KR20230156479A - Cynomorium songaricum extract having an inhibitory effect on dihydrotestosterone production and method for preparing thereof - Google Patents

Abstract

The present invention includes the steps of 1) washing and pulverizing the crushed sheep, 2) immersing the pulverized sheep in an alcohol diluted solution and leaving it at room temperature, 3) adding the alcohol:water to the solution left at room temperature in a ratio of 4:6 to 6. :4, then heating it for 1 to 60 minutes to obtain the primary supernatant; 4) boiling the primary supernatant at 60 to 80°C for 2 to 3 hours; repeating the process to obtain 2 It provides a method for producing a tea extract comprising the step of obtaining a tea supernatant, and 5) freeze-drying the secondary supernatant at a low temperature of -100 to 0 ° C. The tea extract prepared by this method has excellent extraction properties. It is efficient and has the characteristic of containing an active ingredient that inhibits the production of dihydrotestosterone (DHT).

Description

Cynomorium songaricum extract having an inhibitory effect on dihydrotestosterone production and method for preparing the same}

The present invention relates to a plant extract that has an inhibitory effect on the production of dihydrotestosterone and a method for producing the same. Specifically, it relates to a plant extract that can effectively inhibit the production of dihydrotestosterone (DHT), a known cause of male hair loss, and a method for producing the same. It's about how to manufacture it.

Cynomorium songaricum (鎖陽) is a perennial parasitic herbaceous plant of the Cynorrhea family. It is a plant that lives in cold and temperate regions, subtropical regions, the Mediterranean, and deserts, and is known as a medicinal herb that strongly replenishes yang energy. It protects the kidneys, prevents erectile dysfunction and infertility, and forms cellular tissue in the human body. If you eat it when your back and knees are sore, you lack stamina, or you are constipated, it helps relieve symptoms, and the stems of Swayang are a plant used as a medicinal herb to treat tonic and constipation.

Meanwhile, hair loss was considered the exclusive domain of some middle-aged men, but recently it has become a ‘national disease’ for people of all ages. According to the results of the Health Insurance Review and Assessment Service's analysis of alopecia medical records for five years (2009-2013), patients in their 20s and 30s accounted for 43.9%, or nearly half of all hair loss patients.

Oral hair loss treatments require hospital treatment and a prescription, and there are concerns about side effects, so there is a need to develop topical or shampoo-type products that show equal effectiveness.

In particular, male-pattern hair loss due to genetic predisposition occurs when testosterone is converted into dihydrotestosterone (DHT) by 5α-reductase (type II 5α-reductase), and this DHT acts on hair follicles to miniaturize hair follicles and enter the growth phase of the hair cycle. It is reported to be a cause of hair loss by shortening the hair cycle and lengthening the resting period.

Meanwhile, the extract extracted from the above-mentioned dried sheep is known to have the effect of suppressing the production of DHT, but its application to various products is still limited due to its low extraction efficiency.

For example, in Korea Patent Publication No. 2016-0043622, the step of pulverizing dried sheep; And a method for producing a composition containing a sheep extract comprising the step of obtaining a sheep extract from pulverized sheep, the step of obtaining the pulverized sheep extract includes placing the pulverized sheep in purified water and stirring at a temperature of 6 to 72 degrees Celsius at 0 to 10°C. A method for producing a composition containing a sorghum extract comprising the step of extracting for a period of time or adding yeast and sugar to pulverized sorghum extract in purified water and fermenting and extracting the pulverized sorghum extract for 6 to 24 hours at 20 to 40°C, and 50 to 500 ㎍/ A cosmetic composition for skin moisturizing containing active ingredients at a concentration of ml was presented. However, the above patent only describes the effect of suppressing skin moisturization due to the difference between simple extraction and fermentation + extraction process, and does not suggest the DHT suppression effect of the manufactured sorghum extract.

