KR101440372B1 - Food and pharmaceutical composition for anti-inflammation comprising extract of perilla sprout as effective component - Google Patents

Abstract

The present invention relates to a method for curing edema by injecting a food composition and a pharmaceutical composition containing perilla sprout extracts as active ingredients for preventing or curing inflammation and a therapeutic dose of perilla sprout extracts into mammal except for humans. The perilla sprout extracts can be used not only in pharmaceutical compositions for preventing and curing inflammatory diseases but also in functional food such as food and tea.

Description

FIELD OF THE INVENTION The present invention relates to an anti-inflammatory food comprising a perilla sprout extract as an active ingredient and a pharmaceutical composition comprising the perilla sprout as effective component.

The present invention relates to a food composition for preventing or improving inflammation containing perilla sprout extract as an active ingredient, and a method for treating edema by administering a therapeutically effective amount of a perilla sprout extract to mammals other than humans.

Atopic dermatitis is characterized by an intrinsic form (20-30%) due to IgE-mediated sensitization (extrinsic form, 70-80%) and non-IgE-mediated sensitization The clinical characteristics of atopic dermatitis are as follows: collapse of skin barrier due to severe itching, inflammation, increase of serum IgE, infiltration of eosinophil into inflamed area and Th1 (Type I helper T cell) And Th2 (Type II helper T cell) cells. In this process, atopic dermatitis, in the case of acute, Th2 cells develop biased and produce excessive amount of Th2 cytokine, which leads to increase of serum IgE and eosinophil. In addition, when atopic dermatitis is deepened, Th1 cytokine is also increased at the same time and the inflammatory reaction is further aggravated. Mast cells are known to be the primary cells that produce inflammatory disease mediators such as allergies or acute allergic reactions. Acute allergic reactions are caused by the release of histamine by IgE-associated antigens binding to FcεRI receptors in mast cells. After the activation of FcεRI, mast cells accelerate the inflammatory response by progressing the secretion and degranulation of mediators such as arachidonic acid metabolites and inflammatory cytokines.

In particular, itching, a major symptom of atopic dermatitis, is one of the major causes of worsening of the quality of life in patients with atopic dermatitis, as it causes aggravation of skin lesions and severe mental obstructive factors. Scratching of the affected area due to itching causes collapse of the skin barrier, and repeated irritation causes deterioration of the skin inflammatory site. Therefore, effective control of itching is an important target in the treatment of atopic dermatitis. Therefore, recent trend toward the treatment of atopic dermatitis is increasingly interested in the development of therapeutic agents using natural products with few side effects.

Perilla frutescens there is . acuta ) leaves have been used not only as food but also as medicines in Japan and China including Korea, and its efficacy is known to have toxin discharge, vigorous action, antibacterial and antipyretic action.

Korean Patent No. 0809304 discloses a composition for the treatment or prevention of inflammatory diseases, but is different from an anti-inflammatory food composition comprising the perilla sprout extract of the present invention as an active ingredient.

The present invention has been made in view of the above needs, and it is an object of the present invention to disclose various usefulness of the perilla sprout extract by showing the anti-inflammatory and anti-atopic effect by the perilla seed extract.

In order to solve the above problems, the present invention provides a food composition for preventing or improving inflammation, which comprises perilla seed extract as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease containing an extract of perilla spp. As an active ingredient.

The present invention also provides a method of treating edema by administering a therapeutically effective amount of a perilla sprout extract to a mammal other than a human.

According to the present invention, the food for preventing or improving inflammation, which comprises the perilla sprout extract, contains a substance obtained from a plant, so that the proinflammatory cytokines-induced allergy And is effective for treating inflammation and inflammation. Especially, it can be useful as a food and pharmaceutical composition for improving atopic dermatitis.

