liver tissue of the mouse having tissue damage induced by gamma- ray irradiation before the administration of hesperetin

To investigate whether or not the administration of hesperetin after gamma-ray irradiation could be effective in treating damage induced by gamma-ray irradiation and oxidative stress thereby, lipid peroxidation and XO were measured in the liver tissue of the mouse having tissue damage induced by gamma- ray irradiation before the administration of hesperetin.

Particularly, to investigate the lipid peroxidation of the liver tissue by irradiation, liver tissues were extracted from the mice with tissue damage induced by gamma- ray irradiation before the administration of hesperetin selected from each of the 6 experimental groups, according to the method described in Example < 3-l>. The extracted liver tissues were homogenized by the method described in Example <2-4> and the supernatant was obtained. Then, MDA in the supernatant was measured (Figure 15) .

To investigate the changes of XO in the liver tissue induced by irradiation, liver tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation before the administration of hesperetin selected from each of the 6 experimental groups, according to the method described in Example < 3-l>. XO in the extracted liver tissue was measured by the same manner as described in Example <2-4> (Figure 16) . Protein content in the liver tissue was measured according to the method of Bradford (1976) using BSA (bovine serum albumin) as the standard. The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean ± standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 15, MDA of the group irradiated with gamma-ray was significantly increased 7 days after the irradiation, compared with that of the normal control group. In the meantime, MDA of the group irradiated with gamma-ray and treated with hesperetin and hesperidin thereafter was significantly decreased. In particular, the decrease by hesperetin was greater than by hesperidin. The above results indicate that the administration of hesperetin after irradiation was effective in treating cell damage and protecting the cells by suppressing the increase of lipid peroxidation in the liver tissue induced by irradiation (Figure 15) .

As shown in Figure 16, XO of the group irradiated with gamma-ray was significantly increased 7 days after the irradiation, compared with that of the normal control group. In the meantime, in the group treated with 50 mg/kg of hesperetin after gamma-ray irradiation, XO was significantly reduced. From the above results, it was confirmed that the administration of hesperetin after irradiation was effective in treating cell damage and protecting the cells by inhibiting significantly the unnecessary increase of XO in the liver tissue induced by irradiation (Figure 16) . <3-5> Investigation of the changes of antioxidant material and antioxidant enzyme in the liver tissue of the mouse with tissue damage induced by gamma-ray irradiation before the administration of hesperetin

To investigate whether or not the administration of hesperetin after gamma-ray irradiation was effective in treating oxidative damage induced by gamma-ray irradiation, the antioxidant material GHS and the antioxidant enzymes SOD, catalase, and GPx in the liver tissue of the mouse having tissue damage induced by gamma-ray irradiation were measured.

Particularly, liver tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation before the administration of hesperetin selected from each of the 6 experimental groups, according to the method described in Example <3-l>. GSH in the extracted liver tissue was measured by the same manner as described in Example <2-5> (Figure 17) , and so were SOD, catalase, and GPx (Figure 18) . Protein content in the liver tissue was measured according to the method of Bradford (1976) using BSA (bovine serum albumin) as the standard. The test result was examined by one-way ANOVA and the internal comparison was performed by Tukey's multiple comparison test. The measured numbers of all the 7 mice of each group were presented as mean + standard deviation. When P value was less than 0.05 (P <0.05), it was considered as significant.

As a result, as shown in Figure 17, GSH of the group irradiated with gamma-ray was significantly reduced 7 days after the irradiation, compared with that of the normal control group. In the meantime, MDA of the group irradiated with gamma-ray and treated with hesperetin and hesperidin thereafter was significantly increased. The above results indicate that the administration of hesperetin after irradiation was effective in treating cell damage and protecting the cells by suppressing the decrease of antioxidant material in the liver tissue induced by irradiation (Figure 17) .

As shown in Figure 18, SOD, catalase, and GPx of the group irradiated with gamma-ray were significantly decreased 7 days after the irradiation, compared with those of the normal control group. In the meantime, in the group treated with 25 mg/kg of hesperetin after being irradiated with gamma-ray, SOD, catalase, and GPx were significantly increased, compared with those of the gamma-ray irradiation group. In particular, in the groups respectively treated with 50 mg/kg of hesperetin and 100 mg/kg of hesperidin, GPx was significantly increased, compared with that of the gamma-ray irradiation group. Therefore, it was confirmed that the administration of hesperetin after gamma-ray irradiation was effective in treating cell damage and protecting the cells by recovering the decrease of antioxidant enzymes in the liver tissue caused by gamma-ray irradiation (Figure 18) . <3-6> Investigation of the morphological changes of the mouse with tissue damage induced by gamma- ray irradiation before the administration of hesperetin

To investigate the effect of the administration of hesperetin after gamma-ray irradiation on the treatment of cell damage induced by gamma-ray irradiation, sternum tissue of the mouse with tissue damage induced by gamma- ray irradiation before the administration of hesperetin was analyzed.

