KR20200092789A - Antiinflammatory and antibacterial composition comprising manjakani extract - Google Patents
Antiinflammatory and antibacterial composition comprising manjakani extract Download PDFInfo
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- KR20200092789A KR20200092789A KR1020190010150A KR20190010150A KR20200092789A KR 20200092789 A KR20200092789 A KR 20200092789A KR 1020190010150 A KR1020190010150 A KR 1020190010150A KR 20190010150 A KR20190010150 A KR 20190010150A KR 20200092789 A KR20200092789 A KR 20200092789A
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Abstract
본 발명은 만자카니 추출물을 유효성분으로 포함하는 항염증 및 항균용 조성물에 관한 것으로, 본 발명의 일 구체예에 따른 만자카니 추출물은 염증관련 인자의 발현을 억제하며 다양한 세균 및 진균에 대해 항균 활성을 갖고, 히스타민, 아시텔콜린, 물질 P의 생성량을 유의미하게 감소시키며 라디칼 소거능을 갖는 바, 항염증, 항산화, 항소양, 및 항균 효과가 우수하므로 항염증 및 항균용 조성물로서 유용하게 이용될 수 있다.The present invention relates to an anti-inflammatory and antimicrobial composition comprising the Manzacani extract as an active ingredient, the Manzacani extract according to an embodiment of the present invention inhibits the expression of inflammation-related factors and has antibacterial activity against various bacteria and fungi It has a histamine, acitelcholine, and significantly reduces the production amount of the substance P and has a radical scavenging activity, and thus has excellent anti-inflammatory, antioxidant, anti-ulcer, and anti-bacterial effects, and thus can be usefully used as a composition for anti-inflammatory and anti-bacterial properties. have.
Description
본 발명은 만자카니(manjakani) 추출물을 유효성분으로 포함하는 항염증 및 항균용 조성물에 관한 것이다.The present invention relates to an anti-inflammatory and antimicrobial composition comprising a manjakani extract as an active ingredient.
염증반응은 생체조직이 손상을 입었을 때 일어나는 방어적 반응으로서 활성화된 면역세포에 의해 필연적으로 일어나는 일련의 면역반응이다. 염증반응에는 산화질소(nitric oxide, NO) 및 면역세포를 활성화시켜 생산된 전-염증성 사이토카인(pro-inflammatory cytokine)들이 관여하는 것으로 알려져 있다. 또한 COX-2(cyclooxygenase-2)는 아라키돈산(arachidonic acid)으로부터 염증매개물질인 PGE2(prostaglandin E2) 생성을 촉매하는 효소로 염증반응에 중요한 역할을 한다. 따라서 NO의 생성이나 iNOS, COX-2, IL-1β 및 TNF-α의 발현 억제와 염증에 의한 활성산소종의 억제는 염증질환을 치료하는데 있어서 중요한 목표가 된다. An inflammatory reaction is a protective reaction that occurs when a living tissue is damaged, and is a series of immune reactions inevitably caused by activated immune cells. It is known that pro-inflammatory cytokines produced by activating nitric oxide (NO) and immune cells are involved in the inflammatory reaction. In addition, COX-2 (cyclooxygenase-2) is an enzyme that catalyzes the production of inflammatory mediator PGE 2 (prostaglandin E 2 ) from arachidonic acid and plays an important role in the inflammatory reaction. Therefore, the production of NO or suppression of expression of iNOS, COX-2, IL-1β and TNF-α and inhibition of reactive oxygen species by inflammation are important targets in treating inflammatory diseases.
염증 질환의 치료에 사용되는 스테로이드성 소염 약물들은 녹내장, 백내장, 고혈압, 조울증, 체중증가, 당뇨병 및 골다공증과 같은 부작용을 유발한다. 최근에는 이러한 부작용이나 세포독성에 대한 위험이 없거나 적은 천연물질 유래 성분으로, 효과적으로 염증을 억제할 수 있는 물질의 개발이 요구되고 있다.Steroidal anti-inflammatory drugs used in the treatment of inflammatory diseases cause side effects such as glaucoma, cataracts, hypertension, bipolar disorder, weight gain, diabetes and osteoporosis. Recently, there is a need to develop a substance capable of effectively suppressing inflammation as a component derived from a natural substance having little or no risk for side effects or cytotoxicity.
한편, 병원성 미생물에 의해서 식품 산업에서의 식품 유통 과정 중의 부패로 인한 손실, 농산업에서의 농작물에 대한 과량의 화학 살충제의 사용으로 인한 유해성 및 환경오염, 항생제의 오남용으로 인한 항생제 내성 균주의 출현 등과 같이 사회 전반에서 피해가 발생하고 있다. 미생물에 대한 항균작용을 하는 물질인 항균제는 세균 감염에 의한 질병 등을 치료하기 위하여 많이 사용되고 있는데 주로 곰팡이를 비롯한 천연물로부터 분리되거나 화학적으로 합성하는 방법으로 제조되어 왔다. 현재 사용되고 있는 대부분의 항생제는 화학적인 합성을 통해 제조된 것으로서 고비용이 소요되며 부작용을 유발하는 등의 많은 한계를 가지고 있다. On the other hand, due to pathogenic microorganisms, loss due to decay during food distribution in the food industry, harmful and environmental pollution due to the use of excess chemical pesticides for agricultural products in the agricultural industry, the emergence of antibiotic-resistant strains due to misuse of antibiotics, etc. Damage is happening across society. Antimicrobial agents, which are antimicrobial agents for microorganisms, are widely used to treat diseases caused by bacterial infections, and have been mainly prepared by chemically synthesizing or separating from natural products such as fungi. Most of the antibiotics currently being used are manufactured through chemical synthesis, and have a high cost and have many limitations such as causing side effects.
최근에는 화학 항균제의 사용으로 인한 각종 문제점을 해결하기 위해, 천연물 추출물을 이용하여 항균용 조성물을 개발하는 것이 주요한 과제로서 이에 대한 연구가 이루어지고 있으나, 대부분의 천연물 추출물의 경우 항균 효과를 갖는 미생물이 한정적이고, 항균 효과가 있을지라도 매우 높은 농도에서 그 기능이 나타나므로, 추가적인 항균제와 복합적으로 사용되어야 하는 경우가 많다. Recently, in order to solve various problems caused by the use of chemical antibacterial agents, the main task is to develop an antimicrobial composition using natural extracts, but most natural extracts have microorganisms that have antibacterial effects. It is limited, and even if it has an antibacterial effect, its function is exhibited at a very high concentration, and thus it has to be used in combination with an additional antibacterial agent.
따라서 본 출원의 발명자들은 부작용이 없으면서 동시에 우수한 항염 활성 및 다양한 세균 및 진균에 대해 항균 활성을 갖는 만자카니 추출물을 포함하는 조성물에 대한 본 발명을 완성하였다.Therefore, the inventors of the present application have completed the present invention for a composition comprising a manzacani extract having excellent anti-inflammatory activity and antibacterial activity against various bacteria and fungi while having no side effects.
본 발명의 목적은 만자카니 추출물을 유효성분으로 포함하는, 항염증 및 항균용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for anti-inflammatory and antibacterial, comprising the manzacani extract as an active ingredient.
본 발명의 다른 목적은 상기 항염증 및 항균용 조성물을 포함하는, 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a cosmetic composition comprising the anti-inflammatory and antibacterial composition.
본 발명의 또 다른 목적은 상기 항염증 및 항균용 조성물을 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하기 위한 것이다. Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including the anti-inflammatory and antimicrobial composition.
본 발명의 일 양상은 만자카니 추출물을 유효성분으로 포함하는, 항염증 및 항균용 조성물을 제공한다.One aspect of the present invention provides an anti-inflammatory and antimicrobial composition comprising manzacani extract as an active ingredient.
만자카니(manjakani)는 아시아 일부, 그리스, 이란을 원산지로 하는 퀘르쿠스 인팩토리아 올리비에(Querqus infectoria Olivier)라는 작은 나무에 열리는 혹(gall)을 의미한다. 만자카니는 지혈 및 해독의 효력이 있고, 산후관리에 유용하게 사용된다고 알려져 있으며, 탄닌 성분이 풍부한 것으로 알려져 있다. Manjakani means a gall on a small tree called Querqus infectoria Olivier, native to parts of Asia, Greece and Iran. Manzacani is known to have hemostatic and detoxifying effects, useful for postpartum care, and is known to be rich in tannins.
본 발명에서 사용되는 만자카니는 자연에서 채취하거나, 상업적으로 판매되는 것을 구입할 수 있으나, 이에 제한되지 않는다. The manzacan used in the present invention can be purchased from nature or commercially available, but is not limited thereto.
본 발명의 일 구체예에 따르면, 만자카니 추출물을 포함하는 조성물이 (1) NF-κB의 전사활성, (2) 일산화질소(NO)의 생성, (3) 유도형 일산화질소 합성효소(iNOS), 사이클로옥시게나제-2(COX-2), 인터루킨-2(IL-2), 인터루킨-6(IL-6), 인터루킨-1β(IL-1β) 및 종양 괴사 인자-α(TNF-α)로 이루어진 군에서 선택되는 염증관련 인자의 발현을 억제하며, (4) 세균 및 진균에 항균 활성을 갖고 (5) 히스타민, 아세틸콜린(acethylcholine), 물질 P(substance P)의 생성량을 유의미하게 감소시키며 (6) DPPH 및 ABTS 라디칼 소거능을 나타내므로, 본 발명의 일 구체예에 따른 만자카니 추출물을 포함하는 조성물은 항산화, 항소양, 항염증, 및 항균용 조성물로서 유용하게 사용될 수 있다. According to one embodiment of the invention, the composition comprising the manzacani extract (1) NF-κB transcriptional activity, (2) production of nitrogen monoxide (NO), (3) inducible nitrogen monoxide synthetase (iNOS) , Cyclooxygenase-2 (COX-2), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) Inhibits the expression of inflammation-related factors selected from the group consisting of (4) has antibacterial activity against bacteria and fungi (5) significantly reduces the production of histamine, acetylcholine, and substance P (substance P), (6) Since it exhibits DPPH and ABTS radical scavenging activity, the composition comprising the manzacani extract according to one embodiment of the present invention can be usefully used as a composition for antioxidant, anti-pruritic, anti-inflammatory, and antibacterial.
본 발명의 일 구체예에 따르면, 만자카니 추출물을 유효성분으로 포함하는 항소양, 항염증 및 항균용 조성물을 제공할 수 있다.According to one embodiment of the present invention, it is possible to provide an anti-pruritic, anti-inflammatory and antimicrobial composition comprising the manzacani extract as an active ingredient.
본 발명의 일 구체예에 따르면, 만자카니 추출물을 유효성분으로 포함하는 항산화, 항소양, 항염증, 및 항균용 조성물을 제공할 수 있다.According to an embodiment of the present invention, it is possible to provide an antioxidant, anti-pruritic, anti-inflammatory, and antimicrobial composition comprising the manzacani extract as an active ingredient.
본 발명에서, "항균"은 미생물에 저항하는 능력 및 미생물의 생장 또는 증식을 억제하는 능력을 의미하며, 세균이나 곰팡이, 효모 등과 같은 미생물의 작용으로부터 방어하기 위해 이루어지는 모든 기작을 의미한다. In the present invention, "antibacterial" means the ability to resist microorganisms and the ability to inhibit the growth or proliferation of microorganisms, and refers to all mechanisms that are made to defend against the action of microorganisms such as bacteria, fungi, yeast, and the like.
본 발명에서, "항산화"는 산화를 억제하는 작용을 의미하는 것으로, 산화적 스트레스(oxidative stress)가 유발되면 잠재적인 세포손상 및 병리적 질환을 일으키게 된다. 이러한 산화적 스트레스의 직접적 원인이 되는 활성 산소종(reactive oxygen species, ROS)은 불안정하고 반응성이 높아 여러 생체물질과 쉽게 반응하고, 체내 고분자들을 공격하여 세포와 조직에 비가역적인 손상을 일으키거나 돌연변이, 세포독성 및 발암 등을 초래하게 된다. 본 발명의 일 구체예에 따른 만자카니 추출물은 항산화 효과를 달성함으로써, 건강증진에 기여할 수 있다.In the present invention, "antioxidation" refers to the action of inhibiting oxidation, and when oxidative stress is induced, it causes potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of these oxidative stresses, are unstable and highly reactive, so they can easily react with various biological substances, attack the polymers in the body, cause irreversible damage to cells and tissues, or mutate, It causes cytotoxicity and carcinogenesis. Manzacani extract according to an embodiment of the present invention may contribute to health promotion by achieving an antioxidant effect.
본 발명의 일 구체예에 따르면, 만자카니 추출물은 당업계에 공지된 통상의 방법에 따라 제조될 수 있다. 예를 들어, 건조된 만자카니를 분쇄한 후 추출에 통상적으로 사용되는 용매를 각각 첨가하고, 적절한 온도와 압력으로 추출하여 만자카니 추출물을 제조할 수 있다.According to one embodiment of the invention, the manzacani extract can be prepared according to conventional methods known in the art. For example, after pulverizing the dried manzakani, a solvent commonly used for extraction may be added, and the manzakani extract may be prepared by extracting at an appropriate temperature and pressure.
본 발명의 일 구체예에 따르면, 만자카니 추출물은 물, 탄소수 1 내지 4개의 알코올, 프로필렌글리콜, 부틸렌글리콜, 글리세린, 아세톤, 에틸 아세테이트, 부틸 아세테이트, 클로로포름, 디에틸에테르, 디클로로메탄, 헥산, 및 이들의 혼합물로 구성된 군으로부터 선택되는 용매로 추출된 것일 수 있다.According to one embodiment of the present invention, the manzacani extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, And it may be extracted with a solvent selected from the group consisting of mixtures thereof.
본 발명에서, "추출물"은 만자카니의 추출 처리에 의하여 얻어지는 추출액, 추출액의 희석액이나 농축액, 추출액을 건조하여 얻어지는 건조물, 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. In the present invention, the "extract" is an extract obtained by the extraction treatment of manzacani, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a crude product or a purified product of the extract, or a mixture thereof, such as the extract itself and the extract Includes extracts of all formulations formable using.
본 발명의 일 구체예에 따르면, 만자카니 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.According to one embodiment of the present invention, the manzacani extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.
본 발명의 일 구체예에 따르면, 만자카니 추출물은 상술한 추출방법에 의한 추출물뿐만 아니라, 통상적인 정제 과정을 거친 추출물도 포함한다. 예를 들어, 일정한 분자량 컷-오프(cut-off) 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시한 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 추출물에 포함될 수 있다. According to one embodiment of the present invention, the manzacani extract includes not only the extract by the above-described extraction method, but also an extract that has been subjected to a conventional purification process. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the extract of the present invention.
본 발명의 일 구체예에 따르면, 상기 조성물은 이스케리키아 속(Escherichia sp.), 슈도모나스 속(Pseudomonas sp.), 스타필로코쿠스 속(Staphylococcus sp.), 및 칸디다 속(Candida sp.)으로 이루어진 군에서 선택되는 하나 이상의 미생물의 생장을 억제하는 것일 수 있다.According to one embodiment of the present invention, the composition is Escherichia sp., Pseudomonas sp., Staphylococcus sp., and Candida sp. It may be to suppress the growth of one or more microorganisms selected from the group consisting.
