KR20160141093A - Cosmetic composition containing extract fraction or compound of Sorghumdrummondii Nees ex Steud. Millsp. and Chase - Google Patents
Cosmetic composition containing extract fraction or compound of Sorghumdrummondii Nees ex Steud. Millsp. and Chase Download PDFInfo
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Abstract
The present invention relates to the active compounds separated from the ethyl acetate fraction of Sorghum x drummondii (Nees ex Steud.) Millsp. & Chase extract, fraction or Sudan grass, para-Hydroxybenzaldehyde, Since luteoloside, luteolin and apigenin show antioxidative and antimicrobial effects, compositions containing them can be used as cosmetic compositions for antioxidant and antimicrobial use.
Description
The present invention relates to a cosmetic composition comprising Sudanese Grass extract, fraction or compound as an active ingredient.
The human body is in close contact with the external environment, and skin is easily sensitized by environmental pollution, harmful substances, stress and irregular lifestyle. Oxygen is overproduced in the skin exposed by such external environment, and active oxygen is produced. This active oxygen is harmful oxygen in an unsafe state such as attacking the living tissue and damaging the cell. If excess oxygen is produced in the body, it damages the cells and rusts the body, causing various diseases and aging.
On the other hand, the causes of inflammation are numerous, including harmful substances, active oxygen, and biological agents such as bacteria, fungi, and viruses. Acne is caused by bacteria growing and inflammation as sebaceous glands increase in sebaceous glands or sebaceous pores become narrower or clogged and sebum can not be released. The inflamed tissue is a major cause of atopic dermatitis and skin aging as well as skin troubles by destroying protein or genetic material by physiological mechanism of skin.
Recently, there is growing interest in whether you can live younger and healthier regardless of age. As reflecting social trends, there are many issues such as antioxidant, anti-inflammation, antibacterial, etc. in diverse fields such as diet, beauty, and medicine, and since consumers' demand for products that are less mild and irritating to the skin is increasing, The concept of naturalism that uses materials is being strengthened.
It is an object of the present invention to provide a cosmetic composition for antioxidant and antimicrobial containing an extract of Sudanese Grass as an active ingredient.
Another object of the present invention is to provide an antioxidant and antimicrobial cosmetic composition comprising an organic solvent fraction prepared by further extracting a Sudanese Grass extract with an organic solvent as an active ingredient.
It is another object of the present invention to provide a pharmaceutical composition comprising para-hydroxybenzaldehyde, luteoloside, luteolin, apigenin or pharmaceutically acceptable salts thereof, And a cosmetic composition for antioxidative and antimicrobial use which comprises a salt as an active ingredient.
The present invention provides an antioxidant and antimicrobial cosmetic composition containing Sudanese Grass extract as an active ingredient.
Sudanese Grass is a year-old rice-field grass that belongs to the genus Siberia. It is tall and leafy. If cultivation condition is good, it grows up to 1.8 ~ 2.5m, and it is a plant which can be regenerated after cutting. The country of origin is the African tropics including Sudan, and it is recorded that it was cultivated in ancient Egypt. It is also possible to use as hay or silage as well as grazing and livestock (silage, feedstuffs such as grasses with high moisture content, which are stored in vacuum in silo containers and fermented by lactic acid bacteria). Especially, Which is an indispensable forage crop in summer.
The Sudanese Grass is Sorghumx drummondii (Nees ex Steud.) Millsp. & Chase. ≪ / RTI >
The Sudanese Grass may be collected, cultured, or marketed without limitation. For example, the Sudanese Grass may be collected and washed with water to remove foreign matter and salt, followed by drying.
In the present specification, "Sudanese Grass Extract" is not particularly limited as long as it is obtained by extracting the active ingredient from the Sudanese Grass.
