KR101538047B1 - Composition containing polysacchride-removed Paeonia lactiflora extract for improving pregnancy - Google Patents

Abstract

The present invention relates to a composition for promoting pregnancy and a composition for the prevention or treatment of infertility, the composition comprising a peony extract obtained by removing a polysaccharide as an active ingredient. The polysaccharide-free peony extract of the present invention has few side effects in vivo and induces the expression of a leukemia inhibitory factor (LIF) involved in the binding of the embryo to the endothelium, thereby increasing the binding force between placental cells and fetal cells, And thus can be usefully used as a composition for promoting pregnancy such as a pregnancy aid which can promote fertilization and prevent and treat infertility.

Description

[0001] The present invention relates to a composition for promoting pregnancy, which comprises an extract of a Chinese peony with a polysaccharide removed therefrom as an active ingredient,

The present invention relates to a composition for promoting pregnancy or a composition for the prevention or treatment of infertility, which comprises a peony extract obtained by removing a polysaccharide as an active ingredient.

With the recent aging society, infertility is increasing as maternal age increases. In addition, due to environmental pollution caused by industrialization and the advancement of women into society, the stress of women is increasing, and the pregnancy rate is significantly lowered.

Common causes of female infertility include ovulation disorders, transfer of fertilized eggs, and implantation disorders. Infertility due to obstructive ovarian failure and transfer of embryos is largely solved through in vitro fertilization (IVF), but sterilization due to implantation disorder has yet to be clarified.

Embryo implantation is a process in which (i) hatching of the fertilized egg is removed, (ii) inducing apposition of embryo and endometrium, (iii) strong adhesion of embryo and endometrium, And (iv) infiltrating the endometrium into the uterine endothelium. The expression of leukemia inhibitory factor (LIF), which is a cytokine of the IL-6 family, plays an important role in this implantation process. In the case of mice deficient in LIF, the step up to the step (ii) proceeds normally, and the abnormalization of the embryo does not occur since the adhesion of the step (iii) occurs (Nature 359 (6390): 76-9 , 1992; Endocrinology 150 (6): 2915-23, 2009). In addition, when the function of LIF is inhibited by using an antagonist for LIF, no implantation occurs (PNAS 104 (49): 19357-62, 2007; Contraception S0010-7824 (12) 00884-0, 2012) Has been reported, and the LIF has been attracting attention as the most important factor in the conception of the embryo.

On the other hand, peonies ( Paeonia lactiflora Pall) are classified as peptic ulcer and herbal medicines and veterinary medicine. Peonies are known to be distributed widely in the northeast of Jiangsu province in China, Mongolia, southern Far East of Asia and Korea. Peony contains many kinds of sugars, mucilages, organic acids and trace minerals, and it is known to treat malnutrition and to act as a convergence. Peony also works to improve vascular function, strengthen digestive organs, and inhibit pain in the nervous system. In addition, peonies are known to suppress gastric acid secretion, strengthen the movement of the bladder, calm the abdominal pain, and have excellent antimicrobial, anti-inflammatory and antipyretic action.

As described above, peanut is a medicinal substance useful in oriental medicine, and is used for the treatment of various diseases, but its pharmacological mechanism has not yet been clarified.

The inventors of the present invention conducted studies on natural resources having an excellent effect of inducing embryo implantation, and found that the extract of the peony root extract of the peony root had few side effects, and the leukemia inhibitory factor (LIF) involved in the binding of the embryo to the endothelium The present inventors have confirmed that the effect of inducing the expression of the embryo is excellent, thereby increasing the binding force between the placental cell and the fetal cell.

It is an object of the present invention to provide a pharmaceutical composition for promoting pregnancy containing an extract of Peony root which has been obtained by removing a polysaccharide as an active ingredient.

Another object of the present invention is to provide a food composition for promoting pregnancy, which comprises a peony extract obtained by removing a polysaccharide as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating infertility comprising an extract of peony root extract of polysaccharide as an active ingredient.

