FRACTION EXTRACT OF MELISSA OFFICINALIS LEAVES AND NOVEL PHARMACEUTICAL COMPOSITION INCLUDING SAME

Abstract

The present invention relates to a fraction extract of Melissa officinalis leaves, and a novel pharmaceutical composition and a food composition that include same.

Claims

1-28. (canceled)

29. A composition comprising: a fractional extract of Melissa leaf comprising caffeic acid, EDPA, RME and rosmarinic acid and a pharmaceutically acceptable carrier.

30. The composition of claim 29, wherein the fractional extract of Melissa leaf comprising 0.1 to 5% by weight of caffeic acid, 0.05 to 6% by weight of EDPA, 0.01 to 2% by weight of RME, and 5 to 50% by weight of rosmarinic acid, based on the total of a fractional extract of Melissa leaf.

31. The composition of claim 29, wherein the fractional extract of Melissa leaf is obtained by an extraction process comprising extracting and concentrating the Melissa leaf with 50% to 100% alcohol, suspending in water, and fractionating with ethyl acetate, and the fractional extract of Melissa leaf comprises 0.05 to 6% by weight of EDPA (Ethyl 2-(3,4-dihydroxyphenyl) acetate).

32. The composition of claim 29, wherein the composition is a pharmaceutical composition.

33. The composition of claim 29, wherein the composition is a food composition.

34. A method for treating non-alcoholic steatohepatitis, or a non-alcoholic fatty liver disease or an angiogenesis-related diseases or MMP (Matrix metalloproteinase)-mediated disease comprising: administering to a subject in need thereof a therapeutically effective amount of a fractional extract of Melissa leaf.

35. The method of claim 34, wherein the fractional extract of Melissa leaf comprises 0.1 to 5% by weight of caffeic acid, 0.05 to 6% by weight of EDPA, 0.01 to 2% by weight of RME, and 5 to 50% by weight of rosmarinic acid, based on the total of a fractional extract of Melissa leaf.

36. The method of claim 34, wherein the fractional extract of Melissa leaf is obtained by an extraction process comprising extracting and concentrating the Melissa leaf with 50% to 100% alcohol, suspending in water, and fractionating with ethyl acetate, and comprises 0.05 to 6% by weight of EDPA (Ethyl 2-(3,4-dihydroxyphenyl) acetate).

37. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is obesity.

38. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is age-related macular degeneration.

39. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is diabetic retinopathy, Sjogren's syndrome, or glaucoma.

40. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is psoriasis.

41. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is endometriosis.

42. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is cancer growth or cancer metastasis, wherein the cancer is any one of lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, stomach cancer, gastrointestinal cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or eye melanoma, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, Breast cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parasitic adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, childhood Solid tumor, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, contractile tumor, brainstem glioma and pituitary adenoma.

43. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is arthritis, wherein the arthritis is any one of osteoarthritis, degenerative arthritis, dissociative osteochondritis, joint ligament damage, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis.

44. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is inflammatory bowel disease.

45. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is Alzheimer's disease.

46. The method of claim 34, wherein the angiogenesis-related disease or MMP-mediated disease is arteriosclerosis or periodontal disease.

47. The method of claim 34, wherein the composition is a pharmaceutical composition.

48. The method of claim 34, wherein the composition is a food composition.

Description

DESCRIPTION OF DRAWINGS

[0439] FIG. 1 is the result of Picrosirius red staining to detect fibrosis.

[0440] FIG. 2 is a graph showing SMA deposition in the liver.

[0441] FIG. 3 is a graph showing the protein expression level of Col1A2, which is known as a protein associated with liver fibrosis.

[0442] FIG. 4 is a graph showing the protein expression level of TGF beta, which is known as a protein associated with liver fibrosis.

[0443] FIG. 5 is a graph showing the protein expression level of IL-6, which is known as a protein associated with liver fibrosis.

[0444] FIG. 6 is a graph showing the protein expression level of IL-10, which is known as embodiment a protein associated with liver fibrosis.

[0445] FIG. 7 is a graph showing the serum concentrations of AST and ALT.

[0446] FIG. 8 is a graph showing the quantification of the degree of fat accumulation in the liver.

[0447] FIG. 9 shows that single ingredients of rosmarinic acid, caffeic acid, RME and EDPA have angiogenesis inhibitory effects, showing the strong angiogenesis inhibitory effect of EDPA at the same concentration.

[0448] FIG. 10 confirms that, among compositions comprising EDPA, the angiogenesis inhibitory effect significantly increases as the content of EDPA increases.

[0449] FIG. 11 is a graph showing the change in body weight of high-fat diet-induced obese rats.

[0450] FIG. 12 is a graph showing the change in food intake in high-fat diet-induced obese rats.

[0451] FIG. 13 is a graph showing the change in the concentration of triglycerides in the blood of high-fat diet-induced obese rats.

[0452] FIG. 14 is a graph showing the change in the concentration of blood sugar in high-fat diet-induced obese rats.

[0453] FIG. 15 is a graph showing changes in blood total cholesterol concentration in high-fat diet-induced obese rats.

[0454] FIG. 16 is a graph showing the size of a choroidal neovascularization lesion in a laser-induced CNV model.

[0455] FIG. 17 is a graph showing the thickness of the epidermis of the excised ear tissue.

[0456] FIG. 18 is a graph confirming the volume of uterine lesions in the experimental model in which the fractional extract of Melissa leaf of the embodiment was administered to the treatment group and was not administered to the control group.

[0457] FIG. 19 is a graph showing the growth curve of tumor size in nude mice injected with DLD1 cells.

[0458] FIG. 20 is a graph showing the results of measuring and evaluating the formation of atherosclerotic plaques in the aorta in an ApoE-deficient mouse model.

[0459] FIG. 21 is a graph comparing the treatment effect on arthritis in a collagen-induced arthritis (CIA) model.

[0460] FIG. 22 is a graph measuring the weight bearing rate in an osteoarthritis model induced by MIA.

[0461] FIG. 23 is a graph measuring the disease activity index (DAI) in an animal model of inflammatory bowel disease induced by dextran sodium sulfate (DSS).

[0462] FIG. 24 is a graph showing reference memory during a water maze test when the fractional extract of Melissa leaf according to the embodiment was administered.

