KR101400975B1 - Composition containing brazilein of Caesalpinia sappan L. water extract residue for preventing or treating of acne - Google Patents

Abstract

Caesalpinia sappan L. ) Composition for the prevention and treatment of acne of brazilein, an active ingredient of water extract residue

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating acne of brazilein which is an effective ingredient of Caesalpinia sappan L. water extract residue,

The present invention relates to a composition for preventing and treating acne of brazilein which is an active ingredient of Caesalpinia sappan L. water extract residue.

Acne is a chronic inflammatory disease of the epidermis of the hair follicle characterized mainly by facial, chest, back, shoulder, and papillary, papillary, pustular, cystic, and nodular formation. In the past, it occurred mainly in adolescents, but air pollution, . In recent years, it has occurred regardless of age, and the age group of acne is becoming widespread. Although the exact cause of acne is not known, the causes of acne include the increase of sebum secretion, propionibacterium , which breaks down sebum and propagates in androgen and fur branch lines, which are hormones that stimulate sebaceous glands, It is known that the inflammatory process by acnes , abnormal keratinization in hair follicle, and genetic deprivation are caused by complex action. Pilosebaceous unit of the normal skin, Gram-positive, coagulase negative, aerobic bacteria, Staphylococci, Micrococci and lipophilic yeast which are pityrosporum ovale and pityrosporum orbiculare (these Pityrosporum species are reclassified as Malassezia fufure and malassezia species), anaerobic strains of P. acnes and P. granulosum , and the development of acne by the activity of these strains, especially anaerobic strain P. acnes The inflammatory reaction of the sebaceous gland and the hair follicle is induced. Aerobic skin sores are not the primary cause of inflammation, but they have been found to exacerbate inflammation sites and affect acne worsening. Therapeutic purposes of acne usually occur in a complex way. All acne treatments are in suppressing pathogenic factors. Depending on the severity of acne, antibiotics and retinoids may be given orally from topical application. If acne is mild, it can be cured by topical application alone, but in cases of severe acne, topical application and oral therapy are effective. Among the drugs, benzoyl peroxide, zelaic acid, retinoic acid, salicylic acid, sulfur and topical antibiotics are the main drugs used for topical application. Among them, benzoyl peroxide, azelaic acid and retinoic acid, And azathioprine. However, azelaic acid, which was originally developed for the treatment of stinging, was used for moderate to severe acne and was reported to have a good effect because it has side effects such as skin redness or skin dryness during overdosing. It is reported that there is an effect of improving antimicrobial activity and abnormal keratinization of P. acnes . These drugs have been reported to have side effects such as hypofluorescence, dryness of the mucous membranes, erythropoiesis, spots, thrombosis and the like, appearance of antibiotic resistant bacteria and recurrence at the time of discontinuation of treatment. In addition, salicylic acid and sulfur preparations are not only ineffective in treatment, but also cause side effects such as skin seizures or skin ghrepia, which further exacerbates acne. Staphylococcus Aureus is a major cause of hospital infection and community acquired infection, and it causes various diseases such as toxin-related diseases, skin and soft tissue infections, urinary tract infections, pneumonia and bacteremia. Bacteremia caused by S. aureus causes various complications such as osteomyelitis, endocarditis and meningitis, and high mortality, and two-thirds of the causes are known to be related to hospital infection. It has been reported over the last 20 years that colonization of Staphylococcus aureus can aggravate lesions of atopic dermatitis, but there is still much debate as to whether staphylococcal superantigen is involved in the pathogenesis of atopic dermatitis. While the normal antigen is presented as an MHC class antigen through the antigen presenting cell and binds to the binding site of the MHC class molecule peptide of the T cell receptor, the superantigen binds to the MHC class antigen of the antigen cell and the variable region of the T cell receptor chain Direct binding leads to T cell activation. Staphylococcal enterotoxin A (SEA) or B (SEB) or toxin shock syndrome toxin-1 (TSST -1) , such as two seconds acts as an antigen, to bind to various T-cell receptor with non-specific method a large amount of T cell produced by the staphylococci Induce activation, and actually show skin changes such as erythema or induration when the SEB is applied to the skin of a normal person. In addition, the expression of type 1 receptor (FcRI), which has strong specificity - IgE affinity for staphylococcal enterotoxin, is increased in atopic dermatitis, which is a result that superantigen acts as an allergen. Recently, studies for identifying natural products containing strong antimicrobial activity and anti-inflammatory activity without toxicity or side effects have been actively conducted at home and abroad.