It is still another object of the present invention to provide a food composition for preventing or ameliorating infertility, which contains a peony root extract with polysaccharide as an active ingredient.

The present invention provides a pharmaceutical composition for promoting pregnancy, which comprises a crude extract of Peony root with polysaccharide removed as an active ingredient.

In addition, the present invention provides a food composition for promoting pregnancy, which comprises a peony extract obtained by removing a polysaccharide as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating infertility, comprising an extract of Peony root with polysaccharide removed as an active ingredient.

In addition, the present invention provides a food composition for preventing or ameliorating infertility, which contains an extract of Peony root with polysaccharide removed as an active ingredient.

The polysaccharide-free peony extract of the present invention has few side effects in vivo and induces the expression of a leukemia inhibitory factor (LIF) involved in the binding of the embryo to the endothelium, thereby increasing the binding force between placental cells and fetal cells, The composition of the present invention is useful as a composition for promoting pregnancy such as a pregnancy aid which can promote fertilization and prevent or treat infertility.

FIG. 1 is a schematic view illustrating a process for producing a peony root extract of the present invention from which a polysaccharide is removed according to the present invention.
FIG. 2 is a diagram showing the cytotoxicity evaluation of a peony root extract with polysaccharide removed according to the present invention. FIG.
FIG. 3 is a graph showing the results of RT-PCR analysis of the expression of LIF gene in cells treated with the extract of Peony root having the polysaccharide removed according to the present invention.
FIG. 4 is a graph showing the results of Western blot analysis for the expression of LIF protein in cells treated with the extract of Peony root having the polysaccharide removed according to the present invention. FIG.
FIG. 5 is a graph showing the results of Western blot analysis of LIF protein expression in cells treated with a simple hydrothermal extract (polysaccharide-containing) of peony root and polysaccharide-free peony root extract according to the present invention (1. Con: control group; 3. Simple hydrothermal extract of peony: simple peeled hot water extract; and 3. Precipitation per peony: hot water extract of peony root which was precipitated by adding ethanol to peach hydrothermal extract containing polysaccharide.
FIG. 6 is a graph showing the results of RT-PCR analysis of the expression of LIF gene in cells treated with the simple hot-water extract of Peony root and the peony root extract according to the present invention.
FIG. 7 is a graph showing the results of measurement of binding force between placental cells and fetal cells treated with the simple hot-water extract of Peony root and the extract of Peony root with the polysaccharide removed according to the present invention.
FIG. 8 is a schematic diagram showing a process for producing the simple hot-water extract (A), the peony root extract of methanol and the peony root ethanol extract (B) of the present invention.
FIG. 9 is a diagram showing the cytotoxicity evaluation of the simple hot-water extract of Peony root, the peony's methanol extract and the peony root ethanol extract of the present invention.

The present invention provides a composition for promoting pregnancy, comprising a crude extract of Peony root with polysaccharide removed as an active ingredient.

In addition, the present invention provides a composition for preventing or treating infertility, which comprises a peony extract obtained by removing a polysaccharide as an active ingredient.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in detail.

In the composition of the present invention, a conventional extract method may be used for the extract of Peony root, which is an effective ingredient without the polysaccharide, and examples of the conventional extraction method include, but are not limited to, ultrasonic extraction, filtration and reflux extraction.

The peony root extract with the polysaccharide removed according to the present invention can be obtained through the following steps.

First, clean the peony and then dry naturally. The dried peony is put into water, heated and extracted with hot water, and then the peony extract is centrifuged to remove the residue and only the supernatant is separated. The separated supernatant is reacted by adding C ₁ -C ₄ alcohol. At this time, the reaction temperature is preferably 2 to 6 ° C, but is not limited thereto. The above-mentioned alcohol-added supernatant is centrifuged to remove the precipitated polysaccharide, and the supernatant is separated again. The separated supernatant is concentrated under reduced pressure and lyophilized to obtain a peony extract in which powdery polysaccharide is removed.

