KR20050113406A - Composition comprising the extract of mucunae caulis having arresting effect on the g1 phase of the cell cycle - Google Patents

Abstract

The present invention relates to a composition containing an extract of cinnamon, etc. having an effect of inhibiting the G1 cell cycle of the cell cycle circuit as an active ingredient, in detail, the extract of the present invention is a G1 of the cell cycle circuit of uterine smooth myoma cells. Since there is an effect of inhibiting the proliferation of cells by stopping the phase, the composition comprising the same can be used as a pharmaceutical and health functional food for the prevention and treatment of myoma.

Description

Composition containing the extract of Mucunae Caulis having arresting effect on the G1 phase of the cell cycle}

The present invention relates to a composition containing an extract such as cinnamon having an effect of inhibiting the G1 cell cycle of the cell cycle circuit as an active ingredient.

Uterine leiomyoma is a benign tumor of smooth muscle origin, which occurs in about 25% of women of childbearing age and causes 20% to 50% of cases of negative bleeding, pelvic pain, tenderness, infertility, spontaneous abortion, etc. It shows various aspects according to the size and location (Korean Society of Obstetrics and Gynecology, Gynecology, Seoul: Calvin Books, pp472-481, 1987; Iran, et al., Journal of Korean Obstetrics and Gynecology, 37 (11) , pp2216-26, 1994; Buttram VC. , et al., Fertil Steril , 36 , pp 443-45, 1981).

Hysterectomy is commonly used as a treatment for uterine fibroids. Treatments other than hysterectomy include myoma removal and drug therapy. In 51% of myomas, there is a risk of recurrence (Shin Young-woo et al., Korean Journal of Obstetrics and Gynecology, 28 , pp90, 1985; Gambone JC., Et al., Am. J. Obstet.Gynecol. , 63 , pp545-50, 1990 Hormonal therapies, such as the Korean Academy of Gynecology and Rehabilitation Committee, Korean Academy of Gynecology (Sang), Seoul: Book Publishing, pp305, 2001), and GnRh-a, can cause side effects such as osteoporosis, hypercholesterolemia, and cardiovascular disorders. In addition to difficult long-term use, the size of myoma tends to be larger after discontinuation of drug use, which is not suitable for curative therapy (Dong Ho Kim et al., 37 (6) , pp. 1205-16 ) . , 1994; Stewart EA., Et al., Contemp Obstet Gynecol ., 45 , pp 26-35, 2000).

Uterine fibroids are known to be estrogen-dependent tumors and are known to increase estrogen and progesterone receptors (Brandon DD., Et al., J. clin. Endocrinol. Metab. , 80 , pp1876-81, 1995 Sumitani H., et al., Endocrinology, 141 , pp 3852-61, 2000), increase in B cl-2 protein (Matuo H., et al., J. Clin. Endocrinol. Metab ., 82 , pp293-) 9, 1997), transforming growth factor (TGF) -β ( Arici A., et al., Fertil Steril , 73 , pp1006-11, 2000), heparin-binding growth factors (Klagsbrun M. , et al., J. Biol. Chem. , 268 , pp1830-4, 1993), Insulin-like growth factors (Strawn Jr EY., et al., Am. J. Obstet Gynecol ., 172 , pp1837-44, 1995), and recently, cyclin G1 expression has been increased in uterine fibroids (Baek W., et al., Am. J. Obstet Gynecol . 188 , pp63, 2003). Overexpression of genes known to be involved in the cell cycle Suggested that an important factor in the development of fibroids.

Regulation of cell growth and differentiation is essential for normal cell growth, and if this process is impaired by any cause, normal cells undergo an abnormal cell cycle (Seung-Chul Kim, Department of Obstetrics and Gynecology, Ewha Womans University Spring Symposium, pp23-42, 1998). ). Based on this, it is a key point in recent cell cycle-related genetic studies that a drug will be involved in each stage of the cell cycle.