Accordingly, the present inventors have found that brazilein, an active ingredient of Caesalpinia sappan L. water extract residue, exhibits excellent antimicrobial activity in P. acne , a causative organism of acne .

Accordingly, one example of the present invention provides a composition for preventing and treating acne, which comprises at least one member selected from the group consisting of a root extract and a fraction, as an active ingredient.

Another example provides a composition for preventing and treating acne comprising at least one selected from the group consisting of extracts and fractions of alum.

Hereinafter, the present invention will be described in more detail.

There is provided a composition for preventing and treating acne, comprising as an active ingredient a residue and / or a fraction of a carboxymethylcellulose extract.

Caballo ( caesalpinia sappan L. ) is a deciduous shrub plant distributed in tropical Asia, including India, Malaysia and southern China. It is said to be firewood, red tree, and reddish purple. Timber is hard, heartwood is light red, tree bark and fruit have pigment, so it is used as a source of red dye. East, South China and Southeast Asia (Indonesia, Malaysia). Cut the main stem of the tree, cut off the skin and the edge of the woody part, and use only the middle part as a medicinal product. The core contains about 2% of brazilin, a colorless colorant, which is a pale yellow crystal. When brazilin is in contact with air, it is oxidized to become brazilein. Brazilin is a Benz (b) -Indeno (2,1-d) pyran derivative whose structure has been revealed by Perkin et al. In 1982 and has been mainly used as a dye, an acid, an alkali indicator or a food additive. It also contains -L-phellandren, essential oil based on ocimene and tannin. Recent studies have shown that jojoba has effects such as blood coagulation promoting action (Xie et al . , 2000), anticonvulsant action (Baek et al . , 2000), anti-complement effect (Oh et al ., 1998) It acts on the blood stream and smoothes the flow of blood, eliminating the eosinophilia and boils. Especially, it has the function of eliminating the eosinophilia. It has the effect of relieving the pain. Therefore it is used for various symptoms such as bruising, dysmenorrhea, menstrual occlusion, (bae et al ., 2005, Ratu et al ., 2003). Antimicrobial, cytotoxic, antioxidation, hepatoprotective, and muscle contractile activity have been reported. As a study on the components and structure of carob, 3-benzylchroman derivatives and moisoflavonoids have been studied by Saitoh and Namikoshi, and studies on useful components of carob were campesterol, stigmasterol, -sitosterone, hematein, sappanin, volatile oils, protosappanin A, protosappanin B, and protosappanin E.

In the present invention, Caesalpinia sappan L. ) was used for antimicrobial effect to prevent acne.

The joinery used in the present invention is not particularly limited in its form and is characterized in that the composition of the present invention contains a water extract and / or a residue or fraction obtained from the joinery as an active ingredient.

The callus extract may be obtained by a common joinery extraction method using at least one solvent selected from the group consisting of water and C ₁ to C ₄ linear or branched alcohols or mixtures thereof.

       In one embodiment of the present invention, in order to achieve a more excellent antimicrobial effect, the extract may be an ethanol extract obtained by extracting a native tree with 60 to 80% (v / v) ethanol,

The fraction may be obtained by fractionating the extract into at least one selected from the group consisting of hexane, methylene chloride, ethyl acetate and butanol. More preferably, the fraction may be selected from the group consisting of fractions obtained by applying at least one selected from the group consisting of hexane, methylene chloride, ethyl acetate and butanol in the order of polarity, . For example, the hexane fraction obtained by fractionating the ethanol extract with hexane, the methylene chloride fraction obtained by fractionating the hexane fraction, and the remaining aqueous layer fractionated with methylene chloride. The methylene chloride fraction is separated and the remaining aqueous layer is fractionated with ethyl acetate to obtain the ethyl acetate fraction The butanol fraction obtained by separating the ethyl acetate fraction and the remaining aqueous layer with butanol, and the water fraction remaining after separating the butanol fraction.

