KR20090085876A - The extracts and fractions of hippophae rhamnoides l - Google Patents

Abstract

A Hippophae rhamnoides L. extract and its fraction are provided to produce cosmetic or pharmaceutical composition of antioxidation or antiaging, heath food or antibiotics and composition for antidiabetes or health food. A Hippophae rhamnoides L. extract is isolated using water, alcohol or their mixture solvent. The alcohol is C1-C4 low alcohol. An organic solvent fraction of the Hippophae rhamnoides L. extract is produced using organic solvent. The organic solvent is n-hexane, ethyl acetae or n-butanol. The Hippophae rhamnoides L. extract and its fraction comprises pharmaceutical composition for preventing and treating diseases caused by excess accumulation of oxygen free radical, pharmaceutical composition for antiaging, cosmetic product of preventing antiaging and heath food for preventing and treating diabetes.

Description

The extracts and fractions of Hippophae rhamnoides L.

The present invention relates to an antioxidant, an antibiotic and an antidiabetic composition comprising a hawthorn extract or a fraction thereof as an active ingredient.

The recent increase in income has greatly improved the nutritional status of individuals, and the life expectancy is increasing to the level of developed countries due to the development of medical technology. Accordingly, the focus on the prevention of disease rather than the treatment of disease is focused on the demand for health foods of various types. As a biological defense system for preventing diseases, researches on natural products are being actively conducted, and researches on the functionality of natural products and the physiological activity of secondary metabolites contained in natural products are being conducted. It is also a natural plant that can eliminate reactive oxygen species (ROS), which are a direct cause of oxidative stress in vivo, which causes aging and geriatric diseases. Antioxidants present in foods are caused by peroxides. It is expected that it can be developed as an inhibitor and therapeutic agent of various aging diseases.

Hippophae rhamnoides L .; Vitamin tree) is a shrub belonging to Bodhi tree and contains more than 100 kinds of ingredients. Especially, it contains many effective ingredients such as vitamins (A, B, C, E, F, K). It grows well in cold winters and in summers, and has a strong adaptability to grow well even in barren areas. Already, various research fields such as measurement of antioxidant activity of mountain hawthorn, fatty acid content of mountain hawthorn, cancer prevention, immune system, etc. have been made, and it has been reported that hawthorn contains beta carotene. In addition, the active ingredient is measured through the instrumental analysis of the leaves of the mountain, and various foods are made from the mountain, but the research is not actively conducted in Korea.

Therefore, the present inventors measured the electron donating ability, SOD-like activity, antimicrobial and antifungal activity, ferric-thiocyanate activity, α-glucosidase activity of the root and stem extracts of the hawthorn tree and its fractions, and the extract of the present invention. And the fractions thereof can be used as antioxidants, antibiotics and antidiabetic compositions to complete the present invention.

It is an object of the present invention to provide a mountain extract or fractions thereof which exhibit antioxidant activity.

It is also an object of the present invention to provide a mountain extract or fractions thereof which exhibit antibacterial and antifungal activity.

In addition, it is an object of the present invention to provide a mountain extract or fractions thereof that exhibit antidiabetic activity.

In order to achieve the above object, the present invention is extracted from water, alcohol or a mixture of them ( Hippophae rhamnoides L .; Vitamin tree) extract.

In addition, the present invention provides a fraction obtained in each step by extracting the hawthorn extract in the order of n-hexane, ethyl acetate and n-butanol.

In addition, the present invention provides an antioxidant comprising the extract of the hawthorn or fractions thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by the accumulation of excessive amount of active oxygen containing the hawthorn extract or fractions thereof as an active ingredient.

In addition, the present invention provides an anti-aging pharmaceutical composition containing the hawthorn extract or a fraction thereof as an active ingredient.

The present invention also provides a cosmetic for preventing skin aging comprising the extract of the hawthorn or fractions thereof.

In another aspect, the present invention provides an antibiotic comprising the mountain extract or fractions thereof as an active ingredient.

In another aspect, the present invention provides a composition for anti-diabetic comprising the extract of the hawthorn or fractions thereof as an active ingredient.

In addition, the present invention provides a natural preservative comprising the mountain extract or fractions thereof as an active ingredient.

In addition, the present invention provides a health food for preventing and improving diabetes comprising an extract of the hawthorn or fractions thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is a mountain ( hippophae rhamnoides L.) extract.

