KR20120031800A - An antioxidant, antidiabetic and antiaging composition containing mulberry fruit seed extracts - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing oil and a MeOH extract of Morus alba is provided to ensure strong antioxidation and antiaging activities. CONSTITUTION: A pharmaceutical composition for preventing or treating diabetes contains a Morus alba seed extract as an active ingredient. The seed extract is isolated using oil and MeOH. A health food composition for preventing or treating diabetes contains the extract as an active ingredient. A health food composition for antioxidaiton or anti-aging contains the extract as an active ingredient.

Description

Antioxidant, antidiabetic and antiaging composition containing mulberry fruit seed extracts

The present invention relates to an antioxidant, anti-diabetic and anti-aging active compositions containing mulberry fruit seed extract, more specifically, mulberry (Morus alba ) (MA) and Cudramia tricuspidata ) (CT) relates to a pharmaceutical composition for preventing or treating diabetes, a dietary supplement for preventing or improving diabetes, and an antioxidant and anti-aging active cosmetic composition comprising an oil and MeOH extract extracted from a seed of a fruit as an active ingredient. .

Functional foods based on plants have been reported to play an important physiological role in the prevention of several pathological diseases such as cancer, cardiovascular disease, arthritis, cataracts and aging. In particular, it is known that plant seeds obtained as by-products during fruit juice processing may be excellent raw materials for essential fatty acids and functional foods containing important phytochemicals that may be effective for degenerative diseases. Seed residues left after oil extraction are also emerging as a useful source of dietary antioxidants. For these reasons, there is a need for research on the functional components and biological activities of plant seeds in order to use them as a dietary supplement source.

In Korean traditional medicine, two types of mulberry, Morus alba (MA) and Cudramia tricuspidata ) (CT) has been widely used. Audi, the fruit of mulberry (MA), is currently used to treat diabetes, baldness, hangover, high blood pressure and inflammation. Audi is also rich in sugars, organic acids, anthocyanins, etc., which are important in favorite foods such as beverages, jelly and jams, and have 1-deoxynozirimycin (DNJ), which has antidiabetic, antihypercarcinoma, antitumor, antioxidant and anti-aging activities. ), γ-aminobutyric acid (GABA), caffeic acid, rasveratrol derivatives, flavonoids and arylbenzofuran.

On the other hand, kkujippong tree (CT) has recently been studied as an anticancer, anti-inflammatory, anti-neuritis and antimicrobial treatment as well as skin irritation. Several bioactive substances, such as prenylated xanthones and flavonoids, have been reported to be the major cause of important biological effects of CT.

In relation to the physiological activity of Cudrania japonica, Oh et al. Inhibitory effect of histamine release and COX-2 activity by kkujippong glycoprotein in RBL-2H3 cells; Korean Journal of Food Science and Technology, Vol. 41, No. 4, No. 206 (August 2009) pp.405-412 0367-6293] is a substance in which KUJIJUNG glycoprotein has antiallergic effect. It is disclosed that it can be used as a prophylaxis and treatment of diseases, reports of the state, such as [Jun Heon-young, Jeon Ji-taek; Korean Journal of Food Science and Technology, Vol. 41, No. 1, No. 203 (February 2009) pp.93-99 0367-6293], KUJIJUNG glycoprotein is an excellent natural antioxidant that can suppress liver toxicity and inflammatory reactions. Is disclosed.

In addition, Republic of Korea Patent Application No. 10-2002-0030093 'composition comprising a kkujippong extract having inhibitory hepatitis C activity' kkujippong ( Maclura) showing the prevention and treatment effect of hepatitis C polar and nonpolar solvent soluble extracts of tricuspidata CARR.) are disclosed.

In addition, as a technique using kkujippong, kkujippong oil extraction, purification method and device (Korean Patent Application No. 10-2008-0039349), kkujippong oil extraction, purification method and device (Korea Patent Application No. 10-2002-0082420), Kujippong tea and a manufacturing method thereof (Korean Patent Application No. 10-2008-0052258), a manufacturing method of the wax barley bread using various herbal ingredients such as chalbary and jjjippong (Korean Patent Application No. 10-2007-0112507) have.

