KR20170100369A - Composition comprising nelumbo nucifera gaertner extract for anti-oxidant and anti-bacterial effect - Google Patents

Abstract

The present invention relates to an antioxidant and antibacterial composition containing a Nelumbinis Folium fraction as an active ingredient. According to the present invention, the Nelumbinis Folium fraction shows excellent antioxidant effects owing to 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities and 2,2-azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activities, and also exhibits antibacterial functions against Escherichia coli. Since the Nelumbinis Folium fraction can suppress synthesis of melanin through inhibition of tyrosinase, the Nelumbinis Folium fraction can be used as a composition for antioxidant and antibacterial purposes.

Description

TECHNICAL FIELD The present invention relates to an antioxidative and antimicrobial composition containing a lower leaf fraction as an active ingredient,

The present invention relates to an antioxidant and antimicrobial composition containing an extract of lower leaves as an active ingredient.

Kite is a paleontic plant widely distributed in tropical and temperate East Asia, Korea and Japan, mainly in India and China. It is a sacred plant in Buddhism. Flowers have been used as ornamental plants and tea plants. Leaves and roots have been edible. In leaves, the leaves are called lower leaves and are used for diarrhea, headache and dizziness, blood clots, postpartum hemorrhage treatment, enuresis, and detoxification. So it stops the diarrhea and stops the diarrhea by heat and moisture, clears the thirst, clears the wind and heat accumulated in the head and eyes, treats dizziness, and is good for the treatment of various hemorrhages such as blood, nosebleeds, uterine bleeding .

In the oriental literature, it is said that the lotus leaf has a detoxifying effect and is good when it is poisoned by eating sea bream. In addition, the round and large leaf in the lotus leaf is called "buyon (芙蓉)", and the bouquet is a symbol of beauty and leaves are clean, but it is good for skin beauty. Yeonbyeong has antimicrobial and hypotensive effects, strengthens the stomach, and helps with beauty and vitality. In recent years, tea leaves are used to make tea using leaves. In spite of various studies on the physiological activities of the lower leaves, however, the research on the antimicrobial activity, antioxidative activity and whitening activity of the lower leaves is insufficient.

On the other hand, in Korean Patent Laid-Open Nos. 2009-0021644 and 2014-0015662, studies on the pharmacological effects related to metabolic diseases have been carried out on the lower leaves extract. As described above, although the lower leaf extract was widely marketed as a medicinal plant, the effect on prevention and treatment of antioxidant and antibacterial activity was not confirmed.

The present inventors prepared various organic solvent fractions from the lower leaves and evaluated their antioxidant, antibacterial and whitening activity, and found excellent activity in the lower ethyl acetate fraction.

Accordingly, an object of the present invention is to provide an antioxidant and antimicrobial composition containing an extract of a lower leaf as an active ingredient.

Another object of the present invention is to provide a health food composition having an antioxidant and antimicrobial activity, which contains a lower leaf extract as an active ingredient.

Yet another object of the present invention is to provide a cosmetic composition having antioxidant and antimicrobial activity, which contains a lower leaf extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for antioxidant and antimicrobial containing a lower leaf fraction as an active ingredient.

In addition, the present invention provides a health food composition for antioxidant and antimicrobial containing a lower leaf fraction as an active ingredient.

In addition, the present invention provides an antioxidant and antimicrobial cosmetic composition comprising a lower leaf fraction as an active ingredient.

The extract of Nerumbo nucifera GAERTNER according to the present invention exhibits DDPH radical scavenging activity and ABTS radical scavenging activity especially in the ethyl acetate fraction, showing excellent antioxidative effect.

It has also been confirmed that it has antimicrobial activity against E. coli and inhibits the activity of tyrosinase to inhibit melanin synthesis and can be used as a multi-purpose composition having antioxidant, antibacterial and whitening effect.

