A fractional extract of Melissa officinalis leaf and a novel pharmaceutical composition containing the same.

Abstract

The present invention provides compositions for the prevention or improvement of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease, as well as angiogenesis-related diseases / MMP-mediated diseases, such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma. [Solution] A food composition is provided comprising a fractional extract of Melissa leaf containing caffeic acid, EDPA (ethyl 2-(3,4-dihydroxyphenyl) acetate), RME (rosmarinic acid methyl ester), and rosmarinic acid.

Description

Technical Field

[0001] The present invention relates to a fractionated extract of Melissa officinalis leaves and novel pharmaceutical and food compositions containing the same as an active ingredient.

Background Art

[0002] Melissa officinalis is a perennial plant belonging to the Lamiaceae family and is also known affectionately as lemon balm.

[0003] The extract of Melissa officinalis leaves contains flavonoids, triterpenic acids, volatile oils, glycosides of alcohol compounds and phenolic compounds, and coffee acid derivatives. In particular, the flavonoids contained in Melissa officinalis leaves are sinaroside, cosmosiin, rhamnocitrin, isoquercitrin, etc., and the triterpenic acid is ursolic acid. The extract of Melissa officinalis leaves contains hydroxycinnamic acid derivatives, such as rosmarinic acid (one of the non-volatile components that has recently attracted attention), and volatile oils such as geraniol, neral, citronellal, and eugenol.

[0004] Non-alcoholic steatohepatitis (NASH) is a disease in which fat accumulates in the liver, causing ballooning and inflammation of hepatocytes, followed by fibrosis that can lead to cirrhosis, and in some cases, complications such as liver cancer.

[0005] The number of patients with NASH is rapidly increasing, but the etiology of the disease is not clear, and various causes are involved, which makes treatment by managing one mechanism or cause difficult, and there are no approved drugs.

[0006] NASH progresses chronically, so generally there are no specific symptoms, but the liver function gradually deteriorates, for example, there is an increase in the activity of alkaline phosphatase (ALP) or aminotransferase, which are measurements of liver function.

[0007] If left untreated, non-alcoholic steatohepatitis (NASH) progresses to liver fibrosis or cirrhosis, leading to a poor prognosis. Therefore, to prevent NASH from progressing to cirrhosis, it is necessary to suppress fat accumulation in the liver, as well as additional inflammation and fibrosis; however, no such treatment has been suggested. Consequently, there is a need to develop effective treatments for NASH.

[0008] Non-alcoholic fatty liver disease (NAFLD) refers to a condition in which triglycerides accumulate excessively in the liver, regardless of alcohol consumption. Simple fatty liver is defined as NAFLD if the triglyceride content in the liver exceeds the top 95% of healthy, lean individuals, or if the percentage of triglyceride particles in the cytoplasm of liver cells exceeds 5%. NAFLD is reported to occur in 10–24% of the general population and 58–74% of obese individuals.

[0009] Angiogenesis is the process by which new capillaries are formed from existing microvessels. Normal angiogenesis occurs during embryonic development, tissue regeneration and wound healing, and during corpus luteum development, a cyclical change in the female reproductive system, and in these cases it is tightly regulated. [Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int Rev Exp Pathol 16, 207 - 248 (1976)].

[0010] In adults, vascular endothelial cells proliferate very slowly and divide relatively poorly compared to other cell types. However, angiogenesis in adults is induced in a manner dependent on stimulation by angiogenic stimulants, the release of pro-angiogenic cytokines from inflammatory cells, and the activation of hydrolytic enzymes that release angiogenic mediators sequestered within the extracellular matrix.

[0011] Generally, the process of angiogenesis involves the breakdown of the vascular basement membrane by proteases, the migration of vascular endothelial cells, and lumen formation through the proliferation and differentiation of endothelial cells. One of the major phenomena in the process of angiogenesis is the enzymatic breakdown of the basement membrane surrounding the blood vessels, and the most important enzymes for matrix degradation belong to the family of matrix metalloproteinases (MMPs).

[0012] Pathological angiogenesis is known to occur due to dysfunction in the regulation of angiogenesis or high activation of MMPs (important enzymes in angiogenesis), and it is associated with many diseases (References: Polverini PJ, Critical Reviews in Oral Biology, 6(3), 1995, 230-247; Arup Das, et al., Progress in Retinal and Eye Research, 22, 2003, 721-748; Nick Di Girolamo, et al., IOVS, Vol. 42, No.9, August 2001, 1963-1968; Patricia Lee, et al., Survey of ophthalmology, vol 43, No. 3, Nov-Dec 1998, 245-269; DB Holland, et al., British Journal of Dermatology, 150, 2004). 72-81; Anthony H Vagnucci Jr, et al., The Lancet, vol 361, Feb. 15, 2003, 605-608; Berislav V. Zlokovic, Trends in Neuroscience, Vol. 28, No.4, April 2005, 202-208; Jaap G. Neels, et al., The FASEB Journal express article 10. 1096 / fj.03-1101fje. Published online April 14, 2004; DL Crandall, et al., Microcirculation, 4, 1997, 211-232; G. Voros, et al., Endocrinology, 146, 2005, 4545-4554; MA Rupnick, et al., PNAS, 99, 2002, 10730-10735; E. Brakenhielm, et al., Circ. Res., 94, 2004, 1579-1588; HR Lijnen, et al., Arterioscler Thromb Vasc Biol., 22, 2002, 374-379; D. Demeulemeester, et al., Biochem. Biophys. Res. Commun., 329, 2005, 105-110). .

[0013] An example of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease is obesity, in which excess fat accumulates throughout the body due to an imbalance in energy intake and expenditure, which can lead to various health problems. The cause is likely lifestyle factors, such as excessive nutrient intake and lack of physical activity, and in rare cases, it can be a secondary consequence of drug use or other illnesses.

[0014] According to 2014 World Health Organization (WHO) statistics, 1.9 billion adults over the age of 18 were overweight, and of those, 600 million were obese worldwide. Obesity is one of the leading factors that increase the incidence of cardiovascular disease, type 2 diabetes, and several types of cancer, and it leads to more than 2.8 million deaths each year attributable to being overweight or obese.

[0015] As people's living standards improve, obesity has become a global social problem. According to the 2015 National Health and Nutrition Survey, the average fat intake of Koreans increased by only 5.9g over the past decade, and the obesity rate increased by only 1.9% compared to 2005. In particular, the obesity rate among men rose significantly to 39.7%, and the prevalence of hypercholesterolemia increased by 9.9% to 17.9%.

[0016] Several drugs have been developed to improve obesity, and these can be broadly classified into appetite suppressants and fat absorption inhibitors based on their mechanisms of action. However, drugs that suppress appetite, such as phentermine and diethylpropion, have side effects such as increased blood pressure, dizziness, headache, tremor, and dry mouth. In the case of orlistat (a fat absorption inhibitor), the absorption of fat-soluble vitamins is inhibited, and it has side effects such as steatorrhea, fat excretion, frequent bowel movements, and fecal incontinence.

[0017] Therefore, there is a need for the development of safer drugs with fewer side effects to prevent or treat obesity.

[0018] Macular degeneration (an example of neovascularization-related disease or matrix metalloproteinase (MMP)-mediated disease) is a disease that causes visual impairment due to degeneration of the macula, the part of the eye that receives light most clearly and accurately and has a very high density of photoreceptor cells. Macular degeneration is one of the three main causes of blindness, along with glaucoma and diabetic retinopathy. The biggest cause of macular degeneration is age, while other causes include family history, race, and smoking. When the macula is damaged, the eye loses the ability to recognize details, such as small print, facial features, or small objects.

[0019] There are two types of macular degeneration: non-exudative (dry) macular degeneration and exudative (wet) macular degeneration, with 90% of people with macular degeneration having the dry form. In dry macular degeneration, waste products accumulate under the macula, forming yellow deposits called drusen. The presence of drusen interferes with blood flow to the retina, especially the macula. Reduced blood flow leads to decreased nutrient supply to the macula, thereby ceasing or atrophying the efficient function of photosensitive cells. In wet macular degeneration, new, weak blood vessels proliferate in or under the retina, causing fluid and blood to leak into the submacular space.

[0020] Macular degeneration was previously thought to be a disease that frequently occurred among the elderly, but it is now known that the number of patients in their 40s and 50s has been rapidly increasing in recent years. Westernization of dietary habits, such as increased fat intake, has been pointed out as one of the main reasons for the decrease in the age of onset of macular degeneration.

[0021] Lutein is a naturally occurring carotenoid that does not possess vitamin A activity and is found in large quantities in green and yellow vegetables. It is one of the most abundant carotenoids in food and human blood, and is known to play a role in antioxidant activity and protecting plants from ultraviolet radiation. In humans, it is known as a major component of the macula and lens of the eye, playing an important role in improving eye health and vision.

[0022] However, a research team at the Moran Eye Center at the University of Utah reported in JAMA Ophthalmology (the journal of the American Medical Association) that round, yellowish, crystalline substances were found in the fovea of ​​a woman who had taken excessive amounts of lutein over a long period. In this patient's case, it was reported that she had taken 20 mg of lutein supplements daily for the past eight years, as well as consuming lutein-rich foods such as spinach, broccoli, kale, and avocado. Therefore, the research team hypothesized that the lutein precipitated in the eyeball, forming crystals and causing macular degeneration.

[0023] Therefore, while lutein has the effect of improving vision in macular degeneration, side effects, such as macular degeneration, may occur if taken over a long period or in excess. Thus, it is necessary to develop methods to prevent, alleviate, or suppress these side effects.

[0024] Psoriasis is an example of an angiogenesis-associated disorder or matrix metalloproteinase (MMP)-mediated disorder, a chronic inflammatory disease characterized by white or silvery scales and distinct borders of red papules or plaques of varying sizes that recur on the skin of the body. It is a skin disease histologically characterized by epidermal proliferation and dermal inflammation, occurring in 1-2% of the population. It is a chronic skin disease in which small miliary rashes form on the skin, and white, dandruff-like dead skin cells accumulate on these rashes. Therefore, if they spread widely, almost all of the skin on the body can be covered with rashes.

[0025] In psoriasis, the number of skin cells that produce dead skin cells increases rapidly, so flaky dead skin cells accumulate on the skin. The exact cause of psoriasis is not fully understood, but it is thought to be a problem with the immune system. In addition, genetic factors, environmental factors, drugs, skin irritation, dryness, upper respiratory tract inflammation, and mental stress are mentioned as factors that cause or exacerbate psoriasis.

[0026] Vitamin D analogs, narrow-band UVB treatment, psoralen + ultraviolet A (Bath-PUVA), and cyclosporine are used to treat psoriasis. There is still no known causative treatment for psoriasis. This is because the pathological cause of the disease has not been clearly identified. Treatment methods, such as vitamin D derivative ointments, narrow-band UVB treatment, bath PUVA, and cyclosporine, are simply temporary symptomatic treatments, so they cannot be called causative treatments because psoriasis recurs.

[0027] Endometriosis is an example of an angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease in which endometrial or similar tissue occurs outside the uterus depending on an increase in estrogen. It is a benign disease that causes pain (including dysmenorrhea), reduces fertility, and significantly decreases the quality of life of women in social and reproductive activities. In endometriosis, dysmenorrhea seems to be very frequent, and pain symptoms other than menstruation, such as lower abdominal pain, back pain, dyspareunia, and defecation pain, have also been confirmed frequently.

[0028] Many patients with endometriosis will recur or repeat recurrence by menopause unless radical surgery is performed, which requires long-term treatment and management. Drug treatment is often the first choice for the treatment of endometriosis. It is roughly divided into symptomatic treatment and endocrine treatment. In symptomatic treatment, analgesic drugs are mainly used to improve the pain caused by endometriosis. In endocrine therapy, low-dose estrogen-progestin preparations, dienogest, and gonadotropin-releasing hormone (GnRH) agonists are used to suppress estrogen-dependent endometrial growth in addition to pain relief.

[0029] However, the pain associated with endometriosis is said to be uncontrollable by analgesic drugs in 10 - 30% of patients with endometriosis. Also, attention is required for thrombosis and liver dysfunction in the use of low-dose estrogen-progestin preparations. Dienogest has been reported to have a side effect of irregular genital bleeding in 71.9% in long-term administration tests, which can lead to severe anemia. GnRH agonists, in some cases, cannot be administered for more than 6 months in principle due to the decrease in bone density caused by the estrogen-lowering effect.

[0030] As described above, in drug therapy for endometriosis treatment, since each drug has its own side effects, there are many patients who find it difficult to continue administration. Therefore, there is a need for the development of new drugs that can be administered over a long period with reduced side effects.

[0031] Cancer is an example of an angiogenesis-related disease or a matrix metalloproteinase (MMP)-mediated disease and is one of the most common causes of death worldwide. Approximately 10 million new cases occur every year, accounting for about 12% of all deaths and being the third leading cause of death.

[0032] Among cancers, breast cancer is the most common malignancy in women, causing more than 40,000 deaths annually, and early diagnosis is very important. Despite many known anticancer drugs, the survival rate has not been improved when the cancer is very advanced or metastatic.

[0033] Chemotherapy is a typical anticancer treatment and has been used as the most effective treatment for cancer when used alone or in combination with other treatments. However, the effectiveness of anticancer drugs in chemotherapy depends on the ability to kill cancer cells, but there is a problem that the drugs can act not only on cancer cells but also on normal cells, and drugs for efficiently treating cancer are required.

[0034] Atherosclerosis, also known as arteriosclerosis, is an example of angiogenesis-related or matrix metalloproteinase (MMP)-mediated disease in which arteries harden due to thickening or tissue degeneration of the arterial walls. As the inner lining of the arteries thickens, the diameter narrows, obstructing blood flow that supplies oxygen and various nutrients, leading to various diseases. Atherosclerosis is most likely to occur in the cerebral arteries or coronary arteries. In the case of cerebral arteriosclerosis, headaches, dizziness, and mental disorders appear, and cerebral softening occurs. In the case of coronary arteriosclerosis, it is known to cause pain and arrhythmias in the heart, and lead to angina pectoris and myocardial infarction. Furthermore, atherosclerosis is deeply related to the development and progression of diseases such as metabolic syndrome [including trunk obesity, obesity (overweight), thrombosis, hypercoagulation, and thrombotic conditions (arterial and venous)] or dyslipidemia. It also causes or worsens diabetic complications.

[0035] Furthermore, when blood triglyceride levels increase due to obesity or hyperlipidemia, macrophages may transform into foam cells or mortality may increase due to triglycerides. As macrophages die, their immune function and triglyceride metabolic function decline, which reduces their ability to remove foam cells and increases the likelihood of developing or worsening arteriosclerosis. Therefore, to prevent the onset or worsening of arteriosclerosis, it is important to ensure that macrophages maintain their ability to remove foam cells, or to minimize triglyceride-induced death even when blood lipid levels are high.

[0036] Currently, statin drugs, such as rosuvastatin and simvastatin, are primarily used to treat arteriosclerosis. However, it is well known that they have the side effect of reducing the activity of macrophages, which remove foam cells. Furthermore, statin drugs have been criticized for causing side effects such as diabetes, muscular dystrophy, and hyperglycemia. Therefore, there is a need for research into natural products that have fewer side effects.

[0037] Arthritis is an example of angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease, and is broadly divided into rheumatoid arthritis and osteoarthritis (degenerative arthritis). Rheumatoid arthritis is typically a chronic inflammatory multisystem disorder affecting the joints. Symptoms include pain and swelling accompanied by inflammation in various joints, such as fingers, hands, feet, wrists, ankles, and knees, and also cause abnormalities in various organs, such as muscles, skin, lungs, and eyes. Genetic factors, pathogenic infections, and immune abnormalities have been suggested as causes of arthritis, however, these are not precisely known.

[0038] The pathogenesis involves hypersensitivity to autoantigens or foreign antigens, which leads to the release of interleukin-1 (IL-1), tumor necrosis factor (TNF), prostaglandin E2 (PGE2), collagenase, proteinase, and substance P from immune cells. These substances reduce cartilage and collagen, induce synovial fluid proliferation and inflammation, and ultimately lead to the destruction of cartilage and bone.

[0039] Osteoarthritis refers to a degenerative state of the synovial joints and is also called degenerative arthritis. It is a disease that occurs in middle-aged or elderly people as a phenomenon of aging, and causes movement disorders after the age of 65. The main causes are thought to be obesity, hyperactivity syndrome, recurrent dislocation, recurrent hematologic hemorrhage, internal joint dislocation, inflammatory arthritis, and gout.

