KR102413699B1 - Method for producing a isolate having anti-inflammatory activity from a leaf of a paw paw tree in which the acetogenin compounds is isolated - Google Patents
Method for producing a isolate having anti-inflammatory activity from a leaf of a paw paw tree in which the acetogenin compounds is isolated Download PDFInfo
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- KR102413699B1 KR102413699B1 KR1020200100578A KR20200100578A KR102413699B1 KR 102413699 B1 KR102413699 B1 KR 102413699B1 KR 1020200100578 A KR1020200100578 A KR 1020200100578A KR 20200100578 A KR20200100578 A KR 20200100578A KR 102413699 B1 KR102413699 B1 KR 102413699B1
- Authority
- KR
- South Korea
- Prior art keywords
- paw
- isolate
- acetogenin
- leaf extract
- component
- Prior art date
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Abstract
본 발명은 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법에 관한 것으로서, 포포나무 잎 추출물에 극성이 다른 유기용매를 혼합하여 분리물을 제조하고, 상기 제조된 분리물은 실리카겔을 고정상으로 하여 관-크로마토그램 기법을 수행하여 아세토제닌 성분과 항염증 활성 분리물을 각각 분리하는 것을 특징으로 한다.
본 발명에 따른 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법은 항염증 활성을 나타내는 기능성 분리물을 포포나무 잎 추출물로부터 제조하고, 분리물에 존재할 수 있는 신경독성 물질인 아세토제닌 성분을 효율적으로 분리하는 방법을 통해, 기능성 식품, 기능성 화장품 및 의약품 등에 다양하게 적용될 수 있는 효과가 있다.The present invention relates to a method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is separated, and the isolate is prepared by mixing an organic solvent having a different polarity to the paw paw paw tree leaf extract, and the prepared separation Water is characterized in that the acetogenin component and the anti-inflammatory active isolates are separated from each other by performing a tube-chromatogram technique using silica gel as a stationary phase.
In the method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is isolated according to the present invention, a functional isolate exhibiting anti-inflammatory activity is prepared from a paw paw tree leaf extract, and neurotoxicity that may exist in the isolate Through a method of efficiently separating the acetogenin component, which is a substance, there is an effect that can be variously applied to functional foods, functional cosmetics, and pharmaceuticals.
Description
본 발명은 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법에 관한 것으로서, 포포나무 잎 추출물로부터 항염증 활성을 가지는 분리물을 제조하기 위한 최적 조건을 선정하여 분리하고, 상기 분리된 분리물 성분 중 항염증 활성과 관계없고 신경독성을 나타내는 아세토제닌 성분을 분리함으로써, 포포나무 잎 추출물이 더욱 안전한 기능성 식품, 기능성 화장품 및 의약품 등의 원료로 사용될 수 있는 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법에 관한 것이다. The present invention relates to a method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is separated, and selects and separates optimal conditions for preparing an isolate having anti-inflammatory activity from paw paw paw tree leaf extract. , by separating the acetogenin component that is not related to anti-inflammatory activity and exhibits neurotoxicity among the separated components, the acetogenin component that can be used as a raw material for functional foods, functional cosmetics and pharmaceuticals, which is safer for the paw paw tree leaf extract, is The present invention relates to a method for preparing an isolate having anti-inflammatory activity from an isolated paw paw tree leaf extract.
포포나무(Asimina triloba)는 미국에서 paw paw tree로 알려져 있으며, 미국 동부가 원산지이고, 동부의 네브래스카주(Nebraska)에서 중부의 오클라호마주(Oklahoma)에 걸쳐 분포한다. 포포나무의 식물학적 연구를 통해, 그 과일과 씨앗으로부터 오일(oil), 지질(lipid), 지방산(fatty acid), 단백질(protein)이 분리되었고, 타닌(tannin), 시토스테롤(sitosterol), 카페익산(caffeic acid)과 프로시아니딘(procyanidin), 퀘르세틴(quercetin), 퀘르세틴 글리코시드(quercetin glycoside) 같은 플라보노이드(flavonoid)류 그리고 상당수의 이소퀴놀린알칼로이드(isoquinoline alkaloid)가 분리되었다. Paw paw tree ( Asimina triloba ) is known as paw paw tree in the United States, and is native to the eastern United States, and is distributed from Nebraska in the eastern state to the central Oklahoma state (Oklahoma). Through botanical study of paw paw tree, oil, lipid, fatty acid, and protein were isolated from its fruits and seeds, and tannin, sitosterol, and caffeic acid were isolated. Flavonoids such as caffeic acid, procyanidin, quercetin, and quercetin glycoside, and a significant number of isoquinoline alkaloids have been isolated.
