EP3062807A1

EP3062807A1 - Compositions comprising a viola herba extract, or an extract of viola herba, persicae semen, cinnamomi ramulus, and glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases and obesity

Info

Publication number: EP3062807A1
Application number: EP14858980.7A
Authority: EP
Inventors: Yoon Young Sung; Ho Kyoung Kim; Dong Seon Kim; Go Ya Choi
Original assignee: Korea Institute of Oriental Medicine KIOM
Current assignee: Korea Institute of Oriental Medicine KIOM
Priority date: 2013-10-28
Filing date: 2014-10-27
Publication date: 2016-09-07
Also published as: CN105916514A; KR20150050391A; EP3062807A4; KR101672274B1; WO2015064975A1

Abstract

The present invention relates to a pharmaceutical or a food additive composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases or obesity, and a method for the treatment of lipid-related cardiovascular diseases or obesity comprising administering the pharmaceutical composition to a subject in need of the same. The Viola Herba extract, or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. of the present invention is shown to be effective in reducing body weight, body fat, total cholesterol in blood, and lowering blood pressure, and thus can be used for effective prevention or treatment of lipid-related cardiovascular diseases or obesity.

Description

COMPOSITIONS COMPRISING A VIOLA HERBA EXTRACT, OR AN EXTRACT OF VIOLA HERBA, PERSICAE SEMEN, CINNAMOMI RAMULUS, AND GLYCYRRHIZA SPP. FOR THE PREVENTION OR TREATMENT OF LIPID-RELATED CARDIOVASCULAR DISEASES AND OBESITY

The present invention relates to a pharmaceutical composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases or obesity, and a method for prevention of lipid-related cardiovascular diseases or obesity including administering the pharmaceutical composition to a subject in need of the same.

Additionally, the present invention relates to a food additive composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or improvement of lipid-related cardiovascular diseases or obesity.

For the past decade, the dietary fat intake of Korean adults has continuously increased. According to a Korean National Health & Nutrition Examination Survey, the prevalence of obesity as determined by body mass index (BMI) as of 2010 was 36.3% for males, and 24.8% for females. In particular, obesity has continually increased in males over the last decade. According to the results of an investigation conducted in 2005, in Korean adults, the calorie intake from fat exceeded 20%. Because specifically, the increase of animal fat intake has been prominent, and the total amount of fat intake and saturated fatty acid intake have also increased, the amount of dietary fat intake, kinds, and the level of fatty acid intake are important determinant factors that affect the lipid concentration in blood. The change in dietary habits as described above is known to correlate with an increase in the risk of the occurrence of cardiovascular diseases. According to the September 2012 report by Statistics Korea, the number one cause of death was cancers, followed by cerebrovascular diseases, and cardiovascular diseases in this order. It has been known that circulatory diseases such as atherosclerosis, myocardial infarction and stroke are more seriously affected by dietary intake than other chronic diseases. The relevant risk factors known so far include cholesterol level in blood, triglycerides level in blood, hypertension, smoking, thrombosis, diabetes, obesity, etc. Among them, cholesterol level and hypertension are known as the major risk factors causing atherosclerosis and myocardial infarction. Accordingly, the maintenance of the cholesterol concentration in blood to be at a normal level is important to prevent the occurrence of the diseases described above.

Meanwhile, when the cholesterol level is under proper control in the body, cholesterol will not be accumulated in blood plasma or tissues. However, the increase in body cholesterol level may result in hypercholeterolemia leading to a disorder in regulation of cholesterol metabolism, thereby causing a quantitative change in the components of lipoproteins such as low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol. In particular, among cholesterols present in blood, LDL-cholesterol is known as a risk factor for cardiovascular diseases, and the increase in triglycerides concentration level is known to decrease the concentration of HDL-cholesterol and increase chylomicron residues, which are mainly composed of triglycerides, thus becoming a major cause of coronary artery diseases. Additionally, blood lipids can pass through vascular endothelial cells better than other types of lipids and thus can become a major cause of atherosclerosis, and the rapid increase in triglyceride level after a meal is known to be closely related with the occurrence of stroke. In contrast, HDL-cholesterol can remove cholesterol from vascular walls, and is thus known as a preventive factor of cardiovascular diseases. Accordingly, in order to prevent or treat various diseases occurring due to lipid metabolism disorders, it is important to maintain each of the various components of blood lipids comprised of triglycerides, cholesterol, phospholipids, and free fatty acids at normal levels.

Currently, diet therapies recommending the intake of dietary fibers, saponins, vitamin C, vitamin D, and essential amino acids are used as a therapeutic method for proper control of the blood lipid components to thereby prevent and treat lipid metabolism disorders. However, the dietary therapies may not be sustained for a long period because they often accompany a mental discomfort due to limitation in diet. Alternatively, in addition to the dietary therapy, drug therapies have also been used for treating lipid metabolism disorders. However, the drug therapies for lowering blood lipids level must be maintained over the entire course of life, may cause liver dysfunction, and complications such as myopathy. In particular, when the liver function value increases to be at least 3 times that of the normal, the drug therapies can no longer be applied. Therefore, there is a growing need for the discovery of a natural substance useful as an active ingredient for the treatment of lipid-related cardiovascular diseases or obesity.

