KR101312025B1 - Preparation method of extract of Picrasma quassioides and use of the extracts - Google Patents

Abstract

The present invention provides a method of producing a pine tree extract having a new efficacy and effect distinguished from the conventional pine tree extract, or a pine tree extract that is superior to the conventional. The present invention is the addition of the kiwi and pineapple ground grinding solution to the solution of the dispersion of the crushed pine bark bark extract produced by reacting for a period of time the antiangiogenic and interleukin-4 (IL-4), interferon- Gamma (IFN-gamma; IFN-γ) has an inhibitory effect on the production of NF-kB, the cytokine production inhibitory effect is extracted without going through the step of reacting with kiwi and pineapple grinding solution for a certain period of time It is based on the finding that the inhibitory effect is much better.

Description

Preparation Method and Extract of Picrasma quassioides and use of the extracts

The present invention relates to a method of producing a bovine extract. The present invention also relates to medicinal, food and cosmetic uses of the novel pine needle extract.

In medicine, food or cosmetics in which the ingredients of natural materials are included as an active ingredient, natural products in the form of extracts are generally used. The extract is usually cut or crushed natural products such as herbal medicines as it is or appropriately, hot water extraction method of leaching and extracting the active ingredient using water, solvent extraction method using a solvent such as ethanol, methanol, supercritical carbon dioxide extraction method, A steam extraction method, a compression extraction method, a gamma-irradiation extraction method, an ultrasonic extraction method and the like have been manufactured alone or in parallel (Registration No. 670612).

Recently, fermentation extraction methods, including the step of reacting herbal medicines with microorganisms, may be used for the preparation of extracts. For example, a method of fermenting the extract of white baekcho with brown sugar and yeast (registered patent No. 821239), a method of fermenting red ginseng with mold (registered patent No. 884044) and the like. In such a fermentation step, the enzymes contained in the microorganisms act on the herbal medicine, so that new ingredients that were not originally included in the herbal medicine can be produced, or the active ingredient contained in the herbal medicine can be extracted more effectively. Therefore, by using the fermentation extraction method, it is possible to produce an extract having a different efficacy and effect than the herbal extract prepared using the conventional method.

As such, when an additional method is used in addition to the extraction method used in the natural material, it is possible to prepare an extract having an effect different from that of the extract prepared by the conventional extraction method, and thus a new extract suitable for each natural material. Development of manufacturing methods can lead to the development of new applications.

Picrasma quassioides is a broad-leaved arboreous tree belonging to the family Pinaceae. Contains various bioactive substances (Chinese Dictionary). Pine needles have been used for the treatment of dry stomach, insect repellent, digestive agents, and inflammation in Chinese medicine, and have been commonly used as an extract or for external use. In order to diversify the use of pine needle extract, it is necessary to develop a method for preparing a new extract.

Angiogenesis (angiogenesis), on the other hand, refers to the creation of new blood vessels into tissues or organs, which occur under normal physiological conditions, but also cause pathological damage due to unregulated (abnormal) angiogenesis. Various pathological conditions produced due to unregulated (abnormal) angiogenesis lead to 'angiogenesis dependent diseases' or 'angiogenesis related diseases'. Such diseases include tumors, psoriasis, diabetic retinopathy, renal arterial disease, rheumatoid arthritis, osteoarthritis, chronic inflammation, hemangiomas, and the like, which are described in detail in Patent No. 506043 and described in the present invention. It includes the contents described in the patent.

Interferon gamma is produced by Th1 cells, NK cells and the like and is a kind of cytokine involved in immune responses. Interferon gamma participates in defense against viral infections, but also plays an important role in inflammatory responses. Thus, inhibitors of interferongamma can be used as therapeutic agents for inflammatory diseases such as inflammatory bowel disease (US Patent Publication No. US2003 / 0012790). Inhibitors of interferon gamma can also be used to treat wounds, to treat hyperplasia (US Published Patent Application US 2008/0031879).

Interleukin-4 (IL-4) is a type of cytokine that is produced in Th2 cells, mast cells, and the like and promotes IgE production and is involved in immune and inflammatory reactions (International Immunology 2004, 16 (8), p1155-1160). Inhibitors of IL-4 have therapeutic effects such as inflammatory diseases and atopic dermatitis.