[0040] Osteoarthritis primarily involves progressive damage to articular cartilage. As arthritis progresses, the production of inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO) increases, leading to severe pain in the tissues of muscles, tendons, and ligaments.

[0041] Current treatments for arthritis can be broadly categorized into non-medical, pharmacological, and surgical approaches. Non-medical approaches include reducing joint load, patellar fixation, heat therapy, and exercise. Pharmacological approaches include oral medications and intra-articular injections. The drugs used in initial treatment are non-steroidal anti-inflammatory drugs (NSAIDs). While these NSAIDs simply reduce pain and alleviate symptoms, they cannot prevent articular cartilage loss or disease progression. Prolonged use can lead to gastrointestinal, renal, cardiac, and hepatic side effects, as well as heartburn and gastric bleeding due to blood clotting mechanisms. Therefore, there is a need for research into natural products with fewer side effects.

[0042] Inflammatory bowel disease (IBD) is an example of angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease that causes chronic inflammation or ulcers in the mucosa of the large and small intestines. It is an incurable disease characterized by persistent diarrhea and bloody stools, and recurrences over the long term.

[0043] Inflammatory bowel disease (IBD) is more common among Westerners, but it has been rapidly increasing in South Korea since the 1980s. IBD is reported in all age groups between 15 and 35 years of age, with 15% of cases occurring in those over 60 years of age, and approximately 15% of IBD patients have a family history in close relatives.

[0044] Although the exact etiology of inflammatory bowel disease remains unclear, the activation of inflammatory mediators and immune cells, along with environmental or genetic factors, is presumed to be an important etiology contributing to autoimmune diseases.

[0045] Inflammatory bowel disease is classified into two conditions, ulcerative colitis and Crohn's disease, which are clinically similar but differ from each other in histological, endoscopic, and immunological aspects. Activation of inflammatory mediators and immune cells is a well-known important etiology of this disease.

[0046] Sustained or inappropriate activation of the intestinal immune system plays a significant role in the pathophysiology of chronic mucosal inflammation, particularly the infiltration of neutrophils, macrophages, lymphocytes, and mast cells, which ultimately lead to mucosal destruction and ulceration.

[0047] In the pathogenesis of inflammatory bowel disease, inflammatory cytokines such as TNF-α (tumor necrosis factor-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) play major roles.

[0048] In particular, TNF-α is highly expressed in the lumen and epithelial cells of the colon in patients with ulcerative colitis, and recent studies have shown that TNF-α plays an important role in the pathogenesis of ulcerative colitis. Infliximab (an anti-TNF-α antibody) is known to be effective in treating Crohn's disease, which was previously incurable. However, these treatments are expensive and cause side effects in some patients, such as humoral reactions or infectious complications.

[0049] Current treatments for inflammatory bowel disease involve the use of 5-aminosalicylic acid (5-ASA) drugs that block prostaglandin production, such as sulfasalazine and mesalazine, or steroid immunosuppressants. If steroid treatment is ineffective, immunosuppressants such as azathioprine, 6-mercaptopurine, and cyclosporine may also be used, however, there are no drugs that can be expected to cure the disease.

[0050] Alzheimer's disease is an example of angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease, and is the most common cause of dementia, accounting for approximately 60-80% of dementia cases. Around 33.9 million people worldwide have Alzheimer's disease, and it is projected that the incidence will triple in 40 years as life expectancy increases. Currently, there are no drugs that offer a clear improvement in Alzheimer's disease, and because the disease progresses through a deteriorating process over several decades, research worldwide is focusing on preventing Alzheimer's disease.

[0051] Alzheimer's disease is known to be caused by a complex interaction of various risk factors. Along with genetic risk factors, demographic risk factors such as age, sex, and educational background, as well as environmental risk factors such as smoking, alcohol consumption, nutrition, and social activity, are known. Representative genetic markers for Alzheimer's disease include apolipoprotein E (APOE) ε4, however, these genetic markers have not been altered or modified. However, acquired factors (e.g., vascular risk factors, lifestyle) are modifiable. Therefore, in addition to studies investigating how these modifiable factors regulate the onset of dementia, it is possible to reduce or delay the onset of dementia by modulating these factors in reverse.

[0052] Among the drugs used to treat Alzheimer's disease, memantine was developed in the 1980s by Merz Pharmaceuticals in Germany for the treatment of Parkinson's disease and motor disorders, and has also been marketed in Germany for the treatment of dementia. In 1999, trial results were published showing that it is effective in treating severe Alzheimer's disease, and it was approved in Europe in 2002 for the treatment of Alzheimer's disease. In October 2003, it was approved by the US FDA for the treatment of moderate to severe Alzheimer's disease. More recently, it has also been marketed as EBIXA® by Lundbeck in South Korea. Commercially available Evixa contains memantine hydrochloride in tablet or liquid form, and its administration should not exceed the maximum required daily dose of 20 mg, taking into consideration both efficacy and tolerability.

[0053] The mechanism of action of memantine is to block glutamate (an excitatory neurotransmitter). It is known that glutamate excessively stimulates nerves in the brain, causing excessive calcium influx into brain nerve cells, leading to nerve damage and death, and memantine prevents this. It is known that in Alzheimer's disease patients, the calcium ion channel of the NMDA receptor remains open even during quiescence due to excessive glutamate secretion, resulting in excessive calcium influx into cells and associated with nerve death. Memantine blocks calcium influx into cells by acting as a low-affinity antagonist at the calcium channel located at the center of the NMDA receptor. This reduces the excessive excitability of nerve cells caused by excessive calcium influx, and when physiological functions such as memory and learning are required, the calcium exits the calcium channel, normalizing depolarization, stabilizing physiological nerve signals, and normalizing nerve cell function.

[0054] However, side effects of memantine have been reported, including loss of appetite, diarrhea, weight loss, dry mouth, dizziness, sleep disturbances, and fatigue. Rarely, dizziness, headache, constipation, drowsiness, and hypertension have also been reported. Therefore, there is a need for research on natural products with fewer side effects.

[0055] Periodontal disease is an example of angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease, a type of inflammation that occurs in the supporting tissues surrounding the teeth due to toxins, which are metabolites of microorganisms residing in the oral cavity. These periodontal diseases begin as gingivitis in the initial stages, and if left untreated, gingivitis progresses to periodontal disease accompanied by gum swelling, bleeding, and severe bad breath. Furthermore, persistent periodontal disease progresses to progressive periodontal disease, in which the collagen supporting the periodontal ligament is destroyed, the alveolar bone supporting the teeth is dissolved, the periodontal ligaments separate and periodontal pockets are formed, which can lead to tooth damage in severe cases.

[0056] The etiology of periodontal disease varies depending on sex, race, and age. This disease worsens over several years, with remissions occurring as the infection persists. Its incidence is higher in patients with systemic diseases, such as diabetes, AIDS, neutropenia, and Down syndrome.

[0057] The most effective way to prevent periodontal disease can be achieved by using substances that inhibit the activity of these microorganisms and promote blood flow to the inflamed gums. However, among the drugs used for the prevention and / or treatment of periodontal disease, antibiotics such as penicillin, erythromycin, and tetracycline are effective in killing and inhibiting growth, but they have serious side effects, such as causing the emergence of resistant bacteria. In the case of antibacterial agents such as chlorhexidine, it has the effect of inhibiting the formation of tooth decay, but its strong toxicity can be a problem. Therefore, there is a need for research on natural products with fewer side effects.

[0058] Diabetic retinopathy is an example of an angiogenesis-related disease or a matrix metalloproteinase (MMP)-mediated disease, and is more specific to hyperglycemia than other chronic diabetes-related complications.

[0059] Depending on the stage of progression, diabetic retinopathy can be classified into early non-proliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR). Furthermore, if diabetic macular edema (DME) is present, severe visual impairment may occur even at this stage.

[0060] If diabetic retinopathy is detected early, its progression and worsening can be prevented with appropriate management. However, if the condition is not properly managed, it can lead to severe vision loss or blindness. Currently, diabetic retinopathy is considered a leading cause of blindness in adults, and despite its clinical significance, drugs to prevent or treat diabetic retinopathy have not been actively developed.

[0061] Therefore, there is a growing need for the development of drugs to prevent or treat diabetic retinopathy.

[0062] Sjögren's syndrome is a chronic disease characterized by angiogenesis-related disorder or matrix metalloproteinase (MMP)-mediated disorder, resulting in secretory disorders caused by lymphocyte infiltration into the exocrine glands. Characteristic symptoms of this disease include dry eyes and dry mouth due to lymphocyte infiltration in the lacrimal and salivary glands. Characteristic changes in the eyes (known as erythematous keratoconjunctivitis or dry keratoconjunctivitis) and oral cavity (leading to tooth damage, increased oral infections, dysphagia, and stomatitis) associated with the loss of tears and saliva can be painful. Patients may also experience inflammation of the joints (arthritis), muscles (myositis), nerves (neuropathy), thyroid (thyroiditis), kidneys (nephritis), lungs, or other parts of the body, or swelling of the lymph nodes. Patients may also experience fatigue and sleep disturbances. Sjögren's syndrome primarily affects middle-aged women.

[0063] Currently, there are no known treatments for Sjögren's syndrome or effective treatments to restore glandular secretion. Conventional treatments are usually symptomatic and supportive, including hydration therapy (e.g., to alleviate symptoms of dry eye and dry mouth) and various forms of lubrication. Prescription medications are available, including cyclosporine, which helps treat chronic dry eye, and cevimeline or pilocarpine, which helps stimulate saliva flow. Anti-inflammatory agents, such as methotrexate and hydroxychloroquine, are also prescribed for the remission of musculoskeletal symptoms. However, none of the currently available medications are ideal due to their wide range of serious side effects.

[0064] Glaucoma is an example of angiogenesis-related disease or matrix metalloproteinase (MMP)-mediated disease in which the optic nerve, which is involved in the transmission of visual information to the brain, is damaged, leading to optic nerve atrophy and narrowing of the visual field. Known types of glaucoma include primary open-angle glaucoma (POAG), normal-tension glaucoma, primary closed-angle glaucoma, developmental glaucoma, and secondary glaucoma. In addition, ocular hypertension, a condition in which intraocular pressure is chronically high even if the visual field is normal, is one of the risk factors for glaucoma.

[0065] In glaucoma treatment, lowering intraocular pressure and preventing further visual field damage is considered the top priority. Drug therapy, laser treatment, and surgery are used to reduce intraocular pressure; however, these conventional treatments are not satisfactory for all patients. There is a need for glaucoma treatment drugs containing active ingredients with novel mechanisms of action or structures not found in existing therapeutic agents. [Prior art documents] [Patent Documents]

[0066] (Patent Document 1) KP No. 10-1055920 (Patent Document 2) KP No. 10-1292931 [Disclosure of the Invention] [Problems that the invention aims to solve]

[0067] The object of the present invention is to provide a fractional extract of Melissa leaf containing caffeic acid, EDPA (ethyl 2-(3,4-dihydroxyphenyl) acetate), RME (rosmarinic acid methyl ester), and rosmarinic acid.

[0068] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient.

[0069] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a fractional extract of lemon balm leaf as an active ingredient.

[0070] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient containing caffeic acid, EDPA, RME, and rosmarinic acid.

[0071] Another object of the present invention is to provide a food composition containing a fractional extract of Melissa leaf as an active ingredient containing caffeic acid, EDPA, RME, and rosmarinic acid. [Means for solving the problem]

[0072] The inventors recognized the need for research on the prevention or treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease, as well as angiogenesis-related / MMP-mediated diseases such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma. The inventors attempted to achieve the objective of research on natural substances that can prevent or treat these diseases.

[0073] Specifically, the inventors of the present invention have discovered that when a novel fractional extract of Melissa leaf is included as an active ingredient, it has preventive and therapeutic effects on non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and angiogenesis-related / MMP-mediated diseases such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma, thereby completing the present invention.

[0074] The present invention will be described in detail below.

[0075] Melissa leaf extract

[0076] The present invention provides a fractional extract of Melissa leaf containing caffeic acid, EDPA, RME, and rosmarinic acid.

[0077] In one embodiment of the present invention, the fractional extract of lemon balm leaves may contain 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, relative to the whole fractional extract of lemon balm leaves. Specifically, examples include, but are not limited to, (1) 0.2 to 4.0% by weight of caffeic acid, 0.1 to 5.0% by weight of EDPA, 0.02 to 1.5% by weight of RME, and 6 to 40% by weight of rosmarinic acid, (2) 0.3 to 3.0% by weight of caffeic acid, 0.15 to 4.0% by weight of EDPA, 0.03 to 1.0% by weight of RME, and 7 to 35% by weight of rosmarinic acid, and (3) 0.4 to 2.0% by weight of caffeic acid, 0.2 to 3.0% by weight of EDPA, 0.04 to 0.7% by weight of RME, and 8 to 30% by weight of rosmarinic acid. In the present embodiment, unless otherwise specified, the fractional extract of Melissa leaf of the present invention may contain caffeic acid, EDPA, RME, and rosmarinic acid in the weight percentages described above.

[0078] In one embodiment of the present invention, the fractional extract of Melissa leaves may contain 0.05 to 6% by weight of EDPA (ethyl 2-(3,4-dihydroxyphenyl)acetate), specifically (1) 0.1 to 5% by weight of EDPA, (2) 0.15 to 4% by weight of EDPA, and (3) 0.2 to 3% by weight of EDPA, but is not limited to these.

[0079] In one embodiment of the present invention, the pharmaceutical composition containing the highest EDPA content in the fractional extract of Melissa leaves is ALS-T20003 (0.85% by weight), and ALS-T20003 has been confirmed through animal experiments to have the effect of preventing or treating non-alcoholic steatohepatitis and non-alcoholic fatty liver disease.

[0080] The fractional extract of Melissa leaves of the present invention is effective for the prevention and treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease, and has excellent pharmacological effects with low side effects.

[0081] The lemon balm leaf fraction extract of the present invention does not contain rutin.

[0082] In one embodiment of the present invention, a fractional extract of lemon balm leaves can be obtained by extracting lemon balm leaves with alcohol, then concentrating the alcohol extract, suspending the concentrate in water, and then drying the fraction obtained by fractionation with ethyl acetate.

[0083] Specifically, in one embodiment of the present invention, a fractional extract of melissa leaves can be obtained by an extraction process that includes extracting and concentrating melissa leaves with 50% to 100% alcohol, suspending them in water, and fractionating them with ethyl acetate, but is not limited thereto.

[0084] In one embodiment of the present invention, the fraction may be dried using hot air drying, freeze-drying, or spray drying methods. For example, the fraction may be dried with hot air using a hot air dryer, freeze-dried by reducing the air pressure after freezing the fraction, or spray-dried by blowing hot air onto the fraction.

[0085] In the preparation of the lemon balm leaf fraction extract of the present invention, dried or undried lemon balm leaves or mixtures thereof may be used. For effective extraction, the lemon balm leaves may be used after being crushed.

[0086] In one embodiment of the present invention, the alcohol may be 70-80% (v / v) alcohol.

[0087] In this invention, the term "alcohol" refers to a compound in which a hydroxyl group is bonded to a carbon atom of an alkyl or substituted alkyl group, and the term "alkyl" refers to a straight-chain saturated hydrocarbon group or a branched saturated hydrocarbon group. The term "substituted alkyl group" means that the substituent bonded to the carbon of the alkyl group is a hydroxyl, cyano, or halide, etc.

[0088] Alcohol refers to C1-C6 alcohols, including 70-80% (v / v) ethanol and methanol, and is preferably ethanol or methanol.

[0089] In the present invention, the term "C1-C6" means a functional group or main chain having 1 to 6 carbon atoms.

[0090] In one embodiment of the present invention, the alcohol extract may be obtained by the following steps, but is not limited thereto.

[0091] (S1) First extraction step to obtain the first extract by reflux extraction of lemon balm leaves with 50-100% (v / v) alcohol at 80-85°C for 2-6 hours; (S2) A second extraction step to obtain a second extract by reflux extraction of the melissa residue remaining after the first extraction using 50-100% (v / v) alcohol at 80-85°C for 2-6 hours; and (S3) A step of mixing the first extract and the second extract.

[0092] The alcohol extract can be obtained by mixing the first and second extracts obtained through the first and second extraction steps described above.

