KR100771192B1 - Antifungal agent against dermatophytes containing tea tree oil - Google Patents

Antifungal agent against dermatophytes containing tea tree oil Download PDF

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KR100771192B1
KR100771192B1 KR1020050069818A KR20050069818A KR100771192B1 KR 100771192 B1 KR100771192 B1 KR 100771192B1 KR 1020050069818 A KR1020050069818 A KR 1020050069818A KR 20050069818 A KR20050069818 A KR 20050069818A KR 100771192 B1 KR100771192 B1 KR 100771192B1
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최인규
최원실
정의배
장제원
박미진
곽기섭
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재단법인서울대학교산학협력재단
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Abstract

본 발명은 티트리 오일을 함유하는 피부사상균용 항진균제 및 티트리 오일의 항진균제로의 이용방법에 관한 것으로서, 상세하게는, 티트리 오일(tea tree)의 일종인 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 식물성 의약품으로서의 이용 가능성을 탐색하고자 피부사상균인 마이크로스포럼 카니스(Microsporum canis)(KCTC6591), 트리코파이톤 멘타그로파이테스(Trichophyton mentagrophytes)(KCTC6077), 트리코파이톤 루브럼(Trichophyton rubrum) (KCCM60443), 에피더모파이톤 플로코섬(Epidermophyton floccosum)(KCCM11667), 마이크로스포럼 집세움(Microsporum gypseum)에 대한 항진균활성을 평가하고, 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성을 나타내는 화합물을 GC/MS로 확인하여 이러한 항진균 활성성분이나 티트리 오일을 함유하는 피부사상균용 항진균제 및 티트리 오일의 항진균제로의 이용방법에 관한 것이다. The present invention relates to an antifungal agent for dermatophyte containing tea tree oil and a method of using tea tree oil as an antifungal agent. Specifically, Leptospermum petersonii which is a kind of tea tree oil (tea tree) To explore the applicability of oil as a plant medicine, the skin fungus Microsporum canis (KCTC6591), Trichophyton mentagrophytes (KCTC6077), Trichophyton rubrum ( KCCM60443), Epidermophyton floccosum (KCCM11667), Microsporum gypseum to evaluate the antifungal activity of the compounds showing the antifungal activity of Leptospermum petersonii oil Anti-fungal agent for dermatophyte containing these antifungal active ingredients or tea tree oil A method of using tea tree oil as an antifungal agent.

Tea tree oil, L. petersonii oil, 항진균활성, T. mentagrophytes, M. canis, E. floccosum, T. rubrum, M. gypseum Tea tree oil, L. petersonii oil, antifungal activity, T. mentagrophytes, M. canis, E. floccosum, T. rubrum, M. gypseum

Description

티트리 오일을 함유하는 피부사상균용 항진균제{Antifungal agent against dermatophytes containing tea tree oil}Antifungal agent against dermatophytes containing tea tree oil

도 1 은 시험균주로 사용된 피부사상균에 대한 L. petersonii oil의 항진균활성을 나타내는 그래프, 1 is a graph showing the antifungal activity of L. petersonii oil against dermatophytes used as test strains,

도 2 는 TLC plate 에 L. petersonii oil을 전개 용매를 이용하여 분리한 모습을 나타내는 도면, 2 is a view showing a state in which L. petersonii oil is separated on a TLC plate using a developing solvent,

도 3a 내지 도 3c 는 시험 균주에 대한 생육 저지환(clear zone)을 나타내는 도면, 3a to 3c show the growth zone (clear zone) for the test strain,

도 4 는 L. petersonii oil 의 항진균 활성 분획에 대한 GC 분석의 결과를 나타내는 도면, 4 shows the results of GC analysis on the antifungal active fraction of L. petersonii oil,

도 5 는 compound 1, compound 2, compound 3 의 T. mentagrophytes 에 대한 항진균활성 조사 결과를 나타내는 도면, 5 is a view showing the results of antifungal activity of T. mentagrophytes of compound 1, compound 2, compound 3,

도 6 은 compound 1, compound 2, compound 3 의 M. canis 에 대한 항진균활성 조사 결과를 나타내는 도면, 6 is a view showing the results of the antifungal activity of M. canis of compound 1, compound 2, compound 3,

도 7 은 compound 1, compound 2, compound 3 의 M. gypseum 에 대한 항진균활성 조사 결과를 나타내는 도면이다.7 is a diagram showing the results of the antifungal activity of M. gypseum of compound 1, compound 2, compound 3.

본 발명은 티트리 오일을 함유하는 피부사상균용 항진균제 및 티트리 오일의 항진균제로의 이용방법에 관한 것이다.    The present invention relates to an antifungal agent for dermatophyte containing tea tree oil and a method of using tea tree oil as an antifungal agent.

티트리(Tea tree)는 오스트레일리아 원산지로 뉴사우스 웨일스주에서 자생한다. 오스트레일리아 원주민들은 오래 전부터 티트리 나무의 잎으로 베인 상처에 생긴 감염증을 치료해 왔다.    Tea tree is native to Australia and grows in New South Wales. Native Australians have long been treating infections caused by cuts in the leaves of tea tree trees.

