Patent Application
20130171276
The present invention relates to a pharmaceutical composition for suppressing obesity or lowering blood sugar which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract. More particularly, the present invention relates to a pharmaceutical composition for suppressing obesity or lowering blood sugar which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract, a health functional food or food additive composition for suppressing obesity or lowering blood sugar which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract, a tablet having an obesity-suppressing function and a blood sugar-lowering effect which is produced using Cudrania tricuspidata and Coix lachryma-jobi as principal raw materials, and a preparation method thereof.
Recent industrialization progress has increased consumption of western-style foods and convenience of facilities has increased the lack of physical activity, weight gain and society's complexity, leading to changes in disease patterns. Unlike in the past, chronic degenerative diseases such as atherosclerosis, hypertension, cancer, obesity and diabetes have become the leading cause of death. Of these chronic degenerative diseases, especially diabetes, together with obesity, has become a growing concern, because the number of patients has rapidly increased and the age-at-onset of diabetes has become younger.
Diabetes is a disease characterized by the excretion of blood glucose into the urine, and is one of chronic degenerative diseases substantially incurable. Alterations in eating habits due to rapid industrialization progress have prolonged the life expectancy, but have been implicated in the rising prevalence of chronic degenerative diseases such as diabetes or the like. The prevalence of diabetes in Korea is reported to reach approximately 5-10%, and tends to steadily increase. Diabetes is certain to be a serious national health problem in the 21st century. Diabetes is also a global public health crisis, and the third most prevalent disease in the world. Diabetes causes complications in many organs such as the retina, kidney, nerve, and cardiovascular system, and thus continues to be a particularly serious social problem.
Because obesity is a cause of hypertension, diabetes, cardiovascular diseases, and various cancers, and the medical care costs of obesity-related diseases are increasing, investigation of materials having a body fat-reducing effect and demonstration of the effect thereof become a major concern worldwide. Many studies for treating and preventing obesity have been also actively conducted in many countries. Present methods include a diet therapy decreasing food-intake to reduce energy, an exercise therapy increasing energy consumption, surgical therapies such as cutting off parts of inner organs or suction of fat, and drug therapies using a metabolic accelerator, an appetite-suppressant or a digest/absorption-suppressant.
However, no satisfactory method has been reported and the above methods have side effects such as yo-yo effect that obesity becomes more serious after stopping the therapy; unbalanced nutrition condition due to diet therapy; or infections due to decreased immunity. In particular, the drug therapy causes depression, insomnia, indigestion, or the like. Therefore, it is strongly desired to develop a safe and effective method for accelerating body weight loss other than the conventional methods.
Cudrania tricuspidata ((Carr.) Bureau ex Lavallee) is a deciduous tree belonging to the family Moraceae, and is also called Cudrania, Silkworm Thorn, and Chinese Mulberry. Cudrania tricuspidata contains a variety of active ingredients in the root bark, xylem, stem bark, and leaves. Traditionally, the root barks, stems, leaves, stem barks, and fruits of Cudrania tricuspidata have been used in traditional medicines as an antihypertensive, an antitubercular agent, an antifebrile, an antitussive, an expectorant, a diuretic, an antihemorrhagic, and an anti-apoplexy drug, and also used for athlete's foot as an antifungal drug, and for chronic dyspepsia caused by weak digestive systems.
The fruit of Cudrania tricuspidata is a spherical aggregate fruit, and about 2.5 cm in diameter. It ripens to red color in September to October, and the flesh is sweet and edible. Reportedly, the fruit contains useful organic compounds such as vitamin B, B1, B2, C, linolenic acid, glucose, maltose, fructose, malic acid, citric acid, etc., and the stem bark contains a large amount of ingredients effective for lung cancer, colon cancer, skin cancer, and cervix cancer. Cudrania tricuspidata contains flavonoids that are known to have anti-cancer effects, and thus its medicinal uses were reported in traditional medicine books such as DonguiBogam, yaksunggam, and Bonchokangmok
Meanwhile, recent studies have been continuously conducted on the use of Cudrania tricuspidata extracts. There are Korean patents regarding Cudrania tricuspidata, including a Cudrania tricuspidata drink and a preparation method thereof, a method for manufacturing Cudrania tricuspidata granules using sorbitol, functional rice coated with the root extract of Cudrania tricuspidata and a preparation method thereof, etc., but there is no report of the inhibitory effects of Cudrania tricuspidata extract on obesity and diabetes as disclosed in the present invention.
Coix lachryma-jobi (Coicis semen) has the efficacies of invigorating the spleen to eliminate dampness, removing obstruction to stop diarrhea, and clearing up heat and promoting pus discharge, and contains starch, fatty oil, Coixol, sterol, amino acid, vitamin B1, Coixenolide as main ingredients. Coix lachryma-jobi is an annual grass crop belonging to the family Poaceae, and also called adlay, Coix seeds, pearl barley, Job's tears, and semen coicis. It has anti-inflammatory and anti-carcinogenic activities, and cholesterol-lowering, sedative and analgesic effects, and thus used as an injectable formulation for neuralgia or arthralgia. Coix lachryma-jobi is also useful for dyspepsia, arthralgia, and muscle cramps and effective for recovery of fatigue and for the prevention of melasma and freckle to remove facial blemishes. It has an excellent whitening effect, and an effect of expanding and contracting peripheral nerves, and thus is useful for acne, edema, beriberi and neuralgia.
However, there are no reports or descriptions that the mixture of Cudrania tricuspidata and Coix lachryma-jobi extracts is able to suppress obesity and diabetes at the same time.
The present inventors have made many efforts to find a natural substance capable of suppressing obesity and diabetes at the same time. They found that the Cudrania tricuspidata and Coix lachryma-jobi extracts have excellent inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets so as to suppress obesity and diabetes, thereby completing the present invention.