 The root extract may be prepared by a conventional method for producing plant extracts. Preferably, the root extract can be obtained by a pressure extraction method, a reflux extraction method, a warm-up extraction method, an ultrasonic pulverization extraction method and the like, But is not limited thereto.

In addition, the extract prepared as described above can be concentrated or removed by concentration filtration and / or lyophilization in a subsequent vacuum filtration step. Therefore, the aloe extract in the present invention is used in the meaning including a dried product dried by a conventional method and a concentrate concentrated in a conventional manner.

The composition comprising brazilein, which is an active ingredient of Caesalpinia sappan L. water extract residue according to the present invention, is a composition derived from a natural product which is excellent in the prevention and treatment of acne and has no toxicity and side effects, have.

Brief Description of the Drawings Fig. 1 is a diagram showing the yields of water extracts of Boehmeria, fractions, and water extract.
2 is a diagram showing the acne bacteria (P. acnes) Growth Inhibition ring joiner of water extracts and fractions, water extract residue.
FIG. 3 is a graph showing the minimum inhibitory concentration (MIC) of losodidium gingiva in a residue of a water extract of algae.
FIG. 4 is a graph showing the minimum inhibitory concentration (MIC) of oral bacteria in a residue of a water extract of Vinegar.
FIG. 5 is a graph showing the minimum inhibitory concentration (MIC) of oral bacteria in a residue of a water extract of Vinegar.
FIG. 6 is a graph showing the minimum inhibitory concentration (MIC) of Staphylococcus aureus from the extract of Vermiculium extract.
FIG. 7 is a graph showing the ¹ H NMR spectrum analysis result of the residue of the water extract of algae.
FIG. 8 is a graph showing the results of ¹³ C-NMR spectrum analysis of the residue of a water extract of algae.
FIG. 9 is a graph showing the results of the Maldi-tof mass spectrum analysis of the residue of the water extract of algae.
Fig. 10 is a diagram showing the chemical structure of brazilein revealed by structural analysis of the residue of water extract of Bombyx mori.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

Material preparation

In 2010, we purchased and used Indonesian mountain.

Sample Preparation Example 1

The water extracts of V. chinensis were repeated three times from 100, concentrated and then lyophilized to obtain powders. The water extract was fractionated by solvent and fractionated by solvent in the order of methylenechoride layer (MC layer), ethylacetate layer (EtOAc layer), n- Buthnol layer ( n- BuOH layer) and water layer. Each layer was concentrated under reduced pressure, And made into powder and used as a sample.

Sample Preparation Example 2

After removing the residue from the filter paper, the remaining residue was dissolved in 100% ethanol. The filtrate was blown off with a vacuum rotary evaporator (Bchi Rotavapor R-220, Swiss) and the solvent was distilled off with nitrogen gas. .

The initial yields were 1.69% by water extraction from dried beef, and the yield of organic solvent fractions was determined by fractionation of water extracts from polar to nonpolar. The yield was MC fr. 2.17%, EtOAc fr. 29.13%, n-BuOH fr. 11.02%, and water fr. 10.24%, and residue of water extract of corn was 54.92%. Therefore, the yield of the extracts from the algae extract was the highest.

Example 1: Strain, culture medium and culture conditions

The strains used in the present invention include Propionibacterium acnes KCTC3314, Streptococcus mutans KCTC3065, Streptococcus sobrinus KCTC5134, and Staphylococcus aures KCTC1621, which are causative agents of acne, in Korean collections for type cultures (http: //kctc.kribb .re.kr).

Example 2. Antibacterial activity test

The antimicrobial activity was evaluated using a paper disk diffusion method and a minimum inhibitory concentration (MIC).

