KR100993603B1 - Extracts of Paeoniae radix flower with whitening and Manufacturing Method - Google Patents

Abstract

The present invention relates to a peony flower extract having a whitening function and a method of manufacturing the same, and more particularly, to collect peony flowers, pretreatment as a flower or lyophilized, extract with ethanol, and then filter and concentrate, and whitening. Selecting an extract with a function consists of the steps of assaying and analyzing cytotoxicity.

The extract having a whitening function of the present invention and a method for producing the same can be usefully used as a cosmetic composition having excellent whitening efficacy without skin irritation by containing an active ingredient of a side and a peony flower to be recycled.

Whitening, cosmetics, extracts, peony

Description

Peony flower extract with whitening function and its manufacturing method {Extracts of Paeoniae radix flower with whitening and Manufacturing Method}

The present invention relates to a peony flower extract having a whitening function and a method of manufacturing the same, and more particularly, a peony flower is an extract prepared by extracting it with ethanol and then filtering and concentrating it with ethanol. It relates to a cosmetic composition and a method for producing the same, characterized in that no high-functional extract.

Peony (Jakyak, Paeoniae radix ) is one of the most important herbal medicines as the root of the peony and the same plant. The leaves are alternate, the petioles are long, and the effusion leaves are twice. The small leaves are elliptical and there are no sawtooth. Flowers bloom one by one at the end of stem and white flowers bloom from May to June. Herbs are dried roots, and they grow in the shade of trees in the mountains. It is distributed all over Korea, and oriental medicine is collected in March and August and dried in the sun. Alveolar, sheep blood, blood, Ivy, Jitong Used for i). In oriental medicine, it has been used as astringent easing, jingyeong, and analgesics, and it has been used as Gyeji Kahwakyuntang. Fruits are 1-3, with horn-shaped, red immature seeds and black mature seeds. Breeding uses roots or seeds. Peony used for medicine is divided into a herbaceous peony and peony tree I as medicinal plants belonging to the Ranunculaceae (Ranuculaceae) peony (Paeonia). Peonies are grown in Korea, jeokjakyak (Paeonia in the classification plane plant lactiflora Pall, Paeonia obovata Max.) and Paeonia japonica Miyabe et Takeda. It is known that varieties of red peony are cultivated in Korea (Yunpyeongsup, Korea Horticultural Plant Book, 1995).

Peony has been used as an important herbal medicine for more than 1,200 years. In oriental medicine, it is mainly used for the purpose of inflammation, analgesia, and pain relief, such as controlling amenorrhea, treating trauma, nasal bleeding, inflammation, boils and wounds, and relieving pain in the chest and ribs. It has been prescribed for therapeutic purposes. Peony's physiological activity has been reported to be anti-inflammatory, anti-allergic, analgesic, etc., and many reports on the activity of peoniflorin, the main ingredient. Recently, numerous activities have been reported, including immunomodulatory action, anti-allergic action, anti-inflammatory action, cognitive improvement effect, neuromuscular blocking activity, antithrombotic, anticoagulant, anticonvulsive effect.

In general, it is important to determine the black-and-white color of human skin color by melanocytes that produce melanin pigments, which are determined by various external factors (ultraviolet rays, medications, irritating skin externals), and internal factors (hormones, inflammatory factors, cytokines). malanocyte) activity and distribution. In melanocytes in the human body, enzymes such as tyrosinase act and produce a black pigment called melanin through an oxidation reaction based on an amino acid called tyrosine which is always present in a living body. This melanin pigment is widely distributed to the surrounding keratinocytes through the dendritic projections of the pigment cells, thereby determining the color of the skin.

Skin pigment abnormalities such as blemishes, freckles, and senile plaques are caused by abnormally activated melanin production of pigment cells, resulting in excessive formation and deposition. This hyperpigmentation can be a serious mental burden from the point of view of skin beauty and can interfere with normal social activities.

Asian women have long preferred white, fair skin, and have made it an important standard of beauty. There have been many developments of whitening agents for treating skin pigmentation and satisfying cosmetic needs. Ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione and derivatives thereof, including tyrosinase inhibitory activity in cosmetics or medicines to provide cosmetics and medicines with a whitening effect, but safety problems for skin B. There is a problem that its use is limited due to insufficient whitening effect when formulated in cosmetics or pharmaceuticals.

Therefore, the cosmetic composition according to the present invention promotes the whitening function on the skin dull due to pigmentation according to the peony extract, and prevents the occurrence of skin troubles at all by extracting the active ingredient of the peony flower with ethanol. It is an object of the present invention to provide a natural cosmetic composition using a peony extract, and a method for producing the same.

