KR20090094614A - An extract of Nelumbo nucifera's leaves for preventing or treating diabetic complication - Google Patents
An extract of Nelumbo nucifera's leaves for preventing or treating diabetic complication Download PDFInfo
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- KR20090094614A KR20090094614A KR1020080019675A KR20080019675A KR20090094614A KR 20090094614 A KR20090094614 A KR 20090094614A KR 1020080019675 A KR1020080019675 A KR 1020080019675A KR 20080019675 A KR20080019675 A KR 20080019675A KR 20090094614 A KR20090094614 A KR 20090094614A
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- quercetin
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Abstract
Description
본 발명은 연잎에서 추출한 추출물을 함유한 당뇨성 예방 및 치료 효능을 갖는 약학 조성물 및 건강식품에 관한 것으로, 보다 자세하게는 연잎 조추출물, 비극성용매 가용 추출물 또는 그로부터 분리된 플라보노이드류의 화합물을 함유하는 알도즈 환원요소를 억제하여, 당뇨성 합병증에 관한 예방 또는 치료용 약학조성물 및 건강 식품에 관한 것이다.The present invention relates to a pharmaceutical composition and health food having a diabetic prophylactic and therapeutic effect containing an extract extracted from lotus leaf, and more particularly, containing a lotus leaf crude extract, a non-polar solvent soluble extract or a compound of flavonoids isolated therefrom. It relates to a pharmaceutical composition for preventing or treating diabetic complications and health foods by inhibiting the dose reducing element.
당뇨병은 대표적인 만성 성인병의 하나로, 우리나라의 당뇨병 환자는 최소한 250만 명으로 전체 인구의 약 5% 정도로 추정되고 있다. 선진국의 경우에 당뇨병 환자 수가 매년 급증하고 있고, 우리나라도 생활수준의 향상과 더불어 생활양식이 서구화되면서 점차로 환자의 수가 증가될 것으로 생각된다. Diabetes mellitus is one of the representative chronic adult diseases, with at least 2.5 million diabetics in Korea, estimated to be about 5% of the total population. In advanced countries, the number of diabetic patients is increasing rapidly each year, and in Korea, the number of patients is expected to increase gradually as the standard of living is improved and the lifestyle is westernized.
폴리펩타이드성 호르몬인 인슐린은 췌장에 있는 랑게르한스섬의 베타세포에 서 만들어지며, 신체 내의 대부분의 세포가 포도당을 사용하는데 필요한 호르몬이다.Insulin, a polypeptide hormone, is produced from beta cells in the island of Langerhans in the pancreas and is the hormone most cells in the body need to use glucose.
당뇨병은 체세포에서 포도당 대사에 장애가 생겨 혈당치가 증가하여, 혈액에 포도당이 증가됨에 따라 과량의 당분이 소변으로 배설되는 대사성 질환이다.Diabetes is a metabolic disorder in which glucose metabolism in somatic cells increases and blood glucose levels increase, and excess glucose is excreted in the urine as glucose increases in the blood.
췌장 호르몬인 인슐린은 뇌하수체, 부신, 갑상선 등 인슐린에 대항 작용을 하는 호르몬과 체내에서 균형이 이루어졌을 때, 당 조절 기능이 정상으로 유지하게 된다.Insulin, a pancreatic hormone, maintains normal glycemic function when balanced in the body with hormones that act against insulin, such as the pituitary gland, the adrenal glands, and the thyroid gland.
그러나 인슐린 분비량이 부족하거나 인슐린 분비량은 거의 정상이나 대항 호르몬이 상대적으로 많아 인슐린이 부족할 때는 당뇨병이 발병하게 된다.However, insulin secretion is insufficient or insulin secretion is almost normal, but there is a relatively high number of anti-hormones, insulin deficiency causes diabetes.
당뇨병은 그 자체보다는 합병증이 더 위험하기 때문에, 오늘날 당뇨병 치료에 있어서 가장 큰 목표는 당뇨성 합병증의 유발이나 진행을 억제하는 데 있다.Since diabetes is more dangerous than complications per se, the biggest goal in the treatment of diabetes today is to inhibit the development or progression of diabetic complications.
합병증은 당뇨병이 오래 지속되어 나타나는 현상으로 보통 10 내지 15년을 경과한 후에 생기는 만성 합병증이 주이며, 그 대표적인 만성 합병증으로 당뇨성 망막증, 당뇨성 신증, 당뇨성 신경증 등이 있다.Complications are long-term symptoms of diabetes, usually chronic complications occurring after 10 to 15 years, and the typical chronic complications include diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy.
당뇨성 신경증은 당뇨병으로 인해 신경계에 장애가 오는 것으로 말초신경의 장애, 건반사의 소실, 운동신경의 마비, 자율신경 장애 등으로 발바닥이 저릿저릿하고, 화끈거리고, 통증이 심하며, 성기능의 장애가 오고, 뇨나 대변을 가리지 못하는 증상을 가져오기도 한다.Diabetic neuropathy is a disorder of the nervous system due to diabetes, which is caused by disorders of the peripheral nerves, loss of the keyboard, paralysis of motor nerves, autonomic nerve disorders, etc., resulting in tingling, burning, painful, and sexual dysfunction. It can also cause your stool to go away.
당뇨성 신증은 미세혈관 합병증의 하나로 신 사구체 모세혈관의 경화성 병변에 의해 일어나는 것으로 특별한 증상이 없어도 소변검사를 통해 단백질이 나타나 면 신증이 있음을 예측할 수 있다.Diabetic nephropathy is a microvascular complication caused by sclerotic lesions of renal glomerular capillaries.
혈압의 상승은 당뇨병 신증을 악화시키는 요인으로도 작용하는데 보통 10 내지 15년 이상 당뇨병을 앓은 사람들의 약 5% 정도가 당뇨성 신증이 온다.Elevated blood pressure also acts to exacerbate diabetic nephropathy, with about 5% of people who have diabetes for 10 to 15 years or more.