Atopic dermatitis is mostly caused by an immune mechanism associated with IgE, and there are many reports that an immune response caused by T cell abnormality is involved, and the role of helper T cells (Th cell) is known to be important. Many T cells are found in atopic dermatitis lesions, and cytokines secreted by Th2 cells play an important role in the development of atopic dermatitis. In addition to Th2 cell secreting cytokines, interferongamma, a Th1 cell secreting cytokine, plays an important role in atopic dermatitis (Journal of Clinical Investigation 1999, 103 (8), p1103-1111). It is reported that the immune response varies according to the development of atopic dermatitis, Th2 cells are involved in acute atopic dermatitis, and Th1 is involved in chronic atopic dermatitis. In other words, IL-4 and IL-3, which are secreted from Th2, play an important role. Thitch cells increase when itching begins to scratch (Journal of Clinical Investigation 2004, 113, pp651-657). According to Leung et al., Atopic dermatitis caused by house dust mite allergens progresses in two stages, the initial stage of which mainly produces IL-4 by Th2 cells, and interferon gamma by Th1 cells after 24-48 hours ( Lancet 2003, 361, p151-160). Therefore, in order to effectively prevent, treat or improve atopic dermatitis, a substance or composition capable of affecting both Th1 and Th2 cells and inhibiting the production of IL-4 and interferon gamma is effective (Reg. No. 954216).

NF-kB acts on the gene expression mechanism that causes inflammation, tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, IL-8, GM-CSF, inducible It is known as a transcription factor that increases the expression of nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), E-selectin, MHC class I and II molecules. Therefore, inhibitors of NF-kB may exhibit therapeutic effects for chronic and acute inflammatory diseases and atopic dermatitis (Patent No. 954216). In addition, inhibitors of NF-κB are known to have an effect of inhibiting tumor cell proliferation and inducing apoptosis (US Patent Publication No. US 2006/0193784).

It is an object of the present invention to provide a method for producing a pine tree extract having a new efficacy and effect distinguished from the conventional pine tree extract, or a pine tree extract which is more effective than the conventional pine tree extract.

In addition, an object of the present invention is to provide a composition comprising an extract prepared by the method for producing a new pine needle extract.

Furthermore, an object of the present invention is to provide a method of using a composition comprising an extract prepared by the method of producing a new pine needle extract.

In order to achieve the above object, the present invention provides a method of preparing an extract using a conventional extraction method after the step of reacting the pine bark with kiwi and pineapple grinding solution for a certain period of time.

In another aspect, the present invention provides a composition comprising an extract prepared by using a conventional extraction method after the step of reacting the pine bark with a kiwi and pineapple grinding solution for a period of time.

Prepared by the method provided by the present invention, after the reaction for a certain period of time with the kiwi and pineapple grinding solution, the extract of the bovine tree is angiogenesis inhibitory effect, interleukin-4 (IL-4), interferon-gamma (IFN-gamma; IFN-γ) has an inhibitory effect on the production of NF-kB, the inhibitory effect is more than the inhibitory effect of the extract of the bovine extract extracted without going through the reaction for a period of time with kiwi and pineapple grinding solution Excellent

Thus, the extract of the bovine tree prepared by the method provided by the present invention is effective in inhibiting abnormal angiogenesis, the composition of 'angiogenesis dependent diseases' or 'angiogenesis related diseases' such as tumors, psoriasis, diabetic retinopathy, It can be used for the treatment of renal arterial disease, rheumatoid arthritis, osteoarthritis, chronic inflammation, hemangioma and the like.

In addition, the bovine extract produced by the method provided by the present invention is for the inhibition of IL-4, interferon gamma, NF-Kb, or inflammatory diseases, atopic dermatitis, inflammatory diseases, inflammatory bowel disease, wounds, excess fibrosis It can be used for the prevention, treatment and improvement of hyperplasia.