Particularly, sternum tissues were extracted from the mice with tissue damage induced by gamma-ray irradiation before the administration of hesperetin selected from each of the 6 experimental groups, according to the method described in Example <3-l>. The extracted breast bone tissues were fixed in 10% formalin for histopathological analysis. The fixed sternum tissue was washed with ethanol and xylene stepwise, and then embedded in paraffin wax. The paraffin block was sliced in 5 p sections by using a microtome (Leica Microsystems, Germany) . Then, the wax was eliminated, followed by staining with hematoxylin and eosin The sample was observed under microscope.

As a result, as shown in Figure 19, the myelocyte cellularity of the group treated with hesperetin and the normal control was excellent, but the myelocytes of the group irradiated with gamma- ray were almost lost, indicating myelocyte damage. In the meantime, the loss of myelocytes in the group administered with hesperetin after gamma-ray irradiation was less than that in the group irradiated with gamma-ray without being treated with hesperetin. Therefore, it was confirmed that the administration of hesperetin after gamma-ray irradiation was effective in recovering tissue damage caused by gamma- ray irradiation (Figure 19) .

Therefore, it was confirmed that the administration of hesperetin before the gamma-ray irradiation was effective in preventing tissue damage rom gamma-ray irradiation (Figure 11) .

The Manufacturing Examples for the composition of the present invention are described hereinafter.

Manufacturing Example 1: Preparation of pharmaceutical formulations

<!-!> Preparation of powders

Hesperetin or the pharmaceutically acceptable salts thereof 0.1 g

Lactose 1.5 g

Talc 0.5 g

Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders. <l-2> preparation of tablets

Hesperetin or the pharmaceutically acceptable salts thereof 0.1 g

Lactose 7.9 g

Crystalline cellulose 1.5 g

Magnesium stearate 0.5 g

Tablets were prepared by mixing all the above components by direct tableting method.

<l-3> Preparation of capsules

Hesperetin or the pharmaceutically acceptable salts thereof 0.1 g

Corn starch 5 g Carboxycellulose 4.9 g

Capsules were prepared by mixing all the above components, which were filled in hard capsules according to the conventional method for preparing capsules. <l-4> Preparation of injectable solutions

Hesperetin or the pharmaceutically acceptable salts thereof 0.1 g

Sterilized distilled water proper amount pH modifier proper amount Injectable solutions were prepared by mixing all the above components, putting the mixture into 2 mi ampoules by the conventional method for preparing injectable solutions.

<l-5> Preparation of liquid formulations

Hesperetin or the pharmaceutically acceptable salts thereof 0.1 g

Isomerized sugar 10 g

Mannitol 5 g

Purified water proper amount Lemon flavor proper amount

All the above components were dissolved in purified water. After adding lemon flavor, total volume was adjusted to be 100 mi by adding purified water. Liquid formulations were prepared by putting the mixture into brown bottles and sterilizing thereof by the conventional method for preparing liquid formulations.

Manufacturing Example 2 : Preparation of health food

<2-l> Preparation of flour food

0.5 5.0 weight part of hesperetin or the pharmaceutically acceptable salts thereof was added to the flour. Health enhancing foods such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method. <2-2> Preparation of soups and gravies

0.1 ~ 5.0 weight part of hesperetin or the pharmaceutically acceptable salts thereof was added to soups and gravies. Health enhancing meat products, soups and gravies were prepared with this mixture by the conventional method.

<2-3> Preparation of ground beef

Health enhancing ground beef was prepared by mixing 10 weight part of hesperetin or the pharmaceutically acceptable salts thereof with ground beef according to the conventional method.

<2-4> Preparation of dairy products

5 - 10 weight part of hesperetin or the pharmaceutically acceptable salts thereof was added to milk Health enhancing dairy products such as butter and ice cream were prepared with the milk mixture according to the conventional method.

<2-5> Preparation of Sun-Sik

Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders.

Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders.

Hesperetin or the pharmaceutically acceptable salts thereof were concentrated under reduced pressure, spray- dried and pulverized to obtain 60-mesh dry powders.

