WO2012093920A2 - Composition including white morning glory extract for treating leukemia - Google Patents

Composition including white morning glory extract for treating leukemia Download PDF

Info

Publication number
WO2012093920A2
WO2012093920A2 PCT/KR2012/000217 KR2012000217W WO2012093920A2 WO 2012093920 A2 WO2012093920 A2 WO 2012093920A2 KR 2012000217 W KR2012000217 W KR 2012000217W WO 2012093920 A2 WO2012093920 A2 WO 2012093920A2
Authority
WO
WIPO (PCT)
Prior art keywords
leukemia
extract
composition
morning glory
cells
Prior art date
Application number
PCT/KR2012/000217
Other languages
French (fr)
Korean (ko)
Other versions
WO2012093920A3 (en
Inventor
황성연
정경채
Original Assignee
주식회사 한국전통의학연구소
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 한국전통의학연구소 filed Critical 주식회사 한국전통의학연구소
Publication of WO2012093920A2 publication Critical patent/WO2012093920A2/en
Publication of WO2012093920A3 publication Critical patent/WO2012093920A3/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/39—Convolvulaceae (Morning-glory family), e.g. bindweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to a composition for preventing or treating leukemia, including baekchuk extract.
  • Leukemia is divided into acute and chronic depending on the degree of cell differentiation, that is, the rate of deterioration, and is divided into myeloid and lymphocytic depending on the origin of the cell.
  • promyelocytic in acute myeloid leukemia has some morphological differences.
  • HL-60 cells are suspended cells isolated from peripheral white blood cells of promyelocytic leukemia patients. And, there may be a difference in treatment method and effect depending on the nature of the leukemia.
  • Pharbitis Semen is the seed of the white morning glory (Pharbitis nil CHOISY), common to Korea, China and Japan.
  • the morning glory is native to tropical Asia such as India and is known to have about 256 varieties.
  • Black seeds are called black axis and white seeds are called white axis.
  • the main ingredients are Pharbitin, Pharbitic acid, and Tiglic acid. Lower dead ( ⁇ ⁇ ) and diuretic effect is strong, and when the body swells down, chronic pyelonephritis, liver cirrhosis, etc. is used when the ascites. Effective for seawater and asthma and used for stubborn constipation and parasite removal.
  • Intestinal interlocking action as a pharmacological action has been reported to have a strong lower action.
  • the taste is bitter and cold, which acts on the lungs, kidneys, and colon, causing urine to pass through the stool, edema, redness (due to lumps in the stomach due to long weight), and low back pain.
  • Folk remedies are known to be good if you have a lot of leaves attached to the morning glory and cut about 20cm from the root and dry it.
  • the present inventors completed the present invention by confirming that the baekchuk extract can effectively kill leukemia cells while studying the natural products of the baekchuk.
  • the present invention for achieving the above object, provides a composition for the prevention or treatment of leukemia, comprising the active ingredient extracted from the white axis (Pharbitidis Semen; ⁇ ⁇ ) with an organic solvent.
  • the organic solvent is preferably a lower alcohol (C1 to C4), more preferably 95% ethanol solvent.
  • the extraction is preferably extracted for 24 hours at 50 °C, dried under reduced pressure conditions.
  • the white-axis extract active ingredient when treated with HL-60 cells for 48 hours after the extract showing an effect that the EC 50 value determined by the Alamar Blue assay of 91.5 ⁇ g / ml or less, to some extent its efficacy I can guarantee it.
  • composition may be used including a pharmaceutically acceptable carrier or diluent.
  • composition may be suitable for the prevention or treatment of promyelocytic leukemia.
  • the use of a polar solvent selected from lower alcohols or mixed solvents thereof is preferable, among which ethanol is more preferable.
  • the concentration of ethanol may be varied, but preferably 95% ethanol is more preferred.
  • the extraction temperature is preferably 40 ° C. to 60 ° C. because it does not cause denaturation or destruction of the active ingredient, and it is preferable to concentrate and dry under reduced pressure within this range.
  • the ethanol extract of the rat axis kills HL-60 human leukemia cells at 100 ⁇ g / ml at 74.1%, which is unique in that the killing rate is rapidly increased at about 90 ⁇ g / ml. Rapidly increasing mortality means that it can be used as a very useful therapeutic agent for leukemia through proper use control.
  • the killing activity against HL-60 leukemia cells can be expected to show a therapeutic effect against promyeloid leukemia.
  • the EC 50 value of the white axis ethanol extract showing an effective effect was about 91.5 ⁇ g / ml. It can be seen that it contains visible white axis ethanol extract. In other words, it can be seen that the proper extraction process was made.
  • the "pharmaceutically acceptable carrier” is a pharmaceutically acceptable substance such as a liquid or solid filler, diluent, excipient or solvent which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body. , Composition or vehicle.
  • the composition for treating leukemia of the present invention may be prepared as a medicament by adding one or more pharmaceutically acceptable carriers together with the active ingredient.