In addition, Korean Patent No. 10-1964837 proposes a cosmetic composition for improving skin wrinkles containing the ethyl acetate fraction of the ethanol extract of Cynomorium songaricum as an active ingredient. Extract extracted using a mixed solvent; Dilution or concentrate of the extract; Dried product obtained by drying the extract; adjustment offering; or purified products.

The above patent only suggests the effect of inhibiting the activity of elastase by preparing the ethanol extract of sorghum extract as a solvent fraction, but does not suggest the DHT inhibitory effect of the ethanol extract of sorghum extract.

Korean Published Patent 2016-0043622 Korean registered patent 10-1964837

Accordingly, the purpose of the present invention is to provide a method for extracting sheep with high efficiency, which has an inhibitory effect on the production of dihydrotestosterone, which has been found to be a genetic cause of hair loss.

In addition, the purpose of the present invention is to provide a sorghum extract that has high extraction efficiency and an inhibitory effect on dihydrotestosterone production by producing it according to the production method of the present invention.

The method for producing a snail extract according to the present invention includes the following steps: 1) washing and pulverizing the snail, 2) immersing the pulverized snail in a diluted alcohol solution and leaving it to stand at room temperature, 3) adding the alcohol to the solution left at room temperature : mixing water in a volume ratio of 4:6 to 6:4 and then heating it for 1 to 60 minutes to obtain a primary supernatant, 4) heating the primary supernatant at 60 to 80°C for 2 to 60 minutes Obtaining a secondary supernatant by repeating the boiling process for 3 hours, and 5) freeze-drying the secondary supernatant at a low temperature of -100 to 0 ° C to prepare a powdered sorghum extract. .

According to one embodiment of the present invention, the prepared Soyang extract may have an extraction efficiency of 30% or more.

In addition, the Chayang extract prepared by the above production method is characterized by containing an active ingredient that inhibits the production of dihydrotestosterone (DHT).

In addition, according to one embodiment of the present invention, the extract of sorghum extract has an inhibition rate of dihydrotestosterone (DHT) production of 25% or more at a concentration of 2.5 mg/ml.

In the present invention, various conditions were optimized during the manufacturing process to obtain the prickly pear extract with high efficiency. It is pre-immersed in an alcohol diluted solution so that the active ingredient of the dried extract can be dissolved in advance. The mixing ratio of alcohol: water is optimized to increase extraction efficiency, and unlike the conventional method, the extraction process is performed at the optimal temperature and time, resulting in an extraction yield of 30%. The above-mentioned Soyang extract was obtained.

In addition, the extract obtained in this way has the effect of containing an active ingredient that inhibits the production of dihydrotestosterone (DHT), which is known to cause hair loss.

Figures 1 to 3 show HPLC graphs for Testosterone, finasteride, and mixed samples of Testosterone and finasteride, respectively.

The present invention will be described in more detail below.

The terms used herein are used to describe specific embodiments and are not intended to limit the invention.

As used herein, the singular forms include the plural forms unless the context clearly indicates otherwise. Additionally, when used herein, “comprise” and/or “comprising” means specifying the presence of stated features, numbers, steps, operations, members, elements and/or groups thereof. and does not exclude the presence or addition of one or more other shapes, numbers, operations, members, elements and/or groups.

The present invention relates to a method for producing a plant extract that can be produced with relatively high extraction efficiency and optimizes various manufacturing conditions so that the prepared plant extract can effectively suppress the production of dihydrotestosterone, known as a hair loss gene.

The method for producing a snail extract according to the present invention includes the following steps: 1) washing and pulverizing the snail, 2) immersing the pulverized snail in a diluted alcohol solution and leaving it to stand at room temperature, 3) adding the alcohol to the solution left at room temperature : mixing water in a volume ratio of 4:6 to 6:4 and then heating it for 1 to 60 minutes to obtain a primary supernatant, 4) heating the primary supernatant at 60 to 80°C for 2 to 60 minutes Obtaining a secondary supernatant by repeating the boiling process for 3 hours, and 5) freeze-drying the secondary supernatant at a low temperature of -100 to 0 ° C to prepare a powdered sorghum extract. .

