Patent Application
20080044505
Nelumbinis semen is the dried fruit or seed of Nelumbo nucifera Gaertn., a plant of Nymphaeaceae, which is a perennial plant growing in water. Its rhizomes spread sideways, are corpulent and have many nodes, and its leaves sprout from the nodes, are circular in shape and have a pink color. This plant mainly grows in swamps, ponds and shallow lakes. For use in the present invention, between September and October when the fruits are ripened, the fruits are split and dried in sunlight, or the fresh fruits are dried in sunlight after removing the skin. Nelumbinis semen has already been used as a natural medicinal material for the treatment of women's diseases, tonic strengthening, the treatment of premature ejaculation and skin care and used to make the head clear, and has found to have the effects of increasing vigor, removing various diseases, strengthening the five viscera, quenching thirst, stopping diarrhea, and protecting “Gi” and blood. Thus, Nelumbinis semen has been used for the treatment of such diseases.
On the basis of the pharmacological actions of Nelumbinis semen, its various applications have been studied. Korean patent laid-open publication No. 2003-31729 discloses the activities of the Nelumbinis semen extract to protect liver cells and to prevent or treat liver cells. Korean patent laid-open publication No. 2004-26175 discloses a verification method related with the pain-alleviating effect, and a pain-killing preparation containing Nelumbinis semen. Also, the applicant has found that the Nelumbinis semen extract has the effect of treating melanocholia, and filed a patent application concerning a pharmaceutical composition containing it as an active ingredient.(Korean patent laid-open publication No. 2003-79104) However, in the prior art, there is no example of the use of the Nelumbinis semen extract for the prevention or treatment of ischemic heart diseases.
Hereinafter, the present invention will be described in more detail.
The Nelumbinis semen extract according to the present invention can be prepared by extraction with water, lower alcohol, hexane, ethyl acetate or a mixture thereof. More preferably, it can be prepared by an organic solvent extraction process of using an organic solvent to separate volatile or non-volatile substances. The organic solvent extraction process comprises the steps of performing extraction with lower alcohol, hexane, ethyl acetate or a mixture thereof, filtering the extract, concentrating the filtrate, and freeze-drying the concentrate. The lower alcohol is selected from the group consisting of methanol, ethanol and butanol. The extraction processes which can be used in the present invention include percolation extraction, ultrasonic extraction, filtration, reflux extraction, vacuum extraction, and other conventional extraction processes. Moreover, the Nelumbinis semen extract may also be prepared by a hot water extraction process of using high-temperature hot water to separate soluble substances. The hot-water extraction is preferably carried out at a temperature of 80˜100° C. for 1˜3 hours, and comprises the steps of adding water to Nelumbinis semen, filtering and concentrating the solution, and freeze-drying the concentrate. The concentration and freeze-drying steps can be carried out by various conventional methods known in the art.
500 g of a dried powder of Nelumbinis semen was placed in a flask containing 1 liter of triple-distilled water, and extracted with hot water at 100° C. for 1 hour. The extract was filtered through gauze. The filtrate was concentrated with a vacuum filter (Eyela, Japan) and freeze-dried to prepare the inventive Nelumbinis semen extract. As a result, 106 g of the dried extract was obtained.
2 liters of an aqueous ethanol solution was added to 1 kg of a dried powder of Nelumbinis semen and extracted two times with ultrasonic waves for 15 minutes, thus obtaining 3 liters of the extract. The collected extract was filtered through filter paper, and the filtrate was concentrated with a vacuum filter (Eyela, Japan) at 45° C. and 1 atm. The concentrate was freeze-dried to prepare the Nelumbinis semen extract. As a result, 78 g of the dried extract was obtained.
In order to examine the human body safety of the Nelumbinis semen extract, an acute toxicity test was first performed.
Evaluation of Acute Toxicity of the Nelumbinis Semen Extract to Rats
An acute toxicity test was carried out using 6-week specific pathogen-free (SPF) SD rats. The Nelumbinis semen extract of the present invention was suspended in a 0.5% methylcellulose solution and orally administered to groups each consisting of five rats in a single dose of 5 g/kg, 10 g/kg and 20 g/kg. After administration of the extract, death, clinical symptoms and weight change were observed, and a hematological test and hematobiochemical analysis were performed. Upon autopsy, abnormality in abdominal organs and chest organs was visually observed.