  • the carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .
  • Forms of the formulations of the present invention can use any suitable formulation known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA). It can be administered orally at the time of clinical administration and can be used in the form of general pharmaceutical preparations, and when formulated, it is prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. .
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.
  • liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as water for injection Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
  • the dosage of the composition for treating leukemia according to one embodiment of the present invention is determined by the age, sex, condition, It may be determined in consideration of the absorbency and the drug used in combination.
  • the baekchuk extract according to the present invention has a sufficient killing effect on leukemia cells and is valuable as a therapeutic composition for leukemia, and especially since it is an extract from natural products, there is little risk of side effects and more useful for the treatment of promyeloid leukemia. It is thought to be.
  • the efficacy can be expected to be very large according to the quantitative expansion.
  • 1 is a result of Alamar Blue analysis to determine the effect of the introduction of the rat axis in the growth of leukemia cells in human HL-60 cells.
  • the horizontal axis shows the concentration of the extract
  • the vertical axis shows the survival rate of the cells.
  • the white axis is dried in the shade at room temperature for 5 days and ground finely for extraction efficiency.
  • the milled white axis was submerged in 95% ethanol and extracted at 50 ° C. for 24 hours. After filtering this, it dried under 45 degreeC pressure reduction conditions and obtained solid content. It was stored at a low temperature of -20 ° C.
  • Alamar Blue analysis was performed on human leukemia cells HL-60.
  • the Alamar Blue assay is a modified form of the MTT assay, in which a specific enzyme degrades a living cell and then measures the fluorescence intensity of the product as the compound breaks down to determine the relative number of living cells after treatment. I am an experimental method.
  • HL-60 cells the leukemia cell line used in the present invention, were distributed from the Korean Cell Line Bank (KCLB) and used for experiments. Roswell Park Memorial Institute medium containing 20% FBS (fetal bovine serum, fetal bovine serum) (Welgene) and 25 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) ) Incubated in 1640 medium. The cells were maintained at 37 ° C. with 5% CO 2 water and passaged for 2-3 days.
  • FBS fetal bovine serum, fetal bovine serum
  • HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
  • the baekchuk extract extracted in Example 1 confirmed the cell growth inhibitory effect on HL-60 leukemia cells. Specifically, 5.0 ⁇ 10 per well in a 96 well plate 4 The white axis in which HL-60 cells are seeded and dissolved in dimethyl sulfoxide (DMSO) When the ethanol extract was treated at concentrations of 0 to 100 ⁇ g / ml (specifically, concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 ⁇ g / ml, respectively) for 48 hours, the degree of inhibition of cell growth was confirmed. (Table 1).
  • DMSO dimethyl sulfoxide
  • the higher the treatment concentration in the range of the treatment of the baekchuk is more than about 90 ⁇ g / ml shows a tendency of the survival rate of leukemia cells is reduced, from which the baekchuk is prevented or treated It can be seen that there is an effect. In particular, the standard deviation is very small in this vicinity, it can be estimated a certain efficacy.
  • Survival rate of Table 1 shows the relative cell number of the cells after 48 hours according to the treatment concentration of each rat axis extract, when the number of cells of the control group treated with the rat axis extract was 1. In addition, the EC 50 (half maximal effective concentration) was determined to be 91.5 ⁇ g / ml.
  • the baekchuk extract of the present invention has excellent HL-60 apoptosis activity, and further demonstrates the therapeutic and prophylactic activity of leukemia.
  • the white axis used in the present invention was used as a medicinal herb, so it was determined that there was no problem in stability.
  • Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally administered at a dose of 5 g / kg in suspension of the root of the extract of Example 1 of the present invention in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed, and autopsy was performed to observe abdominal and thoracic organ abnormalities.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a composition including a white morning glory extract for preventing or treating leukemia, and the white morning glory extract of the present invention is amply effective in leukemia cell apoptosis, thereby rendering value as the composition for treating leukemia, and above all, is projected to have lower risk of side effects due to the extract having a natural source and to be more useful in treating promyelocytic leukemia. The white morning glory extract can be predicted to have a very significant effect in larger quantities, particularly because the slope of the white morning glory extract for human HL-60 cell apoptosis is very steep at or over a useful density.

