KR20130050698A - Composition containing fermented extract of epicarp of ginkgo biloba for controlling plant diseases - Google Patents
Composition containing fermented extract of epicarp of ginkgo biloba for controlling plant diseases Download PDFInfo
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Abstract
Description
본 발명은 은행 외과피를 포함하는 배지에 발효 미생물을 접종하여 발효한 은행 외과피 발효물을 추출하여 얻은 은행 외과피 발효추출물을 유효성분으로 함유하는 것을 특징으로 하는 식물병 방제용 조성물에 관한 것으로, 본 발명의 식물병 방제용 조성물은 보리 흰가루병, 밀 붉은녹병, 토마토 역병 또는 토마토 잿빛곰팡이병에 매우 뛰어난 방제 효과를 가진다.
The present invention relates to a plant disease control composition comprising the ginkgo biloba fermented extract obtained by extracting the fermented ginkgo biloba fermented by inoculating fermentation microorganisms in a medium containing ginkgo biloba as an active ingredient, the present invention The composition for controlling plant diseases of barley powdery mildew, wheat red rust disease, tomato blight or tomato ash fungus disease has an excellent control effect.
작물 재배시 많은 식물병원균, 해충 및 잡초들이 작물의 생장에 저해를 일으켜, 이들에 대한 방제를 실시하지 않을 경우 작물에 따라서는 30 ~ 100%의 수확량 감소가 야기된다. 이러한 유해 생물을 방제하기 위하여 지금까지 많은 합성농약들이 개발되고 사용되어 왔으나 식물병, 해충 및 잡초방제에 오남용됨에 따라 인축에 대한 독성, 지하수 오염, 환경오염, 생태계 교란, 저항성 유해생물 증가 등 여러 가지 문제점들이 야기되고 있다. 이에 따라 우리나라를 포함한 OECD 가입국들을 중심으로 합성농약의 사용량을 40% 감축하고자 하는 친환경농업정책을 시행 중에 있다. 이러한 정책의 일환으로, 합성농약의 대안으로 떠오르는 기술이 바로 생물농약이다.Many phytopathogens, pests and weeds inhibit crop growth during crop cultivation, resulting in a yield reduction of 30 to 100%, depending on the crop if not controlled. Many synthetic pesticides have been developed and used to control these harmful organisms until now, but as they are misused for controlling plant diseases, pests and weeds, they are toxic to livestock, groundwater pollution, environmental pollution, ecosystem disturbance, and resistance to harmful organisms. Problems are causing. As a result, eco-friendly agricultural policies are being implemented to reduce the use of synthetic pesticides by 40%, especially among OECD member countries including Korea. As part of this policy, bio-pesticides are emerging as an alternative to synthetic pesticides.
생물농약에는 미생물농약과 생화학농약이 있으며, 생물농약은 환경친화적 작물보호제로서 합성농약과는 달리 인축 및 생태계에 안전하여 부가가치가 높은 농산물을 생산할 수 있고, 합성농약으로 방제하기 어려운 병해충을 효과적으로 방제하여 안전한 농작물의 생산을 증대시킴과 동시에 합성농약의 오남용으로 인한 문제점을 해결할 수 있다. 국내의 경우 미생물농약에 대한 연구는 많이 이루어져 등록된 미생물농약이 다수 있지만, 천연물을 이용한 생화학농약의 경우 2005년에 등록규정이 마련되었고, 현재 식물유래 azadirachtin을 이용한 살충제(제품명: 단독) 1개와 식물유래 지방산 perlagonic acid를 이용한 제초제(제품명: 싸이티)가 등록되어 시판되고 있다. 하지만 두 제품 모두 외국에서 개발한 제품을 단지 국내에 등록한 것이며, 자체 개발한 기술은 아니다.Bio pesticides include microbial pesticides and biochemical pesticides, which are environmentally friendly crop protection agents. Unlike synthetic pesticides, biopesticides can produce high value-added agricultural products because they are safe for human beings and ecosystems, and effectively control pests that are difficult to control with synthetic pesticides. Increase the production of safe crops and at the same time solve the problems caused by misuse of synthetic pesticides. In Korea, there are many researches on microbial pesticides, and there are many registered microbial pesticides. However, in the case of biochemical pesticides using natural products, registration regulations were established in 2005. Currently, one pesticide using azadirachtin (product name: single) and a plant Herbicides using the derived fatty acid perlagonic acid (product name: Cyti) are registered and marketed. However, both products are only registered products developed in foreign countries, not their own technology.
전 세계적으로 식물 추출물들을 이용하여 상품화에 성공하여 시판되고 있는 살균제 제품은 2004년 현재 5개이며, 가장 대표적인 것이 밀사나(Milsana™)이다. 이 제품은 왕호장근(Reynouria sachalinensis)의 추출물을 이용하여 개발된 친환경살균제로서, 식물병원균을 직접 치사시키는 효과보다는 식물체에 저항성을 유도함으로써 흰가루병에 대한 방제효과를 발휘하는 것으로 나타났다. 또한, 천연물 살균제로서 등록되어 있는 신남알데하이드(cinnamaldehyde)는 카시아 식물체(Cassia tora)의 종자로부터 추출되는 오일로서 살충활성 뿐만 아니라 버티실리엄(Verticillium) 속, 리족토니아(Rhizoctonia) 속, 피티엄(Pythium) 속 및 후사리움 모니리폼(Fusarium moniliforme var. subglutinans)(근류병: canker disease) 등의 식물병원균들에 대해서도 살균활성이 있는 것으로 보고되어 있다. 또한, 호호바(jojoba) 종자로부터 추출되는 호호바 오일은 살충활성 뿐만 아니라 흰가루병에 대하여 살균활성이 있다. 마클레아이아(Macleaya) 속 추출물은 죽자초(Macleaya cordata) 추출물로서 흰가루병과 알터나리아(Alternaria) 속 잎점무늬병과 셉토리아(Septoria) 속 잎점무늬병 등에 효과가 있다. 또한, 올레산을 주성분으로 하는 지방산도 흰가루병에 효과가 있다.There are five fungicide products on the market that have been successfully commercialized using plant extracts worldwide. As of 2004, Milsana ™ is the most representative. This product is an eco-friendly disinfectant developed using extracts of Reynouria sachalinensis. It has been shown to exert resistance to plant diseases by inducing resistance to plants rather than directly killing plant pathogens. In addition, cinnamaldehyde, which is registered as a natural fungicide, is an oil extracted from seeds of Cassia tora, as well as insecticidal activity, genus Verticillium, Rhizoctonia, and Pythium. It is reported to have bactericidal activity against phytopathogens such as genus and Fusarium moniliforme var.subglutinans (canker disease). In addition, jojoba oil extracted from jojoba seeds is not only insecticidal but also has bactericidal activity against powdery mildew. Macleaya genus extract is Macleaya cordata extract and is effective in powdery mildew, Alternaria leaf spots and Septoria leaf spots. In addition, fatty acids based on oleic acid are also effective in powdery mildew.