Recently, many genes involved in cell cycle regulation have been discovered, and molecular biological regulatory mechanisms among the gene products are rapidly discovered, which has become one of the greatest interests among many research fields of medicine and biology. Molecular biological research on the regulation of G1 checkpoints, the process of deciding to transition to the G1 phase, is very active. This is mainly because it is at this stage whether the cell enters the S phase and proliferates, stays in G1 and differentiates, or whether DNA damage or other disorders are so severe that they die from apoptosis. Et al., The Journal of Commandments, 15 (4) , pp381-93, 1996).

Several signaling agents activated by different growth factors increase the expression of protocogenes Ras, Raf, Myc, Fos, and Jun. The next step is to increase the expression of G1 cyclins responsible for the G1 group. Examples of G1 cyclins that have a specific effect on the G1 / S phase in animal cells include types A, D and E. Cyclin E is expressed at the end of G1 and binds to CDK2 (cyclin dependent kinase), which is the essential factor for G1 / S metastasis by helping CDK2 to reach its highest activity during G1 / S transition (Kim Seung-cheol, Ewha Womans University College of Medicine) Obstetrics and Gynecology Spring Symposium, pp23-42, 1998).

In order to precisely regulate the cell cycle, cells have cyclin dependent kinase inhibitors (CDKI) in addition to positive regulators such as cyclin and CDK, which regulate the cell cycle by inhibiting the CDK activity of the cyclin-CDK complex. One of the essential factors as a negative regulator is p27, p53, p21, which are p21 ^c1p1 family, and p15, p16, p18, p19, p20, which is p16 ^INK4a family (Kim Seung-cheol, Ewha Womans University Department of Obstetrics and Gynecology, Medicine Symposium Proceedings, pp23-42, 1998; seominho et al., Journal of medical commandments, 15 (4), pp381-93, 1996).

In CDKI, p27 is always expressed in a certain amount in the cell and can bind to the cyclin or cyclin-CDK complex. In particular, it binds to the cyclin E-CDK2 complex, inhibits CDK activity and induces arrest of G1 / S phase. To induce apoptosis (Peters G., Nature , 371 , pp204-5, 1994; Sherr CJ., Et al., Cell, 73 , pp1059-65, 1993; Chellappan S., et al., Proc Natl.Acad . Sci. USA., 89 , pp 4549-53, 1992).

p53 acts as a shield for the genome in terms of interrupting the cell cycle to prevent cells with damaged DNA from dividing. If cells with severe DNA damage fail to recover before DNA enters the next cell division cycle, p53 causes apoptosis and acts as a mediator of G1 checkpoints in the G1 / S phase (Seung-Chul Kim, Ewha Womans University School of Medicine). Obstetrics and Gynecology Spring Symposium, pp23-42, 1998). In addition, p53 induces the production of p21, one of CDKI, p21 binds to CDK, induces G1 arrest, and cells repair damaged DNA. At this time, if the repair of damaged DNA fails, p53 causes cell death through apoptosis and blocks abnormal cell proliferation. , 1998; Jae-Hyung Na, Graduate School, Chonnam National University, 1998).

Development of gene therapy for uterine fibroids has recently been actively conducted and studies on proliferation inhibitory effect and gene expression using herbal medicine or herbal medicine (Kim, Jin-Hee et al., Korean Journal of Oriental Medical Gynecology, 14 (2) , pp85-101, 2001; Ho Lee et al., Journal of Oriental gynecology, 15 (2), pp12-24, 2002; Science, Oriental gynecological Yeon et al., 15 (4), pp1-16, 2002; et al yiyoungrim, Oriental Journal of gynecology, 16 (2) , pp1-17, 2003; Kim Dong-cheol et al., Korean Journal of Oriental Medical Gynecology, 16 (2) , pp18-33, 2003).

