KR101782962B1 - Composition for treatment of leukemia and method of manufacturing extract from myrrh - Google Patents

Abstract

The present invention provides a composition for treating leukemia comprising a myrrh extract which is extracted from a dried myrrh powder with an aliphatic polar solvent and which is an effective ingredient and pharmacologically acceptable carriers. The myrrh extract may induce the death and proliferation of leukemia cells by a very small amount, and thus it can be widely applied to various pharmaceutical formulations.

Description

TECHNICAL FIELD The present invention relates to a composition for treating leukemia and a method for producing leukemia,

This technology relates to a therapeutic agent for leukemia, and is a technique relating to a pharmacological composition capable of exerting an excellent killing effect as a trace amount of natural extract. More particularly, it relates to a composition for use as an agent for inducing apoptosis of leukemia cell growth inhibitor, leukemia differentiation treatment agent or leukemia. The present technology also relates to a method for extracting natural extracts capable of exerting a significant killing effect on leukemia factors.

Myrrh (myrrh) is a bitter, fragrant, yellowish reddish brown resin (oleoresin) rubber. It is obtained from a small, thorny flowering arboreum of the Commiphora or a plant of the family Burseraceae such as Commiphora myrrha or Commiphora abyssinica, It is distinguishable from the plant (Commiphora myrrha).

Myrrh is widely used in cosmetics such as fragrance, fragrance, fragrance, fragrance of perfume, medicine and so on. In the ancient Near East, the Middle East and Europe, It was very precious. For the common people, it is famous in the Christian scriptures that the Magi presented gold, frankincense, and myrrh to the baby Jesus.

As a drug, it has been mainly used for the treatment of oral infection and inflammation. Mouthwashes and toothpastes containing myrrh are known to be effective for treating gingivitis. It is said to be used as a topical application, preventing mouth, teeth and eye infections. In Egypt, it was an essential flavor used as a preservative in making mummies. Prior to the first century BC, myrrh was used to prevent wine from becoming fermented and vinegar, while in Greece and Rome it was prescribed as an effective remedy for snakebite. Indians use myrrh for treatment of circulatory disorders, neurological disorders, arthritis, etc. In the West, myrrh has been recognized as a substance that relieves the pain of the body and accelerates the healing process, giving a strong preservative effect.

However, with the advancement of science, the importance of myrrh had gradually decreased. It is currently used mainly as a component of toothpaste, fragrance and irritant tonic, and as a protective agent in the pharmaceutical industry. Myrrh has some antiseptic and astringent effects. It is used as a gastric degreasing agent for medicines. Myrrhic tincture is used for relieving the pain of gums and oral diseases. Essential oils that have been distilled from myrrh have become a component of some strong fragrances. Myrrh can flow from the resin pipe in the bark when the bark cracks or scatters naturally. When exposed to air, the fluid becomes harder and makes droplets and irregular masses, which is called 'tears'.

On the other hand, acute myeloid leukemia ("AML") is a disease that leads to death in a few months without effective treatment. The diagnosis of AML is based on the morphological recognition of immature leukemia cells increased in the bone marrow. In addition, AML cells may exhibit different morphological signal differentiation, including morphological features similar to mature monocytes / macrophages.

Conventional AML leukemia therapy uses chemotherapy with chemicals that are toxic to malignant tumors, but these chemicals have the side effect of destroying normal cells as well. In addition, some leukemia cells are resistant to chemotherapy, making it difficult to remove all of the leukemia cells.

In order to solve such a problem, studies are currently under way to utilize leukemia differentiation therapy having few side effects. In other words, leukemia is a problem in the intermediate process of hematopoietic stem cell differentiation into immune cells or blood cells, and thus, leukocytes and leukocytes, which are not completely differentiated into granulocytes, monocytes, leukocytes and neutrophils, It develops because of the abnormal proliferation of bone marrow. Therefore, cells completely differentiated by the cell differentiation therapeutic agent are very useful because they do not have the ability to self-proliferate and die when the cell life is over in the body. In addition, leukemia differentiation treatment agent can inhibit proliferation of precursor cells by inducing differentiation of leukemia cells. In addition, since the differentiation agent specifically acts on cells that do not undergo differentiation, it has little effect on normal cells in which the differentiation has completely progressed.

Compounds such as retinoic acid, dimethylsulfoxide, phorbol ester and 1,25-dihydroxyvitamin D3 are known as such differentiation therapeutic agents. Among these, retinoic acid can differentiate AML cells into granulocytes, while 1,25-dihydroxyvitamin D3 can differentiate AML cells into monocytes.

However, these known agents are also a kind of chemotherapy, and leukemia cells cause side effects such as resistance to the differentiation agents, and development of a new therapeutic agent is required.

The present invention provides a novel therapeutic composition capable of inducing effective killing of leukemic cells by natural extracts and remarkably inhibiting the growth of the leukemia cells.