As a result, all rats administered with the extract did not show particular clinical symptoms, death, and changes in body weight, as well as toxicity in hematological assay, hematobiochemical analysis and autopsy. As a result, the Nelumbinis semen extract of the present invention exhibited no toxicity even at a dose of 20 g/kg in all rats, and thus had a 50% lethal dose (LD50) higher than 20 g/kg upon oral administration. This result demonstrates that the Nelumbinis semen extract is safe.
The inventive Nelumbinis semen extract was administered orally to white rats according to a conventional method and tested for toxicities in intraabdominal administration and subcutaneous injection. As a result, it was shown that the 50% lethal dose (LD50) of the inventive extract was at least 20 g/kg, indicating that the inventive extract is a safe substance.
Then, in order to examine the ability of the Nelumbinis semen extract to treat ischemic heart disease, changes in blood pressure, aortic output, coronary perfusion rate and cardiac output were measured.
Test of the Ability to Treat Ischemic Heart Disease for Myocardial Infarction-Induced Rats
(1) Preparation of Test Group
(i) Isolation of Heart
Sprague-Dawley male rats weighing about 250˜300 gm were purchased and accommodated in a cage at a temperature of 24˜26° C. and a relative humidity of 50˜60% under a 12-hr light/dark cycle controlled with an automatic power unit while the animals were permitted to free access to water and feed. All test procedures were performed according to the ethics of animal experiments. The animals were fasted for two hours before experiments and injected intraabdominally with 5 mg/100 gm bodyweight of pentobarbital to induce anesthesia. The anesthetized rats were fixed to a test bed by tying the legs and arms, and the inguinal region was incised to expose the femoral veins. Then, the exposed femoral veins were injected with 100 units/100 gm bodyweight of heparin. At 60 seconds after the heparin injection, the rats were subjected to median sternotomy to isolate the heart. The isolated heart was immersed in 4° C. physiological saline to induce the arrest of the heart. When the heart was arrested, the airway and gullet around the heart were removed, and then, catheters were inserted into the aorta and the left atrium and fixed with No. 3 silk suture. After ligating the hilum, the lung tissue was separated, and then, the incision marks were made in the pulmonary aorta to prevent a perfusate from filling in the right atrium.
(ii) Ex Vivo Perfusion of Heart
(Preparation and Principal of Ex Vivo Perfusion System)
An ex vivo perfusion circuit of the white rat heart used in this experiment was prepared by attaching a non-working Langendorff persusion system to a working heart perfusion system designed by Neeley and Chain et al.
The non-working ex vivo perfusion system is countercurrent-perfused from an aortic reservoir placed 100 cm above the heart into the heart under a water pressure of 100-cm H2O, and called the “non-working heart” because it maintains the heart function by coronary perfusion resulting from countercurrent perfusion has no isolation of the heart through the left ventricle. This non-working heart is used for 15 minutes of the initial experimental period and 15 minutes of the first recovery stage after the induction of myocardial infarction, and induces the recovery of the heart from enzyme deficiency in heart isolation and myocardial infarction. The working ex vivo perfusion system refers to a left heart preparation which is perfused from an atrial bubble trap reservoir placed 20 cm above the heart into the left atrium at a water pressure of 20-cm H2O, and a perfusate flowed in the left atrium flows through the left ventricle into the atrial bubble trap reservoir at a height corresponding to a water pressure of 100-cm H2O in a flow rate of 20˜30 ml/min (this amount is “aortic output”) for each heart beat. In the heart beat, an electrical pacing is not used. This working heart is used for 20 minutes before the induction of myocardial infarction and for 60 minutes after the 15-minute use of the Langendorff persusion system after the induction of myocardial infarction and is critical to compare the heart recovery before and after the induction of myocardial infarction. In this test, aortic perfusate and coronary perfusate are not used in reperfusion.
The heart prepared in the part (i) was connected to the modified Langendorff perfusion system prepared as described above, and subjected to Langendorff perfusion for 15 minutes to remove a blood component from the heart and to balance the concentrations of a solution in the extracellular matrix and a substrate in the perfusate. At this time, infusion into the left atrium was also performed. Furthermore, the controls of heart rate, the maximum aortic systolic pressure and coronary blood flow rate were determined.
Then, the Langendorff perfusion continued to perform while perfusing the left atrium, thus converting the heart into the working heart. After 15 minutes of the working heart before the induction of myocardial infarction, the left artial vessels and the aortic vessels were closed.
