KR20150094174A - Extraction method of Hylotelephium ussuriense active component - Google Patents

Abstract

The present invention relates to an extracting method of an active ingredient of Hylotelephium ussuriense having an antioxidant and an antibacterial function. An active ingredient extract from Hylotelephium ussuriense prevents skin aging and treats atopic dermatitis, etc. as well as protecting a skin by applying to a functional health food, a food additive, beverage, cosmetics, atopic dermatitis treating agent, detergent, etc., wherein the method comprises the steps of: (a) collecting Hylotelephium ussuriense except for roots; (b) drying the collected Hylotelephium ussuriense; (c) grinding the dried Hylotelephium ussuriense; (d) extracting an active ingredient of the grinded Hylotelephium ussuriense; (e) isolating supernatant of the extract; and (f) concentrating the supernatant.

Description

{Extraction method of Hylotelephium ussuriense active component} <br> <br> <br> Patents - stay tuned to the technology Extract of Hylotelephium ussuriense active component,

The present invention relates to a method for preventing skin aging and preventing atopic dermatitis, as well as protecting the skin by applying an active ingredient to a healthy functional food, a food additive, a drink, a cosmetic, an atopy treatment agent, The present invention also relates to a method for extracting an effective component of a radish leafy pheasant having an antioxidant and antimicrobial activity.

Hylotelephium ussuriense is a perennial herbaceous plant belonging to the family Staphylococcus aureus. It is a native plant distributed only in the shaded rocks of Gwangwangsan Valley, Cheongsongbuk-do, Gyeongsangbuk-do province. It is a valuable genetic resource belonging to the 2nd class of plant and endangered plants (Kwon and Yoon, 2010).

Leaf is round or oval, edge is irregular, dull sawtooth, flower is from late July to early October, end of stem is similar to flower of artificial flower, (Kwon et al., 2010). It has a high value for use as an ornamental plant and has a high development value as a flower crop.

In addition, the young lion of the round leaf pheasant is used for edible purposes, and the whole plant is also used as a medicament such as blood or blood.

In this study, we investigated the effects of the flowering period on the flowering of the pheasant and the flowering of the pheasant, .

Recently, plant propagation has been reported from leaf and stem cuts and leaf slices and shoots from the trichomes using plant tissue culture techniques (Yoon, 1997; Ahn and Lee, 2004; Kwon and Yoon, 2010).

However, there is no known research on the physiological activity of these proliferated resources or their potential application as functional materials. Modern society is undergoing environmental stress caused by diversity of pathogens and environmental deterioration.

If these stresses continue to increase, oxygen, which is essential for life support, becomes highly reactive and becomes a serious physiological disorder and reactive oxygen species (ROS). These reactive oxygen species promote senescence and act as a cause of various diseases.

The skin aging pattern of these reactive oxygen species not only causes fine wrinkles on the skin, atrophy of the dermis and reduction of subcutaneous fat, but also causes atopic dermatitis and acne skin abnormalities.

In addition, the active oxygen species generated by various kinds of instant eating habits of modern people peroxidize the lipids in the human body to produce harmful substances, causing various diseases such as myocardial infarction, stroke, arteriosclerosis and the like.

To remove these reactive oxygen species, antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are known, but they are synthetic antioxidants and their side effects such as mutation, carcinogenicity and intracellular toxicity have been reported Attempts have been made to develop natural antioxidants (Kang et al 2005).

The present invention relates to a method for extracting an effective ingredient from a bean of a round leaf pheasant having antioxidant and antimicrobial activity using a solvent and extracting the extracted extract with a raw material which requires antioxidant and antibacterial, that is, a health functional food, a food additive, Therapeutic agent, and detergent to protect the skin, as well as to prevent skin aging and to treat atopy.

According to an aspect of the present invention,

(a) collecting the leaf pheasants of a round leaf pheasant except for roots;

(b) drying the pickle of the collected round leaf pheasant;

(c) pulverizing the dried bean curds;

(d) extracting an effective component of the bean curd from the crushed round leaf pheasant;

(e) isolating the supernatant of the extract;

(f) concentrating the supernatant; .

The extract of the effective ingredient of the radish leaf extract of the round leaf pheasant having the antioxidant and antimicrobial activity extracted by the method of the present invention having the above-mentioned structure can be used as a raw material requiring antioxidant and antibacterial, that is, a health functional food, a food additive, , And detergent to protect the skin, as well as to prevent aging of the skin and atopy can be effective.