1 shows results of Chorioallantoic Membrane (CAM) assay. The reaction time of the kiwi and pineapple solution and the pineapple powder dispersion was 0 hours and 1 week, and then the angiogenesis inhibitory effect of the extracted extract was compared.
Figure 2 shows the interferon gamma inhibition rate by the cypress extract.
Figure 3 shows the inhibition rate of IL-4 by pine needle extract.
Figure 4 shows the NF-kB inhibitory effect of the pine needles extract.

The present invention provides a method of producing a pine tree extract having a new efficacy and effect distinguished from the conventional pine tree extract, or a pine tree extract that is superior to the conventional.

The present invention is an anti-angiogenic effect and interleukin-4, interferon-gamma, NF-kB produced by reacting for a certain period of time by adding a kiwi and pineapple grinding solution to the dispersion of the crushed pine wood bark The inhibitory effect of the production of interleukin-4, interferon-gamma, and NF-kB was much higher than that of the extract from the bovine tree without the step of reacting with kiwi and pineapple grinding solution for a certain period of time. Based on what you say is excellent. Kiwi and pineapple are known to contain a large amount of proteolytic enzymes.

The method of manufacturing the pine needle extract of the present invention comprises the steps of making a liquid (A solution) crushed kiwi and pineapple; Pulverizing pine wood bark to make a solution (B solution) dispersed in water; And reacting by mixing the A solution and the B solution. Water can be added when making the kiwi and pineapple ground liquid. The ratio of the kiwi and pineapple used when preparing the A solution can be adjusted as needed. For example, kiwi: pineapple is used in a weight ratio of 1: 0.1-10, preferably 1: 0.1-1, more preferably 1: 0.1-0.5. The amount of water added when preparing solution A may be appropriately controlled, for example, 0 to 100 times may be added to the sum of the weights of kiwi and pineapple. The weight ratio of bovine bark and water used in the preparation of solution B can be properly adjusted as needed, for example, 1 to 100 times the amount of water can be used for the bovine bark.

The temperature at which the A and B solutions are mixed and reacted may be any temperature as long as the activity of the enzymes present in the kiwi and pineapple is maintained. The reaction time can also be appropriately adjusted. For example, it may be reacted for 1 day to 1 month, preferably 7 days. After the reaction by mixing the A solution and B solution may be further included to remove the solids. The completed solution can be concentrated or dried to a powder. Solids removal method, concentration or drying process may be used any method as long as it is widely used in the art.

In addition, the present invention provides an extract or a composition comprising the extract prepared by the method for producing a new pine needle extract.

Prepared by the method provided by the present invention, after the step of reacting for a period of time with the kiwi and pineapple grinding solution for extracting the bovine extract has an neovascular inhibitory effect. Therefore, the pine needle extract prepared by the method provided by the present invention can be used for the preparation of a composition effective for inhibiting abnormal angiogenesis. In addition, the bovine extract produced by the method provided by the present invention is an 'angiogenesis dependent disease' or 'angiogenesis related' diseases such as tumors, psoriasis, diabetic retinopathy, retinal disease of the eyes, rheumatoid arthritis It can be used for the prevention, treatment and improvement of osteoarthritis, chronic inflammation, hemangioma and the like.

The cypress extract provided by the present invention inhibits the production of interleukin-4, interferon-gamma and NF-kB, and the inhibitory effect is extracted without undergoing a step of reacting with kiwi and pineapple grinding solution for a certain period of time. It is much better than the inhibitory effect of pine cone extract. Thus, the pine needle extract, which is prepared by the method provided by the present invention, after the reaction for a certain period of time with the kiwi and pineapple grinding solution, is used for the inhibition of IL-4, interferon gamma, and NF-Kb. Can be. In addition, the botanical extract provided by the present invention can be used for the prevention, treatment, improvement of inflammatory diseases, atopic dermatitis, inflammatory diseases, inflammatory bowel disease, wounds, hyperplasia, psoriasis, rheumatoid arthritis, eczema, etc. .

The composition comprising the bovine extract provided by the present invention can be used as a pharmaceutical composition, cosmetic composition, food composition. Therefore, the present invention includes the pharmaceutical, quasi-drug, food, and cosmetic containing the composition in the scope of the invention.

