KR20170104188A - Anti-inflammatory composition comprising solanum nigrum extract fraction and purified bee venom - Google Patents

Anti-inflammatory composition comprising solanum nigrum extract fraction and purified bee venom Download PDF

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KR20170104188A
KR20170104188A KR1020160026855A KR20160026855A KR20170104188A KR 20170104188 A KR20170104188 A KR 20170104188A KR 1020160026855 A KR1020160026855 A KR 1020160026855A KR 20160026855 A KR20160026855 A KR 20160026855A KR 20170104188 A KR20170104188 A KR 20170104188A
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fraction
bee venom
extract
inflammatory
irritation
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KR1020160026855A
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Korean (ko)
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신장철
박정근
원하영
이종수
김일광
김철구
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주식회사 청진바이오텍
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds

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  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
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  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Insects & Arthropods (AREA)
  • Animal Husbandry (AREA)
  • Alternative & Traditional Medicine (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Plant Substances (AREA)
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Abstract

The present invention relates to a composition for antiinflammation comprising fractions of an insect extract and a tablet bee venom. The fraction of the insect extract comprises the fraction containing steroid alkaloid and the fraction for inhibiting the irritation reaction according to the production method of the present invention.
The antiinflammatory composition of the present invention exhibits an effect of enhancing the anti-inflammatory effect of tablet bee venom and alleviating the inherent irritation toxicity, and thus can be usefully used for prevention and treatment of an inflammatory reaction while minimizing irritation.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an antiinflammatory composition comprising a fragrance of an extract of Angelica keiskei koidz. And a tablet bee venom.

FIELD OF THE INVENTION The present invention relates to an anti-inflammatory composition, and more particularly, to a composition for anti-inflammation comprising fractions of an extract from an extract and a tablet bee venom.

Solanum nigrum L. is widely distributed in annual plants or in the whole country, and is inhabited in mountains, on sunny meadows and on roadside. In the whole grass, steroid alkaloids, atropines, flavonoid rutin, asparagine, solangustin, sitosterin, tannin, saponin, (saponin) and organic acids. The hydrothermal extracts of the outbreaks have an inhibitory effect against Staphylococci, Pseudomonas aeruginosa, Escherichia coli and Shigella, and strong anti-inflammatory effects.

The present invention relates to a cosmetic composition for skin whitening, which is mixed with a soap composition (Korean Patent No. 10-0847478) prepared by forming a polymer with gold or silver and germanium, No. 10-0563547).

Bee Venom is a toxin secreted by the bee's physiology and is composed of complex and various peptides, proteins, enzymes, amines and amino acids. Melittin constitutes about 40 to 50% of the main component thereof, and a peptide component such as 13 to 15% of protein enzyme such as phospholipase, 10 to 21% of apamin, adolapine and MCD (mast cell degranulating) And 12 to 17% of amine, amino acid, saccharide and the like. Bee venom exhibits excellent biological effects such as anti-inflammation, antibacterial sterilization, strengthening of the immune system, and antivirals. However, side effects such as hemolysis, nervous system stimulation, skin irritation, and respiratory irritation also occur. Currently, bee venom is used for the treatment of arthritis, the treatment of skin wounds, skin whitening and wrinkle improvement, and is used for functional cosmetic ingredients, antimicrobial agents for animals, and anti-inflammatory agents.

Currently, the physiological activity of bee venom and purified bee venom on the market plays a leading role in the activity of melittin. However, histamine, phospholipase A2 and hyaluronidase are among the components of bee venom and tablets bee venom, which promote blood cell membrane destruction, blood clotting, vasodilation and hydrolysis of permeable proteins Which leads to strong allergic stimulant toxicity. Therefore, it is known that these stimulants may cause very serious safety problems for users who are hypersensitive to toxicity (Stefan Bogdanov; Bee Venom: Composition, Health, Medicine: A Review, Bee Product Science [2011]).

To use bee venom and tablet bee venom safely and efficiently, it is necessary to separate or remove the irritant components of bee venom or inhibit its activity.

Many of the conventional methods attempted to solve the above problem by the separation and removal method, but the separation process is complicated and the yield is low. Also, considering the separation cost, it is considered that this method is not economical.

Korean Patent No. 10-0847478 (published on July 15, 2008). Korean Patent No. 10-0563547 (published on November 23, 2005). Korean Patent No. 10-1061257 (published on Aug., 2010).