[0093] The alcohol extract may be prepared using 50-100% alcohol (v / v), preferably 70-80% alcohol (v / v), at a ratio of 5-15 times (v / w) of the lemon balm leaf, but is not limited to this.

[0094] According to embodiments of the present invention, the concentrate may be obtained by the following steps, but is not limited thereto.

[0095] (S4) A first concentration step to obtain a first concentrate by concentrating the alcohol extract under temperature conditions of 50-60°C and pressure conditions of -0.066--0.070 MPa for 5-15 hours, and (S5) A second concentration step to obtain a second concentrate by concentrating the first concentrate under temperature conditions of 55 to 60°C and pressure conditions of -0.063 to -0.065 MPa for 3 to 10 hours.

[0096] According to embodiments of the present invention, the following concentration steps may be further included before drying the fraction, but are not limited thereto.

[0097] (S6) A third concentration step to concentrate the fraction.

[0098] A third concentration step involves concentrating the fraction for 3 to 10 hours under temperature conditions of 55 to 60°C and pressure conditions of -0.063 to -0.065 MPa.

[0099] In this invention, the term "fraction" refers to the resulting product obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various components.

[0100] In the present invention, the fractionation method for obtaining a fractional extract of Melissa leaves is not particularly limited and may be carried out according to methods commonly used in the art. Specifically, this includes solvent fractionation carried out by treatment with various solvents, ultrafiltration fractionation carried out by passing through an ultrafiltration membrane having a certain molecular weight cutoff value, chromatographic fractionation by various types of chromatography (prepared for separation according to size, charge, hydrophobicity, or affinity), and any combination thereof. Specifically, there may be a method of obtaining a fraction from an extract by processing an extract obtained by extracting Melissa leaves of the present invention with a predetermined solvent.

[0101] In the present invention, the type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Specifically, ethyl acetate, which is a nonpolar solvent, may be used.

[0102] The fractional extract of lemon balm leaf may be the ethyl acetate fraction of the ethanol extract of lemon balm leaf.

[0103] This method, using 50-100% (v / v) alcohol, efficiently extracts both soluble and insoluble substances, and is effective in extracting insoluble substances that have low solubility in water but high solubility in ethyl acetate.

[0104] The ethyl acetate fraction of the Melissa leaf ethanol extract may contain 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, relative to the total weight of the fraction. Specifically, (1) 0.2 to 4.0% by weight of caffeic acid, 0.1 to 5.0% by weight of EDPA, 0.02 to 1.5% by weight of RME, and 6 to 40% by weight of rosmarinic acid; (2) 0.3 to 3.0% by weight of caffeic acid, 0.15 to 4.0% by weight of EDPA, 0.03 to 1.0% by weight of RME, and 7 to 35% by weight of rosmarinic acid; (3) 0.4 to 2.0% by weight of caffeic acid, 0.2 to 3.0% by weight of EDPA, 0.04 to 0.7% by weight of RME, and 8 to 30% by weight of rosmarinic acid.

[0105] In embodiments of the present invention, the ethyl acetate fraction of the Melissa leaf ethanol extract may contain, but is not limited to, 0.05 to 6% by weight of EDPA (ethyl 2-(3,4-dihydroxyphenyl)acetate), specifically (1) 0.1 to 5% by weight of EDPA, (2) 0.15 to 4% by weight of EDPA, and (3) 0.2 to 3% by weight of EDPA.

[0106] The advantages and features of the present invention, as well as methods for achieving them, will become apparent by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but can be carried out in various different forms, and these embodiments alone make it possible to complete the disclosure of the present invention and are provided to those skilled in the art to which the present invention belongs, fully demonstrating the scope of the invention, and the present invention is defined solely by the claims.

[0107] A pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of lemon balm leaf as an active ingredient.

[0108] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient.

[0109] The fractional extract of lemon balm leaves and its preparation method are the same as those previously discussed.

[0110] According to the present invention, the term "non-alcoholic steatohepatitis" refers to a disease that exhibits a form similar to alcoholic liver disease despite having no history of alcohol consumption.

[0111] According to the present invention, the term "active ingredient" includes, as a means of including the main component, a substance or group of substances (e.g., crude drugs whose pharmacologically active components have not been identified) that is expected to directly or indirectly exert the efficacy of the composition through its inherent pharmacological action.

[0112] According to the present invention, a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient, has the effect of preventing or treating non-alcoholic steatohepatitis by exhibiting a significant inhibitory effect on fibrosis and inhibitory effects on the protein expression levels of Col1A2, TGF-beta, IL-6, and IL-10.

[0113] The pharmaceutical composition of the present invention for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient, further comprising a pharmaceutically acceptable carrier. According to conventional methods, it may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations and sterile injections.

[0114] According to the present invention, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions, such as gastrointestinal disorders, dizziness, or similar reactions, when administered to humans.

[0115] According to the present invention, the term “pharmaceutically acceptable carrier” typically includes a liquid base or a non-liquid base of the pharmaceutical composition. When the pharmaceutical composition is provided in liquid form, the carrier typically includes pyrogen-free water; isotonic saline or buffer (aqueous) solution, such as phosphoric acid or citrate. The injection buffer may be hypertonic, isotonic, or hypotonic in a particular reference medium, i.e., the buffer may have a high, equal, or low salt content in a particular reference medium, but preferably such concentrations of the aforementioned salts (that do not induce cell damage due to osmotic pressure or other concentration effects) may be used.

[0116] The reference medium is generated, for example, as a general buffer or liquid, in a “in vivo” method, such as blood, lymph, cytoplasmic fluid, or other body fluid, or in an “ex vivo” method, in a body fluid that can be used as a reference medium. Such general buffers or liquids are known to those skilled in the art.

[0117] Medicinally acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0118] Furthermore, the pharmaceutical composition of the present invention may include diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other pharmaceutically acceptable additives.

[0119] The pharmaceutical composition of the present invention may be manufactured in the form of a liquid, suspension, powder, granules, tablet, capsule, pill, or extract.

[0120] The compositions of the present invention can be administered orally or parenterally (e.g., by liniment or intravenous, subcutaneous, or intraperitoneal injection).

[0121] According to the present invention, the term "oral administration" refers to a method of injecting a drug by mouth to alleviate pathological symptoms, and according to the present invention, the term "parenteral administration" refers to subcutaneous administration, intramuscular administration, intravenous administration, or intraperitoneal administration using a tube, excluding oral administration.

[0122] Solid forms of medication for oral administration may include powders, granules, tablets, capsules, soft capsules, and pills. Liquid forms of medication for oral administration may include suspensions, oral solutions, emulsions, syrups, and aerosols, and may contain various additives in addition to water and liquid paraffin (a commonly used simple diluent), such as humectants, sweeteners, flavorings, and preservatives.

[0123] For parenteral administration, topical preparations such as sterile aqueous solutions, liquid preparations, non-aqueous solvents, suspending agents, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, etc., and sterile injectable preparations can be formulated and used according to conventional methods. Preferably, but not limited to creams, gels, patches, sprays, ointments, plasters, lotions, liniments, eye ointments, eye drops, pastes, or cataplasma pharmaceutical compositions can be formulated. Compositions for topical administration may be anhydrous or aqueous, depending on the clinical formulation. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspending agents. For suppository bases, vitepsol, macrogol, tween 61, cocoa butter, laurin, glycerogenous gelatin, etc., can be used.

[0124] The pharmaceutically acceptable additive according to the present invention may be included in the composition in an amount of 0.1 to 99.9 parts by weight, specifically 0.1 to 50 parts by weight, but is not limited thereto.

[0125] A pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis, containing a fractional extract of lemon balm leaf as an active ingredient, comprises the fractional extract of lemon balm leaf in an amount of 20% to 80% by weight relative to the total weight.

[0126] According to the present invention, a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient, has the effect of preventing or treating non-alcoholic steatohepatitis by exhibiting a significant inhibitory effect on fibrosis and on the protein expression levels of Col1A2, TGF-beta, IL-6, and IL-10.

[0127] A pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as the active ingredient of the present invention, may be administered in the usual manner via the oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly the oral route.

[0128] A pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as the active ingredient of the present invention, is preferably administered at a dose of 0.001 mg / kg to 50 mg / kg once to several times a day.

[0129] A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of lemon balm leaf as an active ingredient.

[0130] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a fractional extract of lemon balm leaf as an active ingredient.

[0131] The fractional extract of lemon balm leaves and the method for preparing it are the same as those previously studied.

[0132] According to the present invention, the term "non-alcoholic fatty liver disease" refers to a disease that exhibits a form similar to alcoholic fatty liver disease despite having no history of alcohol consumption.

[0133] According to the present invention, a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating non-alcoholic fatty liver disease by demonstrating a significant suppression of body weight, blood triglycerides, blood glucose, and total cholesterol.

[0134] A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, containing a fractional extract of lemon balm leaf as an active ingredient, comprises the fractional extract of lemon balm leaf in an amount of 20% to 80% by weight based on the total weight.

[0135] A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of Melissa leaf as the active ingredient of the present invention, may be administered in the usual manner via the oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly the oral route.

[0136] A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of lemon balm leaf as the active ingredient of the present invention, is administered once to several times a day, preferably in a dose of 0.001 mg / kg to 50 mg / kg.

[0137] Any matters referred to in a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of Melissa leaf as an active ingredient, are equally applicable to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of Melissa leaf as an active ingredient, provided that they do not contradict each other.

[0138] Food composition containing a fractional extract of lemon balm leaf as an active ingredient

[0139] Another object of the present invention is to provide a food composition containing a fractional extract of lemon balm leaf as an active ingredient.

[0140] The fractional extract of lemon balm leaves and the method for preparing it are the same as described above.

[0141] This composition is intended to prevent or alleviate either non-alcoholic steatohepatitis or non-alcoholic fatty liver disease.

[0142] In the food composition according to the present invention, when a fractional extract of Melissa leaf is used as an additive in the food composition, it can be added as is or used together with other foods or food ingredients, and can be used as appropriate according to conventional methods. The amount of active ingredient may be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

[0143] Food compositions can be formulated in any form of a common food or beverage, as well as in powder, granule, pill, tablet, and capsule form.

[0144] There are no specific limitations on the type of food to which the substance can be added. Examples of foods to which the substance can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products (including ice cream), various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all foods in the conventional sense.

[0145] Generally, in the manufacture of food or beverages, a fractional extract of lemon balm leaf may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for health and hygiene or health management, the amount may be lower than the above range. Furthermore, since there are no safety concerns with using fractional extracts from natural products in this invention, it can also be used in amounts exceeding that range.

[0146] The beverage in the food composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, similar to conventional beverages. Natural carbohydrates may include monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners, such as thaumatin and stevia extract, or synthetic sweeteners, such as saccharin and aspartame, or similar substances may be used. The proportion of natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0147] In addition to the above, the food composition according to the present invention contains various nutrients, vitamins, electrolytes, flavorings, colorings, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the food composition containing the lemon balm leaf fraction extract of the present invention as an active ingredient may also contain natural fruit juice and fruit pulp for producing fruit juice and vegetable beverages. These components may be used individually or in combination. The proportions of these additives are not limited, but are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.

[0148] Methods for prevention or treatment

[0149] The object of the present invention is to provide a method for preventing or treating non-alcoholic steatohepatitis by administering a therapeutically effective amount of a fractional extract of Melissa leaves to a subject requiring treatment.

[0150] Another object of the present invention is to provide a method for preventing or treating non-alcoholic fatty liver disease by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject in need of treatment.

[0151] According to embodiments of the present invention, the term "subject requiring treatment" refers to a mammal, including humans, and the term "administration" refers to providing a predetermined substance to a patient by any appropriate method. In the treatment method of the present invention, a pharmaceutical composition containing a fractional extract of Melissa leaf as the active ingredient of the present invention can be administered in the usual manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes.

[0152] Furthermore, in the method for preventing or treating non-alcoholic steatohepatitis of the present invention, the fractional extract of Melissa leaf of the present invention can be administered in the usual manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, and specifically, it can be administered orally.

[0153] Furthermore, in the method for preventing or treating non-alcoholic fatty liver disease of the present invention, the lemon balm leaf fraction extract of the present invention can be administered in the usual manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, and specifically, it can be administered orally.

[0154] According to embodiments of the present invention, the term “therapeutic dose” refers to the amount of an active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as considered by researchers, veterinarians, physicians, or other clinicians, and this includes the amount that elicits remission of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and number of administrations of the active ingredient of the present invention may vary depending on the desired effect. Accordingly, the optimal dose can be easily determined by those skilled in the art and may be adjusted for various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients in the composition, the type of formulation, and the patient’s age, weight, overall health, sex and diet, the time of administration of the composition, the route of administration and the rate of secretion, the duration of treatment, and concomitant drugs.

[0155] In the method for preventing or treating non-alcoholic steatohepatitis of the present invention, the fractional extract of Melissa leaf of the present invention is administered to adults, preferably in doses of 0.001 mg / kg to 50 mg / kg, once to several times a day.

[0156] In the method for preventing or treating non-alcoholic fatty liver disease of the present invention, the fractional extract of Melissa leaf of the present invention is administered to adults, preferably in doses of 0.001 mg / kg to 50 mg / kg, once to several times a day.

[0157] According to the present invention, the term "prevention" refers to any effect of suppressing or delaying non-alcoholic steatohepatitis and non-alcoholic fatty liver disease by administration of the fractional extract of Melissa leaves of the present invention.

[0158] According to the present invention, the term "treatment" refers to any action taken by administering the fractional extract of Melissa leaves according to the present invention to improve or beneficially alter non-alcoholic steatohepatitis and non-alcoholic fatty liver disease.

[0159] Use of pharmaceutical compositions containing a fractionated extract of lemon balm leaf as an active ingredient for prevention or treatment.

[0160] The object of the present invention is to provide a pharmaceutical composition containing a fractionated extract of Melissa leaf as an active ingredient for the prevention or treatment of non-alcoholic steatohepatitis.

[0161] Another object of the present invention is to provide the use of a pharmaceutical composition comprising a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of non-alcoholic fatty liver disease.

[0162] Use of a pharmaceutical composition containing a fractionated extract of Melissa leaf as an active ingredient in the manufacture of pharmaceuticals.

[0163] The object of the present invention is to provide the use of a pharmaceutical composition containing a fractionated extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for the prevention or treatment of non-alcoholic steatohepatitis.

[0164] Another object of the present invention is to provide the use of a pharmaceutical composition comprising a fractional extract of Melissa leaf as an active ingredient for the manufacture of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

[0165] According to embodiments of the present invention, a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical product may be mixed with an acceptable carrier and may further contain other agents.

[0166] [Angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases] Pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases

[0167] The present invention provides a pharmaceutical composition for preventing or treating angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf containing caffeic acid, EDPA, RME, and rosmarinic acid as active ingredients.

[0168] According to the present invention, the term "angiogenesis-related disease" refers to a disease that arises in connection with the pathologically excessive formation of new capillaries from existing microvessels.

[0169] According to the present invention, the term "MMP-mediated disease" refers to a disease caused by excessive activation of matrix metalloproteinases (MMPs).

[0170] According to the present invention, the term "active ingredient" refers to a substance or group of substances (including crude drugs whose pharmacologically active ingredients have not been identified) that is expected to directly or indirectly exert the efficacy of the composition through its inherent pharmacological action, and it means that it includes the main component.

[0171] According to one embodiment of the present invention, a fractional extract of Melissa leaf may contain 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 relative to the whole fractional extract of Melissa leaf. Specifically, it may contain, but is not limited to, (1) 0.2 to 4.0% by weight of caffeic acid, 0.1 to 5.0% by weight of EDPA, 0.02 to 1.5% by weight of RME, and 6 to 40% by weight of rosmarinic acid, (2) 0.3 to 3.0% by weight of caffeic acid, 0.15 to 4.0% by weight of EDPA, 0.03 to 1.0% by weight of RME, and 7 to 35% by weight of rosmarinic acid, or (3) 0.4 to 2.0% by weight of caffeic acid, 0.2 to 3.0% by weight of EDPA, 0.04 to 0.7% by weight of RME, and 8 to 30% by weight of rosmarinic acid. In the following exemplary embodiments, the lemon balm leaf fraction extract of the present invention may contain caffeic acid, EDPA, RME, and rosmarinic acid in the weight percentages described above, unless otherwise specified.