FIG. 1 is a graph comparing DPPH and ABTS radical scavenging activity according to the concentration of perilla sprout extract. FIG.
FIG. 2 is a graph comparing the inhibitory activities of TNF-.alpha. And IL-1β production by addition of LPS-activated RAW 264.7 cells at the concentration of perilla bud extract. # Was compared with the normal group at p <0.001, p <0.05 for **, p <0.01 for *** and *** <0.001 for *** compared to the control group treated with LPS alone do.
FIG. 3 is a graph comparing TNF-.alpha. And IL-1β production inhibitory activities of PMA and A23187-activated RPMCs cells according to addition of perilla sprout extract concentration. # Was compared with the normal group at p <0.001, and there was a significant difference when compared to the control group treated with PMA and A23187 at p <0.05, ** at p <0.01 and *** at p <0.001 .
FIG. 4 is a graph comparing reduction effects of xylene-induced ear edema and carrageenan-induced foot edema with addition of perilla seed extract concentration. * Indicates a significant difference when compared to a control group treated with xylene or carrageenan only at p <0.05, ** at p <0.01 and *** at p <0.001.

In order to accomplish the object of the present invention, the present invention provides a food composition for preventing or improving inflammation comprising perilla seed extract as an active ingredient.

In the food composition for preventing or improving inflammation according to the present invention, the perilla sprout extract may be an extract of perilla seed water, methanol, ethanol, propanol, butanol, ethyl acetate or a mixed solvent thereof, But is not limited thereto.

In the food composition for preventing or improving inflammation according to the present invention, the perilla sprout extract is prepared by pulverizing perilla sprouts dried at 4 to 6 ° C for 20 to 28 hours with a cold air dryer, Deg.] C for 5 to 7 hours, concentrated under reduced pressure, and lyophilized at -65 to -75 [deg.] C. More specifically, the perilla sprouts dried at 4 to 6 [deg.] C for 24 hours Followed by extraction with 80% ethanol at 60 ° C for 6 hours, concentration under reduced pressure, and freeze-drying at -70 ° C.

The food is not particularly limited as long as it can be ingested to increase anti-inflammatory activity.

When the extract of the present invention is used as a food additive, the extract may be directly added, used in combination with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the extract of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

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

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the extract of the present invention.

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

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease containing an extract of perilla spp. As an active ingredient.

In the pharmaceutical composition for the prevention or treatment of inflammatory diseases according to the present invention, the inflammatory disease is selected from the group consisting of atopic dermatitis, edema, dermatitis, allergies, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, pneumonia, gastric ulcer, gastritis, , Sjogren's syndrome and multiple sclerosis, including, but not limited to, rheumatoid arthritis, rheumatoid arthritis, shoulder pain, nephritis, myositis, hepatitis, cystitis, nephritis, rheumatoid arthritis, fibromyalgia, psoriatic arthritis, osteoarthritis, Cirrhosis, &lt; / RTI &gt; but not limited to, &lt; / RTI &gt;

The pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention may comprise 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the above extract, based on the total weight of the pharmaceutical composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the extract of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

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

The present invention also relates to

(a) The perilla sprouts dried at 4 ~ 6 ℃ for 20 ~ 28 hours were pulverized with a cold air dryer and then extracted with 70 ~ 90% ethanol at 55 ~ 65 ℃ for 5-7 hours, Preparing a perilla seed extract by lyophilization at 75 ° C; And

(b) treating the inflammation by administering a therapeutically effective amount of the perilla sprout extract prepared above to mammals other than humans,

Preferably,

(a) The perilla sprouts which were dried at 4 ~ 6 ℃ for 24 hours were crushed and then extracted with 80% ethanol at 60 ℃ for 6 hours. The cells were concentrated under reduced pressure and lyophilized at -70 ℃ to prepare perilla seed extract ; And

(b) A method for treating inflammation by administering a therapeutically effective amount of the above-mentioned perilla sprouts extract to mammals other than humans.

In the method of the present invention, the inflammation is preferably, but not exclusively, edema.

Said "therapeutically effective amount" of the present invention means an amount of therapeutic composition sufficient to exhibit a measurable biological response.