The composition according to the present invention may further contain pharmaceutically, food or cosmetically acceptable ingredients in addition to the pine needle extract. For example, binders, lubricants, disintegrants, excipients, solubilizers, stabilizers, bases, lubricants, preservatives, wetting agents, emulsifiers, suspending agents, viscosity regulators, preservatives, surfactants, antioxidants, moisturizers, fragrances, pigments, etc. It may further contain. In addition, it may further contain a moisturizer such as ceramide or a lipid component, a steroid such as hydrocortisone, a vitamin A derivative such as retinyl palmitate, tocopepol, and other plant extracts, which have been frequently used in conventional atopic skin treatments. The composition of the present invention can be applied orally or topically, tablets, powders, capsules, granules, pills, solutions, suspending agents, ointments, creams, lotion type, lotion type, pack type and the like, as well as soap, face wash It can be manufactured in various forms such as face washes such as foams. It is also possible to make liposomes.

The dosage of the composition according to the present invention can be appropriately adjusted according to the weight, age, sex, health status, digestive status, etc. of the person to which it is applied, and a typical single dosage can be administered at 0.1mg to 100mg per kg of body weight. have. When preparing the composition of this invention in the form of the external preparation apply | coated to skin, the compounding quantity of the bovine extract in a skin external preparation can be 0.05%-50.0 weight% of the whole, Preferably it can be 0.1-10.0 weight%.

The composition of the present invention can be used in a variety of quasi-drugs. In addition, the composition of the present invention can be used in various functional foods. The composition of the present invention can be applied to any formulation that can be taken as a food, and examples thereof include powders, granules, tablets, pills, capsules, soft capsules, or liquids. Examples of the food to which the composition of the present invention can be added include, for example, various foods, meats, beverages such as tea, snacks, confectionery, ramen, other noodles, candy, chocolate, ice cream, gum, vitamin complex, and health. Functional foods;

The present invention also provides a method of using a composition comprising an extract prepared by the method of producing a new pine needle extract.

Hereinafter, the present invention will be described in more detail with reference to examples and the like, but embodiments according to the present invention can be modified in many different forms and the scope of the invention is not limited thereto.

Example  1: Preparation of Pine Tree Extract

100 g of kiwi, 1000 g of water, and 20 g of pineapple were prepared and ground together to form a solution (called A solution). 2000 g of water was added to 100 g of bovine bark crushed powder and dispersed well (this is called B solution).

A solution and B solution were mixed in equal amounts. Some of the samples prepared by mixing were subjected to the extraction process immediately. That is, hot water extraction was performed at 70 ^° C. for 3 hours. After centrifugation, the mixture was filtered (0.2um) and used as a sample for the experiment. This extract is called 'Sample 1'. Some samples of the same amount of A and B solution were mixed for 1 week in a refrigerator (4 ^o C) and reacted with hot water for 3 hours at 70 ^o C for extraction. After centrifugation, filtering (0.2um) was used as a sample for the experiment. This extract is called Sample 2.

Example  2 : CAM  ( chorioallantoic membrane ) assay

In order to observe the ability to inhibit the formation of neovascularization of the bovine tree extract, fertilized eggs were purchased within 1 day of laying, and after 3 days, 5 mL of egg white was removed to make the top of the egg open. The enuretic membrane (CAM) surrounding the chicken embryo On a chorioallantoic membrane, 0.01 mL of pine needle extract (Sample A or Sample B) was added dropwise and covered with a dried coverslip. Three days later, angiogenesis was observed under a microscope.

Normal vascular morphology was observed after 3 days of uncovered coverslip on CAM. The reaction time of the kiwi and pineapple solution and the pineapple powder dispersion was 0 hours and 1 week, and then the angiogenesis inhibitory effect of the extracted extract was compared. In case of dropping 'sample 1', a bovine tree extract that did not react with kiwi and pineapple solution, angiogenesis similar to normal angiogenesis was still observed, but in case of dropping 'sample 2', a bovine tree extract that was subjected to the reaction process Angiogenesis was inhibited (FIG. 1).