[0463] FIG. 25 is a graph showing the improvement rate of the probe test during the underwater maze test when the fractional extract of Melissa leaf according to the embodiment was administered.

[0464] FIG. 26 is a graph showing the RT-PCR result of analyzing the gene expression level of pro-collagen indicating tissue regeneration in a periodontal disease model induced by ligation.

[0465] FIG. 27 is a graph showing the results of testing the retinal blood vessel leakage effect in a rat model of diabetic retinopathy induced by streptozotocin.

[0466] FIG. 28 is a graph analyzing the amount of tear secretion using NOD/ShiLt which is an animal model of Sjogren's syndrome.

[0467] FIG. 29 is a graph showing the effect on lowering intraocular pressure in a hypertonic saline-induced scar glaucoma model.

BEST MODE FOR CARRYING OUT THE INVENTION

[0468] The terms used in the embodiments have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

[0469] Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application.

[0470] Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout the embodiments includes all lower numerical limitations as if the lower numerical limitation was expressly written. Every minimum numerical limitation given throughout the embodiments includes all higher numerical limitations as if the higher numerical limitation was expressly written. Any numerical limitation given throughout the embodiments shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation was expressly written.

[0471] Examples and manufacturing examples are presented to help the understanding of the present invention. The following examples and manufacturing examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples and manufacturing examples.

Examples

Example 1. Preparation of a Fractional Extract of Melissa Leaf

[0472] 400.4 kg of dried Melissa leaves (origin: Europe) were extracted using 4000 L of 75% (v/v) ethanol under reflux at 81° C. for 4 hours, and after 35 minutes of reflux extraction filtered with a 10 μm of cartridge filter for 55 minutes to obtain 3100 L of the first extract.

[0473] The Melissa leaf residue was extracted under reflux at 82° C. for 4 hours using 4000 L of 75 (v/v) % ethanol, and after 35 minutes of reflux extraction filtered with a 10 μm cartridge filter for 55 to 65 minutes to obtain 4100 L of the second extract.

[0474] After mixing the first and second extracts (a total of 7200 L of extract), concentration was performed to obtain 600 L of the first concentrate.

[0475] <The First Concentration Conditions>

[0476] Temperature: 56˜58° C.

[0477] Pressure: −0.066 to −0.070 MPa

[0478] Time: 9 hours

[0479] 600 L of the first concentrate was concentrated to obtain 250 L of the second concentrate.

[0480] <The Second Concentration Conditions>

[0481] Temperature: 56˜58° C.

[0482] Pressure: −0.063 to −0.065 MPa

[0483] Time: 5 hours and 50 minutes

[0484] 250 L of the second concentrate and 200 L of purified water were mixed to obtain a suspension (total 450 L), and 450 L of ethyl acetate were added to the suspension to obtain 900 L of a mixture.

[0485] 900 L of the mixture were stirred for 1 hour, and then left for 2 hours to separate the ethyl acetate layer (450 L) to obtain the first fraction.

[0486] Then, 450 L of ethyl acetate (ratio of mixture and ethyl acetate (v/v)=1:1) were added to the remaining suspension, followed by stirring for 1 hour, and then standing for 2 hours to separate the ethyl acetate layer (450 L) to obtain the second fraction.

[0487] After combining the first fraction and the second fraction (900 L), concentration was performed thereon to obtain 41 L (42.5 kg) of the third concentrate.

[0488] <The third concentration conditions>

[0489] Temperature: 58° C.

[0490] Pressure: −0.064 MPa

[0491] Time: 3 hours and 40 minutes

[0492] The third concentrate was collected in a stainless collector for 20 minutes (42.5 kg).

[0493] Thereafter, after complete drying at 80˜82° C. for 36 hours using a hot air dryer, the dried material (19.1 kg, 44.94% (w/w) of the concentrate) was put into a grinder and pulverized for 3 hours and 50 minutes to obtain a fractional extract of Melissa leaf (yield: 18.9 kg) which is an ethyl acetate fraction of ethanol extract of Melissa leaves.

Example 2. Ingredients of a Fractional Extract of Melissa Leaf

[0494] (1) Analytical Method

[0495] The active ingredients of a fractional extract of Melissa leaf were analyzed using High Performance Liquid Chromatography (HPLC) at the wavelength of 285 nm. The fractional extract of Melissa leaf prepared above was injected into a Zorbax Eclipse Plus C18 (150×4.6 mm) column and mobile phase A (5% aqueous formic acid solution) and mobile phase B (methanol) were delivered at a flow rate of 1 ml per minute according to the ratio shown in Table 1 below

TABLE-US-00001 TABLE 1 Time (min) Mobile Phase A Mobile Phase B 0 100 0 5 100 0 25 0 100 35 0 100 35.1 100 0 45 100 0

[0496] (2) Result

[0497] The fractional extract of Melissa leaf obtained according to Example 1 comprised caffeic acid, EDPA, RME and rosmarinic acid according to the analytical method above and their contents were as follows.

[0498] Caffeic acid: 0.96 wt %

[0499] EDPA: 0.71 wt %

[0500] RME: 0.11 wt %

[0501] Rosmarinic acid: 16.1 wt %

[0502] In addition, rutin was not shown in the HPLC results of the fractional extract of Melissa leaf obtained according to Example 1. Therefore, it was found that the fractional extract of Melissa leaf obtained according to Example 1 did not comprise rutin.

[0503] Experimental Examples

[0504] Experimental Example 1. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Non-Alcoholic Steatohepatitis in Methionine/Choline Deficiency High-Fat Diet Model

[0505] (1) Experimental Method

[0506] The therapeutic effect of the fractional extract of Melissa leaf according to the Example on non-alcoholic steatohepatitis was evaluated in a methionine/choline deficient high-fat diet animal model.

[0507] The study was conducted using C57BI/6 mice fed methionine/choline deficiency high-fat diet (MCD-HFD), which were divided into the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the control group administered with 0.5% CMC.

[0508] For the administration method, while feeding the MCD-HFD, the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 200 mg/kg, and to the control group 0.5% CMC was administered at the same time. The administration period was for 9 weeks. In the normal group, non-alcoholic steatohepatitis was not induced by feeding the normal diet.