In order to achieve the above object, the present invention provides a peony flower extract having a whitening function.

In addition, in order to achieve the above object, the present invention comprises the steps of collecting the peony flowers, pre-treatment as it is or lyophilized flowers; Extracting the active ingredient by immersing the peony flower after pretreatment in ethanol; Filtering the extract obtained through the extraction step; And it provides a method for producing a peony flower extract with a whitening function comprising the step of concentrating the filtrate.

In addition, in order to achieve the above object, the present invention provides a peony flower extract having a whitening function prepared according to the production method of the present invention.

In addition, in order to achieve the above object, the present invention is a peony flower extract having a whitening function prepared according to the method of the present invention peonyflorin as an indicator material peony flower extract, characterized in that it contains more than 20,731 mg per kg powder sample To provide.

In addition, the present invention provides a cosmetic composition for skin whitening, characterized in that it contains 24 to 244 ppm peony flower extract prepared according to the production method of the present invention.

Hereinafter, the present invention will be described in detail for each process.

At this time, if there is no other definition in the technical terms and scientific terms used, it indicates the meanings that are commonly understood by those of ordinary skill in the art.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

First step: raw material pretreatment process

Peony flowers are collected and washed with water to remove foreign substances, dried in the shade, and then subjected to a pretreatment step of the peony flowers as they are or lyophilized.

The pretreatment step of the present invention is a method of using the flowers as it is without lyophilizing the peony flowers or after freeze-freezing at -45 ℃ for 5-8 hours, by continuous operation of the device at 35 ℃, 5 mTorr 48 ~ 72 It is a method of pretreatment using lyophilization system treatment which dries for time.

Step 2: Extraction of Active Ingredients

Peony flowers after the raw material pretreatment step is immersed in ethanol to extract the active ingredient. In general, the extraction solvent is water, C ₁ Lower alcohols of C to C ₃ or a mixed solution thereof are used, and in the present invention, it is preferable to use 50 to 80% of ethanol. The amount of the extraction solvent is added to the pretreated sample 50 to 80% ethanol 4 to 10 times compared to the sample.

The extraction method may be an extraction method such as heat extraction, reflux cooling extraction and the like, in the present invention, it is preferable to extract still in dark conditions for 1 to 5 days at room temperature. Maintaining the temperature of the immersion solution at room temperature is effective for the prevention of decay, discoloration and odor of peony flowers. The immersion time is 1 to 5 days can be sufficiently extracted the active ingredient of peony flowers.

Step 3: Extract Filtration Process

After obtaining the extract of the peony flower, the solid is removed using a filter paper, the suspension is filtered once or twice.

Step 4: Filtrate Concentration Process

The filtered extract is concentrated using a vacuum concentrator. In the concentration step of the present invention, the filtered extract is preferably concentrated under vacuum at 40 to 45 ° C. and 5 to 10 rpm until the powder becomes powder.

Step 5: Cytotoxicity Test

The present invention may further include selecting and analyzing an extract having a whitening function from the concentrate prepared in the fourth step, and analyzing cytotoxicity.

In the present invention, the cytotoxicity test is an experiment to determine the degree of toxicity of a compound to cells, and generally, methods such as Cytotoxicity assay, Neutral red uptake assay, MTT assay, and XTT assay can be used. Among them, the MTT assay is a test method for identifying the formazan of living cells in color, and is the most widely used method among cytotoxicity experiments.

Peony flower extract of the present invention has an effect of providing a cosmetic composition excellent in whitening function without skin irritation by containing a peony flower active ingredient extract and environmentally friendly effect to recycle the discarded peony flowers.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

[ Example  1] Preparation of Peony Flower Extract

Peony flower used in the experiment was collected in the moisture-free state in Namwon Unbong-myeon, Jeonbuk, and used while storing in a freezer at -50 ℃. The extraction solution was selected by comparing and analyzing the conditions for maximum extraction of the active ingredient from natural products. In particular, ethanol, which is widely used for extracting food and cosmetic raw materials, is used because it is intended to produce extracts that are raw materials for cosmetics. It was.

As the extraction method, a static extraction method was used, and the solvent ratio was 4 to 10 times as the conditions, and extracted with 50 to 80% ethanol for 1 to 5 days at room temperature. All experiments were wrapped in aluminum foil to prevent oxidation of natural products. In addition, as a pretreatment, peony flowers were used as they were or were selected by lyophilization.