일반적으로, 랩토스퍼뭄(Leptospermum), 멜라리우카(Melaleuca), 쿤제아(Kunzea) 속을 티트리(tea tree)라 부르며, 세계적으로 랩토스퍼뭄(Leptospermum) 속(genus)에 약 86 종, 멜라리우카(Melaleuca) 속에 약 230여종의 식물이 알려져 있다. 이중 상기 멜라리우카(Melaleuca) 속의 멜라리우카 알터니폴리아 오일(Melaleuca alternifolia oil)이 정유로서 가장 널리 알려져 있는데, 이 정유는 티트리 오일(tea tree oil), TTO 또는 멜라리우카 오일(Melaleuca oil)로 불려지고(Jurgen Reichlin, 2001), 멜라리우카 알터니폴리아 오일(Melaleuca alternifolia oil)은 항미생물, 항염증 특성과 같은 여러 의학적 특성을 갖는 것으로 보고되고 있다(C.F. Carson, 1998).In general, the genus Leptospermum , Melaleuca and Kunzea are called tea trees, and about 86 species of genus Leptospermum around the world, About 230 species of plants are known in the genus Melaleuca . There double the melanoma Liu car (Melaleuca) in the melanoma Liu Ka Alter your polyamic five days (Melaleuca alternifolia oil) best known as the refinery, the refinery is tea tree oil (tea tree oil), TTO or melanoma Liu car five days (Melaleuca oil (Jurgen Reichlin, 2001), Melaleuca alternifolia oil has been reported to have several medical properties such as antimicrobial and anti-inflammatory properties (CF Carson, 1998).

또한, 티트리 오일(tea tree oil)의 에스케리치아 코리(Escherichia coli) (J.E. Gustafson et al, 1998, S.D. Cox et al 1998, M.R. Moreira et al, 2005)), 스타피로코쿠스 아우레우스(Staphylococcus aureus) (C.H. Chan et al, 1998, Christine F. Carson et al, 2002, Linda Halcon et al 2004), 캔디다 알비칸스(Candida albicans)(S.D. Cox et al, 2000), 슈도모나스 아에루지노사(Pseudomonas aeruginosa)(S. Messager et al, 2005, Katgerine A. Hammer et al, 1996), 세라티아 마르세스센스(Serratia marcescens), 크렙시엘라 네우모니에( Klebsiella pneumoniae) (Katherine A. Hammer et al. 1996)에 대한 항미생물 효과가 보고된 바 있으며, 또한, 티트리 오일(tea tree oil)은 오니코마이코시스(onychomycosis)(Buck DS et al, 1994), 티네아 페디스(tinea pedis)(Tong MM et al, 1992, Andrew C Satchell et al, 2002)), 좌창(acne)(Bassett IB et al, 1990), 비듬(dandruff)(Satchell AC et al, 2002)의 치료에 효과적임이 제안된 바 있다.In addition, Escherichia coli of tea tree oil (JE Gustafson et al, 1998, SD Cox et al 1998, MR Moreira et al, 2005), Staphylococcus aureus ( Staphylococcus aureus (CH Chan et al, 1998, Christine F. Carson et al, 2002, Linda Halcon et al 2004), Candida albicans (SD Cox et al, 2000), Pseudomonas aeruginosa (S. Messager et al, 2005, Katgerine A. Hammer et al, 1996 ), Serratia marcescens , Klebsiella pneumoniae (Katherine A. Hammer et al. 1996 Anti-microbial effects have been reported, and tea tree oil is also known as onychomycosis (Buck DS et al, 1994), tinea pedis (Tong MM). et al, 1992, Andrew C Satchell et al, 2002), acne (Bassett IB et al, 1990) and dandruff (Satchell AC et al, 2002). There is a bar.

특히, 피부사상균인 트리코파이톤 루브럼(Trichophyton rubrum)에 대한 항균활성은 평판배지를 이용한 항균활성(Steven Benger et al, 2004)뿐만 아니라 베이퍼 컨택트(vapour contact)에 의한 항균활성(S. Inouye et al, 2001)도 보고되었다.In particular, the antimicrobial activity against trichophyton rubrum , a skin bacterium, is not only antibacterial activity using plate media (Steven Benger et al, 2004) but also antimicrobial activity by vapor contact (S. Inouye et al. , 2001).

그러나, 이러한 티트리 오일(tea tree oil)에 관한 대부분의 연구는 멜라리우카 알터니폴리아 오일(Melaleuca alternifolia oil)을 이용하여 생리활성물질이나 활성 기작에 대한 연구에 치중된 반면, 티트리 오일(tea tree oil)의 한 종류이며 레몬향을 지닌 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)에 대한 연구는 거의 이루어지지 않고 있다. 또한 여러 정유들의 자체 생리활성에 관한 연구는 많이 진행되고 있지만 그 정유에서 생리활성을 나타내는 주요 활성 물질을 탐색하는 연구는 매우 미미한 실정이다.However, most of the research on the tea tree oil has focused on the study of bioactive substances or active mechanisms using Melaleuca alternifolia oil, whereas tea tree oil ( Leptospermum petersonii oil, which is a kind of tea tree oil and has a lemon scent, has been rarely studied. In addition, many studies on the self-physiological activity of the essential oils are progressing, but the search for a major active substance showing a biological activity in the essential oil is very small situation.