Sun-Sik was prepared by mixing the dry powders of the grains, seeds and hesperetin or the pharmaceutically acceptable salts thereof according to the below ratio.

Grains (brown rice: 30 weight part, Yulmu: 15 weight part, barley: 20 weight part),

Seeds (wild sesame: 7 weight part, black soybean: 8 weight part, black sesame: 7 weight part),

Dry powders of hesperetin or the pharmaceutically acceptable salts thereof (3 weight part),

Ganoderma lucidum (0.5 weight part),

Rehmannia glutinosa (0.5 weight part)

<2-6> Preparation of health supplement food

Hesperetin or the pharmaceutically acceptable salts thereof 100 nig

Vitamin complex proper amount

Vitamin A acetate 70 g

Vitamin E 1.0 nig

Vitamin Bl 0.13 nig

Vitamin B2 0.15 nig Vitamin B6 0.5 nig

Vitamin B12 0.2 zg

Vitamin C 10 nig

Biotin 10 μg

Nicotinic acid amide 1.7 nig

Folic acid 50 g

Calcium pantothenate 0.5 nig

Minerals proper amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 nig

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 nig

Potassium phosphate dibasic 55 nig

Potassium citrate 90 nig

Calcium carbonate 100 mg

Magnesium chloride 24.8 nig

Vitamins and minerals were mixed according to the preferable composition rate for health food. However, the composition rate can be adjusted. The constituents were mixed according to the conventional method for preparing health food and then the composition for health food was prepared according to the conventional method.

Manufacturing Example 3 : Preparation of health beverages

Hesperetin or the pharmaceutically acceptable salts thereof 100 nig

Citric acid 100 nig

Oligosaccharide 100 nig

Maesil (Prunus mu e) Extract 2 nig Taurine 100 nig

Purified water up to 500 ml

The above constituents were mixed according to the conventional method for preparing health beverages. The mixture was heated at 85°C for 1 hour with stirring and then filtered. The filtrate was loaded in 1 i sterilized containers, which were sealed and sterilized again, stored in a refrigerator until they would be used for the preparation of a composition for health beverages.

The constituents appropriate for favorite beverages were mixed according to the preferred mixing ratio but the composition ratio can be adjusted according to regional and national preferences, etc

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

[CLAIMS]

[Claim l]

A pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

[Claim 2]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the hesperetin is represented by formula 1 :

[Formula 1]

[Claim 3]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the radiation is selected from the group consisting of UV, X-ray, a-ray, β-ray, γ-ray, electron beam, and neutron beam. [Claim 4]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the cell or tissue is hematopoietic system or liver cell or tissue.

[Claim 5]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by inducing recovery of the body weight which has been reduced by radiation exposure. [Claim 6]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by reducing the activity of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) , the serum marker enzyme which has been increased by radiation exposure.

[Claim 7]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by reducing lipid peroxidation and the activity of XO which have been increased by radiation exposure .

[Claim 8]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by recovering the activity of SOD (superoxide dismutase) , catalase, or GPx (glutathione peroxidase) , the enzymatic antioxidant which has been decreased by radiation exposure .

[Claim 9]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by recovering the activity of GSH (glutathione) , the antioxidant material which has been decreased by radiation exposure.

[Claim 10]

The pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation according to claim 1, wherein the composition is characterized by recovering the number of blood cell which has been decreased by radiation exposure.

[Claim ll]

A pharmaceutical composition for the prevention and treatment of oxidative stress induced by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

[Claim 12]

A radioprotective agent comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient .

[Claim 13]

A health food for the prevention and improvement of cell or tissue damage caused by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

[Claim 14]

A health food for the prevention and improvement of oxidative stress induced by radiation comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

[Claim 15]

A health food for radioprotection comprising hesperetin or the pharmaceutically acceptable salts thereof as an active ingredient.

[Claim 16]

A method for the prevention of cell or tissue damage caused by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

[Claim 17]

A method for the treatment of cell or tissue damage caused by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

[Claim 18]

A method for the prevention of oxidative stress induced by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

Claims

[Claim 19]

A method for the treatment of oxidative stress induced by radiation containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

[Claim 20]

A radioprotective method containing the step of administering a pharmaceutically effective dose of hesperetin or the pharmaceutically acceptable salts thereof to a subject.

[Claim 2l]

A use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition for the prevention and treatment of cell or tissue damage caused by radiation. [Claim 22]

A use of hesperetin or the pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition for the prevention and treatment of oxidative stress induced by radiation. [Claim 23]

A use of hesperetin or the pharmaceutically acceptable salts thereof as a radioprotective agent.