본 발명의 일 구체예에 따르면, 상기 미생물은 대장균(Escherichia coli), 스타필로코쿠스 아우레우스(Staphylococcus aureus), 슈도모나스 에루지노사(Pseudomonas aeruginosa), 및 칸디다 알비칸스(Candida albicans)로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.According to one embodiment of the invention, the microorganism is Escherichia coli , Staphylococcus aureus , Staphylococcus aureus , Pseudomonas aeruginosa , and Candida albicans It may be one or more selected from.
본 발명에서 "이스케리키아 속(Escherichia sp.) 균주"란, 그람 음성 균주로서, 장내세균의 일종으로 식중독 등의 원인 균주이다. 본 발명의 목적상 이스케리키아 속(Escherichia sp.) 균주에는 본 발명에서 제공하는 항균용 조성물의 표적으로 사용될 수 있는 미생물은 제한없이 포함된다. 본 발명에서 이스케리키아 속 균주는 예를 들면 대장균일 수 있으나, 이에 제한되지 않는다. 대장균은 장내 정상세균층을 구성하고 대개는 비병원성이나 담도염, 방광염, 신우염, 요로감염증의 원인균이 될 수 있다. In the present invention, " Escherichia sp. ( Escherichia sp.) strain" is a Gram-negative strain, which is a type of intestinal bacteria and is a causative strain such as food poisoning. For the purposes of the present invention, Escherichia sp. strains include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. In the present invention, the strain of Escherichia genus may be, for example, E. coli, but is not limited thereto. Escherichia coli constitutes a normal bacterial layer in the intestine and can be a causative agent of nonpathogenic or biliary tract infection, cystitis, pyelonephritis, and urinary tract infection.
본 발명의 일 구체예에 따른 조성물을 이용하면 이스케리키아 속 균주의 증식 또는 생장을 억제시킬 수 있으므로 에스케리아 속 균주로 인한 상기 질환을 예방 또는 개선할 수 있다.By using the composition according to one embodiment of the present invention, it is possible to inhibit the growth or growth of the strain of the genus Escherichia, so it is possible to prevent or improve the disease caused by the strain of the genus Escherichia.
본 발명에서 "슈도모나스 속(Pseudomonas sp.) 균주"란, 그람 음성 균주로서, 병원균이나 부패균이 되는 것도 있으나, 아미노산 발효에 의한 아미노산의 제조에 사용되는 것도 있다. 본 발명의 목적상 슈도모나스 속 균주에는 본 발명에서 제공하는 항균용 조성물의 표적으로 사용될 수 있는 미생물은 제한없이 포함된다. 본 발명의 슈도모나스 속 균주는 예를 들면 슈도모나스 에루지노사일 수 있으나 이에 제한되지 않는다. 슈도모나스 에루지노사는 그람음성 호기성 간균으로서 인간에 대한 병원성은 약하지만 널리 자연 환경에 분포 하고 있기 때문에 혼합감염, 2차감염을 일으켜 질병 상태를 악화시킨다. 특히 수술, 화상 등 생체 저항력이 저하되어 있는 경우 감염으로 인해 패혈증을 일으키고, 소화기 점막을 침범해서 설사를 일으키기도 한다. In the present invention, " Pseudomonas sp. ( Pseudomonas sp.) strain" is a gram-negative strain, which may be a pathogen or a rot, but is also used in the production of amino acids by amino acid fermentation. For the purposes of the present invention, the strain of Pseudomonas genus includes, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strain of the genus Pseudomonas of the present invention may be, for example, Pseudomonas aeruginosa, but is not limited thereto. Pseudomonas aeruginosa is a Gram-negative aerobic bacillus, which has weak pathogenicity to humans, but is widely distributed in the natural environment, causing mixed infections and secondary infections to exacerbate the disease state. In particular, when bio-resistance, such as surgery and burns, is lowered, infection causes sepsis and diarrhea by invading the digestive mucosa.
본 발명의 일 구체예에 따른 조성물을 이용하면 슈도모나스 속 균주의 증식 또는 생장을 억제시킬 수 있으므로 슈도모나스 속 균주로 인한 상기 질환을 예방 또는 개선할 수 있다.By using the composition according to an embodiment of the present invention, it is possible to inhibit the growth or growth of the strain of the genus Pseudomonas, thereby preventing or improving the disease caused by the strain of the genus Pseudomonas.
본 발명에서 "스타필로코쿠스 속(Staphylococcus sp.) 균주"란, 미크로콕쿠스과(Micrococcaceae)에 속하며 사람이나 동물의 장관에, 그 외에 피부나 점막에도 존재한다. 사람의 병에서 분리되는 균종에는 S. aureus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. hominis 또는 S. warneri 등이 있다. 본 발명의 목적상 스타필로코쿠스 속 균주에는 본 발명에서 제공하는 항균성 조성물의 표적으로 사용될 수 있는 미생물은 제한없이 포함된다. 본 발명의 스타필로코쿠스 속 균주는 예를 들면 스타필로코쿠스 아우레우스, 스타필로코쿠스 헤모리티쿠스, 스타필로코쿠스 와르너리, 스타필로코쿠스 사프로파이티커스, 스타필로코쿠스 코흐니, 스타필로코쿠스 호미니스, 스타필로코쿠스 자일로서스 또는 스타필로코쿠스 시뮬란스일 수 있으나 이에 제한되지 않는다. 스타필로코쿠스 아우레우스는 그람 양성의 통성혐기성 세균으로서, 건강한 사람이나 가축의 피부와 비강 표면에 일반적으로 존재하고, 내열성인 외독소를 생산하여 식중독을 일으키며, 피부의 화농, 중이염, 방광염 등 화농성 질환을 일으키는 원인균으로 알려져 있다.In the present invention, " Staphylococcus sp. ( Staphylococcus sp.) strain" belongs to the family Micrococcaceae (Micrococcaceae) and is present in the intestinal tract of humans and animals, as well as in the skin and mucous membranes. S. aureus , S. epidermidis , S. saprophyticus , S. haemolyticus , S. hominis, or S. warneri are isolated from human diseases. For the purposes of the present invention, strains of the genus Staphylococcus include, without limitation, microorganisms that can be used as targets of the antimicrobial composition provided by the present invention. The strains of the genus Staphylococcus of the present invention include, for example, Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus warnery, Staphylococcus saprophyticus, Staphylococcus Couscous, staphylococcus hominis, staphylococcus xylos or staphylococcus simulations, but is not limited thereto. Staphylococcus aureus is a gram-positive, anaerobic bacterium, which is present on the skin and nasal surfaces of healthy people or livestock, and produces food poisoning by producing heat-resistant exotoxins, purulent skin, otitis media, cystitis, etc. It is known to cause disease.
본 발명의 일 구체예에 따른 조성물을 이용하면 스타필로코쿠스 속 균주의 증식 또는 생장을 억제시킬 수 있으므로 스타필로코쿠스 속 균주로 인한 상기 질환을 예방 또는 개선할 수 있다.By using the composition according to an embodiment of the present invention, it is possible to inhibit the growth or growth of the strain of the genus Staphylococcus, thereby preventing or improving the disease caused by the strain of the genus Staphylococcus.
본 발명에서 “칸디다 속(Candida sp.) 균주”란 불완전균아문 진균류의 한 속으로 단세포의 구형 또는 난원형이며, 인체나 동물의 입안, 피부 등에 존재하여 정상상태에서는 인체에 무해하나 항생물질을 장기 사용하거나 인체가 면역에 대한 저항력이 약해졌을 때에 체내에서 이상번식을 하여 칸디다증을 일으킨다. 감염빈도가 높은 부위는 입안과 음부(陰部) 등의 점막으로 점막이 짓무르는 가려움이나 통증이 일어난다. In the present invention, " Candida sp. ( Candida sp.) strain" is a genus of incomplete bacteriophage fungi, and is a single cell spherical or oval, and is present in the mouth, skin, etc. of the human body or animal, and is harmless to the human body under normal conditions, but has a long life of antibiotics. When used or when the body's resistance to immunity is weakened, it multiplies abnormally in the body and causes candidiasis. In areas of high infection, the mucous membranes of the mouth and genitals, etc. cause itching or pain that the mucous membranes rub.
본 발명의 목적상 칸디다 속 균주에는 본 발명에서 제공하는 항균성 화합물의 표적으로 사용될 수 있는 미생물은 제한없이 포함된다. 본 발명의 칸디다 속 균주는 예를 들면 칸디다 알비칸스 또는 칸디다 트로피칼리스일 수 있으나 이에 제한되지 않는다. 칸디다 알비칸스는 그람 양성 진균으로 일종의 피부 곰팡이 균으로서 건강한 사람의 구강, 질, 장 및 소화관에도 존재하고 있으나 면역력이 감소하였을 때 증식이 증가하여 수포성 염증을 발생시키며, 소화관 및 구강에서 염증을 일으키기도 하지만 대표적으로는 여성의 음부에 염증을 일으켜서 칸디다성 질염을 발생시킨다. 약 75%의 여성이 한 번 이상의 질과 외음부에 칸디다성 질염을 겪는 것으로 보고된 바 있고, 여성 인구 중 45%가 1년에 2회 이상 재발을 겪는 흔한 질환이다. 칸디다 트로피칼리스는 림프종, 백혈병 및 당뇨병이 있는 사람들에게서 패혈증 및 전파성 칸디다증의 주요 원인균이고, 보통 위장관과 피부 표면에서 발견된다. For the purposes of the present invention, Candida genus strains include, without limitation, microorganisms that can be used as targets of the antimicrobial compounds provided by the present invention. The Candida genus strain of the present invention may be, for example, Candida albicans or Candida tropicalis, but is not limited thereto. Candida albicans is a gram-positive fungus, a type of skin fungus that is present in the oral, vaginal, intestinal, and digestive tracts of healthy people, but when immunity decreases, proliferation increases, causing blistering inflammation, and inflammation in the digestive tract and oral cavity However, representatively, it causes inflammation in the female genitals, resulting in candidal vaginitis. It has been reported that about 75% of women suffer from candidal vaginitis more than once in the vagina and vulva, and 45% of the female population is a common disease that occurs more than once a year. Candida tropicalis is the leading causative agent of sepsis and spread candidiasis in people with lymphoma, leukemia and diabetes, and is usually found on the gastrointestinal tract and skin surface.
본 발명의 일 구체예에 따른 조성물을 이용하면 칸디다 속 균주의 증식 또는 생장을 억제시킬 수 있으므로 칸디다 속 균주로 인한 상기 질환을 예방 또는 개선할 수 있다.By using the composition according to an embodiment of the present invention can suppress the growth or growth of the Candida genus strain, it is possible to prevent or improve the disease caused by the Candida genus strain.
본 발명의 일 구체예에 따른 조성물은 세균 및 진균에 대해 광범위하게 항균 활성을 갖고, 천연물질로부터 추출된 유효성분을 포함하여 화학 합성제품에 비해 독성이나 피부 알레르기 유발, 피부자극, 환경 호르몬으로서의 가능성, 내성균 유발 등과 같은 부작용이 적다.The composition according to one embodiment of the present invention has a wide range of antimicrobial activity against bacteria and fungi, and it is possible to cause toxicity or skin allergies, skin irritation, and environmental hormones compared to chemical synthetic products, including active ingredients extracted from natural substances , Less side effects such as inducing resistant bacteria.
본 발명의 일 구체예에 따르면, 상기 조성물은 피부용, 두피용, 구강용, 안구용 또는 질(膣)용 조성물일 수 있다. 본 발명의 일 구체예에 따르면, 상기 조성물은 샴푸, 바디클렌져, 비누, 폼클렌져, 스킨, 로션, 크림, 연고, 스프레이, 치약, 물티슈, 동물용 물티슈, 주방 세척제, 욕실 세척제, 애완동물용 세정제, 섬유용 항균제, 렌즈 세정제, 여성용 청결제 등으로 사용될 수 있다.According to one embodiment of the present invention, the composition may be a composition for skin, scalp, oral, ocular or vaginal. According to one embodiment of the present invention, the composition is shampoo, body cleanser, soap, foam cleanser, skin, lotion, cream, ointment, spray, toothpaste, wipes, animal wipes, kitchen cleaner, bathroom cleaner, pet cleaner , It can be used as an antibacterial agent for textiles, a lens cleaner, and a female cleaner.
본 발명의 일 구체예에 따르면, 항염증 및 항균용 조성물은 보존제, 방부제, 세정제, 또는 여성용 청결제로 이용되는 것일 수 있다.According to one embodiment of the present invention, the anti-inflammatory and antibacterial composition may be used as a preservative, preservative, detergent, or feminine cleanser.
본 발명의 일 구체예에 따른 조성물은 여성용 청결제로 사용되어 외음부 질 세정, 냉, 가려움, 악취제거 및 질염 개선 효과를 제공할 수 있다.The composition according to an embodiment of the present invention may be used as a feminine cleanser to provide vaginal cleansing, cold, itchiness, odor removal and vaginitis improvement.
본 발명에서 보존제는 세균, 곰팡이 및 효모로 이루어진 군중에서 선택된 1종 이상의 위해 미생물 또는 부패 미생물의 증식을 억제하거나 살균하여 식품, 사료, 화장품 및 의약품의 변질이나 부패, 화학변화를 방지하여 영양가와 신선도를 유지시키기 위하여 사용되는 첨가물을 의미한다. 좁은 의미로는 세균, 곰팡이, 효모 등의 부패 미생물의 증식 또는 생장을 저지하거나 살균할 수 있는 물질을 의미한다. 보존제로서 이상적인 조건으로서 조성물은 독성이 없어야 하며, 미량으로도 효과가 있어야 한다. In the present invention, the preservative inhibits or sterilizes the proliferation of one or more harmful microorganisms or spoilage microorganisms selected from the crowd consisting of bacteria, fungi, and yeast to prevent deterioration or spoilage, chemical changes in food, feed, cosmetics, and medicines, thereby improving nutritional value and freshness. It means the additive used to maintain the. In a narrow sense, it means a substance that can stop or sterilize the growth or growth of decaying microorganisms such as bacteria, mold, and yeast. As an ideal condition as a preservative, the composition should be non-toxic and should be effective even in trace amounts.
본 발명의 일 구체예에서, 보존제는 그 사용 목적 및 용도에 따라 적의 선택된 추가적인 여타의 첨가물을 추가로 더 포함할 수 있으며, 통상적으로 사용되는 첨가물 등 다른 유효성분이나 색소, 계면활성제, 방부제 등의 첨가물과 혼합하여 사용할 수 있다. In one embodiment of the present invention, the preservative may further include additional additional additives appropriately selected according to the purpose and purpose of use, and other active ingredients such as additives commonly used, pigments, surfactants, preservatives, etc. It can be used in admixture with additives.
