KR20060102367A - Natural antimicrobial compound against powdery mildew containing the extract of korean sancho, zanthoxylum schinfolium - Google Patents
Natural antimicrobial compound against powdery mildew containing the extract of korean sancho, zanthoxylum schinfolium Download PDFInfo
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Abstract
본 발명은 산초나무 추출물을 포함하는 흰가루병 방제용 천연 항균조성물에 관한 것으로, 산초 추출물 및 산초 메탄올추출물의 물 분획물은 인체 및 가축에 무해하면서 흰가루병의 발생을 억제하여, 흰가루병 방제를 위한 과도한 화학농약 사용의 문제점을 보완하며, 토마토, 딸기 등의 시설재배작물의 안전성과 상품성을 증대시키는 뛰어난 효과를 가진다. 또한 본 발명의 산초 추출물 및 분획물은 식중독 세균 및 병원성 세균의 성장을 억제시키는 효과를 가지고 있어 상품의 세균성 위해를 방지할 수 있다.The present invention relates to a natural antimicrobial composition for controlling powdery mildew, comprising an extract of the Japanese agar, the water fraction of the extract and the extract of methanol extract of methanol extract is harmless to humans and livestock, while suppressing the occurrence of powdery mildew, using excessive chemical pesticides for the control of powdery mildew Complement the problem, and has an excellent effect of increasing the safety and marketability of plant cultivation crops, such as tomatoes and strawberries. In addition, the extract and fractions of the extract of the present invention has the effect of inhibiting the growth of food poisoning bacteria and pathogenic bacteria can prevent bacterial harm of the product.
산초 추출물, 산초 물 분획물, 흰가루병, 항곰팡이 활성, 항세균 활성 Extracts of Japanese pepper, water extract of Japanese pepper, powdery mildew, antifungal activity, antibacterial activity
Description
도 1은 다양한 천연 식물 추출물(874종)의 포자 발아 억제 활성을 초고속으로 평가하는 96 웰 플레이트 배양 결과를 나타낸 사진도이다. Figure 1 is a photograph showing the results of 96 well plate culture to evaluate the spore germination inhibitory activity of various natural plant extracts (874 species) at ultrafast.
도 2는 산초 추출물 및 상업용 항곰팡이제인 마이코나졸의 흰가루병 곰팡이의 균사 생장 억제 활성을 생육억제환으로 평가한 사진도이다. Figure 2 is a photographic evaluation of the mycelial growth inhibitory activity of powdery mildew fungus of Maichoazole extract and commercial anti-fungal agent.
도 3은 토마토에 흰가루병 곰팡이의 발병 후 산초 추출물 및 상업용 농약을 분무한 결과를 나타낸 사진도이다.Figure 3 is a photograph showing the results of spraying the extract and commercial pesticides after the onset of powdery mildew on tomato.
본 발명은 산초나무 추출물을 포함하는 흰가루병 방제용 천연 항균조성물에 관한 것으로, 보다 상세하게는, 인체에 무해하면서도, 흰가루병 곰팡이의 포자 발아 억제 및 균사 생육 억제를 통해 토마토, 오이, 딸기 등의 시설재배작물에 발생하는 흰가루병을 예방 및 제어하는 산초나무 추출물을 유효성분으로 포함하는 흰가루병 방제용 천연 항균조성물을 제공한다. The present invention relates to a natural antimicrobial composition for controlling powdery mildew, comprising an extract of the Japanese vinegar, and more specifically, harmless to the human body, while cultivating facilities such as tomatoes, cucumbers, strawberries through suppression of spore germination and mycelial growth of powdery mildew fungus It provides a natural antimicrobial composition for powdery mildew control comprising as an active ingredient extracts of the Japanese vinegar to prevent and control powdery mildew occurring in crops.
흰가루병은 토마토, 딸기, 오이, 참외, 호박, 수박, 장미, 작약 등 300 여종의 기주식물에서 발병되며, 스패로세카속(Sphaerotheca), 레베일루나속(Leveilluna), 에리쉬페속(Eryshipe), 푸사리움속(Fusarium)의 곰팡이가 원인균이며(대한민국 특허 공개번호 제10-2004-0066951호, 농업생명공학연구원 http://kacc.rda.go.kr), 시설 내에서 재배되는 여러 작물에서 광범위한 피해를 주고 있다. 흰가루병은 작물에 따라 증상이 다소 차이가 나지만 대부분 줄기, 잎, 열매에 흰가루 모양의 곰팡이가 피며, 병이 진전됨에 따라 식물의 광합성과 호흡을 저해하여 동화작용과 증산작용을 감소시켜 병반조직의 괴사와 함께 급속히 노화되 어 잎이 떨어지게 된다(이상엽 등, 2004, 한국농약과학회지 8, 71-78 ; 이옥희 등, 2001, 원예과학기술지 19, 7-11). 또한 흰가루병 곰팡이는 흰가루병 이외에 뿌리썩음병(근부병)에도 관여하는 것으로 알려져 있다(이순구 등, 2004, 식물병연구 10, 248-259). Powdery mildew is caused by over 300 species of host plants, including tomatoes, strawberries, cucumbers, melons, pumpkins, watermelons, roses, and peonies. Sphaerotheca , Leveilluna , Eryshipe , and Pusa Fusarium fungus is the causative bacterium (Korean Patent Publication No. 10-2004-0066951, Institute of Agricultural Biotechnology http://kacc.rda.go.kr), and extensive damage from various crops grown in the facility Is giving. Although powdery mildew has a slightly different symptom depending on the crop, most of them have white powdery molds on stems, leaves, and fruits. As the disease progresses, necrosis of lesion tissue is reduced by inhibiting photosynthesis and respiration of plants. Aging leaves rapidly fall and leaves fall (Lee Sang-yeop et al., 2004, Korean Journal of Pesticide Science 8, 71-78; Lee Ok-hee et al., 2001, Korean Horticultural Science and Technology Journal 19, 7-11). In addition to powdery mildew, powdery mildew is known to be involved in root rot (root disease) (Lee Soon-gu et al., 2004, Plant Disease Research 10, 248-259).