Description

백축 추출물을 포함하는 백혈병 치료용 조성물Leukemia Therapeutic Compositions Including Leprosy Extract
본 발명은 백축 추출물을 포함하는 백혈병 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating leukemia, including baekchuk extract.
백혈병은 세포의 분화 정도, 즉 악화 속도에 따라 급성(Acute)과 만성(Chronic)으로 나뉘고, 세포의 기원에 따라 골수성(myeloid)과 림프구성(lymphocytic)으로 나뉜다. 또한, 급성 골수성 백혈병 중 전골수성(promyelocytic)은 일부 형태학적 차이를 가진다. 한편, HL-60세포는 전골수구성 백혈병환자의 말초백혈구에서 분리, 수립한 부유세포이다. 그리고, 백혈병의 성격에 따라 치료방법과 효과의 차이가 있을 수 있다. Leukemia is divided into acute and chronic depending on the degree of cell differentiation, that is, the rate of deterioration, and is divided into myeloid and lymphocytic depending on the origin of the cell. In addition, promyelocytic in acute myeloid leukemia has some morphological differences. On the other hand, HL-60 cells are suspended cells isolated from peripheral white blood cells of promyelocytic leukemia patients. And, there may be a difference in treatment method and effect depending on the nature of the leukemia.
백축(Pharbitis Semen)은 흰 나팔꽃(Pharbitis nil CHOISY)의 씨를 말하며, 한국, 중국, 일본이 공통된다. 나팔꽃은 인도 등 열대아시아 원산이며 약 256변종이 있다고 알려져 있으며 검은 씨를 흑축(黑丑), 흰색 씨를 백축(白丑)이라고 한다. 주요 성분으로는 파르비틴(Pharbitin), 파르비틴산(Pharbitic acid), 티글린산(tiglic acid)등이 있다. 사하(瀉下)작용과 이뇨작용이 강하고 기를 잘 내려 몸이 부을 때 , 만성신우신염, 간경화 등으로 복수가 찰 때 사용한다. 해수, 천식에 유효하며 완고한 변비, 기생충제거에도 사용한다. 약리작용으로 장관연동작용으로 강력한 사하작용이 보고되었다. 맛은 쓰고 성질이 차가워 폐와 신장과 대장에 작용하여 대소변을 통하게 하고, 부종, 적취(오랜 체중으로 말미암아 뱃속에 덩어리가 생기는 병), 요통에 효과가 있다. 민간요법으로 나팔꽃에 잎이 많이 붙어 있을 때 뿌리에서 20cm정도 잘라서 말려 두었다가 동상에 걸렸을 때 이것을 달인 물로 환부를 찜질하면 좋다고 알려져 있다. Pharbitis Semen is the seed of the white morning glory (Pharbitis nil CHOISY), common to Korea, China and Japan. The morning glory is native to tropical Asia such as India and is known to have about 256 varieties. Black seeds are called black axis and white seeds are called white axis. The main ingredients are Pharbitin, Pharbitic acid, and Tiglic acid. Lower dead (때 下) and diuretic effect is strong, and when the body swells down, chronic pyelonephritis, liver cirrhosis, etc. is used when the ascites. Effective for seawater and asthma and used for stubborn constipation and parasite removal. Intestinal interlocking action as a pharmacological action has been reported to have a strong lower action. The taste is bitter and cold, which acts on the lungs, kidneys, and colon, causing urine to pass through the stool, edema, redness (due to lumps in the stomach due to long weight), and low back pain. Folk remedies are known to be good if you have a lot of leaves attached to the morning glory and cut about 20cm from the root and dry it.
그러나 아직 백축의 백혈병 억제 효과에 대하여 알려진 바는 없다. 이에 본 발명자들은 백축에 대한 천연물 연구를 하던 중, 백축 추출물이 백혈병 세포를 효과적으로 사멸시킬 수 있음을 확인함으로써 본 발명을 완성하였다. However, there is no known leukemia inhibitory effect. Accordingly, the present inventors completed the present invention by confirming that the baekchuk extract can effectively kill leukemia cells while studying the natural products of the baekchuk.
본 발명의 목적은 백축 추출물을 유효성분으로 함유하는 백혈병 치료 조성물을 제공하는 것이다. It is an object of the present invention to provide a leukemia therapeutic composition containing the baekchuk extract as an active ingredient.
상기 목적을 달성하기 위한 본 발명은, 백축(Pharbitidis Semen; 白丑)을 유기 용매로 추출한 유효성분을 포함하는 백혈병 예방 또는 치료용 조성물을 제공한다. 상기 유기 용매는 저급 알코올(C1 내지 C4)이 바람직하며, 보다 바람직하게는 에탄올 95% 용매인 것이 바람직하다. The present invention for achieving the above object, provides a composition for the prevention or treatment of leukemia, comprising the active ingredient extracted from the white axis (Pharbitidis Semen; 白 丑) with an organic solvent. The organic solvent is preferably a lower alcohol (C1 to C4), more preferably 95% ethanol solvent.
상기 추출은 백축을 50℃에서 24시간 동안 추출하여, 감압 조건하에 건조하는 것이 바람직하다. The extraction is preferably extracted for 24 hours at 50 ℃, dried under reduced pressure conditions.
상기 백축 추출 유효성분은, 이를 HL-60 세포에 48시간 처리한 후 알라머 블루(Alamar Blue) 분석법에 의해 정해진 EC50 값이 91.5 ㎍/㎖ 이하인 효과를 보이는 추출물인 경우, 그 효능을 어느 정도 보장할 수 있다. The white-axis extract active ingredient, when treated with HL-60 cells for 48 hours after the extract showing an effect that the EC 50 value determined by the Alamar Blue assay of 91.5 ㎍ / ㎖ or less, to some extent its efficacy I can guarantee it.
또한, 상기 조성물은 약제학적으로 허용가능한 담체 또는 희석제를 포함하여 사용될 수 있다. In addition, the composition may be used including a pharmaceutically acceptable carrier or diluent.
그리고, 상기 조성물은 전골수성 백혈병의 예방 또는 치료에 적합한 것일 수 있다. And, the composition may be suitable for the prevention or treatment of promyelocytic leukemia.