당뇨성 망막증은 미세혈관의 합병증 중의 하나로 당뇨병 환자에게 실명을 가져오는 심각한 합병증이다. 최근에 당뇨병 조절과 합병증과의 관계연구에 의하면 인슐린 치료를 강화시켜 혈당이 정상화되면 당뇨병 합병증 발생을 크게 감소시킬 수 있는 것으로 나타났다.Diabetic retinopathy is a microvascular complication that is a serious complication that causes blindness in diabetics. Recent studies on the relationship between diabetes control and complications have shown that intensifying insulin therapy can significantly reduce the incidence of diabetes complications.
당뇨성 합병증의 병태는 다양하며, 고혈당에 의한 것으로서 말초신경 장애, 망막증, 신증, 백내장, 각막증 등이 있으며, 그 기전으로 폴리올 패스웨이(polyol pathway) 이상과 혈중의 과량의 당과 단백질의 결합을 들 수 있다.Diabetic complications vary and are caused by hyperglycemia, such as peripheral neuropathy, retinopathy, nephropathy, cataracts, and corneal disease.The mechanisms include polyol pathway abnormalities and excess sugar and protein binding in the blood. Can be mentioned.
상기 폴리올 패스웨이는 포도당 대사경로의 하나로 포도당은 알도즈 환원효소에 의하여 솔비톨로 전환된다.The polyol pathway is one of the glucose metabolic pathways and glucose is converted into sorbitol by aldose reductase.
정상상태에서의 알도즈 환원효소는 포도당에 대한 기질친화성은 매우 낮아 솔비톨을 거의 생성하지 않지만, 당뇨병 상태에서는 수정체, 말초신경, 망막과 같은 인슐린 비감수성의 조직에서의 포도당 농도가 상승하면 위의 경로에 의하여 대량의 솔비톨을 생성하게 되며, 이러한 각 조직에서의 솔비톨 축적이 당뇨성 백내장, 말초신경 장애, 당뇨성 망막증 등의 원인으로 알려져 있다.Aldose reductase at steady state is very low in substrate affinity for glucose and rarely produces sorbitol, but in diabetic state, the glucose pathway in elevated insulin-insensitive tissues such as the lens, peripheral nerve, and retina increases. By generating a large amount of sorbitol, the accumulation of sorbitol in each of these tissues is known to cause diabetic cataracts, peripheral neuropathy, diabetic retinopathy and the like.
또한 당뇨병의 고혈당 상태에서 당의 카르보닐(carbonyl)기와 단백질의 아미노(amino)기가 결합하여 AGEs를 생성하게 되고, 이것은 체내에 축적되어 당뇨성 망막증, 신증, 백내장, 아테롬성 동맥경화증과 같은 당뇨 합병증을 유발하게 된다. In the hyperglycemic state of diabetes, sugar carbonyl group and protein amino group combine to produce AGEs, which accumulate in the body and cause diabetic complications such as diabetic retinopathy, nephropathy, cataracts and atherosclerosis. Done.
그리하여 백내장 등의 당뇨성 합병증 예방 및 치료제로서 알도즈 환원효소 및 최종 당화산물 생성 억제제가 주목을 받고 있으며, 이들 몇몇 억제제가 동물실험 및 임상시험에서 당뇨성 합병증을 개선함이 보고되는 등 합성물질뿐만 아니라 천연물로부터 알도즈 환원효소 및 최종 당화산물 생성 억제성분을 규명하려는 활발한 연구가 이루어지고 있다.As a result, aldose reductase and final glycation inhibitors have attracted attention as agents for the prevention and treatment of diabetic complications such as cataracts, and several of these inhibitors have been reported to improve diabetic complications in animal and clinical trials. Rather, active studies have been conducted to identify aldose reductase and final glycation end product inhibitors from natural products.
그리하여 플라보노이드, 탄닌, 쿠마린, 정유 성분 등이 소나 흰쥐 또는 사람의 여러 조직으로부터 조제된 알도즈 환원효소 활성에 대한 억제 효과가 있음이 보고되었다.Thus, it has been reported that flavonoids, tannins, coumarins, essential oils and the like have an inhibitory effect on aldose reductase activity prepared from various tissues of cattle, rats or humans.
그러나 지금까지 연잎 조추출물의 알도즈 환원효소 억제효과는 보고되지 않았으며, 또한 이에 의한 당뇨병과 그 합병증 치료 효과는 보고된 바가 없다.However, the inhibitory effect of the crude extract of lotus leaf on aldose reductase has not been reported, and there has been no report on the effects of diabetes and its complications.
따라서, 본 발명은 상기와 같은 종래 기술의 제반 단점과 문제점을 해결하기 위한 것으로, 연잎 조추출물, 비극성용매 가용 추출물 또는 그로부터 분리된 플라보노이드류의 화합물을 함유하는 알도즈 환원효소를 억제하여, 당뇨성 합병증에 관한 예방 또는 치료를 할 수 있도록 하는 약학조성물과 건강식품을 제공함에 본 발명의 목적이 있다.Accordingly, the present invention is to solve the above-mentioned disadvantages and problems of the prior art, by inhibiting aldose reductase containing a compound of lotus leaf crude extract, non-polar solvent soluble extract or flavonoids separated therefrom, diabetic It is an object of the present invention to provide a pharmaceutical composition and a health food to enable the prevention or treatment of complications.
본 발명의 상기 목적은 연잎에서 추출한 연잎 조추출물, 연잎에서 추출한 비극성용매 가용추출물 및 상기 연잎 조추출물 또는 비극성용매 가용추출물로부터 분리한 플라보노이드류의 화합물 중 어느 하나 이상을 유효성분으로 함유하여 당뇨성 합병증 예방 또는 치료 작용을 하는 약학 조성물 및 건강식품에 의해 달성된다.The above object of the present invention comprises diabetic complications by containing any one or more of the compounds of lotus leaf crude extract extracted from lotus leaf, nonpolar solvent soluble extract extracted from lotus leaf and flavonoids compounds isolated from the crude extract or nonpolar solvent soluble extract of lotus leaf It is achieved by pharmaceutical compositions and health foods that have a prophylactic or therapeutic action.