The peony extract is characterized in that the polysaccharide is removed by adding C ₁ -C ₄ alcohol to the peach hydrothermal extract.

The C ₁ -C ₄ alcohols include but are not limited to 1-100% (v / v) methanol or 1-100% (v / v) ethanol.

The polysaccharide-free peony extract of the present invention has few side effects in vivo and induces the expression of a leukemia inhibitory factor (LIF) involved in the binding of the embryo to the endothelium, thereby increasing the binding force between placental cells and fetal cells, And thus can be usefully used as a composition for promoting pregnancy such as a pregnancy aid which can promote fertilization and prevent and treat infertility.

The composition of the present invention may contain at least one known active ingredient having the effect of preventing or treating pregnancy promotion and infertility, together with the extract of the fungicide with polysaccharide removed.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the polysaccharide-free peony extract of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, and may be administered once a day or divided into several times.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the promotion of pregnancy, prevention or treatment of infertility.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The composition of the present invention may be added to a health functional food for the purpose of promoting pregnancy or preventing or improving infertility. When the peony extract of the present invention is used as a food additive, the peony extract may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the peony extract in which the polysaccharide of the present invention is removed during the production of food or beverage is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition,

1) adding peony pellets to water and heating at 80 to 100 캜 to obtain peony hydrothermal extract;

2) separating only the supernatant by centrifuging the Abalone hydrothermal extract;

3) adding C ₁ -C ₄ alcohol to the separated supernatant, reacting at a temperature of 2 to 6 ° C, centrifuging the reaction mixture to remove the precipitated polysaccharide, and separating only the supernatant; And

4) a step of concentrating the separated supernatant under reduced pressure and lyophilization to powderize the supernatant, and removing the polysaccharide having excellent pregnancy promoting effect.

As used herein, the term "pregnancy aid" is a generic term meaning pregnancy assistance, which includes pregnancy promotion, prevention of infertility, treatment of menstrual irregularities, treatment of gynecological diseases, treatment of poor uterus, ≪ / RTI > and treatment, and the like.

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, experimental examples and preparation examples.

[Example 1: preparation of peony extract with polysaccharide removed]

100 g of peach was added to 1 L of distilled water, boiled at 100 ° C. for 2 hours, centrifuged at 4,000 rpm for 15 minutes, and only the supernatant was separated to obtain about 600 ml of peach hydrothermal extract. Since the polysaccharide may interfere with the intercellular adhesion in the hydrothermal extract, the ethanol extract is added to the hydrothermal extract to remove the polysaccharide at a final concentration of 70% And centrifugation was then performed twice at 4,000 rpm for 10 minutes to remove the precipitated polysaccharide, and the supernatant was separated. The separated supernatant was concentrated under reduced pressure and lyophilized to give a total of 2.25 g of a dried peony root-like polysaccharide-free peony extract (yield: 2.25%). FIG. 1 shows a process for producing the peony root extract from which the polysaccharide of the present invention is removed.

The resulting extract of the peony root was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg / ml and stored for use in the following experiment.

[Experimental Example 1: Evaluation of cytotoxicity of extract of peony root extract of polysaccharide of the present invention]

In order to examine the cytostability of the extract of peony root extract of the present invention, cytotoxicity of Ishikawa cells was evaluated.

1. Preparation and culture of Ishikawa cells

Ishikawa cells, the human endometrial cell line used in the experiments, were purchased from Dr. Jacques Simard (CHUL Research Center, Quebec, Canada). Cells in a humidified cell incubator with air added with a temperature of _{37 ℃, 5% CO 2,} L- glutamine (200 mg / L), the heat-treated fetal calf serum in 10% (FBS; Gibco) and 1% antibiotics in (Dibbecco's Modified Eagle Medium) (Gibco, Rockville, Md., USA) containing a penicillin / streptomycin (Gibco).

2. Assessment of cytotoxicity

The cytotoxicity of Ishikawa cells prepared in 1 above was confirmed by the conventional MTT method.