Fig. 1 is a photograph showing the antibacterial effect of the bamboo shoot extract of the round leaf pheasant.

Hereinafter, the present invention will be described in detail as follows.

The method of extracting an effective component from a dried leek pheasant having antioxidant and antimicrobial activity of the present invention comprises:

(a) collecting the leaf pheasants of a round leaf pheasant except for roots; (b) drying the pickle of the collected round leaf pheasant; (c) pulverizing the dried bean curds; (d) extracting an effective component of the bean curd from the crushed round leaf pheasant; (e) isolating the supernatant of the extract; (f) concentrating the supernatant; .

A method of extracting an effective ingredient (antioxidant and antibacterial activity) from the above-mentioned leafhopper bean curd lees using an extraction solvent will be described as follows.

(a)

The step (a) of the present invention is a step of collecting the pickle of a round leaf pheasant except the root,

The plants collected from native habitats (Juwangsan, Kyungsangbuk-do) were grown in a greenhouse (day, 252 night, 202, 16 hours, Gwangju, 5000 lux) at Chonnam National University of Science and Technology.

(b)

The step (b) of the present invention is a step of drying the pickle of the collected round leaf pheasant,

The collected leaf pheasants are washed with sterilized distilled water to remove impurities, and the dried leaf pheasants having the impurities removed are subjected to hot air drying at 60 to 70 ° C.

The reason why the above-mentioned pear leaves are heated to 60 to 70 ° C in order to minimize the leakage of the active ingredient which exhibits antioxidant activity and antimicrobial effect contained in the pear leaves of the round leaf pheasant.

(c)

The step (c) of the present invention is a step of pulverizing the dried curd leaf fence,

The reason for crushing the bean curd of the round leaf pheasant is that it is pulverized by using a crusher and the crushing is performed because when the extract is extracted using an extractant, the antioxidant activity and the antimicrobial effect, which are contained in the bean curd, In order to maximize the extraction effect.

 (d)

The step (d) of the present invention is a step of extracting an effective component of the bean curd of the crushed round leaf pheasant,

In a first embodiment of the extraction method, 9 to 11 g of the pulverized raw material is subjected to hot water extraction with 50 to 200 ml, preferably 80 to 100 ml, of sterilized purified water at 60 to 70 ° C for 3 to 5 hours,

In a second embodiment of the extraction method, 9 to 11 g of the pulverized raw material is placed in 50 to 200 ml, preferably 80 to 100 ml, of 80% -95% ethanol and stirred for 24 to 30 hours at room temperature.

In the third embodiment of the extraction method, 9 to 11 g of the pulverized raw material is put into 50 to 200 ml of 80% -95% ethanol and pressure-extracted at 40 to 60 ° C and 1300 to 1600 psi for 3 to 7 hours

In the fourth embodiment of the extraction method, 9 to 11 g of the pulverized raw material is put into 50 to 200 ml of 80% -95% ethanol and subjected to ultrasonic pulverization extraction at 40 to 60 ° C and 10 to 100 kHz.

The extraction conditions and the extraction time in the first to fourth extraction methods are ranges determined in order to obtain the extract at a high yield and to increase the content of the active ingredient having antioxidant activity and antibacterial activity in the extract.

 (e)

The step (e) of the present invention is a step of separating the supernatant of the extract,

The supernatant of the extract is separated by filtration under reduced pressure or centrifuged at 3000 to 5000 rpm for 30 to 60 minutes to recover the supernatant.

(f)

The step (f) of the present invention is a step of concentrating the supernatant,

The supernatant liquid concentrating method comprises concentrating and lyophilizing the supernatant in a rotary vacuum evaporator (N-1000S-WD, EYELA, Tokyo, Japan).

Experimental examples according to the method of extracting the active ingredient of the radish of the round leaf pheasant are as follows.

The berry extract of the round leaf pheasant was washed with sterilized distilled water, dried in a hot air drier at 60 to 70 ° C, and then pulverized with a pulverizer. The pulp of the pulverized round leaf pheasant was put into 80% and 95% ethanol, The extracted extract was filtered and concentrated, lyophilized and extracted with other solvents are shown in Table 1 below

division
Purified water
ethanol 80% 95% Extraction yield 25.2% 35.5% 24.5%

As shown in Table 1, it can be seen that the extracts extracted by the method of the present invention have a higher yield than those extracted by other solvents.

The antioxidant activity of the extracted extract was determined by EDA (eletron donating abilities) measurement, and the electron donating ability was measured according to the Blois method (Kim et al, 2009).