[0509] To confirm the degree of fibrosis in the liver, Picrosirius red staining was performed on liver tissue. For picrosirius red staining, liver tissue samples fixed in 10% formalin were embedded in paraffin, cut into 5 μm sections, and the tissue sections were incubated in 0.5% thiosemicarbazide for 10 minutes. Then, it was stained with 0.1% Sirius Red F3B in saturated picric acid for 1 hour and then washed with 0.5% acetic acid solution. To observe myofibroblasts which are the marker of liver fibrosis, immunohistochemical tests were performed using an alpha-smooth muscle actin (alpha-SMA) antibody. Paraffin-embedded liver tissue was cut into 4 μm sections, attached to a slide glass treated with poly-L-lysine, deparaffinized, and sequentially immersed in ethanol. After 10 minutes of treatment in 3% H2O2/methanol solution to remove endogenous peroxidase, it was reacted with normal goat serum in a moisture chamber for 40 minutes to eliminate non-specific reactions, and then washed with a 0.01 M phosphate-buffered saline solution (PBS, pH 7.4) containing 0.05% non-fat dry milk and 0.3% Triton X-100. The primary antibody was reacted for 1 hour, respectively, and the secondary antibody was reacted for 60 minutes, followed by color development. Western blot was performed to confirm the protein expression levels of Col1A2, TGF beta, IL-6 and IL-10. Specifically, liver tissue was dissolved in a buffer containing a protease inhibitor cocktail, and then the protein was extracted. After the extracted protein was quantified using a BCA assay kit, it was mixed with SDS gel loading buffer, and boiled at 100° C. for 5 minutes for denaturation. Proteins electrophoresed on SDS-PAGE gel were transferred to a nitrocellulose membrane, and then the membrane was left in 5% skim milk for 1 hour to block non-specific protein binding. A general immunoblot was performed by treating antibodies of Col1A2, TGF beta, IL-6, IL-10 and beta-actin as primary antibodies, and then the intensity of the band finally appeared was measured with gel documentation software and graphed.

[0510] (2) Results

[0511] FIG. 1 is a result of Picrosirius red staining to detect fibrosis. Referring to FIG. 1, fibrosis induced by methionine/choline-deficient high-fat diet in non-alcoholic steatohepatitis animal model was inhibited in the treatment group to which the fractional extract of Melissa leaf according to the Example was orally administered.

[0512] FIG. 2 is a graph showing SMA expression in the liver. Referring to FIG. 2, fibrosis induced by methionine/choline-deficient high-fat diet in non-alcoholic steatohepatitis animal model was inhibited in the treatment group to which the fractional extract of Melissa leaf according to the Example was orally administered.

[0513] FIGS. 3 to 6 are graphs showing protein expression levels of Col1A2, TGF beta, IL-6 and IL-10, respectively which are known proteins associated with liver fibrosis. Referring to FIGS. 3 to 6, it was found that all of these proteins increased in the control group, and the increased expression levels of these proteins were inhibited by treatment with the fractional extract of Melissa leaf according to Example in the treatment group.

[0514] Experimental Example 2. Inhibitory Effect of a Fractional Extract of Melissa Leaf on Non-Alcoholic Fatty Liver Disease in High-Fat Diet Model

[0515] (1) Experimental Method

[0516] As experimental animals, thirty SD rats were randomly divided into three groups of ten rats each after acclimatization for one week.

[0517] For each of the three groups, a group fed a standard chow diet (purchased from Saeron Bio) was divided into the normal group, a group fed a high-fat diet (HFD) and administered with the fractional extract of Melissa leaf of the Example was divided into the treatment group, and a group fed a high-fat diet (HFD) and administered with 0.5% CMC was divided into the control group.

[0518] The normal group was fed a standard chow diet. The treatment group was fed a HFD instead of a standard chow diet, and while fed a HFD the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg. The same experiment was performed in the control group as the treatment group except that 0.5% CMC was administered instead of the fractional extract of Melissa leaf of the Example. The administration period was 10 weeks.

[0519] To measure the serum levels of AST and ALT of the normal group, the treatment group, and the control group, respectively blood samples were taken from the abdominal aorta by autopsy of the rats. The collected blood samples were centrifuged to obtain serum. Serum levels of AST and ALT were measured using an automatic biochemical analyzer.

[0520] Three rats were randomly selected from each of the normal group, the treatment group, and the control group to determine the degree of fat accumulation in the liver tissues of each of the normal group, the treatment group, and the control group. After preparing 4 slides stained with Oil red on the frozen liver tissue for each individual of the selected rats, the degree of fat accumulation in the liver tissue was quantified using an analysis program, ImageJ.

[0521] (2) Results

[0522] FIG. 7 is the measured value (unit: IU/L) of serum levels of AST and ALT for the normal group, the treatment group, and the control group. Referring to FIG. 7, it was confirmed fatty liver did not occur in the normal group as the normal group showed low serum levels of AST and ALT. The treatment group administered with the fractional extract of Melissa leaf according to the Example showed lower serum levels of AST and ALT than the control group confirming that fatty liver was inhibited compared with the control group.

[0523] FIG. 8 is a graph showing the quantification of the degree of fat accumulation for each of the treatment group and the control group using an analysis program, ImageJ. Referring to FIG. 8, it was confirmed that the degree of fat accumulation in the liver tissue was insignificant in the normal group. It was confirmed that the treatment group administered with the fractional extract of Melissa leaf according to the Example suppressed the degree of fat accumulation in the liver tissue compared with the control group administered with CMC.

[0524] Experimental Example 3. Inhibitory Effect on Angiogenesis

[0525] To confirm the inhibitory effect of the fractional extract of Melissa leaf according to the Example on angiogenesis, an experiment was conducted using the following method.

[0526] (1) Experimental Method

[0527] A 48-well plate was coated with 200 μl of 10% matrigel, and incubated at 4° C. After 4 hours the temperature was raised to 37° C., and 1 hour later, the test substances listed in Tables 2 and 3 below were added to a final concentration of 50 μg/ml, and then 5×10.sup.4 HUVECs (human umbilical vein endothelial cells) were added to each well, and pictures were taken the next day.