Obesity or obesity symptoms are collective terms referring to the medical condition of a person's body weight being excessively high, and from the pathological point of view, it refers to a case when a peron's body mass index (BMI) is 30 or higher according to WHO standards. However, in Korea, a person with a BMI value of 25 or higher is diagnosed as having obesity. In most cases, obesity refers to the physical condition of a person when his or her body weight is higher than normal body weight. However, it may also refer to a state when the lipid content among body constituents is high, although the body weight itself may not be excessive. Obesity refers to the physical state of a person with excess fat accumulation in the body, which leads to an increase in the number or size of adipocytes in adipose tissues. Obesity may not be a problem in itself, but may become serious when it results in social handicaps induced therefrom and secondary complications due to excessive body fat. For example, obesity may induce major diseases such as hypertension, hyperlipidemia, heat diseases, diabetes atherosclerosis, fatty liver, joint disorders, arthritis, sexual dysfunction, and cancer, and thus has been treated as an important disease. When the body weight of a person increases to 20% over normal body weight, the occurrence rate of diabetes becomes 10 times higher than that for normal people. Further, it may deteriorate his or her cardiac and pulmonary functions, thereby shortening their life expectancy. Complex factors such as hypertension, diabetes, hypercholeterolemia, and atherosclerosis may have an influence on cardiac functions causing heat failure and inducing sudden death. In females, additionally, the occurrence rate of endometrial cancer increases, and also the possibility of cholelithiasis may be higher. In males, obesity increases the risk of colorectal and prostate cancer. Furthermore, the increase in body weight makes people become conscious of the eyes of others around them, and the growing obsession thereof can make them avoid contact with other people, making them more likely to live alone. Additionally, the physical inferiorities may lead to a loss in their self-confidence, and may even influence their ego.

To prevent obesity and maintain a healthy body weight, it is essential to maintain a life-long balanced diet and to regularly exercise. In particular, adipocytes, once produced, cannot be naturally removed and remain permanently in the body, although they may be reduced in size. Therefore, it is important to be careful not to become obese. Accordingly, body weight can be reduced primarily by lowering calorie intake while increasing outdoor activities and the amount of exercise. However, as described above, since the adipocytes are hardly removed once formed, people often take drugs to reduce fat absorption along with a weight-loss dietary intake. However, these drugs may induce adverse reactions such as cardiovascular disease (e.g., hypertension) and stroke, and once administration of the drug is stopped, body weight often starts to increase. For severe obesity, bariatric surgeries may be performed, which may include a surgery to remove body fat, gastroplasty or gastric banding to limit food intake, etc.

Examples of anti-obesity drugs currently used include anorexiants which exert effects by reducing the amount of food intake or regulating body metabolism and increasing exothermic reactions in the body, and is a heat metabolism promoter, a digestive absorption inhibitor, and hormone regulator, etc. Examples of anorexiants include adrenergic drugs, serotonergic drugs, and adrenergic-serotonergic drugs, and of them, sales of serotonergic drugs have been prohibited due to the adverse effects of valvular heart disease. Examples of the heat metabolism promoter include an ephedrine-caffeine combination drug and an adipocyte 3 receptor agonist, and examples of the digestive absorption inhibitor include a lipase inhibitor represented by Xenical, and a low fat food additive called Olestra. The anti-obesity drugs that have been used until now have had numerous reports on their adverse effects including a yo-yo effect, anorexia, headache, blood pressure increase, steatorrhea, diarrhea, pyschonia, etc., which appear to be due to the fact that the products have not been scientifically proven or the effect of reducing body weight with a short period of time has been emphasized. Accordingly, there is a continuing demand for the development of a novel natural substance based on herbal medicines with proven safety having an improved anti-obesity effect with less adverse effects.

The present inventors, while endeavoring to find a natural substance with the effect of preventing or treating lipid-related cardiovascular diseases or obesity but having less adverse effects, have confirmed that a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. has anti-obesity effects such as inhibition of body weight increase, inhibition of the amount of dietary intake, and reduction in body fat, and has an effect of treating lipid-related cardiovascular diseases by reducing triglycerides, which is a neutral fat present in blood, reducing LDL-cholesterol, increasing HDL-cholesterol, increasing Adiponectin mRNA expression within adipose tissues, increasing mitochondrial uncoupling protein-2 (UCP-2) mRNA, and reducing blood pressure, thereby completing the present invention.

An objective of the present invention is to provide a pharmaceutical composition containing a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases or obesity.

Another objective of the present invention is to provide a food additive composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or improvement of lipid-related cardiovascular diseases or obesity.

A further objective of the present invention is to provide a method for treatment of lipid-related cardiovascular diseases or prevention of obesity comprising administering the pharmaceutical composition above to a subject in need of the same.

The Viola Herba extract, or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. of the present invention is effective in reducing body weight, body fat, and total cholesterol in blood, and lowering blood pressure, and thus can be used for effective prevention or treatment of lipid-related cardiovascular diseases or obesity.

Additionally, in an atherosclerotic mouse model, the Viola Herba extract has been shown to be effective in reducing the occurrence of atherosclerotic lesions; and inhibiting lipid accumulation in liver tissues and in blood, and thus can be effectively used for the prevention or treatment of atherosclerosis.

Figure 1 shows a graph illustrating the results of body weight analysis according to the age of the weeks in a normal diet group (normal group) fed with a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp., a control group with high fat diet (HFD), a group with HFD and xenical, a group with HFD and a Viola Herba extract, a group with HFD and an extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp., and a group with HFD and an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp.

Figure 2 shows a graph illustrating the adiponectin mRNA expression in adipose tissues of a control group and in each of the experimental groups of the present invention.

Figure 3 shows a graph illustrating the UCP-2 mRNA expression in adipose tissues of the control group and in each of the experimental groups of the present invention.

Figure 4 shows a graph illustrating the blood pressure measured in the control group and in each of the experimental groups of the present invention.

Figure 5 shows a graph illustrating the inhibitory effect of a Viola Herba extract on increases in body weight in an atherosclerotic mouse model.

Figure 6 shows a graph illustrating the effect of a Viola Herba extract capable of reducing the size of fat cells in liver tissues and the fat deposition in an atherosclerotic mouse model.

Figure 7 shows pictures illustrating the inhibitory effect of a Viola Herba extract on increases in liver weight in an atherosclerotic mouse model.

Figure 8 is a graph illustrating the effect of a Viola Herba extract capable of reducing the occurrence of atherosclerotic lesions in an atherosclerotic mouse model (A: pictures of sections of arterial tissue with dyed fats, B: a graph with measured atherosclerotic lesions).

Figure 9 is a graph illustrating the effect of a Viola Herba extract capable of increasing AMPK activity; and inhibiting expression of Fatty Acid Synthase (FAS) and Sterol Regulatory Element-Binding Protein-1 (SREBP-1) proteins.

In order to accomplish the above objectives, in an aspect, the present invention provides a pharmaceutical composition containing a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases or obesity.

