WO2014025192A1 - Pharmaceutical composition comprising paeonia japonica extract as active ingredient for treating uterine leiomyoma - Google Patents

Pharmaceutical composition comprising paeonia japonica extract as active ingredient for treating uterine leiomyoma Download PDF

Info

Publication number
WO2014025192A1
WO2014025192A1 PCT/KR2013/007084 KR2013007084W WO2014025192A1 WO 2014025192 A1 WO2014025192 A1 WO 2014025192A1 KR 2013007084 W KR2013007084 W KR 2013007084W WO 2014025192 A1 WO2014025192 A1 WO 2014025192A1
Authority
WO
WIPO (PCT)
Prior art keywords
extract
myoma
pharmaceutical composition
cells
earl
Prior art date
Application number
PCT/KR2013/007084
Other languages
French (fr)
Korean (ko)
Inventor
최인호
이은주
전상식
오영은
Original Assignee
영남대학교 산학협력단
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 영남대학교 산학협력단 filed Critical 영남대학교 산학협력단
Publication of WO2014025192A1 publication Critical patent/WO2014025192A1/en

Links

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)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • composition for treating myoma of uterus containing earl extract as an active ingredient
  • the present invention relates to a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
  • Leiomyoma is one of the most frequent tumors in women, it is reported that one of the cells of the smooth muscle of the uterus abnormally proliferate to form a uterine myoma, and is a major cause of hysterectomy. It is estimated that 20% of women aged 30 years or older have such tumors, and in fact, 77% of patients who have had hysterectomy due to other diseases have uterine fibroids. About half of the cases are asymptomatic, and if there are symptoms, various symptoms may occur depending on the location or size of the fibroids. Excessive menstruation is the most common symptom and may include pelvic pain, dysmenorrhea, pain during intercourse, pelvic pressure, and frequent urination.
  • Baekjak is a perennial herb from mountainous regions of Korea. The growing environment grows in high soil fertility, half shade and good drainage. The height is 40 ⁇ 50cni, the leaves are 5 ⁇ 12cm long, 3 ⁇ 7cm wide, the front is green, but the back is white, and 3-4 dogs run alternately and are long oval. Flowers are white, 4 ⁇ 5cni in diameter, hang one by one on the main stem. Calyxes are sweet, 3, petals are upside down, 5-7. There are several surgeries with three to four ovaries. Fruits run in long oval about 2-3cm long in August, and seeds are dark. It is used for ornamental purposes and the root is medicinal for analgesic, dysmenorrhea, and gynecological diseases ([Naver Encyclopedia]).
  • Korean Patent No. 1103481 to which the present inventors have registered a patent discloses a pharmaceutical composition for treating uterine myoma, which contains the herb extract as an active ingredient, but there is no reference to the Earl extract of the present invention.
  • An object of the present invention is to provide a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
  • the present inventors have confirmed that the earl extract is effective in myoma and completed the present invention.
  • the present invention provides a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
  • the extract of Paekjak is characterized in that it is extracted from water, lower alcohols of C1 to C4 or their mixed solvents. More specifically, the earl extract is (i) extracting the earl with alcohol; ( ⁇ ) suspending the bacterium alcohol extract by adding ' alcoholic (containing 3-8% by weight of alcohol) water and then adding nucleic acid; And ⁇ ) is a chloroform fractionation layer obtained by adding chloroform to the water layer containing the remaining alcohol after separating the nucleic acid layer, separating the chloroform layer, and concentrating under reduced pressure.
  • the ear extract extract is a progesterone receptor (PR), ⁇ 4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5), IGF-1R (insulin like growth factor 1 receptor) and C0L4a2 (collagen type) 4 alpha 2) characterized by inhibiting the expression of the gene.
  • the genes are those expressed higher in myoma cells than in normal cells. ⁇ .
  • the present invention provides a use for the preparation of a pharmaceutical composition for the treatment of uterine myoma of the active ingredient.
  • the present invention also provides a method of treating uterine myoma, comprising administering to the subject a therapeutically effective amount of a currant extract.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the earl extract, such a pharmaceutically acceptable carrier is commonly used in pharmaceutical preparations, such as lactose, dextrose, sucrose, Solbi, Manni, Starch, Acacia Rubber, Calcium Phosphate, Alginate, Gelatin, Silicate Sims, Microcrystalline Salose, Polyvinylpyridone, Cellulose, Water, Syrup, Methyl Salose, Methylhydroxybenzo Ate, propylhydroxybenzoate, talc, stearic acid magnesium, mineral oil, and the like, but is not limited thereto.
  • a pharmaceutically acceptable carrier is commonly used in pharmaceutical preparations, such as lactose, dextrose, sucrose, Solbi, Manni, Starch, Acacia Rubber, Calcium Phosphate, Alginate, Gelatin, Silicate Sims, Microcrystalline Salose, Polyvinylpyridone, Cellulose, Water, Syrup, Methy
  • the pharmaceutical composition may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative, and the like as an additive.
  • the pharmaceutical composition is determined in accordance with the degree of symptoms of uterine fibroids, it can be administered to various mammals such as rats, mice, livestock, humans. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous intrauterine dural or intracerebroventricular injection.
  • the dosage (the therapeutically effective amount) of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and preferably a daily dosage of 0.01 to 1,000. mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg. Administration can be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 50% lethal dose (LD50) of the toxicity test has proved to be a safe substance of at least 2 g / kg and its stability is ensured.
  • the pharmaceutical compositions can be prepared in unit dose form or formulated using pharmaceutically acceptable carriers and / or excipients or incorporated into multi-dose containers.
  • the formulation may be in the form of a solution, suspension or emulsion, or may be in the form of an exerciser, extract, powder, granule, tablet, economy, lotion ointment.
  • the present inventors confirmed that the earl extract inhibits the cell proliferation of myoma cells and reduces the expression of genes that are highly expressed in myomas. Therefore, it can be usefully used as a therapeutic agent for fibroids.
  • Figure 1 shows the appearance of myoma cells when treated with Earl extract (1 mg / ml) for 7 days Indicates.
  • Figure 2 is a graph showing the results of the proliferation rate of the cells treated with the white medicinal extract for 7 days using the MTT assay method.
  • the control group was not treated with medicinal herb extracts. When the control value was 100%, the cell proliferation rate in the treatment group was expressed as a relative value (p ⁇ .0001, Tuckey test).
  • Figure 3 is a graph showing the proliferation rate of cells analyzed using MTT assay after incubation for 7 days after treatment with 100 ug / ml, 100 ug / ml, 10 ug / ml.
  • the control group was not treated with medicinal herb extracts, and when the control value was 100%, the cell proliferation in the treatment group was expressed as a relative value ( ⁇ ⁇ 0001, Tuckey test).
  • Figure 4 is a graph showing the proliferation rate of myoblast cell lines C2C12 cells analyzed by ⁇ assay after incubation for 7 days after treatment with 100 ug / ml, 100 ug / ml, 10 ug / ml.
  • the control group was not treated with medicinal herb extracts. When the control group value was 100%, the cell growth rate in the treatment group was expressed as a relative value (p ⁇ .0001, Tuckey test).
  • Figure 5 shows the proliferation rate of human fibroblasts analyzed using ⁇ assay after incubation for 7 days after treatment with 1000 ug / ml Baekje extract. It is a graph.
  • the control group was not treated with medicinal extracts, and when the control value was 100%, the cell proliferation rate in the treatment group was expressed as a relative value (p ⁇ .0001, Tuckey test).
  • Figure 6 is a graph showing the result of analyzing the gene value by processing RNA through extracting the extract of 1 mg / ml of myoma cells and real-time PCR. The relative value of the gene expression value of the treated cells was calculated with the control group value of 1, and ⁇ -act in gene was used for normalization (p ⁇ 0.05, Tuckey test).
  • FIG. 7 is 7 days after treatment with the earl extract Protein expression was observed through immunostaining. Green indicates C0L4a2 ⁇ ⁇ expression and blue indicates nucleus.
  • A) represents normal myoblasts
  • Figure 8 shows the results of treatment of myofibril fraction (ethyl acetate, chloroform, nucleic acid) to the myoma cells for 48 hours.
  • the control group was not treated with medicinal herb extracts, and the results showed the relative cell proliferation rate in the treatment group when the control group was 100% (Tuckey test).
  • Figure 9 shows the appearance of myoma cells when treated with motherwort extract (1 mg / ml) for 7 days.
  • Baekjak extract was prepared at a concentration of 0.1 g / ml, and the Baekjak extract was dissolved in sterile tertiary distilled water, filtered once in a 40 strainer and filtered at 0.2 urn filter and stored frozen at -20 ° C until use.
  • the methanol extract was suspended in 1 liter of 5% methane and then poured into a separatory funnel, followed by pouring 1 liter of nucleic acid (Hexane) and shaking several times. It was then left for 4 hours at room temperature to allow equilibrium between the two solvents. When the nucleic acid layer and the water layer (5% methane is water soluble) were partitioned, the nucleic acid layer was separated and concentrated under reduced pressure to obtain a nucleic acid fraction.
  • the Baekja extract fractions thus obtained were prepared at a concentration of 0.2 g / ml, each fraction was dissolved in DMS0 and refrigerated at 4 ° C.
  • the myoma tissue removed after surgery on the day was soaked in saline and transported. Dip the tissue once again into saline, wash it, cut the tissue into a size of about 3-5m, and then cut the tissue into type I collagenase / KRB (rebs-ringer bicarbonate buffer)]. Put and incubate at 37 ° C for about 30-40 minutes. At this time, the incubation time and the amount of Krebs-Ringer bicarbonate buffer should be changed little by little depending on the state and amount of the sample.
  • the tissues were cultured as they were until they adhered to the culture plate, and the cells emerged from the digested tissues.
  • the cells were filled in the cell culture dish, passage culture was performed, and passage culture was performed with trypsin EDTA.
  • the same cells were dispensed into 6 well culture dishes by cell count measurement before dispensing the cells into the culture dishes.
  • the ⁇ assay method was used to measure the cell proliferation according to the extract treatment, and after 3 hours after the addition of the ⁇ reagent, 500 ⁇ l of DMS0 was added and the cell proliferation was measured at 590 nm. As a result of the measurement, it was observed that the proliferation rate of the cells treated with Baekjak extract was lower than that of the myoma cells of the control group which were not treated with anything. The relative value of the treated cells was obtained with the cell proliferation value of the control group at 100%: in the case of white peony, only 63% of the cells proliferated (FIG. 2).
  • Example 4 Observation of Cell Proliferation According to Concentration of Baek-Pak Extract
  • 1000 ug / ml, 100 ug / ml, and 10 ug / ml were treated. After incubation for 7 days and the growth rate was measured by MTT assay. The results showed that 1000 ug / ml Baekja extract treatment was the most effective in inhibiting cell proliferation compared to control group, and 100 ug / ml and 10 ug / ml treatment did not affect cell proliferation inhibition.
  • Figure 3 Observation of Cell Proliferation According to Concentration of Baek-Pak Extract
  • C2C12 cells which are myoblasts, were used for the toxicity test of the extract. After growing more than about 70% of the cells, the White Pear extract was treated with 1000 ug / ml, 100 ug / ml and 10 ug / ml and incubated for 3 days. After 3 days, the proliferation of cells was measured by MTT assay, and the cells were treated with 100% of untreated cells. As a result of obtaining the relative value, the extract of Baekjak had no significant effect on the proliferation of C2C12 cells (FIG. 4). In addition, human fibroblasts were treated with 1000 ug / ml of Baekja extract for 7 days, and then the proliferation of the cells was observed. As a result of treatment, only 67% of the cells of the Baekjak extract survived the comparison with the control group (FIG. 5).
  • the extract of Pear vulgaris is a gene that is expressed higher in myoma cells than in normal cells, such as PR (progesterone receptor), TPM4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5), IGF-1R (insulin like growth factor 1 receptor) and It was confirmed that C0L4a2 (collagen type 4 al ha 2) gene is effective in suppressing the expression.
  • PR progesterone receptor
  • TPM4 tropomyosin
  • IGFBP5 insulin like growth factor binding protein 5
  • IGF-1R insulin like growth factor 1 receptor
  • the following figure shows the expression of C0L4 a 2 exon in normal uterine myoblasts and myoma cells, and high expression in myoma cells can be observed. After treatment, the decrease in C0L4 a 2 protein expression was observed The earl extract was effective in inhibiting the expression of C0L4 a 2 protein in myoma cells (FIG. 7).
  • motherwort herb which is widely used as a blood-treatment medicine in oriental medicine
  • Fig. 9 there was no significant difference in the proliferation comparison with the cells without the motherwort treatment, and the results of comparing the growth of normal myometrial cells and myoma cells with the motherwort treatment did not show any significant difference. Therefore, it can be seen that the suppression of the proliferation of fibroids is a peculiar effect on the extract of the present invention.