The hawthorn can be used without limitation, such as cultivated or commercially available. In addition, the hawthorn extract according to the present invention may be prepared as follows. The mountain roots and stems are washed thoroughly with water and then dried in the shade and at room temperature for 7 days. The dry mountain roots and stems are pulverized, placed in an extraction container, and extracted with a solvent for a predetermined time at an appropriate temperature. After obtaining the extract of the hawthorn, the solids may be removed using a filter paper, the suspension may be centrifuged, and the supernatant may be filtered under reduced pressure. The extraction solvent is preferably a solvent selected from water, an alcohol or a mixture thereof, preferably a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, more preferably methanol, but is not limited thereto. no. The amount of the extraction solvent is 2 to 10 times, preferably 4 times, the dry weight of the hawthorn. The extraction method may be an extraction method such as immersion extraction, heat extraction, reflux cooling extraction and ultrasonic extraction, and preferably may be extracted 1 to 5 times by immersion extraction method. The temperature during extraction is 20 ° C to 30 ° C, more preferably 20 ° C to 25 ° C at room temperature. The extraction time is 5 to 10 days, preferably 7 days.

In addition, the present invention provides a fraction obtained in each step by extracting the hawthorn extract in the order of n-hexane, ethyl acetate and n-butanol.

The fractional solvent for obtaining the fraction of the hawthorn extract is an organic solvent, preferably selected from the group of n-hexane, ethyl acetate and n-butanol, and fractionated using a separatory funnel. The fraction may be obtained by repeating the fractionation process 1 to 5 times, preferably three times, from the extract, and may be concentrated under reduced pressure after fractionation.

In the embodiment of the present invention, after the suspension of the hawthorn extract in distilled water, and then systematically fractionated in the order of n-hexane, ethyl acetate and n-butanol, this was repeated three times and each solvent fraction was concentrated under reduced pressure.

In addition, the present invention provides an antioxidant comprising the extract of the hawthorn or fractions thereof as an active ingredient.

Electron donating ability is an indicator of antioxidant activity, and it is known that the higher the reducing power, the higher the electron donating ability (Kang YH). et al . , Korean J Food Sci Technol 27: 978-984, 1995). Accordingly, the antioxidant activity of the extract of the hawthorn or fractions thereof was compared with the conventional antioxidants BHA, BHT, α-tocopherol, and showed higher electron donating ability than the control group, in particular, the EtOAc (ethyl acetate) fraction of root and stem. RC ₅₀ at 1.5 ± 0.5 μg / ml and 1.0 ± 0.5 μg / ml, respectively, which showed stronger electron donating ability compared to the control α-tocopherol RC ₅₀ 12 ± 0.5 μg / ml (see Table 1).

In addition, it is an enzyme that oxidizes free radicals in the body, and superoxide dismutase (SOD) -like activity, which is known to have a function of protecting the living body from oxygen injury, is effective in extracting methanol extract from the tree. The concentrations increased in all fractions, including concentration dependent (see Table 2). The extract of the hawthorn extract and its fractions did not show a significant difference in activity at low concentrations, but the inhibition of 65.7% or 73.4% at the high concentration of hexane fraction (1,000 ppm), respectively (see Table 2). The result is Hong HD et al. ( Korean J Food Sci Technol 30: 1484-1487, 1998) in the fruit and SOD-like activity of fruit juices, 14.6% for apple juice, 26.7% for kale concentrate, 27.6% for kiwi juice, and 24.1% for non-juice juice. It showed high SOD-like activity compared to that shown. In addition, the results reported that the higher the content of polyphenols and flavonoids increased the similar activity of SOD (Kwon TD et al . , Kor J Phys Edu 3: 891-899, 2001) and previous findings (Azuma K et. al . , LJ Agric Food Chem 47: 3963-3966, 1999; Lee YS et al . , Korean J Food Based on Preserv 12: 75-79, 2005), the roots and stems of the hawthorn tree are thought to contain a large amount of physiologically active substances including polyphenols and flavonoids.

In addition, as a result of measuring the lipid peroxidation inhibitory activity using a trivalent iron activity measuring method to measure the inhibition of lipid peroxidation according to long-term storage of unsaturated fatty acids, the extract of the present invention and BuOH fraction of the present invention is similar to BHT and BHA It showed high anti-lipid peroxidation ability, and showed a 6 times higher inhibitory effect on lipid peroxidation compared to α-tocopherol (see FIG. 1).

As described above, the extract of the hawthorn or the fraction thereof has strong electron donating ability, SOD-like activity and 6 times higher lipid peroxidation inhibitory effect than α-tocopherol, and as an antioxidant which can effectively remove harmful radicals in vivo. Can be used.