현재까지 이러한 흰가루병 곰팡이의 방제는 주로 디페노코나졸(Difenoconazole), 베노밀(benomyl) 및 만코젭(mancozeb) 등의 화학농약에 의존하여 왔으며(이상엽 등, 2004, 한국농약과학회지 8, 71-78), 이러한 화학농약의 연용은 결국 흰가루병 곰팡이의 약제저항성을 유발시켜 농약 효율성을 떨어뜨리게 되며, 화학농약을 기피하는 소비자들에게는 높은 잔류농약 검출로 인해 농약 사용 과채류가 배척되고 있는 실정이다. 이러한 문제를 해결하기 위해, 최근에는 내성 식물체 개발(이옥희 등, 2001, 원예과학기술지 19, 7-11), 길항 미생물 제제의 개발(이상엽 등, 2004, 한국농약과학회지 8, 71-78), 계면활성제 처리(대한민국 특허 공개번호 2001-0078568; 장경수 등 2001, 한국농약과학회지 5, 51-57), 및 탄산수소나트륨과 폴리옥시에틸렌 솔비탄모노라우레이트(polyoxyethylene sorbitanmonolaurate) 혼용처리에 의한 흰가루병 방제(남명현 등 2003, 원예과학기술지, 21. 98-101) 등 다양한 생화학적 방제법으로 화학농약을 보완, 대치하고자 많은 연구가 이루어지고 있다. To date, the control of powdery mildew fungi has largely relied on chemical pesticides such as Difenoconazole, benomyl and mancozeb (Lee Sang-yeop et al., 2004, Korean Pesticide Science Journal 8, 71-78). In addition, the use of such chemical pesticides induces drug resistance of powdery mildew fungi, which reduces the efficiency of pesticides, and the use of pesticides and vegetables is excluded due to the detection of high residual pesticides. In order to solve this problem, recently developed resistant plants (Lee Ok-hee et al., 2001, Horticultural Science and Technology Journal, 19, 7-11), development of antagonistic microbial agents (Lee Sang-yeop et al., 2004, Korean Journal of Pesticide Science 8, 71-78) Powder treatment by surfactant treatment (Korean Patent Publication No. 2001-0078568; Jang Kyung-Soo et al. 2001, Korean Journal of Agrochemical Sciences 5, 51-57), and a mixture of sodium bicarbonate and polyoxyethylene sorbitanmonolaurate Many studies have been conducted to supplement and replace chemical pesticides with various biochemical control methods such as control (2003, Nam Myung Hyun et al., Horticultural Science and Technology Journal, 21. 98-101).
한편, 화학농약에 비해 상대적으로 안전성이 높은 천연 추출물에 의한 흰가루병 방제 연구도 시도되고 있으며, 최근 식물체(율피, 감, 솔잎, 호프, 감비르나무, 해아다, 암마륵, 커피, 가자, 소목, 지유, 호장, 대황, 조 등)의 탄닌계 추출물을 포함하는 흰가루병 방제용 조성물이 보고되었으며(대한민국 특허 공개번호 제10-2004-0066951호), 생약재인 오배자(Rhus chinensis) 추출물에 의한 흰가루병 방제제(대한민국 특허공개번호 제10-2004-0056061호), 소리쟁이(Rumex crispus) 및 참소리쟁이(Rumex japonicus) 추출물을 함유한 흰가루병 방제 조성물(대한민국 특허공개번호 제2003-0068255호), 소목(Caesalpinia sappan), 황금(Scutellaria baicalensis), 정향(Syringa velutina var kamibayashii), 도두 (Canavalia glaviata), 엿기름을 함유하는 흰가루병 방제 조성물(대한민국 특허공개번호 제10-2004-0066955호)이 각각 알려져 있다. 그러나 많은 종류의 식물추출물을 혼합하여 동시에 사용하는 경우, 제조 및 사용의 어려움이 있으며, 경제적 생산이 용이하지 않으며, 특히 오배자 및 소리쟁이의 경우 원재료 획득 용이성과 잠재적 인체 독성의 문제를 가지고 있다(식품의약품안전청 식품원재료데이터베이스). On the other hand, research on powdery mildew control by natural extracts, which are relatively safer than chemical pesticides, has been attempted. Recently, plants (Julpi, persimmon, pine needles, hops, persimmon tree, Haida, amaranth, coffee, let's go, joiner, Jiyugaoka, polygonaceous, rhubarb, has been reported for powdery mildew control composition comprising a tannin-based extract of the tank, and so on) (Republic of Korea Patent Publication No. 10-2004-0066951 call), a gallnut saengyakjae (Rhus chinensis ) powdery mildew control agent (Korea Patent Publication No. 10-2004-0056061), Rumex powdery mildew control composition ( crispus ) and Rumex japonicus extract (Korean Patent Publication No. 2003-0068255), Caesalpinia sappan ), golden ( Scutellaria baicalensis ), cloves ( Syringa velutina var kamibayashii ), canavalia glaviata , and powdery mildew control composition containing malt (Korean Patent Publication No. 10-2004-0066955) are known, respectively. However, when many kinds of plant extracts are mixed and used simultaneously, they are difficult to manufacture and use, and economical production is not easy, and particularly in the case of gall bladder and raspberry, there are problems of easy raw material acquisition and potential human toxicity. Korea Food Safety Database.