To date, much research has been conducted in screening biologically active compounds from various parts of the two types of mulberry. However, there is little useful information on the functional components and biological activities of seeds obtained as by-products from mulberry juice and wine production.

Therefore, the inventors of the present invention have repeatedly studied to identify functional components and biological activities from the seeds of the mulberry fruit by-products, and to confirm their physiological activity. The present invention has been completed by quantifying the compounds and confirming their physiological activity.

Therefore, it is an object of the present invention to provide a pharmaceutical composition for treating or preventing diabetes containing the mulberry fruit seed extract as an active ingredient.

Another object of the present invention is to provide an antioxidant, anti-diabetic or anti-aging active dietary supplement composition containing the mulberry fruit seed extract as an active ingredient.

Another object of the present invention is to provide an antioxidant or anti-aging active cosmetic composition containing the mulberry fruit seed extract as an active ingredient.

The object of the present invention as described above Mulberry ( Morus alba ) (MA) fruit or Cudramia tricuspidata ) (CT) Prepare oil and MeOH extracts from fruit seeds, analyze their fatty acid composition, tocopherols and phytosterols, determine total polyphenol and total flavonoid content, while their antioxidant, antidiabetic and anti-aging activities It was achieved by confirming that the mulberry and Cudrania fruit seed extracts exhibited high levels of polyphenol and flavonoid content and potent biological activity, which could be very effective for antioxidant, antidiabetic and anti-aging activities.

The present invention provides a pharmaceutical composition for treating or preventing diabetes containing the mulberry fruit seed extract as an active ingredient.

The present invention also provides an antioxidant, antidiabetic or anti-aging active health food composition containing the mulberry fruit seed extract as an active ingredient.

The present invention also provides an antioxidant or anti-aging active cosmetic composition containing the mulberry fruit seed extract as an active ingredient.

In the present invention, the mulberry fruit seeds are mulberry ( Morus alba ) (MA) fruit or Cudramia tricuspidata ) (CT) Fruit seed.

In the present invention, the mulberry fruit seed extract is characterized in that the mulberry fruit seed oil extract or MeOH extract.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a pharmacologically active ingredient or the like, which is expected to express directly or indirectly the efficacy and effect of the drug by intrinsic pharmacological action). It means containing a main component).

In the present invention, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and "functional" means a human body It means the ingestion for the purpose of obtaining a useful effect in health use such as nutrient control or physiological action on the structure and function of.

Pharmaceutical compositions comprising the mulberry fruit seed extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions comprising the mulberry fruit seed extract of the present invention, oral formulations, external preparations, suppositories, and sterile injectables such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, respectively, according to a conventional method Formulated in the form of can be used.

Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the mulberry fruit seed extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate , Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the mulberry fruit seed extract according to the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the mulberry fruit seed extract of the present invention is preferably administered in 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Mulberry fruit seed extract according to the present invention can be administered to various mammals such as mice, mice, livestock, humans. All modes of administration can be expected, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injections and directly to the skin.

In addition, the mulberry fruit seed extract according to the present invention may be added to food or beverage for the purpose of preventing and improving diabetes. Examples of the food to which the mulberry fruit seed extract can be added include various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages. At this time, the amount of the mulberry fruit seed extract in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added in proportion.

The food composition of the present invention is not particularly limited to other ingredients except the mulberry fruit seed extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the mulberry fruit seed extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In addition, the mulberry fruit seed extract of the present invention may contain the flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so important, but the mulberry fruit seed extract of the present invention is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight.

Cosmetic compositions of the present invention further comprise physiologically acceptable media such as fatty substances, preservatives, vitamins, gelling agents, flavoring agents, surfactants, water, antioxidants, fillers, wetting agents, masking agents and mixtures thereof. can do.