FIG. 1 is a graph showing the results of measurement of DPPH radical scavenging activity by concentration according to fractions of lower leaves.
FIG. 2 is a graph showing the results of ABTS radical scavenging activity according to concentration according to fractions of the lower leaf.
FIG. 3 is a graph showing the results of measuring the inhibition rate of tyrosinase activity according to the concentration of the lower leaf fraction.

The present invention provides a pharmaceutical composition for antioxidant and antimicrobial containing an extract of lower leaves as an active ingredient. The composition comprises a food composition and a cosmetic composition.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for preparing a methanol extract from a dry lower leaf for preparing an additional physiological activity of a lower leaf which is used as a food, preparing various organic solvent fractions and water residues of ethyl acetate and butanol, In order to investigate the inhibitory effect of tyrosinase activity which inhibits melanin synthesis, each activity was evaluated.

The antioxidative activity of ethyl acetate showed the highest activity. The antimicrobial activity of the lower leaf fraction was significantly higher than 500 μL / mL. The antimicrobial activity of each sample against S. aureus was in the order of n-BuOH>EtOAc> Water, and the antibacterial activity of each sample against E. coli was not observed. 10, 50, and 100 ppm, respectively, and tyrosinase activity was measured. As a result, it was observed that the inhibition rate of tyrosinase activity was increased in a concentration dependent manner.

The lower leaf extract of the present invention can be prepared using conventional solvents known in the art, that is, under the conditions of ordinary temperature and pressure, using conventional solvents. Examples of the extraction solvent that can be used in the preparation of the lower leaf extract of the present invention include water, an alcohol having 1 to 6 carbon atoms, acetone, ethyl acetate, butyl acetate, 1,3-butylene glycol, etc., , Preferably methanol, but is not limited thereto. The suitable amount of the extraction solvent is about 1 to 10 times, more preferably 5 to 8 times, and most preferably 5 times the dry weight of the lower leaf. As the extraction method, it is possible to use heat extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and the like, and they can be extracted once or repeatedly. The extraction temperature is not particularly limited so long as the effective activity of the useful component of the lower leaf is not removed, but is preferably extracted by immersion at room temperature.

It is preferable that the extract is a methanol extract, and it can be sequentially fractionated from the methanol extract by ethylacetate, n-butanol and water-solvent, and it is confirmed that the fraction has the best antioxidative and antimicrobial activities of ethyl acetate.

The composition of the present invention may contain at least one known active ingredient having antioxidative activity together with the lower leaf extract.

The composition of the present invention may be added to a health functional food for the purpose of enhancing antioxidative and antimicrobial effects. When the lower leaf extract of the present invention is used as a food additive, the lower leaf extract may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the lower leaf extract of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, 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 substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health functional 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. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, 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 0.01-0.20 g, preferably about 0.04-0.10 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition 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 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention can provide antioxidant, antibacterial, and whitening cosmetic compositions containing lower leaf fractions.

The formulation of the cosmetic composition may be at least one selected from the group consisting of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizer cream, a hand cream, a foundation, , A cleansing foam, a cleansing lotion, a cleansing cream, a body lotion or a body cleanser.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

[Example]

Experimental Method

material

The lower leaves (Nelumbo nucifera GAERTNER) used in this experiment were directly harvested from the lotus field in the Hwasan area, Yeongcheon, Kyungbuk province, dried, and pulverized. 70% ethanol was used as the extraction solvent. 300 g of lower leaf was mixed with 3000 mL of extraction solvent and extracted three times at 25 캜 for 24 hours. After extracting, it was filtered with Whatman No.1, concentrated by rotary evaporator (EYELA, Japan) to remove ethanol, and then the extract was fractionated by ethylacetate, n- Butanol and water solvent, and each fraction was concentrated under reduced pressure The concentrated fractions were dissolved in DMSO at a certain concentration and used in the experiment.