The antioxidant may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsions, syrups, etc. by conventional methods It may be formulated as a liquid. Specifically, the composition may be formulated for oral administration, for example stagnation, troches, lozenges, aqueous or dominant suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Formulated). Formulations such as tablets and capsules for lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricants such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above. In addition, the composition may be administered orally or parenterally, it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method when parenteral administration. To formulate into parenteral dosage forms, the extract of the hawthorn or fractions thereof of the present invention is mixed in water with a stabilizer or buffer to prepare a suspension, which is formulated in unit dosage forms of ampoules or vials. The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but in the case of oral administration in general Adults can be administered once to several times in an amount of 0.1 ~ 0.2 g of the hawthorn extract or fractions thereof of the present invention per kg of body weight per day, it is preferable to administer in an amount of 0.1 to 10 mg.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by the accumulation of excessive amount of active oxygen containing the hawthorn extract or fractions thereof as an active ingredient.

In addition, the present invention provides an anti-aging pharmaceutical composition containing the extract of the hawthorn or fractions thereof as an active ingredient.

The extract of the tree of the present invention or a fraction thereof may be usefully used as a pharmaceutical composition for preventing or treating a disease or an anti-aging pharmaceutical composition caused by excessive accumulation of effective active oxygen due to its antioxidant activity.

At this time, the disease caused by the excess accumulation of the active oxygen is characterized in that selected from the group consisting of liver disease, stroke, myocardial infarction, diabetic vascular disorders, hyperlipidemia, acute inflammation, rheumatism, cancer.

The composition may be used alone or in combination with methods of using surgery, hormonal therapy, drug treatment, and biological response modifiers to prevent aging as well as to prevent and treat diseases caused by excessive accumulation of free radicals.

The present invention also provides a cosmetic for preventing skin aging comprising the extract of the hawthorn or fractions thereof.

The skin anti-aging cosmetics include, but are not limited to, lotions, ointments, gels, creams, patches or sprays. In preparing the cosmetic for preventing skin aging containing the extract of the present invention or a fraction thereof, 1 to 15 parts by weight, preferably 2, of the extract of the present invention or a fraction thereof, in the composition for external application for skin Or 10 parts by weight. The skin external preparations of the present invention include fatty acid, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, in addition to the extract of the tree of the present invention or fractions thereof. For surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations. The ingredients may also be introduced in amounts generally used in the field of dermatology.

In another aspect, the present invention provides an antibiotic comprising the mountain extract or fractions thereof as an active ingredient.

The antibiotic is characterized by exhibiting antimicrobial and antifungal activity against pathogenic bacteria and pathogenic fungi. The pathogenic bacterium is Bacillus subtilis ), Staphylococcus Aureus ( Staphylococcus aureus ), Escherichia coli) and Salmonella Thai blood bunch Stadium (Salmonella typhimurium ). The fungus is Pichia jadini jadinii ) and Candida albicans .

In an embodiment of the present invention, Gram (+) bacteria Bacillus subtilis and Staphylococcus Aureus ( Staphylococcus aureus ), Escherichia , Gram (-) coli), Salmonella Thai blood bunch Stadium (Salmonella typhimurium ) , Klebsiella pneumonia ), and fungi Pichia jadinii and Candida albicans , confirmed that the extract of the hawthorn or fractions thereof can be usefully used as an antibacterial and antifungal agent. The Bacillus subtilis is a bacterium that affects the decay of food, Staphylococcus aureus is the causative agent of food poisoning, E. coli is an indicator of food contamination, Salmonella typhimurium is a food poisoning microorganism, Klebsiella neumo Nia is known to cause bacterial pneumonia. Pichia jadini is known to be one of the yeasts isolated from kimchi. Candida albicans, which causes candidasis in the genitals and around the mouth of humans, is a fungus (ie, fungus), which is a normal skin, mucous membrane, feces, sputum, It is a bacterium that can exist in urine. As a result, all of the bacteria except Klebsiella neumonia exhibited growth inhibitory activity (see Table 3). In particular, the ethyl acetate acetate fraction showed a growth inhibitory activity in most bacteria, and in E. coli, all the extracts and its fractions showed activity at ≤500 µg / ml (see Table 3). However, the control group (+)-catechin was found to have a significantly lower antibacterial activity compared to the extract of the present invention and its fraction (see Table 3).

In addition, the present invention provides a natural preservative comprising the mountain extract or fractions thereof as an active ingredient.

Natural preservatives of the present invention showed a wide range of good antimicrobial activity against a variety of microorganisms, in particular bacteria and fungi (see Table 3). Natural preservatives of the present invention may include additional components in addition to the extract of the hawthorn or fractions thereof, so long as the antiseptic activity thereof is not inhibited. Natural preservatives according to the present invention having a broad spectrum of preservation as described above can be widely used to achieve the antiseptic purpose in pharmaceuticals, cosmetics, food, textiles, household goods, etc., these products are used in the safety and quality of products Increase stability When the natural preservative of the present invention is included in the above-mentioned product, its amount is 0.001 to 30% by weight, preferably 0.001 to 20% by weight, more preferably 0.001 to 5% by weight based on the total weight of the product. Range. In order to produce a product comprising the natural preservative of the present invention, it will be possible to select and prepare suitable formulations and additives as is well known to those skilled in the art.