포포나무 열매와 종자를 중심으로 다양한 종류의 아세토제닌(acetogenin) 성분과 관련된 약리학적 연구와 보고가 진행되고 있으며, 아세토제닌(acetogenin) 외에도 카테킨(catechin)이나 에피카테킨(epicatechin) 등의 플라보노이드(flavonoid)류 성분이 확인되었다. 국내에서도 재배가 가능한 것으로 알려지기 시작하면서 최근 전남 보성 등 일부 지역을 중심으로 도입되기 시작함에 따라 국내에서 생산된 포포나무 원료에 대한 연구 및 제품개발이 필요할 것으로 보인다. Pharmacological studies and reports related to various types of acetogenin are in progress, focusing on the fruit and seeds of the paw paw tree. In addition to acetogenin, flavonoids such as catechin and epicatechin component was confirmed. As it is known that it can be cultivated in Korea, it is recently introduced mainly in some areas such as Boseong, Jeollanam-do, so research and product development for raw materials of paw paw tree produced in Korea are expected to be necessary.
특히 이미 활용이 일정 수준 이상으로 이루어지고 있는 열매 부위 외에도 잎이나 가지 등의 부위가 이용 가능한 것으로 알려지고 있으므로 해당 부위에 대한 연구 및 기술 개발이 필요하다. 또한, 포포나무 잎 추출물 중 다량으로 포함되어 있는 루틴은 항산화 및 항균, 항당뇨, 항암 활성 등에 관여하는 물질로서 포포나무 잎을 활용한 제품이나 기술개발에 중요한 지표로서 활용될 수 있다. In particular, it is known that parts such as leaves and branches can be used in addition to the fruit parts, which have already been utilized above a certain level, so research and technology development for the parts are required. In addition, rutin, which is contained in a large amount in the paw paw paw tree leaf extract, is a substance involved in antioxidant, antibacterial, antidiabetic, and anticancer activity, and can be used as an important index for product or technology development using paw paw paw tree leaves.
상기와 같은 생리활성 가능성에도 불구하고 국내에 알려지거나 도입되기 시작한 지 얼마 되지 않아 아직까지 포포나무 관련 기술이 많지 않으므로, 포포나무 잎 추출물로부터 항염증 활성을 가진 안전한 분리물의 개발이 요구된다. Despite the physiological activity potential as described above, as there are not many technologies related to paw paw paw tree as it has not been known or introduced in Korea for a while, the development of a safe isolate with anti-inflammatory activity from paw paw tree leaf extract is required.
본 발명은 상기 종래기술이 갖는 문제점을 해결하기 위해서 안출된 것으로서, 본 발명에서 해결하고자 하는 과제는 포포나무 잎 추출물로부터 항염증 활성을 가지는 분리물을 제조하기 위한 최적 조건을 선정하여 분리하고, 상기 분리된 분리물 성분 중 항염증 활성과 관계없고 신경독성을 나타내는 아세토제닌 성분을 분리함으로써, 포포나무 잎 추출물이 더욱 안전한 기능성 화장품, 의약품 등의 원료로 사용될 수 있도록 함에 그 목적이 있다. The present invention has been devised to solve the problems of the prior art, and the problem to be solved in the present invention is to select and separate optimal conditions for preparing an isolate having anti-inflammatory activity from a paw paw paw tree leaf extract, and The purpose of this is to allow the paw paw paw tree leaf extract to be used as a safer raw material for functional cosmetics and pharmaceuticals by separating the acetogenin component that is not related to anti-inflammatory activity and exhibits neurotoxicity among the separated components.
상기와 같은 목적을 달성하기 위한 본 발명에 따른 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법은 포포나무 잎 추출물에 극성이 다른 유기용매를 혼합하여 분리물을 제조하고, 상기 제조된 분리물은 실리카겔을 고정상으로 하여 관-크로마토그램 기법을 수행하여 아세토제닌 성분과 항염증 활성 분리물을 각각 분리하는 것을 특징으로 한다. The method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is separated according to the present invention for achieving the above object is to prepare an isolate by mixing an organic solvent having a different polarity with paw paw tree leaf extract And, the prepared isolate is characterized in that the acetogenin component and the anti-inflammatory active isolate are separated by performing a tube-chromatogram technique using silica gel as a stationary phase.