Landscapes

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

Abstract

The present invention relates to a pharmaceutical composition comprising Paeonia japonica extract as an active ingredient for treating uterine leiomyoma. More particularly, the present invention provides a pharmaceutical composition comprising Paeonia japonica extract as an active ingredient which may exhibit treatment effects on uterine leiomyoma. The medicinal herb may inhibit the proliferation of myoma tissue and myoma cells while inducing fewer side effects, and therefore, can be used as a safe medicine for uterine leiomyoma.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
백작약 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조성물  Pharmaceutical composition for treating myoma of uterus containing earl extract as an active ingredient
【기술분야】 Technical Field
본 발명은 백작약 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조 성물에 관한 것이다.  The present invention relates to a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
【배경기술】 Background Art
자궁근종 (leiomyoma)는 여성에게 가장 빈번한 종양 중의 하나로서, 자궁의 평활근을 이루는 세포 중 하나가 비정상적으로 증식하여 하나의 자궁근종을 이루는 것으로 보고되고 있으며， 자궁절제의 주요 원인이다. 30세 이상의 여성 증 20%가 이런 종양을 가지고 있는 것으로 보고되고 있으며， 다른 질병에 의해 자궁절제를 한 환자들의 77% 정도가 자궁근종을 가지고 있는 것으로 미루어 보아 실제로 그 빈 도는 더 높을 것으로 추측된다. 증상이 없는 경우가 절반 정도 되고， 증상이 있는 경우에는 자궁근종의 위치나 크기에 따라 다양한 증상이 생길 수 있다. 월경과다가 가장 흔한 증상이며, 골반 통증, 월경통, 성교 시 통증， 골반 압박감， 빈뇨 등이 나타날 수도 있다.  Leiomyoma (leiomyoma) is one of the most frequent tumors in women, it is reported that one of the cells of the smooth muscle of the uterus abnormally proliferate to form a uterine myoma, and is a major cause of hysterectomy. It is estimated that 20% of women aged 30 years or older have such tumors, and in fact, 77% of patients who have had hysterectomy due to other diseases have uterine fibroids. About half of the cases are asymptomatic, and if there are symptoms, various symptoms may occur depending on the location or size of the fibroids. Excessive menstruation is the most common symptom and may include pelvic pain, dysmenorrhea, pain during intercourse, pelvic pressure, and frequent urination.
자궁근종 환자에게서 유전자 재배열, 전이， 삭제 등이 관찰되며, 자궁근종 의 크기와 유전적인 결함 사이에 상관관계가 있는 것으로 알려져 있다. 이런 사실 은 종양 내에 유전적인 결함이 먼저 있은 후에 종양이 진행된다는 것을 시사한다. 그러나, 어떤 유전적인 결함이 종양을 진행시키는지에 대해서는 알려진 바가 없다. 최근 연구에 의하면 정상 자궁근육과 자궁근종 사이에는 유전자 발현의 차이가 있 다는 사실이 보고되고 있다 (Anderson, J. and Barbieri, R.L. (1995) J. Soc. Gynecol. Invest . 2:663-672). 이러한 비정상적인 유전자 발현이 자궁근육의 분화 가 잘못 조절되고 있음에 기인한 것이라고 보고되고 있으며, 특히 임신 시에 높은 발현을 보이는 에스트로겐에 의하여 조절되는 유전자들이 과량으로 자궁근종에서 발현된다는 것이 보고되었다. Gene rearrangement, metastasis, and deletion are observed in myoma and it is known that there is a correlation between the size of myoma and genetic defects. This suggests that the tumor progresses after the genetic defect is present in the tumor first. However, it is not known which genetic defects advance the tumor. Recent studies have reported differences in gene expression between normal uterine muscle and fibroids (Anderson, J. and Barbieri, RL (1995) J. Soc. Gynecol. Invest. 2: 663-672). . These abnormal gene expressions are reported to be due to poorly regulated uterine muscle differentiation, especially during pregnancy It has been reported that genes regulated by estrogens that express them are expressed in uterine fibroids in excess.
백작약은 우리나라 각처의 산지에서 나는 다년생 초본이다..생육환경은 토양 비옥도가 높고 반그늘이며 물 빠짐이 좋은 곳에서 자란다. 키는 40~50cni이고, 잎은 길이 5~12cm, 폭 3~7cm로 앞면은 녹색이지만 뒷면은 흰빛이 돌며 3~4개가 어긋나게 달리고 긴 타원형이다. 꽃은 백색이고 지름은 4~5cni이며, 원줄기 끝에 한 송이씩 달린다. 꽃받침조각은 달갈 모양이며 3개， 꽃잎은 달걀을 거꾸로 세워놓은 모양이 고 5~7개이다. 수술은 여러 개이며 3~4개의 씨방이 있다. 열매는 8월경에 길이 2~3cm 정도의 긴 타원형으로 달리고 종자는 혹색이다. 관상용으로 쓰이며， 뿌리는 약용으로서 진통, 진경, 부인병에 쓰인다 ([출체 네이버 백과사전).  Baekjak is a perennial herb from mountainous regions of Korea. The growing environment grows in high soil fertility, half shade and good drainage. The height is 40 ~ 50cni, the leaves are 5 ~ 12cm long, 3 ~ 7cm wide, the front is green, but the back is white, and 3-4 dogs run alternately and are long oval. Flowers are white, 4 ~ 5cni in diameter, hang one by one on the main stem. Calyxes are sweet, 3, petals are upside down, 5-7. There are several surgeries with three to four ovaries. Fruits run in long oval about 2-3cm long in August, and seeds are dark. It is used for ornamental purposes and the root is medicinal for analgesic, dysmenorrhea, and gynecological diseases ([Naver Encyclopedia]).
한편, 본 발명자들이 특허 등록받은 한국등록특허 제 1103481호에서는 한약재 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조성물에 대해 개시하고 있 으나， 본 발명의 백작약 추출물에 대한 언급은 없다.  On the other hand, Korean Patent No. 1103481 to which the present inventors have registered a patent discloses a pharmaceutical composition for treating uterine myoma, which contains the herb extract as an active ingredient, but there is no reference to the Earl extract of the present invention.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
본 발명의 목적은 백작약 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조성물을 제공하는 데에 있다.  An object of the present invention is to provide a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
【기술적 해결방법】 Technical Solution
상기 목적을 달성하기 위하여， 본 발명자들은 백작약 추출물이 자궁근종에 효과가 있다는 것을 확인하고 본 발명을 완성하였다.  In order to achieve the above object, the present inventors have confirmed that the earl extract is effective in myoma and completed the present invention.
본 발명은 백작약 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조 성물을 제공한다.  The present invention provides a pharmaceutical composition for treating myoma of the uterus containing Baekjak extract as an active ingredient.