An object of the present invention is to provide a pharmaceutical composition for suppressing obesity or lowering blood sugar, which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract.
Another object of the present invention is to provide a health functional food or food additive composition for suppressing obesity or lowering blood sugar, which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract.
Still another object of the present invention is to provide a Cudrania tricuspidata and Coix lachryma-jobi tablet having an obesity-suppressing or blood sugar-lowering effect, which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract.
Still another object of the present invention is to provide a method for preparing a Cudrania tricuspidata and Coix lachryma-jobi tablet, comprising the steps of mixing Cudrania tricuspidata and Coix lachryma-jobi with one or more herbal medicines selected from the group consisting of red ginseng, ginseng, Dioscorea Rhizome, the leaf of Morus alba, the fruit, stem, and leaf of Hovenia dulcis, Glycyrrhiza uralensis, cinnamon bark, Golden thread, Rubus coreanus Miguel, Dodder seed, Cnidium fruit, Lycium fruit, Schizandra fruit and Astragalus root so as to prepare a mixture; adding one or more functional substances selected from the group consisting of vitamins, saccharide, squalene, taurine, herb, octacosanol and sodium alginate to the mixture and mixing them so as to prepare a mixture of Cudrania tricuspidata, Coix lachryma-jobi, herbal medicines, and functional substances; ripening the mixture at 5˜20° C. for 10˜20 hours; preparing tablets using a tabletting machine; and coating the tablets with a hydroxypropyl methylcellulose solution.
In one aspect to achieve the above objects, the present invention provides a pharmaceutical composition for suppressing obesity or lowering blood sugar, which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract.
As used herein, the term “Cudrania tricuspidata” is a deciduous tree belonging to the family Moraceae, and is also called Cudrania, Silkworm Thorn, and Chinese Mulberry. Cudrania tricuspidata contains a variety of active ingredients in the root bark, xylem, stem bark, and leaves. Traditionally, the root barks, stems, leaves, stem barks, and fruits of Cudrania tricuspidata have been used in traditional medicines as an antihypertensive, an antitubercular agent, an antifebrile, an antitussive, an expectorant, a diuretic, an antihemorrhagic, and an anti-apoplexy drug, and also used for athlete's foot as an antifungal drug, and for chronic dyspepsia caused by weak digestive systems. In the present invention, anti-obesity and anti-diabetic effects of Cudrania tricuspidata were investigated for the first time. According to embodiments of the present invention, it was found that the Cudrania tricuspidata extract has anti-obesity and anti-diabetic effects (Tables 1 and 2), and a blood sugar-lowering effect (Table 5). Further, the Cudrania tricuspidata extract was found to suppress lipid accumulation upon adipocyte differentiation (Table 6).
As used herein, the term “Coix lachryma-jobi” is an annual grass crop belonging to the family Poaceae, and also called adlay, Coix seeds, pearl barley, Job's tears, and semen coicis. Reportedly, it has anti-inflammatory and anti-carcinogenic activities. In the present invention, it was found that the Coix lachryma-jobi extract has anti-obesity and anti-diabetic effects (Tables 1 and 2), and a blood sugar-lowering effect (Table 5). Further, the Cudrania tricuspidata extract was found to suppress lipid accumulation upon adipocyte differentiation).
Moreover according to embodiments of the present invention, an equivalent mixture of the Cudrania tricuspidata and Coix lachryma-jobi extracts showed more excellent anti-obesity and anti-diabetic effects than the Cudrania tricuspidata extract and the Coix lachryma-jobi extract alone (Tables 1 and 2), which supports that the mixed extract of the present invention has an obesity-suppressing or blood sugar-lowering effect, and more preferably, both of the obesity-suppressing and blood sugar-lowering effects.
The present invention is characterized in that the Cudrania tricuspidata extract and the Coix lachryma-jobi extract are used as active ingredients. Specifically, each of the Cudrania tricuspidata extract and the Coix lachryma-jobi extract is prepared, and then they are mixed at a proper ratio so as to prepare the mixed extract. A weight ratio of the Cudrania tricuspidata extract and the Coix lachryma-jobi extract may be preferably 1:1.
The Cudrania tricuspidata extract and the Coix lachryma-jobi extract of the present invention may be, but is not limited to, any one of a liquid extract obtained by extraction, a diluted or concentrated liquid extract, a dry product obtained by drying the liquid extract, a crude purified product thereof, and a purified product thereof. In addition, they may be each independently extracted using an extraction solvent typically used in the art, and preferably extracted using water, alcohol having 1 to 6 carbons or a mixture thereof. More preferably, the alcohol may be ethanol, methanol, or butanol, and much more preferably, an ethanol aqueous solution may be used for extraction.
Preferably, the pharmaceutical composition comprising Cudrania tricuspidata and Coix lachryma-jobi of the present invention can be used for prevention or treatment of obesity and diabetes through reduction of blood-sugar level.
When inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets were examined, the Cudrania tricuspidata and Coix lachryma-jobi extracts according to the present invention showed excellent inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets, indicating obesity-suppressing and blood sugar-lowering effects (Table 2). Water and ethanol extracts of Cudrania tricuspidata and Coix lachryma-jobi showed lower glycemic index than the control group glucose, indicating that each of the Cudrania tricuspidata and Coix lachryma-jobi extracts has excellent blood sugar-lowering effect (Table 5).
The Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention may further comprise a pharmaceutically acceptable carrier. For formulation, any one or more selected from a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and a lubricant commonly used may be used, and they may be included in an amount of 0.1-5 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract.