The cosmetic composition according to the present invention may be prepared and used in the formulation of cosmetics, cosmetics, makeup products, essences, creams, lotions, packs, foundations, makeup bases and the like. It can be applied in various forms such as liquid, cream, paste, solid, and can be prepared by a conventional cosmetic preparation method.

The cosmetic composition according to the present invention adds 1 to 10% by weight, preferably 1 to 5% by weight, of the mulberry fruit seed extract as a dry weight when preparing cosmetics.

In the present invention, the oil content, fatty acid, tocopherol and phytosterol composition and content of the seeds of two mulberry fruits, Audi and Cudrania fruit, were measured. In addition, polyphenol compounds and biological activities of the degreased mulberry and Cudrania fruit seed residue extracts were evaluated. The oil contents of the Audi and Cudrania fruit seeds were 29.63% and 16.69%, respectively, and the defatted Audi and Cudrania Fruit Seed residue MeOH extracts were 5.10% and 6.22%, respectively. The two seed oils consisted of 74.37 and 81.4% linoleic acid, 5.75 and 11.39% oleic acid, 8.40 and 10.18% palmitic acid and 3.0 and 3.52% stearic acid and two trace fatty acids linolenic acid and arachidic acid. . Audi seeds contained higher levels of phytosterols (507.59 mg / 100g of oil), tocopherols (99.64 mg / 100g of oil) and total flavonoids (106.50 mg / 100g of oil) than courageous seeds, whereas Contains high levels of total polyphenols. MeOH extract of the mulberry seed residue showed higher antioxidant, antidiabetic and anti-aging activity than the Cudrania fruit seed residue. t-resveratrol (9.62mg / 100g of oil), quercetin (54.83mg / 100g of oil) and 4-prelylamorin (48.70mg / 100g of oil) are found to be the main polyphenolic components of the Audi seed residue MeOH extract lost.

Thus, in the present invention, the mulberry seeds showed higher plant bioactive substance content and biological activity than the Cudrania fruit seeds, and were found to be an excellent source of essential fatty acids in abundant tocopherol and phytosterol amounts. Such high levels of polyphenol and flavonoid content and potent biological activity of the mulberry seed residue may be useful as dietary sources of natural antioxidants. In particular, mulberry seeds consume high amounts of resveratrol, quercetin and 4-prelylacin with high levels of anticancer, antihypertensive and antidiabetic activity, leading to consumption of the mulberry fruit in several pathological diseases, for example, coronary heart disease, diabetes. It may play an important role in preventing atherosclerosis, cancer and aging.

As described above, the mulberry seed extract according to the present invention exhibits a high level of polyphenol and flavonoid content and strong biological activity, which is very effective for antioxidant, antidiabetic and anti-aging activity, and thus is an excellent source of essential fatty acid or natural antioxidant. There is a useful effect as a dietary source.

1 is a diagram showing HPLC chromatograms of seven standard phenolic compounds (A) and MeOH extract of Audi seed residue. 5: t -resveratrol, 6: quercetin, 7: 4-prerylmoracin

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example

Morus ( Morus) in the following examples alba ) (MA) fruit (hereinafter referred to as 'audio') and Cudramia Seeds of tricuspidata ) (CT) fruit (hereinafter referred to as 'Cujippong fruit') were obtained from a large amount of pressed residues produced during juice production. Two kinds of seeds were separated from each residue, washed with running water, and then dried at room temperature.

Free fatty acids, four tocopherol isomers (α-, β-, γ-, and δ-tocopherol), three phytosterols (camphorsterol, stigmasterol, β-sitosterol), 1, l-diphenyl-2-picrylhydrazil (DPPH), α-glucosidase, mushroom tyrosinase, quercetin, rutin, L-dihydroxyphenylalanine (L-DOPA) and t-resveratrol are Signa Chemical Co .; St. Louis, Mo, USA Purchased from Potassium hydroxide, aluminum chloride, Folin-Ciocalteau's phenol reagent and t-butylated hydroxytoluene (BHT) were purchased from Wako Pure Chemicla Ind .; Osaka, Japan. . HPLC solvents were purchased from Merck (Darmstadt, Germany). All other reagents used in the present invention were those of analytical grade.