상세하게는 상기 백작약 추출물은 물， C1 내지 C4의 저급 알코을 또는 이들 의 흔합용매로부터 추출한 것임을 특징으로 한다. 보다 상세하게는， 상기 백작약 추출물은 (i) 백작약을 알코올로 추출하는 단 계; (Π) 상기 백작약 알코올 추출물에' 알코을성 (3-8 증량 %의 알코을 함유) 물을 가하여 현탁시킨 후, 핵산을 첨가하는 단계; 및 ΠΠ) 상기 핵산 층을 분리한 후 남은 알코올을 함유한 물 층에 클로로포름을 첨가하여 클로로포름 층을 분리하고 감압 농축하는 단계를 거쳐 얻은 클로로포름 분획층인 것을 특징으로 한다. 또한， 본 발명에 있어서 백작약 추출물은 근종세포에서 PR (progesterone receptor) , ΤΡΜ4 (tropomyosin) , IGFBP5 (insulin like growth factor binding protein 5)， IGF-1R (insulin like growth factor 1 receptor) 및 C0L4a2 (collagen type 4 alpha 2) 유전자의 발현을 억제하는 것을 특징으로 한다. 상기 유전자들은 정상세포에서 보다 자궁근종 세포에서 높게 발현되는 유전자들이다. ·. 또한， 본 발명은 백작약 추출물을 유효성분의기궁근종 치료용 약학조성물의 제조를 위한 용도를 제공한다. Specifically, the extract of Paekjak is characterized in that it is extracted from water, lower alcohols of C1 to C4 or their mixed solvents. More specifically, the earl extract is (i) extracting the earl with alcohol; (Π) suspending the bacterium alcohol extract by adding ' alcoholic (containing 3-8% by weight of alcohol) water and then adding nucleic acid; And ΠΠ) is a chloroform fractionation layer obtained by adding chloroform to the water layer containing the remaining alcohol after separating the nucleic acid layer, separating the chloroform layer, and concentrating under reduced pressure. In addition, in the present invention, the ear extract extract is a progesterone receptor (PR), ΤΡΜ4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5), IGF-1R (insulin like growth factor 1 receptor) and C0L4a2 (collagen type) 4 alpha 2) characterized by inhibiting the expression of the gene. The genes are those expressed higher in myoma cells than in normal cells. ·. In addition, the present invention provides a use for the preparation of a pharmaceutical composition for the treatment of uterine myoma of the active ingredient.
또한, 본 발명은 치료학적 유효량의 백작약 추출물을 대상체에 투여하는 것 을 포함하는 자궁근종의 치료 방법을 제공한다.  The present invention also provides a method of treating uterine myoma, comprising administering to the subject a therapeutically effective amount of a currant extract.
본 발명에 있어서， 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나， 이들 예에 한정되는 것은 아니다.  In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.
본 발명의 약학적 조성물은 상기 백작약 추출물 이외에 약제학적으로 허용되 는 담체를 포함할 수 있는데， 이러한 약제학적으로 허용되는 담체는 약품 제제 시 에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비를， 만니 를， 전분， 아카시아 고무, 인산 칼슘， 알기네이트， 젤라틴, 규산 칼슴, 미세결정성 샐를로스， 폴리비닐피를리돈, 셀롤로스, 물， 시럽， 메틸 샐를로스， 메틸히드록시벤 조에이트， 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슴， 미네랄 오일 등 을 포함할 수 있으나， 이에 한정되는 것은 아니다. 또한, 상기 약학적 조성물은 첨 가제로서 윤활제， 습윤제, 감미제， 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 상기 약학적 조성물은 자궁근종의 증상 정도에 따라 투여 방법이 결정되는데， 쥐， 생쥐, 가축， 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데， 예를 들면， 경구， 직장 또는 정맥， 근육， 피하ᅳ 자궁 내 경막 또는 뇌혈관 내 (intracerebroventricular) 주사에 의해 투여될 수 있다. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the earl extract, such a pharmaceutically acceptable carrier is commonly used in pharmaceutical preparations, such as lactose, dextrose, sucrose, Solbi, Manni, Starch, Acacia Rubber, Calcium Phosphate, Alginate, Gelatin, Silicate Sims, Microcrystalline Salose, Polyvinylpyridone, Cellulose, Water, Syrup, Methyl Salose, Methylhydroxybenzo Ate, propylhydroxybenzoate, talc, stearic acid magnesium, mineral oil, and the like, but is not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative, and the like as an additive. The pharmaceutical composition is determined in accordance with the degree of symptoms of uterine fibroids, it can be administered to various mammals such as rats, mice, livestock, humans. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous intrauterine dural or intracerebroventricular injection.
또한, 상기 약학적 조성물 중 유효성분의 투여량 (치료학적 유효량)은 투여경 로， 질병의 정도, 환자의 나이， 성별, 체중 등에 따라 달라질 수 있으며, 바람직하 게는 1일 투여량이 0.01 내지 1,000 mg/kg, 구체적으로는 0.1 내지 1,000 mg/kg, 보다 구체적으로는 0.1 내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되 는 것은 아니다.  In addition, the dosage (the therapeutically effective amount) of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and preferably a daily dosage of 0.01 to 1,000. mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg. Administration can be administered once a day or divided into several times, thereby not limiting the scope of the invention.
특히， 본 발명에 따른 백작약 추출물을 인체에 투여하는 경우, 천연 추출물인 관계로 다른 합성 의약품에 비하여 부작용의 우려가 없으며， 본 발명의 백작약 추출 물을 랫트에 경구투여하여 독성 실험을 수행한 결과, 독성시험에 의한 50% 치사량 (LD50)이 적어도 2g/kg 이상인 안전한 물질로 판명되어 그 안정성이 확보되어 있다. 상기 약학적 조성물은 약제학적으로 허용되는 담체 및 /또는 부형제를 이용하 여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때， 제형은 용액, 현탁액 또는 유화액 형태이거나 엘렉시르제, 엑스제, 분말제, 과립제， 정제， 경 제， 로션제 연고제 등의 형태일 수 있다.  In particular, when administering the extract of the present invention according to the present invention to the human body, there is no concern of side effects compared to other synthetic medicines because it is a natural extract, as a result of performing oral administration of the extract of the present invention to rats, The 50% lethal dose (LD50) of the toxicity test has proved to be a safe substance of at least 2 g / kg and its stability is ensured. The pharmaceutical compositions can be prepared in unit dose form or formulated using pharmaceutically acceptable carriers and / or excipients or incorporated into multi-dose containers. In this case, the formulation may be in the form of a solution, suspension or emulsion, or may be in the form of an exerciser, extract, powder, granule, tablet, economy, lotion ointment.
【유리한 효과】 Advantageous Effects
본 발명자들은 백작약 추출물이 근종세포의 세포증식을 억제하고， 근종에서 높게 발현되는 유전자의 발현을 감소시키는 것을 확인하였다. 따라서 이를 이용하 여 자궁근종 치료제로서 유용하게 사용될 수 있다.  The present inventors confirmed that the earl extract inhibits the cell proliferation of myoma cells and reduces the expression of genes that are highly expressed in myomas. Therefore, it can be usefully used as a therapeutic agent for fibroids.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 백작약 추출물 (1 mg/ml)을 7일 동안 처리했을 때 근종세포의 모습을 나타낸다. Figure 1 shows the appearance of myoma cells when treated with Earl extract (1 mg / ml) for 7 days Indicates.
도 2는 백작약 추출물을 7일 동안 처리한 세포를 MTT assay 법을 이용하여 세포의 증식률 결과를 나타내는 그래프이다. 대조군은 약재추출물을 처리하지 않았 으며 , 대조군의 값을 100%로 했을 때 처리구에서의 세포 증식률을 상댓값으로 나타 낸 결과이다 (p<.0001, Tuckey test).  Figure 2 is a graph showing the results of the proliferation rate of the cells treated with the white medicinal extract for 7 days using the MTT assay method. The control group was not treated with medicinal herb extracts. When the control value was 100%, the cell proliferation rate in the treatment group was expressed as a relative value (p <.0001, Tuckey test).