상기 유기 용매의 경우, 저급 알코올 또는 이들의 혼합용매로부터 선택된 극성용매의 사용이 바람직하며, 그 중 에탄올이 더욱 바람직하다. 에탄올의 농도를 다르게 할 수도 있으나, 바람직하게는 95% 에탄올이 더욱 바람직하다. In the case of the organic solvent, the use of a polar solvent selected from lower alcohols or mixed solvents thereof is preferable, among which ethanol is more preferable. The concentration of ethanol may be varied, but preferably 95% ethanol is more preferred.
추출온도는 40℃ 내지 60℃가 유효성분의 변성이나 파괴를 유발하지 않기 때문에 바람직하며, 이러한 범위 내에서 감압조건하에서 농축 및 건조하는 것이 바람직하다. The extraction temperature is preferably 40 ° C. to 60 ° C. because it does not cause denaturation or destruction of the active ingredient, and it is preferable to concentrate and dry under reduced pressure within this range.
본 발명의 구체적인 실시에서 백축의 에탄올 추출물은 HL-60 인간 백혈병세포를 100 ㎍/㎖에서 74.1% 사멸시키는데, 그 경향에서 특이할 점은 약 90㎍/㎖정도에서 급격히 사멸율이 높아진다는 것이다. 급격히 사멸율이 높아진다는 것은 적절한 사용량 조절을 통해, 매우 유용한 백혈병 치료제로 사용가능하다는 것을 의미한다. 한편, HL-60 백혈병 세포에 대한 사멸활성은 전골수성 백혈병에 대한 치료효과를 보일 것으로 예상할 수 있게 한다. 알라머 블루(Alamar Blue) 분석법에 의한 실험에서, 유효한 효과를 보이는 백축 에탄올 추출물의 경우, 그 EC50 값이 91.5 ㎍/㎖ 정도를 나타내었는데, 이 정도 이하의 값을 갖는다면, 충분히 유효한 효과를 보이는 백축 에탄올 추출물이 포함되어 있다고 볼 수 있을 것이다. 즉, 적절한 추출과정이 이루어졌다고 볼 수 있는 것이다. In a specific embodiment of the present invention, the ethanol extract of the rat axis kills HL-60 human leukemia cells at 100 μg / ml at 74.1%, which is unique in that the killing rate is rapidly increased at about 90 μg / ml. Rapidly increasing mortality means that it can be used as a very useful therapeutic agent for leukemia through proper use control. On the other hand, the killing activity against HL-60 leukemia cells can be expected to show a therapeutic effect against promyeloid leukemia. In the experiments by the Alamar Blue assay, the EC 50 value of the white axis ethanol extract showing an effective effect was about 91.5 μg / ml. It can be seen that it contains visible white axis ethanol extract. In other words, it can be seen that the proper extraction process was made.
상기 “약제학적으로 허용가능한 담체”는 신체의 한 기관 또는 부분으로부터 신체의 다른 기관 또는 부분으로 활성 성분을 수송하는 역할을 하는 액체 또는 고체 충진제, 희석제, 부형제 또는 용매와 같은 약제학적으로 허용되는 물질, 조성물 또는 운반체(vehicle)를 의미한다. 본 발명의 백혈병 치료용 조성물은 유효성분과 함께 추가로 약제학적으로 허용되는 1종 이상의 담체를 첨가하여 약제로 제조할 수 있다. 상기 담체로는 식염수, 완충 식염수, 물, 글리세롤 및 에탄올 등이 있으나 이에 한정되지 않으며, 당해 기술 분야에 알려진 적합한 제제(Remingtons's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)를 모두 사용 가능하다.The "pharmaceutically acceptable carrier" is a pharmaceutically acceptable substance such as a liquid or solid filler, diluent, excipient or solvent which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body. , Composition or vehicle. The composition for treating leukemia of the present invention may be prepared as a medicament by adding one or more pharmaceutically acceptable carriers together with the active ingredient. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .
본 발명의 제제의 형태는 당해 기술분야에 알려진 적합한 제제(Remingtons's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)를 모두 사용 가능하다. 임상 투여시에 경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있으며, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드, 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 또한 치료제의 효능 증진을 위해 칼슘이나 비타민 D3를 첨가할 수 있다. 이러한 조성물은 단위-용량(1회분) 또는 다중-용량(수 회분) 용기, 예를 들면, 밀봉된 앰풀 및 바이알에 제시될 수 있고, 사용 직전에 멸균성 액상 담체, 예를 들면, 주사용 수의 부가 만을 요구하는 동결-건조 조건 하에 저장할 수 있다. 즉석의 주사 용제 및 현탁제는 멸균성 산제, 과립제 및 정제로부터 제조할 수 있다.Forms of the formulations of the present invention can use any suitable formulation known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA). It can be administered orally at the time of clinical administration and can be used in the form of general pharmaceutical preparations, and when formulated, it is prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. . Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquids and paraffins, various excipients may be included, such as wetting sweeteners, fragrances, preservatives and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, calcium or vitamin D 3 may be added to enhance the efficacy of the treatment. Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as water for injection Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
일반적으로 세포 실험에서 사용한 농도의 약 100배 내지 200배를 동물실험에서 사용하므로, 본 발명의 일 실시예에 따른 백혈병 치료용 조성물의 투여량은 환자의 연령, 성별, 상태, 체내에서 활성 성분의 흡수도 및 병용되는 약물을 고려하여 결정할 수 있다. In general, about 100 to 200 times the concentration used in cell experiments is used in animal experiments, the dosage of the composition for treating leukemia according to one embodiment of the present invention is determined by the age, sex, condition, It may be determined in consideration of the absorbency and the drug used in combination.
