KR102399804B1 - Composition comprising abeliophyllum distichum extract for prophylaxis and treatment of preventing premature birth as an effective ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing and treating premature birth containing an extract of Miseon tree as an active ingredient.
The extract of Miseon tree of the present invention showed the ability to inhibit TNF-α secretion and IL-1β secretion released as an inflammatory response in an inflammation induction experiment of macrophage cell lines or trophoblast cell lines, By improving the degree of reduction in the number of fetuses, fetal size, and placental size due to inflammation, and significantly lowering the rate of preterm birth, it is possible to prevent and treat premature birth caused by intrauterine inflammatory response.

Description

Composition for the prevention and treatment of premature birth containing Miseon tree extract as an active ingredient

The present invention relates to a composition for preventing and treating premature birth containing a Miseon tree extract as an active ingredient. It relates to a composition for preventing and treating premature birth contained as this active ingredient.

Preterm birth is a birth before 37 weeks of pregnancy and occurs in 5 to 18% of pregnancies. Newborns born prematurely may have cerebral palsy, intellectual disability, visual and hearing impairment due to neurodevelopmental disorders, and the risk of complications increases due to the immaturity of several organs. About 15 million premature babies are born every year worldwide, with the highest rates occurring in Africa and North America. Preterm birth is the leading cause of neonatal death and the second leading cause of death in children under 5 years of age. The incidence of premature babies in Korea was 4.5 per 100 live births in 2003 and is on the rise to 6.5 per 100 live births in 2013.

Preterm birth occurs in about 2/3 of the natural birth process, but other than that, premature birth occurs due to maternal or fetal complications such as preeclampsia or intrauterine growth retardation.

Preterm birth and normal childbirth proceed in the same process: increased contractility of the uterus, dilatation of the cervix, and rupture of the chorioamniotic membranes. In this case, the increase in uterine contractility is caused by the transition from an anti-inflammatory state to a pro-inflammatory state in the uterine fascia. As major factors inducing such an inflammatory state, the chemokines IL-8 and the cytokines IL-1 and IL-6 are known.

In addition, cervical dilatation is caused by changes in extra-cellular matrix proteins, such as loss of collagen cross-linkage or increase in glycosaminoglycans.

The process of chorionic membrane rupture is caused by the lysis and apoptosis of the extracellular matrix by the increase in the activity of MMP-8,9 protease and the increase in the expression of inflammatory cytokines such as TNF-α and IL-1 and chemokines. Prematurity occurs when various diseases activate one or more components of the normal birth process.

The major factors that induce premature birth are known as decreased progesterone activity, cervical diseases, disruption of fetal and maternal immune tolerance, stress, infection, vascular disorders, decidual aging, and uterine hyperinflation.

25% of premature babies are born to mothers infected with amniotic edema. Microorganisms present in the female lower genital tract can be infected with amniotic fluid, and this infection route is known as a frequent route to cause amniotic fluid. Detected, chemokines (IL-8, CCL2) and cytokines (IL-1β, TNF-α) are generated to induce an inflammatory response and cause premature birth.

In particular, it is known that TNF-α induces apoptosis of the decidua and chorionic membrane, and promotes MMP expression, thereby greatly affecting preterm birth. Therefore, the inflammatory response is one of the most important factors of premature birth.

Inflammatory response caused by intrauterine infection before birth not only induces premature birth, but also causes fetal inflammatory response syndrome (FIRS), resulting in neonatal disease morbidity such as neonatal respiratory distress syndrome, pneumonia, intraventricular hemorrhage, cerebral palsy, and retinopathy of prematurity. this rises Currently, prenatal steroids are used to control the inflammatory response, but the need for drug development for the treatment of inflammation is increasing.

In relation to the conventional use for preventing or treating premature birth, Patent Document 1 presents a composition using a pumpkin vine or a main active ingredient of a pumpkin vine as a material that can prevent premature miscarriage and stomach cramps in pregnant women, and Patent Document 2 discloses a gene of a progesterone receptor Methods for diagnosing and preventing premature birth through sequence identification and genetic manipulation thereof are disclosed.