이에 본 발명자들은 천연추출물 및 물질을 이용하여 인체에 무해하면서 환경친화적인 천연물 살균제를 개발하기 위하여 연구 노력한 결과, 은행 외과피 발효추출물이 다양한 식물병에 대하여 높은 방제활성을 나타냄을 발견하고, 이들을 식물병 방제를 위한 천연물 살균제로 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.
Accordingly, the present inventors have conducted research to develop a natural fungicide that is harmless to the human body using natural extracts and materials, and found that ginkgo biloba fermented extract exhibits high control against various plant diseases, and these are plant diseases. The present invention was completed by confirming that it can be used as a natural product disinfectant for control.
본 발명은 보리 흰가루병, 밀 붉은녹병, 토마토 역병 또는 토마토 잿빛곰팡이병에 적용이 가능하고 인체에 무해하여 안전성이 매우 뛰어난 은행 외과피 발효추출물 및 이를 유효성분으로 함유하는 식물병 방제용 조성물을 제공하는 것을 목적으로 한다.The present invention is applicable to barley powdery mildew, wheat rust disease, tomato late blight or tomato ash fungus disease and harmless to the human body to provide a ginkgo biloba fermented extract with excellent safety and a plant disease control composition containing the same as an active ingredient The purpose.
또한, 본 발명은 식물병에 대한 방제활성이 매우 뛰어난 은행 외과피 발효추출물 및 이를 유효성분으로 함유하는 식물병 방제용 조성물을 제공하는 것을 목적으로 한다.
In addition, an object of the present invention is to provide a ginkgo biloba fermented extract having excellent control against plant diseases and a plant disease control composition containing the same as an active ingredient.
상기의 목적을 달성하기 위해, 본 발명은 은행 외과피를 포함하는 배지에 효모, 유산균류 및 바실러스속 균류로 이루어진 군에서 선택되는 1종 이상의 발효 미생물을 접종하여 1~7일간 20~40℃에서 pH 5~7로 유지하며 발효한 은행 외과피 발효물을 추출하여 얻은 은행 외과피 발효추출물을 유효성분으로 함유하는 식물병 방제용 조성물을 특징으로 한다.In order to achieve the above object, the present invention is inoculated with at least one fermentation microorganism selected from the group consisting of yeast, lactic acid bacteria and Bacillus fungi in a medium containing ginkgo biloba blood pH at 20 ~ 40 ℃ for 1-7 days Characterized in that the composition for controlling plant diseases containing ginkgo biloba fermented extract obtained by extracting the fermented ginkgo biloba fermentation fermented to maintain 5 ~ 7 as an active ingredient.
또한, 본 발명에 있어서, 상기 은행 외과피 발효추출물은 상기 은행 외과피 추출물을 정제수, 메탄올, 에탄올, 글리세린, 에틸아세테이트, 부틸렌글리콜, 프로필렌글리콜, 디클로로메탄 및 헥산으로 이루어진 군에서 선택된 1종 이상의 추출용매로 추출한 후 50℃ 이하에서 감압농축 또는 동결건조하여 얻은 감압농출물 또는 동결건조물인 것을 특징으로 한다.In addition, in the present invention, the ginkgo biloba fermentation extract is one or more extracting solvents selected from the group consisting of the ginkgo biloba extract extract from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane After extracting to 50 ℃ or less concentrated or lyophilized dried under reduced pressure, characterized in that the freeze-dried.
또한, 본 발명에 있어서, 상기 은행 외과피 발효추출물은 상기 감압농축물 또는 동결건조물을 정제수, 에탄올, 부틸렌글리콜 및 프로필렌글리콜로 이루어진 군에서 선택된 1종 이상의 용매에 0.001~70.0중량% 함유시킨 것을 특징으로 한다.In addition, in the present invention, the ginkgo biloba fermentation extract is characterized in that the decompression concentrate or lyophilisate contained 0.001 ~ 70.0% by weight in at least one solvent selected from the group consisting of purified water, ethanol, butylene glycol and propylene glycol. It is done.
또한, 본 발명에 있어서, 상기 은행 외과피 발효추출물을 조성물 전체에 대하여 동결건조중량 기준으로 0.0001 내지 30.0 중량% 함유하는 것을 특징으로 한다.In addition, in the present invention, the ginkgo biloba fermented extract is characterized in that it contains 0.0001 to 30.0% by weight based on the lyophilized weight based on the whole composition.
이하, 본 발명의 과제의 해결 수단에 대해 상세히 설명하고자 한다.Hereinafter, the means for solving the problems of the present invention will be described in detail.