산초(Zanthoxylum schinifolium)는 동북아시아권에서 옛날부터 사용되어 온 전통 향신료이며 우리나라, 일본, 중국 등에서 널리 자생하고 있는 운향과(Rutaceae)의 낙엽관목으로서 분지나무 또는 상초나무로도 불린다. 높이는 3미터이고, 잔가지는 가시가 있으며 붉은빛이 도는 갈색이다. 잎은 어긋나고 꽃은 암수 딴그루이고 8∼9월에 흰색으로 피며 가지 끝에 산방꽃차례를 이루며 달린다. 작은 꽃자루에 마디가 있고, 꽃받침은 5개로 갈라지며 갈라진 조각은 달걀 모양의 원형이다. 열매는 삭과이고 둥글며 녹색을 띤 갈색이며 다 익으면 3개로 갈라져서 검은 색의 종자가 나온다. 초피나무와 비슷하지만 잎자루 밑 부분에 가시가 1개 달리고 열매가 녹색을 띤 갈색이며 꽃잎이 있는 것이 다르다. Sancho ( Zanthoxylum schinifolium ) is a traditional spice that has been used in Northeast Asia for a long time. It is a deciduous shrub of Rutaceae , which grows widely in Korea, Japan, China, etc., and is also called branch tree or vinegar. It is 3 meters high, and twigs are spiny and reddish brown. The leaves are alternate, the flowers are male and female, and bloom in white in August-September. A small peduncle has a node, the calyx is divided into 5 pieces, and the fragment is an egg-shaped circle. Fruits are capsules, round, greenish brown, and when they are ripe, they are divided into three and produce black seeds. It is similar to the bark tree, but has one thorn at the bottom of petiole, the fruit is greenish brown and has different petals.
산초는 과실, 껍질, 잎 등을 향신료, 약용 및 제유용으로 이용하며, 우리 정서에 친숙한 식미와 향기를 가져 광범위하게 사용되고 있으며, 구충제, 해독살충 약, 감기약 등에 이용되어 왔다(이종원, 1998, 한국식품영양과학회지, 11, 493-498). 산초의 보고된 성분으로는 리모네네(limonene), 시트로렐랄(citronellal)의 향기 주요성분과, 플라보노이드(flavonoid)계 성분, 카프릭산(caproic acid)등의 지방산 화합물이 있으며(조민구 등, 2003, 한약작지, 11, 40-45; 김정 등, 2000, 농산물저장유통학회지. 7, 189-194), 생리활성으로는 식중독 세균 및 피부사상균에 대한 항균 활성(김정순 등, 2004. 한국식품영양과학회지 33. 500-504; 민경희 1998, 목재공학 26; 78-85; 공개특허 10-2004-0075263)이 보고되어 있다. 그러나 현재까지 산초 추출물에 대한 흰가루병과 같은 식물 곰팡이 병원균 방제효과는 보고된 바 없다. Sancho uses fruits, bark, and leaves for spices, medicinal and oil-reducing oils, and is widely used because of its familiar taste and fragrance. It has been used for insect repellents, detoxification drugs, and cold medicines (Lee Jong-won, 1998, Korea) Journal of Food and Nutrition Science, 11, 493-498. The reported components of Sancho include fatty acid compounds such as flavonene, citronellal, main components of flavonoids, flavonoids, and capric acid (Cho Min-gu et al., 2003). , Korean Journal of Medicinal Crop, 11, 40-45; Kim, Jung et al., 2000, Journal of Agricultural Products Storage and Distribution. 7, 189-194), Physiological Activities of Antimicrobial Activity against Food Poisoning Bacteria and Dermatophytes (Kim, Jung-Soon et al., 2004. Journal 33. 500-504; Min Kyung-hee 1998, Wood Engineering 26; 78-85; Pub. 10-2004-0075263). However, there have been no reports on the control of plant fungal pathogens such as powdery mildew against the extracts of Sancho.
따라서, 본 발명의 목적은 산초나무 추출물을 이용하여 인체에 무해하면서도 작물시설재배 시 발병하는 흰가루병 곰팡이의 생육을 억제하는 천연 항균조성물을 제공하고자 한다.Accordingly, it is an object of the present invention to provide a natural antimicrobial composition that inhibits the growth of powdery mildew fungus that occurs during crop planting, while harmless to the human body by using an extract of the vinegar.
본 발명의 목적은 인간과 가축에 무해한 흰가루병 방제용 천연항균물질을 개 발하기 위해 874종의 천연 추출물로부터 흰가루병 곰팡이의 포자 발아 억제능, 균사 성장 저해능을 평가하여 산초나무 추출물 및 산초나무 메탄올추출물의 물 분획물에서 우수한 방제효과를 확인하였으며, 미비한 약해 효과 및 식물생장조절기에서의 방제효과를 확인함으로써 달성하였다.The purpose of the present invention was to evaluate the spore germination inhibitory activity and mycelial growth inhibitory activity of powdery mildew fungus from 874 natural extracts in order to develop a natural antibacterial substance for controlling powdery mildew, which is harmless to humans and livestock. The excellent control effect was confirmed in the fractions, and was achieved by confirming the weak harmful effect and the control effect in the plant growth regulator.
이하, 본 발명의 구성을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
본 발명은 874종의 천연물 추출물에서 흰가루병 곰팡이균의 포자 발아 억제효과 조사단계; 상기 단계에서 선별된 천연물 추출물의 흰가루병 균사 성장 억제 효과 조사단계; 산초추출물의 조제 및 유기용매에 의한 활성물질 분획단계; 산초 메탄올추출물의 물 분획물의 약해 시험단계; 식물생장조절기에서 산초 물 분획물의 흰가루병 방제시험단계; 산초나무의 메탄올추출물 및 각종 분획물의 식중독세균, 병원성세균의 증식억제효과 실험단계로 구성된다.The present invention is to investigate the spore germination inhibitory effect of powdery mildew fungus from 874 natural products extracts; Investigating the growth inhibition effect of powdery mildew mycelia of the natural product extract selected in the above step; Preparing an active material extract and fractionating the active substance by an organic solvent; Weakness test step of the water fraction of the acidic methanol extract; Powdery mildew control test step of the water fraction of the vinegar in the plant growth regulator; Methanol extracts and various fractions of Japanese vinegar consisted of experimental stages of growth inhibition effect of food poisoning bacteria and pathogenic bacteria.