도 3은 백작약 추출물을 1000 ug/ml, 100 ug/ml, 10 ug/ml을 처리한 후 7일 동안 배양한 후 MTT assay 법을 이용하여 분석한 세포의 증식률을 나타내는 그래프 이다. 대조군은 약재추출물을 처리하지 않았으며， 대조군의 값을 100%로 했을 때 처리구에서의 세포 증식를을 상댓값으로 나타낸 결과이다 (ρ<·0001, Tuckey test). 도 4는 백작약 추출물을 1000 ug/ml , 100 ug/ml, 10 ug/ml을 처리한 후 7일 동안 배양한 후 Μπ assay 법을 이용하여 분석한 근아세포주 C2C12 세포의 증식률 을 나타내는 그래프이다. 대조군은 약재추출물을 처리하지 않았으며, 대조군의 값 을 100%로 했올 때 처리구에서의 세포 증식률을 상댓값으로 나타낸 결과이다 (p<.0001, Tuckey test).  Figure 3 is a graph showing the proliferation rate of cells analyzed using MTT assay after incubation for 7 days after treatment with 100 ug / ml, 100 ug / ml, 10 ug / ml. The control group was not treated with medicinal herb extracts, and when the control value was 100%, the cell proliferation in the treatment group was expressed as a relative value (ρ <· 0001, Tuckey test). Figure 4 is a graph showing the proliferation rate of myoblast cell lines C2C12 cells analyzed by Μπ assay after incubation for 7 days after treatment with 100 ug / ml, 100 ug / ml, 10 ug / ml. The control group was not treated with medicinal herb extracts. When the control group value was 100%, the cell growth rate in the treatment group was expressed as a relative value (p <.0001, Tuckey test).
도 5는 백작약 추출물을 1000 ug/ml을 처리한 후 7일 동안 배양한 후 ΜΊΤ assay 법을 이용하여 분석한 사람섬유아세포의 증식률을 나타내는. 그래프이다. 대 조군은 약재추출물을 처리하지 않았으며， 대조군의 값을 100%로 했을 때 처리구에 서의 세포 증식률을 상댓값으로 나타낸 결과이다 (p<.0001, Tuckey test).  Figure 5 shows the proliferation rate of human fibroblasts analyzed using ΜΊΤ assay after incubation for 7 days after treatment with 1000 ug / ml Baekje extract. It is a graph. The control group was not treated with medicinal extracts, and when the control value was 100%, the cell proliferation rate in the treatment group was expressed as a relative value (p <.0001, Tuckey test).
도 6은 근종세포에 1 mg/ml의 백작약 추출물을 처리한 후 RNA를 추출하여 real-time PCR을 통해 분석해 유전자 값을 분석한 결과를 나타낸 그래프이다. 대조 군의 값을 1로 두고 처리한 세포의 유전자 발현값의 상댓값을 계산하였으며, normalization은 β -act in유전자를 이용하였다 (p < 0.05, Tuckey test).  Figure 6 is a graph showing the result of analyzing the gene value by processing RNA through extracting the extract of 1 mg / ml of myoma cells and real-time PCR. The relative value of the gene expression value of the treated cells was calculated with the control group value of 1, and β-act in gene was used for normalization (p <0.05, Tuckey test).
도 7은 백작약 추출물 처리 7일 후
Figure imgf000007_0001
단백질의 발현을 면역염색법을 통해 관찰한 것이다. 녹색은 C0L4a2^\ 발현을 나타내며 파란색은 핵을 나타낸다. A)는 정상근층세포를 나타내며, B)는 자궁근종세포, C)는 근종세포+백작약추출물 (1 mg/ml)을 처리한 세포의 C0L4 a \ 발현을 나타낸다. 도 8은 백작약 분획 (에틸아세테이드, 클로로포름， 핵산)을 48시간 동안 근종 세포에 처리한 결과를 나타낸다. 대조군은 약재추출물을 처리하지 않았으며, 대조 군의 값을 100%로 했을 때 처리구에서의 세포 증식률을 상댓값으로 나타낸 결과이 다 (Tuckey test ) . 7 is 7 days after treatment with the earl extract
Figure imgf000007_0001
Protein expression was observed through immunostaining. Green indicates C0L4a2 ^ \ expression and blue indicates nucleus. A) represents normal myoblasts, B) uterine myoma cells, C) C0L4 a \ expression of cells treated with myoma cells + peony extract (1 mg / ml). Figure 8 shows the results of treatment of myofibril fraction (ethyl acetate, chloroform, nucleic acid) to the myoma cells for 48 hours. The control group was not treated with medicinal herb extracts, and the results showed the relative cell proliferation rate in the treatment group when the control group was 100% (Tuckey test).
도 9는 익모초 추출물 (1 mg/ml)을 7일 동안 처리했을 때 근종세포의 모습을 나타낸다.  Figure 9 shows the appearance of myoma cells when treated with motherwort extract (1 mg / ml) for 7 days.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하, 하기 실시예를 통해 본 발명을 보다 상세하게 설명한다. 다만， 이러한 실시예에 의해 본 발명이 한정되는 것은 아니다.  Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.
<실시예 1 > 백작약 추출물 제조 Example 1 Preparation of Earl Extract
1. 백작약 추출물 제조  1. Earl extract manufacture
155-200g의 백작약을 이용하여 증류수와 약 3시간 정도 가열한 후 가열하여 얻은 액을 1 麵 크기의 필터에 여과한 후 감압농축을 통하여 동결 건조하여 분말을 얻었다. 이렇게 얻어진 백작약 추출물은 0.1 g/ml 농도로 준비하였으며, 백작약 추 출물은 멸균된 3차 증류수에 녹인 후 40 strainer에 한 번 거른 후 0.2 urn 필터 에 걸러 사용 전까지 -20°C에 냉동 보관하였다. After heating with distilled water for about 3 hours using 155-200 g of white peony, the resulting solution was filtered through a 1 麵 filter and freeze-dried through vacuum concentration to obtain a powder. Thus obtained Baekjak extract was prepared at a concentration of 0.1 g / ml, and the Baekjak extract was dissolved in sterile tertiary distilled water, filtered once in a 40 strainer and filtered at 0.2 urn filter and stored frozen at -20 ° C until use.
2. 백작약 추출물의 각 분획 제조 2. Preparation of each fraction of earl extract
음건하여 세절한 백작약 1 kg을 믹서기를 이용하여 분쇄하였다. 분쇄한 시료 에 메탄올 (MeOH) 4 리터를 가하여 3시간 동안 환류 냉각 열탕추출 하였으며 이를 3 희 반복하였다. 이러한 추출물올 면솜을 이용하여 여과하고， 여과된 추출액을 모아 감압 농축하여 메탄을 추출물을 얻었다.  1 kg of dry and chopped earl was ground using a blender. 4 liters of methanol (MeOH) was added to the ground sample and refluxed under reflux for 3 hours. These extracts were filtered using cotton swabs, and the filtrate was collected and concentrated under reduced pressure to obtain methane extract.
상기 메탄올 추출물에 5% 메탄을성 물 1 리터를 현탁시킨 후 이 현탁액을 분 획깔때기에 붓고 바로 이어 핵산 (Hexane) 1 리터를 부어 수회 흔들어 진탕 흔합한 후 두 용매 사이의 평형이 일어나도록 실온에서 4시간 동안 방치하였다. 핵산층과 물 층 (5% 메탄을 성 물 soluble)이 분배되면 핵산 층은 분리하여 감압 농축하여 핵 산 분획을 얻었다. The methanol extract was suspended in 1 liter of 5% methane and then poured into a separatory funnel, followed by pouring 1 liter of nucleic acid (Hexane) and shaking several times. It was then left for 4 hours at room temperature to allow equilibrium between the two solvents. When the nucleic acid layer and the water layer (5% methane is water soluble) were partitioned, the nucleic acid layer was separated and concentrated under reduced pressure to obtain a nucleic acid fraction.
