KR101151129B1 - Composition for treating or preventing diseases comprising placenta extract - Google Patents

Abstract

The present invention relates to a composition for treating or preventing a disease containing the placenta extract as an active ingredient, and more specifically, a placenta extract, which is a natural product obtained by separating sex hormone containing anabolic steroid from the placenta of a mammal such as a pig, By using it as an osteoporosis treatment, anemia treatment, exhaustive muscle degeneration disease treatment, sexual dysfunction treatment, etc., it is possible to replace the steroids that have been used conventionally, and to treat a wide range of human diseases while reducing side effects of steroids.

Description

Composition for treating or preventing diseases comprising placenta extract}

The present invention is for the treatment or prevention of diseases containing a placenta extract which is a natural product obtained by mass extraction of steroids known to have muscle strengthening effect, growth promoting effect and disease treatment effect from placenta of mammals such as pigs that are disposed of at present. It relates to a composition.

Anabolic steroid is a testosterone or a testosterone derivative synthesized by altering its chemical structure. When used excessively, anabolic steroids reduce the reproductive function of men and cause cardiovascular disease. Banned athletes from taking these anabolic steroids.

In addition, male hormones may be used in males with sexual dysfunction, but male hormones may be converted to dihydrotestosterone by 5α-reductase to cause prostate cancer.

The placenta, on the other hand, is a source of nutrition for the fetus that absorbs nutrient and oxygen-rich blood from the mother's uterine wall and supplies it to the fetus through the umbilical cord. It is rich in nutrients such as amino acids and pharmacologically active peptides, vitamins, nucleic acids and enzymes that are needed for the human body. In addition, the placenta includes various growth factors such as hepatocyte growth factor, neuronal growth factor, epithelial cell growth factor, fibroblast growth factor, insulin growth factor, and growth factor to improve immunity.

To date, proteins, lipids, nucleic acids, glycosaminoglycans, amino acids, vitamins and minerals have been isolated and identified in the placenta and are expected to contain a variety of unknown components.

In the present inventors, a large amount of nandrolone (19-nortestosterone), which is present in the placenta of mammals such as pigs, is unlikely to be converted to dihydrotestosterone by 5α-reductase, unlike general male hormones. In view of the fact that it is possible to reduce the induction of human prostate cancer, we tried to extract the sex hormone including nandrolone most efficiently from the placenta of mammals, such as pigs that are disposed of, and use it as a treatment for human diseases.

Accordingly, an object of the present invention is a natural placenta extract comprising anandrol steroid, a type of anabolic steroid, which is present in a large amount in a placenta of a mammal such as a pig, and a male placenta and a female hormone. It is to provide a composition for treating or preventing a disease that can be utilized to prevent degradation.

In order to achieve the above object, the present invention provides a composition for treating or preventing diseases containing placenta extract as an active ingredient.

The placenta extract may be obtained by extracting the placenta with an extraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, ether, hexane, dichloromethane, and a mixed solvent thereof. As the extraction solvent, a mixed solvent containing an alcohol having 1 to 4 carbon atoms and chloroform may be used.

For 1 g of the placenta, the extraction solvent may be extracted using 10 to 20 mL. If a small amount of the extraction solvent is used outside the above range, the extraction efficiency is low, and the content of nandrolone, which is a kind of anabolic steroid in the placenta extract, may be insufficient, and it may be uneconomical if a large amount of the extraction solvent is used.

In addition, the placenta extract may be separated by the addition of physiological saline solution after extraction of the extraction solvent. After the extraction of the extraction solvent, a basic substance such as sodium hydroxide may be further added, followed by neutralization with an acidic substance such as hydrochloric acid, followed by separation. In addition, the placental extract may be separated by further adding a physiological saline solution after extraction of the extraction solvent, adding a basic substance such as sodium hydroxide, and neutralizing with an acidic substance such as hydrochloric acid.

In addition, the placenta extract is a step of adding an extraction solvent to the placental tissue; Homogenizing placental tissue; Adding a physiological saline solution to the filtrate obtained by filtering the homogeneous solution; Dispensing the separatory funnel to recover the lower layer; Adding an aqueous ethanol solution and shaking; Adding and basing a basic solution; Neutralizing by adding an acidic solution; Dispensing into a separatory funnel; Adding ether; Recovering the ether layer; And concentration under reduced pressure.