Each sample was diluted to a constant concentration and 100 μl of 0.4 mM α, α-diphenyl-picrylhydrazyl (DPPH, Sigma Aldrich Co, MO, USA) was added to 2.0 ml of the sample solution. After stirring for 30 minutes at room temperature, The absorbance (UV-1601 Visible Spectrophotometer, Shimadzu Co., Japan) was measured and expressed as the absorbance reduction rate of each extract.

BHA (2,3-tert-butyl-4-hydroxyanisole, Sigma, USA) was used as a control.

DPPH radical scavenging activity (%) = (Control Absorbance-Sample Absorbance / Control Absorbance) × 100

Examples of the antioxidant activity against DPPH scavenging activity according to the kind of the above-mentioned extraction solvent are shown in Table 2 below.

Antioxidant activity of DPPH extracts of Pinus koraiensis Fraction Concentration (ug / ml) Scavenging effect (%)
Purified water

10 3.53 + - 0.50 50 5.50 + - 0.50 100 10.4 ± 1.50 500 20.5 ± 2.12

80% EtOH
10 2.0 ± 0.22 50 5.4 ± 1.50 100 12.0 ± 0.22 500 25.0 + - 0.71

95% EtOH
10 5.0 ± 3.0 50 7.0 ± 0.25 100 30.3 ± 2.8 500 50.4 ± 2.3

BHA (positive control)
10 5.0 ± 3.0 50 8.0 ± 0.25 100 29.3 ± 1.8 500 45.4 ± 2.3

As shown in Table 2 above, the DPPH scavenging ability of the 95% ethanol extract of the radish extract of the radish leaf pheasant showed the highest antioxidative activity by 50.4%.

The present invention also measured the total polyphenol content, which was measured by the Folin-Denis method for total phenolic compounds (Lee and Kim 2009).

To 25 μl of the sample solution, 0.5 ml of 0.1 N Folin-ciocalteu reagent (Sigma Aldrich Co., MO, USA) was added and left at room temperature for 5 minutes.

After 0.5 ml of 7.5% Na ₂ CO ₃ was added and the mixture was allowed to stand at 30 ° C for 90 minutes, the absorbance was measured at 725 nm.

Amounts were calculated using standard curves prepared with tannic acid equivalents (TAE, Sigma Aldrich Co., MO, USA).

Examples of the total polyphenol content measurement according to the kind of the extract are shown in Table 3 below.

The total polyphenol content of the radish extract of the round leaf pheasant Extraction solvent Total polyphenol ^{1 )} (mg / g) Purified water 50 ± 2.1 80% EtOH 95.3 ± 2.1 95% EtOH 80.20 ± 3.5

As shown in Table 3, the total polyphenol content of the 80% ethanol extract was found to be the highest at 95. mg / g.

The total flavonoid content was measured by the method of Nieva Moreno et al. (2000).

0.1 ml of 10% Aluminum nitrate (Daejung Chemical & Metals Co., Ltd., Shiheung-city, South Korea) and 1 M Potassium acetate (manufactured by Junsei Co., Tokyo) were added to 0.5 ml of a mixture of 100 각 of each sample and 900 쨉 l of 80% , Japan) and 4.3 ml of 80% ethanol were added, and the mixture was allowed to stand at room temperature for 40 minutes, and the absorbance was measured at 415 nm.

At this time, the content of total flavonoid was determined from a standard curve prepared using Quercertin (Sigma Aldrich co., MO, USA).

Table 4 shows examples of total flavonoid contents according to the types of the above-mentioned extraction solvents.

 The total flavonoid content of the radish extract of the round leaf pheasant Extraction solvent Flavonoid (mg / g) Purified water 30.12 ± 0.20 80% EtOH 90.6 ± 0.58 95% EtOH 40.42 + 0.30

As shown in Table 4, the total flavonoid content of the 80% ethanol extract was found to be the highest at 90.6 mg / g.

(Ahn et al., 2009; Lee et al., 2009; Ryu et al., 2010) were used to measure anti-bacterial activity using the extracts of the present invention. .

The anti-bacterial activity was Gram-negative bacteria, Escherichia coli KCTC2571, Pseudomonas aeruginosa KCTC2513 as Gram-positive bacteria and Staphylococcus aureus subsp. Use KCTC1916 aureus, Bacillus subtilis QST713 Tryptic Soy Agar (TSA, Difco Co., MD, USA) in the medium ^{1 × 10 4 colony forming units (} cfu) / ㎖ a smear after a disc of 8mm diameter paper (Adventec, Toyo Roshi Kaisha Ltd., Tokyo, Japan), followed by drying and volatilization of the solvent, followed by incubation at 35 ° C for 18 hours.