In Oriental Medicine, uterine fibroids are regarded as a category of tumors that occur in female genital organs (Sang-Woo Kim et al., Korean Journal of Oriental Gynecology, 4 (1) , pp2-8, 1992; Song Byung-ki, Oriental Gynecology, Seoul: Haenglim Press, pp249-257, 1978) The cause of the punishment is related to external bloating, external warming, gallbladder, phlegm, food, blood, blood Among them, fish blood is regarded as the most important cause, and prescriptions and drugs based on vigor blood pill are being used abundantly (Hee Sang Lee et al., Institute of Oriental Medicine, 6 (2) , pp417 -35, 1998).

Mucunae Caulis is a small branch of Spatholobus suberectus Dun, which belongs to the legumes (Leguminosae), and is used as a medicine. (Hwang, Sang-gu et al., Journal of Oriental Physiological Pathology, 15 (6), pp887-8, 2001; Jiangsu New Medical School, Chinese Medicine Dictionary (1), Shanghai: Shanghai Science and Technology Press, 1979).

Cinnamic blood, etc., is the first drug listed in the Myung-dae College of Herbal Herbs (Cho Hak-min, Herb Herbal Milks, Hyanghang: Commercial Personnel Orthodox Port, pp260-2, 1986). (溫), non-toxic, the ear is the liver, renal, hematopoietic hemostasis (서 行血), stiff muscles (舒筋 活絡) has the effects of anemia, menstrual relief, It has been used to treat pain, dysmenorrhea, habitual pain, arthralgia, etc .. Kang, Byung-Soo, Herbology, Seoul: Younglimsa, pp445-6, 1991; In addition, rPGUF and the like have been described in the change of membrane lipid composition (Wang W., et al., Zhongguo Zhonguo Yao Za Zhi , 16 , pp299-301, 1991), inhibition of HIV-1 protease activity (Lam T., Life Sci. , 67 , pp2889-96, 2000), anti-inflammatory activity effect (Jin-Hoon Lee, Graduate School of Kangwon National University, 1999), hematopoietic and immune action (Hyung-Sook Oh, Graduate School of Daejeon University, 2000), tumor metastasis inhibitory effect (Hyeon-Chul Lee et al. 17 (2) , pp525-8, 2003), Immune Responses to Synovial Cells ( Seoul, et al., Journal of Oriental Physiology and Pathology, 17 (3) , pp780-6, 2003), Cell Cycle Inhibition Effect (Chonam Soo, Graduate School of Wonkwang University) , 2002), but no studies on the effect of uterine fibroids on uterine fibroids have been reported.

Therefore, the present inventors have one of the mechanisms for suppressing the proliferation of uterine smooth myoma cells, such as cinnamon, and treatment of uterine myoma cells in vitro cultured with cinnamon, the effect of inhibiting the proliferation of uterine myoma cells, expression of cell cycle related genes, The present invention was completed by confirming the effect on cell cycle analysis, the expression of genes associated with apoptosis.

An object of the present invention is to provide a composition for the prevention and treatment of myoma of the uterus containing as an active ingredient extracts such as cinnamon having the effect of inhibiting the G1 cell cycle of the cell cycle circuit.

In accordance with the above object, the present invention provides a composition for the prevention and treatment of myoma of the uterus containing as an active ingredient extracts such as cinnamon having the effect of inhibiting the G1 cell cycle of the cell cycle circuit.

In detail, the present invention includes the extract of cinnamon and the like having an effect of inhibiting the G1 cell cycle of the cell cycle circuit as an active ingredient, and the prevention and treatment of uterine myoma comprising a pharmaceutically acceptable carrier, diluent or excipient. To provide a pharmaceutical composition for.

The extract includes a polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably an extract available in water.

Hereinafter, the present invention will be described in detail.