분획깔때기에 남아 있는 물 층에 클로로포름 (CHC13) 1 리터를 가한 후, 위와 같은 방법으로 진탕 흔합한 후 4시간 동안 실온에서 방치하였다. 클로로포름 층을 분리하여 감압 농축하여 클로로포름 분획 6.9 g을 얻었다. 그 후 남아 있는 물 층 에 같은 방법으로 에틸아세테이트 (Ethyl Acetate) 1 리터를 가하여 에틸아세테이트 분획을 얻었다. 1 liter of chloroform (CHC1 3 ) was added to the water layer remaining in the separatory funnel, and the mixture was shaken in the same manner as above and left at room temperature for 4 hours. The chloroform layer was separated and concentrated under reduced pressure to obtain 6.9 g of a chloroform fraction. Thereafter, 1 liter of ethyl acetate (Ethyl Acetate) was added to the remaining water layer to obtain an ethyl acetate fraction.
이렇게 얻어진 백작약 추출물 분획은 0.2 g/ml 농도로 준비하였으며, 각 분 획은 DMS0에 녹인 후 4 °C에 냉장 보관하였다. The Baekja extract fractions thus obtained were prepared at a concentration of 0.2 g / ml, each fraction was dissolved in DMS0 and refrigerated at 4 ° C.
<실시예 2 > 세포 배양 Example 2 Cell Culture
당일 수술 후 제거된 근종 조직을 생리식염수에 담구어 운반하였다. 조직을 다시 한번 생리식염수에 담구어 세정과정을 거치고 조직을 약 3-5m의 크기로 자른 후 조직을 제 1형 콜라게네이즈 /KRB[ type I col lagenase/ RB( rebs-Ringer bicarbonate buffer)]에 넣어 약 30-40 분 정도 37 °C에서 배양하였다. 이때, 배양 시간 및 Krebs-Ringer bicarbonate buffer의 양은 샘플의 상태 및 양에 따라 조금 씩 변형해야 한다. The myoma tissue removed after surgery on the day was soaked in saline and transported. Dip the tissue once again into saline, wash it, cut the tissue into a size of about 3-5m, and then cut the tissue into type I collagenase / KRB (rebs-ringer bicarbonate buffer)]. Put and incubate at 37 ° C for about 30-40 minutes. At this time, the incubation time and the amount of Krebs-Ringer bicarbonate buffer should be changed little by little depending on the state and amount of the sample.
세포분리과정 (digestion)이 끝나면 2500 rpm에서 5분 동안 원심분리하였다. 상층액을 제거한 후 배양배지 (DMEM/10% fetal bovine serum/ 1% penicillin-steptomycin)^! 넣어 준 후 섞어 뭉쳐있는 조직들이 잘 떨어질 수 있도 록 하였고， 조직들을 세포배양 접시에 놓은 후 그곳에 배양배지를 넣었다.  After completion of the cell separation (digestion) was centrifuged for 5 minutes at 2500 rpm. After removing the supernatant, culture medium (DMEM / 10% fetal bovine serum / 1% penicillin-steptomycin) ^! After putting it in, the mixed tissues were allowed to fall well, and the tissues were placed in a cell culture dish and the culture medium was put there.
조직이 배양접시에 붙을 때까지 그대로 배양한 후 소화된 조직에서 세포들이 나오는 것을 관찰하였다. 세포가 세포배양 접시에 가득 차게 되면 계대 배양을 진 행하였고, 계대 배양은 트립신 EDTA를 이용하였다. 세포를 배양 접시에 분주하기 전 세포 수 측정을 통하여 동일한 세포를 6 well 배양 접시에 분주하였다. <실시예 3 > 근종세포와 백작약 처리에 따른 세포증식 관찰 The tissues were cultured as they were until they adhered to the culture plate, and the cells emerged from the digested tissues. When the cells were filled in the cell culture dish, passage culture was performed, and passage culture was performed with trypsin EDTA. The same cells were dispensed into 6 well culture dishes by cell count measurement before dispensing the cells into the culture dishes. Example 3 Observation of Cell Proliferation Following Treatment of Myoma Cells and Earl of Peony
백작약 추출물 처리 전 초기배양 근종세포를 계대배양 하였다. 이 때 약 lxlO4 cells/ml의 근종 세포를 각 배양 접시에 넣은 후 세포흩 70% 까지 성장시켰 다. 70% 이상 성장한 세포에 1 mg/ml 농도의 백작약 추출물을 각 세포에 넣고 7일 동안 세포에 처리한 후 Μπ assay 법을 이용하여 세포의 증식정도를 관찰하였다. 그 결과 아무것도 처리하지 않은 근종세포에 비해 세포들이 많이 떨어진 모습을 관 찰할 수 있었다 (도 1). 추출물 처리에 따른 세포의 증식률 측정을 위해 Μπ assay 법을 이용하였으며， ΜΊΤ 시약을 넣은 후 3시간 후 DMS0 500 ul를 넣은 후 590 nm에 서 세포의 증식를을 측정하였다. 측정결과 아무것도 처리하지 않은 대조군의 근종 세포에 비해 백작약 추출물을 처리한 세포의 증식률이 떨어지는 것을 관찰할 수 있 었다. 대조군의 세포증식 값을 100%로 두고 처리한 세포의 상댓값을 구하였다： 백 작약의 경우 63%의 세포만이 증식하였다 (도 2). Early cultured myoma cells were passaged prior to treatment with Baekjak extract. At this time, about lxlO 4 cells / ml of myoma cells were added to each culture dish and grown up to 70% cell scatter. The cells of 1% mg / ml concentration of Baekjak extract was added to each cell, and the cells were treated for 7 days, and then the proliferation of the cells was observed using Μπ assay method. As a result, it was observed that the cells were far apart compared to the myoma cells that did not process anything (FIG. 1). The Μπ assay method was used to measure the cell proliferation according to the extract treatment, and after 3 hours after the addition of the ΜΊΤ reagent, 500 μl of DMS0 was added and the cell proliferation was measured at 590 nm. As a result of the measurement, it was observed that the proliferation rate of the cells treated with Baekjak extract was lower than that of the myoma cells of the control group which were not treated with anything. The relative value of the treated cells was obtained with the cell proliferation value of the control group at 100%: in the case of white peony, only 63% of the cells proliferated (FIG. 2).
<실시예 4 > 백작약 추출물의 농도별 처리에 따른 세포의 증식 관찰 백작약 추출물의 농도에 따른 세포의 증식를을 관찰하기 위해 근종세포에 1000 ug/ml , 100 ug/ml, 10 ug/ml을 처리한 후 7일 동안 배양하고 MTT assay로 증 식정도를 측정하였다. 측정결과 대조군과 비교 시 1000 ug/ml의 백작약 추출물 처 리가 세포의 증식억제에 가장 효과적이 이었으며， 100 ug/ml과 10 ug/ml 농도 처리 의 경우 세포의 증식억제에는 영향을 미치지 않음을 관찰할 수 있었다 (도 3). Example 4 Observation of Cell Proliferation According to Concentration of Baek-Pak Extract In order to observe the proliferation of cells according to the concentration of Baek-Pak extract, 1000 ug / ml, 100 ug / ml, and 10 ug / ml were treated. After incubation for 7 days and the growth rate was measured by MTT assay. The results showed that 1000 ug / ml Baekja extract treatment was the most effective in inhibiting cell proliferation compared to control group, and 100 ug / ml and 10 ug / ml treatment did not affect cell proliferation inhibition. Could (Figure 3).
< 실시예 5 > 근아세포주인 C2C12 세포와 사람섬유아세포를 이용한 백작약의 독성 관찰 Example 5 Observation of Toxicity of Earl Using C2C12 Cells and Human Fibroblasts