본 발명의 일 구체예에서, 보존제 중 만자카니 추출물의 사용량은 사용목적과 적용형태, 적용부위, 적용대상물품 등에 따라 적절히 사용될 수 있고, 만자카니로부터 추출된 천연성분은 인체에 유해하지 아니하므로, 그 제품의 특성상 적용목적을 달성할 수 있는 경우라면 다양한 양으로 사용 가능하다.In one embodiment of the present invention, the amount of the manzacani extract used in the preservative can be appropriately used according to the purpose of use and application form, application site, and the object to be applied, and the natural components extracted from the manzakani are not harmful to the human body. If the application purpose can be achieved due to the characteristics of the product, it can be used in various amounts.
본 발명의 일 구체예에 따른 조성물은 다양한 미생물에 대하여 우수한 항균 활성 가지므로 합성 방부제를 대체하여 식품, 의약외품 또는 화장료 조성물 등에 첨가되는 방부제로서 이용될 수 있다. Since the composition according to one embodiment of the present invention has excellent antimicrobial activity against various microorganisms, it can be used as a preservative added to food, quasi-drugs or cosmetic compositions by replacing synthetic preservatives.
본 발명의 일 구체예에 따른 조성물은 세정제의 용도로서 이용될 수 있고 세정제는 의류용 세제, 식기 세척제, 식품 세정제, 가전제품 세척제일 수 있다. 본 발명의 일 구체예에 따른 조성물은 생약재로부터 추출된 천연성분을 포함하여 인체에 유해하지 아니하므로, 주방용품 또는 식품세척 등을 포함하는 각종 생활용품의 세척이나 항균성 부여를 목적으로 사용할 수 있다. The composition according to an embodiment of the present invention may be used as the use of a cleaning agent, and the cleaning agent may be a detergent for clothes, a dishwashing agent, a food cleaning agent, a household appliance cleaning agent. Since the composition according to one embodiment of the present invention is not harmful to the human body, including natural ingredients extracted from herbal medicines, it can be used for the purpose of washing or imparting antimicrobial properties to various household products, including kitchenware or food washing.
본 발명의 일 구체예에 있어서, 세정제는 그 사용 목적 및 용도에 따라 적의 선택된 첨가물을 추가로 포함할 수 있으며, 통상적으로 사용되는 세정제 등 다른 유효성분이나 색소, 계면활성제, 방부제 등의 첨가제와 혼합하여 사용할 수 있다. In one embodiment of the present invention, the cleaning agent may further include an appropriately selected additive according to the purpose and purpose of use, and mixed with additives such as other active ingredients, pigments, surfactants, and preservatives, such as commonly used cleaning agents. Can be used.
본 발명의 일 구체예에 있어서, 세정제 중 조성물의 사용량은 사용목적과 적용형태, 적용부위, 적용대상물품 등에 따라 적절히 사용될 수 있으며, 예를 들면 전체 조성물 기준으로 0.01 중량부 내지 50 중량부를 포함할 수 있으나, 상기 항균성 조성물의 함량은 이에 제한되지 않는다. In one embodiment of the present invention, the amount of the composition used in the cleaning agent may be appropriately used according to the purpose of use and the type of application, the application site, the object to be applied, etc. However, the content of the antimicrobial composition is not limited thereto.
본 발명의 일 구체예에 따른 조성물은 청결제의 용도로서 적용될 수 있다. 본 발명의 일 구체예에 따른 청결제는 일상 생활에서 흔히 감염되기 쉬운 병원성 미생물의 체내 감염을 예방 차원에서 수행하기 위하여 간단한 방법으로 손, 구강 또는 여성용 청결제 등의 용도로 사용될 수 있다. 본 발명의 일 구체예에 따른 조성물은 천연성분으로 인체에 유해하지 아니하므로 손, 구강 또는 여성용 청결제로 사용하는 경우에도 안전하게 적용할 수 있다.The composition according to one embodiment of the present invention can be applied as a cleaning agent. The cleaning agent according to one embodiment of the present invention can be used for a hand, mouth or female cleaning agent in a simple manner to perform a systemic infection of a pathogenic microorganism, which is often susceptible to infection in everyday life. Since the composition according to one embodiment of the present invention is a natural ingredient and is not harmful to the human body, it can be safely applied even when used as a hand, mouth or female cleanser.
본 발명의 일 구체예에 따른 조성물은 여성의 음부에 염증을 일으켜 질염, 가려움증 및 악취 등 여성 비뇨생식기계 질환을 일으킬 수 있는 유발균, 예를 들면 칸디다 알비칸스(Candida albicans) 및 대장균(Escherichia coli)에 대해 우수한 항균 활성을 갖고, 피부 가려움을 완화하거나 개선할 수 있으며, 피부자극을 유발하지 않아 여성용 청결제로서 사용될 수 있다.The composition according to one embodiment of the present invention causes inflammation in the female genital organs, which can cause female genitourinary system diseases such as vaginitis, itching and odor, for example Candida albicans and Escherichia coli It has excellent antimicrobial activity against, can alleviate or improve skin itching, and does not cause skin irritation, so it can be used as a cleanser for women.
본 발명의 다른 양상은 상기 조성물을 포함하는, 항염증 및 항균용 화장료 조성물을 제공한다.Another aspect of the present invention provides an anti-inflammatory and antibacterial cosmetic composition comprising the composition.
본 발명의 일 구체예에 따르면, 항염증 및 항균용 화장료 조성물은 피부 가려움을 완화 또는 개선하는 것일 수 있다.According to one embodiment of the present invention, the anti-inflammatory and antibacterial cosmetic composition may be to alleviate or improve skin itching.
본 발명의 일 구체예에 따른 조성물은 히스타민, 아세틸콜린 및 물질 P의 생성량을 억제하는 효과를 갖는 바, 피부 가려움을 완화 또는 개선할 수 있다. The composition according to an embodiment of the present invention has an effect of inhibiting the production amount of histamine, acetylcholine, and substance P, and may reduce or improve skin itching.
본 발명에 따른 화장료 조성물은 용액, 외용연고, 크림, 폼, 영양화장수, 유연화장수, 팩, 유연수, 유액, 메이크업베이스, 에센스, 비누, 액체 세정료, 입욕제, 선 스크린크림, 선오일, 현탁액, 유탁액, 페이스트, 겔, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 패취 및 스프레이로 구성된 군으로부터 선택되는 제형으로 제조할 수 있으나, 이에 제한되는 것은 아니다.The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nutrient makeup, softening makeup, pack, softening water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil, suspension, Emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays can be prepared in a formulation selected from the group consisting of, but not limited to It is not.
본 발명의 일 구체예에 따르면, 화장료 조성물은 일반 피부 화장료에 배합되는 화장품학적으로 허용 가능한 담체를 1 종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면 유분, 물, 계면활성제, 보습제, 저급 알콜, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 제한되는 것은 아니다.According to one embodiment of the present invention, the cosmetic composition may further include one or more cosmetically acceptable carriers to be blended with general skin cosmetics, and for example, oil, water, surfactant, moisturizing agent as a common ingredient , Lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc. may be appropriately blended, but is not limited thereto.
본 발명의 일 구체예에 따르면, 화장료 조성물에 포함되는 화장품학적으로 허용 가능한 담체는 제형에 따라 다양하다.According to one embodiment of the present invention, the cosmetically acceptable carrier included in the cosmetic composition varies depending on the formulation.
본 발명의 일 구체예에 따른 화장료 조성물의 제형이 연고, 페이스트, 크림 또는 젤인 경우에는, 담체 성분으로서 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화아연 또는 이들의 혼합물이 이용될 수 있다.When the formulation of the cosmetic composition according to an embodiment of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite as carrier components , Silica, talc, zinc oxide or mixtures thereof.
본 발명의 일 구체예에 따른 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는, 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록사이드, 칼슘 실케이트, 폴리아미드 파우더 또는 이들의 혼합물이 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진제를 포함할 수 있다.When the formulation of the cosmetic composition according to an embodiment of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component. In particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.
본 발명의 일 구체예에 따른 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는, 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되며, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알콜, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일이 이용될 수 있으며, 특히, 목화씨 오일, 땅콩 오일, 옥수수 배종 오일, 올리브오일, 피마자 오일 및 참깨 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 이용될 수 있다.When the formulation of the cosmetic composition according to an embodiment of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol , Benzyl benzoate, propylene glycol, 1,3-butylglycol oil can be used, in particular cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or sorb Non-fatty acid esters can be used.
본 발명의 일 구체예에 따른 화장료 조성물의 제형이 현탁액인 경우에는, 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알콜, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition according to an embodiment of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorb Suspension agents such as non-esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or trakant, and the like can be used.
본 발명의 다른 양상은 항염증 및 항균용 조성물을 포함하는, 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, including a composition for anti-inflammatory and antibacterial.
본 발명의 일 구체예에 따르면, 상기 염증성 질환은 피부염, 알레르기, 질염, 아토피, 천식, 결막염, 백내장, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 폐혈증, 위궤양, 위염, 크론병, 치질, 강직성 척추염, 루푸스, 섬유근통, 건선, 관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 근육염, 간염, 방광염, 신장염, 다발성 경화증, 당뇨병, 피부경화증, 통풍, 퇴행성 신경질환, 규폐증, 죽상동맥경화증 및 허혈으로 구성된 군으로부터 선택된 하나 이상인 것일 수 있다. According to one embodiment of the present invention, the inflammatory diseases include dermatitis, allergies, vaginitis, atopy, asthma, conjunctivitis, cataract, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, septicemia, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, Ankylosing spondylitis, lupus, fibromyalgia, psoriasis, arthritis, osteoarthritis, rheumatoid arthritis, shoulder periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, diabetes, skin sclerosis, gout, degenerative neuropathy, silicosis, atherosclerosis And it may be one or more selected from the group consisting of ischemia.
본 발명의 약학적 조성물은 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산 알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive is starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate , Lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol and talc can be used.
본 발명의 일 구체예에 따른 약학적 조성물은 약학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 일 구체예에 따른 약학적 조성물에 포함되는 약학적으로 허용되는 담체는 약제의 제조시에 통상적으로 이용되는 것으로써, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 트레할로스, 히알루론산, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 일 구체예에 따른 약학적 조성물은 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(22th ed., 2013)에 상세히 기재되어 있다.The pharmaceutical composition according to an embodiment of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition according to an embodiment of the present invention are those commonly used in the manufacture of pharmaceuticals, lactose, dextrose, sucrose, sorbitol, mannitol, trehalose, hyaluronic acid, Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, Magnesium stearate and mineral oil, and the like, but is not limited thereto. The pharmaceutical composition according to an embodiment of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (22th ed., 2013).
본 발명의 약학적 조성물은 실제 임상 투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 본 발명의 지용성 폴리페놀 성분이 증가된 생약 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calciumcarbonate), 수크로스(Sucrose), 락토오스(Lactose) 또는 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용될 수 있다.The pharmaceutical composition of the present invention may be administered in various formulations, oral and parenteral, during actual clinical administration. When formulated, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants, etc. It can be prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, for example, starch, calcium, in the herbal composition with increased fat-soluble polyphenol component of the present invention. It can be prepared by mixing calcium carbonate, sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate talc may be used in addition to simple excipients.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여할 수 있으며, 비경구 투여시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식을 선택할 수 있다. 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다.The pharmaceutical composition of the present invention may be administered orally or parenterally according to the desired method, and when administered parenterally, an intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method may be used. You can choose. The dosage range varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity.
본 발명의 약학적 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에서 "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 일 구체예에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, drug Sensitivity to, time of administration, route of administration and rate of discharge, duration of treatment, factors including co-drugs, and other factors well known in the medical field. The composition according to an embodiment of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명의 일 구체예에 따른 약학적 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1kg 당 0.1㎎ 내지 200㎎, 바람직하게는 1㎎ 내지 10㎎을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition according to an embodiment of the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.1 mg to 200 mg per 1 kg of body weight, preferably 1 mg to 10 mg Can be administered daily or every other day or divided into 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, and age, the dosage is not limited to the scope of the present invention in any way.
본 발명의 일 구체예에 따른 만자카니 추출물은 염증관련 인자의 발현을 억제하며 다양한 세균 및 진균에 대해 항균 활성을 갖고, 히스타민, 아시텔콜린, 물질 P의 생성량을 유의미하게 감소시키고 라디칼 소거능을 갖는 바 항염증, 항산화, 항소양, 및 항균 효과가 우수하므로 항염증 및 항균용 조성물로서 유용하게 이용될 수 있다. Manzacani extract according to one embodiment of the present invention inhibits the expression of inflammation-related factors and has antibacterial activity against various bacteria and fungi, significantly reduces the amount of histamine, acitelcholine, and substance P and has radical scavenging activity Bars are excellent in anti-inflammatory, antioxidant, anti-ulcer, and anti-bacterial effects, and thus can be usefully used as anti-inflammatory and anti-bacterial compositions.
도 1은 다양한 농도(1 내지 1000㎍/㎖)의 만자카니 증류수 추출물(이하, MDE) 또는 만자카니 에탄올 추출물(이하, MEE) 처리시 DPPH 라디칼 소거능(%)을 나타낸다.
도 2는 다양한 농도(1 내지 1000㎍/㎖)의 MDE 또는 MEE 처리시 ABTS 라디칼 소거능(%)을 나타낸다.
도 3은 다양한 농도(1, 5, 10㎍/㎖)의 MDE 또는 MEE 첨가시 RAW264.7 세포의 생존율을 나타낸다.
도 4는 다양한 농도(1, 5, 10㎍/㎖)의 MDE 또는 MEE 첨가시 MC/9 세포의 생존율을 나타낸다.
도 5는 다양한 농도(1, 5, 10㎍/㎖)의 MDE 또는 MEE 첨가시 PC12 세포의 생존율을 나타낸다.
도 6은 다양한 농도의 MDE 또는 MEE 처리시, NO 생성량을 나타낸다. 정상군(Normal)은 LPS 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 LPS를 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다(명시적인 기재가 없다면 이하 도면에서도 동일하다).
도 7은 RAW264.7 세포에 다양한 농도(1, 5, 10㎍/㎖)의 MDE 또는 MEE 처리시, IL-1β의 발현양(pg/㎖)을 나타내고, 도 8은 IL-2의 발현양(pg/㎖)을 나타내고, 도 9는 IL-6의 발현양(pg/㎖)을 나타내며, 도 10은 TNF-α의 발현양(pg/㎖)을 나타낸다.
도 11은 RAW264.7 세포에서 MDE 또는 MEE 처리시 추출물 농도에 따른 PGE2의 발현양(pg/㎖)을 나타내고, 도 12는 RAW264.7 세포에서 MDE 또는 MEE 처리시 추출물 농도에 따른 LTB4의 발현양(pg/㎖)을 나타낸다.
도 13은 RAW264.7 세포에서 MDE 또는 MEE 처리시 NF-κB의 상대적인 발현양을 나타내고, 도 14는 RAW264.7 세포에서 MDE 또는 MEE 처리시 iNOS의 상대적인 발현양을 나타내며, 도 15는 COX-2의 상대적인 발현양을 나타낸다.
도 16은 MC/9 세포에서 다양한 농도의 MDE 또는 MEE 처리시 히스타민의 생성량을 나타낸다. 도 16에서 정상군(Normal)은 PMA, 이오노마이신(ionomycin) 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 PMA 및 이오노마이신을 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다.