The present invention also provides a method for extracting said natural extract.

The composition for treating leukemia according to an embodiment of the present invention includes a myrrh extract containing an active ingredient extracted by drying a dried myrrh powder in an aliphatic polar solvent and a pharmacologically acceptable carrier do.

When the myrrh extract solution 100 μg / ml was treated in 5.0 × 10 ⁴ HL-60 cells for 48 hours and assayed by Alamar Blue assay, the survival rate of HL-60 cells decreased to less than 6% . In addition, the EC50 value of the myrrh extract may be 32 쨉 g / ml to 36 袖 g / ml.

Drying of the myrrh can be done in the shade. On the other hand, the aliphatic polar solvent may be an alcohol having 1 to 4 carbon atoms, for example, ethanol and the like. The extraction process can be carried out at a temperature of less than 60 < 0 > C.

The therapeutic composition according to one embodiment of the present invention can be used as a therapeutic agent for leukemia, and the leukemia includes acute promyelocytic leukemia.

The method for preparing a mulberry extract according to an embodiment of the present invention comprises the steps of preparing a mulch powder by pulverizing a primary dried myrrh powder in a shade field, adding the pulverized mulch powder to an alcohol solvent having 1 to 4 carbon atoms at a temperature of less than 60 ° C Extracting the active ingredient by submerging the active ingredient in an alcoholic solvent to obtain a solid content of the active ingredient by secondary drying the alcoholic solvent containing the active ingredient, and freezing and storing the solid content of the active ingredient. The secondary drying may be performed under a reduced pressure of less than 60 캜.

The composition for treating leukemia according to an embodiment of the present invention is excellent in the killing and growth inhibitory effect on leukemia cells and the mulberry extract is a natural product and has little side effects and is widely used in various fields such as a food field other than a therapeutic composition . In addition, the mulberry extract may be utilized as an agent for inducing apoptosis of leukemia cell growth inhibitor, leukemia differentiation treatment agent or leukemia.

Furthermore, the mulberry extract according to the present invention can be applied to various formulations in a flexible amount, since the killing effect on leukemic cells is excellent even in a very small amount.

FIG. 1 is a graph showing the survival rate of HL-60 cells corresponding to the experiment performed in Example 2. FIG. The horizontal axis in the figure indicates the concentration of the extract and the vertical axis indicates the survival rate of the leukemia cells.

Hereinafter, the composition for treating leukemia according to the present invention will be described in detail.

The composition for treating leukemia includes myrrh extract and pharmacologically acceptable carriers. To prepare myrrh extract, a step of drying myrrh is first necessary. The drying method of the myrrh is not particularly limited, but the drying can be performed in the shade so that the intrinsic compounds contained in the myrrh can not be denatured by an external strong light source. The dried myrrh powder is pulverized and used in an appropriate form for maximizing the extraction efficiency. The degree of pulverization, that is, the size of the myrrh powder, is independent of the change in chemical properties of the extract. The drying may be performed at room temperature, and the drying period is not particularly limited, but is preferably about several days.

As the aliphatic polar solvent, a solvent excellent in the extraction efficiency of myrrh can be used even at a temperature lower than 80 ° C and furthermore lower than 60 ° C, preferably an aliphatic alcohol having 1 to 4 carbon atoms is used. As the extraction solvent, for example, ethanol can be mentioned. Temperature extraction method such as hot water extraction may cause denaturation or destruction of a compound that is a leukemia-killing factor contained in myrrh. Therefore, the method for extracting myrrh-powdery extract contained in the composition for treating leukemia according to one embodiment of the present invention is a low- desirable.

The myrrh extract may contain a function of a leukemia cell growth inhibitor, a leukemia differentiation treatment agent, or an apoptosis inducing agent of leukemia, and may be effective for promyelocytic acute leukemia, particularly as leukemia.

As described above, the pulverized mulberry can be extracted by taking an organic solvent such as ethanol without taking any additional action. The resulting extract can be dried under a temperature of less than 60 ° C and can be made under reduced pressure to improve the drying efficiency. Upon completion of the drying, a solid myrrh extract can be obtained. The obtained solid mulberry extract is preferably stored at a cryogenic temperature (below -15 DEG C) in order to prevent denaturation of the active ingredient by external conditions such as moisture and ensure stability of the active compound in the extract.

The composition for treating leukemia according to an embodiment of the present invention may include the myrrh extract alone or other pharmacologically acceptable carriers. Examples of the pharmacologically acceptable carrier include water, glycerin, castor oil, starch, saline, and the like.

In addition, the mulberry extract can be used as a composition for preventing or treating leukemia, a medicine, a food or a food additive, and can be used either orally or parenterally when used as a medicine. The formulation of the composition can be variously determined in consideration of the method of use and effectiveness. For example, examples of the composition of the composition include an agent such as a warning agent, a granule, a lotion, a powder, a syrup, a liquid, an injection, a capsule, and a reducing agent.