In another aspect, the present invention provides a composition for anti-diabetic comprising the extract of the hawthorn or fractions thereof as an active ingredient.

The antidiabetic composition is characterized by exhibiting antidiabetic activity by inhibiting α-glucosidase activity. The α-glucosidase is an intestinal digestive enzyme having a role of hydrolyzing carbohydrates ingested after diet into monosaccharides which are in a state for digestion and absorption, and controlling post-prandial blood sugar elevation by inhibiting the enzyme activity and slowing glucose absorption. It is applied to the treatment of diabetes. Therefore, in the embodiment of the present invention, anti-diabetic activity was measured by measuring α-glucosidase inhibitory activity against the extract of the present invention or fractions thereof. As a result, it showed a higher inhibition rate of 79.5% and 80.6% of the control group of diabetic root and stem BuOH fractions than the control group of bolibos 70.4%, but lower inhibition rate compared to another control group of akabos 89%. (See FIG. 2). The results indicate that the extract of the hawthorn or fractions thereof of the present invention can be used as an antidiabetic composition.

In addition, the present invention provides a health food for preventing and improving diabetes comprising an extract of the hawthorn or fractions thereof as an active ingredient.

The hawthorn extract or fractions thereof of the present invention may be added as is or used in combination with other food or food ingredients, and may be suitably used according to conventional methods. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene). Generally, in the preparation of foods or beverages, the hawthorn extract or fractions thereof of the present invention are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

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

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion 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 composition of the present invention.

In addition to the above, the extract of the hawthorn or fractions thereof of the present invention may be various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, Preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the hawthorn extract of the present invention or a fraction thereof or derivative thereof may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually 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 extract of the tree of the present invention or a fraction thereof may be usefully used as an antioxidant or anti-aging cosmetic or pharmaceutical composition by showing an antioxidant effect, and may be useful as an antibiotic or a health food by showing an antibacterial and antifungal effect. By showing anti-diabetic effect, it can be usefully used as an anti-diabetic composition or health food.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

< Example  1> Samples, Instruments and Reagents

<1-1> Hawthorn  Sample Preparation

Hazelwood provided by Samsung Herbal Medicine Co., Ltd. rhamnoides L .; Vitamin Tree) Roots and stems were washed with water, and then dried and ground for 7 days at room temperature in the shade and used.

<1-2> instrument and reagents

Reagents were used for first-class and express, UV / VIS Spectra for absorbance measurements were measured using a Jasco V530 spectrophotometer, and a rotary vacuum evaporator was measured by EYELA (Japan). NE-2001 and AC-1112A were used.

DPPH (1,1-diphenyl-2-picrylhydrazyl), Pyrogallol, Tween 20, linoleic acid, ammonium thiocyanate used for physiological activity, and BHA used as a positive control (tert-butyl hydroxyanisole), BHT (tert-butyl hydroxytoluene), α-tocopherol, ascorbic acid, (+)-catechin, ketoconazol, mycostantin (Mycostantin) Tetracycline was used by Sigma (USA).

< Example  2> Hawthorn  Extracts and Fractions thereof of  Produce

<2-1> Methanol Extract Preparation

Mountain roots (408 g) and stem (115 g) powder prepared by the method of Example 1-1 were immersed in 10-fold 100% methanol, extracted three times at room temperature for 7 days, and filtered through a filter paper. The extract was concentrated using a concentrator under reduced pressure until the solution evaporated completely to obtain a dried extract. As a result of using the above method, extract root 68g and stem 60g could be prepared from the roots of the hawthorn (408 g) and the stem (115 g).

<2-2> Water Extract Preparation

Using water as the extraction solvent, it was extracted in the same manner as the extraction method of Example <2-1> to prepare the extract 7.3 and 1.8 g.

<2-3> Ethanol Extract Preparation

By using 100% ethanol as the extraction solvent, it was extracted in the same manner as the extraction method of Example <2-1> to produce 9.2 and 2.5 g of the extract.

<2-4> Fraction  Produce

The extract obtained in the method of Example 2-1 was suspended in distilled water, and then fractionated in the order of n -hexane, ethyl acetate, butanol. After repeated three times, each solvent fraction was concentrated under reduced pressure.