본 발명에 의한 백축 추출물은 백혈병 세포에 대한 충분한 사멸효과를 갖고 있어 백혈병에 대한 치료용 조성물로서의 가치가 있으며, 특히 천연물로부터의 추출물이기 때문에 부작용의 위험이 적고, 전골수성 백혈병의 치료에 더욱 유용할 것으로 생각된다. 또한, 백축 추출물의 인간 HL-60 세포에 대한 사멸율의 기울기를 고려하면, 양적 확대에 따라 그 효능은 매우 클 것으로 예상할 수 있다. The baekchuk extract according to the present invention has a sufficient killing effect on leukemia cells and is valuable as a therapeutic composition for leukemia, and especially since it is an extract from natural products, there is little risk of side effects and more useful for the treatment of promyeloid leukemia. It is thought to be. In addition, considering the slope of mortality of human HL-60 cells of the baekchuk extract, the efficacy can be expected to be very large according to the quantitative expansion.
도 1은 인간 HL-60 세포에서 백축의 도입이 백혈병 세포의 성장에 미치는 영향을 알아보기 위한 Alamar Blue 분석 결과이다. 도면의 가로축은 추출물의 농도를, 세로축은 세포의 생존률을 나타낸다. 1 is a result of Alamar Blue analysis to determine the effect of the introduction of the rat axis in the growth of leukemia cells in human HL-60 cells. In the figure, the horizontal axis shows the concentration of the extract, and the vertical axis shows the survival rate of the cells.
이하 본 발명을 실시예에 의거하여 보다 상세하게 설명하기로 한다. 그러나, 본 발명의 실시형태에 의해 본 발명이 한정되는 것이 아니라 당업자의 입장에서 본 발명의 기술적 사상을 벗어나지 않는 범위내에서 다양한 변화의 부가 및 변경이 가능함은 물론 균등한 타 실시예가 가능할 것이며, 이는 본 발명의 기술적 사상내의 것이다. 한편, 본 발명을 설명함에 있어, 관련된 공지기술 혹은 구성에 대한 구체적인 설명은 본 발명의 요지를 모호하지 않게 하기 위하여 생략한다. Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the embodiments of the present invention, and various changes can be added and changed within the scope of the technical spirit of the present invention without departing from the spirit of the present invention, as well as other equivalent embodiments. It is within the technical idea of this invention. Meanwhile, in describing the present invention, detailed descriptions of related well-known technologies or configurations are omitted in order not to obscure the subject matter of the present invention.
<실시예 1> 백축 추출물의 제조 Example 1 Preparation of White Axis Extract
백축을 실온의 음지에서 5일간 건조하고, 추출효율을 위해 잘게 분쇄한다. 분쇄된 백축을 95% 에탄올(ethanol)에 침잠시키고 50℃ 에서 24시간 추출하였다. 이것을 여과한 후, 45℃ 감압 조건에서 건조하여 고형분을 수득하였다. 이를 -20℃의 저온에서 보관하였다. The white axis is dried in the shade at room temperature for 5 days and ground finely for extraction efficiency. The milled white axis was submerged in 95% ethanol and extracted at 50 ° C. for 24 hours. After filtering this, it dried under 45 degreeC pressure reduction conditions and obtained solid content. It was stored at a low temperature of -20 ° C.
<실시예 2> 백축 추출물이 백혈병 세포의 성장에 미치는 영향 Example 2 Influence of White Axis Extract on the Growth of Leukemia Cells
상기 실시예 1에서 수득한 백축 추출물이 백혈병 세포의 성장에 미치는 영향을 알아보기 위하여 인간 백혈병 세포인 HL-60에 대하여 Alamar Blue 분석을 시행하였다. Alamar Blue 분석은 MTT 분석의 변형된 형태인데, 특정 효소에 의해서 분해되는 화합물을 살아있는 세포에 처리한 후 화합물이 분해되면서 나오는 생성물의 형광 세기를 측정함으로써 약물을 처리한 후 살아있는 세포의 상대적인 숫자를 알아내는 실험방법이다.In order to examine the effect of the baekchuk extract obtained in Example 1 on the growth of leukemia cells, Alamar Blue analysis was performed on human leukemia cells HL-60. The Alamar Blue assay is a modified form of the MTT assay, in which a specific enzyme degrades a living cell and then measures the fluorescence intensity of the product as the compound breaks down to determine the relative number of living cells after treatment. I am an experimental method.
<2-1> 인간 백혈병 세포주의 준비 및 처리<2-1> Preparation and Processing of Human Leukemia Cell Line
본 발명에 사용된 백혈병 세포주인 HL-60 세포는 한국세포주은행(Korean Cell Line Bank, KCLB)으로부터 분양받아 실험에 이용하였다. 상기 HL-60 백혈병 세포를 20% FBS(fetal bovine serum, 소태아혈청)(Welgene)와 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid )를 포함하는 RPMI(Roswell Park Memorial Institute medium) 1640 배지에서 배양하였다. 상기 세포를 37℃, 5% CO2의 수분이 있는 상태로 유지하고, 2-3일 정도 계대배양하였다.HL-60 cells, the leukemia cell line used in the present invention, were distributed from the Korean Cell Line Bank (KCLB) and used for experiments. Roswell Park Memorial Institute medium containing 20% FBS (fetal bovine serum, fetal bovine serum) (Welgene) and 25 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) ) Incubated in 1640 medium. The cells were maintained at 37 ° C. with 5% CO 2 water and passaged for 2-3 days.
<2-2> HL-60 세포의 세포 생존율 측정<2-2> Cell viability measurement of HL-60 cells