The dose of the composition for treating leukemia according to one embodiment of the present invention may vary depending on the age, sex, condition, and the body of the patient. The degree of absorption and the drug to be used in combination.

Hereinafter, mulberry extract according to one embodiment of the present invention will be described with reference to specific examples. However, the technical idea of the present invention is not limited by the following embodiments.

[Example]

[Example 1] Production of myrrh extract

The obtained myrrh (Chinese origin) was stored at room temperature at room temperature and dried for 5 days. The dried myrrh was crushed to a predetermined size. The pulverized myrrh was immersed in a 95% ethanol solution and subjected to extraction at a temperature of 50 ° C for 24 hours. The mixture of the myrrh extract and the ethanol solution was dried under reduced pressure at a temperature of 45 캜. After drying, a mulberry extract of solid content was obtained. The resulting mulberry extract of solid content was stored at a low temperature of -20 占 폚.

[Example 2]: Effect of myrrh extract on growth of leukemia cells

Alamar blue analysis was performed on HL-60, a human leukemia cell, in order to examine the effect of the mulberry extract obtained in Example 1 on the growth of leukemia cells. Al-Rama analysis is a modified form of MTT assay, which treats compounds that are degraded by specific enzymes in living cells and measures the fluorescence intensity of the products as they decompose to determine the relative number of living cells after treatment It is an experimental method.

The leukemia cells HL-60 cells used in this example were distributed from the Korean Cell Line Bank (KCLB) and tested for their inhibitory effect on cell growth of HL-60, a leukemia cell. HL-60 leukemia cells of 20% FBS and then cultured in RPMI1640 medium containing HEPES 25 mM injection of 5.0 × 10 ⁴ cells per well in a 96-well plate and myrrh extract of Example 1 was dissolved in DMSO 0 to When treated with 100 μg / ml for 48 hours, the degree of inhibition of cell growth was confirmed. After treating each concentration of the extract, 20 μl of AlamarBlue reagent was added to 0.2 ml of the cell culture solution filled in each well in a 96-well plate, and the plate was incubated in an incubator for 2 hours. The plates were gently shaken to uniformly react the cells of each well, and the intensity of fluorescence at 590 nm was measured with a fluorescence microplate reader (Molecular Devices Corp.) while irradiating light at a wavelength of 544 nm. FIG. 1 is a graph showing the survival rate of HL-60 cells corresponding to the experiment performed in this example. Table 1 below shows the experimental results of this embodiment. Each concentration experiment is the result of repeating 5 times, and the change of the average value is shown in Fig.

0
μg / ml 3.125
μg / ml 6.25
μg / ml 12.5
μg / ml 25
μg / ml 50
μg / ml 100
μg / ml Average 1,000 1.048 1.066 0.971 0.684 0.287 0.055 Standard Deviation 0.000 0.025 0.041 0.030 0.013 0.014 0.005

As shown in Table 1 and FIG. 1, as the treatment concentration of mulberry extract was higher, the growth of leukemia cells was decreased. From this, it was confirmed that mulberry extract had therapeutic effect on leukemia.

[Example 3] Measurement of EC50 value

The term EC50 means a concentration showing an effect of 50% when the maximum effect that can be exhibited by the myrrh extract according to the present invention is 100%, and is referred to as 50% effective concentration or 50% efficacy concentration. The EC50 calculation result was 34.0 μg / ml.

[Example 4] Evaluation of stability

The mulberry according to the present invention was divided into herbal medicines, and it was considered that there would be no problem in stability. To confirm the toxicity test at oral administration and intraperitoneal administration.

Acute toxicity tests were carried out using 6-week-old SPF SD rats. The extracts of Example 1 of the present invention were suspended in 0.5% methylcellulose solution and administered to a 2-group animal at a dose of 5 g / kg in a single oral dose. After the administration of the test substance, the mortality, clinical symptoms, and weight changes of the animals were observed, and hematological tests and blood biochemical tests were carried out, and autopsy was performed to observe the abnormalities of the abdominal organs and thoracic organs.

As a result of the test, there were no clinically symptomatic or dead animals in all the animals to which the test substance was administered, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. These results gwandonghwa extract does not represent a change in toxicity both to 5 g / ㎏ in rats administered oral minimum lethal dose (LD ₅₀₎ was determined to be a safe substance less than 5 g / ㎏.

Claims (8)

A composition for preventing or treating leukemia comprising an extract obtained by immersing a pulverized product of myrrh (myrrh) with an active ingredient in an aliphatic polar solvent.
delete delete The method according to claim 1,
Wherein the aliphatic polar solvent is an alcohol having 1 to 4 carbon atoms.
delete The method according to claim 1,
Wherein the leukemia is prolymphocytic acute leukemia.
delete delete

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