The present inventors, while performing studies on various natural substances with a view to find a natural substance capable of preventing or treating lipid-related cardiovascular diseases or obesity with less adverse effects, have discovered the effects of a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp..

The present inventors extracted Viola Herba or a mixture of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. with water, and thereby obtained a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp.. They conducted an experiment on the Viola Herba extract, and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. to examine their effectiveness in the prevention or treatment of cardiovascular diseases or obesity, and confirmed that the Viola Herba extract, and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. have the effects of inhibiting body weight increase, inhibiting the amount of dietary intake, and reducing body fat, and also reducing triglycerides, which is a neutral fat in blood, reducing LDL-cholesterol, increasing HDL-cholesterol, increasing Adiponectin mRNA expression within adipose tissues, increasing UCP-2 mRNA expression, and reducing blood pressure.

Additionally, they also conducted an experiment on the Viola Herba extract to examine its effect on the prevention or treatment of atherosclerosis using an Apolipoprotein E (ApoE) gene-deleted mouse model with atherosclerosis, and confirmed that the Viola Herba extract has the effects of inhibiting body weight increase; inhibiting the liver weight increase and reducing fat deposition; reducing the occurrence of atherosclerotic lesions; reducing the levels of total cholesterol, LDL-cholesterol, triglycerides and glucose; increasing AMPK activity; and inhibiting the expression of FAS and SREBP-1 proteins.

Accordingly, it was confirmed that the Viola Herba extract, and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. can be used for the prevention, treatment or improvement of lipid-related cardiovascular diseases or obesity.

As used herein, the term, "lipid-related cardiovascular diseases", may collectively refer to circulatory vascular diseases, heart diseases, cerebrovascular diseases, etc., specifically at least one selected from the group consisting of atherosclerosis, hypertension, myocardial infarction, stroke, hyperlipidemia, ischemic heart disease, cardiac arrhythmia, heart failure, angina pectoris, endocarditis, cardiac valve disorder, conduction disorder, and ischemic cerebrovascular disease, but is not limited thereto.

As used herein, the term, "circulatory vascular diseases" collectively refers to diseases occurring in various organs including heart involved in the systemic circulation and others associated with pulmonary circulation.

As used herein, the term, "heart diseases" collectively refers to diseases related to the heart in charge of blood supply for the human body, including ischemic heart diseases, cardiac arrhythmia, heart failure, etc. Representative examples of ischemic heart diseases may include angina pectoris, myocardial infarction, etc., and they refer to acute or chronic heart disorders occurring due to the decrease or blocking of blood flow to cardiac muscle along with the progress of coronary artery disease. Other heart diseases include endocarditis, cardiac valve disorder, conduction disorder, cardiac arrhythmia, heart failure, etc. In particular, heart failure refers to a state where the heart cannot sufficiently pump out blood being returned to the heart through a venous system.

As used herein, the term "cerebrovascular disease" refers to a disease that makes people collapse due to sudden brain dysfunction caused by problems with the blood vessels in the brain, which, depending on the type of occurrence, can be subdivided into hemorrhagic cerebrovascular disease due to intracranial hemorrhage with partial rupture of blood vessels and ischemic cerebrovascular disease due to blockage or insufficient blood flow in the blood vessels of the brain, but is not limited thereto.

As used herein, the term "obesity" refers to a medical condition wherein there is an excess of body fat, and from a clinical point of view, it refers to a state with BDI of 25 or higher in Korea, and 30 or higher according to WHO standards. Obesity generally refers to a person's physical body state when his or her body weight exceeds the average body weight, however, it may also refer to a person's physical state when the lipid content among body constituents is relatively high although the body itself may not be excessively overweight. Obesity is a disease occurring in both adults and children.

As used herein, the term "anti-obesity" is a term that is inclusive of prevention, treatment, and improvement of obesity. Accordingly, the term "anti-obesity composition", as used herein, refers to a pharmaceutical composition for the prevention or treatment of obesity as defined above.

As used herein, the term "Viola Herba" refers to the entire plant of Viola mandshurica belonging to Violaceae family or Viola yezoensis belonging to Violaceae family. Viola Herba has a particular smell, is pungent, and tastes bitter and spicy. Additionally, it has the efficacies of fever alleviation and detoxification. It contains saponin, flavonoid compounds, cerotic acid, unsaturated fatty acids, etc. However, the effects of the Viola Herba extract on lipid-related cardiovascular diseases or obesity have not been identified yet.

As used herein, the term "Persicae Semen" refers to seeds (breed) or seeds and branches of Prunus persica or Prunus davidiana belonging to Rosaceaas family. It has almost no smell, is oily, tastes bitter and sweet, and constantly retains the same property without being sided. Since Persicae Semen can remove extravasated blood it can be used for treating abdominal pains, blocked menstruation, dysmenorrhea, contusions, lung abscess, appendicitis, etc. Due to high fat content, it can lubricate intestines and is thus effective for treatment of constipation, and is also useful for the treatment of skin irritation due to blood heat; dry skin; freckles, etc. Its branches can alleviate sudden deep abdominal pain.

As used herein, the term "Cinnamomi Ramulus" refers to branches of bark of a tree belonging to Cinnamomum sp. In detail, it may be a branch or bark of Cinnamomum cassia or Cinnamomum loureirii belonging to Cinnamomum sp., but is not limited thereto. Its pharmacological actions include inhibition of growth of E. coli, Bacillus substilis, Staphylococcus aureus, Streptococcus pneumonia, etc., inhibition of influenza virus, diuretic action, etc.

As used herein, the term "Glycyrrhiza spp." refers to a perennial plant belonging to Glycyrrhiza. In detail, Glycyrrhiza spp. can be Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, or Glycyrrhiza echinata, but is not limited thereto. In the present invention, Glycyrrhiza spp. may be used with its roots or stems attached or after peeling them off.

As used herein, the term "extract" refers to a liquid extract of medicinal herbs obtained using an extraction solvent, although not limited thereto, and may refer to a liquid extract obtained by extraction, a diluent or concentrate of the liquid extract, a dried product obtained by drying the liquid extract, a coarse purified product or purified product obtained therefrom.