Since the composition according to the present invention contains a placental extract containing a large amount of nandrolone, which is a kind of anabolic steroid, as an active ingredient, it is caused by steroid hormone abnormalities selected from the group consisting of sexual dysfunction, osteoporosis, consumer muscle disease, aging and anemia It can be usefully used for the treatment and prevention of disease.

The present invention also provides a contraceptive composition containing the placenta extract as an active ingredient.

In particular, the placenta used in the present invention broadly includes a placenta of a mammal such as a pig, a cow and a horse, and preferably, a placenta of a pig can be used.

The composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients or 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.

In addition, the compositions of the present invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or Prepare by mixing lactose, gelatin and the like.

In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, 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 amount of placenta extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times in an amount of 0.1 to 100 mg / kg.

In addition, the dosage of the placenta extract may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

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

In particular, when the placenta extract according to the present invention is administered to the human body, there is no fear of side effects compared to other synthetic medicines because it is a natural extract, and its stability is secured.

Placenta extract according to the present invention is a natural product containing high concentrations of nandrolone and sex hormones, reducing side effects when using conventional chemically synthesized anabolic steroids and sex hormones, in particular male contraceptives, osteoporosis treatment, anemia treatment It can be very useful for treating exhaustive muscle degenerative diseases and treating male hypogonadism.

1 is a flowchart of a placenta extract manufacturing process according to an embodiment of the present invention,
2 to 3 and 5 to 7 are graphs showing standard curves of estrone, estradiol, nandrolone, testosterone and androstenedione analyzed from placental extracts according to one embodiment of the present invention, respectively.
Figure 4 shows the plate used to analyze the nandrolone content of the placental extract according to an embodiment of the present invention,
Figure 8 illustrates the cell proliferation effect using placenta extract according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are merely to illustrate the invention, the content of the present invention is not limited by these examples.

Example 1 Preparation of Porcine Placenta Extract

For the most efficient separation and purification of steroid hormones in swine placenta, both amniotic fluid and debris were removed and pure placenta were isolated. The isolated placenta was pre-cut into 10-30 g size and stored in a -20 ° C freezer for grinding by a tissue homogenizer (Ultra-Turrax T25, IKA Co. USA).

Chloroform (HPLC grade, SK Chemical Co. Seoul, Korea) / Methanol (HPLC grade, Merck Co. Darmstadt, Germany) (50/50, v / v) mixed solution was used as a solvent to extract the hormone of the placenta. . Place the placenta fragment between 25-50g in a 1000mL beaker (Hanil Chemical, Seoul, Korea), add 8 times the chloroform / methanol solution, and homogenize for 3 minutes. After homogenization, whatman No. 2 The filter paper was removed using a filter paper to remove any residue.

The prepared filtrate was added to the same amount of 0.9% physiological saline solution using a separatory funnel (Hanil Chemical, Seoul, Korea) and gently shaken for about 10 minutes to recover the bottom layer. The recovered material was volatilized with an organic solvent using a rotary pressure reducer, and ethanol / distilled water mixed solution (85/15, v / v) was added to the same amount of the remaining solution, and the contents were mixed well.

An equal amount of 5M sodium hydroxide solution was added thereto, and the mixture was cooled to room temperature after boiling at 80 ° C for 45 minutes. The pH was adjusted to 2-3 with 6N sulfuric acid solution. The contents were divided into a separatory funnel, ether was added by half of the contents, shaken well, mixed well, and allowed to stand still for 10 minutes to separate layers. When the layers were separated cleanly, the lower layer was put in a new separatory funnel and separated once more to extract as much as possible. After that, the lower layer was discarded, and all the ethers of the upper layer were collected, washed with distilled water, purified, and only the ether layer was placed in a rotary vacuum concentrator (1200 type, Eyela Co. Tokyo, Japan), completely dried, and recovered.

Example 2 Steroid Hormone Content Analysis

2-1. Estrone content analysis

Analysis of the content of estrone was performed using the estrone ELISA (DRG. EIA-4174) as follows.