또, 상기 포포나무 잎 추출물은, 포포나무 잎 100중량부에 대하여 50% 에탄올 2,000~3,000중량부를 혼합하여 60~90℃의 온도에서 3~4시간 동안 추출하여 여과한 후, 감압농축하여 동결건조시켜 제조된 것을 특징으로 한다. In addition, the paw paw tree leaf extract is mixed with 2,000 to 3,000 parts by weight of 50% ethanol with respect to 100 parts by weight of paw paw tree leaves, extracted at a temperature of 60 to 90° C. for 3 to 4 hours, filtered, concentrated under reduced pressure, and freeze-dried It is characterized in that it is manufactured.
또, 상기 분리물은, 포포나무 잎 추출물에 순차적으로 n-hexane, chloroform, ethylacetate를 혼합하여 용매별 분획을 실시한 후, 여과와 감압농축을 통해 제조되는 것을 특징으로 한다. In addition, the isolate is characterized in that it is prepared by sequentially mixing n -hexane, chloroform, and ethylacetate with the paw paw tree leaf extract and performing fractionation by solvent, followed by filtration and concentration under reduced pressure.
또, 상기 제조된 분리물을 메탄올에 용해시킨 후, 실리카겔을 고정상으로 하여 관-크로마토그램 기법을 수행하되, n-hexane을 용출 용매로 하여 아세토제닌 성분을 분리한 후, 메탄올을 용출 용매로 하여 항염증 활성 분리물을 분리하는 것을 특징으로 한다. In addition, after dissolving the prepared isolate in methanol, a tube-chromatogram technique is performed using silica gel as a stationary phase, n -hexane as an elution solvent to separate the acetogenin component, and methanol as the elution solvent It is characterized in that the anti-inflammatory active isolate is isolated.
본 발명에 따른 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법은 항염증 활성을 나타내는 기능성 분리물을 포포나무 잎 추출물로부터 제조하고, 분리물에 존재할 수 있는 신경독성 물질인 아세토제닌 성분을 효율적으로 분리하는 방법을 통해, 기능성 식품, 기능성 화장품 및 의약품 등에 다양하게 적용될 수 있는 효과가 있다.In the method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is isolated according to the present invention, a functional isolate exhibiting anti-inflammatory activity is prepared from a paw paw tree leaf extract, and neurotoxicity that may exist in the isolate Through a method of efficiently separating the acetogenin component, which is a substance, there is an effect that can be variously applied to functional foods, functional cosmetics, and pharmaceuticals.
도 1은 본 발명에 따른 포포나무 잎 추출물의 제조공정을 나타낸 도면이다.
도 2는 본 발명에 따른 포포나무 잎 추출물로부터 분리물의 제조공정을 나타낸 도면이다.
도 3은 본 발명에 따른 분리물로부터 아세토제닌 성분의 분리 공정을 나타낸 도면이다.
도 4은 본 발명에 따른 추출 조건별 포포나무 잎 추출물의 항염증 활성을 나타낸 도면이다.
도 5는 본 발명에 따른 분리물의 항염증 활성을 나타낸 도면이다.
도 6는 본 발명에 따른 분리물의 아세토제닌 함량을 나타낸 도면이다.
도 7은 본 발명에 따른 아세토제닌 분리 공정 전후의 아세토제닌 성분 함량을 나타낸 도면이다.
도 8은 본 발명에 따른 아세토제닌 분리 공정 전후의 항염증 활성 분리물의 주요 성분 함량을 나타낸 도면이다. 1 is a view showing a manufacturing process of a paw paw paw tree leaf extract according to the present invention.
2 is a view showing the manufacturing process of the isolate from the paw paw paw tree leaf extract according to the present invention.
3 is a view showing the separation process of the acetogenin component from the isolate according to the present invention.
4 is a view showing the anti-inflammatory activity of the paw paw tree leaf extract for each extraction condition according to the present invention.
5 is a view showing the anti-inflammatory activity of the isolate according to the present invention.
6 is a view showing the acetogenin content of the isolate according to the present invention.
7 is a view showing the acetogenin component content before and after the acetogenin separation process according to the present invention.
8 is a view showing the main component content of the anti-inflammatory active isolate before and after the acetogenin separation process according to the present invention.
이하, 본 발명의 바람직한 실시예를 첨부된 도면을 참조하여 상세히 설명하면 다음과 같다. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
본 발명은 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법에 관한 것이다. The present invention relates to a method for producing an isolate having anti-inflammatory activity from a paw paw tree leaf extract from which an acetogenin component is isolated.