따라서, 본 발명은 티트리 오일(Tea Tree oil)중에서도 특히 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 이용하여 피부사상균에 대한 항진균활성 및 항진균 활성을 나타내는 주요 성분을 조사함으로써 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 티트리 오일을 함유하는 피부사상균용 항진균제 및 항진균제로의 이용 방법을 제공한다. Therefore, the present invention, especially Leptospermum Petersonni by using Leptospermum petersonii oil in the tea tree oil by examining the main components showing antifungal and antifungal activity against dermatophytes Provided are antifungal and antifungal agents for dermatitis containing tea tree oil of Leptospermum petersonii oil.

상기와 같은 목적을 달성하기 위한 본 발명의 구성은,      The configuration of the present invention for achieving the above object,

티트리 오일(Tea tree oil)의 일종인 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)에서 추출한 추출물을 포함하는 피부사상균용 항진균제 조성물을 특징으로 한다. It is characterized by an antifungal composition for dermatophytes comprising an extract extracted from Leptospermum petersonii oil, which is a kind of tea tree oil.

또한, 상기 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 추출물은 시트랄(citral) 성분인 것을 특징으로 한다. In addition, the extract of Leptospermum petersonii oil is characterized in that the citral (citral) component.

또한, 상기의 피부사상균은 에피더모파이톤 플로코섬(E. floccosum), 마이크로스포럼 카니스(M. canis), 트리코파이톤 멘타그로파이테스(T. mentagrophytes), 마이크로스포럼 집세움(M. gypseum), 트리코파이톤 루브럼(T. rubrum) 중 어느 하나인 것을 특징으로 한다.In addition, the above-mentioned dermatophytes are epidermophyton Flocosum ( E. floccosum), M. canis (T. mentagrophytes), M. gypseum (T. mentagrophytes) ), Trichophyton rubrum ( T. rubrum) is characterized in that any one.

또한, 티트리 오일(Tea tree oil)의 일종인 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 피부사상균용 항진균제로 이용하는 방법을 특징으로 한다. In addition, a method of using Leptospermum petersonii oil, a kind of tea tree oil, as an antifungal agent for dermatitis.

또한, 상기 피부사상균은 에피더모파이톤 플로코섬(E. floccosum), 마이크로스포럼 카니스(M. canis), 트리코파이톤 멘타그로파이테스(T. mentagrophytes), 마이크로스포럼 집세움(M. gypseum), 트리코파이톤 루브럼(T. rubrum)중 어느 하나인 것을 특징으로 하는 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 피부사상균용 항진균제로 이용하는 방법을 특징으로 한다. In addition, the dermatophyte is epidermophyton Flocosum ( E. floccosum), M. canis ( M. canis), T. pyrotonta mentagrophytes (T. mentagrophytes), M. gypseum ( M. gypseum) It is characterized by using a leptospermum petersonii oil, characterized in that any one of the trichophyton rubrum ( T. rubrum) as an antifungal agent for dermatophytes.

이하, 본 발명에 따른 티트리오일을 함유하는 피부사상균용 항진균제 및 그 이용 방법을 실시예 및 시험예를 통하여 상세하게 설명한다.     Hereinafter, the antifungal agent for dermatophyte fungi containing tea tree oil and the method of using the same according to the present invention will be described in detail through Examples and Test Examples.

< 실시예 1 > 공시재료 및 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성Example 1 Test Material and Antifungal Activity of Leptospermum petersonii oil

실시예 1 에 사용된 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)은 GRD co. (Australia)에 의해 시판되는 정유를 사용하였으며, 마이크로스포럼 카니스(Microsporum canis)(KCTC6591), 트리코파이톤 멘타그로파이테스(Trichophyton mentagrophytes)(KCTC6077)를 한국생명공학연구원 생물자원센터에서, 트리코파이톤 루브럼(Trichophyton rubrum)(KCCM60443), 에피더모파이톤 플로코섬(Epidermophyton floccosum)(KCCM11667)를 한국미생물보존센터에서, 마이크로스포럼 집세움(Microsporum gypseum)은 충북대학교 수의과대학 동물병원에서 분양받아 공시균주로 사용하였다. 사부로 덱스트로즈 에이거(Sabouraud Dextrose Agar)(Difco, USA) 배지를 사용하여 28℃에서 시험균주를 배양하였다. 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성에 대한 평가는 50, 100, 150, 200 ppm농도에서 한천 희석법을 사용하여 측정하였다. Leptospermum petersonii oil used in Example 1 was GRD co. An essential oil marketed by Australia was used, and Microsporum canis (KCTC6591) and Trichophyton mentagrophytes (KCTC6077) were used at the Korea Research Institute of Bioscience and Biotechnology, Trichophyton Trichophyton rubrum (KCCM60443) and Epidermophyton floccosum (KCCM11667) were collected from the Korea Microbiological Conservation Center and Microsporum gypseum was distributed from the Animal Hospital of Veterinary Medicine, Chungbuk National University. Used as. Test strains were incubated at 28 ° C. using Sabouraud Dextrose Agar (Difco, USA) medium. The antifungal activity of Leptospermum petersonii oil was measured using agar dilution at 50, 100, 150 and 200 ppm concentrations.