(iii) Injection of Nelumbinis Semen Extract and Induction of Myocardial Infarction
After the left atrial vessels and the aortic vessels were closed as in the part (ii), 50 ml of the Nelumbinis semen extract prepared in Example 1 was injected into the coronary artery through an injection cap at a concentration of 1 mg/ml for 3 minutes under a water pressure of 65-cm H2 O and allowed to distribute throughout the heart. Also, to prevent the heart surface from drying, a 37° C. physiological saline was dropped onto the heart surface, and a water jacket of the heart chamber was maintained as a warm jacket using a cardiac local warming method. Also, the temperature of the heart muscle during the entire test process was maintained at 37±1° C., and the left atrial vessels and the aortic vessels were closed and myocardial infarction was induced for 5 minutes.
After the induction of myocardial infarction, the ischemic state of the heart was stopped and the heart was subjected to Langendorff perfusion with a perfusate at 37° C. for 15 minutes. In this case, the coronary perfusate was not subjected to re-perfusion.
After 15 minutes of the non-working perfusion, left atrial perfusion was performed while continuing to perform the Langendorff perfusion, thus the non-working heart to the working heart. The working heart was measured for the recovery of the heart function for 60 minutes. In this case, if the recovery of the heart function was poor due to myocardial injury, the induction of non-working perfusion was not performed so that measurements and observations could be made under the same condition.
(2) Preparation of Control Group
A series of the procedures for preparing the test group were repeated except that myocardial infarction was induced using a perfusate containing no Nelumbinis semen extract.
The test group and the control group, each group consisting of 10 members, were measured for each of blood pressure, aortic output, coronary perfusion rate and cardiac output.
As can be seen from Table 1, the blood pressure, coronary perfusion rate and cardiac output of the test group treated with the Nelumbinis semen extract were statistically significantly increased starting from 10 minutes as compared to those of the control group. Also, the aortic output of the test group were statistically significantly increased starting from 20 minutes as compared to those of the control group.
Also, before and after the induction of myocardial infarction (ischemic shock), the control group and the test groups were measured for blood pressure, aortic output, coronary perfusion rate and cardiac output, and the measurement values before ischemic shock were expressed in terms of percentages of the measurement values before ischemic shock. The results are shown in Table 2 below.
As can be seen from Table 2 above, in the case of the test group treated with the inventive Nelumbinis semen extract, the blood pressure, aortic output, coronary perfusion rate and cardiac output after ischemic shock almost recovered to the levels before ischemic shock. This suggests that the Nelumbinis semen extract is effective in treating ischemic heart diseases.
The present pharmaceutical composition for treating ischemic heart diseases includes the Nelumbinis semen extract which is safe to the human body, and for the prevention or treatment of ischemic heart diseases as an active ingredient. The pharmaceutical composition may be administered orally or parenterally and may be formulated into typical pharmaceutical preparations.
That is, the Nelumbinis semen extract of the present invention may be formulated into various formulations for oral and parenteral administration upon clinical application. In the formulation, diluents or excipients may be used, which are exemplified by fillers, thickeners, binders, humectants, disintegrators, surfactants, etc.
Examples of solid formulations for oral administration include tablets, pills, powders, granules and capsules. The solid formulations may include, in addition to the Nelumbinis semen extract, at least one excipient selected from among starch, calcium carbonate, sucrose, lactose, gelatin, etc. Also, the solid formulations may include, in addition to a simple excipient, a lubricant such as magnesium stearate or talc.
Examples of liquid formulations for oral administration include suspensions, internal solutions, emulsions and syrups. The liquid formulations may include, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients which are exemplified by humectants, sweeteners, aromatics and preservatives.
Examples of preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. In the formulation into non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of suppositories, witepsol, macrogol, Tween 61, cacao fat, lanolin fat, glycerol and gelatin may be used.
The unit dose may, for example, occurs one, two, three or four times, or a half, third or quarter of an individual dose. The individual dose preferably contains the amount of an effective drugs which is given in one administration and usually corresponds to a whole daily dose or a half, third or quarter of the daily dose.
In the pharmaceutical composition for prevention and treatment of ischemic heart diseases, an effective amount of the Nelumbinis semen extract ranges from 30 to 700 mg/kg, and preferably 100 to 500 mg/kg, and may be administered once to six times daily. The dosage for a specific patient may vary according to the patient's weight, age, sex, health state and diet, administration duration, administration routes, excretion rates and severity of the illness.