That is, dispense 50 μl of each control (at room temperature before use, storage at 4 ° C.), sample (diluted with tertiary distilled water), standard (0, 15, 50, 200, 800, 2000 pg / ml) to each well. Then, 100 μl of enzyme conjugate (at room temperature before use, storage at 4 ° C.) was added thereto, and the mixture was left at room temperature for 1 hour. After leaving the plate, the microtitre plate was washed with washing buffer (40-fold concentration, diluted with distilled water) four times, and then allowed to stand for 30 minutes by adding 150 μl of substrate solution (at room temperature before use and storage at 4 ° C.). After 30 minutes 50 μl of the stop solution (at room temperature before use, storage at 4 ° C.) was added and measured at 450 nm, the results of which are shown in Table 1 and Table 2 and FIG. 2.

pg / ml  Standard O.D 0.00 1.97 15.00 1.72 50.00 1.52 200.00 1.17 800.00 0.77 2000.00 0.50

Sample Dilution factor (X) Extract concentration
(ng / g) Average ng / placenta g Placenta 1 1000 47745 51218 512
2000 54690 Placenta 2
2000 110124 122165 1099
6000 134207 Placenta 3
1000 50286 52322 837
3000 54359 Placenta 4
1000 29556 34174 581
2000 38791 Placenta 5 3000 246877 247239 2967
6000 247602

2-2 Estradiol Content Analysis

The content analysis of estradiol was carried out using estradiol ELISA (DRG. EIA-2693) as follows.

That is, 25 μl each of the control (at room temperature before use, storage at 4 ° C.), samples (diluted with tertiary distilled water), standard (0, 25, 100, 250, 500, 1000, 2000 pg / ml) in each well The aliquots were added, and 200 µl each of enzyme conjugate (at room temperature before use, storage at 4 ° C.) was added thereto, and then left at room temperature for 1 hour. After leaving the plate, the microtitre plate was washed three times with washing buffer (40-fold concentration, diluted with distilled water), and then left for 15 minutes by adding 100 μl of substrate solution (at room temperature before use and storage at 4 ° C.). After 15 minutes 50 μl of stop solution (at room temperature before use, storage at 4 ° C.) was added and measured at 450 nm, the results are shown in Table 3 and Table 4 and FIG. 3.

pg / ml  Standard O.D 0.00 2.02 25.00 1.75 100.00 1.02 250.00 0.47 500.00 0.29 1000.00 0.19 2000.00 0.11

Sample Dilution factor (X) Extract concentration
(ng / g) Average ng / placenta g Placenta 1 2000 6085 6319 63 6000 6553 Placenta 2
2000 9515 9494 85 6000 9473 Placenta 3
2000 7659 8197 131 6000 8735 Placenta 4
2000 5396 5743 98 6000 6090 Placenta 5 2000 18385 17992 216 6000 17600

2-3 Analysis of 19-nortestosterone Content

Nandrolone content analysis was performed using 19-nortestosterone-EIA (Euro-Diagnostica B. V. 5082NOR1p) as follows.

That is, after adding 100 μl of zero standard to A1 well in the microtitre plate of FIG. 4, 50 μl of zero standard is added to B1 well, and standard 1-6 to B, C, D, E, F, and GH 1 well, respectively. Added. 50 μl of the sample to be analyzed (diluted with tertiary distilled water) was added to the remaining wells, and the enzyme conjugate solution (in dark, at room temperature before use, and stored at 4 ° C. To use for a long time, 25 µl of -20 ° C) and 25 µl of antibody solution (stored at 4 ° C and dissolved in 4 ml of power dilution buffer) were added. After the above process, the plate was blocked with light paper and placed at 4 ° C. for 1 hour, and then washed three times with a washing buffer. After the plate was washed, 100 μl of substrate solution (in dark, at room temperature before use, storage at 4 ° C.) was added thereto, and left for 30 minutes. Then, 100 μl of stop solution was added, and the optical density was measured at 450 nm. The results are shown in Table 5 and Table 6 and FIG.

ng / ml Standard O.D zero 0.075 zero ' 0.67 0.0625 0.63 0.125 0.54 0.25 0.45 0.5 0.35 One 0.26 2 0.17

Sample Dilution factor (X) Extract concentration
(ng / g) Average ng / placenta g Placenta 1 1000 43097 41384 414 3000 39672 Placenta 2
1000 35218 34879 314 4000 34540 Placenta 3
1000 30783 29774 476 3000 28764 Placenta 4
1000 34695 35893 610 3000 37090 Placenta 5 1000 38813 39392 473 3000 39970

2-4 Testosterone Content Analysis

Testosterone content analysis was performed using Testosterone ELISA (DRG. EIA-1559) as follows.