TABLE-US-00002 TABLE 2 Sample 1 DMSO 2 RME 3 Caffeic acid 4 Rosmarinic acid 5 EDPA

TABLE-US-00003 TABLE 3 Sample Content of EDPA 1 ALS-T20003 0.85% 2 ALS-T20006 0.66% 3 ALS-T20009 0.45%

[0528] (2) Results

[0529] 1) Effect of Single Ingredient (Rosmarinic Acid, Caffeic Acid, RME (Rosmarinic Acid Methyl Ester) and EDPA (Ethyl 2-(3,4-Dihydroxyphenyl Acetate)) on HUVEC Tube Formation, an Angiogenesis Inhibitory Activity Assay

[0530] As shown in FIG. 9, Rosmarinic acid, caffeic acid, RME and EDPA which are single ingredient comprised in the fractional extract of Melissa leaf had an angiogenesis inhibitory activities in HUVEC tube formation assay and EDPA showed the highest angiogenesis inhibitory activity at the same concentration by inhibiting tube formation the most.

[0531] 2) Angiogenesis Inhibitory Activity of a Composition Comprising EDPA

[0532] As shown in FIG. 10 and Table 4, when angiogenesis inhibitory activities were measured with fractional extracts of Melissa leaf having different EDPA contents (referring to the contents of Table 3), it was confirmed that the higher the EDPA content, the more angiogenesis inhibitory activity increased significantly.

TABLE-US-00004 TABLE 4 Concentration Inhibition of No. Sample (μg/ml) Tube Formation (%) 1 DMSO 0 2 ALS-T20003 50 90.6 3 ALS-T20006 50 28.7 4 ALS-T20009 50 14.1

[0533] Experimental Example 4. MMP Inhibitory Activity

[0534] An experiment was conducted to confirm the MMP (Matrix metalloproteinase) inhibitory activity of the fractional extract of Melissa leaf according to the Example with the following method.

[0535] (1) Experimental Method

[0536] The MMP inhibitory activities were measured using a spectrofluorometer (PerkinElmer LS50B) to determine whether the fractional extract of Melissa leaf has an inhibitory effect on MMPs. MMP-2 and MMP-9 were activated with 1 mM APMA (p-aminophenyl mercuric acetate) before use for activity measurement. As substrates for MMP-2 and MMP-9, fluorescent substrate for MMP-2/MMP-9 (Calbiochem) was used. As a control, 2 ml of a buffer solution [50 mM Tricine (pH 7.5), 10 mM CaCl.sub.2), 200 mM NaCl] containing 1 uM of the substrate was added to a 2 ml cuvette, and MMP-2, or −9 was added thereto. Fluorescence intensity was measured by a spectrofluorometer at 2-minute intervals for 20 minutes at room temperature using a 328 nm excitation wavelength and a 400 nm emission wavelength. In the case of the treatment group, each test substance listed in Table 3 was added to the buffer solution containing the substrate and MMP in the same manner as the control group to a final concentration of 20 ug/ml, and then fluorescence intensity was measured at 2-minute intervals for 20 minutes at room temperature.

[0537] (2) Results

TABLE-US-00005 TABLE 5 Concentration MMP-2 inhibition MMP-9 inhibition (20 μg/ml) (%) (%) ALS-T20003 78 89 ALS-T20006 54 62 ALS-T20009 48 53

[0538] Referring to Table 5, it was confirmed that the inhibitory activities of MMP-2 and MMP-9 significantly increased as the content of EDPA increased among the compositions containing EDPA.

[0539] Experimental Example 5. Anti-Obesity Effect in High-Fat Diet-Induced Obese Rats

[0540] (1) Experimental Method

[0541] The anti-obesity effect of the fractional extract of Melissa leaf according to the Example was evaluated in a high-fat diet-induced obese animal model.

[0542] Obesity was induced by feeding a high-fat diet (Saeron Bio Co., Ltd.) to a male Sprague-Dawley (SD) rat (JoongAng Experimental Animals Co., Ltd.), and the study was conducted by dividing into the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the control group administered with 0.5% carboxymethyl cellulose (CMC).

[0543] The control group and the treatment group were acclimatized to the environment with standard diet (Saeron Bio Co., Ltd.) for 1 week before feeding with an experimental high-fat diet, and then fed with an experimental diet for 10 weeks.

[0544] For the administration method, the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg, and 0.5% CMC was administered to the control group. Body weight and food intake were measured twice a week on the same day. For the measurement of food intake, a certain amount of food was filled in a powder feed box at 3 pm and the total weight is measured, and the weight of the reduced food was measured at 3 pm the next day.

[0545] This was calculated as the amount of food consumed for 24 hours, and was carried out constantly until the end of the test. Blood samples were collected at 5 and 10 weeks from the test start date. Blood was collected after anesthesia with ether by ocular bleed method in which a heparin-treated glass capillary tube was inserted into the orbital venous plexus of SD rats. Total cholesterol, triglyceride and insulin concentration were measured from the collected blood.

[0546] (2) Results

[0547] FIG. 11 is a graph showing the change in body weight of high-fat diet-induced obese rats. Referring to FIG. 11, continuous weight gain was observed in the control group, and this weight gain continued until the 10th week of the end of the experiment. But in the treatment group the body weight was significantly reduced in high-fat diet-induced obese rats.

[0548] FIG. 12 is a graph showing changes in food intake in high-fat diet-induced obese rats. Referring to FIG. 12, no significant change in food intake was observed in the control group and the treatment group.

[0549] FIG. 13 is a graph showing changes in triglyceride concentrations in the blood of high-fat diet-induced obese rats. Referring to FIG. 13, in the treatment group blood triglycerides were significantly reduced in high-fat diet-induced obese rats. In particular, the levels of triglycerides in the blood did not increase in the experimental group even after 10 weeks of ingestion of a high-fat diet.

[0550] FIG. 14 is a graph showing changes in the concentration of blood glucose in high-fat diet-induced obese rats. Referring to FIG. 14, in the treatment group blood glucose was significantly reduced in high-fat diet-induced obese rats.

[0551] FIG. 15 is a graph showing changes in blood concentration of total cholesterol in high-fat diet-induced obese rats. Referring to FIG. 15, in the treatment group total cholesterol was significantly reduced in high-fat diet-induced obese rats.

[0552] Experimental Example 6. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Age-Related Macular Degeneration in Laser-Induced Choroidal Neovascularization (CNV) Model

[0553] (1) Experimental Method

[0554] The treatment effect of the fractional extract of Melissa leaf according to Example on age-related macular degeneration was evaluated in a laser-induced choroidal neovascularization (CNV) animal model.