Specifically, when they are formulated in a form of tablet or capsule, any one or more excipients selected from lactose, starch, cellulose, and microcrystalline cellulose; any one or more binders selected from starch, dextrin, honey, syrup, and cellulose; any one or more disintegrants selected from crystalline cellulose, starch, calcium carboxymethyl cellulose, and methylcellulose; and any one or more lubricants selected from magnesium stearate, glyceryl behenate, talc, and hydrogenated vegetable oil may be used.
In the present invention, the Cudrania tricuspidata and Coix lachryma-jobi extracts may be administered in a typical manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intracutaneous route. Once administration dosage for adults is 0.1 to 10 g, and preferably 0.5 to 5 g, and the level of the administration dosage may appropriately vary depending on a patient's gender, age, health conditions, and diet, administration time, administration method, the kind of drugs used in combination, and disease severity. The administration may be performed once or several times a day. Therefore, the scope of the present invention is not limited to the administration dosage in all aspects. The mixture of Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention has been traditionally used as a food and medicine, and thus there is no particular limitation in its administration dosage. The pharmaceutical composition comprising the Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention should be prepared considering its effective range, and administration of a unit dosage formulation prepared thereby can be monitored, if necessary. Further, a specialized administration method may be used according to the expert's decision and patient's request or it may be administered several times at regular intervals.
In another aspect, the Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention may be used as a health functional food or food additive composition for the prevention or treatment of obesity and diabetes.
The Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention may be used in a variety of foods and drinks for the improvement of obesity and diabetes. The type of foods comprising the Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention is not particularly limited, and is exemplified by meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, instant noodles, other noodles, gums, dairy products including ice creams, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes. Normally, the extract may be added to such food or drink in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight. However in the case of long-term intake for the purpose of health and hygiene improvement or health control, a smaller amount may be used.
The food composition of the present invention may further comprise additional ingredients such as a variety of proteins, fats, nutrients, seasoning, flavors, or natural carbohydrates, as in the conventional drinks. The carbohydrate may be exemplified by conventional sugars, such as monosaccharide (e.g., glucose, fructose, etc.), disaccharide (e.g., maltose, sucrose, oligosaccharide, etc.), and polysaccharide (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavor, a natural flavor (e.g., thaumatin, a stevia extract such as rebaudioside A and glycyrrhizin) and a synthetic flavor (e.g., saccharine, aspartame, etc.) may be used.
In addition to the above ingredients, the composition of the present invention may comprise various nutrients, vitamins, electrolytes, a flavor, a coloring agent, pectic acid and a salt thereof, alginic acid and a salt thereof, an organic acid, a protective colloid thickener, a PH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used for a carbonated drink, etc. Also, the composition of the present invention may comprise flesh for the preparation of natural fruit juice, fruit juice drinks, and vegetable drinks. Each of such ingredients may be used independently or in any combination thereof. Although the percentages of such additives are not important, the additives are generally selected in the range of 0.01 to 0.1 parts by weight, based on 100 parts by weight of the composition of the present invention.
In still another aspect, the present invention provides a Cudrania tricuspidata and Coix lachryma-jobi tablet for suppressing obesity or lowering blood sugar, which comprises Cudrania tricuspidata extract and Coix lachryma-jobi extract. Preferably, the tablet of the present invention provides obesity-suppressing and blood sugar-lowering effects at the same time.
Preferably, the tablet may comprise any one or more herbal medicines selected from the group consisting of red ginseng, ginseng, Dioscorea Rhizome, the leaf of Morus alba, the fruit, stem, and leaf of Hovenia dulcis, Glycyrrhiza uralensis, cinnamon bark, Golden thread, Rubus coreanus Miguel, Dodder seed, Cnidium fruit, Lycium fruit, Schizandra fruit and Astragalus root.
The Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention and Cudrania tricuspidata and Coix lachryma-jobi used as main ingredients for the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet may be those obtained by processing any one or more selected from the leaf, stem, and fruit in the form of powder. They may be processed in the form of powder by any method known in the art without limitation, and for example, they may be pulverized in the form of powder using a pulverizer. After pulverization by a pulverizer, they may be further pulverized by passing them through a mesh. Preferably, the powder pulverized by 40˜100 mesh may be used.
The Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention and Cudrania tricuspidata and Coix lachryma-jobi used as main ingredients for the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet may be those obtained by extracting any one or more selected from the leaf, stem, and fruit in a solvent, filtering and spray-drying the obtained Cudrania tricuspidata and Coix lachryma-jobi extract to obtain a solid phase of Cudrania tricuspidata and Coix lachryma-jobi, and then processing the solid in the form of powder. They may be processed in the form of powder by any method known in the art without limitation, and for example, they may be pulverized in the form of powder using a pulverizer. After pulverization, they may be further pulverized by passing them through a mesh. The powder pulverized by preferably 10˜100 mesh, and more preferably 40˜100 mesh may be used.
Hereinafter, the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention and Cudrania tricuspidata and Coix lachryma-jobi used for the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet are defined as Cudrania tricuspidata and Coix lachryma-jobi powders.
Further, the Cudrania tricuspidata and Coix lachryma-jobi may be cut in a predetermined size of 1˜20 mm, preferably 1˜15 mm, more preferably 1˜10 mm, and much more preferably 1˜5 mm.