That is, 50 μl of control (at room temperature before use, storage at 4 ° C.), sample (diluted with tertiary distilled water), standard (0, 0.2, 0.5, 1, 2, 6, 16 ng / ml) was added to each well. Aliquots were added and 100 μl of enzyme conjugate (at room temperature before use, storage at 4 ° C.) was added and allowed to stand at room temperature for 1 hour. After leaving the plate, the microtitre plate was washed three times with a wash buffer solution (40-fold concentration, diluted with distilled water), and then left for 30 minutes by adding 150 μl of substrate solution (at room temperature before use and storage at 4 ° C.). After 30 minutes, 100 μl of the stop solution (at room temperature before use, storage at 4 ° C.) was added and measured at 450 nm. The results are shown in Table 7 and Table 8 and FIG.

ng / ml  Standard O.D 0 2.33 0.2 2.08 0.5 1.83 One 1.52 2 1.14 6 0.62 16 0.30

Sample Dilution factor (X) Extract concentration
(ng / g) Average ng / placenta g Placenta 1 10 648 655 7 30 663 Placenta 2
10 1147 1202 11 30 1256 Placenta 3
10 461 470 8 30 480 Placenta 4
10 2668 2872 49 30 3076 Placenta 5 10 1546 1548 18 30 1550

2-5 Androstenedione Content Analysis

Androstenedione content analysis was performed using an androstenedione ELISA (DRG. EIA-3265) as follows.

That is, 20 μl of the control (at room temperature before use, storage at 4 ° C.), sample (diluted with tertiary distilled water), standard (0, 0.2, 0.5, 1, 2, 6, 16 ng / ml) was added to each well. Aliquots were added and 200 μl of enzyme conjugate (at room temperature before use, storage at 4 ° C.) was added and then allowed to stand at room temperature for 1 hour. After leaving the plate, the microtitre plate was washed three times with a washing buffer (40-fold concentration, diluted with distilled water), and then left for 15 minutes by adding 200 µl of a substrate solution (at room temperature before use and storage at 4 ° C.). After 15 minutes 100 μl of stop solution (at room temperature before use, storage at 4 ° C.) was added and measured at 450 nm. The results are shown in Table 9 and Table 10 and FIG.

ng / ml  Standard O.D. 0.00 1.87 0.10 1.09 0.30 0.87 1.00 0.59 3.00 0.21 10.00 0.09

Sample Dilution factor (X) Extract concentration
(ng / g) Average ng / placenta g Placenta 1 1000 3858 3625 36 3000 3392 Placenta 2
1000 4046 3726 34 3000 3406 Placenta 3
1000 2848 2881 46 3000 2914 Placenta 4
1000 2425 2391 41 3000 2356 Placenta 5 1000 2436 2443 29 3000 2450

The results of separating and purifying steroid hormones extracted from 1 g of pig placenta using different methods according to the examples are shown in Table 11 below.

Placenta 1 Placenta 2 Placenta 3 Placenta 4 Placenta 5 Nandrolone (ng / g) 414 314 476 610 473 Testosterone (ng / g) 7 11 8 49 19 Androstenedione (ng / g) 36 34 46 41 29 Estradiol (ng / g) 63 85 131 98 216 Estrone (ng / g) 512 1099 837 581 2967

Experimental Example 1 Cell Proliferation Using Placenta Extract

In order to examine the effect of placental extract prepared in Example on the proliferation of muscle stem cells (cell line, early cultured cells), the placenta 1 extract prepared in Example in DMEM was 0, 0.4, 4, 40 on the basis of nandrolone. , 400 pg / ml was added and cultured in a 5% CO ₂ , 37 ℃ incubator. After 3 days of culture, the absorbance was measured at 540 nm with an ELISA reader using MTT assay in each well.

As a result, when the culture was added with the extract as shown in Figure 8 it was shown to promote the growth of muscle cells in a concentration-dependent compared to the control group without the extract. Therefore, it was confirmed that the placenta extract according to the present invention is safe for cell culture and effective for the proliferation of muscle cells.

Claims (9)

Containing placenta extract as an active ingredient, the placenta extract is the step of adding an extraction solvent to the placental tissue; Homogenizing placental tissue; Adding a physiological saline solution to the filtrate obtained by filtering the homogeneous solution; Dispensing the separatory funnel to recover the lower layer; Adding an aqueous ethanol solution and shaking; Adding and basing a basic solution; Neutralizing by adding an acidic solution; Dispensing into a separatory funnel; Adding ether; Recovering the ether layer; And a composition for treating or preventing male reproductive function or consumable muscle disease, which is obtained by a step of concentrating under reduced pressure. The method of claim 1, wherein the placenta extract is a composition for treating or preventing male reproductive function or consumer muscle disease, characterized in that it contains nandrolone as an active ingredient. delete delete delete delete delete delete delete

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