For example, the resultant product obtained by putting the Sudanese glass into water or an organic solvent and eluting the active ingredient through means such as setting, stirring, pressing, or heating. It also includes naturally dried or lyophilized material obtained by naturally drying or lyophilizing the liquid extract obtained as described above. It also includes powders obtained by pulverizing such natural products or lyophilized products. In addition, it includes all the extracted extracts regardless of the means available for extracting the active ingredient, including those extracted and then subjected to natural drying or freeze-drying. Other examples include extracts obtained by conventional extraction methods such as extracting by hot water or room temperature, or extracts obtained by conventional extraction methods as described in Korean medicine or textbooks. It also includes a fraction extract obtained through a Stass Otto extraction method or various column chromatography methods, which is a special extraction method for separating specific active compounds.
The Sudanese Grass extract according to the present invention may be one prepared by a conventional extraction method such as an ultrasonic extraction method, a filtration method and a reflux extraction method. Preferably, the Sudan grass may be an extract obtained by extracting C 1 to C 4 lower alcohols or a mixed solvent thereof, more preferably an extract extracted with a C 1 to C 4 lower alcohol, and most preferably methanol Or ethanol extract. For example, the natural dry powder of Sudanese Grass may be dipped in 70% ethanol and extracted at room temperature for 24 hours. The extract residue may be removed and concentrated in vacuo to obtain an extract.
The present invention provides an antioxidant and antimicrobial cosmetic composition comprising an organic solvent fraction prepared by further extracting a Sudanese Grass extract with an organic solvent as an active ingredient.
The organic solvent may be selected from the group consisting of hexane, ethyl acetate, butanol, and water, but is not limited thereto.
The present invention relates to a pharmaceutical composition comprising para-hydroxybenzaldehyde, luteoloside, luteolin, apigenin or a pharmaceutically acceptable salt thereof, isolated from Sudanese Grass extract, The present invention provides an antioxidant and antimicrobial cosmetic composition comprising as an active ingredient.
The para-hydroxybenzaldehyde, luteoloside, luteolin and apigenin may be isolated from the Sudanese gruel extract, especially those isolated from the ethylacetate fractions, such as para-hydroxybenzaldehyde, luteoloside, luteolin, But is not so limited.
The Sudanese Grass extract may be an extract extracted from a C 1 to C 4 lower alcohol or a mixed solvent thereof, more preferably an extract extracted with a C 1 to C 4 lower alcohol, and most preferably methanol or It may be an extract extracted with ethanol.
The cosmetic composition according to the present invention may contain 0.001 to 10 parts by weight per 100 parts by weight of the total composition.
The cosmetic composition may be prepared by dissolving or dispersing at least one compound selected from the group consisting of a fatty substance, an organic solvent, a solubilizer, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, Such as cosmetics or skin, such as fillers, sequestering agents, chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics And may contain adjuvants conventionally used in the scientific field. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.
The external form of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. It may be in any form suitable for topical application, for example, as a solution, a gel, a solid, a paste anhydrous product, an emulsion obtained by dispersing the oil phase in water, a suspension, a microemulsion, a microcapsule, In the form of a non-ionic follicle dispersing agent, or in the form of creams, skins, lotions, powders, ointments, sprays or conical sticks. These compositions may be prepared according to conventional methods in the art. The composition according to the invention may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.
The cosmetic composition containing the active ingredient according to the present invention is not particularly limited in its formulation and may be, for example, softening lotion, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, Cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence.
The cosmetic composition as described above may be applied to the skin, or may be applied to the inside of the skin using a micro needle or the like.
The concentration of para-hydroxybenzaldehyde, luteoloside, luteolin, apigenin or a pharmaceutically acceptable salt thereof in accordance with the present invention may be in the range of 20-100 μg / mL. < / RTI >
In one embodiment of the invention, four compounds were isolated from the Sudanese Grass Ethyl Acetate fractions and the compounds were analyzed using para-Hydroxybenzaldehyde, luteoloside, luteolin, , And apigenin, respectively. The compound is a substance initially separated from Sudanese Grass.