도 17은 PC12 세포에서 다양한 농도의 MDE 또는 MEE 처리시 아세틸콜린의 생성량을 나타낸다.
도 18은 PC12 세포에서 다양한 농도의 MDE 또는 MEE 처리시 아세틸콜린에스터라제의 생성량을 나타낸다.
도 19는 PC12 세포에서 다양한 농도의 MDE 또는 MEE 처리시 물질 P의 생성량을 나타낸다. 도 17 내지 19에서 정상군(Normal)은 NGF 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 NGF를 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다.
도 20a 내지 20e는 만자카니 추출물의 항균 활성을 살펴보기 위하여, 다양한 농도(10, 5, 2.5, 1.25%w/v)의 MDE 또는 MEE 처리시 세균 및 진균에 대해 디스크 확산법을 수행한 결과를 나타낸다. 구체적으로, 도 20a는 대장균(E. coli)에 대한 결과이고, 도 20b는 슈도모나스 에루지노사(P. aeruginosa)에 대한 결과이며, 도 20c는 스타필로코쿠스 아우레우스(S. aureus)에 대한 결과이며, 도 20d는 칸디다 알비칸스(C. albicans)에 대한 결과이고, 도 20e는 아스퍼질러스 나이거(A. niger)에 대해 디스크 확산법을 수행한 결과를 나타낸다.Figure 1 shows the DPPH radical scavenging capacity (%) when treated with various concentrations (1 to 1000㎍ / ㎖) Manzakani distilled water extract (hereinafter, MDE) or Manzakani ethanol extract (hereinafter, MEE).
Figure 2 shows the ABTS radical scavenging capacity (%) in MDE or MEE treatment at various concentrations (1 to 1000 μg/ml).
3 shows the survival rate of RAW264.7 cells when MDE or MEE was added at various concentrations (1, 5, 10 μg/ml).
4 shows the survival rate of MC/9 cells when MDE or MEE was added at various concentrations (1, 5, 10 μg/ml).
5 shows the survival rate of PC12 cells when MDE or MEE was added at various concentrations (1, 5, 10 μg/ml).
Figure 6 shows the amount of NO produced during various concentrations of MDE or MEE treatment. The normal group (Normal) shows the result in cells that did not process LPS and manzacani extract, and the control (Control) shows the result in cells that did not process LPS and manzacani extract (explicit description If not, it is the same in the drawings below).
FIG. 7 shows the expression level of IL-1β (pg/mL) when MDE or MEE treatment of various concentrations (1, 5, 10 μg/mL) in RAW264.7 cells, and FIG. 8 shows the expression level of IL-2 (pg/mL), FIG. 9 shows the expression level of IL-6 (pg/mL), and FIG. 10 shows the expression level of TNF-α (pg/mL).
11 shows the expression level (pg/ml) of PGE 2 according to the extract concentration when treating MDE or MEE in RAW264.7 cells, and FIG. 12 shows LTB 4 according to the extract concentration when processing MDE or MEE in RAW264.7 cells. Expression level (pg/ml) is shown.
FIG. 13 shows the relative expression of NF-κB when treated with MDE or MEE in RAW264.7 cells, FIG. 14 shows the relative expression of iNOS when treated with MDE or MEE in RAW264.7 cells, and FIG. 15 shows COX-2 Represents the relative expression level.
Figure 16 shows the amount of histamine produced in various concentrations of MDE or MEE treatment in MC/9 cells. In FIG. 16, the normal group (Normal) shows the results in cells not treated with PMA, ionomycin and manzacani extract, and the control (Control) processes PMA and ionomycin, and the manzacani extract. Results are shown in untreated cells.
Figure 17 shows the amount of acetylcholine produced in various concentrations of MDE or MEE treatment in PC12 cells.
Figure 18 shows the amount of acetylcholinesterase produced in various concentrations of MDE or MEE treatment in PC12 cells.
Figure 19 shows the amount of production of substance P in various concentrations of MDE or MEE treatment in PC12 cells. In Figures 17 to 19, the normal group (Normal) shows the results in cells not treated with NGF and manzacani extracts, and the control (Control) shows the results in cells treated with NGF and manzacani extracts are not treated. .
20a to 20e show the results of performing a disk diffusion method on bacteria and fungi during MDE or MEE treatment of various concentrations (10, 5, 2.5, 1.25% w/v) in order to examine the antibacterial activity of the manzacani extract. . Specifically, Figure 20a is a result for E. coli , Figure 20b is a result for Pseudomonas aeruginosa ( P. aeruginosa ), Figure 20c is Staphylococcus aureus ( S. aureus ) Results, Figure 20d is a result of Candida albicans ( C. albicans ), Figure 20e shows the results of performing the disk diffusion method for Aspergillus Niger ( A. niger ).
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1. 만자카니 추출물의 제조Example 1 Preparation of Manzacani Extract
만자카니(Manjakani)는 주내물산이 Abdmalek company(Malaysia)로부터 원물 수입하고, 대전대학교 지역혁신센터(TBRC-RIC)에서 정선한 것을 실험에 사용하였다.Manjakani imported raw materials from Abdmalek company (Malaysia), and used them selected by Daejeon University Regional Innovation Center (TBRC-RIC).
만자카니 20g에 각각 23℃의 증류수 또는 99.9% 에탄올을 500㎖씩 첨가하여 3시간 동안 환류추출한 후, 여과액을 회전 진공 농축기(rotary vacuum evaporator; Buchi B-480, Switzerland)를 이용하여 감압 농축하였으며, 농축된 용액을 동결 건조기(freeze dryer; EYELA FDU-540, Japan)로 동결 건조하였다. After adding 500 mL of distilled water or 99.9% ethanol at 23°C to 20 g of manzacani for 3 hours, the filtrate was concentrated under reduced pressure using a rotary vacuum evaporator (Buchi B-480, Switzerland). , The concentrated solution was freeze-dried with a freeze dryer (EYELA FDU-540, Japan).
만자카니 증류수 추출물(Manjakani distilled water extract 이하, MDE) 6.6g(수율 33중량%), 만자카니 에탄올 추출물(Manjakani ethanol extract 이하, MEE) 11.1g(수율 55.5중량%)의 분말을 획득하여 초저온 냉동고(-80℃)에서 보관하고, 실험에 필요한 농도로 증류수에 희석해 사용하였다.Manjakani distilled water extract (MDE) 6.6g (Yield 33% by weight), Manjakani ethanol extract (MEE) 11.1g (Yield 55.5% by weight) of powder was obtained to obtain a cryogenic freezer ( -80°C) and diluted with distilled water to the concentration required for the experiment.
실시예 2. 만자카니 추출물의 항산화 효능Example 2. Antioxidant efficacy of Manzacani extract
실시예 2-1. DPPH 라디칼 소거능 측정Example 2-1. DPPH radical scavenging capacity measurement
DPPH(2,2-diphenyl-1-picrylhydrayl) 분석 방법을 사용하여, 라디칼 소거능(radical scavenging ability)을 측정하여 만자카니 추출물의 항산화 능력을 평가하였다. DPPH는 비교적 안정한 자유 라디칼(free radical)로서, 자유 라디칼이 자유라디칼 제거제에 의해 환원되어 색이 보라색에서 노란색으로 탈색되면, 분광분석을 통하여 라디칼 감소를 측정할 수 있다. Using the DPPH (2,2-diphenyl-1-picrylhydrayl) analysis method, the radical scavenging ability was measured to evaluate the antioxidant capacity of the Manzacani extract. DPPH is a relatively stable free radical, and when the free radical is reduced by a free radical remover to discolor the color from purple to yellow, radical reduction can be measured through spectroscopic analysis.
실시예 1에서 수득한 다양한 농도(1, 10, 100, and 1000㎍/㎖)의 만자카니 증류수 추출물(이하, MDE) 및 에탄올 추출물(이하, MEE)을 로 0.2mM의 DPPH 용액(Sigma, USA) 150㎕와 혼합하여 37℃에서 30분 동안 반응시켰다. 517nm에서 흡광도를 측정하고, 만자카니 추출물 대신 증류수를 처리한 만자카니 추출물 미처리군을 대조군으로 하여 하기 수학식 1에 의해 라디칼 소거능(%)을 계산하였다. 0.2mM DPPH solution with various concentrations (1, 10, 100, and 1000 μg/ml) of Manzacani distilled water extract (hereinafter, MDE) and ethanol extract (hereinafter, MEE) obtained in Example 1 (Sigma, USA) ) It was mixed with 150 µl and reacted at 37°C for 30 minutes. Absorbance was measured at 517 nm, and radical scavenging capacity (%) was calculated by
세 번의 독립적인 실험 결과값으로부터 평균 및 표준편차를 계산하여 결과를 표 1 및 도 1에 나타내었다(이하, 동일하다). The average and standard deviation are calculated from the results of three independent experiments, and the results are shown in Table 1 and FIG. 1 (hereinafter the same).
(㎍/㎖)(Μg/ml)
표 1 및 도 1에 나타난 바와 같이, MDE와 MEE는 모두 농도 의존적으로 DPPH 라디칼 소거능을 증가시켰고, 만자카니 추출물이 항산화 효능을 갖는 것을 확인하였다. As shown in Table 1 and FIG. 1, both MDE and MEE increased DPPH radical scavenging capacity in a concentration-dependent manner, and it was confirmed that the Manzacani extract had antioxidant efficacy.
실시예 2-2. ABTS 라디칼 소거능 측정Example 2-2. ABTS radical scavenging capacity measurement
ABTS[2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] 분석 방법을 사용하여, 라디칼 소거능을 측정함으로써 만자카니 추출물의 항산화 능력을 평가하였다. 과황산 칼륨(photassium persulphate)과의 반응에 의해 생성되는 ABTS+(ABTS radical cation)은 시료 내의 항산화 물질에 의해 제거되어 라디칼 특유의 색인 청록색이 탈색되므로 이를 이용하여 항산화 활성을 측정할 수 있다.The antioxidant scavenging ability of Manzacani extract was evaluated by measuring radical scavenging activity using the ABTS [2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] analysis method. The ABTS+ (ABTS radical cation) produced by the reaction with potassium persulfate (photassium persulphate) is removed by the antioxidant in the sample, and the blue-green color specific to the radical is discolored, so that antioxidant activity can be measured.
7.4mM의 ABTS와 2.6mM의 과황산칼륨(potassium persulphate)을 제조한 후, 암소에 24시간 방치하여 양이온(ABTS+)을 형성시킨 다음 732nm에서 흡광도를 측정하여 흡광도 값이 1.5이하가 나오게 증류수로 희석한 ABTS+용액을 사용하였다.7.4mM ABTS and 2.6mM potassium persulphate were prepared, and then allowed to stand in the dark for 24 hours to form cations (ABTS+), and then measured for absorbance at 732nm, diluted with distilled water to give an absorbance value of 1.5 or less. One ABTS+ solution was used.
상기에서 희석한 ABTS+ 용액 150㎕ 및 실시예 1에서 수득한 다양한 농도(1, 10, 100, and 1000㎍/㎖)의 MDE 또는 MEE를 5㎕씩 혼합하고 실온에서 10분 동안 반응시켰다. 732nm에서 흡광도를 측정하고, 만자카니 추출물 대신 증류수를 처리한 만자카니 추출물 미처리군을 대조군으로 하여 상기 수학식 1에 의해 라디칼 소거능(%)을 계산하였다. 150 μl of the diluted ABTS+ solution and 5 μl of MDE or MEE of various concentrations (1, 10, 100, and 1000 μg/ml) obtained in Example 1 were mixed and reacted at room temperature for 10 minutes. Absorbance was measured at 732 nm, and the radical scavenging capacity (%) was calculated by
표 2 및 도 2에 나타난 바와 같이, MDE와 MEE는 모두 농도 의존적으로 ABTS 라디칼 소거능을 증가시켰다. As shown in Table 2 and FIG. 2, both MDE and MEE increased ABTS radical scavenging capacity in a concentration-dependent manner.
(㎍/㎖)(Μg/ml)
실시예 3. 만자카니 추출물의 세포 독성 평가Example 3. Evaluation of cytotoxicity of manzacani extract
실시예 3-1. 세포 배양Example 3-1. Cell culture
대식세포의 일종인 RAW264.7세포는 ATCC로부터 구입하여, 10% 우태아혈청(fetal bovine serum(FBS); Gibco BRL, U.S.A.)와 1% 페니실린-스트렙토마이신으로 구성된 DMEM 배지(Dulbecco's Modified Eagle's Medium; Gibco BRL, U.S.A.)를 사용하여 배양하였다. RAW264.7 cells, a type of macrophage, were purchased from ATCC and consisted of 10% fetal bovine serum (FBS); Gibco BRL, USA, and 1% penicillin-streptomycin DMEM medium (Dulbecco's Modified Eagle's Medium; Gibco BRL, USA).
비만세포의 일종인 MC/9 세포는 ATCC로부터 구입하여, 10% FBS, 1% 페니실린-스트렙토마이신, 0.055mM 2-머캅토에탄올(Gibco BRL, U.S.A.), 2mM L-글루타민(Gibco BRL, U.S.A.), 10% T-Stim(Corning, U.S.A.)으로 구성된 DMEM 배지를 사용하였으며, 갈색세포종양에서 유래된 PC12 세포는 ATCC로부터 구입하여, 10% 말 혈청(horse serum; Gibco BRL, U.S.A.)과 5% FBS으로 구성된 DMEM 배지를 사용하여 배양하였다. MC/9 cells, a type of mast cell, purchased from ATCC, 10% FBS, 1% penicillin-streptomycin, 0.055 mM 2-mercaptoethanol (Gibco BRL, USA), 2 mM L-glutamine (Gibco BRL, USA) , DMEM medium composed of 10% T-Stim (Corning, USA) was used, PC12 cells derived from brown cell tumor were purchased from ATCC, 10% horse serum (Gibco BRL, USA) and 5% FBS Cultured using DMEM medium consisting of.
모든 세포들은 37℃, 5% CO2 조건이 유지되는 세포배양기(Forma scientific Co., U.S.A.)에서 배양하였으며, 2-3일 주기로 계대 배양하여 추후 실험을 진행하였다.All cells were cultured in a cell incubator (Forma scientific Co., USA) where the condition of 37° C., 5% CO 2 was maintained, and subsequent experiments were performed by subculturing in 2-3 day cycles.
실시예 3-2. 세포 생존율 측정Example 3-2. Cell viability measurement
RAW264.7 세포는 96 웰 플레이트에 1.5×105 세포/웰로 분주하였고, MC/9 세포는 48 웰 플레이트에 2×105 세포/웰로 분주하였으며, PC12 세포는 콜라겐으로 코팅된 48 웰 플레이트에 2×105 세포/웰로 분주하여 24시간 동안 37℃에서 배양하였고, 2-3일 주기로 계대 배양하여 추후 실험을 진행하였다. RAW264.7 cells were dispensed at 1.5×10 5 cells/well in a 96 well plate, MC/9 cells were dispensed at 2×10 5 cells/well in a 48 well plate, and PC12 cells were dispensed in a 48 well plate coated with collagen. × 10 5 cells / well dispensed and were cultured at 37 ℃ for 24 hours and then subculture was performed for a subsequent period of 2 to 3 experiments.