The manufacturing process of the extract is shown in FIG. After static extraction, the filter paper was filtered once or twice, and then concentrated using a vacuum concentrator until it became a powder. 17 g of powder extracts were obtained from 250 g of peony flower raw materials.

[ Example  2]

90 ml of distilled water, 9.8 wt% of 1, 3-butylene glycol and 0.1 to 5.0 wt% of the peony flower extract prepared by the preparation method of the present invention were added to prepare a skin whitening lotion 50 ml.

< Experimental Example  1> Determination of Whitening Function Effect of Peony Flower Extract

Human skin color depends on the amount of melanin, carotene and hemoglobin, of which melanin is the most decisive factor. Melanin is formed in melanocytes and transferred to keratinocytes through melanocyte dendrites. In particular, the skin color is not determined by the number of melanocytes but by the size of melanocytes and their distribution in keratinocytes. Overproduction of melanin can cause melasma and freckles. This melanin is biosynthesized from tyrosine by the oxidation of tyrosinase.

Tyrosinase inhibitory test was used to measure the whitening effect of the peony flower extract. Arbutin, which is commonly used as a control for the whitening function, was used. Experiments confirmed that the peony flower extract extracted for 1 to 5 days was tyrosinase inhibitory activity. In addition, the experiment was performed according to the lyophilization method as a pretreatment for the purpose of long-term storage of the sample.

Arbutin, a control, was doubled in all experiments. Experimental results are shown in Figure 2 and Table 1.

Sample Name Arbutin JY1 (24 hours immersion zone) JY2 (48-hour immersion zone) Concentration (ppm) 50 100 200 50 100 200 50 100 200 % Inhibition 2 28 59 23 100 100 100 100 100

As shown in FIG. 2 and Table 1, JY1 (24 hour immersion zone) and JY2 (48 hour immersion sphere) were found to have superior whitening effect than arbutin in all experiments. In the case of JY1, which was treated for 24 hours, the whitening effect was superior to arbutin at 50 ppm, but the whitening effect was significantly lower than that of JY2. However, at 100 ppm concentration, both JY1 and JY2 showed superior efficacy than the control arbutin, and above 100 ppm concentration, both JY1 and JY2 showed similar results.

As shown in Figure 3, even in the experimental groups treated with lyophilization pretreatment was found to show an excellent whitening effect compared to arbutin at 100ppm or more.

< Experimental Example  2> Indicator Material Analysis of Peony Flower Extract

Peoniflorin with a purity of 98% purchased from Wako was used as a standard, and HPLC analysis was performed using an HPLC grade solvent. First, the standard was diluted to methanol to 20, 50, 70, 100 ppm to prepare the calibration curve. If these samples were taken by HPLC, and the area of the peak is exactly proportional, the straight line can be used to estimate the area of the peak with the same retention value. Each sample diluted to a specific concentration was developed for 30 minutes using HPLC and then peaked at 230 nm. Since the area of the peak increases with the amount of the sample, the area of each peak was integrated and used for calibration.

Based on the integral value, if you derive the calibration formula for the sample quantity according to the integral value, the calibration curve can be represented by a straight line.

The equation is equal to x = 1.45 x 10 ⁴ ｘ + 5.85 x 10 ³ .

The coefficient of determination (R ² ) is 0.9996, which is close to 1, which explains well the integral of the peaks of each sample. Based on the calibration equation, when the sample was analyzed at 230 nm, the amount of peoniflorin in the sample could be calculated as the integral value of the peak where the retention value was 6.883 minutes.

30 mg of the peony flower powder, extracted with 80% ethanol, was dissolved in 10 mL of methanol, filtered through a 45 μm membrane filter, and used as an analytical sample. HPLC (Waters 2695) analysis conditions are shown in Table 2. It was.

Condition Analysis of Indicator Material of Peony Flower Extract Column SYMMETRY C18 (4.6 m I.d. × 150 mm, 5 μm) Detector Photodiode array (PDA, Waters 2996) Eluants Water: Acetonitrile = 85: 15 Flow rate 1 mL / min Injection volume 20 μl Wavelength 230 nm Temperature Ambient

As a result, it was possible to obtain a chromatogram as shown in FIG. 4, and it was confirmed that 20,731 mg peoniflorin was contained per 1 kg of peony flower concentrate powder. In other words, 10.3 mg of peoniflorin was obtained from one peony flower (7 g).