However, access to plant materials effective for suppressing premature birth caused by inflammatory reactions has not yet been reported.

Abeliophyllum distichum is a flowering shrub belonging to the family Oleaceae, one genus, and 1 m in height. The leaves are opposite, ovate or oval-ovate, with narrow ends, flat edges, and short stems. The flower blooms before the leaf and hangs on the raceme, and the shape of the seed resembles a fan, so it is called a Miseon tree.

The Korean Ministry of Environment has designated and protected the Miseon tree, an endemic species in Korea, as a protected wild plant.

Until now, a number of patents using Miseon tree have been reported as anti-inflammatory agents, anticancer agents, compositions for improving atopic dermatitis, and compositions or pharmaceutical compositions for external application to the skin. It has been proposed as a perfume composition that reproduces , a special odor remover for livestock and aquatic products, and a shampoo composition for preventing hair loss.

As an example, Patent Document 3 is for skin care that can achieve antioxidant effect, skin aging prevention effect, wrinkle improvement effect, collagen biosynthesis effect, whitening effect and moisturizing effect by using Miseon tree extract having antioxidant effect extracted from Miseon tree. The composition is disclosed, and Patent Document 4 discloses a composition for improving atopic dermatitis containing the Miseon tree extract for improving inflammatory skin diseases (atopic dermatitis) using the Miseon tree extract.

However, the technology applied to the preventive and therapeutic use of Miseon tree extract is not known.

Accordingly, the present inventors confirmed the ability to inhibit TNF-α secretion and IL-1β secretion, which are released as an inflammatory response in an inflammation induction experiment of macrophage or trophoblast cell lines, as a result of a steady study on the pharmaceutical efficacy of Miseon tree extract, and induce inflammation In a mouse preterm model, it is useful for the prevention and treatment of premature births caused by intrauterine inflammatory reactions from the result of improving the reduction in the number of fetuses, fetal size, and placental size due to inflammation in the group administered with the extract of Miseon tree in the mouse preterm model, and lowering the preterm birth rate. By confirming, the present invention was completed.

Korean Patent Publication No. 2011-0133389 (published on December 12, 2011) US Patent Publication No. 2012/0046261 (published on February 23, 2012) Korea Patent Publication No. 2016-0089257 (published on July 27, 2016) Korea Patent Publication No. 2016-0089256 (published on July 27, 2016)

Journal of the Korean Society for Environmental Health, 2014, Vol. 40, No. 3, 234-244

The present invention is to provide a composition for preventing and treating premature birth containing Miseon tree extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing and treating premature birth in which the Miseon tree extract is contained as an active ingredient.

More specifically, the present invention is to provide a composition for preventing and treating premature birth, in which the extract of Miseon tree is effective for premature birth induced by an inflammatory reaction.

The inflammatory response uses an inflammation induction model for macrophage cell lines or trophoblast cell lines.

More preferably, the Miseon tree extract is to provide a composition for preventing and treating premature birth using the anti-inflammatory effect of the leaf extract.

In the composition of the present invention, the Miseon tree extract is extracted from water, an organic solvent having 1 to 6 carbon atoms, or a mixed solvent thereof, and more preferably an extract extracted with 30% to 95% ethanol.

According to the present invention, the Miseon tree extract lowers the level of inflammatory cytokines including TNF-α and IL-1β that are released as an inflammatory response to control the inflammatory response in the uterus, confirm the efficacy of inhibiting apoptosis, and A composition useful for preventing and treating premature birth caused by an inflammatory reaction can be provided.