상기 실시예 1에서 추출한 백축 추출물이 백혈병 세포인 HL-60에 대한 세포 성장 억제효과를 확인하였다. 구체적으로, 96 웰 플레이트에 각 웰 당 5.0× 104 개의 HL-60 세포를 주입(seeding)하고 DMSO(Dimethyl sulfoxide)에 녹인 상기 백축 에탄올 추출물을 각각 0 내지 100 ㎍/㎖ 농도(구체적으로, 각각 0, 3.125, 6.25, 12.5, 25, 50 및 100 ㎍/㎖ 농도)로 48시간 동안 처리하였을 때, 세포 성장을 저해하는 정도를 확인하였다(표 1). 각 농도의 추출물을 처리한 후, 96-웰 플레이트에서 각 웰에 채워진 0.2 ㎖의 세포 배양액에 20 ㎕의 Alamar Blue 시약을 첨가한 후 플레이트를 인큐베이터에서 2시간 동안 배양하였다. 각 웰의 세포를 고르게 반응시키기 위하여 플레이트를 천천히 흔들고, 544 ㎚의 파장에서 조사광을 조사하면서 590 ㎚에서 형광의 세기를 형광광도계(Fluorescence Microplate Reader; Molecular Devices Corp.)로 흡광도를 측정하였고, 세포의 생존율을 도 1에 나타내었다.The baekchuk extract extracted in Example 1 confirmed the cell growth inhibitory effect on HL-60 leukemia cells. Specifically, 5.0 × 10 per well in a 96 well plate4 The white axis in which HL-60 cells are seeded and dissolved in dimethyl sulfoxide (DMSO) When the ethanol extract was treated at concentrations of 0 to 100 μg / ml (specifically, concentrations of 0, 3.125, 6.25, 12.5, 25, 50 and 100 μg / ml, respectively) for 48 hours, the degree of inhibition of cell growth was confirmed. (Table 1). After treatment of each concentration of extract, 20 μl of Alamar Blue reagent was added to 0.2 ml of cell culture filled in each well in a 96-well plate, and the plates were incubated for 2 hours in an incubator. In order to evenly react the cells in each well, the plate was shaken slowly, and the intensity of fluorescence was measured by Fluorescence Microplate Reader (Molecular Devices Corp.) at 590 nm while irradiating irradiated light at a wavelength of 544 nm. The survival rate of is shown in FIG.
표 1
추출물의 농도(㎍/㎖) 생존률 표준편차
0 1.000 0.000
3.125 1.064 0.008
6.25 1.095 0.032
12.5 1.092 0.036
25 1.150 0.054
50 1.167 0.054
100 0.259 0.002
Table 1
Extract concentration (µg / ml) Survival rate Standard Deviation
0 1.000 0.000
3.125 1.064 0.008
6.25 1.095 0.032
12.5 1.092 0.036
25 1.150 0.054
50 1.167 0.054
100 0.259 0.002
그 결과, 표 1 및 도 1에서 나타난 바와 같이, 백축의 처리 농도가 약 90 ㎍/㎖ 이상의 범위에서 처리 농도가 높을수록 백혈병 세포의 생존률이 떨어지는 경향을 보이고 있으며, 이로부터 백축이 백혈병 예방 내지는 치료 효과가 있음을 알 수 있다. 특히 이 부근의 경우 표준편차가 매우 적어 확실한 효능을 짐작할 수 있다. 표 1의 생존률은 백축 추출물을 처리하지 않은 대조군의 세포의 수를 1로 보았을 때, 각각의 백축 추출물의 처리 농도에 따른 48시간 후의 세포의 상대적 세포수를 나타낸 것이다. 아울러, EC50(half maximal effective concentration)은 91.5 ㎍/㎖로 측정되었다. 이와 같이 본 발명의 백축 추출물은 우수한 HL-60 세포사멸 활성을 가지며, 나아가 백혈병 치료 및 예방 활성을 가진다는 것을 입증한다.As a result, as shown in Table 1 and Figure 1, the higher the treatment concentration in the range of the treatment of the baekchuk is more than about 90 ㎍ / ㎖ shows a tendency of the survival rate of leukemia cells is reduced, from which the baekchuk is prevented or treated It can be seen that there is an effect. In particular, the standard deviation is very small in this vicinity, it can be estimated a certain efficacy. Survival rate of Table 1 shows the relative cell number of the cells after 48 hours according to the treatment concentration of each rat axis extract, when the number of cells of the control group treated with the rat axis extract was 1. In addition, the EC 50 (half maximal effective concentration) was determined to be 91.5 μg / ml. As described above, the baekchuk extract of the present invention has excellent HL-60 apoptosis activity, and further demonstrates the therapeutic and prophylactic activity of leukemia.
<실시예 3> 백축 추출물에 의한 급성독성 시험 Example 3 Acute Toxicity Test by White Axis Extract
본 발명에 이용된 백축은 약재로 이용되고 있어서 안정성에 문제가 없을 것으로 판단하였으나, 경구 투여시 및 복강내 투여시의 독성 실험을 수행하여 이를 확인하고자 하였다.The white axis used in the present invention was used as a medicinal herb, so it was determined that there was no problem in stability.
6주령의 특정병원부재(SPF) SD계 랫트를 사용하여 급성독성실험을 실시하였다. 군당 2 마리씩의 동물에 본 발명의 실시예 1의 대계근 추출물을 각각 0.5% 메틸셀룰로즈 용액에 현탁하여 5 g/㎏의 용량으로 단회 경구투여하였다. 시험물질 투여후 동물의 폐사여부, 임상증상, 체중변화를 관찰하고 혈액학적 검사와 혈액생화학적검사를 실시하였으며, 부검하여 육안으로 복강장기와 흉강장기의 이상여부를 관찰하였다.Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally administered at a dose of 5 g / kg in suspension of the root of the extract of Example 1 of the present invention in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed, and autopsy was performed to observe abdominal and thoracic organ abnormalities.
시험결과, 시험물질을 투여한 모든 동물에서 특기할 만한 임상증상이나 폐사된 동물은 없었으며, 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다. 이상의 결과 대계근 추출물은 모두 랫트에서 5 g/㎏까지 독성변화를 나타내지 않으며 경구 투여 최소치사량 (LD50)은 5 g/㎏이상인 안전한 물질로 판단되었다.As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemical test, autopsy findings, etc. These results for gyegeun extract does not represent a change in toxicity both to 5 g / ㎏ in rats orally minimum lethal dose (LD 50) was determined to be a safe substance less than 5 g / ㎏.