백작약 추출물의 독성실험을 위해 근아세포주인 C2C12 세포를 이용하였다. 세포를 약 70% 이상 성장 시킨 후 백작약 추출물을 1000 ug /ml, 100 ug/ml , 10 ug/ml 을 각각 처리하고 3일 동안 배양하였다. 3일 후 MTT assay 법을 이용하여 세 포의 증식을 측정하였으며 처리하지 않은 세포의 값을 100%로 두고 처리한 세포의 상댓값올 구한 결과 C2C12 세포의 증식에는 백작약 추출물이 큰 영향을 미치지 않 았다 (도 4). 또한 사람섬유아세포에 백작약 추출물 1000 ug/ml을 7일 동안 처리한 후 세포의 증식을 관찰하였다. 처리결과 대조군과의 비교에서 백작약 추출물의 경 우 67%의 세포만이 생존하였다 (도 5). C2C12 cells, which are myoblasts, were used for the toxicity test of the extract. After growing more than about 70% of the cells, the White Pear extract was treated with 1000 ug / ml, 100 ug / ml and 10 ug / ml and incubated for 3 days. After 3 days, the proliferation of cells was measured by MTT assay, and the cells were treated with 100% of untreated cells. As a result of obtaining the relative value, the extract of Baekjak had no significant effect on the proliferation of C2C12 cells (FIG. 4). In addition, human fibroblasts were treated with 1000 ug / ml of Baekja extract for 7 days, and then the proliferation of the cells was observed. As a result of treatment, only 67% of the cells of the Baekjak extract survived the comparison with the control group (FIG. 5).
<실시예 6 > 백작약 추출물 처리에 따른 유전자 발현 관찰 <Example 6> Gene expression observation according to the treatment of earl extract
백작약 추출물을 1 mg/ml을 처리한 후 7일 동안 배양한 세포의 RNA를 추출한 후 cDNA를 합성하여 근종에서 높게 발현되는 PR (progesterone receptor), IGF-1R (insulin like growth factor 1 receptor) , TPM4 (tropomyosin) , TRIM28 (tripartite motif containing 28), IGFBP5 (insulin 1 ike growth factor binding protein 5)， C0L4a2 (collagen type 4 alpha 2)의 유전자 6종과 세포자멸사 (apoptosis, Bax, / /)와 관련 유전자 2종을 분석하였다. 분석 후 β -act in유전자 로 normalization을 진행 한 후 처리하지 않은 세포의 발현 값을 1로 두고 처리한 세포의 상댓값을 계산하였다. 백작약 추출물을 처리한 세포에서는 PR (Fold difference: 0.34)， IGF-1R (Fold difference: 0.19), TPM4 (Fold difference: 0.06), IGFBP5 (Fold difference: 0.08), C0L4a2 (Fold difference: 0.04) 유전자 의 발현이 감소함을 볼 수 있었으며 , 특히 7W와 ^Ω^유전자의 발현이 상당히 많이 감소하였음을 관찰할 수 있었다. p21 (Fold difference: 2.05)유전자의 경우 약간의 발현증가를 보였으나 유전자의 발현에는 큰 영향을 미치지 않는 것으로 관찰되었다 (도 6). 따라서 백작약 추출물은 정상세포에서 보다 근종세포에서 높게 발현되는 유전자인 PR (progesterone receptor), TPM4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5)， IGF-1R (insulin like growth factor 1 receptor) 및 C0L4a2 (collagen type 4 al ha 2) 유전자의 발현억제에 효 과적이라는 것을 확인할 수 있었다. <실시예 6 > 백작약 추출물 처리에 따른 단백질의 발현 관찰 백작약 추출물의 처리에 따른 단백질의 발현을 관찰하기 위해 백작 약 추출물 1 mg/ml을 7일 동안 처리한 후 C( 4a2 단백질의 발현을 면역염색법 (immunocytochemistry)을 통해 관찰하였다. 아래의 그림은 정상자궁 근층세포와 근 종세포에서의 C0L4 a 2엑 발현올 나타내는 결과로 근종세포에서 높은 발현을 관찰할 수 있다. 근종세포에 백작약 추출물을 7일 동안 처리한 후 C0L4 a 2단백질 발현의 감소를 관찰할 수 있었다. 백작약 추출물은 근종세포의 C0L4 a 2 단백질의 발현을 억제하는데 효과적이었다 (도 7). After treatment with 1 mg / ml of Baekjak extract, RNA was extracted from cells cultured for 7 days, followed by synthesizing cDNA, which is highly expressed in myoma, PR (progesterone receptor), IGF-1R (insulin like growth factor 1 receptor), and TPM4. (tropomyosin), TRIM28 (tripartite motif containing 28), IGFBP5 (insulin 1 ike growth factor binding protein 5), C0L4a2 (collagen type 4 alpha 2) genes and apoptosis (apoptosis, Bax, / /) and related genes Two species were analyzed. After analysis, normalization was performed with β -act in gene, and the relative value of the treated cells was calculated with the expression value of untreated cells set to 1. In the cells treated with the earl extract, the genes of PR (Fold difference: 0.34), IGF-1R (Fold difference: 0.19), TPM4 (Fold difference: 0.06), IGFBP5 (Fold difference: 0.08), and C0L4a2 (Fold difference: 0.04) Expression was decreased, and in particular, 7W and ^ Ω ^ gene expression was significantly reduced. p21 (Fold difference: 2.05) gene showed a slight increase in expression, but did not significantly affect the expression of the gene (Fig. 6). Therefore, the extract of Pear vulgaris is a gene that is expressed higher in myoma cells than in normal cells, such as PR (progesterone receptor), TPM4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5), IGF-1R (insulin like growth factor 1 receptor) and It was confirmed that C0L4a2 (collagen type 4 al ha 2) gene is effective in suppressing the expression. Example 6 Observation of Protein Expression According to Baekjak Extract Treatment In order to observe the expression of protein following Baekjak extract treatment, 1 mg / ml of Baekjak extract was treated for 7 days, followed by immunostaining of C (4a2 protein expression. The following figure shows the expression of C0L4 a 2 exon in normal uterine myoblasts and myoma cells, and high expression in myoma cells can be observed. After treatment, the decrease in C0L4 a 2 protein expression was observed The earl extract was effective in inhibiting the expression of C0L4 a 2 protein in myoma cells (FIG. 7).
<실시예 7 > 백작약 분획 처리에 따른 근종세포의 증식 관찰 Example 7 Observation of Proliferation of Myoma Cells Treated with Earl Fraction
백작약에서 에틸아세테이트， 클로로포름, 핵산의 용매를 이용하여 성분별 추 출을 진행하였다. 분획 0.2 mg/ml을 근종세포에 처리하여 48 시간 동안 세포의 증 식을 관찰하였다. 백작약 분획의 경우 시간에 따른 큰 증식효과는 없었으며 추출물 원액에 비해 증식억제효과가 감소함을 관찰할 수 있었다 (도 8).  Extraction of each component was carried out using solvents of ethyl acetate, chloroform, and nucleic acid. Fraction 0.2 mg / ml was treated with the myoma cells to observe the proliferation of the cells for 48 hours. In the case of Baekjak fraction, there was no significant proliferative effect over time and it was observed that the antiproliferative effect was decreased compared to the extract stock solution (FIG. 8).
<비교예 >근종세포와 익모초 처리에 따른 세포증식 관찰 Comparative Example: Observation of Cell Proliferation by Treatment of Myoma Cells and Motherwort
한방에서 어혈치료제로 많이 사용하고 있는 익모초의 경우 실시예 1과 동일 한 방법으로 추출하여 실시예 3과 동일한 조건으로 처리 (lmg/ml을 7일 동안 처리) 한 세포의 증식을 관찰한 결과， 도 9와 같이 익모초를 처리하지 않은 세포 (Control)와의 증식 비교에서 큰 차이를 보이지 않았으며， 또한 익모초 처리에 따 라 정상근층 세포와 자궁근종 세포의 증식을 비교해 본 결과에서도 큰 차이를 나타 내지 않았다. 따라서, 자궁근종의 증식 억제는 본 발명의 백작약 추출물에 특유한 효과임을 알수 있다.  In the case of motherwort herb, which is widely used as a blood-treatment medicine in oriental medicine, it was extracted in the same manner as in Example 1 and observed the proliferation of cells treated under the same conditions as in Example 3 (treated with lmg / ml for 7 days). As shown in Fig. 9, there was no significant difference in the proliferation comparison with the cells without the motherwort treatment, and the results of comparing the growth of normal myometrial cells and myoma cells with the motherwort treatment did not show any significant difference. Therefore, it can be seen that the suppression of the proliferation of fibroids is a peculiar effect on the extract of the present invention.