1 is a cytotoxicity result for macrophage cell line (THP-1) according to the concentration of the leaf extract of Miseon tree of the present invention;
Figure 2 shows the inhibitory effect on the production of TNF-α according to the concentration of the leaf extract of Miseon tree in the macrophage cell line of Figure 1,
3 is a cytotoxicity result for the trophoblast cell line (JEG-3) after 48 hours of treatment time for each concentration of the Miseon tree leaf extract of the present invention;
4 is a cytotoxicity result for the trophoblast cell line (JEG-3) after 72 hours of treatment for each concentration of the Miseon tree leaf extract of the present invention;
5 is an evaluation result of IL-1β secretion inhibitory ability for each concentration of Miseon tree leaf extract of the present invention;
6 is a result of apoptosis of trophoblast cells by TNF-α after 72 hours of treatment for each concentration of the Miseon tree leaf extract of the present invention;
Figure 7 is a model production flow chart for testing the effect of LPS-induced mouse preterm delivery of the extract of Miseon tree leaf extract on the fetus,
8 is an anatomical result comparing the number of fetuses, the size of the fetus, and the size of the placenta on the effect of the extract of the Miseon tree of the present invention on the fetus in the mouse preterm model of FIG. 7;
Figure 9 is a model production flow chart for testing the effect of the extract of Miseon tree leaves on childbirth using a mouse preterm birth model induced by LPS,
10 is a result of evaluation of the effect of the extract of Miseon tree leaf extract of the present invention on childbirth in the mouse preterm model of FIG. 9 .

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention and treatment of premature birth containing the extract of Miseon tree as an active ingredient.

More specifically, since the extract of Miseon tree of the present invention shows the ability to inhibit TNF-α secretion and IL-1β secretion, which are released as an inflammatory response in an inflammation induction experiment of a macrophage cell line or a trophoblast cell line, Valid for premature birth.

Hereinafter, the usefulness of the Miseon tree extract of the present invention as a composition for preventing and treating premature birth will be described with reference to the drawings, and the Miseon tree extract will be described as the Miseon tree leaf extract prepared in Example 1.

1 shows the cytotoxicity results for macrophage cell lines (THP-1) by concentration of the Miseon tree leaf extract of the present invention, even in the group administered with the highest concentration of Miseon tree leaf extract (500 μg/ml) THP- It can be seen that there is no difference in cytotoxicity between the experimental group treated with the basic culture medium and 1 cells.

Figure 2 shows the inhibitory effect on the production of TNF-α for each concentration of the Miseon tree leaf extract in the macrophage cell line. can

3 and 4 show cytotoxicity to the trophoblast cell line (JEG-3) after treatment for each concentration of the Miseon tree leaf extract of the present invention and treatment time for 48 hours and 72 hours, respectively, the Miseon tree leaf extract is It can be confirmed that there is no cytotoxicity to the trophoblast cell line (JEG-3), and based on these results, FIG. 5 is the result of observing the IL-1β secretion inhibitory ability of the Miseon tree leaf extract by concentration, the trophoblast cell line (JEG-3) It can be seen that the production amount of IL-1β in the leaf extract of Miseon tree was reduced in

In addition, FIG. 6 is a result for apoptosis of trophoblast cells by TNF-α after 72 hours of treatment time for each concentration of the Miseon tree leaf extract of the present invention, whereas apoptosis was observed in the group treated with rTNF-α protein. , In the case of the group treated with the rTNF-α protein and the Miseon tree leaf extract, it can be seen that there is no difference in the degree of apoptosis from the group treated with the basic culture medium.

7 and 8 show the results of observing the effect of the extract of Miseon tree leaf on the fetus using the LPS-induced inflammation mouse preterm model. Although the results were all reduced compared to the group treated with the basic culture drug, the group administered orally with the extract of Miseon tree leaf extract showed similarly improved results compared to the group treated with the basic culture drug before induction of inflammation with LPS.

In addition, FIG. 9 is a result of an experiment on the effect of a Miseon tree leaf extract on childbirth using an inflammation-induced mouse premature birth model by LPS. In the group treated only with LPS, more than 80% of preterm birth was administered, while Miseon tree leaf extract was administered. In one group, it can be seen that the premature birth rate was lowered to 30% or less.