Claims (6)

  1. 백축(Pharbitidis Semen; 白丑)을 유기 용매로 추출한 유효성분을 포함하는 백혈병 예방 또는 치료용 조성물 Leukemia prevention or treatment composition comprising an active ingredient extracted from Pharbitidis Semen (白 丑) with an organic solvent
  2. 제 1항에 있어서, The method of claim 1,
    상기 유기 용매는 에탄올 95% 용매인 것을 특징으로 하는 백혈병 예방 또는 치료용 조성물.The organic solvent is a composition for preventing or treating leukemia, characterized in that 95% solvent ethanol.
  3. 제 2항에 있어서, The method of claim 2,
    상기 백축 추출 유효성분은 50℃에서 24시간 동안 추출하여, 감압 조건하에 건조한 것을 특징으로 하는 백혈병 예방 또는 치료용 조성물.The baekchuk extract active ingredient is extracted for 24 hours at 50 ℃, leukemia prevention or treatment composition, characterized in that the dried under reduced pressure conditions.
  4. 제 3항에 있어서, The method of claim 3, wherein
    상기 백축 추출 유효성분은, 이를 HL-60 세포에 48시간 처리한 후 알라머 블루(Alamar Blue) 분석법에 의해 정해진 EC50 값이 91.5 ㎍/㎖ 이하인 효과를 보이는 추출물인 것을 특징으로 하는 백혈병 예방 또는 치료용 조성물.The baekchuk extract active ingredient is treated with HL-60 cells for 48 hours after the leukemia, characterized in that the extract showing the effect of the EC 50 value of 91.5 ㎍ / ㎖ or less determined by the Alamar Blue assay or Therapeutic composition.
  5. 제 1항에 있어서, The method of claim 1,
    상기 조성물은 약제학적으로 허용가능한 담체 또는 희석제를 포함하는 것을 특징으로 하는 백혈병 예방 및 치료용 조성물.The composition is a composition for preventing and treating leukemia, characterized in that it comprises a pharmaceutically acceptable carrier or diluent.
  6. 제 1항 내지 5항의 어느 한 항에 있어서, The method according to any one of claims 1 to 5,
    상기 백혈병은 전골수성 백혈병인 것을 특징으로 하는 백혈병 예방 또는 치료용 조성물. The leukemia is leukemia prevention or treatment composition, characterized in that the promyelocytic leukemia.
PCT/KR2012/000217 2011-01-08 2012-01-09 Composition including white morning glory extract for treating leukemia WO2012093920A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020110002025A KR20120080679A (en) 2011-01-08 2011-01-08 Composition for treatment of leukemia comprising extract of pharbitidis semen
KR10-2011-0002025 2011-01-08