24시간 후, MDE 또는 MEE를 1, 5, 10 ㎍/㎖의 농도로 처리하여 RAW264.7 세포는 24시간, MC/9 세포와 PC12 세포는 48시간 동안 배양하였다. 모든 배양이 끝난 후, 배양액 100㎕당 10㎕의 EZ-Cytox 용액(Daeilab, Korea)을 첨가하고 세포 배양기에서 30분간 반응시켰다. 반응 후, 450㎚에서 흡광도의 변화를 측정하여 만자카니 추출물 대신 증류수를 처리한 만자카니 추출물 미처리군을 대조군에 대한 세포 생존율을 백분율로 나타내었다. After 24 hours, MDE or MEE was treated with concentrations of 1, 5, and 10 μg/ml to incubate RAW264.7 cells for 24 hours, MC/9 cells and PC12 cells for 48 hours. After all the cultures were completed, 10 μl of EZ-Cytox solution (Daeilab, Korea) was added per 100 μl of the culture medium, and reacted for 30 minutes in a cell incubator. After the reaction, the change in absorbance at 450 nm was measured to show the cell viability as a percentage of the untreated group treated with distilled water instead of the manzacani extract.
표 3 및 도 3은 다양한 농도(1, 5, 10㎍/㎖)의 만자카니 증류수 추출물(MDE) 또는 에탄올 추출물(MEE) 첨가시 RAW264.7 세포의 생존율을 나타낸다.Table 3 and FIG. 3 show the survival rate of RAW264.7 cells when various concentrations (1, 5, 10 μg/mL) of Manzacani distilled water extract (MDE) or ethanol extract (MEE) were added.
표 4 및 도 4는 다양한 농도(1, 5, 10㎍/㎖)의 만자카니 증류수 추출물 또는 에탄올 추출물 첨가시 MC/9 세포의 생존율을 나타낸다.Table 4 and FIG. 4 show the survival rate of MC/9 cells when various concentrations (1, 5, 10 μg/ml) of Manzacani distilled water extract or ethanol extract were added.
표 5 및 도 5는 다양한 농도(1, 5, 10㎍/㎖)의 만자카니 증류수 추출물 또는 에탄올 추출물 첨가시 PC12 세포의 생존율을 나타낸다.Table 5 and FIG. 5 show the survival rate of PC12 cells when various concentrations (1, 5, 10 μg/ml) of Manzacani distilled water extract or ethanol extract were added.
(㎍/㎖)(Μg/ml)
표 3 내지 5 및 도 3 내지 5에 나타난 바와 같이, MDE 및 MEE는 10㎍/㎖까지 RAW264.7 세포, MC/9 세포, PC12 세포에 대해 독성이 나타나지 않아 무해한 것을 확인하였다. As shown in Tables 3 to 5 and FIGS. 3 to 5, MDE and MEE were up to 10 μg/ml. RAW264.7 cells, MC/9 cells, PC12 cells were found to be harmless because of no toxicity.
실시예 4. 만자카니 추출물의 항염증 활성Example 4. Anti-inflammatory activity of Manzacani extract
실시예 4-1. NO 생성량 측정Example 4-1. NO production measurement
만자카니 추출물의 항염증 활성을 측정하기 위하여, NO 검출 키트(nitric oxide detection kit; Intron Biotechnology, Korea)를 사용하여 제조사의 매뉴얼에 따라 NO 생성량을 측정하였다.In order to measure the anti-inflammatory activity of the Manzacani extract, the amount of NO produced was measured according to the manufacturer's manual using a NOtric detection kit (Intrical Biotechnology, Korea).
96 웰 플레이트에 RAW264.7 세포를 1.5×105 세포/웰로 분주하여 37℃에서 24시간 동안 배양하였다. 배양 후, 1, 5, 10㎍/㎖ 농도의 MDE 또는 MEE와 1㎍/㎖ LPS를 함께 처리하여 다시 37℃에서 24시간 동안 배양하였다. 배양 후, N1 버퍼를 50㎕씩 각 웰에 처리하여 10분간 상온에서 반응한 후, N2 버퍼를 50㎕씩 각 웰에 처리하고 10분간 반응시켰다. 반응 후, 540㎚에서 마이크로플레이트 리더(Molecular Devices, U.S.A.)를 이용하여 흡광도를 측정하고, 이를 이용하여 NO 생성량을 대조군에 대한 백분율로 나타내어, 그 결과를 표 6 및 도 6에 나타내었다.RAW264.7 cells were plated at 1.5×10 5 cells/well in a 96-well plate and cultured at 37° C. for 24 hours. After incubation, 1, 5, and 10 μg/ml concentrations of MDE or MEE and 1 μg/ml LPS were treated together, followed by incubation at 37° C. for 24 hours. After incubation, 50 µl of N1 buffer was treated in each well to react at room temperature for 10 minutes, and then 50 µl of N2 buffer was treated in each well and reacted for 10 minutes. After the reaction, absorbance was measured using a microplate reader (Molecular Devices, USA) at 540 nm, and the NO production amount was expressed as a percentage of the control using the result, and the results are shown in Table 6 and FIG. 6.
표 6 및 도 6에서 정상군(Normal)은 LPS 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 LPS를 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다(명시적인 기재가 없다면 이하 표 및 도면에서도 동일하다). 표 6 및 도 6에 나타난 바와 같이, MDE와 MEE는 모든 농도에서 대조군에 비해 유의미하게(*; p＜0.05, **; p＜0.01, ***; p＜0.001, 이하 표 및 도면에서도 동일하다) 농도 의존적으로 NO 생성량을 감소시켰다. MDE 대비 MEE가 더 높은 정도로 NO 생성량을 감소시켰다.In Table 6 and Figure 6, the normal group (Normal) shows the results in cells not treated with LPS and manzacani extract, and the control (Control) shows results in cells treated with LPS and manzacani extract is not treated. (It is the same in the following table and drawings unless otherwise specified). As shown in Table 6 and Figure 6, MDE and MEE were significantly (*; p<0.05, **; p<0.01, ***; p<0.001, the same in the following tables and figures) compared to the control group at all concentrations The amount of NO production was decreased depending on the concentration. NO production was reduced to a higher degree than MDE.
실시예 4-2. 만자카니 추출물 처리시 염증성 사이토카인 발현양 측정Example 4-2. Measurement of inflammatory cytokine expression levels when manzacani extract treatment
mouse cytokine milliplex map immunoassay kit(Millipore, U.S.A.)를 사용하여 제조사의 매뉴얼에 따라 만자카니 추출물 처리시 염증성 사이토카인인 IL-1β, IL-2, IL-6, 및 TNF-α의 발현양을 분석하였다.Using the mouse cytokine milliplex map immunoassay kit (Millipore, USA), the expression levels of inflammatory cytokines IL-1β, IL-2, IL-6, and TNF-α were analyzed according to the manufacturer's manual. .
12 웰 플레이트에 RAW264.7 세포를 2×105 세포/웰로 분주하여 37℃에서 24시간 동안 배양하였다. 배양 후, 실시예 1에서 수득한 1, 5, 10㎍/㎖ 농도의 MDE 또는 MEE와 1㎍/㎖ LPS를 함께 처리하여 다시 24시간 동안 배양하였다. 배양액을 1,200rpm에서 5분간 원심분리하여 얻은 상등액과 표준물질(standard)을 96 웰 플레이트에 25㎕씩 분주하고 분석 버퍼(assay buffer) 및 매트릭스 버퍼(matrix buffer), 항체-고정 비드(antibody-immobilized beads)를 각각 25㎕씩 가하여 혼합한 후, 2시간 동안 실온에서 반응시키고 세척 버퍼를 이용하여 2회 세척하였다. 세척 후, 25㎕의 검출 항체(detection antibody)을 가하여 1시간 동안 실온에서 반응시키고 추가로 25㎕의 스트렙타비딘-피코에이트린(Streptavidin-Phycoerythrin)을 가하여 30분 동안 실온에서 반응시킨 뒤 세척 버퍼를 이용하여 2회 세척하였다. 세척 후, 150㎕의 PBS를 첨가하고 5분간 진탕한 후, 루미넥스(Luminex; Millipore, U.S.A.)를 이용하여 측정하고, 염증성 사이토카인 발현의 절대값을 계산하였다.RAW264.7 cells were divided into 2×10 5 cells/well in a 12-well plate and cultured at 37° C. for 24 hours. After incubation, 1, 5, and 10 μg/ml MDE or MEE obtained in Example 1 were treated together with 1 μg/ml LPS to incubate again for 24 hours. The supernatant and the standard obtained by centrifuging the culture solution for 5 minutes at 1,200 rpm were dispensed in 25 µl in a 96-well plate, and assay buffer, matrix buffer, and antibody-immobilized beads beads) were mixed by adding 25 µl each, and then reacted at room temperature for 2 hours and washed twice with a washing buffer. After washing, 25 µl of detection antibody was added to react at room temperature for 1 hour, and further 25 µl of Streptavidin-Phycoerythrin was added to react at room temperature for 30 minutes, followed by washing buffer It was washed twice with. After washing, 150 μl of PBS was added and shaken for 5 minutes, then measured using Luminex (Millipore, USA), and the absolute value of inflammatory cytokine expression was calculated.
RAW264.7 세포에 다양한 농도의 MDE 또는 MEE 처리시, IL-1β의 발현양(pg/㎖)을 각각 표 7 및 도 7에 나타내고, IL-2의 발현양(pg/㎖)을 표 8 및 도 8에 나타내었으며, IL-6의 발현양(pg/㎖)을 표 9 및 도 9에 나타내고, TNF-α의 발현양(pg/㎖)을 표 10 및 도 10에 나타내었다.When treated with various concentrations of MDE or MEE in RAW264.7 cells, the expression level (pg/mL) of IL-1β is shown in Tables 7 and 7, respectively, and the expression level (pg/mL) of IL-2 is shown in Tables 8 and 8, the expression level of IL-6 (pg/mL) is shown in Table 9 and FIG. 9, and the expression level of TNF-α (pg/mL) is shown in Table 10 and FIG. 10.
(㎍/㎖)(Μg/ml)
(㎍/㎖) (Μg/ml)
(㎍/㎖) (Μg/ml)
(㎍/㎖) (Μg/ml)
도 7 내지 10 및 표 7 내지 10에 나타난 바와 같이, MDE 또는 MEE 처리시 모든 농도에서 대조군 대비 염증성 사이토카인인 IL-1β, IL-2, IL-6, 및 TNF-α의 발현양이 농도 의존적으로 유의미하게 감소하였다. MDE 대비 MEE가 더 높은 정도로 IL-1β, IL-6, TNF-α의 발현양을 감소시켰다.As shown in FIGS. 7 to 10 and Tables 7 to 10, the expression levels of the inflammatory cytokines IL-1β, IL-2, IL-6, and TNF-α compared to the control at all concentrations during MDE or MEE treatment are concentration-dependent. Decreased significantly. The expression levels of IL-1β, IL-6, and TNF-α were reduced to a higher degree than MDE.
실시예 4-3. 만자카니 추출물 처리시 PGEExample 4-3. PGE when processing Manzacani extract 22 및 LTB And LTB 44 발현양 측정 Expression level measurement
PGE2(Prostaglandin E2)는 염증 반응의 매개체로서 작용하고, Th2 type 면역반응을 촉진하고 Th1 type의 면역반응은 억제하며, 대식세포에서 TNF-α, IL-1, IL-8, IL-12 등의 염증성 사이토카인의 생성을 억제하고 IL-10과 같은 항염증성 사이토카인의 생성을 촉진하는 면역반응의 조절자로서 역할하는 것이 알려져 있다.PGE 2 (Prostaglandin E 2 ) acts as a mediator of the inflammatory response, promotes the Th2 type immune response, suppresses the Th1 type immune response, and inhibits TNF-α, IL-1, IL-8, IL-12 in macrophages. It is known to act as a modulator of an immune response that inhibits the production of inflammatory cytokines such as IL-10 and promotes the production of anti-inflammatory cytokines such as IL-10.
LTB4(루코트리엔 B4)는 호중구(neutrophils), 산호구(eosinophils), 단핵구(monocytes) 등의 강력한 주화인자(chemoattractant)로써 포식세포(phagocytes)를 혈관벽에 부착시키고 호중구를 탈과립(degranulation)하며 과산화성 음이온(superoxide anions)을 생성시켜 염증을 유발하는 주요인자로 알려져 있다.LTB 4 (Lukotriene B 4 ) is a potent chemoattractant such as neutrophils, eosinophils, and monocytes. It attaches phagocytes to the blood vessel walls and degranulates neutrophils. ) And is known as a major factor causing inflammation by producing superoxide anions.
PGE2 Parameter Assay Kit(R&D systems Co., U.S.A.) 또는 LTB4 Parameter Assay Kit(R&D systems Co., U.S.A.)를 사용하여 제조사의 매뉴얼에 따라, 만자카니 추출물 처리시 PGE2 및 LTB4의 발현양을 분석하였다. Using the PGE 2 Parameter Assay Kit (R&D systems Co., USA) or LTB 4 Parameter Assay Kit (R&D systems Co., USA), according to the manufacturer's manual, the amount of expression of PGE 2 and LTB 4 during the Manzacani extract treatment Analysis.
12 웰 플레이트에 RAW264.7 세포를 2Х105 세포/웰로 분주하여 24시간 동안 배양하였다. 배양 후, 실시예 1에서 수득한 1, 5, 10㎍/㎖ 농도의 MDE 또는 MEE와 1㎍/㎖의 LPS를 함께 처리하여 24시간 동안 배양하였다. 배양액을 1,200rpm에서 5분간 원심분리하여 얻은 상등액과 표준물질을 96 웰 플레이트에 100㎕씩 넣고 37℃에서 90분간 반응시켰다. 반응 후, 세척 버퍼를 이용하여 3회 세척 작업을 진행한 후, 100㎕의 검출 항체(detection antibody)를 넣어 다시 37℃에서 60분간 반응시키고 세척하였다. 세척 후, HRP conjugate를 100㎕씩 넣어 37℃에서 30분간 반응시키고 세척한 뒤 기질 시약(substrate reagent)를 90㎕씩 첨가하여 37℃에서 15분간 반응시키고 50㎕의 정지 용액(stop solution)을 추가하여 ELISA 리더기(MyBioSource, U.S.A.)를 통해 450㎚에서 흡광도를 측정하였으며, 표준 곡선을 기준으로 PGE2 및 LTB4의 발현양을 계산하여 절대 값(pg/㎖)으로 나타내었다. RAW264.7 cells were divided into 2 x 10 5 cells/well in a 12-well plate and cultured for 24 hours. After cultivation, 1, 5, and 10 μg/ml MDE or MEE obtained in Example 1 were treated together with 1 μg/ml LPS to incubate for 24 hours. The culture solution was centrifuged at 1,200 rpm for 5 minutes, and 100 µl of the supernatant and the standard material were added to a 96-well plate and reacted at 37°C for 90 minutes. After the reaction, a washing operation was performed 3 times using a washing buffer, and 100 µl of detection antibody was added to react for 60 minutes at 37° C. and washed. After washing, 100 µl of HRP conjugate was added to react for 30 minutes at 37° C., washed, and 90 µl of substrate reagent was added to react for 15 minutes at 37° C. and 50 µl of stop solution was added. The absorbance was measured at 450 nm through an ELISA reader (MyBioSource, USA), and the expression levels of PGE 2 and LTB 4 were calculated based on a standard curve to represent absolute values (pg/mL).