In one embodiment of the present invention, the Sudanese Grass Ethyl Acetate fraction showed excellent DPPH radical scavenging activity at 91.44 μg / mL compared to the positive control BHT (SC 50 191.64 μg / mL) (FIG. 17).
In one embodiment of the invention, the nitrogen monoxide production in the ethyl acetate fraction at a concentration of 100 μg / mL was reduced to 23.79%, resulting in a nitric oxide production inhibition effect of 38.73% at 100 μg / mL of melasolv, a positive control, (Fig. 19). Furthermore, it was confirmed that luteolin and apigenin exhibit excellent nitric oxide production inhibitory activity (Fig. 22).
The present invention relates to the active compounds separated from the ethyl acetate fraction of Sorghum x drummondii (Nees ex Steud.) Millsp. & Chase extract, fraction or Sudan grass, para-Hydroxybenzaldehyde, Since luteoloside, luteolin and apigenin show antioxidative and antimicrobial effects, compositions containing them can be used as cosmetic compositions for antioxidant and antimicrobial use.
1 is a graph showing the results obtained from Sorghum x drummondii (Nees ex Steud.) Millsp. &Amp; Chase with n- hexane layer, ethyl acetate layer, n- butanol layer, (water layer) is obtained.
Figure 2 is a schematic diagram showing the steps of obtaining
3 is a chemical structural formula of
4 is a graph showing the results of 1 H-NMR (nuclear magnetic resonance spectrometer) spectrometry of
5 is a graph showing the results of 13 C-NMR spectrum analysis for
6 is a chemical structural formula of
7 is a graph showing the results of 1 H-NMR spectrum analysis for
8 is a graph showing the results of 13 C-NMR spectrum analysis for
9 is a chemical structural formula of
10 is a graph showing the results of 1 H-NMR spectrum analysis of
11 is a graph showing the results of 13 C-NMR spectrum analysis for
12 is a chemical structural formula of
3 is a graph showing the results of 1 H-NMR spectrum analysis for
14 is a graph showing the results of 13 C-NMR spectrum analysis for
Figure 15 shows the total polyphenolic compound content in the Sudanese Grass extract and fractions; Extract: Extract, Hex: Hexane fraction, EA: Ethyl acetate fraction, BuOH: Butanol fraction, DW: Water fraction.
Figure 16 shows the total flavonoid compound content in the Sudanese Grass extract and fractions; Extract: Extract, Hex: Hexane fraction, EA: Ethyl acetate fraction, BuOH: Butanol fraction, DW: Water fraction.
Figure 17 shows the DPPH radical scavenging activity of Sudanese Grass extract and fractions; Extract: Extract, Hex: Hexane fraction, EA: Ethyl acetate fraction, BuOH: Butanol fraction, DW: Water fraction, BHT: Positive control, Butylated hydroxytoluene.
Figure 18 shows the ABTS radical cation scavenging activity of Sudanese Grass extract and fractions; Extract: Extract, Hex: Hexane fraction, EA: Ethyl acetate fraction, BuOH: Butanol fraction, DW: Water fraction, BHT: Positive control, Butylated hydroxytoluene.
Fig. 19 is a graph showing nitrogen monoxide (NO) production inhibitory activity of Sudanese Grass extract and fraction; -: untreated, +: LPS (lipopolysaccharide), Extract: Hex: hexane fraction, EA: ethyl acetate fraction, BuOH: butanol fraction, DW: water fraction, Melasolv: positive control, melasolv.
20 is a graph showing the activity of inhibiting nitric oxide (NO) production by concentration of Sudan grass hexane fraction.
Fig. 21 is a graph showing nitric oxide (NO) production inhibitory activity of each concentration of Sudan grass ethyl acetate fraction. Fig.
22 is a graph showing the concentration of nitrogen monoxide in the compound 1 (para-Hydroxybenzaldehyde), Compound 2 (luteoloside), Compound 3 (luteolin) and Compound 4 (apigenin) NO) production inhibitory activity.