[0555] A laser-induced CNV model was prepared with 6-week-old C57BL/6 mice by damaging the retinal optic nerve area using a diode green laser (532 nm, 150 mW, 0.1 sec).

[0556] In the laser-induced CNV model, the study was conducted by dividing into the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the control group administered with 0.5% CMC.

[0557] The fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg two days before laser treatment, and 0.5% CMC was administered to the control group. The dosing period continued for 10 days after laser treatment.

[0558] Ten days after laser treatment, the retina-choroid flat mount was performed to confirm the effect of reducing the size of CNV lesions. Mice were anesthetized and retro-orbital injection of 25 mg/ml of FITC-dextran was performed. After 30 minutes, the mice were euthanized, the eyes were removed, fixed with 10% formalin, and the cornea and lens were removed and flat mounted on a cover glass. The size of the macular degeneration lesion stained with FITC-dextran was measured.

[0559] (2) Results

[0560] FIG. 16 is a graph showing the size of a macular degeneration lesion in a laser-induced CNV model. Referring to FIG. 16, it was shown that the size of the macular degeneration lesion by laser treatment increased in the control group, and the size of the neovascularization was significantly reduced in the treatment group in the laser-induced choroidal neovascularization model.

[0561] Experimental Example 7. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Psoriasis in IL-23 Induced Psoriasis Animal Model

[0562] (1) Experimental method

[0563] The effect of the fractional extract of Melissa leaf according to the Example on psoriasis was evaluated in an IL-23 induced psoriasis animal model according to Ma's method [J. Clin. Cell Immunol. 4:6. (2013)].

[0564] An IL-23 induced psoriasis animal model was prepared in which psoriasis was induced by intradermal injection of 500 ng of IL-23 to the ears of C57BL/6 mice (6 weeks old, Orient Bio) every other day for 16 days.

[0565] The study was conducted in the IL-23 induced psoriasis animal model by dividing into the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the control group administered with 0.5% CMC, and the PBS control group treated with PBS instead of IL-23

[0566] For the administration method, the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg, and 0.5% CMC was administered to the control group. The oral administration started from one day before the IL-23 injection and continued for a total of 16 days, and the experiment was ended on the 17th day after the start of administration.

[0567] After the experiment was finished, the ear thickness of the mice was measured, and the results were shown in FIG. 17.

[0568] (2) Results

[0569] FIG. 17 is a graph showing the thickness of the epidermis of the excised ear tissue. Referring to FIG. 17, in the treatment group, the epidermal thickness was significantly reduced in an IL-23 induced psoriasis animal model.

[0570] Experimental Example 8. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Endometrial Growth in Endometrial Autograft Model

[0571] (1) Experimental Method

[0572] After housing SD rats, in order to match the female estrus cycle, bedding soaked in male urine was placed in a female cage every 5 days, and vaginal cytology was performed daily for a week before surgery to confirm the menstrual cycle.

[0573] Before surgery, the surgical site was shaved with an electric razor and disinfected with 70% ethanol. After making an incision about 1 from 0.5 cm to 1.0 cm from the vaginal opening,

[0574] the left uterus was exposed. Both parts of the exposed left uterus at a position of 6 cm to 8 cm were tightly tied with 5-0 sutures. Cut the uterus between the tied positions and place it in a sterile glass Petri dish with 100 μL of PBS containing penicillin (100U/ml) and streptomycin (100 μg/ml). After removing the fat, the uterus was incised vertically, and cut into three pieces of 2 mm size.

[0575] After finding and pulling out the intestines from the incision site, the intestines were spread on pre-wetted gauze, where three arteries between the mesentery were identified and the uterine fragment was gently sutured. Then, the operated intestine was inserted into the abdominal cavity, the abdominal wall was sewn using a suture, and the skin was closed with an autoclip. After surgery, heat was applied to SD rats to prevent body temperature drop. The surgical site was disinfected with povidone, and acetaminophen, an analgesic, was mixed with drinking water at 2 mg/ml and administered.

[0576] The auto clip was removed 7 days after the operation, and the fractional extract of Melissa leaf according to the Example was orally administered to the treatment group (n=5) once a day for 4 weeks. The administered dose of the fractional extract of Melissa leaf was 100 mg/kg. Group separation was performed so that the average size of uterine lesions in each group was the same.

[0577] 0.5% CMC was orally administered to the control group (n=5).

[0578] Four weeks after surgery, SD rats were euthanized and the peritoneum was incised. Then, endometriotic lesions transplanted to the mesentery were isolated and the size and weight were measured.

[0579] (2) Results

[0580] FIG. 18 is a graph confirming the volume of endometriotic lesions transplanted to the mesentery in the experimental model wherein the fractional extract of Melissa leaf of the Example was administered to the treatment group and was not administered to the control group. In FIG. 18, the average volumes of the endometriotic lesions of the treatment group and the control group were described.

[0581] Table 6 showed the size and volume of endometriotic lesions transplanted into the mesentery of each of the treatment group and the control group.

TABLE-US-00006 TABLE 6 Width (mm) Length (mm) Volume (mm.sup.3) Treatment SD rat 1 1.82 1.51 2.16 group SD rat 2 1.75 1.53 2.13 SD rat 3 1.59 1.39 1.60 SD rat 4 1.63 1.28 1.39 SD rat 5 1.99 1.16 1.39 Control SD rat 1 2.56 2.01 5.38 group SD rat 2 2.39 2.06 5.27 SD rat 3 2.47 1.98 5.04 SD rat 4 2.27 1.82 3.91 SD rat 5 2.45 1.89 4.55

[0582] Referring to Table 6, the average volume of the endometriotic lesions transplanted into the mesentery of the treatment group was about 1.73 mm.sup.3, and the standard deviation was about 0.38. The average volume of the endometriotic lesions of the control group was about 4.83 mm.sup.3 and the standard deviation was about 0.61. Referring to FIG. 18 and Table 6, it was confirmed that the volume of the endometriotic lesions of the treatment group was significantly smaller than that of the control group. As a result of this it could be confirmed that the fractional extract of Melissa leaf according to the Example has an excellent inhibitory effect on endometrial tissue growth.