표 11 및 도 11은 RAW264.7 세포에서 MDE 또는 MEE 처리시 PGE2의 발현양(pg/㎖)을 나타내고, 표 12 및 도 12는 RAW264.7 세포에서 MDE 또는 MEE 처리시 LTB4의 발현양(pg/㎖)을 나타낸다. Table 11 and FIG. 11 show the expression level (pg/ml) of PGE 2 when treated with MDE or MEE in RAW264.7 cells, and Table 12 and FIG. 12 show the expression level of LTB 4 when treated with MDE or MEE in RAW264.7 cells. (pg/ml).
(㎍/㎖)(Μg/ml)
표 11 및 도 11에 나타난 바와 같이, MDE 1㎍/㎖을 처리한 그룹을 제외한 모든 그룹에서 대조군에 비해 유의미하게 농도 의존적으로 PGE2 발현이 감소하였다.As shown in Table 11 and FIG. 11, in all groups except the group treated with
(㎍/㎖) (Μg/ml)
표 12 및 도 12에 나타난 바와 같이, MDE 또는 MEE를 처리한 모든 그룹에서 대조군에 비해 유의미하게 농도 의존적으로 LTB4의 발현이 감소하였다. MDE 대비 MEE가 더 높은 정도로 PGE2 및 LTB4의 발현양을 감소시켰다.As shown in Table 12 and Figure 12, in all groups treated with MDE or MEE, the expression of LTB 4 was significantly decreased in a concentration-dependent manner compared to the control group. The expression levels of PGE 2 and LTB 4 were reduced to a higher MEE than MDE.
실시예 4-4. 만자카니 추출물 첨가에 따른 NF-κB, iNOS, 및 COX-2 유전자 발현양Example 4-4. NF-κB, iNOS, and COX-2 gene expression levels according to the addition of Manzacani extract
염증 반응에 관련된 효소 및 사이토카인의 유전자 발현은 주로 NF-κB가 조절한다. NF-κB는 세포질에서 κB(IκBs) 저해제와 결합하여 불활성화 상태로 존재하고, LPS나 염증성 물질에 의한 자극이 가해지면 NF-κB가 활성화된다. 유리 NF-κB가 핵 안으로 이동하여 κB-결합부위에 결합하게 되면 유전자 전사가 일어나서 염증에 관련하는 효소인 iNOS와 COX-2, 염증성 사이토킨인 TNF-α, IL-1β 등의 유전자의 발현을 조절한다. 만카자니 추출물의 항염증 활성을 살펴보기 위하여 실시간 PCR(real-time PCR)을 수행하여 만자카니 추출물 처리시 NF-κB, 염증 관련 효소 iNOS, COX-2의 발현양을 측정하였다.Gene expression of enzymes and cytokines involved in the inflammatory response is mainly regulated by NF-κB. NF-κB exists in an inactivated state in combination with a κB (IκBs) inhibitor in the cytoplasm, and NF-κB is activated when stimulation by LPS or inflammatory substances is applied. When free NF-κB moves into the nucleus and binds to the κB-binding site, gene transcription occurs to regulate the expression of genes such as iNOS and COX-2, which are inflammation-related enzymes, and TNF-α and IL-1β, inflammatory cytokines. do. In order to examine the anti-inflammatory activity of mankazani extract, real-time PCR (real-time PCR) was performed to measure the expression levels of NF-κB, inflammation-related enzymes iNOS, and COX-2 when manzakani extract treatment.
6 웰 플레이트에 RAW264.7 세포를 1×106 세포/웰로 분주하여 24시간 동안 배양하였다. 배양 후, 실시예 1에서 수득한 1, 5, 10 ㎍/㎖ 농도의 MDE 또는 MEE와 1㎍/㎖ LPS를 함께 처리하여 다시 24시간 동안 배양하였다. 이후, 1,200rpm에서 5분 동안 원심분리하여 얻은 RAW264.7 세포에 easy-Blue™(Intronbio, Korea) 1㎖와 클로로포름 200㎕을 첨가하여 진탕한 후 13,000rpm, 4℃에서 10분 동안 원심분리 하였다. 상층액 400㎕와 결합 버퍼(binding buffer) 400㎕를 실온에서 1분 동안 반응시킨 뒤 반응액 700㎕를 컬럼에 주입하여 13,000rpm에서 30초 동안 원심분리하였다. 컬럼에 세척 버퍼 A를 700㎕ 넣고 13,000rpm에서 30초 동안 원심분리 후, 세척 버퍼 B를 700㎕ 넣고 동일하게 원심분리하였다. 컬럼 하단을 EP 튜브로 교체한 후, 컬럼에 추출 퍼버(elution buffer)를 50㎕ 첨가하고, 1분 동안 반응시킨 뒤 13,000rpm에서 1분 동안 원심분리하여 총 RNA를 추출하였다.RAW264.7 cells were plated at 1×10 6 cells/well in a 6-well plate and cultured for 24 hours. After incubation, 1, 5, and 10 μg/ml concentrations of MDE or MEE obtained in Example 1 were treated together with 1 μg/ml LPS to incubate again for 24 hours. Thereafter, 1 ml of easy-Blue™ (Intronbio, Korea) and 200 µl of chloroform were added to RAW264.7 cells obtained by centrifugation at 1,200 rpm for 5 minutes, followed by shaking, followed by centrifugation at 13,000 rpm and 4°C for 10 minutes. . 400 μl of the supernatant and 400 μl of the binding buffer were reacted for 1 minute at room temperature, and then 700 μl of the reaction solution was injected into the column and centrifuged at 13,000 rpm for 30 seconds. 700 μl of Wash Buffer A was added to the column, and after centrifugation at 13,000 rpm for 30 seconds, 700 μl of Wash Buffer B was added and centrifuged in the same manner. After replacing the bottom of the column with an EP tube, 50 μl of an extraction buffer was added to the column, reacted for 1 minute, and then centrifuged at 13,000 rpm for 1 minute to extract total RNA.
역전사(reverse transcription) 반응은 RT premix kit의 mixture (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT15 primer)를 사용하여 총 RNA가 1㎍이 되도록 DEPC(diethyl pyrocarbonate)가 처리된 증류수(이하, DEPC-DW)에 최종 부피가 20㎕가 되도록 하여 첨가하였다. 이 20㎕의 반응 혼합액을 잘 섞은 뒤 45℃에서 60분 반응시켜 first-strand cDNA를 합성한 후, 95℃에서 5분 동안 방치하여 M-MLV RT를 불활성화 시킨 다음 합성이 완료된 cDNA를 PCR에 사용하였다.For reverse transcription reaction, diethyl pyrocarbonate (DEPC)-treated distilled water (hereinafter referred to as µg) was used to make total RNA of 1 μg using a mixture of RT premix kit (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT15 primer). DEPC-DW) to a final volume of 20 µl. After mixing the 20 µl reaction mixture well, reacting at 45°C for 60 minutes to synthesize first-strand cDNA, incubating at 95°C for 5 minutes to inactivate M-MLV RT, and then synthesizing the completed cDNA to PCR. Used.
실시간 PCR을 수행하여, 만자카니 추출물 처리시 RAW264.7 세포에서 NF-κB, iNOS, 및 COX-2의 상대적인 mRNA 발현양을 확인하였다. 실시간 PCR 전용 튜브에 cDNA 1㎕, 프라이머 2㎕, SYBR Green 10㎕, DEPC-DW 5㎕씩 첨가하여 실시간 PCR을 다음과 같이 진행하였다. 94℃에서 5분 동안 반응한 다음 94℃에서 15초, 60℃에서 30초, 72℃에서 30초를 40회 반복하였다. 각 유전자의 상대적인 발현양은 GAPDH에 의해 정규화시켰다. 각 유전자의 발현양을 확인하기 위하여 사용한 프라이머의 염기서열은 표 13에 기재하였다.Real-time PCR was performed to confirm the relative mRNA expression levels of NF-κB, iNOS, and COX-2 in RAW264.7 cells during Manzacani extract treatment. 1 μl of cDNA, 2 μl of primer, 10 μl of SYBR Green, and 5 μl of DEPC-DW were added to the tube for real-time PCR to perform real-time PCR as follows. After reacting at 94°C for 5 minutes, 40 seconds were repeated at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The relative expression level of each gene was normalized by GAPDH. The base sequences of the primers used to confirm the expression level of each gene are listed in Table 13.
표 14 및 도 13은 RAW264.7 세포에서 MDE 또는 MEE 처리시 NF-κB의 상대적인 발현양을 나타내고, 표 15 및 도 14는 RAW264.7 세포에서 MDE 또는 MEE 처리시 iNOS의 상대적인 발현양을 나타내며, 표 16 및 도 15는 COX-2의 상대적인 발현양을 나타낸다.Table 14 and Figure 13 shows the relative expression of NF-κB when treated with MDE or MEE in RAW264.7 cells, Table 15 and Figure 14 shows the relative expression of iNOS when treated with MDE or MEE in RAW264.7 cells, Table 16 and Figure 15 show the relative expression of COX-2.
(㎍/㎖)(Μg/ml)
표 14 및 도 13에 나타난 바와 같이, MDE 1㎍/㎖을 처리한 그룹을 제외한 MDE 또는 MEE를 처리한 모든 그룹에서 대조군에 비해 유의미하게 농도 의존적으로 NF-κB의 발현이 감소하였다.As shown in Table 14 and FIG. 13, in all groups treated with MDE or MEE except for the group treated with
(㎍/㎖)(Μg/ml)
표 15 및 도 14에 나타난 바와 같이, MDE 또는 MEE를 처리한 모든 그룹에서 대조군에 비해 유의미하게 농도 의존적으로 iNOS의 발현이 감소하였다. MDE 대비 MEE가 더 높은 정도로 NF-κB 및 iNOS의 발현양을 감소시켰다.As shown in Table 15 and FIG. 14, in all groups treated with MDE or MEE, the expression of iNOS was significantly decreased in a concentration-dependent manner compared to the control group. The expression level of NF-κB and iNOS was reduced to a higher degree of MEE than MDE.
(㎍/㎖)(Μg/ml)
표 16 및 도 15에 나타난 바와 같이, MDE 1㎍/㎖ 또는 MEE 1㎍/㎖을 처리한 그룹을 제외하고 MDE 또는 MEE를 처리한 모든 그룹에서 대조군에 비해 유의미하게 농도 의존적으로 COX-2의 발현이 감소하였다. As shown in Table 16 and FIG. 15, the expression of COX-2 was significantly and concentration-dependently compared to the control group in all groups treated with MDE or MEE except for the group treated with
실시예 5. 만자카니 추출물의 항소양 효능Example 5. Anti-tumor efficacy of Manzacani extract
실시예 5-1. 히스타민 생성량 측정Example 5-1. Histamine production
히스타민(histamine)은 세포증식, 분화와 혈구 생성, 염증반응, 조직 재생과 신경전달 등에 관여하는 단백질로서 혈관확장과 혈관의 투과성 증가, 피부신경 자극에 의한 소양증(搔痒症, 피부 가려움증) 등은 H1-히스타민 수용체를 통하여 작용을 나타내는 대표적인 증상이다. 만자카니 추출물의 항소양 활성을 살펴보기 위하여 히스타민 ELISA 키트(MyBioSource, U.S.A.)를 사용하여 제조사의 매뉴얼에 따라 히스타민 생성량을 측정하였다.Histamine is a protein involved in cell proliferation, differentiation and blood cell production, inflammatory reactions, tissue regeneration and neurotransmission, and increases vasodilation and permeability of blood vessels, pruritus caused by skin nerve stimulation (搔痒症, skin itching), etc. -It is a typical symptom that shows action through the histamine receptor. To examine the anti-pruritic activity of the Manzacani extract, histamine production was measured according to the manufacturer's manual using a histamine ELISA kit (MyBioSource, U.S.A.).
12 웰 플레이트에 MC/9 세포를 2×105 세포/웰로 분주하여 24시간 동안 배양하였다. 배양 후, 실시예1에서 수득한 1, 5, 10㎍/㎖ 농도의 MDE 또는 MEE와 50ng/㎖ PMA, 0.5uM 이오노마이신(ionomycin)을 함께 처리하여 다시 48시간 동안 배양하였다. 배양액을 1,200rpm에서 5분간 원심분리하여 얻은 상등액과 표준물질을 96 웰 플레이트에 100㎕씩 넣고 37℃에서 120분간 반응시켰다. 반응 후, 세척 버퍼를 이용하여 3회 세척을 진행하고 Biotin-conjugate를 100㎕씩 넣어 다시 37℃에서 60분간 반응시켰다. 다시 세척을 진행하고 스트렙타비딘-HRP(Streptavidin-HRP)를 100㎕씩 첨가하여 37℃에서 30분간 반응시켰다. 그 후 세척을 진행하고 기질 용액(substrate solution)을 100㎕씩 첨가하여 37℃에서 15분간 반응시켰다. 반응 후, 50㎕의 정지 용액을 추가하여 ELISA 리더기를 통해 450㎚에서 흡광도를 측정하였으며, 표준 곡선(standard curve)을 기준으로 히스타민 생성량을 계산하여 절대 값(uM)으로 나타내었다. MC/9 cells were divided into 2×10 5 cells/well in a 12-well plate and cultured for 24 hours. After incubation, MDE or MEE at concentrations of 1, 5, and 10 μg/ml obtained in Example 1 were treated with 50 ng/ml PMA, 0.5 uM ionomycin, and cultured for 48 hours again. The culture solution was centrifuged at 1,200 rpm for 5 minutes, and 100 µl of the supernatant and the standard material were added to a 96-well plate and reacted at 37°C for 120 minutes. After the reaction, washing was performed 3 times using a washing buffer, and 100 µl of Biotin-conjugate was added thereto, followed by reacting at 37° C. for 60 minutes. After washing again, streptavidin-HRP (Streptavidin-HRP) was added thereto, and the reaction was performed at 37°C for 30 minutes. Thereafter, washing was performed, and 100 µl of a substrate solution was added thereto to react at 37° C. for 15 minutes. After the reaction, 50 μl of the stop solution was added to measure absorbance at 450 nm through an ELISA reader, and histamine production was calculated based on a standard curve and expressed as an absolute value (uM).
표 17 및 도 16은 MC/9 세포에서 MDE 또는 MEE 처리시 히스타민의 생성량을 나타낸다. 정상군(Normal)은 PMA, 이오노마이신 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 PMA, 이오노마이신을 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다.Table 17 and FIG. 16 show the amount of histamine produced during MDE or MEE treatment in MC/9 cells. The normal group (Normal) shows the results in cells not treated with PMA, ionomycin and manzacani extract, and the control (Control) in cells treated with PMA, ionomycin and manzacani extract. Results are shown.