In the present invention, the Cudrania tricuspidata and Coix lachryma-jobi may be extracted by using purified water or ethanol as the solvent. The ethanol may be preferably 50% to 100% ethanol, and more preferably 70% ethanol. If purified water is used as the solvent, Cudrania tricuspidata and Coix lachryma-jobi are added to 3˜50-fold of purified water, preferably 3˜30 fold of purified water, based on the weight of Cudrania tricuspidata and Coix lachryma-jobi, and then extracted at 90˜110° C. until the content of purified water reaches 10˜50%, thereby obtaining the Cudrania tricuspidata and Coix lachryma-jobi extract. If ethanol is used as the solvent, Cudrania tricuspidata and Coix lachryma-jobi are added to 3˜50-fold of ethanol, preferably 3˜10 fold of ethanol, based on the weight of Cudrania tricuspidata and Coix lachryma-jobi, and then extracted at 50˜60° C. for 1˜5 hours, thereby obtaining the Cudrania tricuspidata and Coix lachryma-jobi extract.
The Cudrania tricuspidata and Coix lachryma-jobi extract may be obtained by extraction in the presence of microwave with a wavelength of 1 mm˜1 m in the solvent at 50˜60° C. for 1˜2 hours, and the solvent may be the above mentioned purified water or ethanol.
A minor ingredient used for the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention may be one or more herbal medicines selected from the group consisting of red ginseng, ginseng, Dioscorea Rhizome, the leaf of Morus alba, the fruit, stem, and leaf of Hovenia dulcis, Glycyrrhiza uralensis, cinnamon bark, Golden thread, Rubus coreanus Miguel, Dodder seed, Cnidium fruit, Lycium fruit, Schizandra fruit, Astragalus root, Atractylodes Rhizome white, Poria Sclerotium, Rehmannia root, Paeoniae Radix, Cnidium Rhizome, Korean angelica and Cassia bark, and preferably two or more thereof. According to the specific embodiment of the present invention, two herbal medicines were included to prepare the tablet.
The herbal medicine used as the minor ingredient for the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention may be in the form of powder, and the pulverization process of the herbal medicines may be performed in the same manner as in the above mentioned pulverization process of Cudrania tricuspidata and Coix lachryma-jobi.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, the herbal medicine may be comprised in an amount of 10˜50 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract. Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, if the herbal medicine is used in an amount of less than 10 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract, the herbal medicine functions weakly, and if the herbal medicine is used in an amount of more than 50 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract, the increased content of herbal medicine does not greatly contribute to functional improvement of the herbal medicine. Therefore, it is preferable that the herbal medicine is included in an amount of 10˜50 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract, upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention.
In order to improve the functions of the Cudrania tricuspidata and Coix lachryma-jobi tablet, the present invention may further comprise any one or more functional ingredients, preferably two or more functional ingredients selected from the group consisting of vitamin, saccharide, squalene, taurine, herb, octacosanol, and sodium alginate, in addition to Cudrania tricuspidata, Coix lachryma-jobi, and the herbal medicines. According to the specific embodiment of the present invention, two or more functional ingredients were included to prepare the tablet.
Vitamin may be used as the functional ingredient. At this time, vitamin may be used in an amount of 1˜5 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract. The vitamin may be any one or more selected from vitamin D, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin B13, vitamin C, biotin, folic acid, choline, inositol, vitamin L, and vitamin P.
Saccharide may be used as the functional ingredient. At this time, saccharide may be used in an amount of 1˜10 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract. The saccharide may be any one or more selected from lactose, oligosaccharide, sugar, fructose, glucose, dextrin, xylitol, erythritol, palatinose, mannitol, maltitol, inositol, and quercitol.
Herb may be used as the functional ingredient. At this time, herb may be used in an amount of 1˜10 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract. The herb may be any one or more selected from green tea, gulfweed, aloe, mugwort, chlorella, black tea, oolong tea, Akebia quinata, Aralia elata, and Ulmus davidiana.
Sodium alginate may be used as the functional ingredient. At this time, sodium alginate may be used in an amount of 1˜5 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract.
Two or more functional ingredients selected from vitamin, saccharide, squalene, taurine, herb, octacosanol, and sodium alginate may be used in each content ratio.
Also, in order to improve the functions of the Cudrania tricuspidata and Coix lachryma-jobi tablet, the present invention may further comprise Fenugreek seed powder or ramie leaf powder as a secondary functional ingredient.
The secondary functional ingredient, Fenugreek seed powder or ramie leaf powder may be used in an amount of 0.5˜10 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract. Also, the secondary functional ingredient may be added to 3˜30 fold of purified water, based on the weight of the secondary functional ingredient, and extracted at 90˜110° C. until the content of purified water reaches 5˜50%, preferably 5˜35%, more preferably 5˜30%, and much more preferably 5˜25%, and then filtered and pulverized by spray-drying to control the particle size within the range of 10˜100 mesh.