[0583] Experimental Example 9. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Cancer Growth in Tumorigenicity Model in Nude Mice

[0584] (1) Experimental Method

[0585] The anticancer effect of the fractional extract of Melissa leaf according to the Example was evaluated in a tumorigenicity model in nude mice.

[0586] Colorectal cancer cells (DLD1) were cultured in RPMI-1640 medium, and 6-week-old male nude mice (Orient Bio) were housed in an aseptic facility. The cultured DLD1 cells were resuspended in DMEM medium, and 5×106 cells were injected subcutaneously into the right flank of nude mice, which were divided into the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the control group administered with 0.5% CMC.

[0587] For the administration method, the fractional extract of Melissa leaf was orally administered once a day at a dose of 100 mg/kg from the day of tumor injection, and 0.5% CMC was administered to the control group. The administration period was 28 days, and the tumor size was measured every 3 days with a caliper.

[0588] (2) Results

[0589] FIG. 19 is a graph showing the growth curve of tumor size in nude mice injected with DLD1 cells. In the control rats, tumors grew exponentially until the 28th day, but in the treatment group to which the fractional extract of Melissa leaf according to the Example was orally administered, the tumors significantly decreased in size, thereby exhibiting a strong anticancer effect.

[0590] Experimental Example 10. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Arteriosclerosis in Apolipoprotein E (ApoE)-Deficient Mouse Model [0591] (1) Experimental Method

[0592] The anti-atherosclerotic effect of the fractional extract of Melissa leaf according to the Example was evaluated in an ApoE-deficient mouse model. Mice in which atherosclerosis was induced by a high-fat diet for 16 weeks were selected, and those in which atherosclerosis was not induced by a standard diet instead of a high-fat diet were classified as normal group.

[0593] The control group was administered with 0.5% CMC, and the treatment group was administered with the fractional extract of Melissa leaf according to the Example.

[0594] Mice were given ad libitum access to food and water for 16 weeks, and the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg to the treatment group of mice induced arteriosclerosis by a high-fat diet for 16 weeks, and 0.5% CMC was administered to the control group.

[0595] After 16 weeks, mice were sacrificed, resection from the heart and ascending aorta to the thoracic aorta was performed and fixed in 10% neutral buffered formalin solution. After the fixed tissue is trimmed with a razor, it was embedded in a frozen tissue embedding agent (OCT compound) and frozen in a deep freezer. Slides were prepared by cutting aortic arch into 10 μm thick with a cryostat microtome. For fat staining, slides were immersed in distilled water and treated with absolute propylene glycol for 1 minute, stained in an oil-red solution for 16 hours, and then treated in 85% propylene glycol for 2 minutes. Slides were washed with distilled water, sealed with an aqueous encapsulant, and observed under an optical microscope.

[0596] (2) Results

[0597] FIG. 20 is a graph showing the results of measuring and evaluating the formation of atherosclerotic plaques in the aorta in an ApoE-deficient mouse model. It was confirmed that the formation of atherosclerotic plaques increased in the control group compared to the normal group, and the increase in atherosclerotic plaques was significantly inhibited in the treatment group.

[0598] Experimental Example 11. Efficacy of the fractional extract of Melissa leaf in collagen-induced arthritis animal model [0599] (1) Experimental method

[0600] The efficacy of the fractional extract of Melissa leaf according to the Example was evaluated in a collagen-induced arthritis model.

[0601] The collagen-induced arthritis (CIA) animal model was created by inducing an immune response by injecting collagen, which is considered to be the cause of rheumatoid arthritis.

[0602] 4 mg of chicken type II collagen was mixed with 1 ml of 100 mM acetic acid and dissolved at 4° C. for one day. This was mixed with 1 ml of Freund's complete adjuvant containing 4 mg/ml of Mycobacterium tuberculosis, emulsified, and then 150 μl (300 μg) was intradermally injected into the tail of 6-week-old Lewis rats for immunization. On the 7th day after the primary immunization, a secondary immune (boosting) reaction was induced by intradermal injection of chicken type II collagen again. Arthritis symptoms were confirmed after 1-2 weeks after secondary immunization.

[0603] The control group was administered with 0.5% CMC, and the treatment group was administered with the fractional extract of Melissa leaf according to the Example.

[0604] The fractional extract of Melissa leaf according to the Example was orally administered once a day for 47 days from the next day after induction of the secondary immune response at a dose of 100 mg/kg, and 0.5% CMC was administered to the control group.

[0605] In order to verify the therapeutic effect, the degree of swelling and erythema of the joints and nodes of each rat's paws were observed, and clinical scores were calculated according to the scoring index described in Table 7 below.

TABLE-US-00007 TABLE 7 Score Clinical observations 0 no evidence of erythema and swelling 1 erythema and mild swelling confined to the tarsals or ankle joint 2 erythema and mild swelling extending from the ankle to the tarsals 3 erythema and moderate swelling extending from the ankle to the metatarsal joints 4 erythema and severe swelling encompassing the ankle, foot, and digits

[0606] (2) Results

[0607] FIG. 21 is a graph comparing the treatment efficacy of arthritis in a collagen-induced arthritis (CIA) model. Referring to FIG. 21, arthritis symptoms appeared from about 20 days after the CIA-induced immune response. The treatment group showed a significant therapeutic effect in a collagen-induced arthritis model.

[0608] Experimental Example 12. Efficacy of the Fractional Extract of Melissa Leaf in MIA (Monosodium Iodoacetate)-Induced Osteoarthritis Animal Model

[0609] (1) Experimental Method

[0610] The efficacy of the fractional extract of Melissa leaf according to the Example was evaluated in a MIA-induced osteoarthritis model.

[0611] Osteoarthritis was induced by injection of 50 μl of MIA diluted to a concentration of 60 mg/ml with 0.9% saline into the joint cavity of right hind limb of 7-week-old SD rats and selected.

[0612] 0.5% CMC was administered to the control group, and the fractional extract of Melissa leaf according to the Example was administered to the treatment group.

[0613] For the administration method, the fractional extract of Melissa leaf was orally administered once a day for 21 days at a dose of 100 mg/kg, and 0.5% CMC was administered to the control group. Weight bearing rate was measured at intervals of 7 days after administration. The group that did not induce osteoarthritis was classified as the normal group.