Figure 23 shows the cytotoxicity of the Sudanese grass extract and fractions; +: LPS (lipopolysaccharide), -: untreated, Hex: hexane fraction, EA: ethyl acetate fraction, BuOH: butanol fraction, DW: water fraction.
24 shows the cytotoxicity of the Sudanese Grass hexane fraction by concentration.
25 is a graph showing the cytotoxicity of the Sudan grass ethyl acetate fraction by concentration.
26 shows the cytotoxicity of Compound 1 (para-Hydroxybenzaldehyde), Compound 2 (luteoloside), Compound 3 (luteolin) and Compound 4 (apigenin) Fig.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
< Example 1> Sudanese Grass Extract preparation
Sorghum × drummondii (Nees ex Steud.) Millsp. & Chase) was collected at the morning farmhouse in Wolpyeong-dong, Jeju-do. The ground portion of the collected Sudanese Grass (Sample No. 349) was completely dried naturally in the shade field and used for grinding.
After 1.4 kg of dried Sudanese Grass was pulverized, it was added to 20 L of 70% ethanol and stirred at room temperature for 24 hours. The leached samples were filtered using a filtration apparatus under reduced pressure, and the residue thus separated was further subjected to the same filtration twice. The filtrate thus obtained was concentrated on a rotary vacuum evaporator in a water bath at 40 ° C or lower to obtain 131.85 g of a 70% ethanol extract. The extracts were suspended in distilled water and sequentially fractionated according to the polarity order using a separating funnel to obtain a total of four solvent fraction layers such as a n-hexane layer, an ethyl acetate layer, an n-butanol layer and a water layer ).
< Example 2> Sudanese Grass Isolation and Identification of Compounds from Extracts
≪ 2-1 >
After fractionation of the solvent, 4.76 g of the ethyl acetate layer in each of the obtained fraction layers was subjected to a reversed phase silicagel (C18HS12 + M1946-3, Biotage Co.) column using a medium pressure liquid chromotography The fractions were separated into MP-1 to MP-48 fractions, i.e., a total of 48 fractions, with 10 to 100% methanol (MeOH) for 80 minutes and 100% methanol for 10 minutes.
The fraction separated by MPLC was measured by high-performance liquid chromatography (HPLC) using 20-80% methanol as a method set and a run time of 40 minutes as a method set. The amount was measured.
The same spot fractions were combined through thin layer chromatography (TLC), and the solvent was separated to remove the compound through a Sephadex LH-20 (0.1-0.025 mm) column.
56.9 mg of the MPLC fractions MP-14 to MP-15 were confirmed to have the same spot through TLC and the mobile phase was subjected to Sephadex-
When methanol was added after 182.2 mg of the MPLC fractions MP-18 to MP-20 were concentrated, a powder that did not dissolve but precipitated in a vial was generated. This was recrystallized using methanol to obtain Compound 2 (16.2 mg) Respectively.
The PLC fractions, MP-25 to MP27, 430.7 mg were confirmed to be the same spot through TLC and the mobile phase conditions were subjected to Sephadex LH-20 column chromatography under chloroform: methanol = 8: 1 to obtain
The MPLC fractions MP-29 to MP-30 (303.4 mg) were confirmed to have the same spot through TLC, and the mobile phase conditions were subjected to Sephadex LH-20 column chromatography under chloroform: methanol = 10: Compound 4 (106.6 mg) was isolated.
<2-2> Identification of compound
The nuclear magnetic resonance spectrometer used for the structural analysis was JNM-ECX 400 (FT-NMR system, JEOL) and AVANCE III 500 (FT-NMR system, Bruker) CD 3 OD and pyridine- d 5 were used as NMR-specific solvents.
Apigenin < / RTI > (Table 4, Figs. 12 to 14).
< Example 3> Total polyphenol compound content measurement
The content of the polyphenol compound is determined by the Folin-Denis method (Folin, O. Denis, W. "A colorimetric method for determination of phenols (phenol dericatives) in urine" J. Biol. , 305-308.).