In still another aspect, the present invention provides a method for preparing a Cudrania tricuspidata and Coix lachryma-jobi tablet, comprising the steps of (a) mixing Cudrania tricuspidata and Coix lachryma-jobi with 10˜50 parts by weight of one or more herbal medicines selected from the group consisting of red ginseng, ginseng, Dioscorea Rhizome, the leaf of Morus alba, the fruit, stem, and leaf of Hovenia dulcis, Glycyrrhiza uralensis, cinnamon bark, Golden thread, Rubus coreanus Miguel, Dodder seed, Cnidium fruit, Lycium fruit, Schizandra fruit and Astragalus root, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract so as to prepare a mixture; (b) adding to the mixture one or more functional substances selected from the group consisting of 1˜5 parts by weight of vitamin, 1˜10 parts by weight of saccharide, 0.5˜5 parts by weight of squalene, 0.5˜5 parts by weight of taurine, 1˜10 parts by weight of herb, 0.1˜5 parts by weight of octacosanol and 1˜5 parts by weight of sodium alginate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract, and mixing them so as to prepare a mixture of Cudrania tricuspidata, Coix lachryma-jobi, herbal medicines, and functional substances; (c) ripening the mixture at 5˜20° C. for 10˜20 hours; (d) preparing tablets using a tabletting machine; and (e) coating the tablets with a hydroxypropyl methylcellulose solution.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, mixing of the ingredients for Cudrania tricuspidata and Coix lachryma-jobi tablet may be performed at 10˜20° C. for 20˜60 minutes in order to mix the ingredients uniformly. In this regard, when the ingredients for Cudrania tricuspidata and Coix lachryma-jobi tablet are mixed, Cudrania tricuspidata and Coix lachryma-jobi and herbal medicines may be mixed at once, and when the functional substances are further added, the Cudrania tricuspidata and Coix lachryma-jobi, herbal medicines and functional substances may be mixed at once, or herbal medicines and functional substances may be mixed primarily, and then Cudrania tricuspidata and Coix lachryma-jobi may be added secondarily.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, the ingredients for Cudrania tricuspidata and Coix lachryma-jobi tablet may be ripened at 10˜30° C. for 1˜5 hours, preferably 10˜20° C. for 2˜4 hours, and more preferably at 20° C. for 3 hours, after they are mixed. The ingredients of the Cudrania tricuspidata and Coix lachryma-jobi tablet have different water contents. Thus, it is preferable that the ingredients of the Cudrania tricuspidata and Coix lachryma-jobi tablet are mixed and then aged, in order to maintain water balance between the ingredients of the Cudrania tricuspidata and Coix lachryma-jobi tablet for effective preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, the Cudrania tricuspidata and Coix lachryma-jobi tablet may comprise any one or more selected from an excipient, a binder, a disintegrant, and a lubricant.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, the Cudrania tricuspidata and Coix lachryma-jobi tablet may comprise any one or more selected from an excipient, a binder, a disintegrant, and a lubricant in an amount of 0.1˜5 parts by weight, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract.
The excipient may be any one or more selected from lactose, starch, cellulose, and microcrystalline cellulose.
The binder may be any one or more selected from starch, dextrin, honey, syrup, and cellulose.
The disintegrant may be any one or more selected from crystalline cellulose, starch, calcium carboxymethyl cellulose, and methylcellulose.
The lubricant may be any one or more selected from magnesium stearate, glyceryl behenate, talc, and hydrogenated vegetable oil.
The above mentioned excipient, binder, disintegrant, and lubricant are ingredients commonly used for the preparation of tablets. Thus, if these ingredients are used in the present invention, they may be properly selected and used by those skilled in the art.
Upon the preparation of the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention, the Cudrania tricuspidata and Coix lachryma-jobi tablet is prepared using a tabletting machine, and then the surface of the Cudrania tricuspidata and Coix lachryma-jobi tablet is coated so as to prepare a surface-coated Cudrania tricuspidata and Coix lachryma-jobi tablet. The coating agent applied to the surface of the tablet may be any coating agent, as long as it is not harmful to human and is used as a coating agent in food-related fields. For example, hydroxypropyl methylcellulose (HPMC), Shellac, gelatin, Arabic Gum, cacao, or lecitin may be used to coat the surface of the tablet, and more preferably, a 0.3% hydroxypropyl methylcellulose solution may be used.
When the surface of the Cudrania tricuspidata and Coix lachryma-jobi tablet is coated with the coating agent, the coating agent may be applied to the surface of the Cudrania tricuspidata and Coix lachryma-jobi tablet using a spray system. Meanwhile, the method of coating the surface of Cudrania tricuspidata and Coix lachryma-jobi tablet with the coating agent may be properly selected and used by those skilled in the art.
In still another embodiment, the present invention provides a method for treating obesity or diabetes, comprising the step of administering the pharmaceutical composition to patient with obesity or diabetes.
In the present invention, the patient with obesity or diabetes means all mammals including human with obesity or diabetes or at the risk of obesity or diabetes. The patient can be effectively treated by administering the pharmaceutical composition comprising the Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention to the patient with obesity or diabetes.
As used herein, the term “administration” means introduction of the pharmaceutical composition of the present invention into a patient with obesity or diabetes by any proper method, and the pharmaceutical composition may be administered via the oral or parenteral route, as long as it is able to reach the desired tissue.
The therapeutic method of the present invention may comprise the step of administering the pharmaceutical composition comprising the Cudrania tricuspidata and Coix lachryma-jobi extracts at a pharmaceutically effective amount. The total daily dose should be determined through appropriate medical judgment by a physician, and administered once or several times. With respect to the objects of the present invention, the specific therapeutically effective dose level for any particular patient may vary depending on various factors well known in the medical art, including the kind and degree of the response to be achieved, concrete compositions according to whether other agents are used therewith or not, the patient's age, body weight, health conditions, gender, and diet, the time and route of administration, the secretion rate of the composition, the time period of therapy, other drugs used in combination or coincidentally with the specific composition, and like factors well known in the medical arts.
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples and Experimental Examples.
Based on the data in herbology, Dongui Bogam or the like, 60 kinds of natural products commonly used in the prescription of obesity and diabetes and allowable as food were examined. Among them, 15 kinds of natural products showing inhibitory activities on ATP citrate lyase (anti-obesity) and H2O2-induced oxidative damage of pancreatic islets (anti-diabetes) were selected and examined.
Perilla frutescens, Benincasa hispida, Golden thread, Akebia quinata, ripened pumpkin, Phyllostachydis Folium, Chinese wolfberry root bark, Angelica pubescentis, Ramulus mori, Cornus officinalis, Schizandra fruit, Astragalus root, Coix lachryma-jobi, Cudrania tricuspidata, and broccoli used as samples in this experiment were purchased at Kyung-dong market. All herbal medicines were dried by hot air drying at 45° C., and the dried samples were pulverized using a pulverizer (M20, IKA, Germany), and then sealed in a 0.2 mm PE film, followed by storage in a freezer before use.