Example  3: Preparation of Cell Culture Solution for Cytokine Analysis

Mouse spleen cells were prepared as follows. The mouse was cervical distal bone, sterilized with alcohol, incised abdomen, spleens were collected aseptically, and stored in refrigerated RPMI-complete media with antibiotics. Approximately 2 ml of animal cell medium (RPMI media) per spleen is added and the spleen is completely crushed, and then treated with 0.84% ammonium chloride solution for 10 seconds to remove erythrocytes, followed by erythrocyte haemolysis reaction with medium. After centrifugation, the sunk cells were collected. The obtained cell fraction was used for the experiment after incubation for one day.

Phytohemagglutinin (PHA) Stock Solution was prepared by dissolving 0.5 mg of PHA per 1 ml of sterile phosphate buffer solution PBS. PMA Stock Solution was prepared by dissolving 100 ug of PMA per mL of DMSO.

Peel extracts were prepared by filtering 100 g of bark peels into 2000 g of water, extracting hot water for 3 hours at 70 ^o C, centrifugation, and filtration. The stock solution of the bovine tree extract was used as the sample stock solution prepared in Example 1.

2.5 ml of Jurkat cell culture, mouse splenocytes, EL-4 cell line (1 x 10 ⁶ cells / ml, 1 x 10 ⁷ cells / ml, 5 x 10 ⁶ cells / ml) and 2.5 ml of medium (RPMI 1640 + 10) 10 ml / L penicillin-streptomycin) containing% fetal calf serum was incubated in a 25T flask. After dispensing 150 ul of the cell culture solution into 96 wells, 15 ul of PHA stock solution was added at the same time, and 15 ul of pine needle extract stock solution was added at the same time. After 18 hours of incubation, the supernatant was centrifuged and analyzed for cytokines on the day by ELISA.

The calculation method of cytokine inhibition by a sample is as follows.

 Inhibition rate = {1- (cytokine production in the sample treatment group / cytokine production in the positive control group)} x 100

Example  4: interferon-gamma Assay

The ELISA method was used to determine the inhibitory effect of interferon-gamma activity of pine needles extract. BIOSOURCE's IFN-γ immunoassay kit was used. Each well of a 96-well plate was coated with capture antibody of IFN-γ. Thereafter, 100 ul and 50 ul of Incubation Buffer were added to the appropriately diluted cell culture supernatant, and 50 ul of biotinylated detection antibody (Biotin Conjugate) solution was added thereto and left at room temperature for 2 hours. After completely removing the solution and washing four times, 100 ul of Streptavidin-HRP Working Solution was added and left at room temperature for 30 minutes, and the solution was completely removed and washed. 100 ul of Stabilized Chromogen was added and allowed to stand at room temperature for 30 minutes. Then, 100 Stop Solution was added and data were read at 450 nm.

Figure 2 shows the IFN-gamma inhibition rate of the herbal extracts. 0.56 and 0.14 mean that the sample stock solution contained 0.56% (V / V) and 0.14% (V / V) in the cell culture solution, respectively. Negative control group was not treated with a judo source, positive control group was treated with a judo source.

When the reaction time of the kiwi and pineapple solution and the pineapple powder dispersion was 0 hours, the extract (sample 1) was treated with 0.56% (V / V) and 0.14% (V / V), respectively, at 67.76% and 63.19%, respectively. When the reaction time was 1 week (Sample 2), the inhibition rate was 90.14% and 85.12%, respectively. It was confirmed that the increased. This value was higher than the inhibition rate of 84.36% and 75.74% for 0.56% (V / V) and 0.14% (V / V) treatments of the bark skins (Botan skin extract), which are commonly used as anti-inflammatory agents.

Example  5: interleukin-4 Assay

An ELISA method was used to measure interleukin-4 (IL-4) activity in the cell culture prepared by Example 3. BIOSOURCE's immunoassay kit was used and WHO reference preparation 86/504 (NIBSC, Hertfordshire, UK, EN6 3QG) was observed.

Figure 3 shows the interleukin-4 inhibition rate of the herbal extracts. 0.56 and 0.14 mean that the sample stock solution contained 0.56% (V / V) and 0.14% (V / V) in the cell culture solution, respectively. Negative control group was not treated with a judo source, positive control group was treated with a judo source.