The preparation of the extract of the cinnamon, etc. of the present invention is used to remove cinnamon, etc., without any processing or numerical value, about 1 to 60 times the weight of water, preferably about 20 to 50 times the volume of water, lower alcohols having 1 to 4 carbon atoms, or A mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 3, of about 1 hour to 2 days at an extraction temperature of 20 to 100 ° C, preferably 30 to 70 ° C, Preferably the extraction method such as hot water extraction, reflux cooling extraction, ultrasonic extraction for 2 hours to 1 day, preferably the extraction method of hot water extraction is repeated 1 to 5 times, preferably 2 to 3 times After obtaining, the resultant was filtered under reduced pressure and the filtered extracts were mixed and concentrated under reduced pressure at 20 to 100 ° C., preferably at 50 to 70 ° C., using a rotary vacuum concentrator, and then 1 hour to 36 hours, preferably 12 hours at 70 ° C. or lower. Frozen for from 48 hours Frozen for one to five days, preferably two to four days chulmul dried to can be obtained a crude extract soluble extract according to the water, a lower alcohol or a mixed solvent of 1 to 4 carbon atoms.

The composition of the present invention provides a pharmaceutical composition containing the extract such as cinnamon obtained as an active ingredient.

In addition, the present invention contains an extract of cinnamon such as cinnamon having the effect of inhibiting the G1 cell cycle of the cell cycle circuit obtained through the extraction and separation process as described above as a active ingredient, a pharmaceutically acceptable carrier, diluent Or it provides a pharmaceutical composition for the prevention and treatment of uterine myoma comprising an excipient.

The composition of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

The composition of the present invention is effective in the prevention and treatment of uterine leiomyomas with the effect of inhibiting the expression of cyclin G1, a gene involved in the cell cycle.

The composition containing the extract of the cinnamon, etc. of the present invention may further comprise a suitable carrier, excipient or diluent according to a conventional method.

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising extracts according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used.

In detail, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, 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.

The extract of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once or several times a day. Can be. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Extracts such as cinnamon of the present invention has little toxicity and side effects, so can be used with confidence even during prolonged administration.

In addition, the present invention provides a health functional food for the prevention and treatment of uterine fibroids, including the extract such as cinnamon and food acceptable food supplement additives.

The composition containing the extract of cinnamon, etc. of the present invention can be used in various foods and beverages for the prevention and treatment of myoma by inhibiting the G1 cell cycle of the cell cycle circuit.

Foods to which the extract of cinnamon and the like of the present invention can be added include various foods, for example, candy, chocolate, beverages, gums, teas, vitamin complexes, health functional foods, and the like, powders, granules, tablets, capsules, or the like. It can be used in the form of a drink.

At this time, the amount of the extract in the food or beverage, in the case of the health functional food composition of the present invention can generally be added to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total functional food composition, In the case of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as conventional sugars such as dextrin, cyclodextrin and the like and sugar alcohols such as xylitol, sorbitol, erythritol and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

As other flavors, natural flavors (tauumatin, stevia extract: for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. In addition, the compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.

These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Extracts

Primary distilled water 2000g in 40 g of selected cinnamon without any processing or shame purchased at Oriental Hospital of Daegu Haany University (Daegu, Korea) to manufacture extracts such as cinnamon. 300 ml of the concentrated extract obtained by distilling under reduced pressure with a rotary vacuum evaporator under reduced pressure was concentrated using a rotary vacuum evaporator for 300 hours after freezing at -70 ° C. for at least 24 hours. Lyophilization gave 13 g of cinnamon and the like extract powder. The extract powder, such as cinnamon, was dissolved in tertiary distilled water at a concentration of 0.1 g / ml, and the supernatant was filtered through a 0.2 μm filter (Nalgene, USA).

Experimental Example 1. Cultivation of uterine myoma cells

One-third of the uterine fibroids were taken from the center of the uterine fibroids to the edges of the uterine fibroids after surgery. The uterine fibroids were selected from 12 weeks of gestation and had no secondary degeneration. Was selected. The age of the patients was between 35 and 45 years old. The endometrial cycles of the extracted uterus were 5 proliferative stages and 5 secretory stages. In primary culture, the fresh tissue was chopped as much as possible on a Hank's balanced salt solution (HBSS) solution, transferred to a 15 ml tube, and centrifuged at 1000 rpm for 3 minutes to remove supernatant. The cut tissues were then HEPES (25 mmol / L), penicillin (penicillin, 200 U / mL), streptomycin (200 μg / mL), collagenase type IV (collagenase type IV, 1.5 mg / mL). , DNase (0.2 mg / ㎖) was added to HBSS and incubated for 3-4 hours in a 37 ℃ constant temperature water bath. The sufficiently digested tissues were mixed vigorously using a pipette, separated into single cells, centrifuged again at 2000 rpm for 5 minutes, and the supernatant was removed. This was again washed with phosphate-buffered saline (PBS), centrifuged and the supernatant removed. The collected cells were suspended in DMEM / Ham's F-12 (Dulbecco's modified eagle's medium / Nutrient mixture F-12 ham) medium containing 10% fetal bovine serum and incubated in a 37 ° C, 5% CO ₂ incubator. After 24-48 hours, the cultures were changed.