더 상세하게는, 포포나무 잎 추출물에 극성이 다른 유기용매를 혼합하여 분리물을 제조하고, 상기 제조된 분리물은 실리카겔을 고정상으로 하여 관-크로마토그램 기법을 수행하여 아세토제닌 성분과 항염증 활성 분리물을 각각 분리하는 것이다. More specifically, an isolate is prepared by mixing an organic solvent of different polarity with a paw paw tree leaf extract, and the prepared isolate is performed with a tube-chromatogram technique using silica gel as a stationary phase to obtain an acetogenin component and anti-inflammatory activity isolate each of the isolates.
도 1은 본 발명에 따른 포포나무 잎 추출물의 제조공정을 나타낸 도면이다. 1 is a view showing the manufacturing process of a paw paw paw tree leaf extract according to the present invention.
도 1에 도시된 바와 같이, 포포나무 잎 추출물의 분리물을 제조하기 위하여 먼저 포포나무 잎 추출물을 제조한다. As shown in FIG. 1 , in order to prepare an isolate of the paw paw tree leaf extract, first, a paw paw paw tree leaf extract is prepared.
포포나무 잎은 추출효율을 높이기 위하여, 수분함량이 5% 이하가 되도록 건조하여 10~40mesh의 크기로 분쇄하여 사용한다. In order to increase the extraction efficiency, paw paw tree leaves are dried so that the moisture content is 5% or less, and crushed into a size of 10-40 mesh before use.
포포나무 잎 추출물을 제조하는 방법은 특별히 제한되지 않으며, 예를 들면, 용매 추출, 열수 추출, 환류냉각 추출, 초음파 추출, 여과법 등 통상적인 방법으로 수행될 수 있다. The method for preparing the paw paw tree leaf extract is not particularly limited, and for example, solvent extraction, hot water extraction, reflux cooling extraction, ultrasonic extraction, filtration, and the like may be performed by conventional methods.
본 발명에서는 환류냉각 추출에 의해 추출하는 것으로, 본 발명에서 사용하는 추출용매는 에탄올, 메탄올, 부탄올, 에테르, 에틸아세테이트, 클로로포름 또는 이들 유기용매를 혼합하여 선택될 수 있으며, 바람직하게는 50% 에탄올을 사용한다. In the present invention, extraction is performed by reflux cooling, and the extraction solvent used in the present invention may be selected from ethanol, methanol, butanol, ether, ethyl acetate, chloroform, or a mixture of these organic solvents, preferably 50% ethanol use
더 상세하게는, 상기 포포나무 잎 추출물은 포포나무 잎 100중량부에 대하여 50% 에탄올 2000~3000중량부를 혼합하고, 60~90℃의 온도에서 3~4시간 동안 추출하여 여과한 후, 감압농축하여 동결건조시켜 추출하는 것이다. More specifically, the paw paw tree leaf extract is mixed with 2000 to 3000 parts by weight of 50% ethanol with respect to 100 parts by weight of paw paw tree leaves, extracted at a temperature of 60 to 90° C. for 3 to 4 hours, filtered, and then concentrated under reduced pressure. It is extracted by freeze-drying.
여기서, 상기 추출용매의 혼합량, 추출온도 및 추출시간을 벗어나면 추출효율이 떨어지거나 성분의 변화가 생길 수 있다.Here, when the mixing amount of the extraction solvent, the extraction temperature, and the extraction time are out of the range, the extraction efficiency may decrease or a change in components may occur.
도 2는 본 발명에 따른 포포나무 잎 추출물로부터 분리물의 제조공정을 나타낸 도면이다.2 is a view showing a manufacturing process of an isolate from a paw paw paw tree leaf extract according to the present invention.
도 2에 도시된 바와 같이, 상기의 방법으로 제조된 포포나무 잎 추출물에 대해 물과 섞이지 않는 극성이 다른 유기용매를 활용하여 분리물을 제조한다. As shown in FIG. 2, an organic solvent having a different polarity that does not mix with water is used for the paw paw tree leaf extract prepared by the above method to prepare a separated product.
더 상세하게는, 상기 분리물은 포포나무 잎 추출물을 증류수에 용해 및 분산시킨 후, 순차적으로 n-hexane, chloroform, ethylacetate를 혼합하여 용매별 분획을 실시한 후, 여과와 감압농축을 통해 제조되도록 한다. More specifically, the isolate is prepared by dissolving and dispersing the paw paw paw tree leaf extract in distilled water, sequentially mixing n -hexane, chloroform, and ethylacetate to perform fractionation by solvent, followed by filtration and concentration under reduced pressure. .