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
백작약 추출물을 유효성분으로 함유하는 자궁근종 치료용 약학조성물.  A pharmaceutical composition for treating myoma of the uterus, containing an extract of Paekak as an active ingredient.
【청구항 2】 [Claim 2]
제 1 항에 있어서， 상기 백작약 추출물은 물， 에틸아세테이트， 클로로포름， 핵산， C1 내지 C4의 저급 알코을 및 이들의 흔합용매로부터 이루어진 군에서 선택 된 용매로 추출한 것을 특징으로 하는 자궁근종 치료용 약학조성물.  The pharmaceutical composition for treating myoma of claim 1, wherein the earl extract is extracted with a solvent selected from the group consisting of water, ethyl acetate, chloroform, nucleic acids, lower alcohols of C1 to C4, and a mixed solvent thereof.
【청구항 3】 [Claim 3]
제 1 항에 있어서， 상기 백작약 추출물은' (i) 백작약을 알코을로 추출하는 단계; (ii) 상기 알코올 추출물에 3—8 중량 ¾의 알코올을 함유한 물을 가하여 현탁 시킨 후， 핵산을 첨가하는 단계; 및 (iii) 상기 핵산 층을 분리한 후 남은 알코올 을 함유한 물 충에 클로로포름을 첨가하여 클로로포름 층을 분리하고 감압 농축하 는 단계를 거쳐 얻은 것을 특징으로 하는 자궁근종 치료용 약학조성물. According to claim 1, wherein the earl extract is ' (i) extracting the earl of the earl with alcohol; (ii) adding water containing 3-8 weight ¾ of alcohol to the alcohol extract and suspending it, followed by adding nucleic acid; And (iii) separating the nucleic acid layer and adding chloroform to the water-containing water remaining alcohol to separate the chloroform layer and concentrating under reduced pressure.
【청구항 4】 [Claim 4]
제 1 항에 있어서， 상기 백작약 추출물은 근종세포에서 PR (progesterone receptor) , ΤΡΜ4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5)， IGF-1R (insulin 1 ike growth factor 1 receptor) 및 C0L4a2 (collagen type 4 alpha 2) 유전자의 발현을 억제하는 것을 특징으로 하는 자궁근 종 치료용 약학조성물.  The method of claim 1, wherein the extract of the earl is in the progesterone receptor (PR), ΤΡΜ4 (tropomyosin), IGFBP5 (insulin like growth factor binding protein 5), IGF-1R (insulin 1 ike growth factor 1 receptor) and C0L4a2 ( collagen type 4 alpha 2) A pharmaceutical composition for treating uterine myoma, characterized by inhibiting the expression of the gene.
【청구항 5】 [Claim 5]
백작약 추출물을 유효성분의 자궁근종 치료용 약학조성물의 제조를 위한 용도. 【청구항 6】 Use of Baekjak extract for the preparation of a pharmaceutical composition for treating myoma of the active ingredient. [Claim 6]
치료학적 유효량의 백작약 추출물을 대상체에 투여하는 것을 포함하는 자궁 근종의 치료 방법.  A method of treating uterine fibroids comprising administering to the subject a therapeutically effective amount of a currant extract.
PCT/KR2013/007084 2012-08-07 2013-08-06 Pharmaceutical composition comprising paeonia japonica extract as active ingredient for treating uterine leiomyoma WO2014025192A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20120086362 2012-08-07
KR10-2012-0086362 2012-08-07