[0614] Hind paw weight bearing was measured using an Incapacitance tester. In the holder of the tester, the osteoarthritis-induced rat stood on the normal hind paw without MIA treatment due to pain, so the weight of both paws was out of balance, and the weight of the paw treated with MIA was relatively light compared to the weight of the normal paw. When measuring the weight of the paws, the weight (g) of both paws was measured in the state that the SD rat's belly did not touch the sensor of the device.

[0615] Using the measured weight of the paw, the weight-bearing rate (%) was calculated by Equation 1 below.

Weight bearing rate (%)=[weight of osteoarthritis-induced hind limb/(weight of hind limb of both paws)]×100  [Equation 1]

[0616] (2) Results

[0617] FIG. 22 is a graph measuring the weight bearing rate in the osteoarthritis model induced by MIA. Referring to FIG. 22, it was confirmed that the weight-bearing rate (%) of the control group decreased statistically significantly compared with the normal group as the period elapsed. In contrast, the treatment group showed an excellent weight-bearing effect (therapeutic effect of osteoarthritis) by significantly increasing the reduced weight-bearing rate.

[0618] Experimental Example 13. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Inflammatory Bowel Disease Induced by Dextran Sodium Sulfate (DSS)

[0619] (1) Experimental Method

[0620] The therapeutic effect of the fractional extract of Melissa leaf according to the Example on ulcerative colitis was evaluated in an animal model induced by sodium dextran sulfate.

[0621] Seven-week-old C57BL/6J mice (Central Lab. Animal Inc.) were given ad libitum access to food and water and acclimatized for one week, and then randomly divided into the control group, the normal group, and the treatment group.

[0622] The fractional extract of Melissa leaf according to the Example was orally administered to the treatment group once a day, at a dose of 200 mg/kg for 11 days after group separation and 0.5% CMC was administered to the control group. Inflammatory bowel disease was induced by adding 3% DSS in drinking water instead of water from the third day of group separation. On the other hand, in the normal group, inflammatory bowel disease was not induced because DSS was not administered.

[0623] During the experimental period, the appearance of stool was observed by inducing defecation of mice once a day, and the presence or absence of occult blood was observed using coulter hemoccult single slides, and the scores were indicated according to the criteria in Table 8 below.

TABLE-US-00008 TABLE 8 Score Stool consistency Stool color 0 Normal Normal colored stool 1 Soft but maintains morphology Brown stool 2 Soft Reddish stool 3 Very soft Bloody stool 4 Diarrhea Gross bleeding

DAI(disease activity index)=stool concentration+stool color  [Formula 2] [0624] (2) Results

[0625] FIG. 23 is a graph measuring the disease activity index (DAI) in an animal model of inflammatory bowel disease induced by DSS. At this time, the DAI of the normal group represents 0. As a result of measuring the DAI of the rats with ulcerative colitis induced by DSS, the control group showed high DAI, whereas the treatment group showed a remarkable therapeutic effect with low DAI score.

[0626] Experimental Example 14. The Efficacy of Improving Cognitive Function in Scopolamine-Induced Dementia Animal Model

[0627] (1) Experimental Method

[0628] For cognitive function test, five 9-week-old male ICR mice were used in each group. The treatment group was administered with the fractional extract of Melissa leaf, and the control group was administered with physiological saline for 2 weeks.

[0629] The treatment group was orally administered at a dose of 200 mg/kg seven times a week for two weeks and 60 minutes before the start of the test, scopolamine was orally administered to treatment group and control group, respectively at 1 mg/kg to induce memory impairment, and a water maze test was conducted.

[0630] The reference memory test during the water maze test was conducted after acclimatization by allowing them to swim freely in a water tank without a platform for 60 seconds one day before the start of the test. The reference memory test was performed by measuring the time of escape latency (unit: seconds) to find a platform submerged in water, 4 to 5 times a day for 5 days, and the maximum allowable time was limited to 60 seconds. If the location of the platform was not found until the second day of the test, mice were guided to find the platform within the time limit of 60 seconds and when they climbed on the platform, they were allowed to stay there for 10 seconds.

[0631] 24 hours after the end of the reference memory test, a probe test was performed by removing the platform from the water tank and allowing them to swim freely for 60 seconds to measure the length of time they stayed at the location where the platform was.

[0632] The reference memory test was performed for 5 days after administration of the fractional extract of Melissa leaf according to the Example, and the average of time to find the platform for the treatment group and the control group was compared.

[0633] (2) Results

[0634] Referring to FIG. 24, when the fractional extract of Melissa leaf according to the Example was administered, the effect on improving cognitive function was confirmed (37.2±2.1).

[0635] In the probe test conducted after the reference memory test was completed, the average time of staying on the platform in the control group was compared with the average staying time of the treatment group administered with the fractional extract of Melissa leaf according to the Example, and the improvement rate was expressed.

Probe test improvement rate=(tA−ts)/ts X100(%)  [Formula 3]

[0636] The ts is the average time that the control group stayed at the platform, and to is the average time the treatment group administered with the fractional extract of Melissa leaf according to the Example stayed at the platform.

[0637] Referring to FIG. 25, also in the probe test the treatment group stayed on the platform for a longer time than the control group, and the treatment group (88.3±9.9%) showed an excellent improvement rate compared with the control group.

[0638] Experimental Example 15. Inhibitory Effect on Periodontal Disease in Ligature-Induced Periodontal Disease Model

[0639] (1) Experimental Method

[0640] 7-week-old male SD rats were used as the experimental animals, which were given ad libitum access to general lab animal food and water and after acclimatization for one week they were used in the experiment.

[0641] Periodontal disease was induced by ligating the mandibular first molar with sterile sutures (3-0, nylon thread) after general anesthesia. After confirming only the cervical part, rats that were not ligated were classified as the normal group

[0642] Among the group with periodontal disease induced, the fractional extract of Melissa leaf according to the Example was administered to the treatment group.

[0643] The treatment group was orally administered once a day at a dose of 100 mg/kg and the control group was administered with 0.5% CMC.

[0644] After sacrificing the experimental animals, the surrounding tissues of the excised mandible were removed, and then the gene expression level of procollagen indicating tissue regeneration was confirmed.

[0645] In periodontal disease, regeneration and recovery of periodontal tissue made of collagen is important. The gene expression level of procollagen from the gums was confirmed through RT-PCR.