From the above, in order to confirm the efficacy as a pharmaceutical composition for the prevention and treatment of premature birth by containing the extract of Miseon tree of the present invention as an active ingredient, it was induced with LPS in THP-1, a human macrophage cell line and JEG-3 cell line, a trophoblast cell line. As a result of measuring the effect of preventing and treating premature birth on the induction of inflammation, the extract according to the present invention significantly lowered the level of inflammatory cytokines including TNF-α and IL-1β released as an inflammatory response, resulting in intrauterine inflammation By controlling the response and inhibiting apoptosis, it supports the prevention and treatment of premature birth.

In the present invention, Miseon tree extract refers to extracts from single or two or more parts selected from the group consisting of leaves, stems, fruits, roots, bark and seeds of Miseon tree, preferably selected from Miseon tree leaves or stems. alone or a mixture thereof.

In the embodiment of the present invention, it is described using the leaves of Miseon tree as the most preferred example, but it will not be limited thereto.

The Miseon tree leaf extract of the present invention may use leaves harvested at a specific time or a specific period during the period between spring and autumn, or all of the leaves harvested during the period between spring and autumn.

In particular, the content of sugars such as crude flour, crude protein, crude fat, fructose, glucose, and ascorbic acid in the leaves of Miseon tree are higher than in the stem of Miseon tree. Looking at the fatty acid content of Miseon tree, palmitic acid (palmitic acid) acid), stearic acid, behenic acid, and linoleic acid in the order, and the stem contains linolenic acid, palmitic acid, and linoleic acid in that order. has been The organic acid content is high in the order of malic acid, succinic acid, and citric acid, and it has been reported that the content of the leaf part is higher than that of the stem part. In addition, in the same study, it is reported that the content of total phenolic compounds and flavonoids, which are known as substances exhibiting physiological activity, is about 3.7 to 5 times higher than that of the stem part of the Miseon tree leaf part [Non-Patent Document 1].

In the present invention, extraction of the active ingredient from Miseon tree may be performed by a solvent extraction method.

Specifically, in the composition of the present invention, the Miseon tree extract is extracted from water, an organic solvent having 1 to 6 carbon atoms, or a mixed solvent thereof. Examples of the organic solvent include ethanol, methanol, propanol, butanol, glycerin, ethyl acetate, butylene glycol, propylene glycol glycol), dichloromethane, chloroform, ethyl ether, hexane, and the like. These can be used individually or in mixture of 2 or more, respectively.

In order to obtain the Miseon tree extract in the present invention, the Miseon tree is collected, the desired Miseon tree leaf part is cut, washed, dried and pulverized, and 3 to 30 times the weight of the Miseon tree leaf alcohol pulverized product weight, preferably 10 to 20 The extraction process is performed for at least 1 hour at an extraction temperature of 60° C. to 75° C. with pear water or a lower alcohol (eg, methanol, ethanol, butanol, etc.), or a mixed solvent thereof. At this time, the extraction of alcohol using a lower alcohol is preferably 30% to 95% ethanol. When it is less than 30%, alcohol-soluble active ingredients cannot be obtained substantially, and when it exceeds 95%, it is hardly possible to obtain water-soluble active ingredients.

More preferably, it is 30% to 70% ethanol, and most preferably, it is extracted using 70% ethanol as an extraction solvent.

In addition, the extract of Miseon tree of the present invention can be extracted until the concentration of soluble solids exceeds 0 brix but reaches 30 brix. More preferably, extraction can be carried out until the concentration of soluble solids reaches 20 to 21 brix. At this time, the mixed solvent may be mixed so that the mixing ratio of water and lower alcohol is 1:1 to 1:10, and as an extraction method, hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction, steam distillation extraction, etc. to 5 consecutive extractions. After extraction, it is filtered under a temperature condition of 40° C. to 60° C., and the filtrate is concentrated under reduced pressure and dried with a freeze dryer to obtain a Miseon tree extract.

Since the above extract of Miseon tree according to the present invention is derived from a plant material and has no toxicity or side effects, stable pharmaceutical administration or administration will be possible.

Furthermore, the present invention contains the extract of Miseon tree as an active ingredient, but may include one or more known active ingredients having an effect of preventing and treating premature birth.