또한, 본 발명의 상기 목적은 상기 비극성용매 가용추출물은 디클로로메탄, 에틸아세테이트 및 부탄올 중 어느 하나 이상을 이용하여 연잎으로부터 추출한 추출물인 것을 특징으로 약학 조성물 및 건강식품에 의해서도 달성된다.In addition, the object of the present invention is achieved by the pharmaceutical composition and health food, characterized in that the non-polar solvent soluble extract is an extract extracted from lotus leaf using any one or more of dichloromethane, ethyl acetate and butanol.
본 발명의 연잎에서 추출한 추출물을 함유한 당뇨성 예방 및 치료 효능을 갖는 약학 조성물 및 건강식품은 연잎으로부터 추출한 추출물로 알도즈 환원 효소를 억제하여 당뇨성 합병증을 예방 또는 치료할 수 있는 약학 조성물 및 건강식품을 제공하는 효과가 있다.Pharmaceutical compositions and health foods having an anti-diabetic and therapeutic effect containing extracts extracted from lotus leaf of the present invention is a pharmaceutical composition and health foods that can prevent or treat diabetic complications by inhibiting aldose reductase with extracts extracted from lotus leaves Has the effect of providing.
연(Nelumbo nucifera Gaertn.)은 수련과(Nymphaeceae)에 속하는 다년생 수생식물로서, 인도와 중국을 중심으로 열대 및 온대지역의 동부아시아를 비롯한 한국, 일본 등에 널리 분포하는 고생대의 식물로 알려져 있으며, 이미 오랜 전부터 중국과 인도 등에서는 연을 전통 민간약재로 사용해 왔으며, 우리나라에서도 식용 또는 약용으로 이용되고 있다.The lotus (Nelumbo nucifera Gaertn.) Is a perennial aquatic plant belonging to the family Nymphaeceae, and is known as a paleozoic plant widely distributed in Korea and Japan, including eastern Asia in tropical and temperate regions, mainly in India and China. Yan has been used as a traditional folk medicine in China and India for a long time, and it is also used as food or medicine in Korea.
전통적으로 잎은 하엽이라 하여 해열, 해독작용에 사용하였으며, 종자와 과육은 강장, 지혈약, 야뇨증, 부인병에, 뿌리는 해열독, 소어혈, 일체혈증, 요혈, 장출혈 및 지혈에 사용하여 왔다(김영배, 한약(생약)규격집(KHP), pp500, 2000; 김창민, 중약대사전, pp5956-5959, 1998).Traditionally, the leaves are called the lower lobe and have been used for fever and detoxification. Seeds and flesh have been used for tonic, hemostatic drugs, nocturnal enuresis, and gynecological diseases. Kim, Young-Bae, Herbal Medicines (KHP), pp500, 2000; Kim, Chang-Min, Chinese Medicine Dictionary, pp5956-5959, 1998).
본 발명의 연잎으로부터 조추출물, 비극성용매 가용추출물 및 이로부터 분리되는 플라보노이드류 (flavonoids)의 화합물들은 하기와 같이 수득될 수 있다.Compounds of crude extract, nonpolar solvent soluble extract and flavonoids isolated therefrom from the lotus leaf of the present invention can be obtained as follows.
본 발명의 연잎 조추출물은 연잎을 동결건조 후 마쇄하여 분말화 한 후, 연잎 건조 중량의 약 3 내지 20배, 바람직하게는 약 3 내지 5배에 달하는 부피의 물, 메탄올, 에탄올 및 부탄올과 같은 저급 알콜 또는 이들의 약 1:0.1 내지 1:10의 혼합비를 갖는 혼합용매로, 바람직하게는 에탄올로 20 내지 100℃, 바람직하게는 20 내지 70℃의 추출 온도에서 약 0.5시간 내지 2일, 바람직하게는 1시간 내지 1일 동안 열수 추출, 냉침 추출, 환류 냉각 추출 또는 초음파 추출 등의 추출방법으로 1회 내지 5회, 바람직하게는 3회 연속 추출하여 수득한 후, 감압여과하고 여액을 진공회전농축기로 20 내지 100℃, 바람직하게는 40 내지 70℃에서 감압 농축하여 물, 저급 알콜 또는 이들의 혼합용매에 가용한 연잎 조추출물을 수득할 수 있다.The lotus leaf crude extract of the present invention is powdered by pulverizing the lotus leaf after lyophilization, the volume of water, methanol, ethanol and butanol of about 3 to 20 times, preferably about 3 to 5 times the dry weight of the lotus leaf Lower alcohol or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably about 0.5 hour to 2 days at an extraction temperature of 20 to 100 ° C, preferably 20 to 70 ° C, preferably with ethanol It is preferably obtained by extracting from 1 to 5 times, preferably 3 times consecutively by extraction methods such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for 1 hour to 1 day, and then filtered under reduced pressure and rotating the filtrate under vacuum Concentrated under reduced pressure at 20 to 100 ℃, preferably 40 to 70 ℃ with a concentrator can be obtained crude extract of lotus leaf soluble in water, lower alcohol or a mixed solvent thereof.