Specifically, Ishikawa cell lines were cultured in 24 wells = plate, and then the peony extracts of the indicated concentrations were treated for 24 hours. After the Griess reaction, MTT solution (2.0 mg / mL) was added to each well and reacted in a cell incubator at 37 ° C for 4 hours. Then, the supernatant of the reaction solution was removed, and formazan was measured at 540 nm using a microplate reader. At this time, the measured cell survival rate was calculated as a percentage (%) of the control group, and the results are shown in FIG.

As shown in FIG. 2, the extract of the peony root extract of the present invention without polysaccharide did not show any significant cytotoxicity up to a concentration of 100 μg / ml.

Therefore, the extract of the peony root extract of polysaccharide of the present invention showed cell stability at a concentration of 100 ㎍ / ml or less.

In the following experiment, a peony extract with a polysaccharide concentration of 50 / / ml was used.

EXPERIMENTAL EXAMPLE 2 Effect of the present invention on promoting LIF expression of peony root extract without polysaccharide [

RT-PCR and Western blotting were performed to examine the effect of the present invention on the promoting effect of LIF expression on the extracts of monocotyledonous peel of the present invention, and LIF gene expression and LIF protein expression were measured.

1. Expression of LIF gene

In order to investigate the effect of the extract of the peony root extract of the present invention on the expression of the LIF gene, the expression level of the LIF gene was measured by RT-PCR analysis.

Specifically, the Ishikawa cells of Experimental Example 1 were treated with the peony extracts obtained by removing the polysaccharides prepared in Example 1 at concentrations of 0, 10, 30 and 50 μg / ml, respectively, and cultured for 24 hours. RNA was extracted using Trizol solution (Invitrogen, Carlsbad, Calif., USA) and the same amount of RNA (2 g) was used for reverse transcription using oligo-dT primer and AccuPower RT-PreMix. The synthesized cDNA was subjected to RT-PCR using LIF-specific primers. The primer sequences used for PCR amplification were as follows; 5 '-GATGTCGGCGGTGGCGTTGA-3' (SEQ ID NO: 2) in the forward direction: 5'-ACGCCACCTGTGCCATACGC-3 '(SEQ ID NO: 1), reverse direction: primer sequence used for internal control of β- -CAAGAGATGGCCACGGCTGCT-3 '(SEQ ID NO: 3), reverse direction: 5'-TCCTTCTGCATCCTGTCGGCA-3' (SEQ ID NO: 4). The amplified DNA band was photographed under UV conditions to confirm the expression level. The intensity of the band was measured using a densitometer and plotted. Experiments were repeated three times independently and the results were expressed as mean ± standard deviation. The results are shown in Fig.

As shown in FIG. 3, it was confirmed that the extract of Phytophthora infestans from which the polysaccharide of the present invention was removed increased the concentration of LIF gene in a concentration-dependent manner at a concentration of 10, 30, and 50 / / ml.

In order to compare the effects of the simple hot water extract (polysaccharide - containing) without peeling of the polysaccharide on the LIF gene expression of the peach extract with polysaccharide, Ishikawa cells were inoculated with the same concentration (50 ㎍ / ml) And polysaccharide-free extracts of P. falciparum were treated with RT-PCR to confirm gene expression. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that the peony extract of the present invention in which the polysaccharide of the present invention was removed under the same concentration condition (50 μg / ml) increased the expression of the LIF gene more than that of the simple hot water extract of P. monocytogenes.

Therefore, the extract of the present invention having the polysaccharide of the present invention is excellent in promoting the expression of the LIF gene, and thus, it can be seen that the effect of inducing fertilization of the embryo is excellent.

2. Expression of LIF protein and gene

Western blot analysis was carried out in order to examine the effect of the extract of Phytophthora infestans of the present invention on the expression of LIF protein.