In the present invention, the extract may be obtained by extraction using an extraction solvent, or by fractionation by adding a fractionation solvent to an extract obtained by extraction using an extraction solvent. The extraction solvent may not be limited but water, an organic solvent, or a mixed solvent thereof may be used. Examples of the organic solvent may be C_1-4alcohol, a polar solvent such as ethyl acetate or acetone, a nonpolar solvent such as hexane or dichloromethane, or a mixed solvent thereof. Preferably, water, C_1-4alcohol, or a mixed solvent thereof may be used. In an embodiment of the present invention, the extract was obtained using water as the extraction solvent.

As used herein, the term "an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp." refers to an extract obtained by extracting a combined mixture comprising Viola Herba, and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:1 to 1:5. Peferably, the weight ratio is in the range of 1:2 to 1:4, more preferably 1:3, but is not limited thereto. Additionally, the mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. may be mixed at a weight ratio of 1:1:1 to 6:2:1, specifically, in a weight ratio of 3:2:2, but is not limited thereto.

The Viola Herba extract or the extract of the combined mixture comprising Viola Herba and the mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. may be prepared by a conventional extraction method, and separation and purification methods known in the art. Examples of the extraction method, although not limited thereto, may include hot-bath extraction, hot-water extraction, macerating, reflux extraction, sonication extraction, etc.

In the present invention, the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was obtained by extracting the entire plant for Viola Herba, the seeds of Persicae tree for Persicae Semen, barks of Cinnamomum cassia or Cinnamomum loureirii for Cinnamomi Ramulus, and the entire plant exclusive of bark for Glycyrrhiza spp., but other parts may be used as well.

In an exemplary preparation embodiment of the present invention (Preparation Example 1), the Viola Herba extract of the present invention was obtained by adding 5 to 20 volumes of an extraction solvent to the dry weight of Viola Herba. The extract of a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was obtained by extracting the mixture of the dried Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. in a mixing ratio of 3:2:2 via hot-water extraction. The extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was obtained by extracting a combined mixture containing Viola Herba, and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:3 (25 wt%: 75 wt%) using water as an extraction solvent.

In an exemplary embodiment of the present invention (Example 2), the administration of the Viola Herba extract of the present invention to a high fat diet group was shown to reduce the body weight of mice starting from 11 weeks of age, compared to those in the high fat diet group without treatment, whereas the administration of the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was shown to more significantly reduce the body weight of mice compared to when the Viola Herba extract was administered alone (Figure 1 and Table 1).

In another exemplary embodiment of the present invention (Example 3), the Viola Herba extract was shown to be effective in reducing body weight and inhibiting the amount of dietary intake, and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was shown to more significantly reduce body weight and inhibit the amount of dietary intake (Table 2).

In a further exemplary embodiment of the present invention (Example 4), the effects of the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on blood lipids were examined, and it was confirmed that the administration of the Viola Herba extract or the extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. reduced the levels of total cholesterol and LDL-cholesterol while increasing the level of HDL-cholesterol. The administration of the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was shown to reduce the concentrations of blood glucose, total cholesterol, triglycerides, LDL-cholesterol while increasing the concentration of HDL-cholesterol (Table 3).

In a still further exemplary embodiment of the present invention (Example 5), the administration of the extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on a high fat diet group was shown to significantly increase the expression of Adiponectin mRNA involved in glucose metabolism and lipid decomposition, and the administration of the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on a high fat diet group was also shown to increase the expression of Adiponectin mRNA (Figure 2).

Additionally, the administration of the Viola Herba extract or the extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on a high fat diet group was shown to increase the expression of UCP-2 mRNA, and the administration of the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on a high fat diet group was shown to significantly increase the expression of Adiponectin mRNA (Figure 3).

In a yet further exemplary embodiment of the present invention (Example 6), the administration of both the Viola Herba extract and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. to a high fat diet group was shown to result in a decrease in blood pressure compared to that of the high fat diet control group, thus confirming their effectiveness on the treatment of lipid-related cardiovascular diseases (Figure 4).

In yet another further exemplary embodiment of the present invention, an ApoE gene-deleted mouse model with atherosclerosis was administered with a Viola Herba extract while being fed with high fat diet. Additionally, the control group was fed with general feed and the positive control group was administered with atovastatin (Example 7-1).

As a result, it was confirmed that the adminsitration with the Viola Herba extract is effective on inhibiting body weight increase; inhibiting liver weight; reducing the size of adipocytes and the fat deposition in liver tissues; reducing the incidence of atherosclerotic lesions; lowering the concentrations of total cholesterol, LDL-cholesterol, triglycerides and glucose; increasing AMPK activity; and inhibiting the expression of FAS and SREBP-1 proteins (Examples 7-2 through 7-7).

As used herein, the term "prevention" refers to all kinds of activities that inhibit or delay the occurrence of lipid-related cardiovascular diseases or obesity by administering a pharmaceutical composition according to the present invention, and the term "treatment" refers to all kinds of activities that improve or advantageously change the symptoms of a subject with lipid-related cardiovascular diseases or obesity or a subject suspected of having the same by administering the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to the characteristics of being non-toxic to cells or humans being exposed to the composition. The above carrier may be anything known in the art such as a buffering agent, a preservative, a pain-relieving agent, a solubilizer, an isotonic agent, a stabilizer, an excipient, a lubricant, etc. Additionally, the pharmaceutical compositoin of the present invention may be formulated into preparations for oral administration such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injections. Furthermore, it may be used in the form of an external preparation for skin such as ointments, lotions, sprays, patches, cream, powders, suspensions, gels or dermatologic drugs. The carriers, excipients, and diluents that can be included in the compositon of the present invention are: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. For formulation, commonly used fillers, extenders, binders, humectants, disintegrating agents, diluents such as surfactants or excipients may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. The above solid preparations may be prepared by adding at least one excipient, e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc., to the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. Additionally, lubricants such as magnesium stearate, talc, etc., may be used in addition to the simple excipients. Examples of liquid preparations for oral administration include suspensions, preparations for internal use, emulsifiers, syrups, etc., and various kinds of excipients, e.g., humectants, sweeteners, fragrants, preservatives, etc., may be used in addition to simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsifiers, lyophilized preparations, and suppositories. Examples of non-aqueous solvents or suspensions include propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethylolate, etc. Examples of suppository bases include Macrogol, Tween 61, cacao butter, laurinum, glycerogelatin, etc.