상기 연잎 조추출물은 물에 현탁한 후, 디클로로메탄, 에틸아세테이트, n-부탄올 순으로 용매를 이용하여 추출하여 본 발명의 연잎 비극성용매 가용추출물, 바람직하게는 에틸아세테이트 가용성 분획물을 수득할 수 있고, 더욱 구체적으로는 연잎 조추출물 즉, 연잎 에탄올 추출물에 디클로로메탄:물:에탄올을 일정 비율, 바람직하게는 10:9:1로 혼합하여 디클로로메탄 분획물 및 수가용성 분획물을 수득할 수 있고, 다시 상기 수가용성 분획물에 에틸아세테이트를 가하여 에틸아세테이트 가용성 분획물 및 수가용성 분획물을 수득할 수 있고, 마지막으로 상기 수가용성 분획물을 부탄올로 추출하여 부탄올 가용성 분획물과 수가용성 분획물을 수득할 수 있다. The lotus leaf crude extract is suspended in water, and then extracted with a solvent in the order of dichloromethane, ethyl acetate, n-butanol to obtain a lotus leaf non-polar solvent soluble extract of the present invention, preferably ethyl acetate soluble fraction, More specifically, dichloromethane: water: ethanol may be mixed with a crude extract of lotus leaf, that is, lotus leaf ethanol extract in a ratio, preferably 10: 9: 1, to obtain a dichloromethane fraction and a water-soluble fraction. Ethyl acetate can be added to the soluble fraction to obtain ethyl acetate soluble fraction and water soluble fraction, and finally the water soluble fraction can be extracted with butanol to obtain butanol soluble fraction and water soluble fraction.
상기 비극성 용매인 에틸아세테이트 가용성 분획물에 대하여 디클로로메탄 : 메탄올 혼합용매, 바람직하게는 디클로로메탄 : 메탄올의 6:1 내지 1:1의 혼합용매로 시간당 일정용량, 바람직하게는 1,000 ㎖의 속도로 칼럼 크로마토그래피를 수행하여 F1부터 F6까지 6개의 분획물을 수득할 수 있다.Ethyl acetate soluble fraction, which is a nonpolar solvent, is a dichloromethane: methanol mixed solvent, preferably a 6: 1 to 1: 1 mixed solvent of dichloromethane: methanol at a constant volume per hour, preferably at a rate of 1,000 ml. The chromatography can be carried out to obtain six fractions from F1 to F6.
상기 에틸아세테이트 가용성 분획물은 RP-18 gel로 MPLC(Medium pressure preparative liquid chromatography, Yamazen Co., Osaka, Japan)를 사용하여 각각 1, 2, 3, 4, 5의 화학식으로 표시되는 화합물을 분리 및 동정할 수 있다.The ethyl acetate soluble fraction is an RP-18 gel using MPLC (Medium Pressure Preparative Liquid Chromatography, Yamazen Co., Osaka, Japan) to isolate and identify compounds represented by the formulas of 1, 2, 3, 4, and 5, respectively. can do.
<구조식><Structure Formula>
화학식 1. QuercetinChemical Formula 1. Quercetin
화학식 2. Quercetin 3-O-β-D-glucopyranoside : R1=GlcQuercetin 3-O-β-D-glucopyranoside: R 1 = Glc
화학식 3. Quercetin 3-O-β-D-glucuronopyranoside : R1=Gln Quercetin 3-O-β-D-glucuronopyranoside: R 1 = Gln
화학식 4. Quercetin 3-O-β-D-galactopyranoside : R1=GalQuercetin 3-O-β-D-galactopyranoside: R 1 = Gal
화학식 5. Quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside : R1=Rha-(1→6)-GlcQuercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside: R1 = Rha- (1 → 6) -Glc
본 발명은 연잎으로부터 조추출물, 비극성용매 가용추출물 및 이로부터 분리되는 플라보노이드류 (flavonoids)의 화합물을 분리, 정제하는 방법을 제공한다.The present invention provides a method for separating and purifying crude extracts, nonpolar solvent soluble extracts and compounds of flavonoids separated therefrom from lotus leaves.
본 발명은 상기 방법으로 수득된 연잎 추출물 및 그로부터 분리된 상기 플라 보노이드류(flavonoids)의 화합물을 함유하는 당뇨성 합병증에 대한 예방 및 치료효과를 나타내는 약학조성물을 제공한다.The present invention provides a pharmaceutical composition exhibiting a prophylactic and therapeutic effect against diabetic complications containing the compound of the lotus leaf extract obtained by the above method and the flavonoids isolated therefrom.
상기에서 수득된 연잎 조추출물 또는 그로부터 분리된 플라보노이드류 화합물의 당뇨성 합병증에 대한 예방 및 치료효과를 나타내는 약학조성물로서의 효능을 조사하기 위하여, 알도즈 환원효소 억제활성과 최종 당화산물 생성 억제활성을 검색해 본 결과, 본 발명에 따른 연잎 조추출물 또는 그로부터 분리된 플라보노이드류(flavonoids)의 화합물이 당뇨성 합병증에 대한 예방 및 치료효과를 나타남을 확인하였다.To investigate the efficacy of the crude extract of lotus leaf obtained above or the flavonoid compounds isolated therefrom as a pharmaceutical composition showing the prophylactic and therapeutic effects against diabetic complications, the aldose reductase inhibitory activity and the final glycation product inhibitory activity were investigated. As a result, it was confirmed that the compound of the lotus leaf crude extract or flavonoids isolated therefrom exhibits a prophylactic and therapeutic effect against diabetic complications.
또한 본 발명은 상기 제조방법으로 수득된 연잎 조추출물 또는 그로부터 분리된 플라보노이드류의 화합물을 유효성분으로 하는 당뇨성 합병증에 대한 예방 및 치료효과를 나타내는 약학조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition exhibiting a prophylactic and therapeutic effect against the diabetic complications obtained from the lotus leaf crude extract obtained by the above method or a compound of flavonoids separated therefrom as an active ingredient.
또한 본 발명의 연잎 조추출물 또는 그로부터 분리된 플라보노이드류의 화합물은 당뇨성 합병증에 대한 예방 및 치료효과를 나타내는 약학조성물로서 사용이 가능하다.In addition, the lotus leaf crude extract of the present invention or a compound of flavonoids isolated therefrom can be used as a pharmaceutical composition showing a prophylactic and therapeutic effect against diabetic complications.