[0172] According to embodiments of the present invention, the fractional extract of Melissa leaf may contain, but is not limited to, 0.05 to 6% by weight of EDPA (ethyl 2-(3,4-dihydroxyphenyl)acetate), specifically (1) 0.1 to 5% by weight of EDPA, (2) 0.15 to 4% by weight of EDPA, and (3) 0.2 to 3% by weight of EDPA.

[0173] According to embodiments of the present invention, the pharmaceutical composition with the highest EDPA content in the fractionated extract of Melissa leaves is ALS-T20003 (0.85% by weight), and it can be confirmed that ALS-T20003 used in animal experiments has the effect of inhibiting angiogenesis and preventing or treating angiogenesis and MMP-mediated diseases.

[0174] The pharmaceutical composition of the present invention, which contains a fractional extract of Melissa leaf as an active ingredient, is effective in preventing and treating angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer proliferation or metastasis, atherosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma, and has excellent pharmacological effects with few side effects.

[0175] The fractional extract of lemon balm leaf is an active ingredient contained in the pharmaceutical composition of the present invention and does not contain rutin.

[0176] According to embodiments of the present invention, a fractional extract of melissa leaves may be obtained by extracting melissa leaves under reflux with alcohol, then concentrating the alcohol extract, suspending the concentrate in water, then fractionating with ethyl acetate, and drying the fraction.

[0177] Specifically, according to embodiments of the present invention, a fractional extract of melissa leaves can be obtained by an extraction process that includes extracting melissa leaves with 50% to 100% alcohol, concentrating the extract, suspending it in water, and fractionating it with ethyl acetate, but is not limited thereto.

[0178] According to embodiments of the present invention, the fraction can be dried using hot air drying, freeze-drying, or spray drying methods. For example, the fraction can be hot-air dried using a hot air dryer, or freeze-dried by freezing the fraction and reducing the air pressure, or spray-dried by spraying the fraction and blowing hot air over it.

[0179] In the preparation of the lemon balm leaf fraction extract of the present invention, dried or undried lemon balm leaves or mixtures thereof may be used. For effective extraction, the lemon balm leaves may be finely crushed before use.

[0180] According to one embodiment of the present invention, the alcohol may be, but is not limited to, 70-80% (v / v) alcohol.

[0181] According to the present invention, the term "alcohol" refers to a compound in which a hydroxyl group is bonded to the carbon atom of an alkyl group or a substituted alkyl group, the term "alkyl" refers to a straight-chain saturated hydrocarbon group or a branched saturated hydrocarbon group, and the term "substituted alkyl group" means that the substituent bonded to the carbon atom of the alkyl group is a hydroxyl, cyano, halide, etc.

[0182] Alcohol refers to 70-80% (v / v) ethanol, methanol, and preferably C1-C6 alcohols such as ethanol or methanol.

[0183] According to the present invention, the term "C1-C6" means a functional group or main chain having 1 to 6 carbon atoms.

[0184] According to embodiments of the present invention, the alcohol extract may be obtained by the following steps, but is not limited thereto. (S1) First extraction step: Obtaining a first extract by reflux extraction of lemon balm leaves with 50-100% (v / v) alcohol at 80-85°C for 2-6 hours; (S2) A second extraction step in which a second extract is obtained by reflux extraction of the melissa residue remaining after the first extraction using 50-100% (v / v) alcohol at 80-85°C for 2-6 hours; and (S3) A step of mixing the first extract and the second extract.

[0185] The alcohol extract can be obtained by mixing the first and second extracts obtained through the first and second extraction steps described above.

[0186] The alcohol extract may be prepared using 50-100% alcohol (v / v), preferably 70-80% alcohol (v / v), at a ratio of 5-15 times (v / w) of the lemon balm leaf, but is not limited to this.

[0187] According to embodiments of the present invention, the concentrate may be obtained by the following steps, but is not limited thereto. (S4) A first concentration step to obtain a first concentrate by concentrating the alcohol extract under temperature conditions of 50-60°C and pressure conditions of -0.066--0.070 MPa for 5-15 hours, and (S5) A second concentration step to obtain a second concentrate by concentrating the first concentrate under temperature conditions of 55-60°C and pressure conditions of -0.063--0.065 MPa for 3-10 hours.

[0188] According to embodiments of the present invention, the following concentration steps may be further included before drying the fraction, but are not limited thereto. (S6) A third concentration step to concentrate the fraction.

[0189] A third concentration step involves concentrating the fraction for 3 to 10 hours under temperature conditions of 55 to 60°C and pressure conditions of -0.063 to -0.065 MPa.

[0190] According to the present invention, the term "fraction" refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various components.

[0191] According to the present invention, the fractionation method for obtaining a fractional extract of Melissa leaves is not particularly limited and may be carried out according to methods commonly used in the art. Specifically, this includes solvent fractionation carried out by treatment with various solvents, ultrafiltration fractionation carried out by passing through an ultrafiltration membrane having a certain molecular weight cutoff value, chromatographic fractionation carried out by various chromatography methods (prepared for separation according to size, charge, hydrophobicity, or affinity), and combinations thereof. Specifically, there is a method of obtaining a fraction from an extract by treating an extract obtained by extracting Melissa leaves according to the present invention with a predetermined solvent.

[0192] According to the present invention, the type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used, and specifically, ethyl acetate, which is a nonpolar solvent, may be used.

[0193] The fractional extract of lemon balm leaf may be the ethyl acetate fraction of the ethanol extract of lemon balm leaf.

[0194] This method, by using 50-100% (v / v) alcohol, efficiently extracts soluble and insoluble substances, and is effective in extracting insoluble substances that have low solubility in water but high solubility in ethyl acetate.

[0195] The ethyl acetate fraction of the Melissa leaf ethanol extract may contain 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, relative to the total weight of the fraction. Specifically, (1) 0.2 to 4.0% by weight of caffeic acid, 0.1 to 5.0% by weight of EDPA, 0.02 to 1.5% by weight of RME, and 6 to 40% by weight of rosmarinic acid; (2) 0.3 to 3.0% by weight of caffeic acid, 0.15 to 4.0% by weight of EDPA, 0.03 to 1.0% by weight of RME, and 7 to 35% by weight of rosmarinic acid; (3) 0.4 to 2.0% by weight of caffeic acid, 0.2 to 3.0% by weight of EDPA, 0.04 to 0.7% by weight of RME, and 8 to 30% by weight of rosmarinic acid.

[0196] The pharmaceutical composition of the present invention, for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, further comprising a pharmaceutically acceptable carrier formulated for oral administration, such as powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and for topical application, as well as a sterile injection solution prepared according to conventional methods.

[0197] The contents of the formulation can be equally applied to pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma.

[0198] According to the present invention, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions, such as gastrointestinal disorders, dizziness, or similar reactions, when administered to humans.

[0199] According to the present invention, the term “pharmaceutically acceptable carrier” typically includes a liquid-based or non-liquid-based pharmaceutical composition. When the pharmaceutical composition is provided in liquid form, the carrier typically includes pyrogen-free; isotonic saline or buffer (aqueous) solution, such as phosphoric acid or citric acid. The injection buffer may be hypertonic, isotonic, or hypotonic in a particular reference medium; that is, the buffer may have a high, equal, or low salt content in a particular reference medium, preferably such concentrations of the aforementioned salts (that do not induce cell damage due to osmotic pressure or other concentration effects). The reference medium may be a body fluid that can be used as a reference medium in an “in vivo” method, e.g., blood, lymph, cytoplasmic fluid, or other body fluid, or in an “ex vivo” method, e.g., a general buffer or liquid. Such general buffers or liquids are known to those skilled in the art.

[0200] Medicinally acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0201] Furthermore, the pharmaceutical composition of the present invention may include diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other pharmaceutically acceptable additives.

[0202] The pharmaceutical composition of the present invention may be manufactured in the form of a liquid, suspension, powder, granules, tablet, capsule, pill, or extract.

[0203] The compositions of the present invention may be administered orally or parenterally (e.g., by liniment or intravenous, subcutaneous, or intraperitoneal injection). According to the present invention, the term "oral administration" refers to a method of injecting a drug by mouth to alleviate pathological symptoms, and according to the present invention, the term "parenteral administration" refers to subcutaneous, intramuscular, intravenous, or intraperitoneal administration using a tube, excluding oral administration.

[0204] Solid forms of medication for oral administration may include powders, granules, tablets, capsules, soft capsules, and pills. Liquid forms of medication for oral administration may include suspensions, oral solutions, emulsions, syrups, and aerosols, and may contain various additives in addition to water and liquid paraffin (a commonly used simple diluent), such as humectants, sweeteners, flavorings, and preservatives.

[0205] For parenteral administration, topical preparations such as sterile aqueous solutions, liquid preparations, non-aqueous solvents, suspending agents, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, etc., and sterile injectable preparations can be formulated and used according to conventional methods. Preferably, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, eye ointments, eye drops, pastes, or cataplasma pharmaceutical preparations can be formulated, but are not limited to these. Compositions for topical administration may be anhydrous or aqueous, depending on the clinical formulation. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include vitepsol, macrogol, tween 61, cocoa butter, laurin, glycerogenous gelatin, etc.

[0206] The pharmaceutically acceptable additive according to the present invention may be included in the pharmaceutical composition in an amount of 0.1 to 99.9 parts by weight, specifically 0.1 to 50 parts by weight, but is not limited thereto.

[0207] The advantages and features of the present invention, as well as methods for achieving them, will become apparent by referring to the embodiments described in detail below. However, the present invention is not limited to the exemplary embodiments disclosed below, but can be carried out in various different forms. These exemplary embodiments alone complete the disclosure of the present invention and are provided to fully convey the scope of the invention to those skilled in the art to which the invention belongs, and the present invention is defined solely by the claims.

[0208] Angiogenesis-related diseases or MMP-mediated diseases: Obesity

[0209] According to embodiments of the present invention, angiogenesis-related diseases or MMP-mediated diseases may be obese.

[0210] According to the present invention, the term "obesity" refers to a condition in which body weight has increased beyond the limits of skeletal and physical requirements due to excessive accumulation of fat in the body.

[0211] When angiogenesis-related disease or MMP-mediated disease is associated with obesity, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains a fractional extract of Melissa leaf in an amount of 20% to 80% by weight based on the total weight.

[0212] According to the present invention, a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases is effective in preventing or treating obesity by showing a significant suppression of body weight, blood triglycerides, blood glucose levels, and total cholesterol.

[0213] When angiogenesis-related disease or MMP-mediated disease is associated with obesity, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly by oral administration.

[0214] When angiogenesis-related disease or MMP-mediated disease is associated with obesity, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0215] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to obesity, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0216] Angiogenesis-related diseases or MMP-mediated diseases: Age-related macular degeneration

[0217] According to embodiments of the present invention, the angiogenesis-related disease or MMP-mediated disease may be age-related macular degeneration.

[0218] According to the present invention, the term "macular degeneration" refers to a disease in which the macula changes due to aging, genetic factors, toxicity, inflammation, etc., and in severe cases, leads to complete loss of vision.

[0219] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating age-related macular degeneration by suppressing the size of macular degenerative lesions.

[0220] When the angiogenesis-related disease or MMP-mediated disease is age-related macular degeneration, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0221] When the angiogenesis-related disease or MMP-mediated disease is age-related macular degeneration, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly the oral route.

[0222] When the angiogenesis-related disease or MMP-mediated disease is age-related macular degeneration, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0223] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to age-related macular degeneration, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0224] Angiogenesis-related diseases or MMP-mediated diseases: Psoriasis

[0225] According to embodiments of the present invention, the angiogenesis-related disease or MMP-mediated disease may be psoriasis.

[0226] According to the present invention, the term "psoriasis" refers to a chronic inflammatory skin disease characterized by well-defined papules and plaques covered with silvery-white scales.

[0227] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating psoriasis by suppressing significant epidermal thickness, inflammatory cell infiltration in ear tissue, and mRNA expression levels in ear tissue.

[0228] According to embodiments of the present invention, when the angiogenesis-related disease or MMP-mediated disease is psoriasis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0229] When angiogenesis-related disease or MMP-mediated disease is psoriasis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly by oral administration.

[0230] When angiogenesis-related disease or MMP-mediated disease is psoriasis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0231] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases shall apply equally to psoriasis, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0232] Angiogenesis-related diseases or MMP-mediated diseases: Endometriosis

[0233] According to embodiments of the present invention, the angiogenesis-related disease or MMP-mediated disease may be endometriosis.

[0234] According to the present invention, the term "endometriosis" refers to a disease in which endometrial tissue is present in the ovaries, posterior wall, uterine ligaments, pelvic wall, etc., and various symptoms such as pain and bleeding occur.

[0235] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating endometriosis by significantly reducing the size of uterine lesions.

[0236] According to embodiments of the present invention, when angiogenesis-related disease or MMP-mediated disease is endometriosis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, local, intraocular, or intradermal routes, particularly by oral administration.

[0237] According to embodiments of the present invention, when the angiogenesis-related disease or MMP-mediated disease is endometriosis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0238] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to endometriosis, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0239] Angiogenesis-related diseases or MMP-mediated diseases: Cancer

[0240] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be cancer growth or cancer metastasis.

[0241] According to the present invention, the term "cancer" refers to a disease in which the cell cycle is not regulated and cell division continues.

[0242] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating cancer by significantly reducing the size of cancerous tumors.

[0243] According to embodiments of the present invention, when angiogenesis-related disease or MMP-mediated disease is cancer growth or cancer metastasis, a pharmaceutical composition for preventing or treating angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0244] Cancer may include any one of the following: lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, gastric cancer, gastrointestinal cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, pediatric solid tumors, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, axial contractile tumor, brainstem glioma, or pituitary adenoma, and in particular colorectal cancer.

[0245] According to embodiments of the present invention, when angiogenesis-related disease or MMP-mediated disease is cancer growth or cancer metastasis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, topical, intraocular, or intradermal routes, particularly by oral route.

[0246] When angiogenesis-related disease or MMP-mediated disease is cancer growth or cancer metastasis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0247] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to cancer, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0248] Angiogenesis-related diseases or MMP-mediated diseases: arteriosclerosis

[0249] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be arteriosclerosis.

[0250] According to the present invention, the term "arteriosclerosis" refers to a disease in which cholesterol or triglycerides accumulate in the innermost layer of blood vessels, causing the vessels to narrow, harden, and become blocked.

[0251] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating arteriosclerosis by significantly inhibiting the increase of arteriosclerotic plaques.

[0252] According to embodiments of the present invention, when the angiogenesis-related disease or MMP-mediated disease is arteriosclerosis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0253] When angiogenesis-related disease or MMP-mediated disease is arteriosclerosis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, local, intraocular, or intradermal routes, particularly by oral administration.

[0254] When angiogenesis-related disease or MMP-mediated disease is arteriosclerosis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0255] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to arteriosclerosis, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0256] Angiogenesis-related diseases or MMP-mediated diseases: Arthritis

[0257] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be arthritis.

[0258] According to the present invention, the term "arthritis" refers to a disease in which damage or inflammation occurs in a joint where bones intersect due to multiple causes.

[0259] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating arthritis by exhibiting excellent arthritis-treating effects and excellent load-bearing effects.

[0260] According to embodiments of the present invention, when angiogenesis-related disease or MMP-mediated disease is arthritis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0261] Arthritis can be any one of the following: osteoarthritis, degenerative arthritis, osteochondritis dissecans, articular ligament injury, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis, particularly osteoarthritis and rheumatoid arthritis.

[0262] When angiogenesis-related disease or MMP-mediated disease is arthritis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, topical, intraocular, or intradermal routes, particularly by oral administration.

[0263] When angiogenesis-related disease or MMP-mediated disease is arthritis, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0264] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to arthritis, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0265] Angiogenesis-related diseases or MMP-mediated diseases: Inflammatory bowel disease

[0266] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be inflammatory bowel disease.

[0267] According to the present invention, the term "inflammatory bowel disease" refers to a disease in which abnormal chronic inflammation in the intestinal tract repeatedly improves and relapses.

[0268] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating inflammatory bowel disease by exhibiting a low disease activity index.

[0269] According to embodiments of the present invention, when angiogenesis-related disease or MMP-mediated disease is inflammatory bowel disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0270] Inflammatory bowel disease can be one of the following: ulcerative colitis, Crohn's disease, intestinal tuberculosis, ischemic colitis, or intestinal ulcers due to Behçet's disease, particularly ulcerative colitis.

[0271] When angiogenesis-related disease or MMP-mediated disease is inflammatory bowel disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly by oral administration.