본 발명은 흰가루병 곰팡이의 포자 발아 억제 또는 균사 성장 억제 활성을 갖는 산초나무의 메탄올추출물, 분말 또는 메탄올추출물의 분획물을 유효성분으로 포함하는 식물의 흰가루병 방제용 조성물을 제공한다.The present invention provides a composition for controlling powdery mildew of plants comprising, as an active ingredient, methanol extract, powder or methanol extract of methanol extract having spore germination or mycelial growth inhibitory activity of powdery mildew fungi.
본 발명 식물의 흰가루병 방제용 조성물에서 산초는 산초나무 전체 또는 줄기, 잎, 과실의 일부일 수 있으며, 종자보다는 줄기, 잎의 추출물이 바람직하다. 추출용매는 물 또는 유기용매를 사용할 수 있으나 메탄올 추출이 바람직하며, 통상의 식물추출물 제조방법으로 제조할 수 있다. In the composition for controlling powdery mildew of the plant of the present invention, the herb may be whole or part of stem, leaf, or fruit, and the extract of the stem and leaf is preferable to the seed. The extraction solvent may be water or an organic solvent, but methanol extraction is preferred, it can be prepared by a conventional plant extract manufacturing method.
본 발명의 식물의 흰가루병 방제용 조성물은 산초나무 메탄올추출물은 0.5∼5 중량% 또는 산초나무 메탄올추출물의 물 분획물은 0.1∼1 중량%로 포함하나, 상기 함량은 첨가물의 종류나 조성물의 제형에 따라 적절히 조절할 수 있다. 대표적인 제형으로는 액제, 에어로솔제, 유제, 현탁제이다. Composition for controlling powdery mildew of plants of the present invention contains 0.5 to 5% by weight of methanol extract or 0.1 to 1% by weight of water fraction of methanol extract, the content is depending on the type of additive or the formulation of the composition It can be adjusted appropriately. Representative formulations are liquids, aerosols, emulsions and suspensions.
또한, 본 발명의 식물의 흰가루병 방제용 조성물은 당귀 추출물, 미국가막사리 추출물 및 백선피 추출물 중 어느 하나 이상을 첨가하여 조제될 수 있다.In addition, the composition for controlling powdery mildew of the plant of the present invention can be prepared by adding any one or more of the Angelica extract, American fern extract and ringworm skin extract.
이하 본 발명의 내용을 실시예를 통해 보다 상세히 설명하기로 한다. 다만 이들 실시예는 본 발명의 내용을 설명하기 위한 것일 뿐 본 발명의 권리범위를 제한하는 것은 아니다.Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only for explaining the contents of the present invention and do not limit the scope of the present invention.
[실시예]EXAMPLE
실시예Example 1: 874종의 천연물 추출물로부터 흰가루병 곰팡이의 포자 발아 억제 효과 조사 1: Investigation of Spore Germination Inhibition of Powdery mildew Fungus from 874 Natural Extracts
흰가루병 방제효능을 가진 천연물을 검색하기 위해 874종의 천연물 추출물을 대상으로 오이 및 보리에서 분리된 흰가루병 곰팡이의 포자 발아 억제 효과를 평가하였다. 사용한 천연물은 다양한 식용, 약용 천연물을 다양한 유기용매로 추출, 분획하여 조제된 분말이며 이를 DMSO(dimethylsulfoxide, 미국 시그마사)에 녹여 사용하였다. 사용된 천연물의 종류는 안동대학교 웹 서버에 공개하였다(http://npdb.andong.ac.kr, Materials DB, Natural Products). 포자 발아 억제 효과를 측정하기 위해 푸사리움 그라미네룸(Fusarium graminearum) Schwabe KACC 41040을 PDA(Potato dextrose Agar; 미국 디프코사) 배지에 접종하여 30℃에서 4 주간 배양하여 포자를 형성하게 하고, 이를 회수하여 증류수에 현탁 후 초음파 분쇄기로 균일화한 다음 성장 배지에 일정량 접종하였다. 억제 효과는 96웰에서 실시하였으며, 천연물의 최종농도가 100 ㎍/mL 되도록 첨가한 다음 30℃에서 5일간 배 양하고 생육 정도를 관찰하였다. 포자의 발아 억제 활성이 없는 경우 3일 배양 후 웰에 흰곰팡이가 가득하였으며, 곰팡이가 성장하지 않는 경우 억제 활성을 인정하였다(도 1). 도 1의 화살표는 포자 발아가 억제되어 곰팡이가 자라지 못함을 나타낸다.In order to search for natural products with the control of powdery mildew, 874 kinds of natural extracts were evaluated for the effect of spore germination on powdery mildew isolated from cucumber and barley. The natural product used is a powder prepared by extracting and fractionating various edible and medicinal natural products with various organic solvents, and dissolving it in DMSO (dimethylsulfoxide, Sigma, USA). Types of natural products used were published on the Web site of Andong National University (http://npdb.andong.ac.kr, Materials DB, Natural Products). Fusarium Gras Mine Room (Fusarium spores germinate to measure the inhibitory effect graminearum ) Schwabe KACC 41040 was inoculated in PDA (Potato dextrose Agar; Diphco Co., Ltd.) medium and incubated at 30 ° C. for 4 weeks to form spores. Inoculation. The inhibitory effect was carried out in 96 wells, the final concentration of the natural product was added to 100 ㎍ / mL, then incubated at 30 ℃ for 5 days and observed the growth. When there was no germination inhibitory activity of the spores, the wells were full of white mold after 3 days of culture, and the inhibitory activity was recognized when the fungus did not grow (FIG. 1). Arrows in FIG. 1 indicate that spore germination is inhibited and mold does not grow.