Bogdanov, Stefan. "Bee venom: composition, health, medicine: a review." Peptides 1.0.6 (2012)

The inventors of the present invention have found that, while researching anti-inflammatory compositions, tablets bee venom exhibits an anti-inflammatory effect while exhibiting a risk of irritant toxicity. Accordingly, it is an object of the present invention to provide an anti-inflammatory composition comprising a tablet bee venom and a natural plant extract, which is a natural plant to supplement the bee venom.

According to one aspect of the present invention, an extract or fraction obtained from a sample; And tablet bee venom.

In one embodiment, the extract may be a hot-water extract.

In one embodiment, the fraction may be obtained from a fractionated water layer or an organic solvent layer by fractionating the hot water extract of the can with an organic solvent.

In one embodiment, the fraction comprises: a) fractionating a hot-water extract of the strain with butyl alcohol; And b) eluting the butyl alcohol layer fractionated in step a) with methanol or ethanol. The step b) can be carried out with primary elution of methanol and secondary elution of ethanol.

In one embodiment, the fraction comprises: a) fractionating a hot-water extract of the strain with butyl alcohol; And b) dissolving the water layer fractionated in step a) in methanol or ethanol to obtain a lysate.

In one embodiment, the concentration range of the fraction from the water layer, the fraction from the organic solvent layer, and the tablet bee venom may be 0.5-1.0, 0.5, and 1.0 mg / mL, respectively.

According to the present invention, when the steroid alkaloid-containing fraction and the irritant-inhibiting fraction that are fractionated from the extract of Guar gum act at a certain ratio to the tablet bee-poing, the steroid alkaloid-containing fraction improves the anti-inflammatory effect of bee venom, Inhibition of hypersensitive stimulation of bee venom.

Therefore, the combination of the fractions of the extract and the tablet bee venom exhibits a superior anti-inflammatory effect than the tablet bean poison alone, and thus can be usefully used for treatment and prevention requiring an anti-inflammatory effect.

Brief Description of the Drawings Fig. 1 is a flow chart of a step of separating (A) a steroid alkaloid-containing fraction and (B) a stimulation reaction inhibiting fraction from a rice ganoderma extract.
2 is a graph showing the cytotoxicity test results of the steroid alkaloid-containing fraction prepared by the method of the present invention.
3 is a graph showing the anti-inflammatory effect of the steroid alkaloid-containing fraction prepared by the method of the present invention.
FIG. 4 is a graph showing the effect of inhibiting the irritation reaction by the fraction for inhibiting irritation produced by the method of the present invention.
FIG. 5 is a graph showing the histamine inhibitory effect of each fraction of the rice gruel extract prepared by the method of the present invention.
FIG. 6 is a graph showing the effect of inhibiting stimulation reaction inhibition fractions produced by the method of the present invention to inhibit stimulation induced by tablet bee venom.

In the present specification, the term "Purified Bee Venom (PBV)" means that the bee venom collected from a bee using a bee venom collecting device (Chungjin biotech) is subjected to a purification process such as natural drying, water filtration, sterilization, It means purified bee venom, similar to bee venom, whose physiological activity is similar to bee venom except that water-soluble pollen, dust, carbohydrates and microorganisms are removed from bee venom raw materials.

The present invention relates to an extract or fraction obtained from a strain; And tablet bee venom. The fractions may be obtained by separation or purification for the purpose of increasing the concentration of the anti-inflammatory component in the extract.

In one embodiment of the invention, the extract may be a hot-water extract.

In one embodiment of the present invention, the fraction may be fractionated with an organic solvent and fractionated from a separated water layer or organic solvent layer.

In one embodiment of the invention, the fraction comprises a) fractionally extracting the hot-water extract of the flask with butyl alcohol; And b) eluting the butyl alcohol layer fractionated in step a) with methanol or ethanol and containing steroidal alkaloids. The step b) can be carried out with primary elution of methanol and secondary elution of ethanol.

In one embodiment of the invention, the fraction comprises a) fractionally extracting the hot-water extract of the flask with butyl alcohol; And b) dissolving the water layer fractionated in step a) in methanol or ethanol to obtain a lysate, which may be an effect of inhibiting the irritation reaction.

In one embodiment of the present invention, the concentration ranges of the fraction from the water layer, the fraction from the organic solvent layer, and the tablet bee venom may be 0.5-1.0, 0.5, and 1.0 mg / mL, respectively.

As the concentration of the steroidal alkaloid-containing fraction increases, the anti-inflammatory effect may increase, but when the toxicity to the cells (confirmed by MTT test) is confirmed, the concentration of the fraction is 1.0 mg / mL , The survival rate of the cells may be rapidly reduced, and the steroidal alkaloid-containing fraction is not well dissolved in distilled water (≤ 0.5 mg / mL).