Example 1: Oil and MeOH  Extract manufacturer

In this example, the seeds of Audi and Cudrania fruit were respectively powdered, and the obtained seed powder (10 g) was treated with 0.05% BHT containing petroleum ether (100 mL) for 1 hour in an ultrasonic cleaner (Bransonic 5210R-DTH, USA). Extracted once, filtered through filter paper (Whatman No. 2; Whatman Laboratory Products, Clifon, NL, USA), and finally the solvent was evaporated under reduced pressure. Oil components are shown based on seed dry weight.

Meanwhile, the two seed residues obtained after oil extraction were extracted twice with an ultrasonic cleaner for two hours with an aqueous 90% MeOH solution (200 mL), filtered, and the solvent was evaporated in vacuo. The crude MeOH extract was dissolved in the same solvent and allowed to stand overnight in refrigerated state, and the resulting solution was centrifuged at 3,000 rpm for 30 minutes, and the upper layer was evaporated in vacuo to give a MeOH extract.

Yields (%) of oil and MeOH extract of Audi and Cudrania fruit seeds are shown in Table 1 below.

Recovery of Oil and MeOH Extracts from Audi and Cudrania Seeds Mulberry fruit seeds Yield (%, dry base) Oil extract MeOH Extract Audi seeds 29.36 ± 0.50 ^a 5.10 ± 0.11 ^b Cudrania Fruit Seeds 16.69 ± 1.39 ^b 6.22 ± 0.15 ^a Each figure is the mean ± SD of three analyzes
Subscripted values in columns differ significantly at p <0.05

The yields of oil and MeOH extracts of Audi seeds were 29.36 and 5.10%, respectively, while the yields of oil and MeOH extracts of Cudrania fruit seeds were 16.69 and 6.22%, respectively. From this, it was found that the yield of the oil extract of the Audi seed was significantly higher than the yield of the oil extract of the Cucumis fruit seeds, while the yield of the MeOH extract of the Audi seed residue was somewhat lower than the yield of the MeOH extract of the Cuzipan fruit seed residue. Can be.

These data support the varying yields of oil and MeOH extracts of plant seeds depending on plant type, variety, maturity and processing. It is to be noted that the Audi seeds have a higher oil content than other plant seeds, including Cudrania seeds. The yield of mulberry seeds obtained as a by-product during the processing of mulberry juice was about 3-5% per 100 g of mulberry fruit.

Experimental Example

In the following experimental examples, fatty acid composition analysis, tocopherol analysis, phytosterol analysis, total polyphenol and flavonoid content measurement, antioxidant, antidiabetic and anti-aging activity analysis of the oil and MeOH extracts of the mulberry seed and zigzag fruit seeds obtained in the above examples , Phenolic compound quantification was performed.

All data are presented as mean ± standard deviation (SD) for two samples of each seed, analyzed three times individually. IC ₅₀ values were determined by regression analysis of results obtained at three different concentrations of samples. Statistical analysis was performed using Duncan's multi-range test at p <0.05.

Experimental Example 1 Analysis of Fatty Acid Composition

In the present experimental example, the fatty acid (FA) composition of Audi and Cudrania seed oil was measured by gas chromatography (GC) according to a conventional method. GC conditions were as follows: SupelcowaxTM-10 fusion-silica capillary column (60 m × 0.25 mm Id), FID detector, carrier gas (He, 52.5 mL / min), injector (250 ° C.), oven (190 ° C.), Detector (260 ° C.), thermometer.

The fatty acid composition of the Audi and Cudrania seed oil are shown in Table 2 below.