When the reaction time of the kiwi and pineapple solution and the pineapple powder dispersion was 0 hours, the extract (sample 1) was treated with 0.56% (V / V) and 0.14% (V / V), respectively, 33.176% and 25.98%. When the reaction time was 1 week (Sample 2), the inhibition rate was 60.59% and 42.19% at the same concentrations as above, and the inhibitory effect of interleukin-4 was increased due to the reaction with kiwi and pineapple solution. It was confirmed. This was higher than the inhibition rate of 32.05% and 21.66%, respectively, when treated with 0.56% (V / V) and 0.14% (V / V) in the bark skin (Botan skin extract).

Example  6: NF - kB Assay

For NF-kB analysis, a method using Luciferase (Luciferase) described in Patent Publication No. 506043 was carried out. NIH3T3 from Panomics (Rev A 060820 Stable Cell Line: NIH3T3 / NFkB-luc 1); Stable Cell Line: NIH3T3 / NFkB-luc was used. Put 15 mL of pre-warmed Growth medium in a 100-mm culture dish or T-75 flask, and add the cell line to the prepared culture dish to disperse the cell line well. Place the culture dish in a humidified 37 ^° C. (5% CO ₂ ) incubator and change the medium every 2-3 days. Once the 90% confluency is reached, create a stock to cryopreserve and continue incubating. Cells are cultured according to passage method to preserve cell lines.

NF-kB measurement was performed as follows. Dispense the cells into a 12-well plate at 5 x 10 ⁵ / well level in Initial Growth Media, and incubate for 24 hours in moistened 5% CO ₂ (37 ^o C) to allow cell lines to adhere to the bottom. Replace with fresh medium that does not contain Serum. After incubating in 5% CO ₂ (37 ^o C) incubator for about 8 hours, the medium is removed and 100 ul lysis buffer is added to each well to measure Luciferase activity. For the measurement method, please refer to the Promega manual. (Promega P / N E1500)

NF-kB quantification using 96-Well was performed using trypsin-treated cell lines in the log-growth phase, adjusted to 5 x 10 ⁴ cells / 100 ul, and placed in a 96-well plate. Put together. 5% CO _{2 set} at 37 ^o C Incubate overnight in the incubator, NF-kB-induced medicinal herbs (TNF-alpha) at 15 ul volume level, after removing the medium 50 ul lysis buffer (Promega P / N E1500) in each well and treated. . After treatment at room temperature for 2 minutes with Lysis buffer in place, check if Lysis Buffer is fully dissolved. Rapidly freeze at 0 ^o C, dissolve at room temperature, and mix the solution 2-3 times. 20 ul of each lysate was transferred to a new 96-well, 20 ul of luciferase substrate was added to each well, mixed 2-3 times, and measured using a Luminometer.

Figure 4 shows the effect on the NF-kB production of herbal extracts. The fold of the Y-axis is 1 fold for the amount of NF-kB produced by the negative control group without the induction source. As a result of quantitative determination of NF-kB, the induction rate by induction source was 19.44 times, and 0.56% (V / V) and 0.28 of extract (Sample 1) when the reaction time of kiwi and pineapple solution and pine needle powder dispersion was 0 hour When treated with% (V / V), it was 4.09 times and 7.74 times, respectively, and when the reaction time was 1 week (Sample 2), they were 1.41 times and 3.24 times, respectively, at the same concentration as above, and reacted with kiwi and pineapple solution. It was confirmed that the amount of NF-kB produced was suppressed. In the case of bark skin (Botan bark extract), the production inhibitory effect is greater than that of 0.56% (V / V) and 0.28% (V / V) of 5.49 and 7.12 times, respectively.

The following product was prepared using the pine needle extract prepared according to the present invention, which is an example of an embodiment, and the invention is not limited thereto.

Manufacturing example : cream

1) Milkweed extract of the present invention (sample 2) 1.0% by weight / 2) stearic acid 2.0% by weight / 3) stearyl alcohol 3.0% by weight / 4) 1,3 butylene glycol 5.0% by weight / 5) glycol monostearate 4.0 % By weight / 6) Potassium hydroxide 0.1% by weight / 7) Purified water 77.9% by weight / 8) Lecithin 5.0% by weight / 9) Cholesterol 1.0% by weight

The oil and water components were heated to 75 ^° C., respectively, and then emulsified using a homogenizing mixer in a cream manufacturing mixer, followed by degassing, filtration and cooling to prepare a cream.