[0272] When angiogenesis-related disease or MMP-mediated disease is inflammatory bowel disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0273] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to inflammatory bowel disease, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0274] Angiogenesis-related diseases or MMP-mediated diseases: Alzheimer's disease

[0275] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be Alzheimer's disease.

[0276] According to the present invention, the term "Alzheimer's" refers to the most common cause of dementia, which results in symptoms of dementia caused by degenerative changes in brain cells.

[0277] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of lemon balm leaf as an active ingredient, is effective in preventing or treating Alzheimer's disease by improving cognitive function.

[0278] According to embodiments of the present invention, when the angiogenesis-related disease or MMP-mediated disease is Alzheimer's disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0279] According to the present invention, when angiogenesis-related disease or MMP-mediated disease is Alzheimer's disease, a pharmaceutical composition for preventing or treating angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly by oral administration.

[0280] According to embodiments of the present invention, when the angiogenesis-related disease or MMP-mediated disease is Alzheimer's disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, comprising a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0281] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to Alzheimer's disease, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0282] Angiogenesis-related diseases or MMP-mediated diseases: Periodontal diseases

[0283] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be a periodontal disease.

[0284] According to the present invention, the term "periodontal disease" refers to diseases that appear in the tissues surrounding the teeth, such as the gums, periodontal ligament, and alveolar bone.

[0285] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, which contains a fraction extract of Melissa leaf as an active ingredient, has a significant therapeutic effect on periodontal diseases by effectively regenerating the gingival tissue due to the increased expression of procollagen.

[0286] According to an embodiment of the present invention, when the angiogenesis-related disease or MMP-mediated disease is a periodontal disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, which contains a fraction extract of Melissa leaf as an active ingredient, contains the fraction extract of Melissa leaf in an amount of 20% to 80% by weight based on the total weight.

[0287] When the angiogenesis-related disease or MMP-mediated disease is a periodontal disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, which contains a fraction extract of Melissa leaf as an active ingredient, can be administered by a conventional method via an oral route, a rectal route, an intravenous route, an intra-arterial route, an intraperitoneal route, an intramuscular route, an intrasternal route, a percutaneous route, a topical route, an intraocular route, or an intradermal route, particularly via the oral route.

[0288] When the angiogenesis-related disease or MMP-mediated disease is a periodontal disease, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, which contains a fraction extract of Melissa leaf as an active ingredient, is preferably administered at a dose of 0.001 mg / kg to 50 mg / kg when administered once or several times a day.

[0289] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to periodontal disease, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0290] Angiogenesis-related diseases or MMP-mediated diseases: Diabetic retinopathy

[0291] According to embodiments of the present invention, the angiogenesis-related disease or MMP-mediated disease may be diabetic retinopathy.

[0292] According to the present invention, the term "diabetic retinopathy" refers to a condition in which the blood vessels in the retina of our eyes are damaged by diabetes.

[0293] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating diabetic retinopathy due to its excellent inhibitory effect on retinal vascular leakage.

[0294] When angiogenesis-related disease or MMP-mediated disease is diabetic retinopathy, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0295] When angiogenesis-related disease or MMP-mediated disease is diabetic retinopathy, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, local, intraocular, or intradermal routes, particularly by oral administration.

[0296] When angiogenesis-related disease or MMP-mediated disease is diabetic retinopathy, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0297] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to diabetic retinopathy, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0298] Angiogenesis-related diseases or MMP-mediated diseases: Sjögren's syndrome

[0299] According to embodiments of the present invention, the angiogenesis-related disease or MMP-mediated disease may be Sjögren's syndrome.

[0300] According to the present invention, the term "Sjögren's syndrome" refers to an autoimmune disease in which the body's immune system attacks glands that produce water (body fluids).

[0301] According to the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf as an active ingredient, is effective in preventing or treating Sjögren's syndrome by increasing the amount of saliva and tear secretion.

[0302] When the angiogenesis-related disease or MMP-mediated disease is Sjögren's syndrome, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0303] When the angiogenesis-related disease or MMP-mediated disease is Sjögren's syndrome, a pharmaceutical composition for the prevention or treatment of an angiogenesis-related disease or an MMP (matrix metalloproteinase)-mediated disease containing a fractionated extract of Melissa leaf as an active ingredient can be administered by a conventional method via an oral route, a rectal route, an intravenous route, an intra-arterial route, an intraperitoneal route, a intramuscular route, an intrasternal route, a percutaneous route, a topical route, an intraocular route, or an intradermal route, particularly via the oral route.

[0304] When the angiogenesis-related disease or MMP-mediated disease is Sjögren's syndrome, a pharmaceutical composition for the prevention or treatment of an angiogenesis-related disease or an MMP (matrix metalloproteinase)-mediated disease containing a fractionated extract of Melissa leaf as an active ingredient is preferably administered at a dose of 0.001 mg / kg to 50 mg / kg when administered once or several times a day.

[0305] Matters mentioned in the pharmaceutical composition for the prevention or treatment of an angiogenesis-related disease or a matrix metalloproteinase (MMP)-mediated disease are equally applicable to Sjögren's syndrome, which is an example of an angiogenesis-related disease or an MMP-mediated disease, as long as they do not contradict each other.

[0306] Angiogenesis-related diseases or MMP-mediated diseases: Glaucoma

[0307] According to an embodiment of the present invention, the angiogenesis-related disease or MMP-mediated disease can be glaucoma.

[0308] According to the present invention, the term "glaucoma" refers to a disease that takes the form of optic nerve atrophy and occurs in the optic nerve including retinal nerve plexus cells.

[0309] According to the present invention, a pharmaceutical composition for the prevention or treatment of an angiogenesis-related disease or an MMP (matrix metalloproteinase)-mediated disease containing a fractionated extract of Melissa leaf as an active ingredient is effective in preventing or treating glaucoma by significantly reducing elevated intraocular pressure.

[0310] When angiogenesis-related disease or MMP-mediated disease is glaucoma, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, contains the fractional extract of Melissa leaf in an amount of 20% to 80% by weight relative to the total weight.

[0311] When angiogenesis-related disease or MMP-mediated disease is glaucoma, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, can be administered by conventional methods via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transcutaneous, topical, intraocular, or intradermal routes, particularly by oral administration.

[0312] When angiogenesis-related disease or MMP-mediated disease is glaucoma, a pharmaceutical composition for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, containing a fractional extract of Melissa leaf as an active ingredient, is preferably administered once or several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0313] The matters referred to in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases are equally applicable to glaucoma, which is an example of angiogenesis-related disease or MMP-mediated disease, insofar as they do not contradict each other.

[0314] Food composition containing a fractional extract of lemon balm leaf as an active ingredient

[0315] Another object of the present invention is to provide a food composition containing a fractional extract of Melissa leaf containing caffeic acid, EDPA, RME, and rosmarinic acid as an active ingredient.

[0316] The fractional extract of lemon balm leaves and the method for preparing it are the same as described above.

[0317] According to embodiments of the present invention, this food composition may be used to prevent or improve angiogenesis-related diseases or MMP-mediated diseases.

[0318] According to embodiments of the present invention, angiogenesis-related disease or MMP-mediated disease may be, but is not limited to, any one of the following: obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, or glaucoma.

[0319] Cancer may include, but is not limited to, any one of the following: lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, gastric cancer, gastrointestinal cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, pediatric solid tumors, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, axial contractile tumor, brainstem glioma, or pituitary adenoma, particularly colorectal cancer.

[0320] Arthritis may be, but is not limited to, any one of the following: osteoarthritis, degenerative arthritis, osteochondritis dissecans, articular ligament injury, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis, particularly osteoarthritis and rheumatoid arthritis.

[0321] According to embodiments of the present invention, when a fractional extract of Melissa leaf is used as an additive in a food composition, it can be added as is or used together with other foods or food ingredients, and can be used as appropriate according to conventional methods. The amount of active ingredient may be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

[0322] Food compositions can be formulated in any form of a common food or beverage, as well as in powder, granule, pill, tablet, and capsule form.

[0323] There are no specific limitations regarding the type of food to which this substance can be added. Examples of foods to which this substance can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products (including ice cream), various soups, beverages, tea, alcoholic beverages, and vitamin complexes, and may include all foods in the conventional sense.

[0324] Generally, in the manufacture of food or beverages, a fractional extract of Melissa leaf may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for health and hygiene or health management, the amount may be less than or equal to the above range. Furthermore, since there are no safety concerns regarding the use of fractions from natural products in this invention, it may also be used in amounts exceeding that range.

[0325] The beverage in the food composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, similar to conventional beverages. The natural carbohydrates may include monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners, such as thaumatin and stevia extract, or synthetic sweeteners, such as saccharin and aspartame, or similar substances may be used. The proportion of natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0326] In addition to the above, the food composition according to the present invention contains various nutrients, vitamins, electrolytes, flavorings, colorings, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the food composition according to the present invention, which contains a fractional extract of lemon balm leaf as an active ingredient, may also contain natural fruit juice and fruit pulp for producing fruit juice and vegetable beverages. These components may be used individually or in combination. The proportions of these additives are not limited, but are generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food composition according to the present invention.

[0327] Methods for prevention or treatment

[0328] Another object of the present invention is to provide a method for preventing or treating angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0329] Specifically, depending on the type of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, the following preventive or therapeutic methods are provided.

[0330] According to embodiments of the present invention, a method for preventing or treating obesity is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0331] According to embodiments of the present invention, a method for preventing or treating age-related macular degeneration is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0332] According to embodiments of the present invention, a method for preventing or treating psoriasis is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0333] According to embodiments of the present invention, a method for preventing or treating endometriosis is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0334] According to embodiments of the present invention, a method for preventing or treating cancer is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0335] According to embodiments of the present invention, a method for preventing or treating arteriosclerosis is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0336] According to embodiments of the present invention, a method for preventing or treating arthritis is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0337] According to embodiments of the present invention, a method for preventing or treating inflammatory bowel disease is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0338] According to embodiments of the present invention, a method for preventing or treating Alzheimer's disease is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject in need of treatment.

[0339] According to embodiments of the present invention, a method for preventing or treating periodontal disease is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0340] According to embodiments of the present invention, a method for preventing or treating diabetic retinopathy is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject in need of treatment.

[0341] According to embodiments of the present invention, a method for preventing or treating Sjögren's syndrome is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject in need of treatment.

[0342] According to embodiments of the present invention, a method for preventing or treating glaucoma is provided by administering a therapeutically effective amount of a fractional extract of Melissa leaf to a subject requiring treatment.

[0343] According to embodiments of the present invention, the term "subject requiring treatment" refers to a mammal, including humans, and the term "administration" refers to providing a prescribed substance to a patient by any appropriate method. In the treatment method of the present invention, a pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising the melissa leaf fraction extract of the present invention as an active ingredient, may be administered in the conventional manner by oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes.

[0344] According to embodiments of the present invention, in a method for preventing or treating obesity, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0345] According to embodiments of the present invention, in a method for preventing or treating age-related macular degeneration, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0346] According to embodiments of the present invention, in a method for preventing or treating psoriasis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0347] According to embodiments of the present invention, in a method for preventing or treating endometriosis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0348] According to embodiments of the present invention, in a method for preventing or treating cancer, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0349] According to embodiments of the present invention, in a method for preventing or treating arteriosclerosis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0350] According to embodiments of the present invention, in a method for preventing or treating arthritis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0351] According to embodiments of the present invention, in a method for preventing or treating inflammatory bowel disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0352] According to embodiments of the present invention, in a method for preventing or treating Alzheimer's disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0353] According to embodiments of the present invention, in a method for preventing or treating periodontal disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0354] According to embodiments of the present invention, in a method for preventing or treating diabetic retinopathy, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0355] According to embodiments of the present invention, in a method for preventing or treating Sjögren's syndrome, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0356] According to embodiments of the present invention, in a method for preventing or treating glaucoma, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes, particularly via the oral route.

[0357] According to the present invention, the term “therapeutic dose” refers to the amount of an active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal, or human, as considered by researchers, veterinarians, physicians, or other clinicians, and includes the amount that elicits remission of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration of the active ingredient of the present invention may vary depending on the desired effect. Accordingly, the optimal dose to be administered can be easily determined by those skilled in the art, but may be adjusted by various factors (such as the type of disease, the severity of the disease, the content of the active ingredient and other ingredients in the composition, the type of formulation, as well as the patient’s age, weight, general health, sex and diet, administration time, route of administration and secretion rate of the composition, duration of treatment, and concomitant drugs).

[0358] In a method for preventing or treating obesity, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0359] In a method for preventing or treating age-related macular degeneration, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0360] In a method for preventing or treating psoriasis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0361] In a method for preventing or treating endometriosis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0362] In a method for preventing or treating cancer, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0363] In a method for preventing or treating arteriosclerosis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0364] In a method for preventing or treating arthritis, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0365] In a method for preventing or treating inflammatory bowel disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0366] In a method for preventing or treating Alzheimer's disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0367] In a method for preventing or treating periodontal disease, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0368] In a method for preventing or treating diabetic retinopathy, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0369] In a method for preventing or treating Sjögren's syndrome, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered to adults at a dose of 0.001 mg / kg to 50 mg / kg once or several times a day.

[0370] In adults, for a method of preventing or treating Sjögren's syndrome, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf extract of the present invention is preferably administered once to several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0371] In adults, in a method for preventing or treating glaucoma, which is an example of angiogenesis-related disease or MMP-mediated disease, the lemon balm leaf fraction extract of the present invention is preferably administered once to several times a day at a dose of 0.001 mg / kg to 50 mg / kg.

[0372] According to the present invention, the term "prevention" refers to any effect by which the administration of the fractional extract of Melissa leaves of the present invention suppresses or delays angiogenesis-related diseases or MMP-mediated diseases, such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma.

[0373] According to the present invention, the term "treatment" refers to any effect by which the administration of the fractional extract of Melissa leaf of the present invention improves or beneficially alters angiogenesis-related diseases or MMP-mediated diseases, such as obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma.

[0374] Use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases.

[0375] The present invention provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases.

[0376] Specifically, it provides the use of pharmaceutical compositions for preventing or treating angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, depending on the type of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease described below.

[0377] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of obesity, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0378] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of age-related macular degeneration, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0379] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of psoriasis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0380] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of endometriosis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0381] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of cancer, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0382] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of arteriosclerosis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0383] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of arthritis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0384] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of inflammatory bowel disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0385] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of Alzheimer's disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0386] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of periodontal disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0387] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of diabetic retinopathy, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0388] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of Sjögren's syndrome, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0389] According to embodiments of the present invention, the aim is to provide the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of glaucoma, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0390] Use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases.

[0391] The present invention provides the use of a pharmaceutical composition containing a fractionated extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases.

[0392] Specifically, it provides the use of a pharmaceutical composition containing a fractionated extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for the prevention or treatment of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, depending on the type of angiogenesis-related disease or MMP (matrix metalloproteinase)-mediated disease, as described below.

[0393] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating obesity, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0394] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating age-related macular degeneration, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0395] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a medicament for preventing or treating psoriasis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0396] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating endometriosis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0397] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating cancer, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0398] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating arteriosclerosis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0399] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating arthritis, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0400] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmacopoeia for the prevention or treatment of inflammatory bowel disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0401] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating Alzheimer's disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0402] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating periodontal disease, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0403] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating diabetic retinopathy, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0404] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating Sjögren's syndrome, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0405] According to embodiments of the present invention, it provides the use of a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmaceutical for preventing or treating glaucoma, which is an example of angiogenesis-related disease or MMP-mediated disease.

[0406] According to one embodiment of the present invention, a pharmaceutical composition comprising a fractional extract of Melissa leaf as an active ingredient for the manufacture of a pharmacopoeia for the prevention or treatment of angiogenesis-related disease or MMP-mediated disease may be mixed with an acceptable carrier or similar and may further contain other agents.

[0407] The pharmaceutical compositions, food compositions, treatment methods, and matters referred to in the use of the present invention shall apply equally to each other, insofar as they do not contradict each other.

[0408] [Advantageous effect]

[0409] The fractional extract of Melissa leaf of the present invention exhibits superior efficacy in the prevention and treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease compared to extracts of Melissa leaf obtained by other extraction methods. Therefore, compositions containing the fractional extract of Melissa leaf of the present invention as an active ingredient may be useful for the prevention or treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease, while having significantly fewer side effects and exhibiting superior pharmacological effects.