Fatty Acid Content of Audi and Cudrania Seed Oil fatty acid Content (% by weight) Audi seeds Cudrania Fruit Seeds Palmitic acid (C _{16 : 0} ) 8.40 ± 0.01 10.18 ± 0.00 Palmitoleic acid (C _{16 : 1} ) ND ^{1 )} 0.15 ± 0.00 Stearic Acid (C _{18 : 0} ) 3.52 ± 0.00 3.00 ± 0.01 Oleic Acid (C _{18 : 1} ) 5.75 ± 0.00 11.39 ± 0.01 Linoleic Acid (C _{18 : 2} ) 81.40 ± 0.00 74.37 ± 0.01 Linolenic acid (C _{18 : 3} ) 0.63 ± 0.00 0.58 ± 0.00 Arachidic acid (C _{20 : 0} ) 0.30 ± 0.00 0.33 ± 0.02 ∑SFA ^{2 )} 12.22 ± 0.00 13.51 ± 0.02 ∑USFA ^{3 )} 87.78 ± 0.00 86.49 ± 0.02 Each figure is the mean ± SD of three analyzes
^{1) not} detected
²⁾ total saturated fatty acid
³⁾ Total unsaturated fatty acid

The Audi seed oil consisted of 8.40% palmitic acid, 3.52% stearic acid, 5.75% oleic acid, 81.40% linoleic acid, 0.63% linolenic acid and 0.30% arachidonic acid. Cudrania fruit seed oil consisted of 10.18% palmitic acid, 0.15% palmitoleic acid, 3.0% stearic acid, 11.39% oleic acid, 74.37% linoleic acid, 0.58% linolenic acid and 0.33% arachidonic acid. From this, a similar oil composition was observed although the fatty acid composition was slightly different between the two mulberry seed oils. Of the fatty acid compositions detected, linoleic acid was the main fatty acid, followed by palmitic acid and oleic acid, and other fatty acids such as stearic acid, linolenic acid and arachidonic acid were minor oil components. In addition, total saturated fatty acid (SFA) content ranged from 12.22% in Audi seed oil to 13.51% in Cudrania seed oil, and total unsaturated fatty acid (USFA) content ranged from 86.49% in Cudrania seed oil to 87.78% in Audi seed oil. It was.

Experimental Example  2: Tocopherol Analysis

In this experimental example, the quantification of four tocopherol isomers in the Audi and Cudrania fruit seed oils was performed according to a conventional method. HPLC conditions were as follows: LiChrosorb DIOL column (5 μm, 3 × 100 mm, Merck Co., Chrompack, Palo Alto, CA, USA); 295 nm UV detector (Younglin Absorbance, Seoul, Korea); Fluidized bed, n-hexane-acetic acid (1000: 1, v / v); Flow rate 0.5 mL / min.

The contents of the three tocopherol isomers, α-, γ-, and β-tocopherols of the Audi and Cudrania fruit seeds are shown in Table 3 below.

Tocopherol Content in Audi and Cudrania Seed Oil Mulberry fruit seeds Tocopherol (mg / 100g, seed oil) α-tocopherol γ-tocopherol δ-tocopherol synthesis Audi seeds 6.23 ± 0.34 17.96 ± 0.76 75.45 ± 1.82 99.64 ± 0.97 Cudrania Fruit Seeds 4.45 ± 0.39 7.46 ± 0.28 4.16 ± 0.18 16.07 ± 0.28 Each figure is the mean ± S.D. of three analyzes

Audi seed oil had a total tocopherol content of 99.64 mg%, of which 6.23 mg% for α-tocopherol, 17.96 mg% for γ-tocopherol and 75.45 mg% for β-tocopherol. On the other hand, the Cudrania fruit seed oil had a total tocopherol content of 16.07 mg%, of which 4.45 mg% of α-tocopherol, 7.46 mg% of γ-tocopherol and 4.16 mg% of β-tocopherol. The three tocopherol levels of the OD seeds were significantly higher than the KUJIGDON fruit seeds, in particular the γ- and β-tocopherol contents of the OD seeds were about 2.4 and 18 times higher than those of the KUJIDGE fruit seeds. It was found that Audi seeds could be an excellent source of vitamin E, a natural antioxidant.