The amount of the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. according to an embodiment of the present invention may not be particularly limited, but is preferably contained in the amount of 0.01 to 100 wt%, and more preferably 1 to 100 wt%, relative to the total weight of the final composition.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "administration" refers to the introduction of a particular substance into a subject by an appropriate method, and the administration route of the composition may be any conventional route that enables delivery of the composition to the target tissue, for exmaple, intraperitoneal adminstration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal adminsitration, oral administration, local administration, intransal adminstration, intrapulmonary administration, and intrarectal administration, but is not limited thereto.

As used herein, the term "subject" refers to all kinds of animals such as rats, mice, livestock and humans, preferably mammals including humans, which already have the lipid-related cardiovascular disease(s) or obesity or have the potential of having the same.

As used herein, the term "a pharmaceutically effective amount" refers to an amount sufficient for the treatment of a disease at a reasonable benefit/risk ratio but without any adverse effects, thereby being applicable to medical treatment, and the level of the effective dosage may be easily determined by a skilled person in the art in consideration of sex, age, body weight, health status of a subject, type of a disease, severity of a disease, drug activities, drug sensitivities, methods of administration, duration of adminstration, drug efflux rate, duration of treatment, combination of drugs or factors including drugs used in combination, and other factors well known in the medical field. Preferably, the pharmaceutical composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. of the present invention may be administered daily in the amount of 0.0001 to 1000 mg/kg of body weight, more preferably 0.001 to 500 mg/kg of body weight. The administration may be performed once daily or in divided doses per day.

Since the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. of the present invention is prepared using natural herb materials it will have much fewer adverse effects than the genenral synthetic compounds and thus can be used safely.

In another aspect of the present invention, there is provided a food additive composition for the prevention or improvement of lipid-related cardiovascular diseases or obesity containing the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. as an active ingredient.

The tersms of Viola Herba, Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp., extract, lipid-related cardiovascular diseases, obesity, and prevention are the same as defined above.

As used herein, the term "improvement" refers to all kinds of activities that can improve or advantageously change the symptoms of lipid-related cardiovascular diseases or obesity by administering the above composition.

As used herein, the term "food additive composition" means a composition intended to be added to any food for providing desired effects.

The Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. of the present invention is not only shown to be effective in the prevention or treatment of lipid-related cardiovascular diseases or obesity but also has been shown to be safe because it is derived from natural substances. Accordingly, they can be used in a food additive composition to manufacture a health functional food for the prevention or improvement of obesity.

When used as a food additive as such, the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. may be directly used by adding them or may be used in combination with other foods or food components, and may be appropriately used according to a conventional method. The amount of active ingredients may be appropriately determined according to their intended use (prevention, health or therapeutic treatment), and auxiliary food additive(s) acceptable from the food nutritional point of view may be further added thereto. Since the Viola Herba extract or the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is derived from natural substances their safety is ensured, and thus the amount to be added into the food additive composition may not be particularly limited, but is preferably in the range of 0.001 to 100 wt%, more preferably 0.01 to 80 wt% relative to the weight of the total composition. Additionally, the composition may further include other components that are conventionally used in food compositions to improve smell, taste, visual flavor, etc., for example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Additionally, minerals such as Zn, Fe, Ca, Cr, Mg, Mn, and Cu may be included. Additionally, amino acids such as lysine, tryptophane, cysteine, and valine may be included. Additionally, food additives such as a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a germicide (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), a coloring agent (tar color, etc.), a color fixing agent (sodium nitrite, etc.), a bleaching agent (sodium sulfite), a seasoning (MSG, sodium glutamate, etc.), a sweetenre (dulcin, cyclamate, saccharin, sodium, etc.), a flavor (vanillin, lactones, etc.), a swelling agent (alum, D-potassium bitartate, etc.), a reinforcing agent, an emulsifier, a thickener (adhesive paste), a film-forming agent, a gum base agent, an antifoaming agent, a solvent, an improver, etc., may be added. The additives may be selected according to the food types, and used in an appropriate amount.

The food additive composition of the present invention, being an additive added during the manufacture of conventional foods, may be used in the manufacture of foods effective in prevention or improvement of lipid-related cardiovascular diseases or obesity. The foods having the food additive composition may be interchangeably used with terms such as functional foods, health functional foods, etc., well known in the art.

As used herein, the term "functions foods" refers to foods manufactured or processed using material(s) or component(s) with useful functions according to the Korean Law No. 6727 for health functional foods, and as used herein, the term "functional" refers to the activity of an intake for the purpose of obtaining useful effects on health-related uses such as regulating nutrients or physiological actions regarding the structure and functions of human body.

Additionally, as used herein, the term "health functional foods" refers to foods manufactured or processed via extraction, concentration, purification, mixing, etc., of particular components as raw materials or particular components contained in the food materials for the purpose of health supplement, and the foods designed and processed to sufficiently exhibit the biological regulatory functions such as biological defense and biological rhythm, prevention and recovery of diseases, etc., to a living subject by the above components.

The food types in which the composition of the present invention can be added thereto may include all kinds of foods such as special nutritious foods (e.g., milk formula, infant formula, etc.), processed meat products, fish meat products, soybean curd, starch jelly, noodles (e.g., ramens, fine noodles, etc.), health supplementary foods, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed soybean paste, etc.), sauces, confectioneries (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchies, pickled vegetables, etc.), beverages (e.g., fruits, vegetable drinks, soybean milk, fermented drinks, etc.), natural seasonings (e.g., ramen soup, etc.), etc.

To accomplish the above-identified objectives, in another aspect of the present invention, there is provided a method for treating lipid-related cardiovascular diseases or obesity including administering the above pharmaceutical composition to a subject in need of the same.