In addition, the composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and if necessary, an antioxidant; Other conventional additives such as buffers and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

In the composition for preventing and treating premature birth in which the Miseon tree extract according to the present invention is contained as an active ingredient, the Miseon tree extract is 0.001 to 90% by weight, preferably 0.01 to 15% by weight, more preferably 0.01 based on the total composition to 10% by weight.

The composition for the prevention and treatment of premature birth containing the extract of Miseon tree of the present invention as an active ingredient is a preservative, stabilizer, wetting agent or emulsification accelerator, salt or buffer for osmotic pressure control, disintegrant, binder, lubricant so that it is suitable for pharmaceutical use. , may further contain pharmaceutical adjuvants such as diluents and other therapeutically useful substances, and may be formulated in various oral or parenteral dosage forms according to conventional methods.

Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. , mannitol, sorbitol, cellulose and glycine, lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycol. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt It may contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods. In addition, a representative formulation for parenteral administration is an injection formulation, preferably an isotonic aqueous solution or suspension.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors, such as the severity of symptoms, the route of administration selected, the age, sex, weight, and health status of the subject.

The appropriate dosage of the composition for preventing and treating premature birth containing the extract of Miseon tree according to the present invention as an active ingredient varies depending on various factors such as the degree of obesity progression of the subject to be administered, the onset time, age, health status, complications, etc. Based on an adult, 1 to 500 mg/kg of the composition, preferably 30 to 200 mg/kg, may be administered in divided doses 1 to 2 times a day, and the dosage is not intended to limit the scope of the present invention in any way. .

Hereinafter, an embodiment according to the present invention will be described in detail.

However, the following examples are provided only for easier understanding of the present invention, and the present invention is not limited by the examples.

<Example 1> Preparation of Miseon tree leaf extract 1

40L of 70% ethanol was added to 2 kg of minced Miseon tree leaves, dried in the shade, and extracted at 75°C for 4 hours. Thereafter, after filtration with filter paper, the extract was concentrated to 2 L at 50° C. under reduced pressure. The concentrated extract was dried with a freeze dryer to obtain 1.02 kg of 70% ethanol extract of Miseon tree leaves.

<Example 2> Preparation 2 of extract of Miseon tree leaf

40L of distilled water was added to 2 kg of minced Miseon tree leaves, dried in the shade, extracted at 100°C for 4 hours, filtered through a filter paper, and the extract was concentrated to 2L at 50°C under reduced pressure. The concentrated extract was dried with a freeze dryer to obtain 0.75 kg of hot water extract from Miseon tree leaves.

<Experimental Example 1> Culturing of cell lines

THP-1, a human macrophage cell line, and JEG-3, a human trophoblast cell line, were mixed with 10% FBS (Fetal Bovine Serum) in Dulbecco's modified Eagle's medium, 100 U/ml penicillin, 0.1 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin. B was added and used for culture experiments. The THP-1 cell line was treated with 100 nM of PMA (Phorbol 12-myristate 13-acetate), which induces PKC (protein kinase C) activity, for 24 hours to activate the THP-1 cell line before the experiment. was used.

<Experimental Example 2> Inflammation inhibition evaluation in macrophage cell lines

1. Cytotoxicity measurement

In order to measure the cytotoxicity according to the concentration of the Miseon tree leaf extract of Example 1, the macrophage THP-1 was treated with the Miseon tree leaf extract for each concentration to determine the degree of cytotoxicity and observed cytotoxicity. . Specifically, the group (CNT) treated with PMA 100 nM for 24 hours and the activated THP-1 cells were treated with the basal culture solution, the same amount of solvent as the amount of solvent used when 500 μg/ml of Miseon tree leaf extract was treated. The group (Sol.), the group (100, 500) treated with the Miseon tree leaf extract at a concentration of 100 and 500 μg/ml in the culture medium were treated for 24 hours, respectively.