본 발명의 연잎 조추출물 또는 그로부터 분리된 화합물은 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 사용시에도 안심하고 사용할 수 있다. The lotus leaf crude extract of the present invention or a compound isolated therefrom has little toxicity and side effects, and thus can be used safely for long-term use for prophylactic purposes.
본 발명의 연잎 조추출물 및 그로부터 분리된 화합물을 포함하는 약학조성물의 적용량 및 적용방법은 제형 및 사용목적에 따라 다를 수 있다.The application amount and application method of the pharmaceutical composition comprising the lotus leaf crude extract of the present invention and a compound separated therefrom may vary depending on the formulation and the purpose of use.
<실시 예 1> 연잎 조추출물의 제조<Example 1> Preparation of lotus leaf crude extract
본 발명에 사용한 연잎은 전남 무안에서 구입하여 사용하였다. 연잎은 동결건조 후 마쇄하여 얻은 분말 300g을 에탄올 3ℓ를 넣고 70℃에서 일정시간 간격 (12h, 6h, 3h)으로 3회 반복하여 환류 냉각 추출한 후 여지(와트만사, 미국)로 감압 여과한 다음, 여과 추출물은 진공회전농축기로 40℃에서 에탄올을 제거한 후 추출된 잔사로서 연잎 조추출물 53.7g을 수득하였으며, 이중 2 g을 취하여 억제활성 검색을 위한 시료로 사용하였다. The lotus leaf used in the present invention was purchased and used in Muan, Jeonnam. After lotus leaf lyophilized, 300 g of the powder obtained by grinding was put into 3 l of ethanol, and refluxed and extracted at 70 ° C. three times at regular intervals (12 h, 6 h, 3 h) for cooling under reduced pressure and filtered under a reduced pressure (Watman, USA). The filtrate extract was obtained by removing the ethanol at 40 ℃ in a vacuum rotary concentrator, 53.7 g of the crude extract of the lotus leaf as an extracted residue, of which 2 g was used as a sample for screening inhibitory activity.
<실시예 2> 연잎 디클로로메탄 가용추출물의 제조Example 2 Preparation of Lotus Leaf Dichloromethane Soluble Extract
상기 <실시 예 1>에서 얻은 조출물을 물 2ℓ에 녹여 얻어진 수가용성 층에 디클로로메탄 2ℓ를 가하여 혼합한 후 3 내지 4차례 분획하여 수가용성 분획물 2ℓ 및 디클로로메탄 가용성 분획물 2ℓ를 얻은 후, 상기 디클로로메탄 가용성 분획물을 건조하여 디클로로메탄 가용추출물 16.1g을 수득하였으며, 이중 2g을 취하여 억제활성 검색을 위한 시료로 사용하였다.2 liters of dichloromethane was added to the water-soluble layer obtained by dissolving the crude product obtained in Example 1 in 2 liters of water, and then fractionated 3 to 4 times to obtain 2 liters of water-soluble fractions and 2 liters of dichloromethane-soluble fractions. The methane soluble fraction was dried to give 16.1 g of dichloromethane soluble extract, of which 2 g was used as a sample for screening inhibitory activity.
<실시예 3> 연잎 에틸아세테이트 가용추출물의 제조Example 3 Preparation of Lotus Leaf Ethyl Acetate Soluble Extract
상기 <실시 예 2>에서 얻은 수가용성 분획물 2ℓ에 에틸아세테이트 2ℓ를 가하여 혼합한 후 3 내지 4차례 분획하여 수가용성 분획물 2ℓ 및 에틸아세테이트 가용성 분획물 2ℓ를 얻은 후, 상기 에틸아세테이트 분획물을 건조하여 에틸아세테이트 가용추출물 3.7g을 수득하여 시료로 사용하였으며, 이중 2g을 취하여 억제활성 검색을 위한 시료로 사용하였다.2 liters of ethyl acetate was added to 2 liters of the water-soluble fraction obtained in Example 2, followed by mixing 3 to 4 times to obtain 2 liters of water-soluble fractions and 2 liters of ethyl acetate-soluble fractions, and then dried the ethyl acetate fractions to obtain ethyl acetate. Soluble extract 3.7g was obtained and used as a sample, of which 2g was used as a sample for screening inhibitory activity.
<실시예 4> 연잎 부탄올 가용추출물의 제조Example 4 Preparation of Lotus Leaf Butanol Soluble Extract
상기 <실시 예 3>에서 얻은 수가용성 분획층 2ℓ에 부탄올 2ℓ를 가하여 혼합한 후 3 내지 4차례 분획하여 수가용성 분획물 2ℓ 및 부탄올 가용성 분획물 2ℓ를 얻은 후, 수가용성 분획물 및 부탄올 가용성 분획물을 건조하여 수가용 추출물 및 부탄올 가용추출물 6.9g을 수득하여 시료로 사용하였으며, 이중 2g을 취하여 억제활성 검색을 위한 시료로 사용하였다.2 L of butanol was added to 2 L of the water-soluble fraction obtained in Example 3, followed by mixing 3 to 4 times to obtain 2 L of water-soluble fraction and 2 L of butanol-soluble fraction, and then the water-soluble fraction and butanol-soluble fraction were dried. Soluble extract and 6.9 g of butanol soluble extract were obtained and used as a sample, of which 2 g was used as a sample for screening inhibitory activity.