은행은 10월경에 은행나무 암그루에 지름 2cm 정도의 황색 열매로 열려서 익게 되는데, 열매 바깥쪽의 외과피(外果皮)는 다육성이고 외과피의 안쪽에 2개의 모서리가 있는 흰색의 단단한 중과피를 가진 달걀 모양 원형의 종자가 있는데, 이것을 은행 또는 백자(白子)라 한다. 중과피 안쪽에 갈색피막의 내종피가 있고 이속에 청록색의 배젖이 있다. 은행은 맛과 향이 뛰어나고 영양이 풍부하여 각종 음식의 부재료로 많이 쓰이고 있고, 생리활성 성분인 지베렐린, 사이토키닌, 카로틴, 비타민B2, 칼륨, 인, 철 뿐만 아니라 엘고스테린, 비타민B12를 만드는 데 필요한 시안(cyanogen)을 포함하고 있는 청산배당체 등의 성분 함유하고 있어 폐의 보호 기능강화, 피부진정효과, 가래, 천식, 숙취, 전신 피로 회복, 여성 대하증, 어린이 야뇨증 치료 등에 활용된다고 알려져 있다.The ginkgo is ripened in October by ginkgo biloba with yellow fruit about 2cm in diameter. The outer skin of the fruit is fleshy, and the egg shape has white hard membranous skin with two corners inside the surgical skin. There are circular seeds, which are called ginkgo or white porcelain. Inside the mesenchyme, there is a brown mesothelial epidermis, and there is a cyan belly. Ginkgo biloba is excellent in taste, aroma, and rich in nutrition, and it is widely used as an ingredient in various foods, and it is used to make bioactive ingredients such as gibberellin, cytokinin, carotene, vitamin B2, potassium, phosphorus, iron, as well as elgosterin and vitamin B12 It contains ingredients such as cyanogen, which contains necessary cyanogen, and is known to be used for strengthening the protective function of the lungs, soothing effects of skin, sputum, asthma, hangover, recovery from systemic fatigue, fevers and children's enuresis.
본 발명에서 사용되는 은행 외과피는 긴고릭산(ginkgolic acid)을 함유하고 있어 결핵균에 대한 항균 활성을 가지지만, 피부 알레르기를 일으키는 독성이 있는 바이로볼(bilobol)(C21H34O2)과 독성은 없지만 역시 피부 알레르기를 일으키는 긴콜(ginkgol)을 함유하고 있고, 특유의 떫은 맛과 암모니아 냄새와 유사한 특유의 악취가 있어 이용되지 않고 버려지고 있다. Bank ginseng used in the present invention contains ginkgolic acid (ginkgolic acid) has antimicrobial activity against Mycobacterium tuberculosis, but it is not virulent (bilobol) (C21H34O2) and toxic but also skin allergic to skin allergies It contains ginkgol, which has a peculiar astringent taste and a characteristic odor similar to that of ammonia and is discarded without being used.
현재까지 은행 외과피 발효추출물의 식물병 방제효과 등에 관한 연구는 이루어지지 않았으며 더욱이 본 발명에서와 같이 미생물 발효를 통한 은행 외과피 발효추출물이 기존 은행 외과피 추출물보다 피부 자극이 없으면서 우수한 효과를 갖는다는 것을 기재한 사실은 없다.To date, no research has been conducted on plant disease control effects of ginkgo biloba fermented extracts, and furthermore, as described in the present invention, it is described that ginkgo biloba fermented extracts through microbial fermentation have superior effects without skin irritation than conventional ginkgo biloba extracts. There is no fact.
본 발명에 있어서, 발효 추출의 재료가 되는 은행 외과피는 이를 건조하여 사용하거나 또는 그대로 사용할 수 있으며, 바람직하게는 건조하여 사용하는 것이다. 건조법으로는 자연 건조, 음지 건조, 열풍 건조, 진공건조 또는 동결 건조법을 이용할 수 있다. 또한, 추출을 용이하게 하기 위하여 은행 외과피는 분쇄하여 사용할 수도 있다.In the present invention, the ginkgo scavenger serving as the material for fermentation extraction may be dried or used as it is, and preferably dried and used. As a drying method, natural drying, shade drying, hot air drying, vacuum drying or freeze drying may be used. In addition, in order to facilitate extraction, the ginkgo biloba may be ground and used.
또한, 본 발명에서 사용하는 발효 미생물은 효모, 유산균류 및 바실러스 속 균류로 이루어진 군에서 선택되는 1종 이상이고, 사카로미세스(Saccharomyces) 속[Saccharomyces cerevisisae KCTC 7904] 균주가 바람직하다. 발효는 1~7일간 20~40℃에서 pH 5~7로 유지하며 발효하는 것이 바람직하다.In addition, the fermentation microorganism used in the present invention is one or more selected from the group consisting of yeast, lactic acid bacteria and Bacillus genus fungi, Saccharomyces genus [Saccharomyces cerevisisae KCTC 7904] strain is preferred. Fermentation is preferably maintained at pH 5-7 at 20-40 ° C. for 1-7 days.
또한, 본 발명에 사용되는 추출용매는 특정의 종류에 한정하는 것은 아니나, 바람직하게는 정제수, 메탄올, 에탄올, 글리세린, 에틸아세테이트, 부틸렌글리콜, 프로필렌글리콜, 디클로로메탄, 헥산 및 그의 혼합물로부터 선택된 것이 좋다.In addition, the extraction solvent used in the present invention is not limited to a specific kind, but is preferably selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane and mixtures thereof good.
또한, 본 발명에 사용된 은행 외과피 발효추출물은 추출액을 일부 또는 전부 농축하여 얻은 농축물, 또는 다시 그 농축물을 건조시켜 제조한 추출 엑기스 및 추출액 중에 함유되고 있는 주 효과를 발휘하는 화학물질 그 자체를 포함한다.In addition, the ginkgo biloba fermented extract used in the present invention is a concentrate obtained by partially or fully concentrating the extract, or an extract extracted by drying the concentrate and a chemical substance having the main effect contained in the extract itself. It includes.