The terms of subject, administration, lipid-related cardiovascular diseases, obesity and treatment are the same as defined above.

The present invention will be explained in further detail herein below with reference to Examples, however, they are disclosed for illustrative purposes and should not be construed as limiting the scope of the present invention.

Preparation Example 1. Preparation of Extracts

Viola Herba was purchased from Omniherb Co.(Daegu, KOREA), and extracted via hot-water extraction by adding 10 volumes of water. Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. were purchased from Kwangmyungdang Medicinal Herbs (Ulsan, KOREA). The dried herb materials were mixed at a weight ratio of 3:2;2 between Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp., and extracted via hot-water extraction. The extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. was obtained by extracting a combined mixture containing Viola Herba, and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:3 (25 wt%: 75 wt%) using water as an extraction solvent.

Example 1. Construction of a high fat diet (HFD)-inducing animal model

Male C57bl/6 mice were used after purchasing them from the Jackson Laboratory (Bar Harbor, Maine, USA). Of them, healthy 10 week-old mice were subjected to a maintenance experiment under Specific Pathogenic Free (SPF) condition, and allowed to freely intake foods and water. A high fat diet (HFD) (Rodent diet D12492, Research Diets, New Brunswick, NJ, USA) consisting of proteins (20%), carbohydrates (20%), and fats (60%) was used after purchasing it from Central Lab. Animal Inc. (Seoul, KOREA).

The experimental animals were adapted to basic feeds (AIN-76A diet) for 2 weeks, and then co-administered with HFD diet and herb drugs from 8 weeks of age for 7 weeks. The experimental groups were a total of 6 consisting of: a normal group (Normal Diet) administered with saline while being fed with general feeds, a control group (HFD) administered with saline while being fed with HFD, a positive control group (15.6 mg/kg) administered xenical while being fed with HFD, and 3 groups administered with drug (200 mg/kg) while being fed with HFD. All test materials were suspended in saline and orally administered at the same time dailly using a mouse sonde.

Example 2. Effects of a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on reducing body weight and inhibition of food intakes

The body weight and the amount of feed intake of the experimental animals were measured using an electronic scale (GP5202, Sartorius, Germany), analyzed, and the feed efficiency ratio (EFR) was calculated based on the ratio of the body weight gain over the amount of feed intake.

The HFD group administered with a Viola Herba extract (HFD-Viola Herba) showed a decrease in their body weight starting from the age of 11 weeks compared to that of the HFD-control group. The HFD group administered with an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) showed a significant decrease in their body weight gain compared to that administered with the Viola Herba extract alone (p<0.05)(Figure 1).

Table 1

Group	Feed intakes (g/day)	Body weight gain (g/day)	Feed efficiency ratio (FER,%)
Normal Diet	3.52	0.161±0.01	4.57±0.30
HFD (HFD-control group)	3.14	0.441±0.03 ###	14.06±0.83 ###
HFD-xenical	2.86	0.230±0.01 ***	8.04±0.37 ***
HFD-Viola Herba	2.89	0.239±0.01 ***	8.27±0.49 ***
HFD-Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.	3.03	0.305±0.03 **	10.06±0.99 *
HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)	2.99	0.227±0.01 ***	7.59±0.44 ***

###: p<0.001 compared with the normal diet

* : p<0.05 compared with the HFD-control group

** : p<0.01 compared with the HFD-control group

*** : p<0.001 compared with the HFD-control group

As can be seen in Table 1 above, the body weight gain in the HFD group (HFD-control group) was significant compared to that of Normal Diet group (normal group), and the HFD group administered with the Viola Herba extract (HFD-Viola Herba) showed a significant decrease in the body weight gain (p<0.05). Additionally, the HFD group administered with the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) showed a significant decrease in the body weight gain.

Additionally, the HFD group administered with the Viola Herba extract (HFD-Viola Herba) showed a significant decrease in the FER was compared to that of the HFD group, and the HFD group administered with the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) the FER showed a more significant decrease in the FER (p<0.05)(Table 1).

From the above results, it was confirmed that the Viola Herba extract is effective in reducing body weight and inhibiting the amount of dietary intake, and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is more effective than the Viola Herba extract in reducing body weight and inhibiting the amount of dietary intake.

Example 3. Effect of a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp. on reducing body fat weight

Upon analysis of the result of Example 1, the mice were sacrificed and the amount of their abdominal fat was compared. As a result, as can be seen in Table 2, the HFD group (HFD-control group) showed an increase in the weight of Subcutaneous fat, fat tissues around epididymis, and liver tissues. The group administered with the Viola Herba extract (HFD-Viola Herba) showed a significant decrease in the weight of Subcutaneous fat, intestinal fat tissues, and liver tissues.

Additionally, the HFD group administered with the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) showed a more significant decrease in the weight of Subcutaneous fat, intestinal fat tissues, and liver tissues compared to that administered with the Viola Herba extract.

Table 2

Group	Tissue weight (g)
Group	Subcutaneous fat	Intra-abdominal fat	Liver
Normal Diet	0.286±0.077	0.280±0.032	1.036±0.068
HFD (HFD-control group)	1.916±0.120 ###	3.786±0.273 ###	1.116±0.036
HFD-xenical	1.567±0.115 *	2.837±0.116 **	1.009±0.043 *
HFD-Viola Herba	1.548±0.084 *	3.108±0.304	0.922±0.031 ***
HFD-Persicae Semen,Cinnamomi Ramulus, Glycyrrhiza spp.	1.318±0.234 **	2.848±0.097 **	0.900±0.037 ***
HFD-Viola Herba+(Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)	1.267±0.081 *	2.640±0.084 *	0.836±0.030 ***

###: p<0.001 compared with the normal diet

* : p<0.05 compared with the HFD-control group

** : p<0.01 compared with the HFD-control group

*** : p<0.001 compared with the HFD-control group

Example 4. Effect of a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on lipid in blood

Blood samples collected from mice were left at room temperature for 30 minutes, and centrifuged at 3000 rpm for 15 minutes to obtain blood sera. The contents of glucose, total cholesterol, triglycerides, LDL, and HDL in blood sera were measured and analyzed via blood biochemical analyzer (Table 3).