< 실시예 2 > 항진균활성 분획 단리     Example 2 Antifungal Activity Fraction Isolation

상기 실시예 1 의 공시 재료를 사용하여 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성 성분을 분획 단리하였다.The antifungal active ingredient of Leptospermum petersonii oil was fractionated using the test material of Example 1 above.

항진균 활성성분의 검출에는 TLC 활성법을 이용하였는데, TLC 플레이트(plate)에 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 n-헥산(hexane) : 아세톤(acetone) = 4 : 1의 전개용매를 이용하여 전개 영역을 6부분(A, B, C, D, E, F)으로 분리한 뒤 트리코파이톤 멘타그로파이테스(Trichophyton mentagrophytes), 마이크로스포럼 집세움(Microsporum gypseum), 에피더모파이톤 플로코섬(Epidermophyton floccosum)을 도말한 사브로 덱스트로즈 에이거(Sabouraud Dextrose Agar)(SDA) 평판 배지에 올려놓고 배양하여 생육 저지환의 유무와 크기로 항진균활성을 평가하였다. TLC활성법의 결과 항진균활성을 갖는 분획을 단리하고자 컬럼 크로마토그래피(column chromatography)를 이용하였다. 컬럼 충진물질은 실리카겔, 전개용매는 n-헥산(hexane) : 아세톤(acetone) = 8 : 1을 사용하여 TLC assay에서 항진균활성을 보였던 부분과 일치하는 분획을 단리하였다. TLC activity was used for the detection of the antifungal actives, using a developing solvent of Leptospermum petersonii oil in n-hexane: acetone = 4: 1 on a TLC plate. After separating the development area into 6 parts (A, B, C, D, E, F), Trichophyton mentagrophytes, Microsporum gypseum , Epidermophyton flocosum ( Epidermophyton floccosum) was placed on a plate of Sabouraud Dextrose Agar (SDA) plated medium and cultured to evaluate antifungal activity in the presence and absence of growth inhibiting ring. As a result of the TLC activity method, column chromatography was used to isolate fractions having antifungal activity. The column packing material was silica gel and the developing solvent was n-hexane (acetone): acetone (acetone) = 8: 1 to isolate the fraction consistent with the antifungal activity in the TLC assay.

< 실시예 3 > 활성분획 주성분 단리와 각 성분에 대한 항진균활성     <Example 3> Isolation of Active Fraction Main Components and Antifungal Activity against Each Component

실시예 2 의 컬럼(column)을 이용하여 단리한 분획은 Prep-HPLC를 이용하여 활성분획의 주성분을 분리하였다. HPLC는 Thermo Separation Products P2000모델로 컬럼은 Waters 의 19×300 ㎜ 실리카 겔 컬럼(silica gel column)을 사용하였으며 UV 디텍터(detector)를 사용하여 320 ㎚에서 검출하였다. 플로우트 레이트(Flow rate) 4㎖/min로 용매는 n-헥산(hexane) : 에틸 아세테이트(ethyl acetate) = 92 : 8을 사용하여 분취하여 활성분획의 주 피크 3개를 각각 단리하였다. 단리한 3개의 성분에 대한 항진균 활성은 페이퍼 디스크(paper disc) 법을 이용하여 조사하였는데, 트리코파이톤 멘타그로파이테스(T. mentagrophytes), 마이크로스포럼 카니스(M. canis), 마이크로스포럼 집세움(M. gypseum), 에피더모파이톤 플로코섬(E. floccosum)을 도말한 SDA 평판배지 위에 단리한 세 성분을 2㎕ 씩 8㎜ 페이퍼 디스크(paper disc)에 각각 흡수시켜 클리어 존(clear zone)의 크기로 항진균활성을 조사하였다.The fraction isolated using the column of Example 2 separated the main components of the active fraction using Prep-HPLC. The HPLC was a Thermo Separation Products P2000 model, and the column was water 19 × 300 mm silica gel column, and was detected at 320 nm using a UV detector. At a flow rate of 4 ml / min, the solvent was aliquoted using n-hexane: ethyl acetate = 92: 8 to isolate three main peaks of the active fraction. Antifungal activity of the three isolated components was investigated using a paper disc method: Trichophyton T. mentagrophytes , M. canis , M. canis collection Three zones of M. gypseum and epidermophyton Flocosum ( E. floccosum) isolated on SDA plate media were absorbed by 2µl each on 8mm paper discs for clear zone. The antifungal activity was investigated by the size of.