 Primary antimicrobial screening;紙

The paper disc diffusion method was applied to the search for the antimicrobial activity of the fractional algae extract. Antibacterial activity After P. acnes (acne strain) was incubated for 3 days in RCA solid medium, activated acne bacteria were inoculated on RCA medium (10 mL) by one colony. Then, anaerobic jar was used for Gaspack system . After incubation for 3 days in anaerobic incubator, S. mutans and S. sobrinus were inoculated into the medium and incubated in anaerobic jar with Gaspack system (Merck. Anaerocult Gaspack system, Germany), anaerobically incubated for 1 day in 37 incubator, S. aureus was inoculated in medium and incubated in 37 incubator for 1 day. The broths were inoculated on each agar plate with a sterile cotton swab. After the medium containing the broth was fixed on the surface of the plate, the paper disc (8 mm, adventec, USA) was placed on the surface of the plate inoculated with the strain. Each sample was dissolved in DMSO and the concentration was adjusted to 50 L / disc Absorbed. The agar plate treated with each sample was incubated in an anaerobic jar using a gaspack system for 3 days, and the antimicrobial activity was measured at the size of the growth inhibition ring around the disc. As a positive control, the efficacy of triclosan, which is currently used as an antibiotic against acne bacteria, was compared in the same way.

The antimicrobial activity of P. acnes , C. mutans , S. mutans and S. sobrinus was investigated . The extracts of P. fructus were 93.3 mm in diameter, Water extract residue 163.3 mm, MC fr. 6 mm, EtOAc fr. 4 mm in order of antimicrobial activity. In the case of S. mutans , C. mutans showed an antimicrobial effect of 6.3 mm in the residue of water extract, 2.3 mm in water extract, 1 mM in MC and EtOAc fr., And 4 mm in water extract residue in S. sobrinus . Effect. S. aureus showed 8.3 and 4.3 mm antimicrobial effects in the order of water extract and water extract, respectively.

<Table 1> Size of the inhibitory effect on the growth of Gram-positive bacilli in water extracts, fractions,

Sample Strains Concentration
(g / mL ) Inhibition  zone ( mm ) One st 2 nd 3 rd Average C. sappan  L.
water ex . P. acnes
50
60 110 110 93.3 residue 160 170 160 163.3 MC fr . 3 2 3 2.6 EtOAc fr . 4 4 4 4 n- Butanol fr . ND ND ND ND water fr . ND ND ND ND Triclosan 160

* ND (not dectected): no antibacterial effects

* Triclosan: Positive control

<Table 2> Size of inhibitory growth inhibition of S. mutans in water extracts, fractions and water extract residues

Sample
Strains
Concentration
(g / mL )
Inhibition  zone ( mm )
One st
2 nd
3 rd
Average
C. sappan  L.
water ex .
S. mutans
50
2
3
2
2.3
residue
6
7
6
6.3
MC fr .
One
One
One
One
EtOAc fr .
One
One
One
One
n- Butanol fr .
ND
ND
ND
ND
water fr .
ND
ND
ND
ND
Triclosan
7

* ND (not dectected): no antibacterial effects

* Triclosan: Positive control

<Table 3> Size of inhibitory growth of S. sobrinus in water extracts, fractions,

Sample
Strains
Concentration
(g / mL )
Inhibition  zone ( mm )
One st
2 nd
3 rd
Average
C. sappan  L.
water ex .
S. sobrinus
50
ND
ND
ND
ND
residue
4
4
4
4
MC fr .
ND
ND
ND
ND
EtOAc fr .
ND
ND
ND
ND
n- Butanol fr .
ND
ND
ND
ND
water fr .
ND
ND
ND
ND
Triclosan
6

* ND (not dectected): no antibacterial effects

* Triclosan: Positive control

<Table 4> joiner water extracts and fractions, staphylococcus of water extract residue (S. aureus) Growth Inhibition ring size

Sample
Strains
Concentration
(g / mL )
Inhibition  zone ( mm )
One st
2 nd
3 rd
Average
C. sappan  L.
water ex .
S. aureus
50
4
4
5
4.3
residue
8
9
8
8.3
MC fr .
2
3
4
3
EtOAc fr .
3
3
3
3
n- Butanol fr .
ND
ND
ND
ND
water fr .
ND
ND
ND
ND
Triclosan
ND