또한, 본 발명의 은행 외과피 발효추출물의 제조방법의 일 예는 다음과 같다. 본 발명의 은행 외과피는 충분히 세척한 다음 분쇄한 후 100메시 크기의 체를 이용하여 미세하게 만든다. 이것을 100g/L되게 정제수에 은행 외과피 분쇄물을 섞어주고, 사카로미세스(Saccharomyces) 속[Saccharomyces cerevisisae KCTC 7904] 균주 배양액에 첨가한다. 여기에 탄소원으로는 포도당 1~4%를 사용하며 바람직하게는 1~2%를 첨가하고, 펩톤 0.1~1%를 첨가하며 바람직하게는 0.2~0.5%를 첨가하여 배양한다. 배양은 5L 발효조를 이용하여 1~7일간, 37℃, pH 5-7로 유지하며 배양한다. 배양 후 배양액을 원심분리하여 배양균을 1차 제거한다. 발효 후 배양균을 1차 제거한 은행 외과피 발효물을 최종 70%(V/V) 에탄올 수용액이 되도록 에탄올을 첨가하여 5시간씩 3회 환류추출하고 냉침한 후, 와트만(whatman) #10 여과지로 여과한다. 이렇게 얻은 여과액을 다시 0.25uM의 여과기를 이용하여 최종 여과하고 여과가 완료되면 상기 추출단계에서 얻어진 추출액을 농축조로 이송하여 50℃ 이하에서 감압농축 또는 동결 건조하였다. 이렇게 얻은 감압농축물 또는 동결건조물이 0.001~70.0 중량% 함유되게 정제수, 에탄올, 부틸렌글리콜, 프로필렌글리콜 중에서 선택된 1종 이상의 용매를 사용하여 혼합추출물을 제조한다.In addition, an example of a method for producing ginkgo biloba fermented extract of the present invention is as follows. Ginkgo bleeding blood of the present invention is sufficiently washed and then ground to make fine using a 100 mesh sieve. This is mixed with 100 g / L of purified ginkgo crushed groundwater in purified water and added to Saccharomyces (Saccharomyces cerevisisae KCTC 7904) strain culture. Here, 1 to 4% of glucose is used as a carbon source, preferably 1 to 2% is added, 0.1 to 1% of peptone is added, and preferably 0.2 to 0.5% is added to the culture. Cultivation is carried out using a 5L fermenter for 1-7 days at 37 ℃, pH 5-7. After incubation, the culture medium is centrifuged to remove the culture medium first. After fermentation, the ginkgo septic fermentation product from which culture was first removed was refluxed three times for 5 hours by adding ethanol to the final 70% (V / V) ethanol aqueous solution, and cooled, and then washed with Whatman # 10 filter paper. Filtered. The filtrate thus obtained was finally filtered again using a filter of 0.25 uM, and when the filtration was completed, the extract obtained in the extraction step was transferred to a concentration tank and concentrated under reduced pressure or freeze-dried at 50 ° C. or lower. The mixed extract is prepared by using at least one solvent selected from purified water, ethanol, butylene glycol, and propylene glycol so that the obtained vacuum concentrate or lyophilized product is contained in an amount of 0.001 to 70.0% by weight.
또한, 본 발명의 은행 외과피 발효추출물은 특정의 범위에 한정하는 것은 아니나, 식물병 방제용 조성물 총중량에 대해 동결건조중량 기준으로 0.0001~30중량% 함유되는 것이 바람직하다. 본 발명의 은행 외과피 발효추출물이 0.0001중량% 미만으로 첨가하는 경우에는 그 효과를 기대하기 힘들고, 30중량%를 초과하여 첨가되는 경우에는 뚜렷한 효능·효과의 상승이 보이지 않는다.In addition, ginkgo biloba fermented extract of the present invention is not limited to a specific range, it is preferably contained 0.0001 to 30% by weight based on the lyophilized weight relative to the total weight of the composition for plant disease control. When the ginkgo biloba fermentation extract of the present invention is added at less than 0.0001% by weight, the effect is hard to expect, and when added in excess of 30% by weight, no obvious increase in efficacy and effect is seen.
또한, 본 발명에 따른 식물병 방제용 조성물은 상기의 추출물 외에 본 발명이 목적으로 하는 주 효과를 손상시키지 않는 범위 내에서 바람직하게는 주 효과에 상승효과를 줄 수 있는 다른 성분 등을 함유하는 것도 무방하다.In addition, the composition for controlling plant diseases according to the present invention, in addition to the above-mentioned extract, may also contain other components, etc., which can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect of the present invention. It's okay.
한편, 본 발명의 방제용 조성물은 통상적으로 이용되는 살충제 또는 살균제에 함유되는 물질을 추가로 포함할 수 있으며, 상기 활성성분 이외에 부형제로 약제학적으로 허용 가능한 고체 담체, 액체 담체, 액체 희석제, 액화된 기체 희석제, 고체 희석제, 또는 기타 적당한 보조제, 예를 들면 유화제, 분산제 또는 기포제 등의 계면활성제를 추가로 포함할 수 있다. 활성성분과 상기 부형제를 혼합한 방제용 조성물을 농약분야에 공지된 다양한 제형으로 제제화시켜 사용할 수 있으며, 제제화를 위해서는 농약분야에서 통상적으로 사용되는 제제화 방법을 어느 것이나 사용할 수 있다.On the other hand, the control composition of the present invention may further include a substance contained in a conventionally used insecticide or fungicide, and in addition to the active ingredient, a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied as an excipient Surfactants such as gas diluents, solid diluents, or other suitable auxiliaries, such as emulsifiers, dispersants or foaming agents. The composition for controlling the active ingredient and the excipients may be formulated into various formulations known in the pesticide field. For formulation, any formulation method commonly used in the pesticide field may be used.
본 발명의 방제용 조성물은 바람직하게는 수화제, 입제, 분제, 유제, 스프레이상, 연막제, 캅셀형 및 젤상의 제형으로 제제화될 수 있고, 제제의 부력을 위해 도넛형과 같은 제형을 통한 접촉제로서 제공되는 것이 바람직하다.The control composition of the present invention may preferably be formulated in a hydrated, granulated, powdered, emulsified, sprayed, smokescreen, capsule and gelled formulation and contacted through a formulation such as a donut for buoyancy of the formulation. Preferably provided as.
본 발명의 방제용 조성물은 보리 흰가루병, 밀 붉은녹병, 토마토 역병 및 토마토 잿빛곰팡이병을 효과적으로 방제하기 위하여 은행 외과피 발효추출물은 식물병 방제용 조성물에 0.0001 내지 30.0중량%의 농도로 포함하는 것이 바람직하나, 이는 작물의 종류, 생육 정도, 경작지 환경, 식물병의 발병 정도 등을 고려하여 적절하게 조절할 수 있다.The control composition of the present invention, in order to effectively control powdery mildew, wheat red rust, tomato blight and tomato ash fungal disease, ginkgo biloba fermented extract is preferably included in a concentration of 0.0001 to 30.0% by weight in the composition for plant disease control This may be appropriately adjusted in consideration of the type of crop, the degree of growth, the cultivated environment, and the degree of development of plant diseases.