Publications (1)

Publication Number Publication Date
WO2014025192A1 true WO2014025192A1 (en) 2014-02-13

Family

ID=50068362

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2013/007084 WO2014025192A1 (en) 2012-08-07 2013-08-06 Pharmaceutical composition comprising paeonia japonica extract as active ingredient for treating uterine leiomyoma

Country Status (1)

Country Link
WO (1) WO2014025192A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030033473A (en) * 2001-10-23 2003-05-01 주식회사 바이오딕스 Compositions comprising medicinal herb extract for preventing and improving women's diseases
KR100823123B1 (en) * 2000-09-13 2008-04-18 지앙수 카니온 파마수티컬 씨오., 엘티디. A herbal pharmaceutical composition and its process
KR100950564B1 (en) * 2008-03-03 2010-04-01 동아대학교 산학협력단 Anti-Cancer Composition Containing an Extract of Chinese Herb as an Effective Ingredient
KR20110015172A (en) * 2009-08-07 2011-02-15 최장현 A composition of plants extract for dysmenorrhea in women

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100823123B1 (en) * 2000-09-13 2008-04-18 지앙수 카니온 파마수티컬 씨오., 엘티디. A herbal pharmaceutical composition and its process
KR20030033473A (en) * 2001-10-23 2003-05-01 주식회사 바이오딕스 Compositions comprising medicinal herb extract for preventing and improving women's diseases
KR100950564B1 (en) * 2008-03-03 2010-04-01 동아대학교 산학협력단 Anti-Cancer Composition Containing an Extract of Chinese Herb as an Effective Ingredient
KR20110015172A (en) * 2009-08-07 2011-02-15 최장현 A composition of plants extract for dysmenorrhea in women

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PARK, JUN-SIK ET AL.: "Case Reports of 6 Outpatients Complaining of Uterine Myoma", THE JOURNAL OF ORIENTAL OBSTETRICS & GYNECOLOGY, vol. 18, no. 4, 2005, pages 230 - 241 *
PARK, YOUNG SUN ET AL.: "The effect of p-sitosterol proliferation and appoptosis in human uterine leiomyoma cells", THE JOURNAL OF ORIENTAL OBSTETRICS & GYNECOLOGY, vol. 18, no. 1, 2005, pages 181 - 191 *

Similar Documents

Publication Publication Date Title
CN103687606B (en) The therapeutic composition and application thereof of specific herbal medicinal product
WO2014110936A1 (en) Periplaneta americana extract, preparation method therefor and use thereof
US20220409671A1 (en) Composition for preventing or treating ocular diseases comprising amniotic epithelial cell derived exosomes
KR101662719B1 (en) A composition for the prevention or treatment of prostate-related disease comprising Ponciri Fructus extract
Olowofolahan et al. Ameliorative potentials of methanol extract and chloroform fraction of Drymaria cordata on MSG-induced uterine hyperplasia in female wistar rats
KR100686950B1 (en) A composition for improving generative of male
EP4218782A1 (en) Composition for prevention and treatment of benign prostatic hyperplasia, comprising fruit extracts of elaeagnus multriflora thunb. as active ingredient
US10588928B2 (en) Anti-obesity composition comprising pine needle juice powder as effective component
KR101193540B1 (en) Composition comprising the extract of Astragalus membranaceus BGE.,Cinnamomum cassia and Phellodendron amurensis for preventing and treating of osteoporesis and bone disease
KR101103481B1 (en) Pharmaceutical composition for treating uterine leiomyoma comprising herb extract
WO2014025192A1 (en) Pharmaceutical composition comprising paeonia japonica extract as active ingredient for treating uterine leiomyoma
JP2021176888A (en) Therapeutic pharmaceutical composition comprising purple corn extract for prevention or treatment of skin disease
EP1200107B1 (en) Composition having steroidal estrogen effect without increasing the risk of breast cancer
CN110279728A (en) Herba Visci extract improves the purposes in gonad granulocyte activity tcm product in preparation
WO2018223922A1 (en) Use of pharmaceutical composition in preparing drug against helicobacter pylori
JP2008303199A (en) Anti-neovascularization composition containing lipid extract of grifola frondosa and genistein
WO2023090542A1 (en) Pharmaceutical composition containing hot water extract of fruit of vaccinium bracteatum thunb. as active ingredient for preventing or treating benign prostatic hyperplasia
CN115919934B (en) Peony active substance with effect of inhibiting prostatic hyperplasia, extraction method and application
KR102063321B1 (en) Composition for Preventing or Treating Uterine Myoma Comprising Orostachys japonicus Extract
KR102551879B1 (en) Composition for Prevention or Treatment of Prostatic Desease Comprising Sargassum Horneri Extract
KR102119812B1 (en) Composition for Preventing or Treating Uterine Myoma Comprising Rhus verniciflua Stokes Extract
KR102121969B1 (en) Pharmaceutical Composition Comprising Fraction of Melissa Leaf Extract
CN1899313B (en) Use of wasp&#39;s nest in preparing medicine for preparing endometriosis
KR20230172710A (en) Health functional food composition for improving menopausal men&#39;s health containing Perilla frutescens var. acuta Kudo extract
CN116077562A (en) Application of rose in preparation of products for resisting prostatic hyperplasia/hypertrophy

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: 13827249

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13827249

Country of ref document: EP

Kind code of ref document: A1