Table 3

Group	Glucose(mg/dL)	Total cholesterol(mg/dL)	Triglyceride(mg/dL)	LDL(mg/dL)	HDL(mg/dL)
Normal Diet	101.4±7.3	158.0±3.2	82.8±19.6	42.2±2.6	66.2±3.9
HFD (HFD-control group)	157.2±26.5	214.2±7.2 ###	529.8±31.7 ###	56.4±1.9 ##	68.4±3.7
HFD-xenical	117.0±11.0	173.8±2.0 **	483.6±44.8	48.4±2.7	84.3±1.5 **
HFD-Viola Herba	93.4±11.2	180.6±7.1 *	454.0±53.8	38.6±2.5 ***	79.2±2.1 *
HFD-Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.	93.5±11.8	176.8±5.8 **	477.0±46.3	36.8±1.5 ***	80.6±2.5 *
HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)	88.4±8.3 *	171.8±7.6 **	330.3±11.2 **	35.4±2.1 ***	78.3±1.2 *

##: p<0.01 compared with the normal diet

###: p<0.001 compared with the normal diet

* : p<0.05 compared with the HFD-control group

** : p<0.01 compared with the HFD-control group

*** : p<0.001 compared with the HFD-control group

The analysis of haematological index revealed that the HFD group (HFD-control group) showed a higher increase in the concentrations of total cholesterol, LDL-cholesterol, and triglycerides in blood than the Normal Diet group (normal group), and the HFD group administered with the Viola Herba extract or the extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. showed a decrease in the concentrations of total cholesterol and LDL-cholesterol while showing an increase in the concentration of HDL-cholesterol (p<0.05).

Additionally, when the HFD group was administered with an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp., the concentrations of glucose, total cholesterol, triglycerides, and LDL-cholesterol, and triglycerides were significantly decreased while the concentration of HDL-cholesterol was increased (p<0.05).

Example 5. Effects of a Viola Herba extract or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. on increase of the expression of Adiponectin and Mitochondrial Uncoupling Protein-2 (UCP-2) genes

An analysis was performed via real time PCR using the intestinal fat tissues of HFD-induced mice, and the result revealed that the HFD group administered with a Viola Herba extract (HFD-Viola Herba) or an extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. showed a significant increase in the mRNA expression of Adiponectin involved in glucose metabolism and fat decomposition, and the HFD group administered with an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) also showed an increase in mRNA expression of Adiponectin (Figure 2).

The HFD group administered with the Viola Herba extract (HFD-Viola Herba) or the extract of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. showed an increase in mRNA expression of UCP-2, and the HFD group administered with the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) also showed a significant increase in mRNA expression of UCP-2 (Figure 3).

Example 6. Effect of a Viola Herba extract and an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp. on reduction of blood pressure

Blood pressure was measured on the tails of mice via CODA non-invasive blood pressure systems (Kent Scientific Co., Torrington, CT, USA). A total of 4 measurements were made, and represented as an average blood pressure (Hg) between systolic and diastolic blood pressures (Figure 4). Both groups administered with the Viola Herba extract ((HFD-Viola Herba) and the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp. (HFD-Viola Herba + (Persicae Semen, Cinnamomi Ramulus, Glycyrrhiza spp.)) showed a higher reduction in blood pressure than the HFD-control group.

Example 7. Effect of a Viola Herba extract on inhibition of atherosclerosis

Example 7-1. Construction of an animal model with HFD-induced atherosclerosis

As experimental model animals with atherosclerosis, 6 week-old ApoE gene-deleted mice were provided by Jackson Laboratory (Bar Harbor, Maine, USA) and allowed to adapt for 2 weeks while being freely supplied with a basic feed (AIN-76A diet) and water. Upon their adaptation, HFD feeds were used after purchasing from Central Lab. Animal Inc. (Seoul, Korea), and the feeds contained 45% of fats and 0.15% of cholesterol.

The experimental animals were subjected to a combined administration of both HFD and drugs for 16 weeks starting from the 8 weeks of age. The experimental groups were a total of 4 consisting of: a normal group administered with saline while being fed with general feeds, a HFD group administered with saline while being fed with HFD, a drug-treated group (v. mandshurica, 150 mg/kg) administered with a drug while being fed with HFD, and a positive control group administered with atovastatin as a drug while being fed with HFD. All test materials were suspended in saline and administered orally on the same predetermined time daily via a mouse sonde.

Example 7-2. Effect of a Viola Herba extract on inhibition of body weight gain

The body weight of experimental animals was measured using an electronic scale at the same predetermined time once a week. As a result, it was confirmed that the HFD group administered with a Viola Herba (v. mandshurica) showed a more significant inhibition of body weight gain starting from the 3^rdweek compared to the HFD group, and in particular, showed a continuous inhibition of body weight gain compared to the positive control group treated with atovastatin (p<0.05) (Figure 5).

Example 7-3. Effect of a Viola Herba extract on inhibition of liver weight gain

Mice administered with a Viola Herba extract for 16 weeks were autopsied, and the liver tissues obtained therefrom were measured for their weight. As a result, it was confirmed that the HFD group showed an increase in liver weight compared to the control group, but the liver weight of the drug-treated group administered with a Viola Herba (V. mandshurica) extract, although fed with HFD, was similar to that of the normal group which was fed with general feeds (p<0.05) (Figure 6). Accordingly, it was confirmed that the Viola Herba extract is effective in inhibiting the liver weight gain.

Example 7-4. Effect of a Viola Herba extract on inhibition of lipid deposition on liver tissues

The experimental mice administered with a Viola Herba extract along with HFD for 16 weeks were autopsied, and the liver tissues obtained therefrom were sliced into 6 ㎛-thick specimen, and the presence of lipid deposition on the liver tissues was examined via hematoxylin-eosin staining, and oil red O staining method for specific staining of lipids. As a result, it was confirmed that there was a progress of a lipid deposition in the liver tissues of the mice fed with HFD compared to those of the normal group, and also that the size of adipocytes and lipid deposition in the liver tissues of the drug-treated group administered with Viola Herba (V. mandshurica) were reduced (p<0.05)(Figure 7).