[0646] cDNA was synthesized using RT-PCR (reverse transcriptase-polymerase chain reaction) from 10 μg of RNA obtained by pulverizing the extracted gum tissue with a homogenizer using the Trizol method. The nucleotide sequences of the PCR primers for each gene are shown in Table 9 below.

TABLE-US-00009 TABLE 9 Primer sequences Gene Forward primer Reverse primer GAPDH ggc atg gac tgt ggt cat ga ttc acc acc atg gag aag gc Pro-collagen tct act ggc gaa acc tgt atc cg caa gga agg gca ggc gtg at

[0647] (2) Results

[0648] FIG. 26 is a graph showing the RT-PCR result of analyzing the gene expression level of pro-collagen indicating tissue regeneration in a periodontal disease model induced by ligation. Referring to FIG. 26, the administration of the fractional extract of Melissa leaf according to the Example increased the expression of pro-collagen to regenerate the gum tissue effectively, thereby showing a significant therapeutic effect on periodontal disease.

[0649] Experimental Example 16. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Diabetic Retinopathy in Streptozotocin (STZ)-Induced Diabetic Retinopathy Rat Model

[0650] (1) Experimental Method

[0651] The therapeutic effect of the fractional extract of Melissa leaf according to the Example was evaluated in a streptozotocin-induced diabetic retinopathy rat model.

[0652] A streptozotocin solution (100 mM) dissolved in citrate buffer (100 mM, pH 4.5) was injected into the abdominal cavity of rats at 150 mg/kg, and 10% sucrose was sufficiently supplied to prevent hypoglycemic shock. After 2 days, blood glucose level was measured using a blood glucose meter, and a diabetic animal in which non-fasting blood glucose was maintained over 300 mg/dl within 1 to 2 weeks were used.

[0653] Among the diabetes-induced rats, 0.5% CMC was administered to the control group, and the fractional extract of Melissa leaf according to the Example was administered to the treatment group, and diabetes was not induced in the normal group.

[0654] For the administration method, diabetes-induced rats were selected and the fractional extract of Melissa leaf according to the Example was orally administered once a day at a dose of 100 mg/kg for 16 weeks from 2 weeks after streptozotocin administration, and 0.5% CMC was administered to the control group. After 16 weeks to analyze retinal vascular leakage quantitatively, 1.25 mg of 500-kDa FITC-dextran (Sigma-Aldrich) was injected into the left ventricle of a rat, and stained by circulating blood for 5 minutes. Eyes were enucleated and immediately fixed with 4% paraformaldehyde for 45 minutes. The retina was incised from the fixed eyeball, cut in the shape of a Maltese cross, and the cut retina was placed on a slide glass, and then observed using a confocal microscope. Retinal vascular leakage was quantified by measuring the strength of FITC-dextran exuding from the entire retinal tissue.

[0655] (2) Results

[0656] FIG. 27 is a result of testing the inhibitory effect on retinal vascular leakage in the streptozotocin-induced diabetic retinopathy rat model. Referring to FIG. 27, it was confirmed that retinal vascular leakage was high in the control group, and in the treatment group retinal vascular leakage was significantly inhibited in the streptozotocin-induced diabetic retinopathy rat model.

[0657] Experimental Example 17. Efficacy of the Fractional Extract of Melissa Leaf in Sjogren's Syndrome Animal Model

[0658] (1) Experimental Method

[0659] Experiments were performed using NOD/ShiLt mice, an animal model of Sjogren's syndrome.

[0660] In NOD/ShiLt, an animal model of Sjogren's syndrome, 0.5% CMC was administered to the control group, and the fractional extract of Melissa leaf according to the Example was administered to the treatment group.

[0661] For the administration method, the tear secretion of the mice was analyzed after administering the fractional extract of Melissa leaf at 100 mg/kg for 12 weeks. After anesthetizing NOD/ShiLt mice with isoflurane, the tear secretion was measured by using cotton wool soaked in phenol red according to the method of Zoukhri et al. (Exp Eye Res. 2007; 84:894-904).

[0662] (2) Results

[0663] FIG. 28 is a graph analyzing tear secretion when the fractional extract of Melissa leaf according to the Example was administered using NOD/ShiLt mice, an animal model of Sjogren's syndrome. Referring to FIG. 28, tear secretion was significantly increased in the treatment group.

[0664] With the above result, it was confirmed that Sjogren's syndrome was improved by administering the fractional extract of Melissa leaf according to the Example to increase tear secretion.

[0665] Experimental Example 18. Inhibitory Effect of the Fractional Extract of Melissa Leaf on Glaucoma in a Hypertonic Saline-Induced Scar Glaucoma Model

[0666] (1) Experimental Method

[0667] The efficacy of the fractional extract of Melissa leaf according to the Example was evaluated in a hypertonic saline-induced scar glaucoma model.

[0668] Rats were anesthetized by injecting ketamine-xylazine intraperitoneally, and injected with 50 μl of 1.8 M hypertonic saline into the episcleral veins of the left eye to induce scar tissue in the trabecular meshwork, which induced elevation of intraocular pressure due to resistance of the aqueous humor outflow pathway. The right eye was used as a control, and intraocular pressures was measured in the left and right eyes before glaucoma induction and at intervals of 2 days after glaucoma induction for 2 weeks.

[0669] After selecting the rats induced with glaucoma, 0.5% CMC was administered to the control group, and the fractional extract of Melissa leaf according to the Example was administered to the treatment group, and glaucoma was not induced in the normal group.

[0670] For the administration method, the fractional extract of Melissa leaf according to the Example was orally administered to treatment group once a day for 2 weeks from the glaucoma induction date at a dose of 100 mg/kg, and 0.5% CMC was administered to the control group.

[0671] (2) Results

[0672] FIG. 29 is a graph showing that the fractional extract of Melissa leaf according to the Example reduced intraocular pressure in a scar glaucoma model induced by hypertonic saline. Referring to FIG. 29, it was confirmed that in the control group, the intraocular pressure reached the maximum value on the 6th day after the hypertonic saline injection and continued to be maintained, and in the treatment group, the elevated intraocular pressure was significantly alleviated in a glaucoma model with scar in the trabecular meshwork induced by hypertonic saline.