그 결과, 산초, 당귀, 미국가막사리, 까마중, 오수유, 윤판나물, 미치광이풀, 때죽나무, 백선, 방기, 정향, 멍석딸기, 호장근, 천황련, 작약, 대황, 모란, 달맞이꽃, 대극, 개박하 추출물의 20 종을 1차 선별하였다. As a result, Sancho, Angelica, Barley, Camellia japonica, Fructus, Yunpanmul, Maniac, Staphylococcus, Ringworm, Bangui, Cloves, Jackass, Blackberry, Peony, Peony, Peony, Rhubarb, Peony, Evening primrose, Dramatically, Catnip extract 20 species of were selected first.
실시예Example 2: 1차 선별된 천연물 추출물의 흰가루병 곰팡이의 균사 성장 억제 효과 조사 2: Investigation of Mycelial Growth Inhibitory Activity of Powdery Mildew of Primary Screened Natural Products Extracts
실시예 1에서 1차 선별된 20종의 추출물의 항균성 활성은 디스크 방법(Davidson, P.M and Parish, M.E. 1989. Food Technology, 43, 148-155)을 이용하였다. PDA 고체배지에 푸사리움 그라미네룸 곰팡이의 현탁액을 손분무기로 고르게 분사한 후 상온에서 건조하고, 이후 멸균된 종이디스크(Filter paper No.2, 영국 와트만사)를 PDA 배지 위에 올린 후 각각의 추출물 10 ㎕(디스크 당 농도 100 또는 200 ㎍)를 디스크 위에 떨어뜨려 건조하고 30℃에서 3일간 배양하여 디스크 주위의 생육억제환 생성 유무를 조사하였다. 이때 대조구로는 DMSO와 임상에서 항진균제로 사용하는 마이코나졸(miconazole, 미국 시그마사)을 동일한 방법으로 종이디스크에 가하여 측정하였으며, 시판 농약 4종(푸르겐-Difenoconazole, 다이센-Mancozeb, 톱신-Thiopanate methyl, 빈크로졸린)의 분말을 녹여 종이디스크 당 250 또는 500 ㎍ 농도가 되도록 가하여 배양하였다. The antimicrobial activity of the 20 extracts selected first in Example 1 was used by the disk method (Davidson, PM and Parish, ME 1989. Food Technology , 43, 148-155). Evenly spray the suspension of Fusarium Graminerum fungus onto PDA solid medium with a hand sprayer and dry at room temperature. Then, sterilized paper disc (Filter paper No. 10 μl (
+++: 성장억제환이 20 mm 이상인 경우, ++: 성장억제환이 10~19 mm 경우, +: 성장억제환이 9 mm 이하인 경우, -: 성장억제환이 없는 경우+++: When growth inhibition ring is 20 mm or more, ++: When growth inhibition ring is 10 to 19 mm, +: When growth inhibition ring is 9 mm or less,-: When there is no growth inhibition ring
표 1에 나타난 바와 같이, 추출물 농도 100 ㎍/종이디스크에서 산초, 당귀, 미국가막사리, 백선피에서 곰팡이 성장억제 효과가 있음을 나타내며, 마이코나졸을 제외한 시판농약은 500 ㎍/종이디스크에서 성장억제효과가 나타나지 않음을 나타낸다. As shown in Table 1, the extract concentration of 100 ㎍ / paper disk shows that the fungus growth inhibitory effect in the pepper, Angelica, American creeper, ringworm skin, commercial pesticides except myconazole, growth inhibitory effect in 500 ㎍ / paper disk Does not appear.
도 2는 산초추출물과 마이코나졸 처리구에서의 생육저지환을 나타내었다. 도 2(가)는 산초의 메탄올추출물(100, 200㎍)을 처리한 경우를, 도 2(나)는 시약급 항진균제인 마이코나졸(10, 20㎍)을 처리한 경우를 나타내었다. 도 2에 나타난 바와 같이, 산초추출물을 종이디스크에 가한 후 생육저지환을 확인하였으며, 마이코나졸을 처리한 경우에는 10㎍ 및 20㎍ 모두에서 커다란 생육저지환이 생성되어 강력한 항균력을 확인하였다. 그러나 마이코나졸의 경우 1그람당 45,000원의 가격으로 실제적 적용의 어려움과 경제성이 인정되지 않고 있다. Figure 2 shows the growth hypoglycemic ring in the extract and myconazole treatment. FIG. 2 (A) shows the case of treating methanol extracts (100, 200 μg) of acid salt, and FIG. 2 (B) shows the case of treating myconazole (10, 20 μg), which is a reagent grade antifungal agent. As shown in Figure 2, after adding the acid extract to the paper disk to confirm the growth-lowering ring, when treated with myconazole, a large growth-lowering ring was generated in both 10㎍ and 20㎍ confirmed the strong antibacterial activity. However, in the case of myconazole, the price of 45,000 won per gram is not recognized for its practical application and economic feasibility.