Experimental Example 3: Phytosterol Analysis

In this experimental example, three phytosterol quantifications of the Audi and Cudrania fruit seed oils were performed according to a conventional method. Seed oil (0.1 g) was saponified with 2N KOH in EtOH and then injected into GC. Conditions for GC are as follows: Ultra 2 fusion-silica capillary column (60m X 0.25mm Id), FID detector, carrier gas (He, 52.5mL / min), injector (300 ° C), oven (285 ° C), detector (300 ℃), thermometer.

Phytosterol content of the Audi and Cudrania fruit seeds are shown in Table 4.

Phytosterol Content of Audi and Cudrania Seed Oil Mulberry fruit seeds Phytosterol (mg / 100g, seed oil) Campestrol Stigmasterol β-sitosterol synthesis Audi seeds 36.64 ± 7.20 10.29 ± 1.12 460.66 ± 8.27 507.59 ± 5.53 Cudrania Fruit Seeds 24.54 ± 0.22 16.47 ± 2.52 410.94 ± 9.74 451.95 ± 4.16 Each figure is the average S.D. of three analyzes

Three phytosterol derivatives, campestrol, stigmasterol and β-sitosterol were found, of which β-sitosterol was the main component. Audi seed oils were 34.64 mg% camphorsterol, 10.29 mg% stigmasterol and 460.66 mg% β-sitosterol, with a total phytosterol content of 507.59 mg%. Cudrania fruit seed oil was 24.54 mg% camphorsterol, 16.47 mg% stigmasterol and 410.94 mg% β-sitosterol, with a total phytosterol content of 451.95 mg%. From these results, it was found that the total phytosterol level of the mulberry seeds was higher than that of the Cudrania fruit seeds. This was higher than the Cudrania fruit seeds.

Experimental Example  4: Determination of total polyphenol and flavonoid content

In the present experimental example, the total polyphenol content of the mulberry extract and the zigzag fruit seed MeOH extract were measured by using a Pauline-Siocalceu phenol reagent by modifying a conventional method such as Singleton. Seed extract (1 mL) and Pauline-Siocalteu reagent (0.5 mL) were placed in a 100 mL volumetric flask. After 3 minutes, 20% Na ₂ CO ₃ solution (2 mL) was added and filled with distilled water. After 1 hour, the absorbance was measured at 760 nm using a UV-vis spectrophotometer (Sinco, Seoul, Lorea). The total polyphenol content is expressed in gallic acid equivalents (mg / seed 100 g) in a standard calibration curve.

Flavonoids content was determined by modifying the colorimetric assay, such as laver. The sample solution (0.5 mL) was mixed with 0.1 mL of 10% aluminum nitrate nonhydrate, 0.1 mL of 1.0 M potassium acetate, and 4.3 mL of 80% MeOH, followed by vigorous vortexing. After incubation at room temperature for 40 minutes, the absorbance of the reaction mixture was measured at 415 nm. As a blank group, 10% aluminum nitrate was replaced with an equal amount of distilled water. The total flavonoid content in the standard assay curve is expressed as rutin equivalent (mg / seed 100 g).

The polyphenols and flavonoids content of the Audi and Cudrania fruit seed residues are shown in Table 5 below.

Total Polyphenols and Flavonoids Content of MeOH Extracts from Audi and Cudrania Fruit Seeds Mulberry fruit seeds Total polyphenols
(mg / 100g, dry base) Total polarvonoids
(mg / 100g, dry base) Audi seeds 367.26 ± 7.89 106.50 ± 3.40 Cudrania Fruit Seeds 500.09 ± 11.46 74.20 ± 1.72 Each figure is the mean ± S.D. of three analyzes

The total polyphenol contents of the mulberry and Cudrania seed MeOH extracts were 367.26 and 500.09 mg%, respectively, and the total flavonoid contents were 106.52 and 74.26 mg%, respectively. Therefore, the total polyphenol content was higher in the case of odyssey seeds than in the case of Cudrania fruit seeds, and the total flavonoid content was higher in the case of Cudrania fruit seeds. It should be noted that although the total phenolic content of the mulberry seed MeOH extract was lower than that of the Cudrania fruit seeds, it had a higher total flavonoid content.