Publications (2)

Publication Number Publication Date
WO2012093920A2 true WO2012093920A2 (en) 2012-07-12
WO2012093920A3 WO2012093920A3 (en) 2012-11-29

Family

ID=46457899

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2012/000217 WO2012093920A2 (en) 2011-01-08 2012-01-09 Composition including white morning glory extract for treating leukemia

Country Status (2)

Country Link
KR (1) KR20120080679A (en)
WO (1) WO2012093920A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019046311A1 (en) 2017-08-28 2019-03-07 Revance Therapeutics, Inc. Transmucosal botulinum toxin compositions, kits, and methods for treating bladder disorders

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014069687A1 (en) * 2012-10-31 2014-05-08 주식회사 한국전통의학연구소 Composition for treating renal cancer and cosmetic composition containing pharbitidis semen extract

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100313832B1 (en) * 1999-06-14 2001-11-15 배일주 Health supplementary food using pharbitis nilchoisy and pharbitis semon
KR20090110401A (en) * 2008-04-18 2009-10-22 닥터후 주식회사 Pharmaceutical composition for preventing or treating periodontal disease comprising extracts of Pharbitidis Semen

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100313832B1 (en) * 1999-06-14 2001-11-15 배일주 Health supplementary food using pharbitis nilchoisy and pharbitis semon
KR20090110401A (en) * 2008-04-18 2009-10-22 닥터후 주식회사 Pharmaceutical composition for preventing or treating periodontal disease comprising extracts of Pharbitidis Semen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KANG, TAK LIM STUDY ON ANTI-CANCER ACTIVITY OF VARIOUS HERB MEDICINES vol. 3, no. 2, 1995, pages 315 - 321 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019046311A1 (en) 2017-08-28 2019-03-07 Revance Therapeutics, Inc. Transmucosal botulinum toxin compositions, kits, and methods for treating bladder disorders

Also Published As

Publication number Publication date
KR20120080679A (en) 2012-07-18
WO2012093920A3 (en) 2012-11-29

Similar Documents

Publication Publication Date Title
KR20070026901A (en) An anticancer composition comprising obovatol or obovatal
WO2012093920A2 (en) Composition including white morning glory extract for treating leukemia
KR101745718B1 (en) Composition for treatment of Leukemia comprising extract of Buddleiae Flos
WO2011008052A2 (en) Composition for the prevention or treatment of bone diseases comprising colforsin daropate
RU2443415C2 (en) Anthracenedione compounds
WO2015190872A1 (en) Pharmaceutical composition containing spirulina maxima extract as active ingredient for treating and preventing obesity
KR101327282B1 (en) Improved pharmaceutical preparation containing Yellow-popular bark extract as active ingredient
KR20210052377A (en) Composition for the prevention or treatment of prostate-related disease comprising Rhodiola sachalinensis root extract containing kaempferol and epicatechin gallate
WO2015156498A1 (en) Pharmaceutical composition containing oenothera odorata extract as active ingredient for preventing or treating muscle atrophy
WO2019098811A2 (en) Composition for preventing, alleviating, or treating bone loss diseases, comprising cyclo(his-pro) (chp)
US10420808B2 (en) Seaweed extract and composition useful against cancer cells
KR20130094487A (en) Pharmaceutical composition containing yellow-popular bark extract as active ingredient
WO2021187905A1 (en) Pharmaceutical composition for cancer and resistant cancer comprising trichosanthes kirilowii maxim, dictamnus dasycarpus turcz and morus alba l.
KR20150045127A (en) A composition for anti-cancer comprising extract of Pinus Koraiensis SIEB
KR20110041710A (en) Pharmaceutical composition for inhibiting aging comprising herb extract
TW202139995A (en) Use of ovatodiolide against sars-cov-2
KR20100118667A (en) The pharmaceutical composition for arthritis therapy having anti-inflammation activity
WO2018226060A1 (en) Composition for preventing or treating kidney disease including cudratricusxanthone a having kidney-protecting activity as active ingredient
WO2012093919A2 (en) Composition for treating leukemia and method for preparing myrrh extract
WO2012134214A2 (en) Composition containing extracts of the fruit of hovenia dulcis thunb as an active ingredient for preventing and treating bone diseases
WO2012108743A2 (en) Composition and cosmetic composition for treating brain cancer comprising white pharbitis seed extract
WO2022154444A1 (en) Composition including decursinol as active ingredient for preventing or treating smooth muscle cell proliferative diseases
KR20140044223A (en) A pharmaceutical composition comprising extract of uv-induced rice for preventing or treating a colon cancer
KR102311887B1 (en) Composition comprising Sargassum Horner extract for preventing or treating liver disease
KR101785096B1 (en) Composition for Treatment of Brain cancer and Functional Whitening cosmetic Comprising Extract of TRIGONELLAE SEMEN

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12732322

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12732322

Country of ref document: EP

Kind code of ref document: A2