In order to examine inhibitory effects of 15 kinds of natural products selected in Example 1 on ATP-citrate lyase activity (anti-obesity), the following experiment was performed.
12-week-old Sprague-Dawley male rats were fed with high carbohydrate diet (63% sucrose, 30% casein, 4% salt mixture, 2% cellulose powder, 1% vitamin mixture, and 0.1% choline chloride) for 3 days. After 3 days, the male rats were sacrificed by cervical dislocation. The liver was excised and homogenized in ice-cold buffer (0.25 M sucrose, 50 mM Tris-HCl, pH 8.2, 10 mM MgCl2, 5 mM DTT (dithiothreitol) and 0.1 mM PMSF (phenylmethylsulfonyl fluoride)). Next, the homogenate was precipitated with 40% saturated ammonium sulfate solution, and centrifuged at 4° C. and 17,500×g for 15 minutes. The supernatant was filtered and high-speed centrifuged (at 4° C. and 100,000×g for 1 hour), followed by fractionation. The resulting fraction was stored at −80° C. and used as an ATP-citrate lyase sample. ATP citrate lyase activity of the liver of the male rat was measured by malate dehydrogenase-catalyzed reduction of oxaloacetate by NADH.
In order to examine inhibitory effects of 15 kinds of natural products selected in Example 1 on oxidative damage of pancreatic islets (anti-diabetes), the following experiment was performed.
The pancreas was excised from the rat, and treated with a collagenase V solution (2 mg/ml) at 37° C. for 15 minutes, and digestion was stopped by adding cold Krebs-ringer bicarbonate buffer. After washing three times with the same buffer, islets were handpicked under a stereomicroscope. The cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100/ml streptomycin at 37° C., 95% O2 and 5% CO2 for 24 hours. Pancreatic islets were transferred to 96 well plates at a density of 10 islets/well in 150 μl RPMI1640 medium and pre-incubated overnight. After pre-incubation, the natural product samples prepared in Example 1 were added to each well, and incubated at 37° C. for 48 hours. Then, 500 μM H2O2 was added to each well, and incubated for 2 hours. 10 μl of MTT solution was added thereto, and further incubated at 37° C. for 4 hours. The supernatant was discarded, and 100 μl of DMSO (dimethylsulphoxide) was added into each well to dissolve the dark blue crystal formazan. The absorbance was determined at 570 nm to analyze the inhibitory effects on oxidative damage of pancreatic islets.
Based on the results of Examples 2 and 3, 15 kinds of natural products showing excellent inhibitory activities on ATP citrate lyase and H2O2-induced oxidative damage of pancreatic islets were selected from 60 kinds of natural products, and their IC50 values were shown in the following Table 1.
9.8 ± 1.6a
Among 15 natural product samples, as shown in Table 1, H5 (ripened pumpkin), H14 (Coix lachryma-jobi), and H15 (Cudrania tricuspidata) showed superior inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets, H2 and H11 showed superior inhibitory activity on ATP citrate lyase, and H9 showed superior inhibitory activity on H2O2-induced apoptosis of pancreatic islets.
Each of H14 and H15 showing the most excellent inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets or a blended mixture with equal amounts thereof was used to measure their inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets, and the results are shown in the following Table 2.
As shown in Table 2, inhibitory activities on ATP citrate lyase and H2O2-induced apoptosis of pancreatic islets were remarkably increased in the blended mixture with equal amounts thereof than in each of them, leading to selection of two functional substances, Cudrania tricuspidata and Coix lachryma-jobi (H14, H15) capable of suppressing obesity and diabetes at the same time.
The results of a study in collaboration with the diabetic food company showed that 12 diabetic patients provided with the blend of H14 and H15 for 2 months showed 10% reduction in the blood glucose level, indicating that the blend of Cudrania tricuspidata and Coix lachryma-jobi has the anti-diabetic effect. Further, the results of Tables 1 and 2 indicate that the blend of Cudrania tricuspidata and Coix lachryma-jobi has the anti-obesity and anti-diabetic effects.
Cudrania tricuspidata and Coix lachryma-jobi were naturally dried in the shadow, and then pulverized using a pulverizer, resulting in 80-mesh sized powders. Each 20 g of Cudrania tricuspidata and Coix lachryma-jobi powders were added to 1 L of purified water which was approximately 50-fold of the weight of Cudrania tricuspidata and Coix lachryma-jobi powders, and extracted at a boiling point for 2 hours so as to obtain Cudrania tricuspidata and Coix lachryma-jobi extracts. The Cudrania tricuspidata and Coix lachryma-jobi extracts were concentrated, and vacuum freeze-drying was performed to obtain Cudrania tricuspidata and Coix lachryma-jobi powder extracts.
Cudrania tricuspidata and Coix lachryma-jobi were naturally dried in the shadow, and then pulverized using a pulverizer, resulting in 80-mesh sized powders. Each 20 g of Cudrania tricuspidata and Coix lachryma-jobi powders were added to 1 L of 70% ethanol which was approximately 50-fold of the weight of Cudrania tricuspidata and Coix lachryma-jobi powders, and extracted at 80° C. for 2 hours so as to obtain Cudrania tricuspidata and Coix lachryma-jobi extracts. The Cudrania tricuspidata and Coix lachryma-jobi extracts were concentrated, and vacuum freeze-drying was performed to obtain Cudrania tricuspidata and Coix lachryma-jobi powder extracts.
The yields of purified water or 70% ethanol extracts of Cudrania tricuspidata and Coix lachryma-jobi prepared in Examples 4 and 5 were determined, and the experiment was repeated three times. The results are shown in the following Table 3.