First, the ingredients for extracting hayang are prepared by thoroughly washing commercially available products and grinding them finely.

Next, the pulverized powdered material is immersed in a diluted solution of about 75% alcohol and left at room temperature. In the present invention, the drying material is first dissolved in alcohol and then immersed in the alcohol dilution solution to increase the extraction efficiency of the active ingredient. In the case of dried sheep, it does not dissolve well in water and is very sensitive to the extraction solvent used. Therefore, in the present invention, it is important to ensure that the active ingredient is dissolved as much as possible in the following extraction process through immersion in an alcohol solution as much as possible.

The immersion time in the alcohol dilution is preferably 48H to 96H in order to maximize the yield of the extract, and leaving it to stand at room temperature (r.t.) around 18 to 25°C is preferable to prevent damage to the extract. In addition, when the immersion solution uses a solvent other than an alcohol diluent containing water, it is preferable to use a diluent of an alcohol solvent because the active ingredient does not dissolve well. The alcohol used is methanol with a purity of 99%, There are ethanol, isopropyl alcohol, etc., but among these, ethanol is the most preferable.

The third step is a process of mixing alcohol:water in a volume ratio of 4:6 to 6:4 with the solution left at room temperature, then heating and extracting the mixture for 1 to 60 minutes to obtain the first supernatant. In this process, water is added to ensure that the alcohol solvent does not evaporate quickly during the extraction process and that extraction occurs efficiently under boiling water conditions. At this time, because the extraction efficiency varies greatly depending on the volume ratio of alcohol: water, it is important to optimize the volume ratio. If the volume ratio of alcohol: water is outside of 4:6 to 6:4 and the alcohol content is too low or too high, the extraction efficiency This is undesirable because it is impossible to obtain an extract containing the desired active ingredient.

The extracted solution is prepared by centrifuging to remove residues and taking only the supernatant (first supernatant).

The next step is to repeat the process of boiling the first supernatant at 60 to 80°C for 2 to 3 hours to obtain a second supernatant excluding precipitates. In the present invention, in order to solve the problem of reduced extraction efficiency due to the use of only hot water or alcohol extraction when extracting natural products in the past, the extraction efficiency of the finally produced Saeyang extract is increased by repeating the boiling process for a certain period of time at a relatively low temperature. I was able to confirm.

At this time, since the temperature or time of decoction has a very slight effect on extraction efficiency, it is very important to maintain the condition of performing a single decoction process at 60 to 80°C for 2 to 3 hours. If the temperature of the decoction process is less than 60℃ or exceeds 80℃, there is a problem of denaturation of the extract. If the time is less than 2 hours, the extraction effect decreases, and if it exceeds 3 hours, by-products increase or the efficacy of the extract decreases. It is not desirable.

It is preferable to repeat the decoction process two or more times to increase extraction efficiency, and each time the decoction process is performed, it is desirable to perform an additional decoction process by removing only the supernatant, excluding the residue.

Lastly, the supernatant that has gone through the decoction process is freeze-dried at a low temperature of -100 to 0°C to produce a powdered Yangyang extract. This can prevent the extract from being damaged due to drying at high temperature, and can be preferably used to obtain it in powder form.

When this process is used, the Saeyang extract of the present invention has the characteristic of having a high extraction efficiency of 30% or more.

In addition, it was confirmed that the extract prepared according to the present invention contains an active ingredient that can effectively inhibit the production of dihydrotestosterone (DHT), which is known to cause hair loss in men.

Hereinafter, preferred embodiments of the present invention will be described in detail. The following examples are only for illustrating the present invention, and should not be construed as limiting the scope of the present invention by these examples. In addition, although the examples below are exemplified using specific compounds, it is obvious to those skilled in the art that equivalent effects can be achieved even when equivalents thereof are used.