1) Antioxidant activity of lower leaves extract

(1) DPPH radical scavenging activity

M DPPH 용액 100μL 을 넣어 10초간 잘 혼합한 후, 상온에서 30분간 방치한다. 그리고 파장525nm에서 UV/VIS spectrophotometer를 사용하여 흡광도를 측정하였다. DPPH radical 제거능은 시료용액의 첨가구와 무첨가구 사이의 흡광도의 차이를 백분율로 나타내었다.DPPH (a, a-diphenyl-β-picrylhydrazyl) radical scavenging activity was measured by modified Blois (5). The lower leaf fractions were dissolved in dimethylsulfoxide (DMSO) at concentrations of 100, 200, 500, 1000 and 2000 ppm, respectively.

M DPPH solution, mix well for 10 seconds, and allow to stand at room temperature for 30 minutes. The absorbance was measured using a UV / VIS spectrophotometer at a wavelength of 525 nm. DPPH radical scavenging ability was expressed as a percentage difference between the absorbance of the sample solution and that of the sample solution.

DPPH radical scavenging acitivity (%) = (1- (A _test -A _blank / B _control )) 100

A _test : absorbance of sample added sphere

A _blank : absorbance of blank

B _control : absorbance of sample without added sample

2) ABTS radical scavenging activity

0.02가 되도록 조정하였다. 소거능은 ABTS 용액 100μL와 100, 200, 500, 1000, 2000 ppm 농도별 하엽 분획물 100μL를 혼합하여 6분간 반응 후, 파장 700nm에서 흡광도를 측정하였다. ABTS radical 제거능은 시료 용액의 첨가구와 무첨가구 사이의 흡광도의 차이를 백분율로 나타냈다.The antioxidant activity of ABTS (2,2-azino-bis-3-ethylbonzo-thiazoline-6-sulfonic acid) was measured by Re (5). ABTS was dissolved in distilled water at a concentration of 7 mM, and 2.4 mM potassium persulfate was added thereto to allow the formation of ABTS radicals. The incubation was carried out at room temperature for 20 to 24 hours. Radical ABTS solution was diluted with 94% ethanol and absorbance at 700nm was 0.70

0.02. 100 μL of ABTS solution and 100 μL of lower leaf fraction at 100, 200, 500, 1000, 2000 ppm concentrations were mixed for 6 minutes and absorbance was measured at a wavelength of 700 nm. ABTS radical removability was expressed as a percentage of absorbance difference between the addition of the sample solution and no addition.

ABTS radical scavenging acitivity (%) = (1- (A _test -A _blank ) / B _control )) 100

A _test : absorbance of sample added sphere

A _blank : absorbance of blank

B _control : absorbance of sample without added sample

3) Antibacterial effect

 (1) Strain used

Strains Strains No. Escherichia coli KCTC1682 Staphylococcus aureus KCTC1916

Among the causative organisms of food poisoning, two gram - positive Escherichia coli (E. coli) and Staphylococcus aureus were purchased from the Korea Biological Resource Center (KCTC). The species and number of each microorganism strain used in the experiment are shown in Table 1 above.

The cultures of the strains were sterilized in an autoclave at 121 ° C for 30 minutes and then cooled to 25 ° C. The cells were lyophilized using a loop in a solid medium and cultured in Incubator (SANYO, Japan) for 24 hours . Then, 100 mL of the liquid medium was placed in a 250 mL Erlenmeyer flask, sterilized, and then a single colony of the bacteria grown in the solid medium was taken and incubated in a liquid medium for 24 hours in a shaking incubator at 30 ° C.

(2) Antibacterial effect

The antimicrobial activity of each sample was measured by the Disc paper method using E.coli and S. aureus using a solid medium with a high accuracy and a low contamination rate. Each of the subculture strains was prepared by laminating 1 mL each of the subcultured strains using a sterile spread stick. The samples were absorbed in 8 mm disc paper (ADVANTEC, Japan) with sterilized tweezers to 10 mg per disc and dried. Dried and cultured in Incubator (SANYO, Japan) at 37 ° C for 24 hours in a state in which they were in close contact with each other on the plate.