Specifically, the Ishikawa cells of Experimental Example 1 were treated with the extracts of Peony root, which had been prepared by removing the polysaccharide prepared in Example 1, at concentrations of 0, 10, 30 and 50 / / ml, respectively, and then cultured for 72 hours. Proteins were extracted from the cells and subjected to electrophoresis. The proteins separated by electrophoresis were transferred to a nitrocellulose membrane. LIF-specific antibodies (SantaCruz) and HRP-conjugated secondary antibodies were sequentially bound to the membrane, followed by sensitization with ECL solution Protein bands were measured. The intensity of the measured band was measured and plotted using a density meter. Experiments were repeated three times independently and the results were expressed as mean ± standard deviation. The results are shown in FIG.

As shown in FIG. 5, it was confirmed that the extract of the peony root extract of the present invention without polysaccharide significantly increased the expression of LIF protein in a concentration-dependent manner.

In order to compare the effects of the simple hot water extract (polysaccharide - containing) without peeling of the polysaccharide on the LIF gene expression of the peach extract with polysaccharide, Ishikawa cells were inoculated with the same concentration (50 ㎍ / ml) And polysaccharide-free peony extracts were treated and RT-PCR analysis was used to confirm gene expression. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the peony extract of the present invention in which the polysaccharide of the present invention was removed under the same concentration condition (50 μg / ml) increased the expression of the LIF gene compared to the simple hot water extract of P. monocytogenes.

Therefore, the extract of the present invention having the polysaccharide of the present invention is excellent in promoting the expression of LIF protein, and therefore, it can be seen that the effect of inducing fertilization of the embryo is excellent.

[Experimental Example 3: Improvement effect of the extract of peony root extract of polysaccharide of the present invention on endometrial cell adhesion ability]

In order to examine the effect of the peony root extract of the present invention on the endometrial cell adhesion ability, the following procedure was performed.

Specifically, the extract of the peony root which had been obtained by removing the polysaccharide prepared in Example 1 was administered to a fetal cell line (JAR cell), and the adherence ability to the endometrial cell line (Ishikawa cell) was measured. Ishikawa cells of the human endometrium-derived cell line of Experimental Example 1 were cultured in a monolayer so as to be in an 80% confluence state. Then, the extract of the peony root was treated at a concentration of 50 μg / ml for 72 hours Lt; RTI ID = 0.0 > 100% < / RTI > Then, JAR cells, which were pre-cultured fetal-derived cell lines, were detached and well suspended to give a single cell. Then, 4 ', 6-diamidino-2-phenylindole, (DAPI). The cells were then sprinkled on Ishikawa cells and cultured at 37 ° C for 30 minutes in an incubator with stirring at a speed of about 150 rpm. The cells were then washed twice with phosphate-buffered saline (PBS) to remove unattached cells and fixed with 5% formalin solution to the adherent cells. Fixed cells were measured by fluorescence microscopy. At this time, the cells that emit light by DAPI mean the number of JAR cells attached to the bottom Ishikawa cells. The total number of cells counted in photographs from four or more different areas was compared to the control group without drug treatment, and the mean and standard deviation of the three replicates were plotted. The results are shown in Fig.

As shown in FIG. 7, it was confirmed that the peony extract of the present invention, in which the polysaccharide was removed, showed a 2.5-fold increase in the binding between the placenta-derived cell line and the fetal-derived cell line as compared to the control group.

Therefore, it was confirmed that the peony extract of the present invention, which removed the polysaccharide of the present invention, increased the binding force between the placenta and the fetus, and therefore, the effect of inducing fertilization of embryos is excellent.

[Experimental example 4: Measurement of cytotoxicity of solvent of peony extract of the present invention]

In order to specify the solvent of the peony extract of the present invention, the cytotoxicity of each of the extracted solvents was evaluated to confirm safety.

1. Preparation of simple hot water extract

1 g of distilled water was added to 100 g of pea pod, boiled under reduced pressure for 2 hours, and centrifuged to remove the residue to obtain 600 ml of a simple hydrothermal extract of peony root. Thereafter, the obtained simple hydrothermal extract was concentrated under reduced pressure and lyophilized to obtain 10.12 g of a simple hot water extract (containing polysaccharide) of peony root (yield: 10.12%) (see FIG. 8A).