<실시예 5> 플라보노이드류 화합물의 분리Example 5 Isolation of Flavonoid Compounds
상기 <실시 예 3>의 연잎 에틸아세테이트 추출물 3.7g을 전개용매로서 디클로로메탄 : 메탄올을 6:1 내지 1:1의 혼합용매를 사용하여 키에셀 겔(Kiesel gel 60, 230-400 매쉬, Merck, Germany) 칼럼 크로마토그래피를 수행하여 6개의 하부 분획물(EF01∼EF06)로 나누었다.3.7 g of the lotus leaf ethyl acetate extract of <Example 3> was developed as a developing solvent using a mixed solvent of dichloromethane: methanol 6: 1 to 1: 1 (
상기 하부 분획물 중 EF01(1.14g)을 90% 메탄올로 RP-18 gel(LiChroprep RP-18, 40-63 μm, Merck, Darmstadt, Germany)로 MPLC를 수행하여 화합물 1(25mg)을 분리하였다.EF01 (1.14 g) in the lower fractions was subjected to MPLC with RP-18 gel (LiChroprep RP-18, 40-63 μm, Merck, Darmstadt, Germany) with 90% methanol to separate compound 1 (25 mg).
상기 EF03과 EF04를 혼합한 분획물(2.78g)을 75% 메탄올로 RP-18 gel을 이용하여 MPLC를 수행하여 화합물 2(450mg)와 4(66mg)를 분리하였다.Compounds 2 (450 mg) and 4 (66 mg) were separated by MPLC using ERP03 and EF04 mixed fractions (2.78 g) with 75% methanol using RP-18 gel.
상기 EF05(0.89 g)와 EF06(0.86 g)을 각각 50% 메탄올로 RP-18 gel을 고정상으로 MPLC를 수행하여 화합물 3(320mg)과 5(25 mg)를 분리하였다. 각각의 화합물은 NMR을 측정하여 구조를 결정하였고, 결과는 하기와 같다.Compounds 3 (320 mg) and 5 (25 mg) were separated by MPLC of EF05 (0.89 g) and EF06 (0.86 g), respectively. Each compound was determined by measuring NMR to determine the structure, and the results are as follows.
물질의 성상 : 무정형 노란색 분말. Appearance of substance: Amorphous yellow powder.
물질의 분자식 : C15H10O7 Molecular formula of substance: c 15 h 10 o 7
1H-NMR (400 MHz, DMSO-d6) δ : 6.19(1H, d, J = 2.0Hz, H-6), 6.44(1H, d, J=2.0Hz, H-8), 6.88 (1H, d, J=8.0Hz, H-5′), 7.57(1H, dd, J=2.0, 9.0Hz, H-6′), 7.59(1H, d, J=2.0 Hz, H-2′), 12.47(1H, brs, 5-OH). 1 H-NMR (400 MHz, DMSO-d 6 ) δ: 6.19 (1H, d, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H , d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 12.47 (1H, broad singlet, 5-OH).
13C-NMR (100 MHz, DMSO-d6) δ : 175.9 (C-4), 163.9 (C-7), 161.7(C-5), 156.5(C-9), 148.5(C-4′), 146.8(C-2), 144.8(C-3′), 133.3(C-3), 121.6(C-6′), 121.0(C-1′), 116.2(C-5′), 115.2(C-2′), 104.1(C-10), 98.2(C-6), 93.5(C-8). 13 C-NMR (100 MHz, DMSO-d 6 ) δ: 175.9 (C-4), 163.9 (C-7), 161.7 (C-5), 156.5 (C-9), 148.5 (C-4 ′) , 146.8 (C-2), 144.8 (C-3 '), 133.3 (C-3), 121.6 (C-6'), 121.0 (C-1 '), 116.2 (C-5'), 115.2 (C -2 '), 104.1 (C-10), 98.2 (C-6), 93.5 (C-8).
물질의 성상 : 무정형 노란색 분말. Appearance of substance: Amorphous yellow powder.
물질의 분자식 : C21H20O12 Molecular formula of substance: c 21 h 20 o 12
1H-NMR (400 MHz, DMSO-d6) δ : 5.47(1H, d, J=7.3Hz, H-1″), 6.21(1H, J=2.0Hz, H-6), 6.44(1H, d, J=2.0Hz, H-8), 6.88(1H, d, J=8.0Hz, H-5′), 7.57(1H, dd, J=2.0, 9.0Hz, H-6′), 7.59(1H, d, J=2.0Hz, H-2′), 12.62 (1H, brs, 5-OH). 1 H-NMR (400 MHz, DMSO-d 6 ) δ: 5.47 (1H, d, J = 7.3 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 ′), 12.62 (1H, brs, 5-OH).
13C-NMR (100 MHz, DMSO-d6) δ : 177.6(C-4), 164.2(C-7), 161.3(C-5), 156.5(C-9), 156.4(C-2), 148.5(C-4′), 144.8(C-3′), 133.3(C-3), 121.6(C-6′), 121.0(C-1′), 116.2(C-5′), 115.2(C-2′), 104.1(C-10), 100.9(C-1″), 98.8(C-6), 93.7(C-8), 77.6(C-5″), 76.5(C-3″), 74.3(C-2″), 70.0(C-4″), 60.9(C-6″). 13 C-NMR (100 MHz, DMSO-d 6 ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 100.9 (C-1 "), 98.8 (C-6), 93.7 (C-8), 77.6 (C-5"), 76.5 (C-3 "), 74.3 (C-2 ″), 70.0 (C-4 ″), 60.9 (C-6 ″).
물질의 성상 : 무정형 노란색 분말. Appearance of substance: Amorphous yellow powder.
물질의 분자식 : C21H18O13 Molecular formula of substance: c 21 h 18 o 13
1H-NMR (400 MHz, DMSO-d6) δ : 12.30(1H, s, 5-OH), 9.72(1H, brs, H-3′), 8.10(1H, s, H-2), 7.40(1H, dd, J=2.0, 8.4Hz, H-6′), 6.82(1H, d, J=8.6Hz, H-5′), 6.34(1H, d, J=1.8Hz, H-8), 6.15(1H, d, J=2.1Hz, H-6), 5.26(1H, d, J=6.5Hz, H-1″). 1 H-NMR (400 MHz, DMSO-d 6 ) δ: 12.30 (1H, s, 5-OH), 9.72 (1H, brs, H-3 ′), 8.10 (1H, s, H-2), 7.40 (1H, dd, J = 2.0, 8.4Hz, H-6 ′), 6.82 (1H, d, J = 8.6Hz, H-5 ′), 6.34 (1H, d, J = 1.8Hz, H-8) , 6.15 (1H, d, J = 2.1 Hz, H-6), 5.26 (1H, d, J = 6.5 Hz, H-1 ″).