The cultured cells were examined by immunohistochemistry using a smooth muscle actin antibody to confirm that the primary cultured cells were smooth muscle cells grown in myoma. ).

Experimental Example 2 Observation of Proliferation Inhibition of Uterine Myoma Cells

Uterine fibroids were cultured in a 60 mm tissue culture dish at 2 × 10 ⁵ cells / dish and cultured for 24 hours, followed by 100, 200, 300 ㎍ / Treatment was carried out at a concentration of ㎖ and then transfected. The cultured cells were washed with PBS solution for 24-48 hours, resuspended with 1 × trypsin-EDTA, and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed, washed with PBS solution and resuspended. 20 μl of cell suspension and the same amount of trypan blue solution were mixed and allowed to stand for 1 minute, followed by a hemocytometer on a hemocytometer. As a result of measuring only the number of living cells repeated three times, the effect of inhibiting cell proliferation after 24 hours increased in a concentration-dependent manner, and proliferation of 65% was inhibited at 300 µg / ml (see FIG. 2).

In addition, uterine myoma cells were divided into 4 × 10 ³ cells in a 96 well plate using a multi pipet, and cultured in an incubator for 24 hours, followed by treatment of extracts such as cinnamon at 100, 200, and 300 ㎍ / ml. Cell titer 96 ^® single aqueous solution (Cell titer 96 ^® AQueous One Solution, Promega, Ma, WI, USA) (stored at -20 ° C) was left at room temperature for 90 minutes or at 37 ° C for 10 minutes to measure MTS. . 200 μl of the medium contained in each well was pipetted with a multipipette to retain 100 μl per well after the cells were suspended. 20 μl of the above-described cell titer 96 ^® single aqueous reagent was added to each well, incubated in a 37 ° C., 5% CO ₂ incubator, and MTS was measured at 1 hour intervals for 1 to 4 hours. Absorbance at 490 nm was measured using a plate reader (Bio-Rad Model 550, Japan).

Experimental Example 3. Observation of effects on cell cycles such as cinnamon through flow cytometric analysis

Uterine fibroids were cultured in a 60 mm tissue culture dish at 3 × 10 ⁵ cells / dish and cultured in the incubator for 24 hours, and then the extracts such as cinnamon were treated at concentrations of 100, 200, and 300 ㎍ / ml. . Cells were incubated for 24-48 hours, washed with PBS, resuspended with 1x trypsin-EDTA, transferred to 1.5 ml test tubes and centrifuged at 1000 rpm for 3 minutes. The supernatant was removed, resuspended in PBS solution, and centrifuged again at 1000 rpm for 3 minutes. The supernatant was removed, re-suspended in 1 ml of refrigerated pure ethanol (cold absolute EtOH), and the cells were fixed at 4 ° C. for at least 1 hour. After centrifugation at 1000 rpm for 3 minutes, the supernatant was removed, washed with PBS solution, and then 0.1% of sodium citrate (Trisodium citrate, igma, MO, USA) and 0.1% of IGEPAL-Ca-630 (Sigma, MO, USA) 5 mg / ml RNase A and propidium iodide (Sigma, MO, USA) solution were added to the solution and stained for 1 hour in the dark at 4 ° C., followed by DNA analysis using a flow cytometer (FACS). According to the cell distribution was measured.