As a result, n -hexane [root (3.9 g), stem (5.1 g)], ethyl acetate [root (2 g), stem (4.4 g)], butanol [root (14 g), stem (11.1 g), respectively. ] Solvent fractions were obtained. As a result of using the above method, extract root 68g and stem 60g could be prepared from the roots of the hawthorn (408 g) and the stem (115 g).

< Experimental Example  1> Electron donating ability  Research

Xiong Q etc. ( Biological and Pharmaceutical The electron donating ability by DPPH free radical scavenging method presented by Bulletin 19: 1580-1585, 1996) was used.

Specifically, as a test group obtained by the method of Example 2 in a test tube containing 4 ml of methanol, the root and stem extracts and their fractions, positive control (BHA, BHT, α-tocopherol) and negative control (no addition) Samples were prepared by adding each concentration. After adding 1 ml of DPPH (0.15 mM) solution to the sample, the mixture was left to react at room temperature for 30 minutes, and absorbance was measured at 517 nm with a UV / VIS spectrophotometer. In this case, the concentration of the sample (RC ₅₀ ; μg / ml) required for the sample to inhibit 50% of the DPPH free radicals was expressed as the concentration of the sample necessary to reduce the DPPH free radical value of the negative control by 50%.

Comparing the antioxidant activity of the experimental group with the existing antioxidants BHA, BHT, α-tocopherol, as shown in Table 1, the extract and all fractions of all the roots and stems of the hawthorn showed high electron donating ability, in particular the root and stem RC ₅₀ in the EtOAc (ethyl acetate) fractions of 1.5 ± 0.5 μg / ml and 1.0 ± 0.5 μg / ml, respectively, which showed stronger electron donating ability as compared to the control α-tocopherol RC ₅₀ 12 ± 0.5 μg / ml.

Antioxidant activity Extracts and Fractions thereof RC ₅₀ ^{1 )} (µg / ml) Root stem MeOH Extract 5 ± 0.5 2.7 ± 0.5 Water extract 10.9 ± 0.43 4.1 ± 0.24 EtOH Extract 4.3 ± 0.56 2.6 ± 0.32 Hexane Fraction 3 ± 1.0 3.2 ± 0.23 EtOAc fraction 1.5 ± 0.29 1 ± 0.35 BuOH fraction 2.5 ± 0.58 1.4 ± 0.2 Water soluble fraction 9.5 ± 0.2 3.3 ± 0.2 BHT 34 ± 0.58 BHA 14 ± 0.5 α-tocopherol 12 ± 1.15

¹⁾ RC ₅₀ : The amount required for 50% reduction of DPPH after 30 minutes, each value being average ± standard deviation by three repeated tests.

< Experimental Example  2> free radical scavenging enzyme ( Superoxide dismutase ; SOD ) Similar activity investigation

Marklund et al. (Marklund S & Marklund G, Eur J Biochem 47: 468-474, 1975), SOD-like activity was measured.

Specifically, the experimental group obtained by the method of Example 2 as a concentration of (100, 1,000, 5,000 and 10,000 ppm) hawthorn root and stem extract and its fractions and control group (additive-free group) 0.2 ml each of Tris-HCl buffer solution 0.2 mL of 7.2 mM pyrogallol was added to a 2.6 mL mixed sample (50 mM Tris + 10 mM EDTA, pH 8.5). After the reaction was allowed to stand at 25 ° C. for 10 minutes, 0.1 mL of 1.0 N HCl was added to stop the reaction, and the amount of oxidized pyrogallol in the reaction solution was measured at 420 nm. SOD-like activity is a value expressed as a percentage (%) of the absorbance difference between the experimental group and the control group according to the following equation (1).

SOD-like activity (%) = {1- {control absorbance / experiment absorbance}} × 100

As a result, as shown in Table 2, as the concentration increases in all fractions, including methanol extract in the hawthorn, superoxide dismutase (SOD) -like activity increased in a concentration-dependent manner. The root and stem extracts of the hawthorn and their fractions showed no significant difference in activity at low concentrations (100 ppm), respectively, but inhibited 65.7% or 73.4% at 1,000 ppm of hawthorn root or stem hexane fractions, respectively.