13C-NMR (100 MHz, DMSO-d6) δ : 177.4(C-4), 172.6(C-6″), 165.2(C-7), 160.9(C-5), 157.2(C-9), 156.5(C-2), 148.5(C-4′), 144.8(C-3′), 133.9(C-3), 120.9(C-1′), 120.5(C-6′), 117.6(C-5′), 115.4(C-2′), 103.5(C-10), 102.7(C-1″), 99.0(C-6), 93.8(C-8), 76.5 (C-3″), 74.3(C-5″), 74.0(C-2″), 71.8(C-4″). 13 C-NMR (100 MHz, DMSO-d 6 ) δ: 177.4 (C-4), 172.6 (C-6 ″), 165.2 (C-7), 160.9 (C-5), 157.2 (C-9) , 156.5 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.9 (C-3), 120.9 (C-1 '), 120.5 (C-6'), 117.6 (C -5 '), 115.4 (C-2'), 103.5 (C-10), 102.7 (C-1 "), 99.0 (C-6), 93.8 (C-8), 76.5 (C-3"), 74.3 (C-5 ″), 74.0 (C-2 ″), 71.8 (C-4 ″).
물질의 성상 : 무정형 노란색 분말. Appearance of substance: Amorphous yellow powder.
물질의 분자 : C21H20O12 Molecule Of Material: C 21 H 20 O 12
1H-NMR (400 MHz, DMSO-d6) δ : 5.41(1H, d, J=7.7Hz, H-1″), 6.21(1H, J=2.0Hz, H-6), 6.44(1H, d, J=2.0Hz, H-8), 6.88(1H, d, J=8.0Hz, H-5′), 7.57(1H, dd, J=2.0, 9.0Hz, H-6′), 7.59(1H, d, J=2.0Hz, H-2′), 12.62(1H, brs, 5-OH). 1 H-NMR (400 MHz, DMSO-d 6 ) δ: 5.41 (1H, d, J = 7.7 Hz, H-1 ″), 6.21 (1H, J = 2.0 Hz, H-6), 6.44 (1H, d, J = 2.0 Hz, H-8), 6.88 (1H, d, J = 8.0 Hz, H-5 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 7.59 ( 1H, d, J = 2.0 Hz, H-2 '), 12.62 (1H, brs, 5-OH).
13C-NMR (100 MHz, DMSO-d6) δ : 177.6(C-4), 164.2(C-7), 161.3(C-5), 156.5(C-9), 156.4(C-2), 148.5(C-4′), 144.8(C-3′), 133.3(C-3), 121.6(C-6′), 121.0(C-1′), 116.2(C-5′), 115.2(C-2′), 104.1(C-10), 101.7(C-1″), 98.7(C-6), 93.7(C-8), 75.8(C-5″), 73.1(C-3″), 71.2(C-2″), 67.9(C-4″), 60.2(C-6″). 13 C-NMR (100 MHz, DMSO-d 6 ) δ: 177.6 (C-4), 164.2 (C-7), 161.3 (C-5), 156.5 (C-9), 156.4 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 121.0 (C-1'), 116.2 (C-5 '), 115.2 (C -2 '), 104.1 (C-10), 101.7 (C-1 "), 98.7 (C-6), 93.7 (C-8), 75.8 (C-5"), 73.1 (C-3 "), 71.2 (C-2 ″), 67.9 (C-4 ″), 60.2 (C-6 ″).
(Quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside) (Quercetin 3-O-α-L-rhamnopyranosyl- (1 → 6) -β-D-glucopyranoside)
물질의 성상 : 무정형 노란색 분말. Appearance of substance: Amorphous yellow powder.
물질의 분자식 : C27H30O16 Molecular formula of substance: C 27 H 30 O 16
1H-NMR(400MHz, DMSO-d6) δ : 12.64(1H, s, 5-OH), 7.59(1H, d, J=2.0Hz, H-2′), 7.57(1H, dd, J=2.0, 9.0Hz, H-6′), 6.42 (1H, d, J=1.8Hz, H-8), 6.19(1H, d, J=2.1Hz, H-6), 5.47(1H, d, J=7.3Hz, H-1″), 4.40(1H, brs, H-1′′′), 0.98(3H, d, J=6.2Hz, H-6′′′). 1 H-NMR (400 MHz, DMSO-d 6 ) δ: 12.64 (1H, s, 5-OH), 7.59 (1H, d, J = 2.0 Hz, H-2 ′), 7.57 (1H, dd, J = 2.0, 9.0 Hz, H-6 ′), 6.42 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 2.1 Hz, H-6), 5.47 (1H, d, J = 7.3 Hz, H-1 "), 4.40 (1H, brs, H-1 ''), 0.98 (3H, d, J = 6.2 Hz, H-6 '').