After 24 hours treatment with extracts of cinnamon and the like on the leiomyoma cells, the cell cycle was analyzed after 24 hours. The control group showed 1.07% of cell distribution according to the DNA content in the subG1 phase. In one group, quantitative evidence of apoptosis that delayed subG1 phase was observed at 23.49%, and the delay of subG1 phase was prolonged in a concentration-dependent manner (see Table 1 and FIG. 3).

Cell cycle Cell distribution (%) Control 100 μg / ml 200 μg / ml 300 μg / ml sub G1 1.07 1.69 6.85 23.49 G1 56.8 60.57 58.45 46.80 S flag 7.8 4.55 3.88 5.00 G2 / M machine 33.58 32.03 30.53 24.52

Experimental Example 4. Observation of apoptosis induction effect through DNA fragmentation analysis

Uterine fibroids were cultured in a 60 mm tissue culture dish at 3 × 10 ⁵ cells / dish and cultured in the incubator for 24 hours, and then treated with extracts such as cinnamon at concentrations of 100, 200 and 300 ㎍ / ml. After 24 hours, a buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100 was treated and dissolved in ice for 30 minutes. The dissolved lysates were sufficiently mixed and centrifuged at 10,000 rpm for 20 minutes. DNA fragments fragmented in the supernatant were extracted with a solvent of the same amount of neutral phenol: chloroform: isoamyl alcohol in a 25: 24: 1 mixture, and then 0.1 µg / ml It was observed after electrophoresis on a 2% agarose gel containing ethidium bromide.

As a result of analyzing the DNA fragments by electrophoresis, it was observed that the segmentation phenomenon of the DNA increased as the concentration of the cinnamon and the like increased compared to the control group (see FIG. 4).

Experimental Example 5 Observation of the Expression of Genes Involved in the Cell Cycle by Western Blot Analysis

Uterine fibroids were cultured in a 60 mm tissue culture dish at 3 × 10 ⁵ cells / dish and cultured in the incubator for 24 hours, and then treated with extracts such as cinnamon at concentrations of 100, 200 and 300 ㎍ / ml. After 24 hours, expression differences of p27, p53, p21, p16, pro-caspase 3, and PARP gene proteins related to cyclin E and cell apoptosis were confirmed, and the cell cycle was observed. Expression of genes involved in the circuit was observed. Lysis buffer (Lysis Buffer, 10 mM Tris-Cl (pH 7.4), 5 mM EDTA (pH 8.0), 130 mM NaCl, 1% Triton X-100), 0.2 M phenyl-methyl-sulphate in the ground cells Phenyl fluoride (phenyl-methyl-sulfonyl fluoride), proteinase inhibitor mixture (cocktail), put on ice for 30 minutes and centrifuged to take the upper layer and Biorad protein assay kit (Biorad protein assay kit, Protein was extracted using Bio-Rad Laboratories Inc., PA, USA, and the protein was quantified by measuring the absorbance at 595 nm of a spectrophotometer (Du ^® 650, Beckman Coulter Inc., USA). The obtained protein fraction was electrophoresed and subjected to electrotransfer with nitrocellulose membranes (nitrocellulose paper, Immobilin Milipore, Bedford, UK). The electrophoretic membranes were dissolved in a blocking solution (TBS-T buffer 5). In a% skim dry milk and shaken in a cold chamber for 12 hours, the fixed solution was removed and the primary antibodies p27, p53, p21, p16, cyclin E, PARP (Santa) Cruz, CA, USA) was reacted with a fixed solution diluted at 1: 1,000 (room temperature) for 3 hours, followed by 1 × TBS-T solution (20 mmol / L Tris, 137 mmol / L NaCl, 0.5 Nitrocellulose membrane was added with 20%% Tween, and washed three times for 10 minutes, and a fixed solution of 1,1,000 diluted goat secondary polyclonal IgG (Santa Cruz, CA, USA). Into the nitrocellulose membrane and allowed to react for 2 hours to bind the antibody 1 × TBS-T solution The non-specifically bound antibody was removed by washing the nitrocellulose membrane three times for 10 minutes, after which the TBS-T remaining on the membrane was removed and washed with an ECL western blotting detection reagent (Amersham Biosciences, NJ, USA). Detected.