First, gallic acid standard solution was used as a standard curve for the determination of polyphenol compounds. To prepare a standard curve, 1 mg of gallic acid was dissolved in 1 mL of a 1: 1 solution of dimethyl sulfoxide (DMSO): ethanol (EtOH) to prepare a stock solution. The solution was adjusted to 0, 31.25, 62.5, 125, 250 and 500 μg / mL, and absorbance was measured at 700 nm using an ultraviolet-visible spectrophotometer to prepare a standard curve. Each sample was dissolved in a solvent at a concentration of 1 mg / mL, and then 100 μL of this solution was taken in a microtube and 900 μL of distilled water was added thereto to dilute the total volume to 1 mL. To this, 100 μL of Folin-ciocalteu's phenol reagent was added, mixed well and left at room temperature for 3 minutes. Add 200 μL of 7% sodium carbonate (Na 2 CO 3 ) solution to this solution, dilute to a total volume of 2 mL with 700 μL of distilled water, leave at room temperature for 1 hour, take the supernatant, nm, and the polyphenol compound content was converted.
The total polyphenol compound content of the Sudanese Grass extract and fractions was expressed as the amount of gallic acid (GAE) contained per 1 mg of the extract. As a result, 24 μg GAE / 1 mg of the extract and 135.9 μg GAE / 1 mg of ethyl acetate in the fraction were obtained. mg, indicating a relatively high polyphenol compound content (Fig. 15).
< Example 4> Determination of total flavonoid compound content
The total flavonoid content side of Davis (Davis) method (Davis, FB; Middleton, E .; Davis, PJ; Blas, SD, "Inhibition by quercetin of thyroid hormone stimulation in vitro of human red
The total flavonoid content of Sudanese Grass extract and fractions was calculated as 43.92 μg quercetin / 1 mg in the extract, 50.92 μg quercetin in the hexane, ethyl acetate, butanol and water fractions, respectively, / 1 mg, 186.75 μg quercetin / 1 mg, 51.75 μg quercetin / 1 mg, 36.75 μg quercetin / 1 mg, and the ethyl acetate fraction showed a relatively high flavonoid content (FIG.
< Example 5> DPPH Radical scavenging activity experiment
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity test was performed to evaluate the free radical scavenging ability or the hydrogen donating ability of the sample. The DPPH radical scavenging activity experiment was applied to Blois method (Blois, M. S. "Antioxidant determination by the use of a stable free radical" Nature, 1958, 181, 1199-1200).
180 μL of a solution of 20 μL (ethanol solvent (in EtOH)) and 0.2 mM DPPH (ethanol solvent (in EtOH)) diluted to concentrations of 400, 200, 100 and 50 μg / mL in a 96 well plate And incubated at room temperature for 25 minutes. Absorbance was measured at 515 nm using an enzyme-linked immunosorbent assay reader (ELISA Reader). The radical scavenging rate (%) was calculated by the following equation, and the sample concentration (SC 50 ) when the scavenging activity percentage of each sample was 50% was determined. As a positive control, butylated hydroxytoluene (BHT) was used.
Radical scavenging activity (%) = {1- (A sample -A blank ) / A control } × 100
A sample : absorbance of DPPH at 515 nm
A blank : Absorbance of sample and DPPH reaction solution at 515 nm
A control : the absorbance of the sample itself at 515 nm
As a result, Sudan grass ethyl acetate fraction showed excellent DPPH radical scavenging activity at 91.44 μg / mL as compared to the positive control group BHT (SC 50 191.64 μg / mL) (FIG. 17).