Example 1: Preparation of Soyang Extract

100 g of well-washed and pulverized dried sheep was prepared, immersed in 500 ml of 75% (v/v) ethanol diluted solution, and left at room temperature (25°C) for 48 hours. Here, the same volume of the ethanol diluted solution and the same amount of hot water were mixed, extracted for 10 minutes, and then centrifuged to obtain the first supernatant. The obtained primary supernatant was boiled at a temperature of 60°C for 2 hours, and the process of boiling it again at a low heat of 70°C for 2 hours was repeated twice to obtain a washed supernatant. The supernatant of the dried sheep was freeze-dried at a low temperature of -80°C to prevent damage to the extract, thereby obtaining a powdered sheep extract.

The extraction efficiency of the obtained extract was calculated by the following equation, and the extraction efficiency was 31.2%.

Extraction efficiency (%) = Weight of powder after freeze-drying (g) / Weight of crushed sheep before extraction (g) x 100

Examples 2-3, and Comparative Examples 1-3

In Example 1, a decoction extract was obtained while changing the conditions of the decoction process as shown in Table 1 below. Examples 2 and 3 were carried out by changing the alcohol immersion time and the mixing ratio of ethanol: water, changing the temperature and time conditions in the decoction process, or changing the freeze-drying temperature.

In addition, Comparative Example 1 did not undergo the process of immersion in the alcohol dilution of the present invention, Comparative Example 2 is an example in which the alcohol:water mixing ratio is outside the scope of the present invention, and Comparative Example 3 is an example of the conditions of the decoction process in Example 2. This is an example outside the scope of the present invention, and Comparative Example 4 was manufactured using the conventional high-temperature drying method without freeze-drying in Example 3.

Alcohol
immersion
(day) Ethanol:water
mixing ratio decoction process dry Extraction Efficiency
(%) extract state Primary temperature
(℃) Primary hour
(hr) Secondary Secondary Example 2 2 4:6 70 2 Freeze drying
(-85℃) 35.9 Good 70 2 Example 3 3 6:4 80 3 Freeze drying
(-90℃) 33.4 Good 60 2 Comparative Example 1 - 5:5 60 2 Freeze drying
(-80℃) 17.6 Good 70 2 Comparative Example 2 2 3:7 70 2 Freeze drying
(-85℃) 20.8 Good 70 2 Comparative Example 3 2 4:6 50 2 Freeze drying
(-90℃) 25.3 denaturalization 85 2 Comparative Example 4 3 6:4 80 3 High temperature drying
(90℃) 21.6 Increased by-products, denaturation 60 4

Referring to the results in Table 1, it can be seen that the extraction efficiencies of all of the Saeyang extracts prepared according to the conditions of Examples 1 to 3 of the present invention can be obtained with an extraction efficiency higher than 30%, and the extracts are also in good condition.

However, in the case of Comparative Example 1, which did not undergo the alcohol immersion process, it can be seen that the extraction efficiency is very low even if other conditions are the same as in Example 1. This is because the process of sufficiently dissolving the active ingredient from the bleaching material through the alcohol immersion process It can be seen as a result of not going through it.

In addition, even when the mixing ratio of ethanol:water is outside the scope of the present invention, it can be seen that the extraction efficiency is very low compared to Example 2 where all other conditions are the same.

In addition, when the temperature in the decoction process is less than 60°C or exceeds 80°C, the efficiency is somewhat higher than that of other comparative examples, but is still less than 30%, and there is a problem of denaturation of the extract, making it difficult to use for the purpose of the present invention. there is a problem.

Lastly, it can be seen that high-temperature drying as in the conventional method is undesirable because not only does by-products increase in the extract, but the extract is denatured.

Experimental Example 1: Analysis of dehydrotestosterone (DHT) inhibition effect of extract

In this experiment, HPLC was used to confirm whether each of the above extracts had a DHT inhibitory effect. However, since there is still no method to directly detect DHT, finasteride, whose performance is well defined, was identified as a provider of type II 5α-reductase, which is known to be involved in converting testosterone to dehydrotestosterone (DHT).

That is, Figures 1 to 3 show HPLC graphs for Testosterone, finasteride, and mixed samples of Testosterone and finasteride, respectively.