SOD-like activity Concentration (ppm) SOD-like activity ¹⁾ MeOH Extract Water extract EtOH Extract Hexane Fraction EtOAc fraction BuOH fraction Water soluble fraction Ascorbic acid Root 100 3.4 ± 0.1 1.5 ± 0.2 4.4 ± 0.4 7.3 ± 0.2 2.9 ± 0.3 1.1 ± 0.3 0.5 ± 0.3 32.5 ± 0.5 1,000 12.8 ± 1 10.8 ± 1.2 21.4 ± 1.8 65.7 ± 0.12 25.7 ± 1.4 11.7 ± 0.4 11.3 ± 2.0 68.5 ± 0.5 5,000 44.3 ± 2.0 20.1 ± 1.0 66.1 ± 2.2 92.4 ± 0.3 67.9 ± 2.0 41.1 ± 2.0 24.1 ± 1.5 <99.0 10,000 66.6 ± 0.1 30.1 ± 0.5 75.2 ± 0.2 95.1 ± 0.1 80.4 ± 2.0 58.7 ± 2.0 33.7 ± 2.0 <99.0 stem stem 100 2.1 ± 0.4 1.6 ± 0.3 2.3 ± 0.5 9.1 ± 1.4 2.2 ± 0.5 8.0 ± 0.5 2.6 ± 0.4 32.5 ± 0.5 1,000 25.4 ± 1.5 20.1 ± 1.4 28.3 ± 0.5 73.4 ± 1.0 31.4 ± 1.5 21.3 ± 2.0 21.8 ± 0.4 68.5 ± 0.5 5,000 69.8 ± 1.4 35.7 ± 0.5 53.5 ± 0.7 92.9 ± 0.2. 54.0 ± 0.3 58.5 ± 0.5 39.7 ± 1.7 <99.0 10,000 72.6 ± 1.4 45.3 ± 1.2 58.6 ± 1.0 94.6 ± 0.3 58.0 ± 2.0 61.2 ± 0.5 48.5 ± 1.2 <99.0

¹⁾ All values were average ± standard deviation by three repeated tests.

< Experimental Example  3> Investigation of lipid peroxidation inhibitory activity

Inatani R etc. ( Agricultural and Biological Lipid peroxidation inhibitory activity was measured using a trivalent iron activity assay according to Chemistry 47: 521-528, 1983). The trivalent iron activity measuring method is an antioxidant measuring method for measuring the inhibition of lipid peroxidation according to long-term storage of unsaturated fatty acids.

Specifically, 20 containing unsaturated fatty acids linolenic acid (2.51%), ethanol (2.0 mL), 0.05 M phosphate buffer (pH 7.0, 4.0 mL), H ₂ O (1.9 mL) and 10% Tween 20 (0.1 mL) In a 10 ml test tube, the total volume was 10 ml, and the roots and stem extracts and their fractions, the positive control (BHA, BHT, α-tocopherol) and the control (no addition group) were each added so that the final concentration was 0.005%. A dark batch at &lt; RTI ID = 0.0 &gt; To 0.1 ml of the mixed solution sample, 75% ethanol (9.7 ml) and 30% ammonium thiocyanate (0.1 ml) were added, followed by the addition of 2 x 10 ^-2 M ferric chloride 3.5% hydrochloric acid solution (0.1 ml). After 3 minutes absorbance was measured with a UV / VIS spectrophotometer at 500 nm. Subsequently, a total of 35 days was measured every 48 hours. Lipid peroxide production inhibitory capacity (%) was calculated according to the following equation (2).

% Inhibition of lipid peroxide formation = {(1- (control absorbance-experiment absorbance)} × 100

As a result, as shown in FIG. 1, α-tocopherol was rapidly oxidized after 17 days, resulting in lower anti-lipid peroxidation ability than BHT and BHA. BuOH fraction and MeOH extract showed high antilipid peroxidation ability similar to the BHT and BHA positive control in mountain roots (FIG. 1A), and the stem (FIG. Similarly, it showed high antilipid peroxidation ability. In addition, BuOH fraction and MeOH extract showed a 6 times higher lipid peroxidation inhibitory effect than α-tocopherol in stem and root.

< Experimental Example  4> antimicrobial activity

Kobayasi A et al. ( Zeitschrift fur Naturforsch 51: 527-533, 1996), the antimicrobial activity was measured by serial 2-fold dilution.

<4-1> Subject

Bacillus, a Gram (+) bacterium subtilis ) and Staphylococcus Aureus ( Staphylococcus aureus ), Escherichia , Gram (-) coli), Salmonella Thai blood bunch Stadium (Salmonella typhimurium ) , Klebsiella pneumonia , and the fungus Pichia jadini jadinii ) and Candida albicans .

<4-2> Culture and Activity Measurement

Staphylococcus aureus (micrococcus) was incubated in nutrient YM medium and inoculated with 10 ml of the cell suspension in a 25 mm diameter test tube, followed by shaking culture (100 rpm) for 12 hours at 37 ° C and 30 ° C, respectively. The culture solution was diluted 100-fold in each medium and used for the antibacterial test. Samples were placed in the first sphere of a 96 well microanalysis plate and the prepared cell suspensions were aliquoted and diluted twice.