도 3은 본 발명에 따른 분리물로부터 아세토제닌 성분의 분리 공정을 나타낸 도면이다.3 is a view showing the separation process of the acetogenin component from the isolate according to the present invention.
도 3에 도시된 바와 같이, 상기의 방법으로 제조된 분리물을 메탄올에 용해시킨 후, 실리카겔(silica-gel)을 고정상으로 하여 관-크로마토그램 기법을 수행하되, n-hexane을 용출 용매로 하여 아세토제닌 성분을 분리시킨 후, 메탄올을 용출 용매로 하여 항염증 활성 분리물을 분리시키도록 한다.As shown in FIG. 3, after dissolving the isolate prepared by the above method in methanol, a tube-chromatogram technique is performed using silica-gel as a stationary phase, n -hexane as the elution solvent, After separation of the acetogenin component, the anti-inflammatory active product is separated using methanol as the elution solvent.
상기의 방법으로 분리된 항염증 활성 분리물은 신경독성을 나타낼 수 있는 아세토제닌 성분이 제거된 안전한 조성물로서, 기능성 식품, 기능성 화장품 및 의약품 등에 다양하게 적용될 수 있다. The anti-inflammatory active isolate separated by the above method is a safe composition from which acetogenin, which may exhibit neurotoxicity, is removed, and can be variously applied to functional foods, functional cosmetics, and pharmaceuticals.
전체 조성물에 대하여, 상기의 방법으로 분리된 항염증 활성 분리물은 1~30중량%를 유효성분으로 포함하는 것이 바람직하다.With respect to the entire composition, the anti-inflammatory active isolate separated by the above method preferably contains 1 to 30% by weight as an active ingredient.
이는 최소한의 항염증 활성 성분 효과를 달성할 수 있도록 조성물의 함량이 상기 최소치 이상인 것이 바람직하며, 또한 과량 첨가에 따른 사용감 저하 및 각종 제형에의 적용가능성을 고려하여 조성물의 함량이 상기 최대치 이하인 것이 바람직하다.It is preferable that the content of the composition is above the above minimum value to achieve the minimum anti-inflammatory active ingredient effect, and the content of the composition is preferably below the above maximum value in consideration of the decrease in feeling of use due to excessive addition and applicability to various formulations do.
이하에서 실시예를 통하여 본 발명을 더욱 구체적으로 설명한다. 그러나 하 기의 실시예는 본 발명을 구체적으로 예시하기 위한 것일 뿐, 본 발명의 권리범위를 제한하는 것이 아님은 통상의 기술자에게 있어서 명백한 사실이다. 즉, 본 발명의 단순한 변형 내지 변경은 통상의 기술자에 의하여 용이하게 실시될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.Hereinafter, the present invention will be described in more detail through examples. However, it is clear to those skilled in the art that the following examples are only for specifically illustrating the present invention, and do not limit the scope of the present invention. That is, simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.
실시예 1 : 추출물 제조 Example 1: Preparation of extract
1) 원료1) Raw material
실험에 사용된 포포나무 잎은 전남 보성군에서 재배되고 있는 것을 제공받은 것이다. The paw paw tree leaves used in the experiment were provided from those grown in Boseong-gun, Jeollanam-do.
2) 추출물 제조2) Preparation of extract
건조 후 분쇄된 포포나무 잎에 표 1과 같은 조건을 적용하여 추출물을 제조하였다. 가열 온도는 각 조건의 용매가 끓을 수 있도록 하였으며, 가열 시간은 용매가 끓기 시작한 후부터 3시간으로 하였다. 추출방법 및 조건에 따른 추출물은 도 1과 같은 과정을 거쳐 얻어진 추출물 양을 바탕으로 수율을 산출하였다.An extract was prepared by applying the conditions shown in Table 1 to the pulverized paw paw tree leaves after drying. The heating temperature allowed the solvents of each condition to boil, and the heating time was set to 3 hours after the solvent started to boil. The extract according to the extraction method and conditions was calculated based on the amount of extract obtained through the process as shown in FIG. 1 .
실시예 2 : 분리물 제조Example 2: Preparation of isolates
도 2와 같이 항염증 활성이 가장 우수한 50% 에탄올 추출물을 증류수에 분산시킨 후 순차적으로 n-hexane, chloroform, ethylacetate를 혼합하여 용매별 분획을 실시한 후 여과와 감압농축을 거쳐 분리물을 확보하였으며, 각 분리물의 분리 수율은 표 2와 같다.As shown in Figure 2, after dispersing the 50% ethanol extract with the best anti-inflammatory activity in distilled water, sequentially mixing n -hexane, chloroform, and ethylacetate to carry out fractionation by solvent, followed by filtration and concentration under reduced pressure to obtain a separated product, The separation yield of each isolate is shown in Table 2.