Coix lachryma-jobi (Alday)
Cudrania tricuspidata
As shown in Table 3, when Cudrania tricuspidata and Coix lachryma-jobi were extracted using purified water, the Cudrania tricuspidata and Coix lachryma-jobi powder extracts were obtained in a yield of 22.41% and 31.72%, respectively. When Cudrania tricuspidata and Coix lachryma-jobi were extracted using ethanol, the Cudrania tricuspidata and Coix lachryma-jobi powder extracts were obtained in a yield of 21.12% and 2.86%, respectively.
Cudrania tricuspidata and Coix lachryma-jobi were naturally dried in the shadow, and then pulverized using a pulverizer, resulting in 80-mesh sized powders. The Cudrania tricuspidata and Coix lachryma-jobi powders were added to purified water in a volume of approximately 50-fold of the weight of Cudrania tricuspidata and Coix lachryma-jobi powders, and extracted in the presence of microwave with a wavelength of 500±5 mm at 100° C. for 1 hour so as to obtain Cudrania tricuspidata and Coix lachryma-jobi extracts. The Cudrania tricuspidata and Coix lachryma-jobi extracts were spray-dried, and the particle size was controlled to obtain 80-mesh sized Cudrania tricuspidata and Coix lachryma-jobi powders.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Dioscorea Rhizome, 9 parts by weight of the leaf of Morus alba, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were primarily mixed at 20° C. for 40 minutes using a Hobart mixer, and then Cudrania tricuspidata and Coix lachryma-jobi powders were added thereto, and mixed well. In this regard, all of Dioscorea Rhizome, the leaf of Morus alba, xylitol, vitamin C, octacosanol and magnesium stearate were those that were respectively pulverized using a pulverizer to have the same particle size with Cudrania tricuspidata and Coix lachryma-jobi powders.
The mixture containing the Cudrania tricuspidata and Coix lachryma-jobi powders was ripened at 20° C. for 3 hours, and tabletted using a tabletting machine at a pressure of 8 kg/cm2 and an instantaneous temperature of 80° C. so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets having a weight of 0.3 g per tablet.
The surface of the Cudrania tricuspidata and Coix lachryma-jobi tablet was spray-coated with a 3% hydroxypropyl methylcellulose (HPMC) solution so as to prepare HPMC-coated Cudrania tricuspidata and Coix lachryma-jobi tablets.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Glycyrrhiza uralensis, 9 parts by weight of cinnamon bark, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Golden thread, 9 parts by weight of Astragalus root, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Atractylodes Rhizome white, 9 parts by weight of Poria Sclerotium, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Rehmannia root, 9 parts by weight of Paeoniae Radix, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Dioscorea Rhizome, 9 parts by weight of the leaf of Morus alba, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, 0.2 part by weight of magnesium stearate, and 3 parts by weight of Fenugreek seed, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 6 were used.
27 parts by weight of Dioscorea Rhizome, 9 parts by weight of the leaf of Morus alba, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, 0.2 part by weight of magnesium stearate, and 2.5 parts by weight of ramie leaf, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
Cudrania tricuspidata and Coix lachryma-jobi were naturally dried in the shadow, and then pulverized using a pulverizer, resulting in 80-mesh sized powders. The Cudrania tricuspidata and Coix lachryma-jobi powders were added to 70% ethanol in a volume of approximately 50-fold of the weight of Cudrania tricuspidata and Coix lachryma-jobi powders, and extracted in the presence of microwave with a wavelength of 500±5 mm at 80° C. for 2 hours, and concentrated under reduced pressure so as to obtain ethanol-removed Cudrania tricuspidata and Coix lachryma-jobi extracts. The Cudrania tricuspidata and Coix lachryma-jobi extracts were spray-dried, and the particle size was controlled to obtain 80-mesh sized Cudrania tricuspidata and Coix lachryma-jobi powders.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 14 were used.
27 parts by weight of Dioscorea Rhizome, 9 parts by weight of the leaf of Morus alba, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 14 were used.
27 parts by weight of Glycyrrhiza uralensis, 9 parts by weight of cinnamon bark, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 14 were used.
27 parts by weight of Golden thread, 9 parts by weight of Astragalus root, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 14 were used.
27 parts by weight of Atractylodes Rhizome white, 9 parts by weight of Poria Sclerotium, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
The Cudrania tricuspidata and Coix lachryma-jobi powders obtained in Example 14 were used.
27 parts by weight of Rehmannia root, 9 parts by weight of Paeoniae Radix, 4 parts by weight of xylitol, 3.8 parts by weight of vitamin C, 1 part by weight of octacosanol, and 0.2 part by weight of magnesium stearate, based on 100 parts by weight of Cudrania tricuspidata extract and Coix lachryma-jobi extract powders, were used so as to prepare Cudrania tricuspidata and Coix lachryma-jobi tablets in the same manner as in Example 7.
Sensory evaluation including taste, flavor, color, appearance, chewness, and preference was performed with respect to the Cudrania tricuspidata and Coix lachryma-jobi tablets prepared in Examples 7 to 11 in accordance with a 9-point hedonic scale, and the results are shown in the following Table 4. The sensory evaluation was performed by 20 participants (each 10 of male and female participants) who have been engaged in food-related fields for 3 years or longer and experienced in sensory evaluation. The results are shown in the following Table 4.
The marks in the sensory test items of Table 4 are average values (rounded off to two decimal places) obtained by dividing the total scores by the number of panel members, and higher score value in the sensory test items represents more excellent sensory properties.
As shown in Table 4, the Cudrania tricuspidata and Coix lachryma-jobi tablets prepared in Examples 7 to 11 presented 7 points or higher in taste, flavor, color, appearance, texture, and preference, indicating excellent sensory properties.