(㎍/㎖)(Μg/ml)
표 17 및 도 16에 나타난 바와 같이, LPS의 처리에 의하여 대조군에서 히스타민 생성량이 현저하게 증가하였고, MDE 또는 MEE 처리시 농도 의존적으로 히스타민 생성량이 감소하였다.As shown in Table 17 and FIG. 16, histamine production amount was significantly increased in the control group by treatment of LPS, and histamine production amount was decreased depending on the concentration during MDE or MEE treatment.
실시예 5-2. 아세틸콜린, 아세틸콜린에스터라제 및 물질 P의 생성량 측정Example 5-2. Measurement of the amount of acetylcholine, acetylcholinesterase and substance P
아세틸콜린은 히스타민 민감성(histamine-sensitive) 및 히스타민 비민감성(histamine-insensitive)과 상관없이 C-섬유에서 가벼운 가려움증을 유발하는 신경전달물질로 알려져 있다. 또한 가려움증은 C-섬유말단의 유해 감수기를 통해 무수구심신경으로 전달되며, C-섬유 말단에서 분비되는 푸루리토젠(pruritogen)인 물질 P(substance P), CGRP(calcitonin-gene related peptide) 등은 각각의 수용체와 결합하여 유발되는 것으로 알려져 있고, 이들 푸루리토젠들은 비만세포(mast cell)를 활성화시켜 가려움증의 또 다른 중재자인 히스타민의 방출을 유도하는 것으로 보고되었다.Acetylcholine is known as a neurotransmitter that causes mild itching in C-fibers regardless of histamine-sensitive and histamine-insensitive. In addition, itching is transmitted to the anhydrous centripetal nerve through a harmful receptor at the C-fiber end, and substances P (substance P) and CGRP (calcitonin-gene related peptide) that are secreted from the C-fiber end are It is known to be associated with each receptor, and these pururitogens have been reported to induce the release of histamine, another mediator of itching by activating mast cells.
만자카니 추출물의 항소양 활성을 살펴보기 위하여 아세틸콜린 ELISA 키트(MyBioSource, U.S.A.), 아세틸콜린에스터라제 ELISA 키트(MyBioSource, U.S.A.), 물질 P ELISA 키트(MyBioSource, U.S.A.)를 사용하여 제조사의 매뉴얼에 따라 만자카니 추출물 처리시 아세틸콜린(Acetylcholine), 아세틸콜린에스터라제(acetylcholinesterase) 및 물질 P(substance P)의 생성량을 측정하였다.In order to examine the anti-pruritic activity of Manzacani extract, the acetylcholine ELISA kit (MyBioSource, USA), acetylcholinesterase ELISA kit (MyBioSource, USA), and the substance P ELISA kit (MyBioSource, USA) were used in the manufacturer's manual. Accordingly, the amount of acetylcholine, acetylcholinesterase, and substance P (substance P) was measured when manzacani extract treatment.
콜라겐으로 코팅된 12 웰 플레이트에 PC12 세포를 2×105 세포/웰로 분주하여 24시간 동안 배양하였다. 배양 후, 실시예 1에서 수득한 1, 5, 10 ㎍/㎖ 농도의 MDE 또는 MEE와 50ng/㎖의 NGF를 함께 처리하여 다시 48시간 동안 배양하였다. 이후, 배양액을 1,200rpm에서 5분간 원심분리하여 얻은 상등액과 표준물질을 96 웰 플레이트에 100㎕씩 넣고 conjugate를 50㎕씩 추가하여 37℃에서 60분간 반응시켰다. 반응 후, 세척 버퍼를 이용하여 5회 세척 작업을 진행하고 기질(substrate) A와 B를 각각 50㎕씩 넣어 37℃에서 15분간 반응시켰다. 반응시킨 후 50㎕의 정지 용액을 추가하여 ELISA 리더기를 통해 450㎚에서 흡광도를 측정하였으며, 표준 곡선을 기준으로 절대 값으로 나타내었다.PC12 cells were divided into 2 x 10 5 cells/well in a 12-well plate coated with collagen and cultured for 24 hours. After cultivation, MDE or MEE at a concentration of 1, 5, 10 µg/ml obtained in Example 1 and 50 ng/ml NGF were treated together, and cultured again for 48 hours. Subsequently, the culture solution was centrifuged at 1,200 rpm for 5 minutes, and 100 µl of the supernatant and the standard were added to a 96-well plate, and 50 µl of the conjugate was added thereto, followed by reaction at 37°C for 60 minutes. After the reaction, washing was performed 5 times using a washing buffer, and 50 µl of substrates A and B were added, respectively, and reacted at 37° C. for 15 minutes. After the reaction, 50 μl of the stop solution was added to measure absorbance at 450 nm through an ELISA reader, and the absolute values were expressed based on a standard curve.
표 18 및 도 17은 MDE 또는 MEE 처리시 아세틸콜린의 생성량을 나타내고, 표 19 및 도 18은 다양한 농도의 MDE 또는 MEE 처리시 아세틸콜린에스터라제의 생성량을 나타내며, 표 20 및 도 19는 다양한 농도의 MDE 또는 MEE 처리시 물질 P의 생성량을 나타낸다. 표 18 내지 20 및 도 17 내지 19에서, 정상군(Normal)은 NGF 및 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타내고, 대조군(Control)은 NGF를 처리하고, 만자카니 추출물을 처리하지 않은 세포에서의 결과를 나타낸다. Table 18 and FIG. 17 show the production amount of acetylcholine when treated with MDE or MEE, Table 19 and FIG. 18 show the production amount of acetylcholinesterase when treated with MDE or MEE at various concentrations, and Table 20 and FIG. 19 show various concentrations It represents the amount of material P produced during the MDE or MEE treatment. In Tables 18 to 20 and FIGS. 17 to 19, the normal group shows results in cells not treated with NGF and manzacani extract, and the control group is treated with NGF and manzacani extract is not treated Results are shown in cells.
(㎍/㎖) (Μg/ml)
표 18 및 도 17에 나타난 바와 같이 MDE 또는 MEE 처리시 농도의존적으로 아세틸콜린 생성량이 감소되었고, 10㎍/㎖의 MDE 또는 MEE 처리시 대조군에 비해 유의미하게 아세틸콜린 생성량이 감소되었다. MDE 대비 MEE가 더 높은 정도로 아세틸콜린의 생성량을 감소시켰다.As shown in Table 18 and FIG. 17, the concentration-dependent production of acetylcholine was decreased when treated with MDE or MEE, and the production of acetylcholine was significantly decreased compared to the control group when treated with MDE or MEE at 10 μg/ml. The production amount of acetylcholine was reduced to a higher degree than MDE.
(㎍/㎖) (Μg/ml)
표 19 및 도 18에 나타난 바와 같이, MDE 또는 MEE 처리시 미미하지만 추출물의 농도 증가에 따라 아세틸콜린에스터라제 생성량이 감소하는 것으로 나타났다.As shown in Table 19 and Figure 18, MDE or MEE treatment was insignificant, but the amount of acetylcholinesterase production decreased as the concentration of the extract increased.
(㎍/㎖)(Μg/ml)
표 20 및 도 19에 나타난 바와 같이 MDE 또는 MEE 처리시 농도 의존적으로 물질 P의 생성량이 감소되었고, 10㎍/㎖의 MDE 또는 10㎍/㎖, 5㎍/㎖의 MEE 처리시 대조군에 비해 유의하게 물질 P의 생성량이 감소되었다. MDE 대비 MEE가 더 높은 정도로 물질 P의 생성량을 감소시켰다.As shown in Table 20 and FIG. 19, the production amount of the substance P was decreased depending on the concentration of MDE or MEE, and compared to the control group when treating 10 μg/ml MDE or 10 μg/ml, 5 μg/ml MEE. The production amount of substance P was reduced. The amount of production of substance P was reduced to a higher degree of MEE than MDE.
실시예 6. 만자카니 추출물의 항균 활성Example 6. Antibacterial activity of Manzacani extract
실시예 6-1. 균주 및 배양 조건Example 6-1. Strains and culture conditions
만자카니 추출물의 항균 활성을 살펴보기 위해, 시험 균주로서 스타필로코쿠스 아우레우스(Staphylococcus aureus; 이하, S. aureus), 슈도모나스 에루지노사(Pseudomonas aeruginosa; 이하, P. aeruginosa), 대장균(Escherichia coli; 이하, E. coli), 칸디다 알비칸스(Candida albicans; 이하, C. albicans), 및 아스퍼질러스 나이거(Aspergillus niger; 이하, A. niger)를 미국 세포주 은행(ATCC)으로부터 분양받아 실험에 사용하였다. 사용한 균주의 종류 및 균주의 번호를 하기 표 21에 기재하였다. To investigate the antimicrobial activity of the swastika crab extract, as the test organism Staphylococcus aureus (Staphylococcus aureus; or less, S. aureus), Pseudomonas Rouge labor (Pseudomonas aeruginosa; hereinafter, P. aeruginosa), E. coli (Escherichia coli; below, E. coli), Candida albicans (Candida albicans; hereinafter, C. albicans), and Aspergillus and this (Aspergillus niger; or less, A. niger) for experiment received pre-sale from the American cell Line bank (ATCC) Used for. The type of strain used and the number of the strain are shown in Table 21 below.
(Bacteria)Germ
(Bacteria)
(Yeast)leaven
(Yeast)
스톡으로 동결 보관된 균주를 사면 한천배지(Agar slant)에 하기 표 22에 기재된 조건하에서 계대 배양한 후 냉장고 5±3℃에 보관하였다. 단 최초 균주에서 5세대가 지난 균주는 사용하지 않도록 하였다.The strains stored frozen as stocks were passaged in agar medium (Agar slant) under the conditions described in Table 22 and stored in a refrigerator at 5±3°C. However, strains older than 5 generations from the first strain were not used.
세균 및 효모의 균액을 제조하기 위하여, 약 5g의 유리 구슬이 포함된 멸균 시험관에 10㎖의 멸균생리식염수 희석액을 분주하였다. 상기 표 22에 기재된 배양 조건에서 배양된 각 한천 배지에서 세균 또는 효모의 콜로니를 4 내지 5개 취하여 10㎖의 희석액에 넣고 전체적으로 균질화되도록 최대 3분간 진탕(Vortexing)하였다. 이 때 맥팔란드 탁도 0.5로 맞추어 세균(S. aureus, P. aeruginosa, 또는 E. coli)은 1x107 내 지1x108cfu/㎖가 되도록 하고, 효모(C. albicans)는 1x106 내지 1x107cfu/㎖가 되도록 하였다. In order to prepare a bacterial and yeast bacterial solution, 10 ml of sterile physiological saline dilution was dispensed into a sterile test tube containing about 5 g of glass beads. 4 to 5 colonies of bacteria or yeast were taken from each agar medium cultured in the culture conditions described in Table 22 above, placed in a dilution of 10 ml, and shaken for up to 3 minutes so as to be homogenized as a whole. At this time, the McFarland turbidity is adjusted to 0.5 so that bacteria ( S. aureus , P. aeruginosa , or E. coli ) are 1x10 7 to 1x10 8 cfu/ml, and yeast ( C. albicans ) is 1x10 6 to 1x10 7 cfu /Ml.
A. niger 균액을 만들기 위해 상기 표 22에 기재된 조건으로 배양된 스톡(Stock culture)에10㎖의 0.01%의 폴리소르베이트 용액(polysorbate solution)을 첨가한 후 스폐츌러나 유리 막대로 배지 표면을 1분 동안 긁어준 후, 멸균된 유리필터나 거즈에서 걸러주었다. 현미경 검사(x400)를 통해 미성숙 포자나 균사체가 있는지 확인하였다. 미성숙 포자 발생시에는 희석액을 폐기하였다. 균사체가 1/10이상 발견시 2,000g, 2분 이상 원심 분리한 후 두 번 이상 폴리소르베이트 용액으로 재 희석과 원심분리를 반복하였다. 최종 농도가 1x106 내지 1x107cfu/㎖가 되도록 희석액을 첨가하여 조절하도록 하였다. A. After adding 10 ml of 0.01% polysorbate solution to the stock culture cultured under the conditions described in Table 22 above to make a niger bacterial solution, the surface of the medium was removed with a spatula or glass rod. After scraping for a minute, it was filtered through a sterile glass filter or gauze. Microscopic examination (x400) confirmed the presence of immature spores or mycelium. In the case of immature spores, the dilution was discarded. When the mycelium was found to be 1/10 or more, centrifugation was performed for 2,000 g or more for 2 minutes or more, and re-dilution and centrifugation were repeated twice or more with a polysorbate solution. The final concentration was adjusted to 1x10 6 to 1x10 7 cfu/ml by adding a diluent.
세균의 경우, 정치 배양을 위해 TSA(Tryptic Soy Agar; Difco, USA)를 사용하였으며, 액상 배지는 TSB(Tryptic Soy Broth; Difco, USA)를 사용하였다. 진균(효모 및 곰팡이)의 경우, 정치 배양을 위해 PDA(Potato Dextrose Agar; Difco, USA)를 사용하였으며, 액상 배지는 PDB(Potato Dextrose Broth)를 사용하였다(표 23).For bacteria, TSA (Tryptic Soy Agar; Difco, USA) was used for static culture, and TSB (Tryptic Soy Broth; Difco, USA) was used for the liquid medium. For fungi (yeast and fungi), PDA (Potato Dextrose Agar; Difco, USA) was used for stationary culture, and PDB (Potato Dextrose Broth) was used for the liquid medium (Table 23).
최초의 접종 균 농도를 측정하기 위하여 각 희석액에서 순차적으로 10배 희석한 후 혼합 평판법(pour plate)을 이용하여 하기 표 24의 조건으로 배양하였다.In order to measure the concentration of the first inoculated bacteria, each diluted solution was sequentially diluted 10-fold, and then cultured under the conditions shown in Table 24 using a pour plate.
실시예 6-2. 디스크 확산법(Disc diffusion method)Example 6-2. Disc diffusion method
만자카니 추출물의 항균 활성을 살펴보기 위하여 시험균주를 배양 접시에 배양한 뒤 일정 면적에 일정량의 항균 작용을 하는 물질을 떨어뜨려 배양 후 플라크(plaque)의 지름을 측정함으로써 활성 정도를 확인하는 디스크 확산법을 수행하였다.In order to examine the antimicrobial activity of the Manzacani extract, after culturing the test strain on a culture dish, drop a substance having a certain amount of antimicrobial activity on a certain area and measure the diameter of the plaque after cultivation. Was performed.