Manufacturing example : Lotion

1) Milkweed extract of the present invention (sample 2) 0.1% by weight / 2) 1,3-butylene glycol 8.0% by weight / 3) glycerin 2.0% by weight / 4) xanthan gum 0.02% by weight / 5) citric acid 0.01% % / 6) Sodium citrate 0.1 wt% / 7) Ethanol 5.0 wt% / 8) Methyl paraoxybenzoate 0.1 wt% / 9) Polyoxyethylene hardened castor oil (40E.O.) 0.1 wt% / 10) Perfume 0.1 wt% % / 11) 84.47 wt% of purified water

The components 1-6, 11, and 7-10 were melt | dissolved uniformly, respectively, and both were mixed and filtered.

Manufacturing example : Ointment

1) Milkweed extract of the present invention (sample 2) 1.0% by weight / 2) polyoxyethylene cetyl ether (30 EO) 2.0% by weight / 3) glycerin monostearate 10.0% by weight / 4) liquid paraffin 5.0% by weight / 5) Cetanol 6.0 wt% / 6) Propylene glycol 10.0 wt% / 7) Methyl paraoxybenzoate 0.1 wt% / 8) Purified water 64.9 wt%

Mix components 2-5 by dissolution by heating, and make oily phase at 70 ^° C. Components 1 and 6-7 are dissolved by heating and mixed, and maintained at 75 ^° C. to form an aqueous phase. Emulsify the oil phase and the water phase together and cool the product to 30 ^o C.

Manufacturing example : Bath Solvent

1) Milkweed extract of the present invention (Sample 2) 5.0 wt% / 2) Sodium bicarbonate 50.0 wt% / 3) Yellow No. 202 0.05 wt% / 4) Flavor 0.25 wt% / 5) Anhydrous sodium sulfate 44.7 wt%

Mix 1-5 components evenly.

Manufacturing example : beverage

1) pine needle extract of the present invention (sample 2) 1.0% by weight / 2) citric acid 5.0% by weight / 3) flavor 0.1% by weight

Water is mixed with components 1-3 to make 100% by weight in total.

Manufacturing example : refine

1) Milkweed extract of the present invention (sample 2) 2.5% by weight / 2) 20.0% by weight per tablet / 3) carboxymethyl cellulose 27.5% by weight / 4) microcrystalline cellulose 35.0% by weight / 5) polyvinylpyrrolidone 5.0 % By weight / 6) Talc 10.0% by weight

After mixing components 1-4, the aqueous solution of component 5 is added as a binder and granulated by a conventional method. The compound 6 is blended here as a lubricant, and then compressed into tablets.

Claims (7)

Preparing a liquid (A solution) crushed kiwi and pineapple;
Pulverizing pine wood bark to make a solution (B solution) dispersed in water; And
Mixing and reacting the solution A and solution B, so that the enzyme of the kiwi and pineapple grinding solution to act on the components of the pine wood bark pulverization comprising the step of extracting the pine needles. The method of claim 1, further comprising the step of reacting the solution A and solution B, followed by hot water extraction. Pine tree extract prepared by the method of claim 1. A pharmaceutical composition effective for suppressing abnormal angiogenesis, comprising the extract of pine needles of claim 3. Tumor, psoriasis, diabetic retinopathy, renal artery disease, rheumatoid arthritis, osteoarthritis, chronic inflammation, hemangioma, atopic dermatitis, inflammatory disease, inflammatory bowel disease, wound, including fibrous extract of claim 3 Pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of hyperplasia and eczema. Tumor, psoriasis, diabetic retinopathy, renal artery disease, rheumatoid arthritis, osteoarthritis, chronic inflammation, hemangioma, atopic dermatitis, inflammatory disease, inflammatory bowel disease, wound, including fibrous extract of claim 3 Food composition for the prevention or amelioration of a disease selected from the group consisting of hyperplasia and eczema. A cosmetic composition for alleviating or ameliorating dermatitis or atopic dermatitis, comprising the botanical extract of claim 3.

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