HPLC measurement conditions for each sample were performed under the same conditions as follows.

go. HPLC conditions

- Column: C18 column (250mm x 4.6 mm, 5 μm)

- Column temp: RT

- Mobile phase A: Me-OH B: water

- Flow rate: 1.0 ml/min

- Detection: UV at 242 nm

By comparing the HPLC graph of FIG. 1 measuring Testosterone alone and the HPLC graph of the mixed sample of Testosterone and finasteride and FIG. 3, it can be seen that the peak due to Testosterone was significantly reduced as finasteride was mixed. In other words, it can be confirmed that finasteride was effectively used to convert Testosterone into dehydrotestosterone (DHT). From these results, the effect of type II 5α-reductase can also be traced back.

Below, the dehydrotestosterone (DHT) inhibitory effect of each extract prepared above was measured using this measurement principle.

1) Measurement of conversion rate of Testosterone

First, from the HPLC graph measured using Testosterone, the area values of the peak were calculated when Testosterone was 100% (0 min) and when Testosterone was converted to dehydrotestosterone (DHT) to some extent (30 min) after 30 minutes, and the area was calculated. The conversion rate was calculated according to the following equation and the results are shown in Table 2 below.

Conversion rate (%) = {(Peak area of Testosterone at 0 min - Peak area of Testosterone at 30 min)/Peak area of Testosterone at 0 min} x 100

Peak Area of Testosterone Over Time 0 min 819.507 30min 318.527 Conversion rate (%) 61.30

2) Measurement of conversion and inhibition rates of finasteride

Next, from the HPLC graph measured using finasteride, the area value of the finasteride peak at 100% Testosterone (0 min) and at 30 minutes was calculated, and the conversion rate was calculated from the area according to the following equation, and the results are shown in the following table. It is shown in 3.

Conversion rate (%) = (Peak area of Testosterone at 0 min - Peak area of finasteride at 30 min)/Peak area of Testosterone at 0 min x 100

In addition, based on the conversion rate of Testosterone of 61.3% in Table 2, the inhibition rate (%) for each concentration was calculated using the conversion rate of finasteride according to each concentration above, and the results are shown in Table 3 below.

Inhibition rate (%) = {(Conversion rate of Testosterone in 30min - Conversion rate of finasteride in 30min)/Conversion rate of Testosterone in 30min} x 100

concentration of finasteride 0.02μM 0.1μM 0.5μM 1.0μM finasteride average
Peak area (30min) 233.559 316.821 384.676 418.03 Conversion rate (%) 71.50% 61.34% 53.06% 48.99% Inhibition rate (%) -16.96% -0.34% 13.20% 19.86%

From the results in Table 3 above, it can be seen that the inhibition rate of finasteride, whose performance is well defined as a provider of type II 5α-reductase, gradually increases as the concentration increases. From these results, it can be confirmed that finasteride is effective in inhibiting type II 5α-reductase, which is known to be involved in converting testosterone to dehydrotestosterone (DHT).

3) Measurement of conversion and inhibition rate of Soyang extract

The conversion rate and inhibition rate were measured according to the method of 2) above, using the area of each peak in each HPLC graph measured in each of the dried sheep extracts prepared according to the above Examples and Comparative Examples, and the results are shown in Table 4 below. indicated.

In the case of the sorghum extract in Table 4 below, the exact molecular weight and structure cannot be confirmed, and accordingly, it cannot be written in molar concentration like finasteride, so it has a different concentration range of mg/ml, but as in the results in Table 3 above, the concentration of finasteride It can be seen that the inhibition rate improves as the concentration increases from 0.02 μM to 1.0 μM, and in this experiment of the present invention, the inhibition rate tends to improve as the concentration of the Soybean extract increases from 0.01 mg/ml to 2.5 mg/ml. From this, it was judged to be effective.