뮐러 힌튼 아가 배지(Mueller Hinton Agar; Difco USA) 및 포테이토 덱스트로오즈 아가 배지(Potato Dextrose Agar; Difco USA)를 사용하여 평판 배지를 만들었다. 상기 실시예 6-1에서 제조한 균액 0.5㎖을 45℃로 식힌 각각의 배지 5㎖에 섞은 후 상기의 평판배지에 추가로 부어서 균주별 배지를 준비하였다. 각 균주에 사용된 배지의 종류는 표 25에 기재하였다.Flat plates were made using Mueller Hinton Agar (Difco USA) and Potato Dextrose Agar (Difco USA). After mixing 0.5 ml of the fungus prepared in Example 6-1 with 5 ml of each medium cooled to 45° C., the medium was prepared by strain by further pouring into the plate medium. Table 25 shows the types of media used in each strain.
균주별 배지 위에 페이퍼 디스크(Paper disc)(ø; 8mm, ADVANTEC, Japan)를 올려놓고 실시예 1에서 수득한 만자카니 에탄올 추출물(MEE) (10, 5, 2. 5, 1.25 %w/v) 만자카니 증류수 추출물(MDE) (10, 5, 2.5, 1.25 %w/v), 또는 메트로니다졸(Metronidazole)(10, 5, 2.5, 1.25 %w/v)을 각각 3㎕씩 페이퍼 디스크에 흡수시켰다. 치주염, 질염 등에 항생제로 사용되는 메트로니다졸을 양성대조군으로서 사용하였다. Manzacani ethanol extract (MEE) obtained in Example 1 with a paper disc (ø; 8 mm, ADVANTEC, Japan) on a strain-specific medium (10, 5, 2. 5, 1.25% w/v) Manzacani distilled water extract (MDE) (10, 5, 2.5, 1.25% w/v), or metronidazole (10, 5, 2.5, 1.25% w/v) were each absorbed in 3 μl of paper disc. Metronidazole used as an antibiotic for periodontitis and vaginitis was used as a positive control.
표 25에 기재된 바와 같이, 각 시료가 처리된 플레이트를 배양기에서 세균은 37℃에서 24시간 동안 배양하고, 진균은 25℃에서 72시간 동안 배양 후 형성된 클리어존을 관찰하여 도 20a내지 도 20e에 나타내었다. 클리어존(clear zone(mm))은 하기의 수학식 2를 이용하여 계산하고, 계산된 클리어존의 크기를 기준으로 만자카니 추출물의 각 균주에 대한 항균 활성을 표 26 에 나타내었다.As shown in Table 25, in the incubator, the plate treated with each sample is cultured at 37°C for 24 hours, and the fungus is shown in FIGS. 20A to 20E by observing the clear zone formed after incubation at 25°C for 72 hours. Did. Clear zone (clear zone (mm)) was calculated by using the following equation (2), and based on the calculated size of the clear zone, Table 26 shows the antibacterial activity for each strain of the Manzacani extract.
표 26및 도 20a에 나타난 바와 같이, E. coli(ATCC 8739)에 대한 디스크 확산 시험 결과 다양한 농도(10, 5, 2.5, 1.25%w/v)의 MDE 또는 MEE 처리시 모든 농도의 추출물 처리군에서 E. coli에 대해 항균력을 나타내는 것을 알 수 있었다. 메트로니다졸의 경우, 10%(w/v)의 농도에서 E. coli에 대해 항균 활성을 나타내었으나 나머지 농도(5, 2.5, 1.25%w/v)에서는 항균 활성이 나타나지 않았다. MDE 및 MEE 모두 단일 화합물인 메트로니다졸 보다 현저히 우수한 항균 활성을 갖는다. As shown in Table 26 and Figure 20a, the results of the disk diffusion test for E. coli (ATCC 8739) various concentrations (10, 5, 2.5, 1.25% w / v) of MDE or MEE treatment at all concentrations of the extract treatment group It was found that it exhibited antibacterial activity against E. coli . Metronidazole showed antibacterial activity against E. coli at a concentration of 10% (w/v), but did not show antibacterial activity at the remaining concentrations (5, 2.5, 1.25% w/v). Both MDE and MEE have significantly better antibacterial activity than the single compound metronidazole.
표 26및 도 20b에 나타난 바와 같이, P. aeruginosa(ATCC 9027)에 대한 디스크 확산 시험 결과 다양한 농도(10, 5, 2.5, 1.25%w/v)의 MDE 또는 MEE 처리시 모든 농도의 추출물 처리군에서 P. aeruginosa에 대해 항균 활성을 나타내었고, MEE 처리군이 MDE 처리군 보다 더 우수한 항균 활성을 나타내었다. 메트로니다졸의 경우 모든 농도의 처리군에서 클리어 존의 크기가 증가하지 않았으므로, 메트로니다졸의 경우 P. aeruginosa 에 대해 항균 활성을 보이지 않는 것으로 나타났다. As shown in Table 26 and FIG. 20B, the results of disk diffusion test for P. aeruginosa (ATCC 9027) treated with extracts of all concentrations when MDE or MEE treatment of various concentrations (10, 5, 2.5, 1.25% w/v) In P. aeruginosa showed antibacterial activity, the MEE treatment group showed better antibacterial activity than the MDE treatment group. In the case of metronidazole, the size of the clear zone did not increase in the treatment group at all concentrations, so it was shown that the metronidazole did not show antibacterial activity against P. aeruginosa .
표 26및 도 20c에 나타난 바와 같이, S. aureus(ATCC 6538)에 대한 디스크 확산 시험 결과 다양한 농도(10, 5, 2.5, 1.25%w/v)의 MDE 또는 MEE 처리시 모든 농도의 추출물 처리군에서 P. aeruginosa에 대해 항균 활성을 나타내었고, MEE 처리군이 MDE 처리군 보다 더 우수한 항균 활성을 나타내었다. 메트로니다졸의 경우, 10%(w/v)의 농도에서 S. aureus 에 대해 항균 활성을 나타내었으나 나머지 농도(5, 2.5, 1.25%w/v)에서는 항균 활성이 나타나지 않았다.As shown in Table 26 and Fig. 20c, disk diffusion test results for S. aureus (ATCC 6538) resulted in extracts of all concentrations when treated with MDE or MEE at various concentrations (10, 5, 2.5, 1.25% w/v) In P. aeruginosa showed antibacterial activity, the MEE treatment group showed better antibacterial activity than the MDE treatment group. Metronidazole showed antibacterial activity against S. aureus at a concentration of 10% (w/v), but did not show antibacterial activity at the remaining concentrations (5, 2.5, 1.25% w/v).
표 26및 도 20d에 나타난 바와 같이, C. albicans(ATCC 10231)에 대한 디스크 확산 시험 결과 10%(w/v)의 MDE 또는 10%의 MEE 처리시에 C. albicans 에 대한 항균 활성이 나타났다. 메트로니다졸의 경우 모든 농도의 처리군에서 클리어 존의 크기가 증가하지 않았으므로, 메트로니다졸의 경우 C. albicans에 대해 항균 활성을 보이지 않는 것으로 나타났다. As shown in Table 26 and FIG. 20D, the disk diffusion test results for C. albicans (ATCC 10231) showed antibacterial activity against C. albicans upon treatment with 10% (w/v) MDE or 10% MEE. In the case of metronidazole, the size of the clear zone did not increase in the treatment group at all concentrations, so it was found that metronidazole did not show antibacterial activity against C. albicans .
표 26및 도 20e에 나타난 바와 같이, A. niger(ATCC 16404)에 대한 디스크 확산 시험 결과 MDE 또는 MEE 처리시에 A. niger에 대해 항균 활성을 나타내지 않았다. As shown in Table 26 and FIG. 20E, disc diffusion test results for A. niger (ATCC 16404) did not show antibacterial activity against A. niger upon MDE or MEE treatment.
(%w/v)
density
(%w/v)
ATCC 8739 E. coli
ATCC 9027 P. aeruginosa
ATCC6538 S. aureus
ATCC6538
ATCC 10231 C. albicans
ATCC 16404 A. niger
※ 클리어존 크기: +<10mm, 10mm<++<15mm, 15mm<+++<20mm, 20mm<++++<25mm, 25mm<+++++<30mm, 30mm<++++++<35mm, 35mm<+++++++<40mm , 40mm<++++++++<45mm, 45mm<+++++++++<50mm , 50mm<++++++++++<60mm, 60mm<+++++++++++※ Clear Zone Size: +<10mm, 10mm<++<15mm, 15mm<+++<20mm, 20mm<++++<25mm, 25mm<+++++<30mm, 30mm<+++++ +<35mm, 35mm<+++++++<40mm, 40mm<++++++++<45mm, 45mm<+++++++++<50mm, 50mm<+++++ +++++<60mm, 60mm<+++++++++++
- : Not Detected-: Not Detected
메트로니다졸의 경우 10%(w/v) 농도 처리시에 E. coli및 S. aureus에 대해서만 항균 활성을 보였으나 만자카니 추출물(MDE 또는 MEE)의 경우, 세균인 E. coli, S. aureus, S. aureus, 및 진균인C. albicans에 대해서 메트로니다졸 보다 우수한 항균 활성을 갖는 것을 알 수 있었다.Metronidazole showed antibacterial activity only against E. coli and S. aureus when treated with 10% (w/v) concentration, but in the case of Manzacani extract (MDE or MEE), bacteria E. coli , S. aureus, S Aureus, and the fungus C. albicans were found to have better antimicrobial activity than metronidazole.
실시예 6-3. 최소 억제 농도(Minimum inhibition concentration(MIC)) 평가Example 6-3. Evaluation of minimum inhibition concentration (MIC)
만자카니 추출물의 항균 활성을 살펴보기 위하여, 서로 다른 농도의 항균물질을 첨가한 후 세균은 TSB 배지 그리고 효모 및 진균은 PDB 배지에서 시험세균을 배양하여 농도별 증식여부를 육안으로 관찰하여 처음으로 증식하지 않은 시점의 농도인 최소억제농도(MIC)를 측정하였다.In order to examine the antibacterial activity of the Manzacani extract, after adding different concentrations of antibacterial substances, the bacteria are grown in TSB medium and yeast and fungi in the PDB medium to test the bacteria for growth. The minimum inhibitory concentration (MIC), which was the concentration at the time of not doing, was measured.
실시예 1에서 수득한 만자카니 증류수 추출물(MDE)을 해당배지로 각 농도별(5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1 %(w/v)) 희석한 후 상기 실시예 6-1에서 준비한 균액을 최종 농도의 1%가 되도록 접종하였다. 각 농도별로 접종된 세균은 TSB에 접종후 37℃/24시간, 진균은 PDB에 접종 후 25℃/3일 배양하였으며 증식여부를 육안으로 관찰하여 농도별 MIC 값을 측정하였다 필요시 세균은 TSA에 도말후 37℃/ 24시간, 진균은 PDA에 도말 후 25℃/3일 배양하여 균 발생 유무를 플레이트 안에서 재확인하였다.After diluting the Manzacani distilled water extract (MDE) obtained in Example 1 by the respective medium (5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1% (w/v)) The bacterial solution prepared in Example 6-1 was inoculated to be 1% of the final concentration. Bacteria inoculated for each concentration were cultured at 37°C/24 hours after inoculation into TSB, and fungi were cultured at 25°C/3 days after inoculation in PDB, and the proliferation was visually observed to measure the MIC value for each concentration. After smearing at 37° C./24 hours, the fungus was cultured on a PDA at 25° C./3 days for re-confirmation of the occurrence of bacteria in the plate.
만자카니 증류수 추출물 첨가시 S. aureus 및 P. aeruginosa에 대한 MIC는 0.1%(w/v), E. coli 에 대해서는 0.3%(w/v)으로 측정 되었다. 그러나 C. albicans 및 A. niger 에 대해서는 5%(w/v) 이상 MIC 값을 나타내는 것을 알 수 있었다. When adding Manzacani distilled water extract, the MIC for S. aureus and P. aeruginosa was 0.1% (w/v) and 0.3% (w/v) for E. coli . However, it was found that C. albicans and A. niger showed a MIC value of 5% or more (w/v).
(Bacteria)Germ
(Bacteria)
<110> DAEJEON UNIVERSITY Industry-University Cooperation Foundation <120> Antiinflammatory and antibacterial composition comprising manjakani extract <130> PN190010 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NF-k(kappa)B_Forward <400> 1 ggatcacatt tgctttgtgt tgtt 24 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NF-k(kappa)B_Reverse <400> 2 cacaacttac agtagatggc tagaaagg 28 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> iNOS_Forward <400> 3 cgaaacgctt cacttccaa 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS_Reverse <400> 4 tgagcctata ttgctgtggc t 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX-2_Forward <400> 5 aaccgcattg cctctgaat 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_Reverse <400> 6 catgttccag gaggatggag 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Forward <400> 7 catcactgcc acccagaaga ctg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Reverse <400> 8 atgccagtga gcttcccgtt cag 23 <110> DAEJEON UNIVERSITY Industry-University Cooperation Foundation <120> Antiinflammatory and antibacterial composition comprising manjakani extract <130> PN190010 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NF-k(kappa)B_Forward <400> 1 ggatcacatt tgctttgtgt tgtt 24 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NF-k(kappa)B_Reverse <400> 2 cacaacttac agtagatggc tagaaagg 28 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> iNOS_Forward <400> 3 cgaaacgctt cacttccaa 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> iNOS_Reverse <400> 4 tgagcctata ttgctgtggc t 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX-2_Forward <400> 5 aaccgcattg cctctgaat 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_Reverse <400> 6 catgttccag gaggatggag 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Forward <400> 7 catcactgcc acccagaaga ctg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Reverse <400> 8 atgccagtga gcttcccgtt cag 23
Claims (9)
An anti-inflammatory and antimicrobial composition comprising manzacani extract as an active ingredient.
The method according to claim 1, The manzakani extract is water, alcohol having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butyl acetate, chloroform, diethyl ether, dichloromethane, hexane, and their Anti-inflammatory and antibacterial composition extracted with a solvent selected from the group consisting of mixtures.
The method according to claim 1, wherein the composition is selected from the group consisting of Escherichia sp., Pseudomonas sp., Staphylococcus sp., and Candida sp. Anti-inflammatory and antimicrobial composition that inhibits the growth of one or more microorganisms.
The method according to claim 3, wherein the microorganism is Escherichia coli , Staphylococcus aureus ( Staphylococcus aureus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), and one selected from the group consisting of Candida albicans ( Candida albicans ) The anti-inflammatory and antibacterial composition.
The composition according to any one of claims 1 to 4, wherein the composition is used as a preservative, preservative, detergent, or feminine cleanser.
The anti-inflammatory and antibacterial cosmetic composition comprising the composition of any one of claims 1 to 4.
The method according to claim 6, The cosmetic composition is an anti-inflammatory and antibacterial cosmetic composition that alleviates or improves itching of the skin.
A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases, comprising the composition of claim 1.
The method according to claim 8, The inflammatory diseases are dermatitis, allergies, vaginitis, atopy, asthma, conjunctivitis, cataract, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, septicemia, gastric ulcer, gastritis, Crohn's disease, hemorrhoids, ankylosing spondylitis, lupus , Fibromyalgia, psoriasis, arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hayitis, myositis, hepatitis, cystitis, nephritis, multiple sclerosis, diabetes, skin sclerosis, gout, degenerative neuropathy, silicosis, atherosclerosis and ischemia Pharmaceutical composition that is at least one selected from the group.
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