[0410] Specifically, the pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of non-alcoholic steatohepatitis, and is effective in preventing or treating non-alcoholic steatohepatitis by significantly inhibiting fibrosis and the protein expression levels of Col1A2, TGF-beta, IL-6, and IL-10.

[0411] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of non-alcoholic fatty liver disease, and is effective in preventing or treating non-alcoholic fatty liver disease by exhibiting inhibitory effects on liver fat accumulation, blood triglycerides, blood glucose, and total cholesterol.

[0412] As an active ingredient in pharmaceutical compositions for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, the fractional extract of Melissa leaf of the present invention is superior to fractional extracts of Melissa leaf extracted by other extraction methods in preventing or treating obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, and glaucoma. Therefore, a pharmaceutical composition containing a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases according to the present invention may be useful for preventing or treating obesity, age-related macular degeneration, psoriasis, endometriosis, cancer growth or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, or glaucoma, while having significantly fewer side effects and exhibiting superior pharmacological effects.

[0413] Specifically, the pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating obesity by showing a significant suppression of body weight, blood triglycerides, blood glucose, and total cholesterol.

[0414] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating age-related macular degeneration by suppressing the size of macular degenerative lesions.

[0415] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating psoriasis by suppressing significant epidermal thickness, inflammatory cell infiltration in ear tissue, and mRNA expression levels in ear tissue.

[0416] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating endometriosis by significantly reducing the size of uterine lesions.

[0417] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating cancer by significantly reducing the size of cancerous tumors.

[0418] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating arteriosclerosis by significantly inhibiting its increase in arteriosclerotic plaques.

[0419] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating arthritis by exhibiting excellent arthritis treatment effects and excellent weight-bearing effects.

[0420] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating inflammatory bowel disease by exhibiting a low disease activity index.

[0421] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating Alzheimer's disease by improving cognitive function.

[0422] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating periodontal diseases by effectively regenerating gingival tissue due to increased expression of procollagen.

[0423] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating diabetic retinopathy due to its excellent inhibitory effect on retinal vascular leakage.

[0424] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating Sjögren's syndrome by increasing the amount of saliva and tear secretion.

[0425] The pharmaceutical composition according to the present invention contains a fractional extract of Melissa leaf as an active ingredient for the prevention or treatment of angiogenesis-related diseases or matrix metalloproteinase (MMP)-mediated diseases, and is effective in preventing or treating glaucoma by significantly lowering elevated intraocular pressure. [Brief explanation of the drawing]

[0426] [Figure 1] Figure 1 shows the results of picrosilius red staining for detecting fibrosis. [Figure 2] Figure 2 is a graph showing SMA deposition in the liver. [Figure 3] Figure 3 is a graph showing the protein expression levels of Col1A2, which is known as a protein associated with liver fibrosis. [Figure 4] Figure 4 is a graph showing the protein expression levels of TGF-beta, which is known as a protein associated with hepatic fibrosis. [Figure 5] Figure 5 is a graph showing the protein expression levels of IL-6, which is known as a protein associated with liver fibrosis. [Figure 6] Figure 6 is a graph showing the protein expression levels of IL-10, which is known as a protein associated with hepatic fibrosis. [Figure 7] Figure 7 is a graph showing serum concentrations of AST and ALT. [Figure 8] Figure 8 is a graph showing the quantification of the degree of fat accumulation in the liver. [Figure 9] Figure 9 shows that rosmarinic acid, caffeic acid, RME, and EDPA as single components have angiogenic inhibitory effects, demonstrating the potent angiogenic inhibitory effect of EDPA at the same concentration. [Figure 10] Figure 10 confirms that among compositions containing EDPA, the angiogenesis inhibitory effect significantly increases as the EDPA content increases. [Figure 11] Figure 11 is a graph showing the changes in body weight of obese rats induced by a high-fat diet. [Figure 12] Figure 12 is a graph showing the changes in food intake in high-fat diet-induced obese rats. [Figure 13] Figure 13 is a graph showing the changes in blood triglyceride concentration in high-fat diet-induced obese rats. [Figure 14] Figure 14 is a graph showing the changes in blood glucose concentration in high-fat diet-induced obese rats. [Figure 15] Figure 15 is a graph showing the changes in total blood cholesterol concentration in high-fat diet-induced obese rats. [Figure 16] Figure 16 is a graph showing the size of choroidal neovascular lesions in a laser-induced CNV model. [Figure 17] Figure 17 is a graph showing the thickness of the epidermis of the excised ear tissue. [Figure 18]Figure 18 is a graph showing the volume of uterine lesions in an experimental model in which the fractional extract of melissa leaf according to the embodiment was administered to the treatment group and not to the control group. [Figure 19] Figure 19 is a graph showing the tumor size growth curve in nude mice injected with DLD1 cells. [Figure 20] Figure 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. [Figure 21] Figure 21 is a graph comparing the effects of treatments on arthritis in a collagen-induced arthritis (CIA) model. [Figure 22] Figure 22 is a graph showing the weight-bearing ratio measured in an osteoarthritis model induced by MIA. [Figure 23] Figure 23 is a graph showing the disease activity index (DAI) measured in an animal model of inflammatory bowel disease induced by dextran sulfate sodium (DSS). [Figure 24] Figure 24 is a graph showing reference memory during a water maze test when administered a fractional extract of Melissa leaf according to the embodiment. [Figure 25] Figure 25 is a graph showing the improvement rate of the probe test during the water maze test when the fractional extract of Melissa leaf according to the embodiment was administered. [Figure 26] Figure 26 is a graph showing the results of RT-PCR analyzing the gene expression levels of procollagen, which indicates tissue regeneration in a periodontal disease model induced by ligation. [Figure 27] Figure 27 is a graph showing the results of testing the retinal vascular leakage effect in a rat model of diabetic retinopathy induced by streptozotocin. [Figure 28] Figure 28 is a graph analyzing the amount of tear secretion using NOD / ShiLt, an animal model of Sjögren's syndrome. [Figure 29] Figure 29 is a graph showing the effect of hypertonic saline-induced scarring glaucoma on reducing intraocular pressure. [Modes for carrying out the invention]

[0427] The terminology used in the embodiments has been selected as far as possible as commonly used terms, taking into account the function of the present invention; however, these terms may vary depending on the intent of those skilled in the art, precedents, the emergence of new technologies, etc. In certain cases, there are terms arbitrarily selected by the applicant, in which case their meaning will be described in detail in the corresponding description of the invention. Therefore, the terminology used in the present invention should be defined based on the meaning of the terms and the overall content of the invention, rather than simply by their names.

[0428] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as they would be generally understood by those skilled in the art to whom this invention relates. Terms, such as those defined in commonly used dictionaries, should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not in an ideal or overly formal sense unless expressly defined herein.

[0429] A numerical range includes the values ​​defined within that range. All maximum numerical limits given through embodiments include all lower numerical limits as if lower numerical limits were explicitly stated. All minimum numerical limits given through embodiments include all higher numerical limits as if higher numerical limits were explicitly stated. Any numerical limit given through embodiments shall include all numerical ranges within a wider numerical range as if narrower numerical limits were explicitly stated.

[0430] Examples and manufacturing examples are provided to aid in understanding the present invention. The following examples and manufacturing examples are provided solely for a simpler understanding of the present invention and do not limit the scope of the present invention.

[0431] Examples

[0432] Example 1. Preparation of fractional extract of lemon balm leaf

[0433] 400.4 kg of dried lemon balm leaves (origin: Europe) were extracted under reflux at 81°C for 4 hours using 4000 L of 75% (v / v) ethanol. After 35 minutes of reflux extraction, the mixture was filtered through a 10 μm cartridge filter for 55 minutes to obtain 3100 L of the first extract.

[0434] Melissa leaf residue was extracted under reflux at 82°C for 4 hours using 4000 L of 75 (v / v) ethanol. After 35 minutes of reflux extraction, the mixture was filtered through a 10 μm cartridge filter for 55–65 minutes to obtain a 4100 L second extract.

[0435] 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.

[0436] <First concentration condition> Temperature: 56~58℃ Pressure: -0.066 to -0.070 MPa Time: 9 hours The first concentrate of 600 L was concentrated to obtain a second concentrate of 250 L.

[0437] <Second concentration condition> Temperature: 56~58℃ Pressure: -0.063 to -0.065 MPa Duration: 5 hours 50 minutes

[0438] 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 was added to this suspension to obtain a 900 L mixture.

[0439] 900 L of this mixture was stirred for 1 hour, then left to stand for 2 hours to separate the ethyl acetate layer (450 L) and obtain the first fraction.

[0440] Next, 450 μL of ethyl acetate (ratio of mixture to ethyl acetate (v / v) = 1:1) was added to the remaining suspension and stirred for 1 hour, then allowed to stand for 2 hours to separate the ethyl acetate layer (450 L) and obtain the second fraction.

[0441] After combining the first and second fractions (900 L), concentration was carried out on the mixture to obtain a third concentrated solution of 41 L (42.5 kg).

[0442] <Third concentration condition> Temperature: 58℃ Pressure: -0.064 MPa Duration: 3 hours 40 minutes

[0443] The third concentrate was collected in a stainless steel collector over a period of 20 minutes (42.5 kg).

[0444] Subsequently, 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 placed in a pulverizer and finely ground for 3 hours and 50 minutes to obtain a fractional extract of melissa leaves (yield: 18.9 kg), which is the ethyl acetate fraction of the ethanol extract of melissa leaves.

[0445] Example 2. Components of the fractionated extract of Melissa leaf

[0446] (1)Analysis method

[0447] The active ingredients of the fractionated extracts of lemon balm leaves were analyzed using high-performance liquid chromatography (HPLC) at a wavelength of 285 nm. The fractionated extracts of lemon balm leaves prepared above were injected into a Zorbax Eclipse Plus C18 (150 × 4.6 mm) column, and mobile phase A (5% formic acid aqueous solution) and mobile phase B (methanol) were delivered at a flow rate of 1 ml per minute according to the ratios shown in Table 1 below. [Table 1]

[0448] (2) Results

[0449] The fractional extract of Melissa leaves obtained according to Example 1 contained caffeic acid, EDPA, RME, and rosmarinic acid according to the analytical method described above, with their content being as follows: Caffeic acid: 0.96% by weight EDPA: 0.71% by weight RME: 0.11% by weight Rosmarinic acid: 16.1% by weight

[0450] Furthermore, rutin was not detected in the HPLC results of the melissa leaf fraction extract obtained according to Example 1. Therefore, it was found that the melissa leaf fraction extract obtained according to Example 1 does not contain rutin.

[0451] Experimental example

[0452] Experimental Example 1. Inhibitory effect of lemon balm leaf extract on non-alcoholic steatohepatitis in a methionine / choline-deficient high-fat diet model.

[0453] (1) Experimental method

[0454] The therapeutic effect of fractionated lemon balm leaf extract, prepared according to the examples, on non-alcoholic steatohepatitis was evaluated in a methionine / choline-deficient, high-fat diet animal model.

[0455] This study was conducted using C57Bl / 6 mice fed a methionine / choline-deficient high-fat diet (MCD-HFD). The mice were divided into a treatment group administered with a fractional extract of lemon balm leaf as described in the example, and a control group administered with 0.5% CMC.

[0456] Regarding the administration method, MCD-HFD was fed while a fractional extract of Melissa leaf, prepared according to the example, was orally administered once daily at a dose of 200 mg / kg. The control group received 0.5% CMC at the same frequency. The administration period lasted for 9 weeks. In the normal group, non-alcoholic steatohepatitis was not induced by feeding a normal diet.

[0457] To assess 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 incubated in 0.5% thiosemicarbazide for 10 minutes. These sections were then stained with 0.1% Sirius Red F3B in saturated picric acid for 1 hour, followed by washing with 0.5% acetic acid. To observe myofibroblasts, a marker of liver fibrosis, immunohistochemical tests were performed using alpha-smooth muscle actin (alpha-SMA) antibody. Paraffin-embedded liver tissue was cut into 4 μm sections, mounted on poly-L-lysine-treated glass slides, deparaffinized, and sequentially immersed in ethanol. After treatment in 3% H2O2 / methanol solution for 10 minutes to remove endogenous peroxidase, the samples were reacted with normal goat serum in a humidifier for 40 minutes to eliminate nonspecific reactions, and then washed with 0.01 M phosphate-buffered saline (PBS, pH 7.4) containing 0.05% skim milk powder and 0.3% Triton X-100. Primary antibodies were reacted for 1 hour each, and secondary antibodies for 60 minutes, followed by subsequent color development. Western blotting 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 proteins were extracted. The extracted proteins were quantified using a BCA assay kit, mixed with SDS gel loading buffer, and boiled at 100°C for 5 minutes for denaturation. Proteins were electrophoresed on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was then left in 5% skim milk for 1 hour to block nonspecific protein binding. Standard immunoblotting was performed by treating the proteins with antibodies against Col1A2, TGF-beta, IL-6, IL-10, and beta-actin as primary antibodies. The intensity of the resulting bands was then measured and graphed using gel documentation software.

[0458] (2) Results

[0459] Figure 1 shows the results of picrosilius red staining to detect fibrosis. Referring to Figure 1, fibrosis induced by a methionine / choline-deficient, high-fat diet in an animal model of non-alcoholic steatohepatitis was inhibited in the treatment group that received oral administration of a fractional extract of Melissa leaf as described in the example.

[0460] Figure 2 is a graph showing SMA expression in the liver. Referring to Figure 2, fibrosis induced by a methionine / choline-deficient, high-fat diet in an animal model of non-alcoholic steatohepatitis was inhibited in the treatment group that received oral administration of a fractional extract of Melissa leaf as described in the example.

[0461] Figures 3-6 are graphs showing the protein expression levels of Col1A2, TGF-beta, IL-6, and IL-10, which are known proteins associated with liver fibrosis. Referring to Figures 3-6, it was found that all of these proteins were increased in the control group, and that the increased expression levels of these proteins were inhibited in the treatment group by treatment with the melissa leaf extract according to the example.

[0462] Experimental Example 2. Inhibitory effect of lemon balm leaf extract on non-alcoholic fatty liver disease in a high-fat diet model.

[0463] (1) Experimental method

[0464] As experimental animals, 30 SD rats were randomly divided into three groups of 10 rats each after a one-week acclimatization period.

[0465] For each of the three groups, the group fed a standard solid diet (purchased from Saeron Bio) was divided into a normal group, the group fed a high-fat diet (HFD) and administered the fractionated extract of melissa leaf used in the example was divided into a treatment group, and the group fed a high-fat diet (HFD) and administered 0.5% CMC was divided into a control group.

[0466] The normal group was fed a standard solid diet. The treatment group was fed HFD instead of the standard solid diet, and while being fed HFD, they were orally administered a fractional extract of Melissa leaf, as described in the example, once daily at a dose of 100 mg / kg. The same experiment was performed on the control group, except that 0.5% CMC was administered instead of the fractional extract of Melissa leaf as described in the example. The administration period was 10 weeks.

[0467] To measure serum AST and ALT levels in the normal, treatment, and control groups, blood samples were collected from the abdominal aorta by rat necropsy. The collected blood samples were centrifuged to obtain serum. Serum levels of AST and ALT were measured using an automated biochemical analyzer.

[0468] Three rats were randomly selected from each of the following groups: a normal group, a treatment group, and a control group. The degree of fat accumulation in the liver tissue of each group was determined. For each selected rat, four slides stained with oil red were prepared from frozen liver tissue, and the degree of fat accumulation in the liver tissue was quantified using the ImageJ analysis program.

[0469] (2) Results

[0470] Figure 7 shows the measured serum levels of AST and ALT (unit: IU / L) for the normal group, treatment group, and control group. Referring to Figure 7, the normal group showed low serum levels of AST and ALT, confirming that fatty liver did not occur in the normal group. The treatment group, which was administered the fractionated extract of Melissa leaves according to the example, showed lower serum levels of AST and ALT than the control group, confirming that fatty liver was inhibited compared to the control group.

[0471] Figure 8 is a graph showing the quantification of the degree of fat accumulation in the treatment group and the control group, respectively, using the analysis program ImageJ. Referring to Figure 8, it was confirmed that the degree of fat accumulation in liver tissue was not significant in the normal group. The treatment group administered with the fractionated extract of Melissa leaves according to the example showed suppressed fat accumulation in liver tissue compared to the control group administered with CMC.