3) Measurement of tyrosinase activity

Tyrosinase activity was measured by modifying the method of Masanoto et al. (1), which measures DL-β-3,4-dihydroxypheyl alanine (DOPA) chrome produced by the action of tyrosinase by colorimetry. Tyrosina, a melanin-forming enzyme

In order to inhibit the production of melanin pigment and to inhibit the production of reactive oxygen species in the skin, the activity of tyrosinase of the lower leaf fraction was measured. The tyrosinase activity was measured using 96 wells, and 170 μL of DL-DOPA solution,

20 μL of tyrosinase was added to 10 μL of spate buffer and 10 μL of sample solution. After reacting at 35 ° C. for 20 minutes, the absorbance was measured at 490 nm and Tyrosinase inhibition rate was calculated according to the following equation.

Experiment result

1) Antioxidant activity of lower leaves fraction

(1) DPPH radical scavenging activity

DPPH radical is a simple and reliable method for measuring electron donating ability which is an index of antioxidative activity against phenol acid, flavonoids and other phenol substances. When DPPH radical reacts with electron donating antioxidants, .

Fig. 1 shows the measurement results of DPPH radical scavenging activity by concentration according to fractions of lower leaves.

As can be seen in FIG. 1, water generally exhibited a low radical scavenging ability, while n- BuOH and EtOAc showed high radical scavenging ability. n- BuOH and EtOAc showed 56% and 75% at 2000 ppm, respectively.

(2) ABTS radical scavenging activity

ABTS radical scavenging activity is a relatively stable free radical, which is highly correlated with DPPH radical scavenging activity. It is used to measure antioxidative activity by the decolorization of unique cyan color by reacting with antioxidative substances such as polyphenols in the sample.

Fig. 2 shows the results of ABTS radical scavenging activity by concentration according to the fraction of lower leaves.

ABTS also showed low effect in water and EtOAc and n - BuOH showed high effect. For EtOAc and n- BuOH, 99.5% and 98.4% of EtOAc and 99.9% and 94.5% of n- BuOH were respectively present at 2000PPM and 1000PPM, respectively. It can be seen that the activity of EtOAc and n- BuOH is higher than that of the control BHA of 94% at 1000 ppm.

2) The antimicrobial effect of the lower leaves fraction

E. coli and S. aureus were tested for their antibacterial activity by disk paper method. The antimicrobial activity was measured as the size of growth inhibition ring formed when the lower leaves fraction was treated with 100, 200, 500 mg / As a result, in the E. coli strain, the EtOAc layer showed an antimicrobial activity effect at 500 ppm at 16 mm, an antimicrobial activity effect at 19 mm at the n- BuOH layer, and an antimicrobial activity effect at 13 mm in the water layer. In the case of S. aureus strain, the antibacterial effect was not confirmed as a whole.

As a result of this example, the antimicrobial activity of each fraction layer was different according to each strain. Overall, the results of the present invention showed excellent antibacterial activity against E. coli strains.

3) Measurement of tyrosinase activity

In this example, the inhibitory effect of tyrosinase on the inhibition of melanin synthesis was examined by an in vitro method. The inhibition rate of tyrosinase activity was increased as the concentration of lower leaf fraction increased. The inhibition rate of tyrosinase activity according to the concentration of the lower leaf fraction is shown in FIG.