* ND (not dectected): no antibacterial effects

* Triclosan: Positive control

 Secondary antimicrobial screening; MIC method

The minimum inhibitory concentration (MIC) of the selected substances was determined by a first round of search using the Paper disc method. The minimum inhibitory concentration (MIC) was determined by partially modifying the broth dilution method using a 96-well plate. Various strains were cultured in liquid medium to activate them and used as a bacterial solution. The culture broth was inoculated with 20 L of each well in a 96-well plate containing 180 L of culture medium. Two-fold serial dilutions were performed using a liquid medium to obtain a 96-well culture medium containing the strain and the medium at a concentration of 1: 1, and the water-extracted fraction and the water extract residue (10 mg / mL) Add 20 L to the plate and mix well. Triclosan was used as a positive control. The 96-well plate inoculated with the bacterial and sample samples was anaerobically / aerobically cultured at 37 for 3 days or 24 hours, and the absorbance was measured at 620 nm.

The minimum inhibitory concentration of various strains tested with the fraction showing efficacy in the primary antimicrobial screening and the water extract residue showed IC ₉₀ value of 0.02 g / mL in acne strain P. acnes . mutans , and S. sobrinus were 0.24 and 0.05 g / mL IC ₉₀ values, respectively. Staphylococcus aureus, S. aureus IC ₅₀ of 0.02 g / mL And the highest antimicrobial activity was obtained from the extracts of Bombyx mori.

<Table 5> Minimal Inhibitory Concentrations of Acne Bacteria ( P. acnes ) in Water Extracts, Fractions and Water Extract Residues

Sample
Strains
MIC  ( IC ₉₀  : g / mL )
C. sappan  L.
water ex.
P. acnes
0.06
residue
0.02
MC fr.
ND
EtOAc fr.
ND
Triclosan
0.03

* Triclosan: Positive control

<Table 6> Minimal Inhibitory Concentration of Salmonella ( S. mutans ) in Water Extracts, Fractions and Water Extract Residues

Sample
Strains
MIC  ( IC ₉₀  : g / mL )
C. sappan  L.
water ex.
S. mutans
0.34
residue
0.24
MC fr.
1.03
EtOAc fr.
0.94
Triclosan
0.004

* Triclosan: Positive control

<Table 7> joiner water extracts and fractions, guganggyun (S. sobrinus) The minimum inhibitory concentration of the water extract residue

Sample
Strains
MIC  ( IC ₉₀  : g / mL )
C. sappan  L.
water ex.
S. sobrinus
ND
residue
0.05
MC fr.
ND
EtOAc fr.
ND
Triclosan
ND

* Triclosan: Positive control

<Table 8> joiner water extracts and fractions, staphylococcus of water extract residue (S. aureus) The minimum inhibitory concentration

Sample
Strains
MIC  ( IC ₅₀  : g / mL )
C. sappan  L.
water ex.
S. aureus
0.03
residue
0.02
MC fr.
0.04
EtOAc fr.
0.06
Triclosan
0.004

* Triclosan: Positive control

Example  3. Carbohydrate extract Residue  Structure analysis

From the above results, the best antimicrobial activity was obtained from the residue of the water extracts of corn borer. ^{In the 1} H-NMR (FIG. 7), the hydrogen information of the benzene ring can be known at 6 to 8 ppm, and it can be seen that the hydrogen 1, 11, 2, 4 and 8 are sequentially emitted. There was no singlet. No. 6 In the hydrogen integrated value is found that the number of introduction can be combined as about 12 individuals and were superimposed the integral of seven times the hydrogen is so be introduced the number two as about 2 singlelet, ¹³ C-NMR (Fig. 8 ), The peak at 180 ppm showed C = O at the 9th peak, the 6th and 6a were connected to CO-, and the 7th CH ₂ peak overlapped with the solvent. The remaining peaks were the remaining carbon peaks of the brazilein, consistent with the data of the standard. As a result of checking the molecular weight with Maldi-tof mass (FIG. 9), the molecular weight of this compound was 284, which is shown as [M + H] 285, which is exactly the same. Based on all these results, the chemical structural formula was analyzed and found to be brazilein (Fig. 10).

Claims (6)

After extracting the marine algae, the residues remaining in the filter paper were dissolved in 100% ethanol.
A composition for preventing and treating acne characterized by having an antibacterial activity against Propionibacterium acnes . delete delete delete delete delete

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