[0472] Experimental Example 3. Inhibitory effect on angiogenesis

[0473] To confirm the inhibitory effect of the fractional extract of Melissa leaves on angiogenesis, as described in the examples, experiments were conducted using the following method.

[0474] (1) Experimental method

[0475] 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 after 1 hour, the test substances listed in Tables 2 and 3 below were added to a final concentration of 50 μg / ml, followed by 5 × 10⁻⁶ 4 Each HUVEC (human umbilical vein endothelial cell) was added to a well, and photographs were taken the following day. [Table 2] [Table 3] (2) Results

[0476] 1) Effects of single components (rosmarinic acid, caffeic acid, RME (methyl rosmarinic acid), and EDPA (ethyl 2-(3,4-dihydroxyphenyl acetate)) on HUVEC tube formation and angiogenesis inhibitory activity assays.

[0477] As shown in Figure 9, rosmarinic acid, caffeic acid, RME, and EDPA, which are single components contained in the fractionated extract of Melissa leaves, all exhibited angiogenesis inhibitory activity in the HUVEC tube formation assay. However, EDPA showed the highest angiogenesis inhibitory activity at the same concentration by most effectively inhibiting tube formation.

[0478] 2) Angiogenesis inhibitory activity of compositions containing EDPA

[0479] As shown in Figure 10 and Table 4, when angiogenesis inhibitory activity was measured in fractional extracts of Melissa leaves with different EDPA content (see Table 3), it was confirmed that the higher the EDPA content, the more significantly the angiogenesis inhibitory activity increased. [Table 4]

[0480] Experimental Example 4. MMP Inhibitory Activity

[0481] To confirm the MMP (matrix metalloproteinase) inhibitory activity of the fractional extract of Melissa leaf according to the examples, the following experiment was conducted.

[0482] (1) Experimental method

[0483] MMP inhibitory activity was measured using a spectrofluorometer (PerkinElmer LS50B) to determine whether the fractional extract of Melissa leaf had an inhibitory effect on MMPs. MMP-2 and MMP-9 were activated with 1 mM APMA (p-aminophenylmercury acetate) before use for activity measurement. The fluorescent substrate for MMP-2 / MMP-9 (Calbiochem) was used as the substrate for MMP-2 and MMP-9. As a control, 2 ml of buffer [50 mM tricine (pH 7.5), 10 mM CaCl2, 200 mM NaCl] containing 1 μM of the substrate was added to a 2 ml cuvette, to which MMP-2 or -9 was added. Fluorescence intensity was measured at 2-minute intervals over 20 minutes at room temperature using a spectrofluorometer with an excitation wavelength of 328 nm and an emission wavelength of 400 nm. In the treatment group, each test substance listed in Table 3 was added to a buffer containing the substrate and MMP in the same manner as in the control group until a final concentration of 20 μg / ml was reached. The fluorescence intensity was then measured at 2-minute intervals over 20 minutes at room temperature. (2) Results [Table 5]

[0484] Referring to Table 5, it was confirmed that in compositions containing EDPA, the inhibitory activity of MMP-2 and MMP-9 increased significantly as the EDPA content increased.

[0485] Experimental Example 5. Anti-obesity effect in high-fat diet-induced obese rats.

[0486] (1) Experimental method

[0487] The anti-obesity effect of the fractional extract of Melissa leaf prepared according to the examples was evaluated in an animal model of obesity induced by a high-fat diet.

[0488] Obesity was induced in male Sprague-Dawley (SD) rats (JoongAng Experimental Animals Co., Ltd.) by feeding them a high-fat diet (Saeron Bio Co., Ltd.). The rats were then divided into a treatment group administered a fractional extract of Melissa leaf as described in the examples, and a control group administered 0.5% carboxymethylcellulose (CMC).

[0489] The control and treatment groups were acclimatized to the environment for one week on a standard diet (Saeron Bio Co., Ltd.) before being fed the experimental high-fat diet, and then fed the experimental diet for 10 weeks.

[0490] For administration, a fractional extract of Melissa leaf, as described in the examples, was orally administered once daily at a dose of 100 mg / kg, while 0.5% CMC was administered to the control group. Body weight and food intake were measured twice a week on the same day. For food intake measurement, a fixed amount of food was filled into a powder food box at 3 p.m., the total weight was measured, and the weight of the reduced food was measured at 3 p.m. the following day.

[0491] This was calculated as the amount of food consumed over a 24-hour period and was performed consistently throughout the test. Blood samples were collected at 5 and 10 weeks from the start of the test. Blood was collected by ocular hemorrhage, in which a heparinized glass capillary was inserted into the orbital venous plexus of SD rats after anesthesia with ether. Total cholesterol, triglycerides, and insulin concentrations were measured from the collected blood.

[0492] (2) Results

[0493] Figure 11 is a graph showing the changes in body weight in high-fat diet-induced obese rats. Referring to Figure 11, continuous weight gain was observed in the control group, and this weight gain continued until week 10, the end of the experiment. However, in the treatment group, body weight significantly decreased in high-fat diet-induced obese rats.

[0494] Figure 12 is a graph showing changes in food intake in high-fat diet-induced obese rats. Referring to Figure 12, no significant changes in food intake were observed in either the control or treatment group.

[0495] Figure 13 is a graph showing the changes in blood triglyceride concentrations in high-fat diet-induced obese rats. Referring to Figure 13, blood triglyceride levels were significantly reduced in the treatment group in high-fat diet-induced obese rats. In particular, blood triglyceride levels did not increase in the experimental group, even after 10 weeks of high-fat diet intake.

[0496] Figure 14 is a graph showing the changes in blood glucose concentration in high-fat diet-induced obese rats. As shown in Figure 14, blood glucose levels were significantly lower in the treatment group of high-fat diet-induced obese rats.

[0497] Figure 15 is a graph showing the changes in total cholesterol blood concentration in high-fat diet-induced obese rats. As shown in Figure 15, total cholesterol was significantly reduced in the treatment group in high-fat diet-induced obese rats.

[0498] Experimental Example 6. Inhibitory effect of lemon balm leaf extract on age-related macular degeneration in a laser-induced choroidal angiogenesis (CNV) model.

[0499] (1) Experimental method

[0500] The therapeutic effect of a fractional extract of Melissa leaf, as described in the examples, on age-related macular degeneration was evaluated in a laser-induced choroidal neovascularization (CNV) animal model.

[0501] A laser-induced CNV model was created in 6-week-old C57BL / 6 mice by damaging the retinal optic nerve region using a diode green laser (532 nm, 150 mW, 0.1 sec).

[0502] In the laser-induced CNV model, the study was conducted by dividing participants into a treatment group administered with a fractional extract of Melissa leaf as described in the examples, and a control group administered with 0.5% CMC.

[0503] A fractional extract of Melissa leaf, prepared according to the examples, was orally administered once daily at a dose of 100 mg / kg two days prior to laser treatment, while 0.5% CMC was administered to the control group. The treatment period continued for 10 days after the laser treatment.

[0504] Ten days after laser treatment, retinal-choroidal flat mounts were performed to confirm the effect of reducing the size of CNV lesions. Mice were anesthetized and injected retroorbitally with 25 mg / ml of FITC-dextran. After 30 minutes, the mice were euthanized, the eyes were removed, fixed in 10% formalin, the cornea and lens were removed, and flat-mounted on coverslips. The size of macular degeneration lesions stained with FITC-dextran was measured.

[0505] (2) Results

[0506] Figure 16 is a graph showing the size of macular degeneration lesions in the laser-induced CNV model. As shown in Figure 16, the size of macular degeneration lesions increased in the control group after laser treatment, and the size of neovascularization was significantly reduced in the treated group in the laser-induced choroidal neovascularization model.

[0507] Experimental Example 7. Inhibitory effect of lemon balm leaf extract on psoriasis in an IL-23-induced psoriasis animal model.

[0508] (1) Experimental method

[0509] The effects of the fractionated lemon balm leaf extract, prepared according to the examples, on psoriasis were evaluated in an IL-23-induced psoriasis animal model according to Ma's method [J. Clin. Cell Immunol. 4:6.(2013)].

[0510] An IL-23-induced psoriasis animal model was prepared, in which psoriasis was induced by intradermal injection of 500 ng of IL-23 into the ear of C57BL / 6 mice (6 weeks old, Orient Bio) every other day for 16 days.

[0511] This study was conducted in an IL-23-induced psoriasis animal model by dividing the subjects into three groups: a treatment group administered with a fractional extract of Melissa leaf as described in the examples, a control group administered with 0.5% CMC, and a PBS control group treated with PBS instead of IL-23.

[0512] For administration, a fractional extract of Melissa leaf, as described in the examples, was orally administered once daily at a dose of 100 mg / kg, while 0.5% CMC was administered to the control group. Oral administration began one day before IL-23 infusion and continued for a total of 16 days, concluding the experiment on the 17th day after the start of administration.

[0513] After the experiment was completed, the thickness of the mice's ears was measured, and the results are shown in Figure 17.

[0514] (2) Results

[0515] Figure 17 is a graph showing the epidermal thickness of excised ear tissue. Referring to Figure 17, epidermal thickness was significantly reduced in the treatment group in the IL-23-induced psoriasis animal model.

[0516] Experimental Example 8. Inhibitory effect of lemon balm leaf extract on endometrial proliferation in an endometrial autotransplantation model.

[0517] (1) Experimental method

[0518] After raising the SD rats, bedding soaked in male urine was placed in the females' cages every five days to coincide with the females' estrous cycle. Vaginal cytology was performed daily for one week prior to surgery to confirm the menstrual cycle.

[0519] Before the surgery, the surgical site was shaved with an electric razor and disinfected with 70% ethanol. An incision was made approximately 0.5 cm to 1.0 cm from the vaginal opening to expose the left uterus. The two sections of the exposed left uterus, 6-8 cm from the opening, were tightly tied together with 5-0 sutures. The uterus was cut between the tied sections and placed in a sterile glass petri dish with 100 μL of PBS containing penicillin (100 U / ml) and streptomycin (100 μg / ml). After removing the fat, the uterus was cut longitudinally and divided into three 2 mm sections.

[0520] After locating and withdrawing the intestine from the incision site, the intestine was spread out on pre-moistened gauze, the three intermesenteral arteries were identified, and the uterine fragment was gently sutured. Next, the surgically removed intestine was inserted into the abdominal cavity, the abdominal wall was sutured with suture thread, and the skin was closed with autoclips. Postoperatively, heat was applied to the SD rats to prevent hypothermia. The surgical site was disinfected with povidone, and acetaminophen (an analgesic) was administered at a concentration of 2 mg / ml mixed with drinking water.

[0521] Autoclips were removed on the 7th day post-surgery, and a fractional extract of Melissa leaf, prepared according to the example, was orally administered once daily for 4 weeks to the treatment group (n=5). The dose of the fractional extract of Melissa leaf was 100 mg / kg. The groups were divided to ensure that the average size of uterine lesions was the same in each group.

[0522] 0.5% CMC was orally administered to the control group (n=5).

[0523] Four weeks after surgery, the SD rats were euthanized and their peritoneum was incised. Next, the endometriotic lesions transplanted into the mesentery were isolated and their size and weight were measured.

[0524] (2) Results

[0525] Figure 18 is a graph showing the volume of endometriotic lesions transplanted into the mesentery in an experimental model in which the fractional extract of Melissa leaves from the example was administered to the treatment group and not to the control group. Figure 18 shows the average volume of endometriotic lesions in the treatment group and the control group.

[0526] Table 6 shows the size and volume of endometriotic lesions transplanted into the mesentery in the treatment group and the control group, respectively. [Table 6]

[0527] Referring to Table 6, the average volume of endometriotic lesions transplanted into the mesentery in the treatment group was approximately 1.73 mm². 3 The standard deviation was approximately 0.38. The mean volume of endometrial lesions in the control group was approximately 4.83 mm³. 3 The standard deviation was approximately 0.61. Referring to Figure 18 and Table 6, it was confirmed that the volume of endometriotic lesions in the treatment group was significantly smaller than that in the control group. As a result, it was confirmed that the fractional extract of Melissa leaves prepared according to the example has an excellent inhibitory effect on the proliferation of endometrial tissue.

[0528] Experimental Example 9. Inhibitory effect of lemon balm leaf extract on cancer growth in a tumorigenesis model in nude mice.

[0529] (1) Experimental method

[0530] The anticancer effect of the fractional extract of Melissa leaf prepared according to the examples was evaluated in a tumorigenicity model using nude mice.

[0531] Colorectal cancer cells (DLD1) were cultured in RPMI-1640 medium, and 6-week-old male nude mice (Orient Bio) were housed in a sterile facility. The cultured DLD1 cells were resuspended in DMEM medium and 5 × 10⁻⁶ cells were used. 6 Individual cells were subcutaneously injected into the right flank of nude mice, and these mice were divided into a treatment group administered with a fractional extract of Melissa leaf as described in the example, and a control group administered with 0.5% CMC.

[0532] For administration, a fractional extract of Melissa leaf was orally administered once daily at a dose of 100 mg / kg starting from the day of tumor injection, while 0.5% CMC was administered to the control group. The administration period was 28 days, and tumor size was measured every 3 days using calipers.

[0533] (2) Results

[0534] Figure 19 shows a graph illustrating the tumor size growth curve in nude mice injected with DLD1 cells. In control rats, tumors grew exponentially until day 28; however, in the treatment group treated with oral administration of a fractional extract of Melissa leaves as described in the example, tumor size was significantly reduced, demonstrating a strong anti-cancer effect.

[0535] Experimental Example 10. Inhibitory effect of lemon balm leaf extract on arteriosclerosis in an apolipoprotein E (ApoE)-deficient mouse model.

[0536] (1) Experimental method

[0537] The anti-atherosclerotic effect of lemon balm leaf fraction extracts prepared according to the examples was evaluated in an ApoE-deficient mouse model. Mice in which atherosclerosis was induced by a 16-week high-fat diet were selected, and mice in which atherosclerosis was not induced by a standard diet instead of the high-fat diet were classified as the normal group.

[0538] The control group was administered 0.5% CMC, while the treatment group was administered a fractional extract of lemon balm leaf prepared according to the example.

[0539] Mice were given free access to food and water for 16 weeks. A fractional extract of Melissa leaf, prepared according to the example, was orally administered once daily at a dose of 100 mg / kg to the treatment group of mice in which arteriosclerosis had been induced by a high-fat diet for 16 weeks, while 0.5% CMC was administered to the control group.

[0540] After 16 weeks, the mice were sacrificed, and the heart and ascending aorta to the thoracic aorta were excised and fixed in 10% neutral buffered formalin solution. After trimming the fixed tissue with a razor, it was embedded in a cryogenic tissue embedding medium (OCT compound) and frozen in a deep freezer. Slides were prepared by cutting the aortic arch into 10 μm thick sections using a cryostat microtome. For fat staining, the slides were immersed in distilled water, treated with anhydrous propylene glycol for 1 minute, stained in oil red solution for 16 hours, and then treated with 85% propylene glycol for 2 minutes. The slides were washed with distilled water, sealed with aqueous mounting medium, and observed under a light microscope.

[0541] (2) Results

[0542] Figure 20 is a graph showing the results of measuring and evaluating the formation of atherosclerotic plaques in the aorta of 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 that the increase in atherosclerotic plaques was significantly inhibited in the treatment group.

[0543] Experimental Example 11. Efficacy of Melissa Leaf Fractionation Extract in an Animal Model of Collagen-Induced Arthritis

[0544] (1) Experimental method

[0545] The efficacy of the lemon balm leaf fraction extract prepared according to the examples was evaluated in a collagen-induced arthritis model.

[0546] A collagen-induced arthritis (CIA) animal model was created by inducing an immune response through injection of collagen (which is thought to be a cause of rheumatoid arthritis).

[0547] 4 mg of chicken type II collagen was mixed with 1 ml of 100 mM acetic acid and dissolved at 4°C for 1 day. This was then 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 tails of 6-week-old Lewis rats for immunization. Seven days after primary immunization, a secondary immunization (boost) response was induced by another intradermal injection of chicken type II collagen. Symptoms of arthritis were observed 1-2 weeks after secondary immunization.

[0548] The control group was administered 0.5% CMC, while the treatment group was administered a fractional extract of lemon balm leaf prepared according to the example.

[0549] A fractional extract of Melissa leaf, prepared according to the examples, was orally administered once daily at a dose of 100 mg / kg for 47 days starting the day after the induction of a secondary immune response, while 0.5% CMC was administered to the control group.