상기와 같이 제형화된 본 발명의 방제용 조성물을 방제가 필요한 식물체 또는 지역에 처리함으로써 보리 흰가루병, 밀 붉은녹병, 토마토 역병 및 토마토 잿빛곰팡이병을 방제할 수 있다. 바람직한 방법은 상기 방제용 조성물을 식물 병원균과 직접 접촉할 수 있도록 적용하여 방제하는 것으로, 토양에 혼화 처리하거나 작물에 직접 분무하는 방법이 가장 바람직하다.
Barley powdery mildew, wheat red rust, tomato blight and tomato gray mold disease can be controlled by treating the control composition of the present invention formulated as described above to plants or areas requiring control. The preferred method is to apply the control composition so as to be in direct contact with the plant pathogens to control, the most preferred is a method of mixing the soil or spraying directly to the crop.
본 발명은 보리 흰가루병, 밀 붉은녹병, 토마토 역병 또는 토마토 잿빛곰팡이병에 적용이 가능하고 인체에 무해하여 안전성이 매우 뛰어난 은행 외과피 발효추출물 및 이를 유효성분으로 함유하는 식물병 방제용 조성물을 제공할 수 있다.The present invention can be applied to barley powdery mildew, wheat rust disease, tomato late blight or tomato ash fungus disease and can provide a composition for controlling ginkgo biloba fermentation extract having excellent safety and harmless to human body and plant disease containing the same as an active ingredient. have.
또한, 본 발명은 식물병에 대한 방제활성이 매우 뛰어난 은행 외과피 발효추출물 및 이를 유효성분으로 함유하는 식물병 방제용 조성물을 제공할 수 있다.
In addition, the present invention can provide a ginkgo biloba fermented extract having excellent control against plant diseases and a plant disease control composition containing the same as an active ingredient.
이하, 본 발명에 대해 하기 실시예에서 더욱 상세히 설명하지만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 이와 등가의 기술적 사상의 변형까지 모두 포함한다.Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes all modifications of equivalent technical spirit.
제조예Manufacturing example 1: 은행 1: bank 외과피Surgical 발효추출물의 제조 Fermentation Extract
은행 외과피는 충분히 세척한 다음 모두 분쇄한 후 100메시의 체를 이용하여 미세하게 만들었다. 이것을 100g/L되게 정제수에 은행 외과피 분쇄물을 섞어주고, Saccharomyces 속 (Saccharomyces cerevisisae KCTC 7904) 균주 배양액에 첨가하였다. 여기에 탄소원으로 포도당 1~4%를 첨가하였으며, 펩톤 0.2~0.5%를 추가로 첨가하여 배양하였다. 배양은 5L 발효조를 이용하여 1~7일간, 37도, pH 5-7로 유지하며 배양하였다. 배양 후 배양액을 원심분리하여 배양균을 1차 제거하였다.The ginkgo bleeding was thoroughly washed and then pulverized and then finely made using a 100 mesh sieve. This was mixed with 100 g / L of purified ginkgo crushed water in purified water and added to Saccharomyces (Saccharomyces cerevisisae KCTC 7904) strain culture. 1 to 4% of glucose was added as a carbon source, and 0.2 to 0.5% of peptone was further added and cultured. The culture was maintained for 1-7 days, 37 degrees, pH 5-7 using a 5L fermenter. After the culture, the culture medium was centrifuged to remove the culture medium first.
발효 후 배양균을 1차 제거한 은행 외과피 발효물을 최종 70%(V/V) 에탄올 수용액이 되도록 에탄올을 첨가하여 5시간씩 3회 환류추출하고 냉침한 후, 와트만(whatman) #10 여과지로 여과하였다. 이렇게 얻은 여과액을 다시 0.25uM의 여과기를 이용하여 최종 여과하였다. 여과가 완료되면 상기 추출단계에서 얻어진 추출액을 농축조로 이송하여 50℃ 이하에서 감압농축 또는 동결 건조하였다. 감압농축물 또는 동결건조물이 0.001~70.0 중량% 함유되게 정제수, 에탄올, 부틸렌글리콜, 프로필렌글리콜 중에서 선택된 1종 이상의 용매를 사용하여 혼합추출물을 제조하였다.
After fermentation, the ginkgo septic fermentation product from which culture was first removed was refluxed three times for 5 hours by adding ethanol to the final 70% (V / V) ethanol aqueous solution, and cooled, and then washed with Whatman # 10 filter paper. Filtered. The filtrate thus obtained was finally filtered using a filter of 0.25 uM. After filtration was completed, the extract obtained in the extraction step was transferred to a concentration tank and concentrated under reduced pressure or freeze-dried at 50 ° C or lower. Mixed extracts were prepared using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol to contain 0.001-70.0 wt% of the reduced pressure concentrate or lyophilisate.
비교 compare 제조예Manufacturing example 1: 은행 1: bank 외과피Surgical 추출물의 제조 Preparation of extract
상기 제조예 1과 동일한 은행 외과피를 시료로 선별하여 세척한 다음 분쇄한 후 100메시의 체를 이용하여 미세하게 만들었다. 은행 외과피 분쇄물을 100g/L가 되도록 70% 에탄올을 첨가하여 5시간씩 3회 환류추출하고 냉침한 후, 와트만(whatman) #10 여과지로 여과하였다. 이렇게 얻은 여과액을 다시 0.25uM의 여과기를 이용하여 최종 여과하였다. 여과된 추출물을 50℃ 이하에서 감압농축 및 동결 건조하였다. 감압농축물 및 동결건조물이 0.001~70.0 중량% 함유되게 정제수, 에탄올, 부틸렌글리콜, 프로필렌글리콜 중에서 선택된 1종 이상의 용매를 사용하여 혼합 추출물을 제조하였다.
The same bank surgical skin as in Preparation Example 1 was selected and washed as a sample, and then pulverized and finely made using a 100 mesh sieve. The ginkgo pulverized crushed powder was refluxed three times for 5 hours by adding 70% ethanol to 100 g / L, cooled, and filtered through a Whatman # 10 filter paper. The filtrate thus obtained was finally filtered using a filter of 0.25 uM. The filtered extract was concentrated under reduced pressure and freeze dried at 50 ° C or lower. The mixed extract was prepared using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol such that the vacuum concentrate and the lyophilisate contained 0.001 to 70.0 wt%.