< 실시예 4 > GC/MS분석      Example 4 GC / MS Analysis

상기 실시예 3 에 의해 단리된 3개의 항진균활성 물질을 알아보고자 한국기초과학지원연구원에 의뢰하여 GC/MS분석을 하였으며 그 조건은 다음과 같다.     In order to determine the three antifungal active substances isolated by Example 3, GC / MS analysis was conducted by the Korea Institute of Basic Sciences and the conditions were as follows.

GC(model-Agilent 6890)분석을 위해 HP5 컬럼(column)을 사용하였다. 캐리어 가스(Carrier gas)는 헬륨을 사용하였고 온도 조건은 인젝터 온도(injector temp). 240℃, 디텍터 온도(detector temp). 280℃, 오븐(oven) 온도는 초기온도 60℃에서 3분간 유지시킨 후 10℃/min씩 승온하여 최종온도 280℃까지 올린 후 5분간 유지시켜서 분석하였다. MS는 model Agilent 5973을 사용하였고 EI mode로 분석하였다. 얻어진 시료 피크의 매스 데이터(mass data)와 표준 라이브러리 데이터(library data)와의 비교를 통하여 피크의 화합물 구조를 동정하였다.     HP5 columns were used for GC (model-Agilent 6890) analysis. The carrier gas used helium and the temperature condition was the injector temp. 240 ° C., detector temp. The oven temperature was maintained at 280 ° C. for 3 minutes at 60 ° C. initial temperature, then heated at 10 ° C./min, raised to a final temperature of 280 ° C., and maintained for 5 minutes. MS was model Agilent 5973 and analyzed in EI mode. The compound structure of the peak was identified through comparison between the mass data of the obtained sample peak and the standard library data.

< 시험예 1 > 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성<Test Example 1> Antifungal Activity of Leptospermum petersonii oil

피부사상균에 대한 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성은 도 1 에 도시된 바와 같이 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)은 시험균주에 따른 약간의 활성차이는 있지만 피부사상균에 대해 매우 높은 항진균활성을 보였다. 50 ppm과 100 ppm 농도에서는 항진균활성이 낮았으며 oil의 농도 증가에 따른 활성의 증가는 보이지 않았다. 오히려 50 ppm보다 100 ppm 농도에서 활성이 약간 감소하는 것을 볼 수 있었다. 그러나 100 ppm 이상의 농도에서는 oil 농도와 항진균활성과의 관계가 비례함을 볼 수 있었다. 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 200 ppm 농도에서 에피더모파이톤 플로코섬(E. floccosum), 마이크로스포럼 카니스(M. canis), 트리코파이톤 멘타그로파이테스(T. mentagrophytes)의 균사 생장은 전혀 관찰되지 않았으며, 마이크로스포럼 집세움(M. gypseum)과 트리코파이톤 루브럼(T. rubrum) 의 경우 플러그(plug)에 약간의 균사생장만이 관찰되었다. 대표적인 티트리 오일(tea tree oil)이며 뛰어난 항미생물 효과가 알려져 있는 멜라리우카 알터니폴리아 오일(Melaleuca alternifolia oil)의 피부사상균에 대한 항진균 활성을 조사하여 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 활성과 비교한 결과 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)이 피부사상균에 대한 항진균활성이 훨씬 뛰어났다. 또한 Griffin SG et al(2000)에 의하면 트리코파이톤 루브럼(T. rubrum)에 대한 티트리 오일(tea tree oil)(Melaleuca alternifolia)의 MIC는 1.0% v/v이며 트리코파이톤 멘타그로파이테스(T. mentagrophytes)에 대한 MIC는 0.3-0.4% v/v 라고 보고하였으나, 이는 이 균들에 대한 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 MIC보다 높은 농도로 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 활성이 매우 높음을 알 수 있었다.Antifungal activity of Leptospermum petersonii oil against skin fungus is shown in Figure 1 Leptospermum petersonii oil Leptospermum petersonii oil, although there is a slight difference in activity according to the test strain Showed very high antifungal activity against. The antifungal activity was low at 50 ppm and 100 ppm, and there was no increase in activity with increasing oil concentration. Rather, the activity was slightly decreased at the concentration of 100 ppm rather than 50 ppm. However, at concentrations above 100 ppm, the relationship between oil concentration and antifungal activity was found to be proportional. At 200 ppm concentration of Leptospermum petersonii oil, epidermophyton Flocosum, E. floccosum, M. canis, Trichophyton Tta mentagrophytes Mycelial growth was not observed at all. Only slight mycelial growth was observed in the plugs of M. gypseum and T. rubrum . Leptospermum petersonii oil was investigated by investigating the antifungal activity of Melaleuca alternifolia oil, a representative tea tree oil and known for its excellent antimicrobial effect against dermatitis. Leptospermum petersonii oil showed much better antifungal activity against dermatophytes. In addition, according to Griffin SG et al (2000), the MIC of tea tree oil ( Melaleuca alternifolia ) against T. rubrum is 1.0% v / v and tricophyton mentagrophytes ( T). The MIC for mentagrophytes has been reported to be 0.3-0.4% v / v, but this is higher than the MIC of Leptospermum petersonii oil for these bacteria, but Leptospermum petersonii oil. ) Activity was very high.