< Example 6> ABTS Radical Cationic scavenging activity experiment
In order to measure the total antioxidant activity (TAC) of the sample, a radical cation-scavenging activity test was performed on ABTS [2,2'-Azino-bis (3-ethyl benzothiazoline-6-sulfonic acid)]. The ABTS radical cationic scavenging activity experiment was carried out by the method of Re, Pellegrin et al. (Re .; Pellegrini, N .; Proteggente, A. Pannala, A .; Yang, M .; Rice-Evans, , "Free Radical Biology and Medicine, 1999, 26, 1231-1237".
To prepare the ABTS radical cation, a solution of 7.0 mM ABTS (in DW) and 2.45 mM potassium persulfate (in DW) solution were mixed 1: 1 and reacted in the dark for 16 hours. . The reacted ABTS solution and ethanol were diluted 1:60 and adjusted to an absorbance of 0.78 ± 0.002 at 700 nm. 20 μL of the sample solution (in ethanol (in EtOH)) diluted by 100, 50, and 25 μg / mL was mixed with 180 μL of ABTS solution in a 96-well plate and reacted in a dark place for 15 minutes. An enzyme immunoassay reader Absorbance at 700 nm. The radical scavenging rate (%) was calculated by the following equation, and the sample concentration (SC 50 ) when the scavenging activity percentage of each sample was 50% was determined. As a positive control, butylated hydroxytoluene (BHT) was used.
Radical scavenging activity (%) = {1- (A sample -A blank ) / A control } × 100
A sample : absorbance of ABTS at 700 nm
A blank : Absorbance of sample and ABTS reaction solution at 700 nm
A control : the absorbance of the sample itself at 700 nm
As a result, the Sudanese Grass Ethyl Acetate fraction showed an ABTS radical cation-scavenging activity of 10.24 μg / mL (FIG. 18).
< Example 7> Antitumor activity test using cell line
<7-1> Cell culture
RAW 264.7 cell line, a murine macrophage cell line, was purchased from Korean cell line bank (KCLB, Korean Cell Line Bank, Seoul, Korea) and cultured with 1% penicillin- streptomycin and 10% fetal bovine serum (CO 2 ) at 37 ° C in DMEM (Dulbecco's Modified Eagle Medium, GIBCO, Grand Island, NY, USA) supplemented with fetal bovine serum (FBS) Subculture was performed.
<7-2> Nitric oxide ( Nitric oxide ) Production inhibition evaluation
To evaluate the inhibitory activity against nitric oxide production, RAW 264.7 cell line was divided into 6 well plates at 3 x 105 cells / mL. The cultured cells were cultured for 18 hours at 37 ℃ under 5% carbon dioxide incubation. The pre-cultured cells were exchanged with a medium containing 1 μg / mL LPS, and the samples were added for each concentration and cultured for 24 hours. The amount of NO produced was determined using the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine, 2.5% phosphoric acid) in the cell culture medium And measured in the form of nitrite ions (NO 2 - ). 100 μL of the cell culture supernatant and 100 μL of the grease reagent were mixed and reacted on a 96-well plate for 10 minutes, and the absorbance was measured at 540 nm. The amount of nitrogen monoxide (NO) produced was compared using a calibration curve using sodium nitrite (NaNO 2 ).
As a result, the nitrogen monoxide production in the ethyl acetate fraction of 100 μg / mL was reduced to 23.79%, and the inhibitory effect on nitrogen monoxide production was superior to that of 38.73% in the positive
On the other hand, the results of experiments on para-hydroxybenzaldehyde, luteoloside, luteolin, apigenin isolated from Sudanese Grass extract, luteolin, And apigenin showed excellent inhibitory activity against nitrogen monoxide production (Fig. 22).
<7-3> Evaluation of cytotoxicity
To evaluate cytotoxicity, MTT [3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay was performed. RAW 264.7 cells were plated at 6 × 10 5 cells / mL in a 6-well plate and cultured at 37 ° C. under 5% carbon dioxide condition for 18 hours. Then, 1 μg / mL lipopolysaccharide (LPS) And cultured for 24 hours. After 24 hours of incubation, MTT was added at a concentration of 500 μg / mL and reacted at 37 ° C for 3 hours, and then the supernatant was removed. The formazan precipitate formed by reacting with live cells was added to dimethyl sulfoxide (DMSO), and the resultant was transferred to a 96-well plate and absorbance was measured at 570 nm. Cell viability was calculated by the following equation.