After 24 hours of treatment, each well was replaced with a culture solution containing 10% WST-8, and left in an incubator for 1 hour and 30 minutes. Thereafter, the absorbance was measured at 450 nm and analyzed. 1 shows the results of cytotoxicity in macrophage cell lines by concentration of the Miseon tree leaf extract of the present invention, and there was no difference in cytotoxicity between the CNT group and the 500 μg/ml group, which is the highest concentration of the Miseon tree leaf extract.

2. Evaluation of TNF-α secretion inhibitory ability

When the human macrophage cell line (THP-1) was treated with LPS, an experiment was carried out to measure the amount of TNF-α in order to confirm the effect of inhibiting the production of TNF-α in the leaf extract of Miseon tree.

Specifically, THP-1 cells activated by treatment with PMA (Phorbol 12-myristate 13-acetate) 100 nM for 24 hours were simultaneously treated with LPS 100 ng/ml and Miseon tree leaf extract 20, 100, and 500 μg/ml, 24 After time, the amount of TNF-α in the supernatant was measured by ELISA (enzyme-linked immunosorbent assay).

2 is a result showing the inhibitory effect on the production of TNF-α according to the concentration of the Miseon tree leaf extract in the macrophage cell line. In the group treated with the Miseon tree leaf extract, TNF produced in THP-1 cells compared to the group treated with only LPS. It was confirmed that the amount of -α was reduced.

<Experimental Example 3> Inflammation inhibition evaluation in trophoblast cell lines

1. Cytotoxicity measurement

In order to confirm the degree of toxicity of the trophoblast cell line JEG-3 according to the concentration of the Miseon tree leaf extract of Example 1, a cytotoxicity measurement experiment was performed.

Specifically, the group in which the trophoblast cell line was treated with the basic culture solution (CNT), the group treated with the same amount of solvent as the amount of the solvent used when 500 μg/ml of the Miseon tree leaf extract was treated (Sol.), 100 to 500 in the culture medium The group treated with the Miseon tree leaf extract at a concentration of μg/ml was treated for 48 and 72 hours, respectively. After 48 and 72 hours of treatment, each well was replaced with a culture solution containing 10% WST-8, and then left in an incubator for 1 hour and 30 minutes. Thereafter, the absorbance was measured at 450 nm and analyzed.

3 shows the cytotoxicity results in the trophoblast cell line after 48 hours of treatment according to the concentration of the Miseon tree leaf extract of the present invention, there was no difference in cytotoxicity between the CNT group and the 500 μg/ml group, which is the highest concentration of the Miseon tree leaf extract. .

4 shows the cytotoxicity results in the trophoblast cell line after 72 hours, and there was no difference in cytotoxicity between the CNT group and the CNT group even in the highest concentration group of 200 μg/ml of 70% ethanol extract of Miseon tree leaves.

As a result of the cytotoxicity test for JEG-3, a trophoblast cell line, according to the concentration of the Miseon tree leaf extract of FIGS. 3 and 4, it was confirmed that the Miseon tree leaf extract had no cytotoxicity to JEG-3.

2. Evaluation of IL-1β secretion inhibitory ability

In order to confirm the effect of inhibiting the production of IL-1β of the Miseon tree leaf extract in the trophoblast cell line (JEG-3), the supernatant of the macrophage line treated with LPS and Miseon tree leaf extract was treated to the trophoblast cell line to reduce the level of IL-1β. An experiment was conducted to measure the amount. Specifically, 100 ng/ml of LPS and 20, 100, and 500 μg/ml of LPS leaf extract 20, 100, and 500 μg/ml of THP-1 cells activated by treatment with PMA 100 nM for 24 hours were simultaneously treated, and the supernatant was collected after 24 hours and placed in a centrifuge. It was treated at 3,000 RPM for 10 minutes, and the supernatant except for the precipitate was treated in the trophoblast cell line for 48 hours. After 48 hours of treatment, the amount of IL-1β in the supernatant of the trophoblast cell line was measured by ELISA.

5 is a result of evaluation of IL-1β secretion inhibitory ability by concentration of the Miseon tree leaf extract of the present invention, and it was confirmed that the production amount of IL-1β decreased in the group treated with the Miseon tree leaf extract after 48 hours of treatment.