< 시험예 2 > 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil) 항진균활성분획Test Example 2 Antifungal Activity Fraction of Leptospermum petersonii oil

렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균 활성분획을 조사하고자 도 2 에 도시된 바와 같이, 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 TLC 플레이트(plate)에 전개하여 A, B, C, D, E, F의 6개 분획으로 나눈 후 마이크로스포럼 집세움(M. gypseum), 트리코파이톤 멘타그로파이테스(T. mentagrophytes), 에피더모파이톤 플로코섬(E. floccosum)을 도말한 SDA 평판배지 위에 올려 생육저지환의 유무를 관찰하였다. 이때, 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)에 대한 생육 저지환의 크기가 50 mm 이상으로 나타난 경우 생육 저해활성을 가지는 것으로 판단하였다. To deploy the rep monohydrate peomum Peter Sony oil, rep monohydrate peomum Peter Sony five days (Leptospermum petersonii oil) as shown in Figure 2 to examine the antifungal activity fractions (Leptospermum petersonii oil) on TLC plate (plate) A, B, After dividing into six fractions of C, D, E, and F, smeared the M. gypseum , trichophyton mentagrophytes, and epidermophyton Flocosum. On the SDA plate medium, the presence of growth-lowering ring was observed. At this time, when the size of the growth inhibitory ring for Leptospermum petersonii oil (50 mm or more) appeared to have a growth inhibitory activity.

그 결과, 도 3a 내지 도 3c 에 나타낸 바와 같이, 시험한 균주인 마이크로스포럼 집세움(M. gypseum), 트리코파이톤 멘타그로파이테스(T. mentagrophytes), 에피더모파이톤 플로코섬(E. floccosum)에 대하여 생육 저지환 크기의 차이는 있지만 모두 B 분획에서 항진균 활성을 보였다. As a result, as shown in Figures 3a to 3c, M. gypseum , trichophyton Mentagrophytes ( T. mentagrophytes) , epidermophyton Flocosum ( E. floccosum) as the strains tested Although there was a difference in the size of the growth inhibitory ring, all showed antifungal activity in the B fraction.

< 시험예 3 > 활성분획의 주성분들에 대한 항진균활성     <Test Example 3> antifungal activity of the main components of the active fraction

TLC assay 에서 활성을 보였던 B 분획은 실시예 2 와 같은 방법에 의해 단리되었고, GC 분석결과 도 4 에 나타낸 바와 같이 활성분획에 3개의 주성분이 함유되어 있음을 알 수 있었다. 이 성분들은 GC 크로마토그램(chromatogram)상의 RT(retention time)에 따라 편의상 각각 콤파운드(compound) 1, 콤파운드(compound) 2, 콤파운드(compound) 3으로 명기하였다. GC분석결과 콤파운드(compound) 1은 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil) 내에 약 21%, 콤파운드(compound) 2는 21%, 콤파운드(compound) 3은 28%정도 함유되어 있었다.The B fraction showing activity in the TLC assay was isolated by the same method as in Example 2, and GC analysis showed that the active fraction contained three main components, as shown in FIG. 4. These components are designated as Compound 1, Compound 2, and Compound 3 for convenience according to the retention time (RT) on the GC chromatogram. GC analysis showed that Compound 1 contained about 21% in Leptospermum petersonii oil, Compound 2 about 21%, and Compound 3 about 28%.

실시예 3 과 같은 방법에 의해 활성분획에서 prep-HPLC를 이용하여 3개의 주성분을 단리했으며 단리된 3개의 화합물은 GC분석결과 토탈 에어리어 퍼센트(total area percentage)가 95%이상의 순도를 갖는 화합물들이었다. 이렇게 단리된 3성분들, 즉, 콤파운드(compound) 1, 콤파운드(compound) 2, 콤파운드(compound) 3을 페이퍼 디스크(paper disc)법을 이용하여 피부사상균에 대한 항진균활성을 조사하였다. 그 결과 균주에 따른 각 성분의 항진균활성의 차이가 있음을 볼 수 있었다.    Three main components were isolated from the active fraction by prep-HPLC in the same manner as in Example 3, and the three isolated compounds were those having a total area percentage of greater than 95% purity by GC analysis. . Three isolated components, ie, Compound 1, Compound 2, and Compound 3, were investigated for antifungal activity against dermatophytes by using a paper disc method. As a result, it was found that there is a difference in the antifungal activity of each component according to the strain.