Oral glucose tolerance test was performed with respect the Cudrania tricuspidata and Coix lachryma-jobi extracts prepared in Examples 4 and 5. The results are shown in the following Table 5.
Coix lachryma-
jobi (water)
Coix lachryma-
jobi (ethanol)3)
Cudrania
tricuspidata
Cudrania
tricuspidata
1)2)glycemic index (mg/dL) after 120 min (0 min vs 120 min), as percentage (%)
3)data is insufficient and thus not shown,
ns not significant
As shown in Table 5, the Cudrania tricuspidata and Coix lachryma-jobi extracts showed lower glycemic index than the control group, glucose, indicating that the Cudrania tricuspidata and Coix lachryma-jobi extracts have excellent blood glucose-lowering effects.
In order to measure anti-obesity effect of the Cudrania tricuspidata and Coix lachryma-jobi extracts prepared in Examples 4 and 5, their effects on lipid accumulation upon differentiation of a preadipocyte cell 3T3-L1 into adipocyte were tested. The specific method is as follows.
3-1: Culture of 3T3-L1 Preadipocyte Cell
The preadipocyte cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 1% P/S (penicillin-streptomycin) and 10% BCS (bovine calf serum) at 37° C. and 5% CO2. The cells were cultured at 3 day-intervals. When the cells reached 70% confluence in a 100Φ plate, they were washed with PBS (phosphate buffered saline), and then left in a 0.25% trypsin-EDTA solution at 37° C. and 5% CO2 for 3 minutes. The detached cells were maintained by subculturing.
3-2: Induction of 3T3-L1 Preadipocyte Cell Differentiation
In order to induce differentiation of preadipocyte cells, cells were seeded in DMEM supplemented with 10% BCS and 1% P/S in a 6-Well plate at a density of 1.25×105 cells/well. After 2 days, the medium was replaced, and cells were completely fused at 3˜4 days. Differentiation of the fused cells was induced in a differentiation medium MDI (IBMX, Dexa, Insulin). At this time, in order to examine the effect of Cudrania tricuspidata and Coix lachryma-jobi extracts on adipocyte differentiation, the extract sample was added to the differentiation medium at a concentration of 1 mg/ml. At 2 days after differentiation induction, the medium was replaced with DMEM containing the sample, 10% FBS, 1% P/S, and 5 μg/ml insulin. From 4 days after differentiation induction, the medium was replaced with DMEM containing 10% FBS and 1% P/S once every 2 days to induce differentiation.
3-3: Oil-Red O Staining
Oil-red O staining and quantification were performed using cells at 7˜9 days of differentiation. After removing 10% FBS DMEM, cells were washed with PBS twice, and fixed in 10% formalin at 4° C. for 1 hour, and then washed with distilled water three times. The washed cells were stained for 1 hour with an oil-red O solution specific to lipid droplets produced in cells. After staining, the staining solution was removed, and the cells were washed with distilled water three times. Then, each well was filled with distilled water, and examined under a microscope. For quantification of neutral lipids, distilled water was removed from the well, and 2 ml of 100% iso-propyl alcohol was added to the completely dried well to elute oil-red O. Absorbance was measured at 490 nm using an ELISA reader.
In order to examine the effect of each sample on lipid accumulation during differentiation of 3T3-L1 adipocytes, each sample was diluted with the differentiation medium at a concentration of 1 mg/ml to perform Oil-red O staining. As compared to the control, the Coix lachryma-jobi ethanol extract showed 92.9% lipid accumulation, the Cudrania tricuspidata water extract showed 60.9% lipid accumulation, and the Cudrania tricuspidata ethanol extract showed the lowest 20.5% lipid accumulation (Table 6).
These results support that Coix lachryma-jobi and Cudrania tricuspidata extracts suppress adipocyte differentiation directly involved in obesity, indicating that Coix lachryma-jobi and Cudrania tricuspidata extracts have anti-obesity effect.
Cudrania tricuspidata water extract
Cudrania tricuspidata ethanol extract
Coix lachryma-jobi water extract
Coix lachryma-jobi ethanol extract
Based on 1 kg of raw materials, 40% (400 g) of Cudrania tricuspidata powder extract, 40% (400 g) of Coix lachryma-jobi powder extract, 12% (120 g) of dextrin, and 5% (50 g) of vitamin C were mixed, and then 3% (20 g) of HPMC was blended thereto so as to prepare a Cudrania tricuspidata and Coix lachryma-jobi tablet product (Table 7).
Cudrania tricuspidata powder extract
Coix lachryma-jobi powder extract
Since the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention comprises herbal medicines, the Cudrania tricuspidata and Coix lachryma-jobi tablet having improved functions by herbal medicines and a preparation method thereof are provided. The Cudrania tricuspidata and Coix lachryma-jobi tablet further comprising functional substances and a preparation method thereof are also provided. Further, the Cudrania tricuspidata and Coix lachryma-jobi extracts of the present invention exhibit excellent inhibitory effects on ATP citrate lyase and H2O2-indclued apoptosis of pancreatic islets, and excellent anti-obesity and blood sugar-lowering effects, and thus can be used as a pharmaceutical composition or functional food for the prevention or treatment of obesity and diabetes. Furthermore, the Cudrania tricuspidata and Coix lachryma-jobi tablet of the present invention is easy to carry, and store Cudrania tricuspidata and Coix lachryma-jobi, and intake Cudrania tricuspidata and Coix lachryma-jobi, because Cudrania tricuspidata and Coix lachryma-jobi are processed in a form of tablet.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2010-0089122 | Sep 2010 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2011/006694 | 9/9/2011 | WO | 00 | 3/8/2013 |