The effect of the lower leaf fraction was confirmed through the above examples. The antioxidative activity of ethyl acetate showed the highest activity. The antimicrobial activity of the lower leaf fraction was significantly higher than 500 μL / mL. The antibacterial activity of each sample against S. aureus was also excellent. In addition, tyrosinase activity was measured by treatment with 10, 50, and 100 ppm, respectively. As a result, it was observed that the inhibition rate of tyrosinase activity was increased in a concentration dependent manner.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation Example 1: Preparation of pharmaceutical preparations

1. Manufacturing of powder

200 mg of lower leaves

Lactose 100 mg

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

200 mg of lower leaves

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

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

3. Preparation of capsules

200 mg of lower leaves

100 mg of corn starch

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

200 mg of lower leaves

100 mg mannitol

Na ₂ HPO _{4 12} H ₂ O 2 mg

Sterile sterilized water for injection

Injection was prepared by mixing the above components per ampoule (2 mL) according to the usual injection preparation method.

Formulation Example 2: Preparation of food

Foods containing the lower leaf extract of the present invention were prepared as follows.

1. Preparation of cooking seasoning

The safflower for health promotion was prepared by 20 to 95% by weight of lower leaf extract.

2. Manufacture of tomato ketchup and sauce

0.2-1.0 wt.% Of lower leaf extract was added to tomato ketchup or sauce to make healthy tomato ketchup or sauce.

3. Manufacture of flour food

0.5-5.0 wt% of lower leaf extract was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare foods for health promotion.

4. Manufacture of soups and gravies

0.1-5.0 wt.% Of lower leaf extract was added to soups and juices to prepare health promotion meat products, noodle soups and juices.

5. Manufacture of ground beef

10% by weight of lower leaf extract was added to ground beef to prepare ground beef for health promotion.

6. Manufacture of dairy products

5-10% by weight of lower leaf extract was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 3: Preparation of beverage

1. Manufacture of carbonated beverages

Syrup was prepared by mixing 10-15% of lower leaf extract, 5-10% of sugar, 0.05-0.3% of citric acid, 0.005-0.02% of caramel and 0.1-1% of vitamin C and 75-80% of purified water . The syrup was sterilized at 85-98 ° C for 20-180 seconds, mixed with cooling water at a ratio of 1: 4, and 0.5-0.82% carbon dioxide gas was injected to prepare a carbonated drink containing the lower leaf extract.

2. Manufacture of health drinks

(70%, 0.12%), vitamin C (0.02%), calcium pantothenate (0.02%), licorice extract (Solid content: 65%, 0.01%) were uniformly blended, instant sterilized, and packaged in small containers such as glass bottles and plastic bottles to produce health drinks.

3. Manufacture of vegetable juice

Healthy vegetable juice was prepared by adding 0.5g of low - leaf extract to 1,000ml of tomato or carrot juice.

4. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1 g of lower leaf extract to 1,000 ml of apple or grape juice.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (9)

A pharmaceutical composition for antioxidant and antimicrobial use containing a fraction of Nelumbo nucifera GAERTNER as an active ingredient.
The pharmaceutical composition for antioxidant and antimicrobial activity according to claim 1, wherein the lower leaf fraction is a fraction obtained by sequentially fractionating lower leaf extracts with ethyl acetate, butanol, and water.
The pharmaceutical composition according to claim 2, wherein the lower leaf fraction is an ethyl acetate fraction.
A health food composition for antioxidant and antimicrobial, comprising a fraction of Nelumbo nucifera GAERTNER as an active ingredient.
[Claim 5] The composition according to claim 4, wherein the lower leaf fraction is a fraction obtained by sequentially fractionating lower leaf extracts with ethyl acetate, butanol, and water.
5. The composition of claim 4, wherein the lower leaf fraction is an ethyl acetate fraction.
An antioxidant and antimicrobial cosmetic composition comprising a fraction of Nerumbo nucifera GAERTNER as an active ingredient.
8. The cosmetic composition for antioxidant and antimicrobial activity according to claim 7, wherein the lower leaf fraction is a fraction obtained by sequentially fractionating lower leaf extracts with ethyl acetate, butanol, and water.
8. The cosmetic composition according to claim 7, wherein the lower leaf fraction is an ethyl acetate fraction.

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