[0550] To verify the therapeutic effect, the degree of swelling and erythema of the joints and nodules of each rat's leg was observed, and a clinical score was calculated according to the score index shown in Table 7 below. [Table 7]

[0551] (2) Results

[0552] Figure 21 is a graph comparing the effects of treatment on arthritis in a collagen-induced arthritis (CIA) model. Referring to Figure 21, symptoms of arthritis appeared approximately 20 days after the CIA-induced immune response. The treatment group showed a significant therapeutic effect in the collagen-induced arthritis model.

[0553] Experimental Example 12. Efficacy of Melissa Leaf Fractionation Extract in an Animal Model of MIA (Monosodium Iodoacetate)-Induced Osteoarthritis

[0554] (1) Experimental method

[0555] The efficacy of the fractional extract of Melissa leaf prepared according to the examples was evaluated in a model of MIA-induced osteoarthritis.

[0556] Osteoarthritis was induced and selected in 7-week-old SD rats by injecting 50 μl of MIA diluted to a concentration of 60 mg / ml in 0.9% saline into the joint cavity of the right hind limb.

[0557] 0.5% CMC was administered to the control group, and a fractional extract of lemon balm leaf prepared according to the example was administered to the treatment group.

[0558] For administration, a fractional extract of lemon balm leaf was orally administered once daily at a dose of 100 mg / kg for 21 days, while 0.5% CMC was administered to the control group. Body weight load was measured at 7-day intervals after administration. The group that did not develop osteoarthritis was classified as the normal group.

[0559] The weight-bearing capacity of the hind legs was measured using an Incapacitance tester. In the tester holder, osteoarthritis-induced rats stood on their normal hind legs, which were not treated with MIA due to pain. As a result, the weight balance of both legs was disrupted, and the weight of the leg treated with MIA was relatively lighter compared to the weight of the normal leg. When measuring leg weight, the weight (g) of both legs was measured with the abdomen of the SD rat not touching the device's sensor.

[0560] The weight load percentage (%) was calculated using the measured leg weights according to Equation 1 below.

[0561] [Equation 1] Weight load percentage (%) = [Weight of the hind limb that induces osteoarthritis / (Weight of both hind limbs)] × 100

[0562] (2) Results

[0563] Figure 22 is a graph showing the weight-bearing rate measured in an osteoarthritis model induced by MIA. As shown in Figure 22, the weight-bearing rate (%) in the control group decreased statistically significantly over time compared to the normal group. In contrast, the treatment group showed a superior weight-bearing effect (therapeutic effect on osteoarthritis) by significantly increasing the reduced weight-bearing rate.

[0564] Experimental Example 13. Inhibitory effect of lemon balm leaf extract on dextran sulfate sodium (DSS)-induced inflammatory bowel disease.

[0565] (1) Experimental method

[0566] The therapeutic effect of lemon balm leaf fraction extracts, prepared according to the examples, on ulcerative colitis was evaluated in an animal model induced by dextran sulfate sodium.

[0567] Seven-week-old C57BL / 6J mice (Central Lab. Animal Inc.) were given free access to food and water for one week to allow for acclimatization, and then randomly divided into a control group, a normal group, and a treatment group.

[0568] A fractional extract of Melissa leaf, prepared according to the examples, was orally administered to the treatment group at a dose of 200 mg / kg once daily for 11 days after group assignment, while 0.5% CMC was administered to the control group. Inflammatory bowel disease was induced by adding 3% DSS to drinking water instead of water starting from the third day after group assignment. On the other hand, inflammatory bowel disease was not induced in the normal group because DSS was not administered.

[0569] During the experimental period, the appearance of the stool was observed once a day by inducing defecation in the mice, and the presence or absence of occult blood was observed using a Coulter Hemocult single slide, with the score indicated according to the criteria in Table 8 below. [Table 8] [Formula 2] DAI (Disease Activity Index) = Stool concentration + Stool color

[0570] (2) Results

[0571] Figure 23 is a graph showing the disease activity index (DAI) measured in an animal model of inflammatory bowel disease induced by DSS. In this case, the normal group's DAI is represented as 0. When the DAI of rats with ulcerative colitis induced by DSS was measured, the control group showed a high DAI, while the treatment group showed a significant therapeutic effect with a low DAI score.

[0572] Experimental Example 14. Efficacy of improving cognitive function in an animal model of scopolamine-induced dementia.

[0573] (1) Experimental method

[0574] For cognitive function testing, five 9-week-old male ICR mice were used in each group. The treatment group was administered a fractional extract of lemon balm leaves, while the control group was administered physiological saline for two weeks.

[0575] The treatment group received an oral dose of 200 mg / kg seven times a week for two weeks, 60 minutes before the start of the test. Both the treatment and control groups received oral scopolamine at a dose of 1 mg / kg to induce memory impairment, and then underwent the water maze test.

[0576] The reference memory test during the water maze test was conducted after an adaptation period of 60 seconds of free swimming in a tank without a platform, one day prior to the start of the test. The reference memory test was performed by measuring the escape latency (in seconds) to find the submerged platform 4-5 times a day for 5 days, with a maximum allowable time limited to 60 seconds. If the platform location was not found by the second day of the test, the mouse was guided to find the platform within the 60-second time limit, and once the mouse was on the platform, it was allowed to remain there for 10 seconds.

[0577] Twenty-four hours after the completion of the reference memory test, the probe test was performed by removing the platform from the tank, allowing the mice to swim freely for 60 seconds, and measuring the time the mice stayed in the area where the platform had been.

[0578] A reference memory test was administered over 5 days following administration of the melissa leaf fraction extract according to the example, and the average time to find the platform was compared for the treatment group and the control group.

[0579] (2) Results

[0580] Referring to Figure 24, administration of the fractional extract of Melissa leaves prepared according to the example demonstrated an effect on improving cognitive function (37.2 ± 2.1).

[0581] In the probe test conducted after the reference memory test was completed, the average time spent on the platform in the control group was compared with the average time spent in the treatment group administered the melissa leaf fraction extract according to the example, and the improvement rate was expressed. [Formula 3] Probe test improvement rate = (tA - ts) / ts × 100 (%)

[0582] ts is the average time the control group stayed on the platform, and tA is the average time the treatment group, administered the fractional extract of Melissa leaves according to the example, stayed on the platform.

[0583] Referring to Figure 25, 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 a superior improvement rate compared to the control group.

[0584] Experimental Example 15. Inhibitory effect on periodontal disease in a ligation-induced periodontal disease model.

[0585] (1) Experimental method

[0586] Seven-week-old male SD rats were used as experimental animals. They were given free access to general laboratory food and water, and after a one-week acclimatization period, they were used in the experiments.

[0587] Periodontal disease was induced by ligating the mandibular first molar with sterile suture (3-0 nylon thread) after general anesthesia. After examining only the neck area, rats that were not ligated were classified as the normal group.

[0588] Among the periodontal disease induction group, the treatment group was administered a fractional extract of Melissa leaf prepared according to the example.

[0589] The treatment group received oral administration at a dose of 100 mg / kg once daily, while the control group received 0.5% CMC.

[0590] After slaughtering the experimental animals, the surrounding tissue of the excised mandible was removed, and then the gene expression levels of procollagen, which indicates tissue regeneration, were examined.

[0591] In periodontal disease, the regeneration and restoration of periodontal tissue made of collagen is important. The gene expression levels of procollagen from the gums were confirmed by RT-PCR.

[0592] cDNA was synthesized using RT-PCR (reverse transcriptase polymerase chain reaction) from 10 μg of RNA obtained by homogenizing extracted gum tissue using the Trizole method. The nucleotide sequences of the PCR primers for each gene are shown in Table 9 below. [Table 9]

[0593] Figure 26 is a graph showing the results of RT-PCR analyzing the gene expression levels of procollagen, which indicates tissue regeneration in a periodontal disease model induced by ligation. Referring to Figure 26, administration of the fractional extract of Melissa leaves according to the example increased procollagen expression and effectively regenerated gingival tissue, thereby showing a significant therapeutic effect against periodontal disease.

[0594] Experimental Example 16. Inhibitory effect of lemon balm leaf extract on diabetic retinopathy in a streptozotocin (STZ)-induced diabetic retinopathy rat model.

[0595] (1) Experimental method

[0596] The therapeutic effects of the lemon balm leaf fraction extract prepared according to the examples were evaluated in a rat model of streptozotocin-induced diabetic retinopathy.

[0597] Streptozotocin solution (100 mM) dissolved in citrate buffer (100 mM, pH 4.5) was injected into the peritoneal cavity of rats at a dose of 150 mg / kg, and 10% sucrose was adequately supplied to prevent hypoglycemic shock. Two days later, blood glucose levels were measured using a blood glucose meter, and diabetic animals that maintained a non-fasting blood glucose level of 300 mg / dl or higher for 1-2 weeks were used.

[0598] In diabetic rats, 0.5% CMC was administered to the control group, and a fractional extract of Melissa leaf prepared according to the example was administered to the treatment group. Diabetes was not induced in the normal group.

[0599] For the administration method, diabetic rats were selected and orally administered a fractional extract of Melissa leaf, as described in the examples, once daily at a dose of 100 mg / kg for 16 weeks starting 2 weeks after streptozotocin administration. 0.5% CMC was administered to the control group. After 16 weeks, to quantitatively analyze retinal vascular leakage, 1.25 mg of 500 kDa FITC-dextran (Sigma-Aldrich) was injected into the left ventricle of the rats and stained with circulating blood for 5 minutes. The eyes were excised and immediately fixed with 4% paraformaldehyde for 45 minutes. The retina was cut from the fixed eyeball, cut in the shape of a Maltese cross, placed on a glass slide, and then observed using a confocal microscope. Retinal vascular leakage was quantified by measuring the intensity of FITC-dextran exuding from the entire retinal tissue. (2) Results

[0600] Figure 27 shows the results of testing the inhibitory effect on retinal vascular leakage in a rat model of streptozotocin-induced diabetic retinopathy. As can be seen in Figure 27, retinal vascular leakage was high in the control group, and it was confirmed that retinal vascular leakage was significantly inhibited in the treatment group in the rat model of streptozotocin-induced diabetic retinopathy.

[0601] Experimental Example 17. Efficacy of Melissa Leaf Fractionation Extract in an Animal Model of Sjögren's Syndrome

[0602] (1) Experimental method

[0603] The experiment was conducted using NOD / ShiLt mice (an animal model of Sjögren's syndrome).

[0604] In NOD / ShiLt (an animal model of Sjögren's syndrome), 0.5% CMC was administered to the control group, and a fractional extract of Melissa leaf, prepared according to the example, was administered to the treatment group.

[0605] Regarding the administration method, tear secretion in mice was analyzed after administering a fractional extract of lemon balm leaf at a dose of 100 mg / kg for 12 weeks. NOD / ShiLt mice were anesthetized with isoflurane, and tear secretion was measured using cotton wool soaked in phenol red according to the method of Zoukhri et al. (Exp Eye Res. 2007; 84:894-904).

[0606] (2) Results Figure 28 is a graph analyzing tear secretion in NOD / ShiLt mice (an animal model of Sjögren's syndrome) after administration of a fractional extract of Melissa leaf as described in the example. As shown in Figure 28, tear secretion significantly increased in the treatment group.

[0607] Based on these results, it was confirmed that Sjögren's syndrome can be improved by administering a fractional extract of Melissa leaves as described in the example, thereby increasing tear secretion.

[0608] Experimental Example 18. Inhibitory effect of lemon balm leaf extract on glaucoma in a hypertonic saline-induced scar glaucoma model.

[0609] (1) Experimental method

[0610] The efficacy of the lemon balm leaf fraction extract prepared according to the examples was evaluated in a hypertonic saline-induced scarring glaucoma model.

[0611] Rats were anesthetized by intraperitoneal injection of ketamine-xylazine, and 50 μl of 1.8 M hypertonic saline was injected into the superior scleral vein of the left eye to induce scar tissue in the trabecular network, thereby inducing an increase in intraocular pressure due to resistance in the aqueous humor outflow pathway. The right eye was used as a control, and intraocular pressure was measured in both the left and right eyes at 2-day intervals before and after glaucoma induction for 2 weeks.

[0612] After selecting rats in which glaucoma was induced, 0.5% CMC was administered to the control group, and a fractional extract of Melissa leaf prepared according to the example was administered to the treatment group. Glaucoma was not induced in the normal group.

[0613] Regarding the administration method, a fractional extract of Melissa leaf, as described in the examples, was orally administered to the treatment group once daily at a dose of 100 mg / kg for two weeks from the day of glaucoma induction, while 0.5% CMC was administered to the control group.

[0614] (2) Results Figure 29 is a graph showing that the fractional extract of Melissa leaves, prepared according to the examples, reduced intraocular pressure in a hypertonic saline-induced scarring glaucoma model. Referring to Figure 29, in the control group, intraocular pressure reached its maximum value on day 6 after hypertonic saline injection and remained thereafter, while in the treatment group, the elevated intraocular pressure was significantly reduced in a hypertonic saline-induced glaucoma model with scarring in the trabecular network.

Claims

1. A fractional extract of lemon balm leaf containing caffeic acid, EDPA, RME, and rosmarinic acid.

2. The fractional extract of melissa leaf according to claim 1, 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, relative to the total fractional extract of melissa leaf.

3. The pharmaceutical composition according to claim 1, wherein the fractional extract of melissa leaf is obtained by an extraction process comprising extracting and concentrating melissa leaf with 50% to 100% alcohol, suspending it in water, and fractionating it with ethyl acetate, and the fractional extract of melissa leaf contains 0.05 to 6% by weight of EDPA (ethyl 2-(3,4-dihydroxyphenyl)acetate).

4. A pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, comprising a fractional extract of lemon balm leaf as an active ingredient.

5. A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a fractional extract of lemon balm leaf as an active ingredient.

6. The pharmaceutical composition according to any one of claims 4 or 5, wherein the fractional extract of melissa leaves contains 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, relative to the total fractional extract of melissa leaves.

7. A pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases or MMP (matrix metalloproteinase)-mediated diseases, comprising a fractional extract of Melissa leaf containing caffeic acid, EDPA, RME, and rosmarinic acid as active ingredients.

8. The pharmaceutical composition according to claim 7, wherein the fractional extract of Melissa leaf contains 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.

9. The pharmaceutical composition according to claim 7, wherein the fractional extract of melissa leaf is obtained by an extraction process comprising extracting and concentrating melissa leaf with 50% to 100% alcohol, suspending it in water, and fractionating it with ethyl acetate, and contains 0.05 to 6% by weight of EDPA (ethyl 2-(3,4-dihydroxyphenyl)acetate).

10. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is obese.

11. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is age-related macular degeneration.

12. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is psoriasis.

13. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is endometriosis.

14. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is cancer growth or cancer metastasis.

15. The pharmaceutical composition according to claim 14, wherein the cancer is one of the following: lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, gastric cancer, gastrointestinal cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, esophageal cancer, laryngeal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, multiple myeloma, chronic or acute leukemia, pediatric solid tumor, lymphoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, contractile tumor, brainstem glioma, or pituitary adenoma.

16. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is arteriosclerosis.

17. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is arthritis.

18. The pharmaceutical composition according to claim 17, wherein the arthritis is one of osteoarthritis, degenerative arthritis, osteochondritis dissecans, articular ligament injury, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis.

19. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is inflammatory bowel disease.

20. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is Alzheimer's disease.

21. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is a periodontal disease.

22. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is diabetic retinopathy.

23. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is Sjögren's syndrome.

24. The pharmaceutical composition according to claim 7, wherein the angiogenesis-related disease or MMP-mediated disease is glaucoma.

25. A food composition containing a fractional extract of Melissa leaf, which contains caffeic acid, EDPA, RME, and rosmarinic acid as active ingredients.

26. The food composition according to claim 25, 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 fractional extract of lemon balm leaf.

27. The food composition according to claim 25, wherein the food composition is for preventing or improving non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, or angiogenesis-related disease or MMP-mediated disease.

28. The food composition according to claim 27, wherein the angiogenesis-related disease or MMP-mediated disease is any of the following: obesity, age-related macular degeneration, psoriasis, endometriosis, cancer proliferation or metastasis, arteriosclerosis, arthritis, inflammatory bowel disease, Alzheimer's disease, periodontal disease, diabetic retinopathy, Sjögren's syndrome, or glaucoma.

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