Cell viability (%) = A sample / A control × 100
A sample : Absorbance of sample treated with solvent and LPS at 570 nm
A control : Absorbance of the sample and LPS-treated control at 570 nm
As a result, 80% or more cell viability was observed in the n-hexane fraction at a concentration of 100 μg / mL (FIG. 23). In addition, it was confirmed that the cell viability was reduced in a concentration-dependent manner in the n-hexane and ethyl acetate fractions at the concentrations of 100, 50 and 25 μg / mL (FIGS. 24 and 25).
On the other hand, in the cytotoxicity test results of para-hydroxybenzaldehyde, luteoloside, luteolin and apigenin isolated from Sudanese Grass extract, luteolin ) Did not show cytotoxicity, but apigenin showed cytotoxicity (Fig. 26).
< Example 8> Antibacterial activity test using cell line
<8-1> Diffusion in paper discs Paper disc diffusion ) Measure
In order to measure the antimicrobial activity of Sudanese Grass extract and fractions, Propionibacterium acnes ) CCARM 0081, CCARM 9009, CCARM 9010 and Staphylococcus epidermis CCARM 3709, CCARM 3710, CCARM 3711 (antibiotic-resistant strains) were measured by the paper disc diffusion method.
P. acnes was prepared by adding 200 μL of a liquid medium containing 104 CFU / mL P. acnes to a GAM solid medium (Nissui Pharmaceutical, Japan) containing 1.5% of agar and then smearing with a smear stick. When the liquid medium was sufficiently absorbed into the medium, the 8 mm diameter paper disk containing the sample solution was placed on the disk and the circular growth inhibition circle formed after anaerobic incubation at 37 ° C for 48 hours was measured.
In S. epidermidis , 200 μL of liquid medium containing 105 CFU / mL of S. epidermidis was added to TSA (Tryptic Soy Agar) solid medium containing 1.5% of agar, and then stained with a smear rod. When the liquid medium was sufficiently absorbed into the solid medium, a paper disk having a diameter of 8 mm containing the sample solution was placed on the disk and incubated at 37 ° C for 24 hours to measure the size of the circular growth-inhibitory circle formed around the disk.
As a result, the circular growth inhibition diameter values of P. acnes and S. epidermidis were found to be 19 to 20 mm for the Sudan grass hexane fraction and 19 to 21 mm for the ethyl acetate fraction (Table 5, Table 6). In the positive control group Erythromycin, P. acnes CCARM 9010 and S. epidermidis CCARM 3710 showed no growth retardation, but hexane and ethyl acetate fractions showed growth inhibition effect.
<8-2> Minimum inhibitory concentration ( MIC , minimum inhibitory concentration ) Measure
The same manner as in Example 8-1 with P. acnes CCARM 0081, CCARM 9009 and CCARM 9010 were cultured. After adding 150 μL of medium to each well of a 96-well plate, 150 μL of 2 mg / mL sample was added to make a concentration of 1000 μg / mL. 150 μL of the solution was inoculated into a well containing 150 μL of the medium mu] g / mL. Samples were continuously diluted in the same manner and diluted to a concentration of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.63 μg / mL, 7.81 μg / mL, 3.91 μg / mL, μg / mL, and the last well was used as a control. To the diluted samples, 15 μL of 104 CFU / mL P. acnes was added, and after 48 hours, the concentration of the microorganism did not grow and the MIC was determined.
As a result, it was confirmed that the MIC was 500 μg / mL in the
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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KR20140135870A (en) | 2013-05-16 | 2014-11-27 | (주)에이씨티 | Antiseptic compositions comprising Magnolia grandiflora extract and cosmetic compositions containing the same |
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