In addition, the present invention provides a health food for treating ischemic heart diseases, comprising the Nelumbinis semen extract as an active ingredient. In the case of using the present extract as a food, the present extract may be added as it exists or in combination with other food or food ingredients, and may be used suitably according to general methods. Mixed amounts of active ingredients may be suitably determined according to the intended use (preventive, health or therapeutic purposes). Typically, the present extract may be added in an amount of 0.01 to 1 wt %, and preferably 0.1 to 1 wt %, based on the total weight of raw materials used in preparing a food or drink. An effective amount of the present extract may be determined based on an effective amount of the pharmaceutical composition. When consumed for a long period of time for health and sanitary purposes or health control, the present extract may be used in an amount lower than the range. Also, it is apparent that the present extract can be used in an amount higher than the range because the active ingredient carries no safety risk.
The type of the food is not particularly limited. Examples of foods to which the present extract can be added include meats, sausages, breads, chocolates, candies, snacks, confectionary, pizza, instant noodles, other noodles, gums, dairy products including ice creams, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, as well as traditional therapeutic preparations for use as an antianemic, a body function-strengthening agent, a skin whitening agent, and the like. Also, it can be used in various herbal medicinal formulations, such as Yul-Da-Han-So-Tang, Chung-Shim-San-Yak-Tang, and Tae-Eum-Jo-Wea-Tang.
Pharmaceutical formulations and health foods containing the Nelumbinis semen extract were prepared in Formulation Examples 1-7 below, but the scope of the present invention is not limited to these Examples.
A soft capsules were prepared according to a soft capsule preparation method described in General Rules for Preparation in a guidebook, Korean Pharmacopoeia, using 100.0 mg of the Nelumbinis semen extract prepared in Example 1, 175.0 mg of soybean oil, 45.0 mg of cera flava, 127.5 mg of hydrogenated palm oil, 21.0 mg of soybean phospholipids, 212.0 mg of gelatin, 50.0 mg of glycerin (gravity: 1.24), 76.0 mg of di-sorbitol, 0.54 mg of methyl-paraoxybenzoate, 0.90 mg of propyl-paraoxybenzoate, 0.56 mg of methylvanillin, and a proper amount of yellow no. 203.
100.0 mg of the Nelumbinis semen extract prepared in Example 1, 90.0 mg of corn starch, 175.0 mg of lactose, 15.0 mg of L-hydroxypropylcellulose, 5.0 mg of polyvinylpyrolidone 90 and a proper amount of ethanol were homogeneously mixed, g ranulated by wet granulation, mixed with 1.8 mg of magnesium stearic acid, and forced into 400 mg tablets.
100.0 mg of the Nelumbinis semen extract prepared in Example 1, 83.2 mg of corn starch, 175.0 mg of lactose and 1.8 mg of magnesium stearic acid were homogeneously mixed, and filled into capsule shells at 360 mg per capsule.
Chewing gum was prepared according to a general method using 0.24˜0.64% of the Nelumbinis semen extract prepared in Example 1, 20% of gum base, 1% of a fruit aromatic, 2% of water and the balance of sugar.
Ice cream was prepared according to a general method using 0.24˜0.64% of the Nelumbinis semen extract prepared in Example 1, 10.0% of milk fat, 10.8% of SNF (Solids Not Fat), 12.0% of sugar, 3.0% of starch syrup, 0.5% of an emulsion stabilizer (span), 0.15% of an aromatic (strawberry) and the balance of water.
A beverage was prepared according to a general method using 0.48˜1.28 mg of the Nelumbinis semen extract prepared in Example 1, 522 mg of honey, 5 mg of thioctic acid amide, 10 mg of nicotinic acid amide, 3 mg of riboflavin hydrochloride sodium, 2 mg of pyridoxine hydrochloride, 30 mg of inositol, 50 mg of orotic acid and 200 ml of water.
Sausage was prepared according to a general method using 0.24˜0.64% of the Nelumbinis semen extract prepared in Example 1, 27.5% of chicken, 3.5% starch, 1.7% of soybean proteins, 1.62% of edible salt, 0.5% of glucose, 0.94˜1.34% of another additive (glycerin) and the balance of pork,
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2004-0042190 | Jun 2004 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR05/01739 | 6/9/2005 | WO | 00 | 12/8/2006 |