도 5 에 나타낸 바와 같이, 트리코파이톤 멘타그로파이테스(T. mentagrophytes) 에 대한 세 성분의 항진균활성 조사 결과 배양 4 일째에는 세 성분을 처리한 처리구 모두 균사의 생장이 전혀 관찰되지 않았으나, 배양 7 일째에는 콤파운드(compound 1)은 약간의 생육 저지환이 존재하였으며, 처음의 항진균활성을 유지하는 것은 콤파운드(compound) 3 임이 관찰되었다. 이러한 결과로 볼 때 콤파운드(compound) 1과 콤파운드(compound) 2는 트리코파이톤 멘타그로파이테스(T. mentagrophytes) 에 대해 살균(fungicidal)이 아닌 균의 생장 억제(fungistatic)의 효과를 나타내는 것으로 파악된다. As shown in Figure 5, the antifungal activity of the three components of the T. mentagrophytes ( T. mentagrophytes) as a result of the investigation of the mycelial growth was not observed at all on the 4th day treatment, all three treatments, The compound (compound 1) had a slight growth inhibition ring, and it was observed that the compound (compound) to maintain the first antifungal activity. These results suggest that Compound 1 and Compound 2 show the effect of fungistatic, but not fungicidal, on trichophyton T. mentagrophytes. .

마이크로스포럼 카니스(M. canis)에 대한 항진균활성의 결과는 도 6 에 나타낸 바와 같다. 콤파운드(Compound) 1에서는 균사 생육저지환이 관찰되지 않았으며 콤파운드(compound) 2와 콤파운드(compound) 3에서는 어떠한 균사생장도 관찰되지 않았다. 또한, 도 7 에 나타낸 바와 같이 마이크로스포럼 집세움(M. gypseum)의 경우는 콤파운드(compound) 3에서만 강한 항진균활성을 보였다. Results of antifungal activity against M. canis are shown in FIG. 6. Mycelial growth was not observed in Compound 1 and no mycelial growth was observed in Compound 2 and Compound 3. In addition, as shown in FIG. 7, M. gypseum showed strong antifungal activity only in compound 3.

렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)을 페이퍼 디스크(paper disc)에 2㎕ 처리 했을 때의 항진균 활성은 콤파운드(compound) 3과 같이 직경 60㎜ 페트리 디쉬(Petri dish)에서는 균사의 생장을 관찰할 수 없었다. 이 결과, 피부사상균에 대한 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성은 콤파운드(compound) 1이나 콤파운드(compound) 2도 영향을 미치지만 주로 콤파운드(compound) 3에 의해 활성이 나타나는 것으로 파악된다. Antifungal activity when 2 μl of Leptospermum petersonii oil was applied to a paper disc was observed in the Petri dish, 60 mm in diameter, as in compound 3. I could not. As a result, the antifungal activity of Leptospermum petersonii oil against dermatophytes affects Compound 1 or Compound 2, but is mainly caused by Compound 3. Is figured out

< 시험예 4 > GC/MS분석    Test Example 4 GC / MS Analysis

피부사상균에 대한 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균 활성 분획의 성분들인 콤파운드(compound) 1, 콤파운드(compound) 2, 콤파운드(compound) 3에 대한 성분을 확인하고자 실시예 4 와 같이 GC/MS분석을 실시한 결과를 하기의 표 1 로서 나타내었다. 콤파운드(compound) 1은 시트로넬라(citronella), 콤파운드(compound) 2는 네랄(neral), 피부사상균에 대해 활성이 가장 뛰어났던 콤파운드(compound) 3은 게라니알(geranial)로 확인되었다. 이러한 결과는 멜라리우카 알터니폴리아 오일(M. alternifolia oil)의 항미생물 활성을 나타내는 성분으로 알려진 테르피넨-4-올(terpinen-4-ol)(Andrew C Satchell, 2002)과는 확실히 다른 성분들이다.As shown in Example 4 to identify the components for compound 1, compound 2, and compound 3, which are components of the antifungal active fraction of Leptospermum petersonii oil against dermatophytes The results of the GC / MS analysis are shown in Table 1 below. Compound 1 was identified as citronella, compound 2 as neral, and compound 3 as geranial, which was the most active against dermatophytes. This result is clearly different from terpinen-4-ol (Andrew C Satchell, 2002), which is known as an antimicrobial activity of M. alternifolia oil. admit.

Active antifungal compounds of L. petersonii oil identified by GC/MS analysis Active antifungal compounds of L. petersonii oil identified by GC / MS analysis Compound identified by GC/MS analysis  Compound identified by GC / MS analysis Mass peaks 주 1)             Mass peaks note 1) Compound 1  Compound 1 Citronella Citronella 41, 69, 95, 55, 121 (BP 주2) : 41, MP 주3) : 154)             41, 69, 95, 55, 121 (BP Note 2): 41, MP Note 3): 154) Compound 2  Compound 2 Neral Neral 41, 69, 94, 84, 109 (BP : 41, MP : 152)             41, 69, 94, 84, 109 (BP: 41, MP: 152) Compound 3  Compound 3 Geranial  Geranial 69, 84, 94 (BP : 69, MP : 152)             69, 84, 94 (BP: 69, MP: 152)