Experimental Example 5 Analysis of Biological Activity of MeOH Extract

In the present experimental example, the antioxidant, antidiabetic and anti-aging activities of the mulberry seedlings and the zigzag fruit seed MeOH extract were measured using DPPH radical, α-glucosidase and trocinase, respectively, in an in vitro assay system.

Experimental Example 5-1: Antioxidant Activity Assay

In the present experimental example, the antioxidant activity of the mulberry seedlings and the kkujippong fruit seed MeOH extract was measured by the DPPH radical by modifying the method of Blyze. MeOH solution (200 μL) of samples with varying concentrations (0.1-10 mg / mL) was added to 0.1 mM methanol solution (4 mL) of DPPH and shaken vigorously. The reaction mixture was allowed to stand at room temperature for 10 minutes and the absorbance was measured at 517 nm. Radical scavenging activity is expressed as percent inhibition of free radicals by the sample and was calculated using the following formula:

DPPH radical scavenging activity (%) = (control OD-sample OD / control OD) × 100

Experimental Example 5-2: Antidiabetic Activity Analysis

In the present experimental example, the anti-diabetic activity of the mulberry seedlings MeOH extract was measured by using the α-glucosidase by modifying the method such as lamb. Sample solution [50 μL, 0.01-1.0% in MeOH (5% final concentration)] was added to α-glucosidase (0.15 unit / mL) (50 μL) in the test tube. The reaction mixture was allowed to stand at 25 ° C. for 10 minutes, then 3 mM p-nitrophenyl-α-D-glucopyranoside (pNPG, 100 μL) in phosphate buffer (pH 6.8) was added to initiate the reaction. After the reaction was performed at 37 ° C. for 15 minutes, the reaction was stopped by addition of 0.1 M Na ₂ CO ₃ (750 μL). α-glucosidase inhibitory activity was evaluated by measuring the release rate of p-nitrophenol from pNPG at 405 nm.

Experimental Example 5-3: Anti-aging Activity Measurement

In the present experimental example, the anti-aging activity of the mulberry seedlings MeOH extract was measured using mushroom tyrosinase according to the method such as L-DOPA as a substrate. The reaction mixture (3 mL) using L-DOPA as a substrate containing 1 mL of 1.5 mM L-DOPA solution, 0.1 mL of sample containing (or without) DMSO and 1 / 15M phosphate buffer solution (pH6.8) was 25 ° C. Incubated for 10 minutes at. 0.1 mL of mushroom tyrosinase in aqueous solution was further added to the mixture to immediately determine the initial linear increase in optical density at 475 nm. Control reactions were performed with DMSO. The percent tyrosinase inhibition was calculated by the following formula:

% Inhibition = (A-B) / A X 100

Wherein A is the absorbency of the test solution and B is the absorbency of the control solution.

The results of Examples 5-1 to 5-3 are shown in Table 6 below.

Comparison of Biological Activity of MeOH Extracts from Audi and Cudrania Fruit Seeds Mulberry fruit seeds Inhibitory activity (IC ₅₀ , mg / mL) DPPH RSA ^{1 )} α-glucosidase Tyrosinase Audi seeds 0.15 ± 0.01 0.87 ± 0.01 1.01 ± 0.10 Cudrania Fruit Seeds 0.74 ± 0.03 2.79 ± 0.05 2.54 ± 0.13 Each figure is the mean ± SD of three analyzes
¹⁾ radical scavenging activity

As shown in Table 6, the mulberry extract and mulberry seed MeOH extract showed a high radical scavenging activity and inhibitory activity in a dose-dependent manner, 50% inhibition concentration (IC ₅₀ ) of each extract was calculated from the results. Odysed seeds had higher clearance for DPPH radicals (IC ₅₀ = 0.15 mg / mL), α-glucosidase (IC ₅₀ = 0.87 mg / mL) and tyrosinase (IC ₅₀ = 1.01 mg / mL) compared to Kudjippon fruit seeds Activity and inhibitory activity. These results indicate that the Audi seed MeOH extract serves to prevent skin aging due to diabetes and lipid peroxidation, and contains an antioxidant polyphenol compound as shown in Table 5 above.