2. Preparation of peanut methanol extract and peony root ethanol extract

To 100 g of peony was added 1 L of methanol or ethanol, allowed to stand at room temperature for 16 hours, and shaken occasionally. After repeating this process twice, the extract solution was collected and centrifuged to remove the residues, and 1200 ml of each of the crude methanol extract and the crude ethanol extract were obtained. The obtained crude extracts of methanol and crude extracts of ethanol were concentrated under reduced pressure and lyophilized to obtain 2.3 g of peony root extract (yield 2.3%) and 3.04 g of peony root ethanol extract (yield 3.04%) (see Fig. 8B).

3. Assessment of cytotoxicity

The Ishikawa cells prepared in Experimental Example 1 were treated with the simple hot-water extract, peony root extract and peony root extract prepared in the above 1 and 2, and the cytotoxicity was evaluated using the conventional MTT method. At this time, the cytotoxicity evaluation by the MTT method was performed in the same manner as described in Experimental Example 1, and the results are shown in FIG.

As shown in FIG. 9, both the methanol extract of Peony root and the ethanol extract of Peony root showed significant cytotoxicity at a concentration of 10 μg / ml. At a concentration of 100 μg / ml, only about 25% of the cells survived in both extracts .

On the other hand, the extracts of P. monocytogenes showed significant cytotoxicity at a concentration of 10 μg / ml. Especially, at a concentration of 100 μg / ml, only about 70% of the cells survived.

Therefore, the simple hot-water extract of the present invention has lower cytotoxicity than the peanut-methanol extract and the peanut ethanol extract, but it is more cytotoxic than the peanut extract without the polysaccharide.

Hereinafter, the pharmaceutical composition or food composition for promoting pregnancy, the pharmaceutical composition for the prevention or treatment of infertility or the preparation of food composition containing the extract of Peony root without polysaccharide of the present invention will be described. However, This is specifically intended to explain.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

Polysaccharide-free Peony Extract 20 ml

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Polysaccharide-free Peony Extract 10 ml

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

Polysaccharide-free Peony Extract 10 ml

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

Polysaccharide-free Peony Extract 10 ml

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

Polysaccharide-free Peony Extract 20 ml

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

[Formulation example 2. Preparation of food preparation]

1. Manufacture of health food

Polysaccharide-free peony extract 100 ml

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary 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 mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

Polysaccharide-free peony extract 100 ml

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (7)

A pharmaceutical composition for promoting pregnancy, comprising a peony extract obtained by removing a polysaccharide as an active ingredient.
[Claim 2] The pharmaceutical composition for promoting pregnancy according to claim 1, wherein the polysaccharide is removed from the crude extract by adding C ₁ -C ₄ alcohol to the crude extract.
The pharmaceutical composition for promoting pregnancy according to claim 2, wherein the C ₁ -C ₄ alcohol is 1 to 100% (v / v) methanol or 1 to 100% (v / v) .
A food composition for promoting pregnancy, comprising a peony extract obtained by removing a polysaccharide as an active ingredient.
A pharmaceutical composition for preventing or treating ovarian non-implantation infertility comprising an extract of peony root extract of polysaccharide as an active ingredient.
A food composition for preventing or ameliorating the infertility caused by oocyte non-implantation containing a peony extract obtained by removing polysaccharide as an active ingredient.
1) adding peony pellets to water and heating at 80 to 100 캜 to obtain peony hydrothermal extract;
2) separating only the supernatant by centrifuging the Abalone hydrothermal extract;
3) adding C ₁ -C ₄ alcohol to the separated supernatant, reacting at a temperature of 2 to 6 ° C, centrifuging the reaction mixture to remove the precipitated polysaccharide, and separating only the supernatant; And
And 4) powdering the separated supernatant by vacuum concentration and lyophilization to remove the polysaccharide.

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