실시예 3 : 아세토제닌 성분 분리를 위한 관-크로마토그래피 Example 3: Tube-Chromatography for Separation of Acetogenin Components
비극성 용매에 선택성을 가지는 아세토제닌 성분의 특성을 고려하여 메탄올에 용해시킨 항염증 활성이 높은 분리물을 극성의 silica-gel을 고정상으로 채운 관(column)에 로딩한 후, 비극성 용매인 n-hexane을 1차 용출 용매로 하여 아세토제닌 성분을 분리하였다. 이후 메탄올을 2차 용출 용매로 하여 항염증 활성 성분을 용출하였으며, 각 용출액은 감압농축을 실시한 후 최종 활성 분리물과 아세토제닌 성분 분리물을 각각 획득하였으며, 각 과정은 도 3과 같이 실시하였다. In consideration of the properties of acetogenin components having selectivity to non-polar solvents, the isolate with high anti-inflammatory activity dissolved in methanol was loaded into a column filled with polar silica-gel as a stationary phase, and then, n -hexane, a non-polar solvent, was loaded. was used as the primary elution solvent to separate the acetogenin component. Thereafter, the anti-inflammatory active ingredient was eluted using methanol as a secondary elution solvent, and each eluate was concentrated under reduced pressure to obtain a final active isolate and an acetogenin component separated, respectively, and each process was carried out as shown in FIG. 3 .
실험 1 : Nitric oxide 생성 억제 효과 측정을 통한 항염증 활성의 측정Experiment 1: Measurement of anti-inflammatory activity by measuring the inhibitory effect of nitric oxide production
Nitric oxide(산화질소)는 생체 내에서 합성 효소에 의해 L-arginine(엘-아르기닌)으로부터 생성되는 자유 라디칼이며, 염증반응의 매개체로써 시간이 지나면 nitrite(아질산염), nitrate(질산염) 등의 안정한 화합물로 존재하며, 이 과정에서 형성되는 peroxynitrite anion(퍼옥시니트리트 음이온)으로 인해 혈관투과성, 부종 등의 염증반응을 촉진시킬 뿐 아니라, cyclooxygenase(시클로옥시게나아제)를 활성화하여 prostaglandin(프로스타글란딘)과 같은 염증 매개체의 생합성을 촉진함으로써 염증을 심화시키는 것으로 알려져 있다. 추출물 및 분리물 시료의 nitric oxide 생성억제 효과를 측정하기 위하여 미리 배양된 Raw 264.7 세포를 96 well plate에 2*104 cells/well이 되도록 분주하여 24시간 동안 배양한 후, 각 시료를 4∼200㎍/㎖의 농도로 처리한 후, lipopolysaccharide(리포 다당류, LPS, 2㎍/㎖)를 혼합하여 24시간 동안 추가로 배양하였다. 배지 상등액 100㎕와 Griess reagent(그리스 시약) 100㎕를 혼합하여 상온에서 15분간 반응시킨 다음 microplate reader를 이용하여 540㎚에서 흡광도를 측정하였다. 시료 대신 PBS를 처리한 대조구의 흡광도를 기준으로 시료별 NO 생성억제 활성을 산출하였다.Nitric oxide (nitric oxide) is a free radical generated from L-arginine by synthetase in the living body, and as a mediator of the inflammatory reaction over time, stable compounds such as nitrite and nitrate It not only promotes inflammatory reactions such as vascular permeability and edema due to the peroxynitrite anion formed in this process, but also activates cyclooxygenase, such as prostaglandin, It is known to intensify inflammation by promoting the biosynthesis of inflammatory mediators. To measure the nitric oxide production inhibitory effect of extracts and isolates, pre-cultured Raw 264.7 cells were dispensed to a 96-well plate at 2*10 4 cells/well and cultured for 24 hours. After treatment at a concentration of ㎍ / ㎖, lipopolysaccharide (lipopolysaccharide, LPS, 2㎍ / ㎖) was mixed and further cultured for 24 hours. 100 μl of the medium supernatant and 100 μl of Griess reagent were mixed and reacted at room temperature for 15 minutes, and then absorbance was measured at 540 nm using a microplate reader. The NO production inhibitory activity for each sample was calculated based on the absorbance of the control treated with PBS instead of the sample.