실시예Example 3: 산초추출물의 조제 및 유기용매에 의한 활성물질 분획 3: Preparation of Acid Extract and Fractionation of Active Substance by Organic Solvent
안동 재래시장에서 구입한 뿌리부분을 제외한 산초나무 음건물(열매껍질, 잎, 가지, 줄기)을 파쇄하고, 각 파쇄물에 1: 10 중량비율로 메탄올, 주정, 에틸아세테이트, 핵산을 각각 가하여 상온에서 24시간 활성물질의 추출을 시도하였다. 물 추출의 경우에는 18시간 동안 상온에서 추출하였으며, 열수추출의 경우에는 100℃에서 4시간 동안 추출하였다. 추출액은 여과지(와트만 여과지 No.2)로 여과 후 감압농축하여 분말화하였다. 감압농축 시 온도는 60 내지 80℃를 유지하며 냉각관을 부착한 감압농축기를 사용하며 시간은 추출용매에 따라 적절히 조절할 수 있다. 조제된 추출물을 실시예 2에서 기술한 디스크법으로 흰가루병 곰팡이의 성장 억제 효과를 측정하였으며, 표 2에는 각각의 추출용매별 추출효율과 종이디스크당 50 ㎍의 추출물을 떨어뜨린 경우 메탄올추출물에서 억제 활성이 나타남을 표시하였다. 따라서 흰가루병 방제용 산초추출물의 조제는 추출효율이 우수하며, 방제효과도 우수한 메탄올추출물이 바람직하다. Crush the vinegar dry matters (berry bark, leaves, branches, stems) except the roots purchased from Andong traditional market, and add methanol, spirit, ethyl acetate and nucleic acid to each crushed substance at a ratio of 1:10 by weight. Attempted extraction of the active material for 24 hours. In the case of water extraction was extracted at room temperature for 18 hours, in the case of hot water extraction was extracted for 4 hours at 100 ℃. The extract was filtered through filter paper (Watman filter paper No. 2), concentrated under reduced pressure, and powdered. Temperature under reduced pressure concentration is maintained at 60 to 80 ℃ using a reduced pressure condenser with a cooling tube, the time can be appropriately adjusted according to the extraction solvent. The prepared extracts were measured for the growth inhibitory effect of powdery mildew fungus by the disk method described in Example 2. Table 2 shows the inhibitory activity in methanol extract when the extraction efficiency of each extractant and 50 ㎍ of extract per paper disk were dropped. It is indicated that this appears. Therefore, methanol extract having excellent extraction efficiency and excellent control effect for powdery mildew extract control is preferable.
메탄올추출물의 활성분획을 얻기 위해 통상의 분획 방법으로 메탄올추출물을 1: 10 중량비로 물에 현탁한 후, 현탁액: 핵산을 1: 5 부피비가 되도록 첨가하여 반복 분획하였다. 이후 핵산 분획을 회수하고, 에틸아세테이트, 부탄올을 각각 사용하여 순차적으로 분획한 후 최종적으로 물 분획을 회수하였다. 회수된 분획물은 상기 기술한 방법으로 감압농축하여 분말화하였고, 이를 DMSO 및 물에 녹여 디스크법으로 흰가루병 곰팡이 성장억제 효과를 측정하였다. In order to obtain an active fraction of the methanol extract, the methanol extract was suspended in water at a ratio of 1: 10 by weight in a conventional fractionation method, and then the fraction was repeatedly added by adding a suspension: nucleic acid at a ratio of 1: 5 by volume. Thereafter, the nucleic acid fractions were recovered, fractionated sequentially using ethyl acetate and butanol, respectively, and finally water fractions were recovered. The recovered fractions were concentrated under reduced pressure by the method described above to be powdered and dissolved in DMSO and water to measure the effect of powdery mildew growth inhibition by the disk method.
표 3에 나타난 바와 같이, 각각의 분획용매별 분획수율과 종이디스크당 50 ㎍의 추출물을 떨어뜨린 경우 물 분획에서 억제 활성이 나타났다. 따라서 흰가루병 방제를 위한 천연물로는 산초나무의 메탄올추출물 및 메탄올추출물의 물 분획물이 가장 바람직함을 확인하였다. As shown in Table 3, when the fraction yield of each fraction solvent and 50 ㎍ of extract per paper disc were dropped, the water fraction showed inhibitory activity. Therefore, as a natural product for the control of powdery mildew, it was confirmed that the methanol extract of the Japanese vinegar and the water fraction of the methanol extract were the most preferable.
+: 성장억제환이 9 mm 이하인 경우, -: 성장억제환이 없는 경우 +: When growth inhibition ring is 9 mm or less,-: When growth inhibition ring does not exist
+: 성장억제환이 9 mm 이하인 경우, -: 성장억제환이 없는 경우 +: When growth inhibition ring is 9 mm or less,-: When growth inhibition ring does not exist
실시예Example 4: 산초 메탄올추출물의 물 4: water from methanol extract 분획물의Fraction 약해 시험 A weak test
토마토 종자(감복제품-완숙토마토, 대목:헬파-M)를 부농상토 3호(부농산업사 제품)에 파종하여, 5엽기까지 온실조건(온도 20∼40℃, 자연광, 자연습도, 수돗물 분무 관수)에서 재배한 다음, 식물생장 조절기(대한랩테크, LS-103-2)로 옮겨 실험에 사용하였다. 이때 식물생장 조절기의 조건은 25∼27℃, 12시간 채광(2,000 Lux, 06:00∼18:00, 암조건 18:00∼06:00), 습도 70%로 조절하였다. 산초 메탄올추출물의 물 분획물을 증류수에 용해하여 500 ㎍/mL, 1,000 ㎍/mL, 10,000 ㎍/mL의 농도로 조제하여 수압식 손분무기를 이용하여 흘러내릴 정도로 분무하고 식물생장조절기에서 재배하였다. 분무 7일 후 약해 정도와 증상을 조사하고 결과를 표 4에 나타내었다. 이때 처리당 반복수는 5반복으로 하였다. Sowing tomato seeds (absorbed products-ripe tomatoes, large tree: Helpah-M) in Bu-Nan-Sang No. 3 (product of Bu-Nong Industrial Co., Ltd.), and greenhouse conditions up to 5 leaves (temperature 20-40 ℃, natural light, natural humidity, tap water spraying) After cultivating in the plant growth regulator (Daelab Lab, LS-103-2) was used for the experiment. At this time, the conditions of the plant growth regulator were adjusted to 25 ~ 27 ℃, 12 hours of light (2,000 Lux, 06:00 ~ 18:00, dark conditions 18:00 ~ 06:00), humidity 70%. Water fractions of the methanol extracts of the vinegar were dissolved in distilled water, prepared at concentrations of 500 ㎍ / mL, 1,000 ㎍ / mL, 10,000 ㎍ / mL, sprayed to a flow rate using a hydraulic hand sprayer, and cultivated in a plant growth regulator. After 7 days of spraying, the degree of weakness and symptoms were investigated and the results are shown in Table 4. The number of repetitions per treatment was 5 repetitions.