Example 7-5. Effect of a Viola Herba extract on improvement of blood lipids

The experimental mice administered with a Viola Herba extract along with HFD for 16 weeks were autopsied, and the arterial tissues obtained therefrom were sliced into 6 ㎛-thick specimen via rapid-freeze fixation, and the state of atherosclerotic lesions formed in blood vessels was examined via oil red O staining method. As a result, it was confirmed that there was a progress of lipid deposition in the arterial tissues of the mice fed with HFD compared to those of the normal group, and also that the thickness of arterial wall was reduced due to lipid deposition along with the reduction in lipid deposition in the drug-treated group administered with a Viola Herba (V. mandshurica) extract were reduced (p<0.05)(Figure 8). Accordingly, it was confirmed that the Viola Herba extract is effective in reducing the generation of atherosclerotic lesions.

Example 7-6. Effect of a Viola Herba extract on improvement of blood lipids

Blood samples collected from mice were left at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes to obtain blood sera. The contents of glucose, total cholesterol, triglycerides, LDL, and HDL in the blood sera were measured via blood biochemical analyzer (Table 4).

Table 4

Group	Glucose (mg/dL)	Total cholesterol(mg/dL)	Triglyceride(mg/dL)	LDL(mg/dL)	HDL(mg/dL)
Normal Diet (control)	160.43±7.59	462.29±25.80	56.86±6.40	60.00±2.26	17.00±2.06
HFD	402.00±26.55 ###	1381.57±58.18 ###	178.00±17.49 ###	377.86±17.73 ##	9.00±1.15 ##
Viola Herba extract (V. mandshurica)	114.67±19.17 ***	864.17±42.74 ***	68.17±11.90 ***	227.67±24.38 ***	11.67±1.43
Positive Control (statin)	275.67±20.12 ***	1184.00±62.00 *	77.33±7.50 ***	262.00±21.14 ***	14.33±1.56 **

##: p<0.01 compared with the normal diet

###: p<0.001 compared with the normal diet

* : p<0.05 compared with the HFD

** : p<0.01 compared with the HFD

*** : p<0.001 compared with the HFD

The analysis of the haematological indices revealed that the HFD group has higher concentrations of total cholesterol, LDL-cholesterol, triglycerides, and glucose in blood while having a lower concentration of HDL-cholesterol in blood than the Normal Diet (control group). The HFD group administered with a Viola Herba extract showed a decrease in the concentrations of total cholesterol, LDL-cholesterol, triglycerides, and glucose in blood (p<0.05). Accordingly, it was confirmed that the Viola Herba extract is effective in inhibiting atherosclerosis through the improvement of blood lipids.

Example 7-7. Effect of a Viola Herba extract on inhibition of factors involved in lipid synthesis in liver tissues

Upon eviseration of liver tissues, the activity of AMP-activated protein kinase (AMPK), which plays an important role in glucose and lipid metabolism, and the expression amount of factors involved in lipid synthesis were measured. As a result, the mice fed with HFD showed a decrease in AMPK activities, and an increase in expression of Fatty Acid Synthase (FAS), Sterol Regulatory Element-Binding Protein (SREBP-1), compared to the normal group. Meanwhile, the Viola Herba extract (V. mandshurica) showed an inhibition of the expression of FAS and SREBP-1 proteins along with an increase in AMPK activity, and was thus confirmed to be effective in inhibiting atherosclerosis (Figure 9).

From the foregoing, a skilled person in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.

Claims

A pharmaceutical composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases or obesity.
The pharmaceutical composition of claim 1, wherein the extract is obtained by extraction using a solvent selected from the group consisting of water, C_1-4alcohol, ethyl acetate, acetone, hexane, dichloromethane, and a mixed solvent thereof.
The pharmaceutical composition of claim 1, wherein the extract is obtained by extraction using water as a solvent.
The pharmaceutical composition of claim 1, wherein the extract is a hot-water extract.
The pharmaceutical composition of claim 1, wherein the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is obtained by extracting a combined mixture comprising Viola Herba and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:1 to 1:5.
The pharmaceutical composition of claim 1, wherein the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is obtained by extracting a combined mixture comprising Viola Herba and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:3.
The pharmaceutical composition of claim 1, wherein the extract improves obesity by inhibiting the increase in body weight and body fat due to high fat diet (HFD) and lowering the levels of triglycerides, total cholesterol, and low density lipoprotein (LDL)-cholesterol in blood while increasing the level of high density lipoprotein (HDL)-cholesterol in blood.
The pharmaceutical composition of claim 1, wherein the lipid-related cardiovascular diseases are at least one selected from the group consisting of atherosclerosis, hypertension, myocardial infarction, stroke, hyperlipidemia, ischemic heart disease, cardiac arrhythmia, heart failure, angina pectoris, endocarditis, cardiac valve disorder, conduction disorder, and ischemic cerebrovascular disease.
A food additive composition comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or improvement of lipid-related cardiovascular diseases or obesity.
The food additive composition of claim 9, wherein the extract is obtained by extraction using a solvent selected from the group consisting of water, C_1-4alcohol, ethyl acetate, acetone, hexane, dichloromethane, and a mixed solvent thereof.
The food additive composition of claim 9, wherein the extract is a hot-water extract.
The food additive composition of claim 9, wherein the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is obtained by extracting a combined mixture comprising Viola Herba and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:1 to 1:5.
The food additive composition of claim 9, wherein the extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. is obtained by extracting a combined mixture comprising Viola Herba and a mixture of Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. at a weight ratio of 1:3.
The food additive composition of claim 9, wherein the lipid-related cardiovascular diseases are at least one selected from the group consisting of atherosclerosis, hypertension, myocardial infarction, stroke, hyperlipidemia, ischemic heart disease, cardiac arrhythmia, heart failure, angina pectoris, endocarditis, cardiac valve disorder, conduction disorder, and ischemic cerebrovascular disease.
A method for the treatment of lipid-related cardiovascular diseases or obesity comprising administering the pharmaceutical composition of claim 1 to a subject in need of the same.