As a result of observing the expression of genes involved in the cell cycle, p27, p53, p21, and p16 genes increased in a concentration-dependent manner, but cyclin E did not change expression (see FIG. 5).

In addition, as a result of measuring the expression of pro-caspase 3 and PARP genes in order to determine the path of apoptosis, a quantitative decrease in the inactive form of pro-caspase 3 protein was observed in proportion to the treatment concentration of extracts such as cinnamon, The expression of PARP decreased with increasing concentration, and the expression of PARP cleavage was increased depending on the concentration (see FIG. 6).

Experimental Example 6. Acute Toxicity Test

6-1. Oral administration

       The composition of the present invention was divided into four groups of 10 ICR mice (weight 25 wh 5 g) and Sprague-dawly rats, respectively, at doses of 100, 250, 500 and 1000 mg / kg, respectively. Two weeks after the administration, no toxicities were observed in all four groups, and no symptoms were apparent in the control group.

6-2. Intraperitoneal administration

       ICR-based mice (weight 25 × 5 g) and Sprague-dawly rats were divided into four groups of 10 rats each, and the composition of the present invention was intraperitoneally administered at doses of 25, 50, 100 and 200 mg / kg, respectively. After 24 hours of administration, no toxicities were observed in all four groups and no symptoms were apparent in the control group.

       As a result, it was confirmed that the composition of the present invention has little acute toxicity.

Extracts such as cinnamon of the present invention may be prepared in the following formulations, the following formulation examples are merely to illustrate the present invention, thereby not limiting the content of the present invention.

Formulation Example 1 Preparation of Powder

300 mg of cinnamon light extract

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

50 mg of extract such as cinnamon

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

50 mg of extract such as cinnamon

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

50 mg of extract such as cinnamon

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

1000 mg of extract such as cinnamon

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 6 Preparation of Healthy Food

1000 mg of extract such as cinnamon

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Substrate mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

1000 mg of extract such as cinnamon

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and stored in the refrigerator and then Used to prepare the healthy beverage composition of the invention.

As described above, a composition containing an extract of cinnamon and the like as an active ingredient having an effect of inhibiting the G1 cell cycle of the cell cycle circuit according to the present invention inhibits the expression of genes and inhibits the proliferation of leiomyoma tumor cells. It can be used as a medicine and health functional food for the prevention and treatment of.

1 is a photograph observing the proliferation inhibitory effect of the extract of cinnamon on uterine myoma cells by immunohistochemical staining (immunohistochemistry),

Figure 2 is a diagram showing the observation of the proliferation inhibitory effect on uterine myoma cells according to the administration concentration of the extract such as cinnamon by measuring the number of cells,

3 is a diagram observing the effect on the cell cycle (cell cycle) of the extract such as cinnamon through flow cytometric analysis,

Figure 4 is a photograph observed through the DNA fragment analysis using electrophoresis of the extract of cinnamon, etc.,

5 is a photograph showing the expression of genes involved in the cell cycle by treating extracts such as cinnamon on uterine myoma cells using Western blot method,

FIG. 6 is a photograph illustrating the expression of genes involved in the path of apoptosis by treating extracts such as cinnamon on uterine myoma cells using Western blot.

Claims (5)

A pharmaceutical composition for the prevention and treatment of uterine myoma, comprising as an active ingredient an extract such as cinnamon having an effect of inhibiting the G1 cell cycle of the cell cycle circuit, and comprising a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition of claim 1, wherein the extract is extracted with a polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 2, wherein the extract is extracted with water. A health functional food for the prevention and treatment of uterine fibroids containing as an active ingredient extracts such as cinnamon having an effect of inhibiting the G1 cell cycle of the cell cycle circuit, as an active ingredient. The dietary supplement of Claim 4 which is a healthy beverage.