실시예Example 5: 식물생장 조절기에서의 산초 메탄올추출물의 물 5: Water of Methanol Extract of Sancho Extract in Plant Growth Regulator 분획물의Fraction 흰가루병 방제 시험 Powdery mildew control test
실시예 4에서 기술한 식물성장조절기에서 토마토 묘목을 이용하여 산초 물 분획물의 흰가루병 방제 활성을 검토하였다. 먼저 흰가루병 곰팡이 현탁액을 수압식 손분무기로 1일 간격으로 5회 분무한 후 식물생장 조절기에서 계속 재배하여 흰가루병을 유발하였다. 한편 산초 물 분획물을 증류수에 용해하여 500 ㎍/mL의 농도로 조제한 다음, 상기 곰팡이 현탁액을 5회 분무한 토마토 묘목에 수압식 손분무기를 이용하여 흘러내릴 정도로 분무하고 식물생장 조절기에서 계속 재배하였다. 대조구로는 다이센 엠 500 ㎍/mL를 처리한 경우 증류수를 분무한 경우로 하였다. 재배 10일 후 흰가루병 발생 정도를 산초 물 분획물 처리구와 다이센 엠 처리구와 비교하였으며, 처리당 반복수는 5반복으로 하였다. 표 5에는 산초 물 분획물과 시판 농약의 흰가루병 방제 효과를, 도 3에는 토마토 잎의 흰가루병 곰팡이의 성장 억제 효과를 나타내었다. 도 3(가)는 흰가루병 곰팡이 분무 후 증류수 분무한 경우(화살표는 곰팡이 균사를 나타냄)를, 3(나)는 흰가루병 곰팡이 분무 후 산초 물 분획물(500 ㎍/mL)을 분무한 경우를, 3(다)는 흰가루병 곰팡이 분무 후 다이센 엠(화학농약 500 ㎍/mL)을 분무한 결과를 나타냈다.In the plant growth regulator described in Example 4, tomato seedlings were used to examine the powdery mildew control activity of the water fraction of the cabbage. First, powdery mildew suspension was sprayed five times at a daily interval with a hydraulic hand sprayer and then cultivated continuously in a plant growth regulator to cause powdery mildew. On the other hand, the acidic water fraction was dissolved in distilled water to prepare a concentration of 500 ㎍ / mL, and then sprayed to the tomato seedlings sprayed 5 times using a hydraulic hand sprayer and continued to grow in a plant growth regulator. As a control, the case of treatment with Daisen M 500 μg / mL was the case of spraying distilled water. The incidence of powdery mildew after 10 days of cultivation was compared with that treated with Sancho water fraction and Daisen M treatment, and the number of repetitions per treatment was 5 repetitions. Table 5 shows the powdery mildew control effect of the water fraction and the commercial pesticides, and Figure 3 shows the growth inhibitory effect of powdery mildew fungus of tomato leaves. Figure 3 (a) is a case of spraying distilled water after spraying powdery mildew fungi (arrow indicates the fungus mycelia), 3 (b) is a case of spraying the water of the acetic acid water (500 ㎍ / mL) after spraying powdery mildew fungi, 3 ( C) shows the result of spraying Daisen M (chemical pesticide 500 ㎍ / mL) after powdery mildew spray.
방제가(%)=(1-처리구에서의 병반면적율/무처리구에서의 병반면적율)x100 Control value (%) = (Length area ratio in 1-treatment / Lesion area ratio in untreated) x100
표 5에 나타난 바와 같이, 산초 물 분획물과 농약을 살포한 경우에는 발병이 거의 나타나지 않았다. 또한, 도 3에서는 잎을 육안 및 현미경 관찰한 경우 증류수를 분무한 대조구에서만 곰팡이가 확인되었다. As shown in Table 5, the incidence was rarely observed when sprayed with the water fraction and the pesticides. In addition, the mold was confirmed only in the control sprayed distilled water when the leaves were visually and microscopically observed in FIG.