In the present invention, the mammal may be a mammal including, but not limited to, a human.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

1. Materials and Methods

(1) Material

A) Reagent

Tumor necrosis factor-α and IL-1β (interleukin-1β) were purchased from R & D Systems (Minneapolis, USA). PMA (Phorbol 12-myristate 13-acetate), calcium ionophore, A23187, xylene, aspirin, carrageenan, percoll, hematoxylin & eosin, RPMI 1640 medium, fetal bovine serum (FBS) and penicillin / streptomycin were purchased from Gibco BRL (Grand Islang, NY, USA), and were purchased from Sigma-Aldrich Respectively.

B) Production of perilla sprout extract

The seeds were stored at 4 ℃ for 2 ~ 10 months. Then, each seed was dipped in distilled water for 2 hours and then sterilized with 70% ethanol for 30 seconds. All the germination conditions were germinated in light of 20 ~ 25 ℃, humidity was maintained, and grown for 10 days in dark condition to promote length growth. The harvested sprouts vegetables were dried with a cold air dryer (MEX-230A, MST Lab. Co. Ltd., Korea). The cold air dryer was kept at 4 ~ 6 ℃ and dried for 24 hours. The dried sample was pulverized with a pulverizer (FM-681C, Hanil Electric., Korea) and reflux-cooled for 6 hours at 80 ° C in an ethanol solvent. The extract was depressurized with a rotary vacuum concentrator (Eyela A-1000S, Tokyo Rikakikai Co., Tokyo, Japan) using a filter paper (Advantec No. 2, Toyo Roshi Kaisha Ltd., Japan) Japan) to obtain 15 g of the extract, which was then stored at -20 ° C for use in the experiment.

C) Experimental animals

Seven week old male Balb / c mice and 10 week old Sprague Dawley rats were housed in an aseptic environment and purchased from Central Laboratory Animals (Seoul, Korea). They were purified for one week while feeding sufficient feed and water. Respectively. The incubation environment consisted of day and night cycles for 12 hours, and the temperature (20 ~ 22 ℃) and humidity (50 ~ 60%) were kept constant and tested according to the regulations of the Laboratory Animals Committee.

(2) Experimental method

A) Measurement of DPPH radical scavenging activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was measured by the Blois method. 100 μl of each sample was injected into a 96-well plate at the final concentration of 15, 30, 60, 125, 250 and 500 μg / ml, and 100 μl of 0.3 mM DPPH was added to the sample to obtain a total amount of 200 Lt; / RTI &gt; After incubation at room temperature for 10 minutes, the absorbance was measured at 540 nm using an ELISA reader (Molecular Devies, USA). The DPPH radical scavenging activity was expressed as a percentage of absorbance difference between the addition group and the no addition group of the sample solution.

DPPH radical scavenging activity (%) = {1- (absorbance of addition group / absorbance of no addition group)} x 100

B) Measurement of ABTS radical scavenging activity

The ABTS radical scavenging activity was obtained by mixing 7.4 mM of ABTS and 2.6 mM of potassium persulphate. The mixture was allowed to stand at room temperature for 24 hours to form radicals. The ABTS solution was incubated at 732 nm with an absorbance of 0.70 ± 0.03 (mean SE ) Was diluted with methanol and used. To 50 μl of the extract (1 mg / mL), 950 μl of ABTS solution was added, reacted in a dark place for 30 minutes, and the absorbance was measured at 732 nm.

ABTS radical scavenging activity (%) = {1- (absorbance of addition group / absorbance of no addition group)} x 100

C) RAW 264.7 cell and extract treatment

Rodent RAW 264.7 macrophages were purchased from fetal bovine serum (FBS), penicillin G (100 IU / mL) and streptomycin (100 μg / mL) from the American Type Culture Collection (ATCC, Rockville, using the RPMI 1640 medium with addition of CO ₂ to be moist enough to maintain 37 ℃ And cultured in an incubator (5% CO ₂ and 95% air). RAW 264.7 macrophages (1 × 10 ⁶ / mL) were treated with various concentrations of bud extract (50-200 μg / mL) for 2 hours and then cultured for 24 hours with LPS (1 μg / mL) Cytokines were measured.