The fraction for inhibiting the irritation reaction is preferably used at a concentration of 0.5 to 1.0 mg / mL. This is because it exhibits the highest effect in inhibiting histamine and related stimuli induced by tablets bee venom 1.0 mg, and the effect is not improved even if the concentration is increased higher.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

<Examples>

1. Preparation of extract

4.8 kg of the dried outpox (Korean, Chungcheongbuk-do) was washed with cold water and subjected to hot-water extraction at 120 ° C for 3 hours using an ultra-high vacuum low-temperature concentrator (COSMOS 660, Kyonsu Machinery Industry). After the extract and the sample were separated, the extraction was carried out twice more by the same method. After mixing the extracted extracts three times, 2.98 kg of the extract powder was obtained through low-temperature concentration and freeze-drying.

2. Preparation of steroidal alkaloid-containing fractions from citron extract

 2.98 kg of the extract powder obtained in Example 1 was dissolved in 6 L of distilled water and fractionated with butyl alcohol using the partitioning property of butyl alcohol and water. The obtained butyl alcohol layer and distilled water layer were subjected to vacuum rotary concentration and freeze drying to obtain 1.55 kg and 1.42 kg of powder, respectively. The extraction process is as shown in Fig.

Chromatography was used here to prepare steroidal alkaloid containing fractions. 5 g of the butyl alcohol fraction sample was adsorbed on an SPE column packed with a C18 resin, the polar component was eluted with 10% methanol, and the eluent was discarded. Again, the steroidal alkaloid component adsorbed to the column with 65% methanol was first eluted. The first eluant was dried and adsorbed onto an SPE column packed with a silica gel resin, followed by secondary elution with 80% ethanol to obtain about 3.3 g of a steroid alkaloid-purified fraction (FIG. 1A).

The steroidal alkaloid purified fraction obtained by the above method was measured using a colorimetric method and found to contain a total saponin content of 48.3% (compared with dioscin) and a total alkaloid content of 8.6% (as compared with solanidine) It can be judged that it can exhibit the inflammation effect.

3. Preparation of fractions for inhibiting irritation reaction from extracts

1.42 kg of brown water-soluble powder was obtained by freeze-drying the distilled water layer fraction of the organic solvent fraction extract. The dried powder was put into methanol or ethanol and dissolved by ultrasonic wave at room temperature for 30 minutes. After 30 minutes, the precipitate and the supernatant were separated through filtration. Then, the same solvent was added to the precipitate again, and the solution was dissolved by the above-mentioned method and then filtered. The filtrate through two filtrations was mixed and methanol or ethanol was removed using a vacuum rotary condenser to obtain 370 g of pale yellow powder (FIG. 1B).

<Test Example>

1. Assessment of cytotoxicity of steroidal alkaloid-containing fractions

(Dulbecco's modified Eagle's medium, Gibco, Co.) containing 10% fetal bovine serum (FBS), penicillin 100 μg / ml and streptomycin 100 U / USA) were transplanted into a 96-well plate at a density of 2.0 × 10 4 cells per well and cultured for 24 hours. Steroidal alkaloid containing fractions were treated by concentration according to Example 2 (final measured concentrations are 0, 0.125, 0.25, 0.5, 1.0, 2.0, and 2.5 mg / mL). After incubation for 18-24 hours, MTT solution was added to induce the reduction reaction for 4 hours. After the reaction, the sample and the medium were removed by suction, and cells stained with DMSO (dimethyl sulfoxide) were selected and lysed to measure the absorbance at 540 nm and the cell survival rate was calculated.

As shown in FIG. 2, when treated with a low concentration steroid alkaloid-containing fraction of 0 to 1.0 mg / mL, cytotoxicity was not observed but rather the effect of promoting cell growth was realized. As the fraction concentration increased (≥1.0 mg / mL), the survival rate of the cells showed a drastic decrease. To determine the anti-inflammatory effects of steroidal alkaloid-containing fractions in surviving cells, the concentration range of the anti-inflammatory assay was set at 0-1.0 mg / mL.

2. Evaluation of anti-inflammatory effects of steroidal alkaloid-containing fractions

RAW 264.7 cells cultured in a medium (Dulbecco's modified Eagle's medium) containing 10% FBS, penicillin 100 μg / ml and streptomycin 100 U / ml were maintained in a 96-well plate at 37 ° C. under a 5% 2.0 × 10 4 cells, and cultured for 24 hours. Cells treated with 1.0 μg / ml of LPS 2 hours beforehand were treated with steroid alkaloid-containing fractions (final concentrations: 0, 0.125, 0.25, 0.5 and 1.0 mg / ml) for 24 hours. The amount of NO produced from RAW 264.7 cells was measured in the form of NO 2 - present in the cell culture medium using a Griess reagent kit. The mixture was incubated in a 96-well plate for 10 minutes and absorbance was measured at 540 nm.