Concentration of each sage extract 0.01mg/ml 0.1mg/ml 1.0mg/ml 2.5mg/ml Example 1 Average peak area of thyme extract (30min) 383.559 416.011 434.386 458.013 Conversion rate (%) 53.20% 49.24% 46.99% 44.11% Inhibition rate (%) 12.98% 19.46% 23.13% 27.84% Example 2 Average peak area of thyme extract (30min) 375.072 391.282 434.426 452.567 Conversion rate (%) 54.23% 52.25% 46.99% 44.78% Inhibition rate (%) 11.29% 14.52% 23.13% 26.76% Example 3 Average peak area of thyme extract (30min) 404.583 418.678 455.699 501.378 Conversion rate (%) 50.63% 48.91% 44.39% 38.82% Inhibition rate (%) 17.18% 19.99% 27.38% 36.50% Comparative Example 1 Average peak area of thyme extract (30min) 320.822 356.456 366.824 384.121 Conversion rate (%) 60.85% 56.50% 55.24% 53.13% Inhibition rate (%) 0.46% 7.57% 9.64% 13.09% Comparative Example 2 Average peak area of thyme extract (30min) 314.522 356.987 367.825 386.475 Conversion rate (%) 61.62% 56.44% 55.12% 52.84% Inhibition rate (%) -0.80% 7.68% 9.84% 13.56% Comparative Example 3 Average peak area of thyme extract (30min) 314.668 321.866 356.796 358.246 Conversion rate (%) 61.60% 60.72% 56.46% 56.29% Inhibition rate (%) -0.77% 0.67% 7.64% 7.93% Comparative Example 4 Average peak area of sorghum extract (30min) 345.556 355.625 356.897 356.472 Conversion rate (%) 57.83% 56.61% 56.45% 56.50% Inhibition rate (%) 5.40% 7.41% 7.66% 7.57%

Referring to the results in Table 4 above, it can be seen that the inhibition rate increases in a similar trend to that of finasteride as the concentration increases in the dried sheep extract prepared according to Examples 1 to 3 of the present invention. As a result, it can be confirmed that the sorghum extracts of Examples 1 to 3 prepared according to the present invention are also effective in inhibiting type II 5α-reductase, which is known to be involved in converting testosterone to dehydrotestosterone (DHT). In Examples 1 to 3 of the present invention, the inhibition rate all satisfied more than 25% at a concentration of 2.5 mg / ml of the sorghum extract, which shows a higher inhibitory effect than the reference substance finasteride and has an excellent DHT production inhibition effect at the concentration or higher. It can be judged by having it.

However, in the case of the Soyang extracts according to Comparative Examples 1 to 4 that are outside the conditions of the present invention, there is a tendency for the inhibition rate to increase as the concentration increases, but it can be seen that the inhibitory effect is very poor compared to the Examples of the present invention. In addition, the results were found to fall short of the 25% inhibition rate at a concentration of 2.5 mg/ml of the Soyang extract, which is the standard for inhibition rate.

Claims (5)

1) Step of washing and pulverizing the dried sheep,
2) immersing the pulverized sheep in an alcohol diluted solution and leaving it at room temperature,
3) mixing the alcohol diluted solution:water in a volume ratio of 4:6 to 6:4 with the solution left at room temperature, then heating the mixture for 1 to 60 minutes to obtain a first supernatant,
4) Obtaining a secondary supernatant by repeating the process of boiling the primary supernatant at 60-80°C for 2-3 hours, and
5) A method for producing a prickly pear extract comprising the step of freeze-drying the secondary supernatant at a low temperature of -100 to 0°C to produce a powdery prickly pear extract.
According to claim 1,
A method for producing a plant extract, wherein the extract has an extraction efficiency of 30% or more.
Soyang extract prepared by the manufacturing method of claim 1.
According to claim 3,
The sauerkraut extract contains an active ingredient that inhibits the production of dihydrotestosterone (DHT).
According to claim 4,
The dried sheep extract has an inhibition rate of dihydrotestosterone (DHT) production of 25% or more at a concentration of 2.5 mg/ml.