The sample was incubated at 37 ° C. and 30 ° C. under dark conditions for 24 hours to confirm bacterial growth by visually measuring the degree of turbidity. Antimicrobial activity was determined by the minimum concentration (MIC) to inhibit the growth of bacteria (Kim MJ & Hyun JO, Kor J Vreed 29: 115-123, 1997).

As a result, as shown in Table 3, it showed the growth inhibitory activity as a whole in the bacteria except Klebsiella neumonia. In particular, ethyl acetate fractions of the roots and stems of the hawthorn showed growth inhibiting activity in most bacteria. Among E. coli, all of the extracts of cotyledon and its fractions showed activity at ≦ 500 μg / ml. The control group (+)-catechin was found to have a significantly lower antibacterial activity compared to the extract of the present invention and its fractions.

Antimicrobial activity Extracts and Fractions thereof MIC ¹⁾ (μg / ml) Germ Fungus      Gram (+) Gram (-) B.s. ^One) S.a. ^One) E.c. ^One) S.t. ^One) K.p. ^One) P.j. ^One) C.a. ^One) Root MeOH Extract 1000 500 500 1000 1000 125 125 Water extract 500 1000 500 500 1000 125 125 EtOH Extract 500 250 250 500 1000 125 125 Hexane Fraction 1000 1000 500 1000 1000 125 125 EtOAc fraction 500 250 250 500 1000 125 125 BuOH fraction 500 125 500 500 1000 125 125 Water soluble fraction 500 1000 500 500 1000 125 125 stem MeOH Extract 500 250 500 1000 1000 125 62 Water extract 500 500 500 500 500 125 125 EtOH Extract 500 250 250 500 1000 125 62 Hexane Fraction 1000 250 500 500 1000 62 62 EtOAc fraction 1000 125 250 500 1000 62 62 BuOH fraction 125 500 500 500 500 125 125 Water soluble fraction 500 500 500 500 500 125 125 (+)-Catechin 1000 < 1000 < 1000 < 1000 < 1000 < 500 500 Ketoconazol - - - - - 250 250 Mycostantin - - - - - 500 500 Tetracycline 8 8 8 8 8 - -

1) Bs: Bacillus subtilis , Sa: Staphylococus aureus , Ec: Escherichia coli , St: Salmonella typhimurium , Kp: Klebsiella pneumonia , Pj: Pichia jadinii and Ca: Candida albicans .

< Experimental Example  5> Antidiabetic  Active investigation

α-glucosidase is an intestinal digestive enzyme that has a role of hydrolyzing carbohydrates taken after diet into monosaccharides that are in a state for digestion and absorption. A method of controlling post-prandial blood sugar elevation by treating glucose is being applied to the treatment of diabetes. Antidiabetic activity was measured by measuring the α-glucosidase inhibitory activity.

After extracting the root and stem extracts from the method of Example 2 with 0.3 M KPB buffer solution (pH 7.0) to a concentration of 1 mg / ㎖, α-glucosidase enzyme solution (Sigma Chemical, USA) 100 Μl was added and mixed for 30 minutes at 37 ° C. After stopping the reaction by placing the reaction solution at 100 ° C. for 5 minutes, 200 μl of glucose kit (Lot.R658, Asan Pharmaceutical Co., Ltd.) was added to 40 μl of the reaction solution, followed by color development at 37 ° C. for 5 minutes, followed by 496 nm. Absorbance was measured. At this time, acarbose and aggolibos, which are diabetic drugs, were used as positive controls for absorbance of the extract of the hawthorn and its fractions. Acarbose, a drug marketed as an α-glucosidase inhibitor, is known to inhibit the activity of maltase in the mucosa of small intestine cells (Carrascosa JM et al. , Diabetes) . Obes Metab 3: 240-248, 2001). Inhibitory activity (inhibition rate) for α-glucosidase was calculated according to the equation (3).

% Inhibition = {(1- (control absorbance-experiment absorbance)} × 100

As a result, as shown in FIG. 2, the root and stem BuOH fractions of the hawthorn showed high inhibition rates of 79.5% and 80.6%, respectively, compared to the control vogliose 70.4%, but showed a lower inhibition rate than the 89% acarbose.

< Formulation example  1> Preparation of Pharmaceutical Formulations

1. Preparation of powder

Hawthorn extract or fraction 2 g

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

Mountain berry extract or fraction 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

Mountain berry extract or fraction 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

Hawthorn extract or fraction 1 g

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

5. Preparation of Granules

Mountain berry extract or fraction 150 mg

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Formulation example  2> Cosmetic  Produce

<2-1> Preparation of flexible lotion

Formulation example of the flexible lotion containing a mountain extract or fraction as an active ingredient was prepared as shown in Table 4.