실시예Example 6: 산초나무의 메탄올추출물 및 각종 6: Methanol extract and various 분획물의Fraction 식중독세균, 병원성 세균의 증식억제 효과 Growth Inhibition Effect of Food Poisoning Bacteria and Pathogenic Bacteria
산초나무의 메탄올추출물과 추출물의 다양한 분획물을 대상으로 항세균 활성을 검토하였다. 식중독균인 스타필로코커스 아우레우스(Staphylococcus aureus) KACC 10196, 살모넬라 티피무리움(Salmonella typhimurium) KCTC 1926, 프로테우스 불가리스(Proteus vulgaris) KCTC 2433, 에스케리치아 콜라이(Escherichia coli) H7;O157 균과 아토피 관련 병원성균인 슈도모나스 애어로기노사(Pseudomonas aeroginosa) KACC 10186, 충치원인균인 스트렙토코커스 무탄스(Streptococcus mutans) JC-2(원광대학교 치과대학 구강미생물 실험실에서 분양)의 Nutrient broth(미국 디프코사)배양액을 원심집균하여 수세하고, 이들 각각 균의 증류수 현탁액(세포농도: ∼109/mL)을 조제하여 각각 Nutrient Agar(미국 디프코사)배지에 도말하여 건조 후, 상기의 디스크 방법과 동일하게 멸균 종이 디스크를 올린 후 각각의 추출물, 분획물을 DMSO 또는 물에 적당한 농도로 녹여 종이디스크당 250 ㎍가 되도록 떨어뜨려 37℃에서 2일간 배양하여 생육억제환 크기를 측정하였다. 대조구로는 스트렙토마이신 설페이트(미국 시그마사)를 종이디스크에 10 ㎍가 되도록 떨어뜨렸다. The antibacterial activity of methanol extracts and various fractions of extracts from Japanese vinegar was examined. Sikjungdokgyun of Staphylococcus aureus (Staphylococcus aureus) KACC 10196, Salmonella typhimurium (Salmonella typhimurium) KCTC 1926, Proteus vulgaris (Proteus vulgaris) KCTC 2433, Escherichia coli (Escherichia coli ) H7; O157 Nutrient broth of bacteria and atopy-related hospital seonggyunin Pseudomonas kids fishing machine labor (Pseudomonas aeroginosa) KACC 10186, dental caries pathogen is Streptococcus free Tansu (Streptococcus mutans) JC-2 (pre-sale at Wonkwang University College of Dentistry, Oral microbiology laboratory) ( Centrifugal centrifugation was carried out to wash the culture medium of the US Dipco Co., distilled water suspensions (cell concentration: ˜10 9 / mL) of these bacteria were prepared, each plate was plated on a Nutrient Agar medium (Dipco Co., Ltd.) and dried, and then the above disk method. As in the same manner, after raising the sterile paper disk, each extract, fractions were dissolved in DMSO or water at a suitable concentration drop to 250 ㎍ per paper disk and cultured for 2 days at 37 ℃ to measure the growth inhibitory ring size. As a control, streptomycin sulfate (Sigma, USA) was dropped to 10 [mu] g on a paper disk.
-: 무활성 -: Inactive
표 6에 나타난 바와 같이, 메탄올추출물과 다양한 분획물이 상기의 병원성세균, 식중독세균의 생육을 억제함을 나타내며, 특히 핵산 분획물과 물 분획물에서 광범위한 항세균 활성을 나타내었다. 이러한 결과는 산초추출물을 사용하는 경우 흰가루병의 방제와 함께 재배작물의 과실의 세균성 위해를 제고할 수 있음을 나타낸다.As shown in Table 6, methanol extracts and various fractions inhibit the growth of the pathogenic bacteria, food poisoning bacteria, and showed a wide range of antibacterial activity, especially in nucleic acid fractions and water fractions. These results indicate that the use of the herbaceous extract can enhance the bacterial risk of fruit crops along with the control of powdery mildew.
상기 실시예를 통해 살펴본 바와 같이, 본 발명은 산초나무 추출물을 포함하는 흰가루병 방제용 천연 항균조성물에 관한 것으로, 산초 추출물 및 산초 메탄올 추출물의 물 분획물은 인체 및 가축에 무해하면서 흰가루병의 발생을 억제하여, 흰가루병 방제를 위한 과도한 화학농약 사용의 문제점을 보완하며, 토마토, 딸기 등의 시설재배작물의 안전성과 상품성을 증대시키는 뛰어난 효과를 가진다. 또한 본 발명의 산초 추출물 및 분획물은 식중독세균 및 병원성세균의 성장을 억제시키는 효과를 나타내어 상품의 세균성 위해를 방지할 수 있어 원예 및 시설재배산업상에 매우 유용한 발명이다.As described through the above embodiment, the present invention relates to a natural antimicrobial composition for powdery mildew disease control comprising an extract of the vinegar, water fractions of the extract and the methanol extract of methanolic acid extracts are harmless to humans and livestock while suppressing the occurrence of powdery mildew. In addition, it supplements the problem of excessive use of chemical pesticides to control powdery mildew, and has an excellent effect of increasing the safety and marketability of plant cultivation crops such as tomatoes and strawberries. In addition, the extract and fractions of the present extract of the present invention exhibits the effect of inhibiting the growth of food poisoning bacteria and pathogenic bacteria can prevent bacterial risks of the product is a very useful invention for horticulture and facility cultivation industry.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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KR101386228B1 (en) * | 2012-10-04 | 2014-04-17 | 국민대학교산학협력단 | Extracts from Angelica gigas using methanol and ethyl acetate for inhibition of biofilm formation of microbes |
CN112521443A (en) * | 2021-01-13 | 2021-03-19 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
SE2030378A1 (en) * | 2020-12-28 | 2022-06-29 | Taha Adam | The Ultimate solution for the Powdery mildew |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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KR101386228B1 (en) * | 2012-10-04 | 2014-04-17 | 국민대학교산학협력단 | Extracts from Angelica gigas using methanol and ethyl acetate for inhibition of biofilm formation of microbes |
SE2030378A1 (en) * | 2020-12-28 | 2022-06-29 | Taha Adam | The Ultimate solution for the Powdery mildew |
SE545328C2 (en) * | 2020-12-28 | 2023-07-04 | Taha Adam | A new fungicde formulation and method for treating powdery mildew |
CN112521443A (en) * | 2021-01-13 | 2021-03-19 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
CN112521443B (en) * | 2021-01-13 | 2024-03-26 | 昆明理工大学 | Preparation method and application of pseudo-ginseng flower protein |
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