포포나무 잎 추출물과 분리물의 항염증 활성을 확인하기 위한 nitric oxide 생성 억제 효과를 측정한 결과를 각각 도 4 및 도 5에 나타내었다. Nitric oxide 생성 억제 효과에서 50% 생성을 억제하는 농도(SC50)를 산출하여 각 시료의 활성을 비교하였으며, 결과값이 낮을수록 우수한 활성을 가지는 것으로 나타났다. 각 추출물의 측정 결과에서 50% 에탄올 추출물과 75% 에탄올 추출물이 각각 0.309mg/mL와 0.317mg/mL의 SC50 값을 나타냄으로써 다른 추출 조건에 비해 우수한 항염증 활성을 가지는 것으로 확인되었다. 가장 활성이 높았던 50% 에탄올 추출물을 도 2와 같은 과정을 통해 분리한 1차 분리물의 nitric oxide 생성억제 효과 측정 결과에서 ethyl acetate 분리물(EF)의 SC50 값이 0.106mg/mL를 나타냄으로써 다른 분리물에 비해 현저히 높은 항염증 활성을 가지는 것으로 확인되었다.The results of measuring the nitric oxide production inhibitory effect to confirm the anti-inflammatory activity of the paw paw paw tree leaf extract and isolates are shown in FIGS. 4 and 5, respectively. In the nitric oxide production inhibitory effect, the concentration (SC 50 ) that inhibits 50% production was calculated and the activity of each sample was compared. The lower the result, the better the activity. In the measurement results of each extract, 50% ethanol extract and 75% ethanol extract showed SC 50 values of 0.309 mg/mL and 0.317 mg/mL, respectively, and it was confirmed that they had superior anti-inflammatory activity compared to other extraction conditions. The SC 50 value of the ethyl acetate isolate (EF) was 0.106 mg/mL in the measurement result of the nitric oxide production inhibitory effect of the primary isolate obtained by separating the 50% ethanol extract with the highest activity through the same process as in FIG. It was confirmed to have significantly higher anti-inflammatory activity than the isolate.
실험 2 : 추출물 및 분리물 중 성분 분석 조건Experiment 2: Conditions for component analysis in extracts and isolates
신경독성을 유발할 수 있는 아세토제닌 성분과 항염증 활성을 가지는 분리물의 주요 성분인 quercetin, rutin의 함량 비교 분석을 위해 표 3과 같은 조건이 적용된 액체크로마토그래피-질량분석기(LC-MS/MS)를 사용한 분석을 실시하였다. 아세토제닌 성분 분석을 위한 세부 질량분석 조건은 표 4와 같았으며, 항염증 활성 성분인 quercetin과 rutin의 분석을 위한 세부 질량분석 조건은 표 5와 같았다.Liquid chromatography-mass spectrometry (LC-MS/MS) to which the conditions shown in Table 3 were applied for comparative analysis of the contents of acetogenin, which can cause neurotoxicity, and quercetin and rutin, which are main components of isolates with anti-inflammatory activity, were used. The analysis used was performed. Detailed mass spectrometry conditions for the analysis of acetogenin components are shown in Table 4, and detailed mass spectrometry conditions for the analysis of quercetin and rutin, which are anti-inflammatory active ingredients, are shown in Table 5.
이동상 조건
mobile phase conditions
B : Acetonitrile
B: Acetonitrile
분리물을 대상으로 실시한 아세토제닌 성분 분석 결과 도 6과 같이 나타났다. 가장 높은 함량을 가지는 것으로 확인된 n-hexane(HF) 분리물의 함량을 100%로 하여 산출한 상대 함량에서 항염증 활성이 높게 나타났던 ethyl acetate 분리물(EF)의 아세토제닌 성분 함량이 5.62% 수준으로 확인되었다.As a result of the analysis of the acetogenin component performed on the isolate, it was shown in FIG. 6 . The acetogenin component content of the ethyl acetate isolate (EF), which showed high anti-inflammatory activity, was 5.62% at the relative content calculated with the content of the n -hexane (HF) isolate, which was confirmed to have the highest content, as 100%. was confirmed as
또한, 아세토제닌 성분의 소량으로도 신경독성 등을 유발할 수 있는 것으로 알려져 있으므로 추출물이나 분리물에서 제거하여 안전성을 확보할 필요가 있다. 상기 분리물 중 항염증 활성이 높은 것으로 확인된 ethyl acetate 분리물(EF, before CC)과 도 3과 같은 공정을 실시하여 얻은 최종 활성 분리물(EF, after CC)의 아세토제닌 성분의 비교 분석 결과는 도 7에 나타내었다. 분리 정제 공정 전 함량 수준(EF, before CC)을 100%로 하여 비교한 결과, 정제 후의 함량이 0.94% 수준으로 나타남에 따라 99% 이상의 아세토제닌 성분 분리 효과를 나타낸 것을 확인할 수 있다.In addition, since it is known that even a small amount of acetogenin can induce neurotoxicity, it is necessary to secure safety by removing it from the extract or isolate. Comparative analysis result of the acetogenin component of the ethyl acetate isolate (EF, before CC) confirmed to have high anti-inflammatory activity among the isolates and the final active isolate (EF, after CC) obtained by performing the same process as in FIG. 3 is shown in FIG. 7 . As a result of comparing the content level (EF, before CC) before the separation and purification process as 100%, it can be confirmed that the content after purification was 0.94%, indicating that the acetogenin component separation effect was greater than 99%.