시험예Test Example 1: 세포 생존율 측정 1: Cell viability measurement
상기 제조예 1 및 비교 제조예 1에서 수득한 은행 외과피 발효추출물과 은행 외과피 추출물이 세포에 미치는 독성을 측정하기 위해 실시되었다.Ginkgo biloba fermented extract obtained in Preparation Example 1 and Comparative Preparation Example 1 and ginkgo biloba extract was performed to determine the toxicity on the cells.
세포 독성은 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide(MTT) 시약을 이용하여 세포 생존율을 측정하는 Mosmann의 방법을 변형하여 실시하였다.Cytotoxicity was performed by modifying Mosmann's method of measuring cell viability using 3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide (MTT) reagent.
HDF를 2×104셀/웰 농도로 96-웰 플레이트에 접종한 후, 각 웰에 시료(제조예 1, 비교 제조예 1)를 각 농도별(7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 ㎍/㎖)로 각각 첨가하여 혼합하여 CO2 배양기에서 24시간 배양하였다. MTT 용액(5 ㎍/㎖)을 첨가하고 4시간 후 원심분리하여 상등액을 제거하고, 100 ㎕ 산-이소프포판올(이소프로판올에 0.04 N HCl)을 첨가한 후 푸른색의 포마잔(formazan)이 용출되도록 하여 마이크로 플레이트 리더(micro plate reader, BIO-TEK Instruments, USA)로 565 nm에서 흡광도를 측정하였다.After HDF was inoculated into 96-well plates at 2 × 10 4 cells / well concentration, samples (Preparation Example 1, Comparative Preparation Example 1) were added to each well for each concentration (7.81, 15.63, 31.25, 62.5, 125, 250). , 500, 1000 ㎍ / ㎖) were added and mixed respectively and incubated in a CO 2 incubator for 24 hours. MTT solution (5 μg / ml) was added and centrifuged after 4 hours to remove supernatant, and 100 μl acid-isofopanol (0.04 N HCl in isopropanol) was added followed by elution of blue formazan. Absorbance was measured at 565 nm with a micro plate reader (BIO-TEK Instruments, USA).
측정 결과 제조예 1은 모든 농도에서 세포 독성은 나타나지 않았지만 비교 제조예 1은 125 ㎍/㎖이하에서 현저한 세포 독성을 나타내었다. 이로부터 은행 외과피 추출물이 발효과정을 통해 세포 독성이 없어지는 것을 알 수 있었으며 또한, 본 발명의 은행 외과피 발효추출물은 인체에 무해하며 안정성이 매우 우수함을 확인할 수 있었다.
As a result, Preparation Example 1 showed no cytotoxicity at all concentrations, but Comparative Preparation Example 1 showed significant cytotoxicity at 125 µg / ml or less. From this, it can be seen that the ginkgo biloba extract is cytotoxic through the fermentation process, and furthermore, the ginkgo biloba fermented extract of the present invention was found to be harmless to humans and very excellent in stability.
시험예Test Example 2: 2: 식물병에In plant diseases 대한 방제활성 Control activity
상기 제조예 1 및 비교 제조예 1에서 수득한 은행 외과피 발효추출물과 은행 외과피 추출물의 식물병에 대한 방제활성을 평가하기 위하여, 벼 도열병(Magnaporthe grisea), 벼 잎집무늬마름병(Rhizoctonia solani), 토마토 잿빛곰팡이병(Botrytis cinerea), 보리 흰가루병(Erysiphe graminis f. sp. hordei), 토마토 역병(Phytophthora infestans), 밀 붉은녹병(Puccinia recondita) 및 고추 탄저병(Colletotrichum coccodes)의 7가지 식물병에 대한 in vivo 항균활성을 조사하였다. 각각의 시료를 메탄올(최종농도, 5%)로 용해한 후 계면활성제 트윈 20을 250 ㎍/㎖ 수준으로 포함하는 증류수에 희석하였다. 이때 대조구는 5% 메탄올과 250㎍/㎖의 트윈 20을 함유하는 증류수를 사용하였다. 각 식물병당 2개의 포트를 이용하였고, 활성성분 시료를 엽면에 분무 살포한 후 24시간 동안 풍건한 다음 각각의 식물 병원균을 접종하였다.In order to evaluate the control activity against the plant diseases of ginkgo biloba fermented extract and ginkgo biloba extract obtained in Preparation Example 1 and Comparative Preparation Example 1, rice blast (Magnaporthe grisea), rice leaf blight (Rhizoctonia solani), tomato grayish In vivo antibacterial against fungal disease (Botrytis cinerea), barley flour disease (Erysiphe graminis f. Sp.hordei), tomato late blight (Phytophthora infestans), wheat rust disease (Puccinia recondita) and pepper anthrax (Colletotrichum coccodes) The activity was investigated. Each sample was dissolved in methanol (final concentration, 5%) and then diluted in distilled water containing surfactant Tween 20 at a level of 250 μg / ml. At this time, the control group was using distilled water containing 5% methanol and 250 μg / ㎖ Tween 20. Two pots were used for each plant disease, and the active ingredient samples were spray-sprayed on the foliar and air-dried for 24 hours before inoculating each plant pathogen.