Experimental Example  6: Quantitative determination of polyphenolic compounds

In the present experimental example, the degreased seed residue (10 g) obtained from the mulberry fruit and mulberry fruit was extracted twice with an 80% EtOH aqueous solution (200 mL) in an ultrasonic grinder, filtered and the solvent was evaporated under reduced pressure. The EtOH extract was again dissolved in 100 mL of an 80% aqueous solution of EtOH and left overnight. The solution was centrifuged at 5,000 rpm for 20 minutes and the top layer was charged with 100 mL of the same solvent. Soluble extract (2 mL) was diluted twice, passed through a 0.45 μm membrane filter (Gelman, USA) and then injected into an HPLC system for polyphenol compound quantification. HPLC was performed on a Waters e2690 / 5 HPLC system with a 2998 photodiode array detector and a SM7 autosampler in a 10 μL loop. HPLC analysis was performed using a YMC-Pack Pro C ₁₈ column (46 mm Id × 250 mm, YMC Inc., USA) with Guard-Pak C18 precolumn inserts. H ₂ O of _{0.05% v / v H 3 PO} 4 ( solvent A) to MeOH-CH ₃ CN-H ₂ O using a linear gradient of (1:: 1 1, v / v, solvent B) for 60 min. Separation was performed by detecting at 250, 310 and 350 nm at 0.8 mL / min flow rate. Elution profiles are as follows: 0-5 min, 70% A, 30% B: 10-20 min, 50% A, 50% B; 30-40 minutes, 30% A, 70% B; 45-50 minutes, 0% A, 100% B; 55-60 minutes, 70% A, 30% B. The column was reduced to initial conditions for 10 minutes before the next injection. The retention times of the individual polyphenol compounds were compared by comparison with the retention times of three standard polyphenol compounds (resveratrol, quercetin and 4-prerylmoracin) isolated from the two mulberry fruits in the previous report. Peaks were identified by co-chromatography with conventionally isolated native samples. The linear correlation coefficient for each polyphenol compound was greater than 0.999. The concentration of the polyphenolic compound was determined by the calibration curve of the three standard polyphenolic compounds and expressed in mg% of dry weight. The recovery of resveratrol, 4-prenylmoracin and quercetin was 97%, 98% and 91%, respectively.

1 shows the HPLC chromatogram of the standard polyphenol compound (A) and the MeOH extract (B) of the Audi seed residue. Three major polyphenol compounds, for example resveratrol, quercetin and 4-prerylmoracin, were found in the MeOH extract of the Audi seed residue, their contents being 9.62, 54.83 and 48.70 mg per 100 g of seed residue, respectively. These results show that the three polyphenolic compounds contribute to the strong antioxidant, antidiabetic and anti-aging activity of the mulberry seed residue.

Claims (6)

A pharmaceutical composition for preventing or treating diabetes containing mulberry fruit seed extract as an active ingredient. The composition of claim 1, wherein the mulberry fruit seeds are Morus alba (MA) fruit or Cudramia tricuspidata (CT) fruit seeds. The composition of claim 1, wherein the mulberry fruit seed extract is a mulberry fruit seed oil extract or a MeOH extract. Health functional food composition for preventing or improving diabetes containing mulberry fruit seed extract as an active ingredient. Antioxidant or anti-aging active health food composition containing mulberry fruit seed extract as an active ingredient. Antioxidant or anti-aging active cosmetic composition containing mulberry fruit seed extract as an active ingredient.

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