아세토제닌 성분을 분리하는 과정에서 항염증 활성 성분의 소실이 어느 정도 발생되었는지 확인하기 위해 1차 ethyl acetate 분리물(EF, before CC)과 분리 공정 후 ethyl acetate 분리물(EF, after CC)의 quercetin과 rutin 함량을 비교 분석하여 도 8에 나타내었다. Quercetin의 경우 100%에서 95.6%로 4.4% 수준의 감소를 보였으며, rutin은 100%에서 99.4%로 0.6% 수준의 감소를 나타냈다. 이와 같은 수준의 미량의 활성 성분 감소는 아세토제닌 성분 제거 수준을 감안하면 양호한 것으로 판단된다.In order to determine to what extent the loss of the anti-inflammatory active ingredient occurred in the process of separating the acetogenin component, the quercetin of the primary ethyl acetate isolate (EF, before CC) and the ethyl acetate isolate (EF, after CC) after the separation process It is shown in FIG. 8 by comparing and analyzing the rutin content. Quercetin showed a decrease of 4.4% from 100% to 95.6%, and rutin showed a decrease of 0.6% from 100% to 99.4%. This level of reduction of trace amounts of active ingredients is considered good considering the level of removal of the acetogenin component.
Claims (4)
상기 포포나무 잎 추출물은,
포포나무 잎 100중량부에 대하여 50% 에탄올 2,000~3,000중량부를 혼합하여 60~90℃의 온도에서 3~4시간 동안 추출하여 여과한 후, 감압농축하여 동결건조시켜 제조되고,
상기 분리물은,
포포나무 잎 추출물에 순차적으로 n-hexane, chloroform, ethylacetate를 혼합하여 용매별 분획을 실시한 후, 여과와 감압농축을 통해 제조되며,
상기 제조된 ethylacetate 분리물을 메탄올에 용해시킨 후, 실리카겔을 고정상으로 하여 관-크로마토그램 기법을 수행하되, n-hexane을 용출 용매로 하여 아세토제닌 성분을 분리한 후, 메탄올을 용출 용매로 하여 항염증 활성 분리물을 분리하고,
전체 조성물에 대하여, 상기의 방법으로 분리된 항염증 활성 분리물은 1~30중량%를 유효성분으로 포함하는 것을 특징으로 하는 아세토제닌 성분이 분리된 포포나무 잎 추출물로부터 항염증 활성을 가진 분리물의 제조방법.An isolate was prepared by mixing an organic solvent of different polarity with the paw paw tree leaf extract, and the prepared isolate was separated from the acetogenin component and the anti-inflammatory active material by performing a tube-chromatogram technique using silica gel as a stationary phase. but,
The paw paw tree leaf extract,
It is prepared by mixing 2,000 to 3,000 parts by weight of 50% ethanol with respect to 100 parts by weight of paw paw tree leaves, extracting it at a temperature of 60 to 90° C. for 3 to 4 hours, filtering, concentrating under reduced pressure, and freeze-drying,
The isolate is
It is prepared by sequentially mixing n -hexane, chloroform, and ethylacetate with the paw paw tree leaf extract and performing fractionation by solvent, followed by filtration and concentration under reduced pressure.
After dissolving the ethylacetate isolate prepared above in methanol, a tube-chromatogram technique was performed using silica gel as a stationary phase, and acetogenin component was separated using n -hexane as an elution solvent, and then, methanol was used as an elution solvent. isolate the inflammatory active isolate,
With respect to the entire composition, the anti-inflammatory active isolate separated by the above method contains 1 to 30% by weight as an active ingredient. manufacturing method.
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