D) Cell separation and drug treatment of RPMCs

Separation of rat peritoneal mast cells (RPMCs) from a healthy male Sprague Dawley rats was anesthetized with ether, and 10 mL of calcium-free HEPES-Tyrode buffer was added to the abdominal cavity to gently massage the abdominal cavity for about 90 seconds After the abdominal cavity was carefully opened, the cell suspension was taken using a Pasteur pipette and RPMCs were obtained by percoll density gradient method. To obtain sufficient cells, peritoneal mast cells obtained from 5 to 10 rats were mixed and used. Sprout extracts (50 ~ 200 ㎍ / mL) were pretreated with RPMCs (2 × 10 ⁵ ) for 10 min and then stimulated with PMA and A23187 for proinflammatory cytokine assay.

E) MTT analysis

As described above, rodent RAW 264.7 macrophages and RPMCs treated with bud extract were measured by MTT assay based on MTT reduction, which turned into purple formazan product by mitochondrial dehydrogenase of dense cells after 24 hours of culture.

F) Measurement of cytokine

RAW 264.7 macrophages were treated with LPS (1 ㎍ / mL) for 2 hours, and then incubated for 24 hours. After incubation for 24 hours, culture supernatants were obtained. RPMCs were stimulated simultaneously with PMA (30 ng / mL) and A23187 (10 μM) after 10 min of treatment with or without treatment of extracts (50-200 μg / mL) TNF-α and IL-1β were measured by ELISA method provided by R & D company. Briefly, each cell supernatant (100 μl) was injected into a plate coated with anti-mouse TNF-α antibody or IL-1β, reacted, washed well and incubated with horseradish peroxidase (Molecular Devices, USA). The quantification of each substance was performed by treating each substance with a concentration and reacting it to obtain a standard curve And the amount of the substance contained in the cell supernatant or serum was calculated.

G) Measurement of xylene induced ear edema

(50 ~ 200 mg / kg) and aspirin (50 mg / kg) were orally administered to Balb / c mice for 3 days once a day for 12 hours, and then the perilla seed extract And 30 μl of xylene was applied to the right ear and the lower surface of the left ear. After 1 hour, the mice were sacrificed without applying xylene to the left ear, and ear punches with a diameter of 7 mm were used. And weighed to measure the degree of ear edema.

A) Carrageenan (Induction of foot swelling)

(50 ~ 200 mg / kg) and aspirin (50 mg / kg) were orally administered to Balb / c mice for 3 days, respectively, in the same manner as the induction of xylene ear edema. After fasting time, carrageenan [300 ㎍ / paw (foot)] was injected into the right footpad after oral administration of the extract and aspirin in the same amount, and the left footpad remained steady. Foot edema was measured using verner digital caliper (Mitutoyo, Japan) 24 hours after the edema reached its peak.

Therefore,

All data were expressed as mean ± SD. Statistical analysis was performed with ANOVA and Student's t-test.

Example  1: Antioxidative effect of perilla sprout extract

To investigate the antioxidative effect of perilla sprout extract, DPPH and ABTS radical scavenging activities were investigated. First, we investigated the DPPH radical scavenging activity of perilla sprouts compared with BHT, which is well known as a synthetic antioxidant. As a result, as shown in FIG. 1, DPPH radical scavenging activity was higher than that of BHT at a concentration of less than 250 ㎍ / mL, and the effect was similar at 500 ㎍ / mL and 1,000 ㎍ / mL. To investigate the ABTS radical scavenging activity of perilla sprouts, we compared them with Trolox, which is well known as an antioxidant. As a result, as shown in FIG. 1, ABTS radical scavenging activity was lower than that of Trolox at a concentration of less than 500 μg / mL, but showed an ABTS radical scavenging activity such as Trolox at 1,000 μg / mL.

These results suggest that DPPH and ABTS scavenging activities of perilla sprout extracts are similar to those of BHT and Trolox, which are known synthetic antioxidants.