Raw material Content (parts by weight) Hawthorn extract or fraction 10.00 1,3-butylene glycol 1.00 Disodium ID 0.05 Allantoin 0.10 Dipotassium glycylizate 0.05 Citrix Acid 0.01 Sodium citrate 0.02 Glyceres-26 1.00 Arbutin 2.00 Hydrogenated Castor Oil 1.00 ethanol 30.00 Preservative a very small amount coloring agent a very small amount Flavor a very small amount Purified water Remaining amount

<2-2> Preparation of nutrition cream

Formulation example of nutrition cream containing the extract or fractions of the hawthorn was prepared as shown in Table 5 below.

Raw material Content (parts by weight) Hawthorn extract or fraction 10.0 1,3-butylene glycol 7.0 glycerin 1.0 D-panthenol 0.1 Plant extracts 3.2 Magnesium Aluminum Silicate 0.3 PEG-40 Stearate 1.2 Stearic acid 2.0 Polysorbate 60 1.5 Lipophilic glyceryl stearate 2.0 Sorbitan sesquioleate 1.5 Cetearyl Alcohol 3.0 Mineral oil 4.0 Squalane 3.8 Carlylic / Capric Triglycerides 2.8 vegetable oil 1.8 Dimethicone 0.4 Dipotassium glycylizate a very small amount Allantoin a very small amount Sodium hyaluronate a very small amount Tocopheryl Acetate Quantity Triethanolamine Quantity Preservative Quantity Flavor Quantity Purified water Remaining amount

< Formulation Example 3> Preparation of Food

Food containing the extract or fraction of the present invention was prepared as follows.

1. Preparation of Cooking Seasonings

20 ~ 95 parts by weight of the mountain extract or fraction of the present invention was prepared for health promotion cooking seasoning.

2. Manufacturing of Flour Foods

0.5 to 5.0 parts by weight of the extract or fraction of the present invention is added to the flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

3. Manufacture of dairy products

5-10 parts by weight of the extract of the hawthorn or fraction of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

4. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphanated by a known method to distribute the dried ones, and then prepared into a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The grains, seeds and the dry powder of the extract or fraction of the present invention prepared in the above were formulated in the following ratio.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Hawthorn extract or fraction (3 parts),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Formulation example  4> Manufacture of beverage

1. Manufacture of health drinks

       Mountain berry extract or fraction 1000 mg

       Citric acid 1000 mg

       100 g oligosaccharides

       Plum concentrate 2 g

       1 g of taurine

       Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

1 is a diagram showing the antioxidant effect of the extract of the hawthorn and fractions thereof measured by the trivalent iron activity assay:

a: root; And,

b: stem.

2 is a diagram showing the α-glucosidase activity of the hawthorn extract and fractions thereof:

a: root; And,

b: stem.

Claims (15)

Extract of Hippophae rhamnoides L. extracted with water, alcohol or a mixture thereof as a solvent. The extract of claim 1, wherein the alcohol is a C ₁ to C ₄ lower alcohol. The organic solvent fraction prepared by extracting the extract of claim 1 with an organic solvent. The fraction as claimed in claim 3, wherein the organic solvent is n-hexane, ethyl acetate or n-butanol. Antioxidant comprising the extract of claim 1 or the fraction of claim 3 as an active ingredient. A pharmaceutical composition for preventing and treating diseases caused by excessive accumulation of active oxygen, including the extract of claim 1 or the fraction of claim 3 as an active ingredient. 7. The disease according to claim 6, wherein the disease caused by excessive accumulation of free radicals is any one selected from the group consisting of liver disease, stroke, myocardial infarction, diabetic vascular disorder, hyperlipidemia, acute inflammation, rheumatism, and cancer. Pharmaceutical compositions. Anti-aging pharmaceutical composition comprising the extract of claim 1 or the fraction of claim 3 as an active ingredient. Claim 1 skin or cosmetic for preventing skin aging comprising the extract of claim 3 as an active ingredient. An antibiotic comprising the extract of claim 1 or a fraction of claim 3 as an active ingredient. The antibiotic according to claim 10, which exhibits antimicrobial activity against pathogenic bacteria or fungi. The antibiotic according to claim 10, which exhibits antifungal activity against fungi. Natural preservative comprising the extract of claim 1 or the fraction of claim 3 as an active ingredient. An antidiabetic composition comprising the extract of claim 1 or a fraction of claim 3 as an active ingredient. Claim 1 or claim 3 diabetes prevention and improvement health food comprising the fraction of claim 3 as an active ingredient.

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