실험에 사용한 벼, 토마토, 보리 및 밀은 지름 4.5㎝의 플라스틱 포트에 수도용 상토 또는 원예용 상토를 70%정도 채운 후, 종자를 파종하여 25±5℃의 온실에서 1주 내지 4주간 재배하였다. 벼 도열병은 3 ~ 4엽기의 유묘에 도열병의 원인균인 마그나포르테 그리세아(Magnaporthe grisea)의 포자 현탁액(5×105 spores/㎖)을 분무 접종하고, 25℃의 습실상에서 하루 동안 습실처리한 후, 25℃의 항온실에서 5일간 배양하여 발병을 유도하였다. 벼 잎집무늬마름병은 잎집무늬마름병의 원인균인 라이족토니아 솔라니(Rhizoctonia solani)를 배지(밀기울 90g, 왕겨 15g 및 증류수 100㎖)에서 7일간 배양하여 얻은 배양물을 5엽기 유묘에 접종하고 25℃의 습실상에서 4일간 습실처리한 후, 25℃의 항온실에서 4일간 배양하여 발병을 유도하였다. 토마토 역병은 3 ~ 4엽기 토마토 유묘에 역병의 원인균인 파이토프토라 인페스탄스(Phytophthora infestans)의 유주자낭(105 sporangia/㎖)에서 나출된 유주자 현탁액을 분무 접종한 후 25℃의 습실상에서 2일간 습실처리하고 25℃의 항온항습실에서 1일간 배양하여 발병을 유도하였다. 토마토 잿빛곰팡이병은 토마토 3 ~ 4엽기 유묘에 잿빛곰팡이병의 원인균인 보트라이티스 시네리아(Botrytis cinerea)의 포자 현탁액(106 spores/㎖)을 처리한 후, 20℃의 습실상에서 3일간 배양하여 발병을 유도하였다. 밀 붉은녹병은 1엽기 유묘에 활물기생균으로 알려진 녹병의 원인균인 퍽시니아 리콘디타(Puccinia recondita)의 포자를 250 ㎍/㎖의 트윈 20 용액에 0.67 g spores/ℓ의 양으로 현탁하여 분무 처리하고 20℃의 습실상에서 하루 동안 습실처리한 후 20℃의 항온실로 옮겨 6일간 배양하여 발병을 유도하였다. 보리 흰가루병은 보리의 1엽기 유묘에 숙주 식물에서 계대배양된 흰가루병의 원인균인 에리시페 그래미니스 폼 스피시스 호르데이(Erysiphe graminis f. sp. hordei)의 포자를 털어서 접종하고 20℃의 항온실에서 7일간 배양하여 발병을 유도하였다. 벼 도열병, 밀 붉은녹병, 보리 흰가루병은 접종 7일 후, 벼 잎집무늬마름병은 접종 8일 후, 그리고 토마토 잿빛곰팡이병과 토마토 역병은 접종 3일 후에 병반 면적율을 조사하였다.Rice, tomatoes, barley and wheat used in the experiments were filled with water or soil for horticulture 70% in plastic pots with a diameter of 4.5 cm, and then seeded and grown in a greenhouse at 25 ± 5 ℃ for 1 to 4 weeks. Rice blasts were sprayed with spore suspensions (5 × 10 5 spores / ml) of Magnaporthe grisea, the causative agent of blasts, in 3 ~ 4 leaf seedlings, and then wet-treated in a 25 ° C. chamber for one day. Thereafter, the incubation was induced by incubation for 5 days in a constant temperature room at 25 ℃. Rice leaf sheath blight was inoculated in 5-leaf seedlings by incubating the culture obtained by incubating Rhizoctonia solani, the causative agent of leaf blight, in medium (90 g of wheat bran, 15 g of chaff and 100 ml of distilled water) for 7 days. After 4 days of treatment in a wet room, the culture was incubated for 4 days in a constant temperature room at 25 ℃ to induce the onset. Tomato plague was sprayed with 3-4 leaf tomato seedlings inoculated with the distilled suspension from the larvae (10 5 sporangia / ml) of Phytophthora infestans, the causative agent of late blight. Treatment was induced daily and incubated in a constant temperature and humidity room at 25 ° C. for 1 day to induce onset. Tomato gray mold disease was treated with spore suspension (10 6 spores / mL) of Botrytis cinerea, a causative agent of gray mold disease, in tomato seedlings of 3-4 leaves, and then in a humidified environment at 20 ° C for 3 days. Incubation was induced by incubation. Wheat rust is sprayed by suspending spores of Puccinia recondita, a causative agent of rust, known as lycosis, in a single-leafed seedling, suspended in 250 µg / ml Tween 20 solution in an amount of 0.67 g spores / l. After a day of treatment in a humid room of ℃ ℃ was transferred to a constant temperature room of 20 ℃ was incubated for 6 days to induce the onset. Barley powdery mildew is inoculated with spores of Erysiphe graminis f. Sp. Incubation was induced by incubation for 7 days. After 7 days of inoculation, 8 days after inoculation of rice sheath blight, and 3 days after inoculation with tomato gray mold and tomato blight,
한편, 고추 탄저병에 대한 방제활성 실험을 위해서는, 지름 7.0㎝의 플라스틱 포트에 원예용 상토를 70% 정도 채운 후 최아된 고추 종자를 파종하고, 이를 온실에서 3 ~ 4엽기까지 키운 다음 상기에서 준비된 천속단의 메탄올 추출물 시료를 엽면 살포하였다. 시료 살포 후 24시간 동안 상온에서 건조시킨 다음 고추 탄저병원균인 콜레토트리쿰 코코데스(Colletotrichum coccodes)의 포자 현탁액(4×105 포자/㎖)을 분무 접종하였다. 습실상에서 2일간 발병시킨 후 25℃의 항온실에 1일간 방치하였고, 접종 3일 후에 병반면적율을 조사하였다.On the other hand, for the control activity test for pepper anthracnose, 70% of horticultural soil is filled in a plastic pot with a diameter of 7.0 cm, sowing the seeds of the most pepper, growing them in the greenhouse to 3 to 4 leaves, and then prepared cloth Methanol extract samples of fast-acting were foliar sprayed. After spraying the sample was dried at room temperature for 24 hours, and then spray inoculated with a spore suspension (4 × 10 5 spores / ml) of the pepper anthrax bacterium, Colletotrichum coccodes. After 2 days of onset, it was allowed to stand in a constant temperature room at 25 ℃ for 1 day.
상기로부터 얻은 병반면적율을 이용하여 다음과 같은 식에 따라 방제가를 계산하였다.
Using the area area ratio obtained from the above, the control value was calculated according to the following formula.