Example  2: activated RAW  In 264.7 cells, the perilla sprout extract Proinflammatory  Cytokine ( proinflammatory cytokines ) Inhibitory effect

To investigate the effect of perilla sprout extract on the production of proinflammatory cytokines in RAW 264.7 cells activated by LPS, TNF-α and IL-1β were investigated. As a result, the levels of TNF-α and IL-1β in the control group were significantly increased to 1856 ± 142 pg / mL and 804 ± 82 pg / mL, respectively, as compared with the normal group. However, treatment with perilla sprout decreased in a dose - dependent manner. In the case of TNF-α, there was no significant difference in the treatment with 50 ㎍ / mL perilla sprout extract compared to the control, but the effect was significantly suppressed in the 100 ㎍ / mL and 200 ㎍ / mL treatment groups (p <0.05 and p < 0.01). IL-1β was significantly inhibited (p <0.05, p <0.01 and p <0.001) in all of the perilla seed extracts compared to the control. Especially at the concentration of 200 ㎍ / mL extract, the effect of aspirin was similar.

Example  3: activated RPMCs  Of perilla sprout extract from cells Proinflammatory  Cytokine ( proinflammatory cytokines ) Inhibitory effect

TNF-α and IL-1β were investigated to determine the effect of perilla sprout extract on the proinflammatory cytokine production of PMA and A23187-activated RPMCs cells. As a result, as shown in FIG. 3, TNF-α and IL-1β in the control group were significantly increased to 1,118 ± 112 pg / mL and 383 ± 35 pg / mL, respectively, as compared with the normal group. However, treatment with perilla sprout decreased in a concentration - dependent manner. In the case of TNF-α, there was no significant difference in the treatment with 50 ㎍ / mL perilla sprout extract compared to the control, but the effect was significantly suppressed in the 100 ㎍ / mL and 200 ㎍ / mL treatment groups (p <0.05 and p < 0.01). IL-1β was significantly inhibited in all perilla seed extracts compared to the control (p <0.0, p <0.01 and p <0.001). Especially at the concentration of 200 ㎍ / mL extract, the effect of aspirin was similar.

Example  4: Inhibitory effect of perilla sprout extract on ear edema and foot edema

The effects of perilla sprout extract on xylene - induced ear edema and carrageenan - induced foot edema were investigated. As a result, the ear edema and foot edema in the control group were significantly increased to 7.43 ± 1.31 mg and 0.551 ± 0.081 g / mL, respectively, as compared with the normal group as shown in FIG. However, treatment with perilla sprout decreased in a concentration - dependent manner. In the 100 ㎍ / mL and 200 ㎍ / mL treatment groups, ear swelling and foot edema were significantly inhibited (p <0.05 and p &Lt; 0.01). Especially at the concentration of 200 ㎍ / mL extract, the effect of aspirin was similar.

Claims (5)

A food composition for preventing or improving inflammation, comprising perilla sprout extract as an active ingredient. The perilla seed extract according to claim 1, wherein the perilla seed extract is ground in a cold air dryer at 4 to 6 ° C for 20 to 28 hours, pulverized into perilla sprouts, extracted with 70 to 90% ethanol at 55 to 65 ° C for 5 to 7 hours, Concentrated and then freeze-dried at -65 to -75 ° C. A pharmaceutical composition for preventing or treating an inflammatory disease containing an extract of perilla sprouts as an active ingredient. 4. The method of claim 3, wherein the inflammatory disease is selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, Wherein the disease is selected from the group consisting of spondylitis, rheumatic fever, rheumatoid arthritis, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder circumflex, tendinitis, hay fever, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of inflammatory diseases. (a) The perilla sprouts dried at 4 ~ 6 ℃ for 20 ~ 28 hours were pulverized with a cold air dryer and then extracted with 70 ~ 90% ethanol at 55 ~ 65 ℃ for 5-7 hours, Preparing a perilla seed extract by lyophilization at 75 ° C; And
(b) administering to the mammal a therapeutically effective amount of the perilla sprouts extract to treat edema.

Images