< Experimental Example  3> Cytotoxicity Test of Peony Flower Extract

Cytotoxicity test is a test to determine the degree of toxicity of a compound (natural and other chemicals) to cells, such as Cytotoxicity assay, Neutral red uptake assay, MTT assay, XTT assay. Among them, MTT assay is the most widely used method among cytotoxicity experiments. The MTT assay is a test to measure the activity of mitochondria. When MTT is applied to living cells, MTT is formed by a reductase in mitochondria, forming a crystal called formazan, and in the dead cells, the mitochondria are already broken. Because there is no reductase. Therefore, MTT assay is an experiment that can confirm the formazan of living cells by color.

1. Incubate HaCaT cells.

2. When cells are about 80% grown, passaging (96 well plate).

3. Discard the supernatant of cells collected by centrifuge and add a new solvent to loosen slowly.

4. Drop 12µl onto Haemocytometer and perform cell counting under microscope.

5. Add cells to 2 * 10 ⁵ cells / 500µl per well.

6. Incubate the cells for 24 hours.

7. After 24 hours, wash with 1X PBS, mix the sample to be treated with the solvent according to the concentration, mix well, and incubate for 2 hours.

8. Remove the medium and sample and wash with PBS. The MTT solution is treated and then covered with aluminum foil and stored in the incubator for 3-4 hours.

9. Remove the MTT solution, add 50 μl of DMSO, mix for 20 minutes in a shaking incubater, and measure by ELISA.

The experimental results are shown in FIG. 5 and Table 3.

Concentration (ppm) 0 0.48 2.4 4.8 24 48 244 480 Amount of cells 0.438 0.432 0.430 0.436 0.461 0.449 0.452 0.429

As shown in FIG. 5, the Y-axis is the amount of living cells, and the unit is (* 10 ⁴ cells). The higher the value, the higher the number of living cells. X-axis is the concentration of the peony flower extract, 0 is the untreated portion of the peony flower extract, and then treated with a concentration of 0.48, 2.4, 4.8, 24, 48, 244, 480ppm. This concentration was chosen because the extract was dissolved in DMSO. DMSO appears to be toxic to cells, but treatment with a certain concentration (less than 0.4%) does not act as a toxicity to cells. Therefore, the concentration of DMSO 0.4% was set to the highest concentration of 480ppm, and the experiment was carried out while diluting to 1/2 and 1/5.

Compared with the untreated peony flower extract, there is no difference in the concentration of 0.48ppm, 2.4ppm, 4.8ppm. However, the peony flower extract concentrations of 24ppm, 48ppm, 244ppm increased the viable cells, which is not cytotoxicity, but rather a factor that can promote the growth of cells. However, if you look at the graph, you can see that the value (0.429) falls at 480ppm compared to the negative control. This can act as a toxic because the amount of peony flower extract inhibits the growth of cells. Based on the above experimental data, at 24 ~ 244ppm, peony flower extract can be seen as a range of concentrations available for functional experiments.

The present invention is to provide a cosmetic composition with excellent whitening function without skin irritation by containing the side of recycling peony flowers discarded after cultivation and the extract of the active flower of peony flowers, can be widely used for securing raw materials and activating product production industrially have.

1 is a flow chart showing a method of manufacturing peony flower extract of the present invention.

Figure 2 is a graph showing the tyrosinase inhibitory effect of the extract extracted as a peony flower of the present invention.

Figure 3 is a graph showing the tyrosinase inhibitory effect of the extract prepared by lyophilizing the peony flowers of the present invention.

Figure 4 shows the index chromatogram of peony flower extract according to the present invention.

Figure 5 is a graph showing the cytotoxicity test results of the peony flower extract according to the present invention.

Figure 6 is a sample photograph of the skin whitening lotion containing peony flower extract prepared by Example 2 of the present invention as an active ingredient.

Claims (9)

delete (a) freezing frozen peony flowers for 5 to 8 hours at −45 ° C., then preparing dried peony flowers for 48 to 72 hours at 35 ° C. and 5 m Torr; (b) adding 50 to 80% of ethanol to the peony flower prepared in step (a) 4 to 10 times as compared to the peony flower, and then extracting the mixture under dark conditions for 1 to 5 days at room temperature; (c) filtering the extract obtained through step (b); (d) vacuum concentration of the filtrate of step (c) under conditions of 40 to 45 ℃, 5 to 10 rpm until it becomes a powder; And (e) a method of producing a peony flower extract with a whitening function, comprising the step of selecting and analyzing the extract having a whitening function in the concentrate of step (d) and analyzing cytotoxicity. delete delete delete delete delete delete delete

Images