(㎍/㎖)density
(Μg / ml)
RCB: 벼 도열병; RSB: 벼 잎집무늬마름병; TGM: 토마토 잿빛곰팡이병; TLB: 토마토 역병; WLR: 밀 붉은녹병; BPM: 보리 흰가루병; PAN: 고추 탄저병
RCB: rice blast; RSB: rice leaf blight; TGM: tomato gray mold; TLB: tomato late blight; WLR: wheat rust; BPM: wheat flour; PAN: Chilli Anthrax
상기 표 1에 나타난 바와 같이, 은행 외과피 발효추출물(제조예1)은 7가지 식물병에 대해 방제활성을 나타내었으며 특히 밀 붉은녹병과 보리 흰가루병에 각각 87%와 94%의 강한 항균효과를 보여주었다. 또한, 은행 외과피 발효추출물(제조예1)은 은행 외과피 추출물(비교제조예1)과 비교해 7가지 식물병 모두에 대해 보다 강한 방제활성을 나타내었다.
As shown in Table 1, the fermented ginkgo biloba extract (Preparation Example 1) showed control activity against seven plant diseases, and showed particularly strong antibacterial effects of 87% and 94%, respectively, against wheat rust and barley flour. . In addition, ginkgo biloba fermented extract (Production Example 1) showed a stronger control activity against all seven plant diseases compared to ginkgo biloba extract (Comparative Example 1).
Claims (5)
식물병 방제용 조성물.
Characterized in that it contains a fermented product or fermented extract by the fermentation microorganism of ginkgo biloba as an active ingredient,
Composition for controlling plant diseases.
상기 발효 미생물은 효모, 유산균류 및 바실러스속 균류로 이루어진 군에서 선택되는 1종 이상의 발효 미생물임을 특징으로 하는,
식물병 방제용 조성물.
The method of claim 1,
The fermentation microorganism is characterized in that at least one fermentation microorganism selected from the group consisting of yeast, lactic acid bacteria and Bacillus fungi,
Composition for controlling plant diseases.
상기 발효물은, 상기 발효물을 정제수, 메탄올, 에탄올, 글리세린, 에틸아세테이트, 부틸렌글리콜, 프로필렌글리콜, 디클로로메탄 및 헥산으로 이루어진 군에서 선택된 1종 이상의 추출용매로 추출한 후 감압농축 또는 동결건조하여 얻은 감압농출물 또는 동결건조물인 것을 특징으로 하는,
식물병 방제용 조성물.
3. The method according to claim 1 or 2,
The fermented product is extracted with one or more extractants selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane, and then concentrated under reduced pressure or lyophilized. Characterized in that the obtained reduced pressure extract or freeze-dried,
Composition for controlling plant diseases.
상기 추출물을 조성물 전체에 대하여 동결건조중량 기준으로 0.0001 내지 30.0 중량% 함유하는 것을 특징으로 하는,
식물병 방제용 조성물.
3. The method according to claim 1 or 2,
Characterized in that the extract contains 0.0001 to 30.0% by weight based on the total freeze-drying weight of the composition,
Composition for controlling plant diseases.
상기 식물병이 보리 흰가루병, 밀 붉은녹병, 토마토 역병 또는 토마토 잿빛곰팡이병인 것을 특징으로 하는,
식물병 방제용 조성물.
The method of claim 1,
The plant disease is characterized in that the wheat flour, wheat rust, tomato blight or tomato gray mold disease,
Composition for controlling plant diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020110115907A KR20130050698A (en) | 2011-11-08 | 2011-11-08 | Composition containing fermented extract of epicarp of ginkgo biloba for controlling plant diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020110115907A KR20130050698A (en) | 2011-11-08 | 2011-11-08 | Composition containing fermented extract of epicarp of ginkgo biloba for controlling plant diseases |
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| Publication Number | Publication Date |
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| KR20130050698A true KR20130050698A (en) | 2013-05-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| KR1020110115907A KR20130050698A (en) | 2011-11-08 | 2011-11-08 | Composition containing fermented extract of epicarp of ginkgo biloba for controlling plant diseases |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101534827B1 (en) * | 2013-12-24 | 2015-07-09 | 예산군 | A composition for preventing sheath blight of rice containing an extract of ginkgo spermoderm |
| WO2016085039A1 (en) * | 2014-11-28 | 2016-06-02 | (주)농예가 | Mixed strains for inhibiting seed germination of plant, and use thereof as herbicide |
| CN108184836A (en) * | 2017-11-23 | 2018-06-22 | 北京联合大学 | A kind of combined fermentation object for accelerating crops degrading pesticide residue |
| KR20180073818A (en) * | 2016-12-23 | 2018-07-03 | 이용순 | A method for manufacturing a crust of a bank envelope and a crushing powder of a bank manufactured by the method for producing the same, and a pharmaceutical composition for enhancing the antimicrobial activity and sterilizing power using the same |
| CN108713558A (en) * | 2018-05-12 | 2018-10-30 | 湖南科技学院 | The growth-promoting preparation of general flavone content in a kind of promotion ginkgo leaf |
-
2011
- 2011-11-08 KR KR1020110115907A patent/KR20130050698A/en not_active Application Discontinuation
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101534827B1 (en) * | 2013-12-24 | 2015-07-09 | 예산군 | A composition for preventing sheath blight of rice containing an extract of ginkgo spermoderm |
| WO2016085039A1 (en) * | 2014-11-28 | 2016-06-02 | (주)농예가 | Mixed strains for inhibiting seed germination of plant, and use thereof as herbicide |
| KR20180073818A (en) * | 2016-12-23 | 2018-07-03 | 이용순 | A method for manufacturing a crust of a bank envelope and a crushing powder of a bank manufactured by the method for producing the same, and a pharmaceutical composition for enhancing the antimicrobial activity and sterilizing power using the same |
| CN108184836A (en) * | 2017-11-23 | 2018-06-22 | 北京联合大学 | A kind of combined fermentation object for accelerating crops degrading pesticide residue |
| CN108184836B (en) * | 2017-11-23 | 2020-12-01 | 北京联合大学 | Combined fermentation product for accelerating degradation of residual pesticide on crops |
| CN108713558A (en) * | 2018-05-12 | 2018-10-30 | 湖南科技学院 | The growth-promoting preparation of general flavone content in a kind of promotion ginkgo leaf |
| CN108713558B (en) * | 2018-05-12 | 2020-09-01 | 湖南科技学院 | Growth promoting preparation for increasing content of total flavonoids in ginkgo leaves |
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