The diameter of the growth inhibition ring was measured using a digital caliper (PDIC-301, Stolz, USA), and the mean value was used after 3 repeated experiments.

Examples of antibacterial effects according to the kind and method of the above extract are shown in FIG. 1 and Table 5 below.

Antimicrobial activity of dried leafy pheasant extract
Strain
Clear zone (mm) ^* Content of extract (mg / disc) Extracts 0.1 0.5 1.0 2.5 5.0 Gram  (-) bacteria Escherichia coli
KCTC2571 80% ethanol - - - - - 95% ethanol - - - - - Pseudomonas aeruginosa
KCTC2513 80% ethanol - - - 13.69 + - 0.53 28.00 ± 0.55 95% ethanol - - - - - Gram  (+) bacteria Staphylococcus aureus
KCTC1916 80% ethanol - - - 15.29 ± 1.25 30.28 ± 1.38 95% ethanol - - - - - Bacillus subtilis
QST713 80% ethanol - - - 13.69 + - 0.53 28.00 0.25 95% ethanol - - - - -

As shown in Table 5, when 5 mg of 80% ethanol extract was treated with Gram negative bacteria strain Pseudomonas aeruginosa Clear zone 28 mm for KCTC2513, Staphylococcus aureus KCTC1916 clear zone 30 mm for Gram positive bacteria strain was confirmed to have the highest antimicrobial activity.

The active ingredient extract of Bacillus thuringiensis having antioxidant and antimicrobial activities extracted by the method of the present invention as described above can be used as a raw material which requires antioxidant and antibacterial, that is, a health food, a food additive, a drink, a cosmetic, It protects the skin by applying it, and it also has the advantage of preventing skin aging and treating atopy.

Claims (9)

(a) collecting the leaf pheasants of a round leaf pheasant except for roots;
(b) drying the pickle of the collected round leaf pheasant;
(c) pulverizing the dried bean curds;
(d) extracting an effective component of the bean curd from the crushed round leaf pheasant;
(e) isolating the supernatant of the extract;
(f) concentrating the supernatant;
Wherein the antimicrobial activity of the antimicrobial activity of the antimicrobial antimicrobial agent of the present invention is not less than 10%. The method according to claim 1,
The method for extracting the effective components of a radish pheasant having an antioxidant and antimicrobial activity, wherein the radish drying of the radish leaves of the step (b) is carried out by hot air drying at 60 to 70 ° C. The method according to claim 1,
In the step (d), 9 to 11 g of the pulverized raw material is extracted with 50 to 200 ml of sterilized purified water by heating at 60 to 70 ° C for 3 to 5 hours. The antioxidant and antimicrobial activity Extracting Method of Active Components from. The method according to claim 1,
In the step (d), 9 to 11 g of the pulverized raw material is put into 50 to 200 ml of 80% -95% ethanol, and the mixture is stirred at room temperature for 24 to 30 hours to extract the antioxidant and antimicrobial activity. Extracting Method of Active Components from. The method according to claim 1,
In step (d), 9 to 11 g of the pulverized raw material is put into 50 to 200 ml of 80% -95% ethanol and pressure-extracted for 3 to 7 hours under the conditions of 40 to 60 ° C and 1300 to 1600 psi Wherein the antimicrobial activity of the antimicrobial agent is not less than 10%. The method according to claim 1,
In the step (d), 9 to 11 g of the pulverized raw material is put into 50 to 200 ml of 80% -95% ethanol and ultrasonically pulverized and extracted at 40 to 60 ° C and 10 to 100 kHz. Antioxidant and Antimicrobial Activity of Leaf Pheasant in Round Leaf. The method according to claim 1,
In step (e), the supernatant is separated by filtration under reduced pressure, or the supernatant is separated by centrifugation at 3000 to 5000 rpm for 30 to 60 minutes. Antioxidant and Antimicrobial Activity of Leaf Pheasant in Round Leaf. The method according to claim 1,
In step (f), the supernatant is concentrated or lyophilized in a rotary vacuum evaporator (N-1000S-WD, EYELA, Tokyo, Japan) Extracting the active ingredient from the bran. 8. An extract of an effective component of the bark extract of a round-leaved pheasant having antioxidant and antimicrobial activity extracted by an extraction method according to any one of claims 1 to 8.

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