As shown in FIG. 3, the cells treated with steroidal alkaloid-containing fractions showed high NO emission due to the induction reaction by LPS. On the other hand, the release of NO from cells treated with steroidal alkaloid-containing fractions (treated with the same LPS) showed a tendency to decrease with concentration of fractions. Especially at 0.5 mg / mL and 1.0 mg / mL concentrations.

3. Evaluation of efficacy of fractions for inhibiting irritation

A histamine enzyme assay (Histamine Enzymatic Assay) was performed to evaluate the effect of the fractions for inhibiting the stimulation reaction. RBL-2H3 cells cultured in a medium (Dulbecco's modified Eagle's medium) containing 10% FBS, 100 μg / ml of penicillin and 100 U / ml of streptomycin were maintained in a 96-well plate at 37 ° C. under 5% 2.0 x 10 &lt; 5 &gt; cells per well, and cultured for 24 hours. After treatment with the histamine-inducing substance C48 / 80 (50 μg / ml), the cells were immediately treated with 10 μl of the 20 mg / ml stimulation-inhibiting fraction. And the amount of histamine released from RBL-2H3 cells at 450 nm was measured by ELISA using a histamine assay kit (MaxSignal ® Histamine Enzymatic Assay Kit, BIOO SCIENTIFIC, USA) for about 10 to 15 minutes.

As shown in FIG. 4, when RBL-2H3 cells were treated with tablet bee venom, histamine was released in a larger amount than in C48 / 80 treatment. However, when treated with the mixed composition of fractions for inhibiting irritation, it was confirmed that histamine release amount was remarkably reduced and the irritation reaction inhibitory effect was exhibited.

5, the steroid alkaloid-containing fraction or the fractions for inhibiting the irritation reaction were added to the histamine solution (2.8 μg / mL) contained in the histamine assay kit so as to have a total concentration of 2.0 mg / mL, The histamine inhibitory effect was confirmed by measuring the absorbance according to the reaction time. The fractions for the inhibition of irritation did not only inhibit the stimulation reaction induced by tablet bee venom but also inhibited by direct reaction with histamine. Therefore, it is possible to mitigate the stimulation reaction when it occurs.

It is most preferable that the fractions for inhibiting the irritation reaction are composed at a ratio of 0.5 to 1 mg / mL to the tablet bee venom. The inhibitory effect is most effective when 0.5 to 1.0 mg / mL of the mixed composition of histamine contained in the tablet bee venom 1.0 mg and the fractions for inhibiting irritation in the irritation reaction induced therefrom is added. It was confirmed that the effect was no longer remarkably improved even when it was added in excess (FIG. 6).

Claims (7)

Extracts or fractions obtained from yeast; And tablet bee venom.
The anti-inflammatory composition according to claim 1, wherein the extract is a hot-water extract.
The anti-inflammatory composition according to claim 1, wherein the fraction is obtained from a fractionated water layer or an organic solvent layer by fractionating the hot-water extract of the extract by an organic solvent.
4. The process according to claim 3, wherein said fraction
a) fractionating the hot water extract of Yonggi with butyl alcohol; And
b) eluting the butyl alcohol layer fractionated in step a) with methanol or ethanol
Lt; RTI ID = 0.0 &gt; anti-inflammatory &lt; / RTI &gt;
5. The composition according to claim 4, wherein step b) is carried out with a first elution of methanol and a second elution of ethanol.
4. The process according to claim 3, wherein said fraction
a) fractionating the hot water extract of Yonggi with butyl alcohol; And
b) dissolving the water layer fractionated in step a) in methanol or ethanol to obtain a lysate
&Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof.
The anti-inflammatory composition according to claim 3, wherein the concentration ranges of the fraction from the water layer, the fraction from the organic solvent layer, and the tablet bee venom are 0.5 to 1.0, 0.5 and 1.0 mg / mL, respectively.
KR1020160026855A 2016-03-07 2016-03-07 Anti-inflammatory composition comprising solanum nigrum extract fraction and purified bee venom KR20170104188A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101994256B1 (en) 2019-03-05 2019-06-28 주식회사해림 Seaweed fastening device for sea forest composition excluding use of air tacker

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101994256B1 (en) 2019-03-05 2019-06-28 주식회사해림 Seaweed fastening device for sea forest composition excluding use of air tacker

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