3. Measurement of apoptosis according to treatment of Miseon tree leaf extract

In order to confirm that the degree of apoptosis of trophoblast cells caused by TNF-α can be improved by extracts from Miseon tree leaves, rTNF-α (recombinant human tumor necrosis factor-α) protein was directly treated on trophoblast cells. The trophoblast cell line was treated with 1,000 ng/ml of rTNF-α protein and 1, 10, and 100 μg/ml of Miseon tree leaf extract and cultured for 72 hours. After 72 hours, each well was replaced with a culture solution containing 10% WST-8, and then left in the incubator for 1 hour and 30 minutes. Thereafter, the absorbance was measured at 450 nm and analyzed.

From the results of Figure 6, when treated for 72 hours, the group treated with rTNF-α protein 1,000ng / ㎖ confirmed that apoptosis occurred compared to the CNT group. It was confirmed that there was no difference in the degree of apoptosis between the group treated with rTNF-α protein at 1,000 ng/ml and the Miseon tree leaf extract 100 μg/ml together with the CNT group.

<Experimental Example 4> Inflammation-induced mouse premature birth model experiment by LPS

1. Assessment of the effect on the fetus

In order to confirm the effect of the leaf extract of Example 1 on the fetus in a mouse preterm model induced by LPS, PMSG 5 I.U/100 μl and HCG before mating 2 days before mating in 6-week-old C57BL/6 female mice After intraperitoneal administration of 5 I.U/100 μl, they were mated with C57BL/6 male mice of the same age for 2 days. From the 7th day of the first mating start date, the Miseon tree leaf extract was administered by oral administration at 2 mg/100 μl for 10 days. On the 17th day from the start of mating, 0.5 μg/100 μl of LPS was administered intraperitoneally to induce an inflammatory response, and dissection was performed on the 18th day.

7 is a flowchart showing the production of a mouse preterm model induced by LPS for this experiment, and through the mouse dissection results of FIG. 8, in the group treated only with LPS, the number of fetuses, the size of the fetuses, and the size of the placenta were CNT It was confirmed that the results were all reduced compared to the group. However, it was confirmed that the level of improvement was similar to that of the CNT group in the group to which the extract of Miseon tree leaf extract was orally administered.

2. Assessment of impact on childbirth

In order to confirm the effect of the leaf extract of Example 1 on childbirth in a mouse preterm model induced by LPS, PMSG 5 I.U/100 μl and HCG before mating 2 days before mating in 6-week-old C57BL/6 female mice After intraperitoneal administration of 5 I.U/100 μl, they were mated with C57BL/6 male mice of the same age for 2 days. From the 3rd day of the first mating start date, the leaf extract of Miseon tree was administered by oral administration at 2 mg/100 μl for 15 days. On the 18th day of the mating start date, LPS was administered intraperitoneally with 0.5 μg/100 μl to induce an inflammatory response, and then the birth of mice was confirmed until the 20th day.

9 is a flowchart showing the production of a mouse preterm model induced by LPS for this experiment, and as a result of confirming the birth of mice through FIG. 10, more than 80% of the LPS-treated group gave birth prematurely, In the group administered for 15 days, it was confirmed that the rate of premature birth fell to 30% or less.

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical spirit of the present invention, and it is natural that such variations and modifications belong to the appended claims.

Claims (5)

It is a composition for preventing and treating premature birth containing Miseon tree extract as an active ingredient,
The Miseon tree extract is,
It is obtained by washing, drying and pulverizing the leaves of the Miseon tree, and performing the extraction process at an extraction temperature of 60° C. to 75° C. for at least 1 hour with 30% to 95% ethanol of 10 to 20 times the weight of the pulverized Miseon tree leaf. ,
A composition for preventing and treating premature birth, which is extracted until the concentration of soluble solids reaches 20 to 21brix. [Claim 2] The composition for preventing and treating premature birth according to claim 1, wherein the extract of Miseon tree extract is effective for premature birth induced by an inflammatory reaction in the uterus. delete delete delete

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