주 1) Major mass peaks of the corresponding compound Note 1) Major mass peaks of the corresponding compound

주 2) BP : base peak Note 2) BP: Base Peak

주 3) MP : molecular ion peak Note 3) MP: molecular ion peak

네랄(Neral)과 게라니알(geranial)은 분리하기 어려워서 이들 혼합물을 하나의 물질로 간주하여 시트랄(citral)로 부르며, 게라니알(geranial)과 네랄(neral)은 각각 시트랄(citral) A와 시트랄(citral) B로 알려져 있는 물질이다(A. A. Newman, 1972). 천연 시트랄(citral)은 거의 항상 이들 아이소머(isomer)의 혼합형태로 나타나며 레몬-그래스 오일(lemon-grass oil)에 85%이상이 함유되어 있는 것으로 보고되고 있다(Kurt Bauer et al, 1990). 또한, 시트랄(citral)은 일반적으로 항염, 항바이러스, 진정효과를 지닌 물질로 알려져 있다.      Nerral and geranial are difficult to separate, so these mixtures are regarded as a substance and are called citral, and geranial and neural are citral, respectively. A substance known as A and citral B (AA Newman, 1972). Natural citral almost always appears as a mixture of these isomers and is reported to contain more than 85% in lemon-grass oil (Kurt Bauer et al, 1990). . Citral is also commonly known as an anti-inflammatory, antiviral, and sedative material.

이상으로 상술한 바와 같이, 본 발명은 티트리오일(tea tree oil)의 한 종류이며 레몬향이 나는 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 피부사상균에 대한 항진균활성과 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil) 중 항진균활성을 나타내는 성분을 조사하였다. As described above, the present invention is a kind of tea tree oil and lemon-flavored leptospermum petersonii oil anti-fungal activity against leuktospermum petersonii oil and leptospermum peter sony oil The components showing antifungal activity in ( Leptospermum petersonii oil) were investigated.

한천희석법을 이용하여 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 항진균활성을 조사하였으며, 대조구에 대한 처리구의 균사 생장 억제율로 항진균활성을 계상하였을때 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)은 매우 높은 항진균활성을 보였다. 50 ppm과 100 ppm에서는 40%이하의 낮은 활성을 보였으나 200 ppm에서는 트리코파이톤 루브럼(T. rubrum)을 제외한 모든 균에 대해 90%이상의 항진균활성을 보였으며, 특히 트리코파이톤 멘타그로파이테스( T. mentagrophytes), 마이크로스포럼 카니스(M. canis), 그리고 에피더모파이톤 플로코섬(E. floccosum)의 균사생장이 완전 억제되는 것을 볼 수 있었다.The antifungal activity of Leptospermum petersonii oil was investigated by the agar dilution method. When antifungal activity was estimated by the inhibition of mycelial growth of the control, the Leptospermum petersonii oil was Very high antifungal activity. At 50 ppm and 100 ppm, the activity was lower than 40%, but at 200 ppm, the antifungal activity was higher than 90% against all the bacteria except T. rubrum. In particular, tricophyton mentagrophytes ( Mycelial growth of T. mentagrophytes , M. canis , and E. floccosum was completely suppressed.

이렇게 피부사상균에 대한 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)의 높은 항진균활성은 주로 시트로넬라(citronella), 게라니알(geranial), 네랄(neral) 성분에 의해 발현되는 것으로 나타났으며, 특히 게라니알(geranial)의 항진균활성이 뛰어났다.The high antifungal activity of Leptospermum petersonii oil against dermatophytes is mainly expressed by citronella, geranial and neural components. In particular, the antifungal activity of geranial was excellent.

이러한 결과로 볼때, 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil)은 레몬향을 이용한 향기제품으로의 개발 및 의약품 개발에 유용한 효과가 있다. 또한, 피부사상균에 대한 항균제 개발시 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil) 뿐만 아니라 이것의 활성성분인 시트랄(citral)도 이용가능한 효과를 가진다.As a result, Leptospermum petersonii oil has a useful effect in the development of fragrance products using lemon flavor and drug development. In addition, Leptospermum petersonii oil (citral) as well as its active ingredient in the development of an antibacterial agent against dermatophytes has an effect available.

Claims (5)

삭제delete 삭제delete 렙토스퍼뭄 피터소니 오일(Leptospermum petersonii oil) 추출물을 포함하는, 에피더모파이톤 플로코섬(Epidermophyton floccosum), 마이크로스포럼 카니스(Microsporum canis), 마이크로스포럼 집세움(Microsporum gypseum)으로 구성된 군으로부터 선택되는 피부사상균용 항진균제 조성물. Epidermophyton floccosum , comprising Leptospermum petersonii oil extract, Microsporum canis , An antifungal composition for dermatophytes selected from the group consisting of Microsporum gypseum . 삭제delete 삭제delete
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Flavour and Fragrance Journal 15(5), 342-51 (2000)
Microbios 89(358), 39-46 (1997)

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