13C-NMR(100MHz, DMSO-d6) δ : 177.3(C-4), 164.2(C-7), 161.2(C-5), 156.3(C-9), 156.1(C-2), 148.5(C-4′), 144.8(C-3′), 133.3(C-3), 121.6(C-6′), 120.9(C-1′), 116.2 (C-5′), 115.2 (C-2′), 104.1 (C-10), 101.1(C-1″), 100.6(C-1′′′), 98.7(C-6), 93.6(C-8), 77.5(C-3″), 75.8(C-5″), 74.2(C-2′′), 71.8(C-4′′′), 70.6(C-4′′), 70.3(C-3′′′), 69.9(C-2″), 68.3 (C-5 ′′′), 66.9(C-6″), 17.2(C-6′′′). 13 C-NMR (100 MHz, DMSO-d 6 ) δ: 177.3 (C-4), 164.2 (C-7), 161.2 (C-5), 156.3 (C-9), 156.1 (C-2), 148.5 (C-4 '), 144.8 (C-3'), 133.3 (C-3), 121.6 (C-6 '), 120.9 (C-1'), 116.2 (C-5 '), 115.2 (C- 2 ′), 104.1 (C-10), 101.1 (C-1 ″), 100.6 (C-1 ′ ′ ′), 98.7 (C-6), 93.6 (C-8), 77.5 (C-3 ″) , 75.8 (C-5 ″), 74.2 (C-2 ′ ′), 71.8 (C-4 ′ ′ ′), 70.6 (C-4 ′ ′), 70.3 (C-3 ′ ′ ′), 69.9 (C -2 ″), 68.3 (C-5 ′ ′ ′), 66.9 (C-6 ″), 17.2 (C-6 ′ ′ ′).
<실험 예 1> 연 부위 조추출물의 총 페놀성 성분의 정량Experimental Example 1 Determination of Total Phenolic Components of Crude Extracts
연 부위 조추출물의 총 페놀성 성분의 정량은 폴린-시오칼토(Folin-Ciocalteu)법을 이용하였으며, Iqbal 등이 고안한 방법을 변형하여 측정하였다(Iqbal et al., J. Food Comp. Anal., 19, pp544-551, 2006). The total phenolic component of the crude extract of the soft area was determined using the Folin-Ciocalteu method, and was modified by a method devised by Iqbal et al. (Iqbal et al., J. Food Comp. Anal. , 19, pp 544-551, 2006).
0.4에서 2mg/ml 농도의 시료 250μl와 폴린-시오칼토 반응시약 750μl를 첨가한 반응액을 실온에서 5분간 방치한 후, 7.5% Na2CO3 2ml을 첨가하였다.250 μl of the sample at a concentration of 0.4 to 2 mg / ml and 750 μl of the Pauline-Siocalto reaction reagent were added at room temperature for 5 minutes, and then 2 ml of 7.5% Na 2 CO 3 was added.
그 후 탈이온수로 총 부피가 7ml이 되도록 희석하였다.It was then diluted with deionized water to a total volume of 7 ml.
반응액은 반응이 일어나도록 1시간 동안 암실에 방치한 후, UV/Visible spectrophotometer(Ultrospec 2100pro, Amersham Biosciences, USA)를 이용하여 765nm에서 흡광도를 측정하였다. 모든 실험은 갈산(gallic acid, Sigma, St. Louis, MO, USA)를 CS(calibration standard)로 사용하였으며, 각각의 결과는 건조 추출물 g당 갈산의 당량으로 표시하였다[GAE(gallic acid equivalent), mg/g of each extract or fraction].The reaction solution was left in the dark for 1 hour to allow the reaction to occur, and then absorbance was measured at 765 nm using a UV / Visible spectrophotometer (Ultrospec 2100pro, Amersham Biosciences, USA). All experiments used gallic acid (gallic acid, Sigma, St. Louis, MO, USA) as the calibration standard (CS), and each result was expressed as the equivalent of gallic acid per gram of dry extract [gallic acid equivalent (GAE), mg / g of each extract or fraction].
그 결과, 표 1에서 나타낸 바와 같이 연 부위 조추출물 중에서 총 페놀성 성분함량이 177.71±1.23mg/g extract로 연잎 조추출물에서 페놀성 성분의 함량이 가장 높은 것으로 나타났다.As a result, as shown in Table 1, the total phenolic content of 177.71 ± 1.23mg / g extract was the highest in the lotus leaf crude extract.
수율 : 각각의 생시료 300g에서 에탄올 추출물의 퍼센트.Yield: Percent of ethanol extract in 300 g of each raw sample.
<실험 예 2> 연잎 분획물의 총 페놀성 성분의 정량Experimental Example 2 Quantification of Total Phenolic Components of Lotus Leaf Fractions
연잎 분획물의 총 페놀성 성분의 정량은 폴린-시오칼토법을 이용하였으며, Iqbal 등이 고안한 방법을 변형하여 측정하였다. Quantitative determination of the total phenolic components of the lotus leaf fraction was carried out using the Pauline-Siocalto method, which was measured by modifying the method devised by Iqbal et al.
0.4에서 2mg/ml 농도의 시료 250μl와 폴린-시오칼토 반응시약 750μl를 첨가한 반응액을 실온에서 5분간 방치한 후, 7.5% Na2CO3 2ml을 첨가하였다.250 μl of the sample at a concentration of 0.4 to 2 mg / ml and 750 μl of the Pauline-Siocalto reaction reagent were added at room temperature for 5 minutes, and then 2 ml of 7.5% Na 2 CO 3 was added.
그 후 탈이온수로 총 부피가 7ml이 되도록 희석하였다.It was then diluted with deionized water to a total volume of 7 ml.
반응액은 반응이 일어나도록 1시간 동안 암실에 방치한 후, UV/Visible spectrophotometer를 이용하여 765 nm에서 흡광도를 측정하였다. 모든 실험은 갈산을 CS로 사용하였으며, 각각의 결과는 건조 추출물 g당 갈산의 당량으로 표시하였다.The reaction solution was left in the dark for 1 hour to allow the reaction to occur, and the absorbance was measured at 765 nm using a UV / Visible spectrophotometer. All experiments used gallic acid as CS, with each result expressed in equivalents of gallic acid per gram of dry extract.
그 결과, 표 2에서 나타낸 바와 같이 연잎 에틸아세테이트 가용성 분획물이 총 페놀성 성분함량이 418.19±2.35mg/g fraction로 연잎 분획물들 중에서 총 페놀성 성분의 함량이 가장 높은 것으로 나타났다.As a result, as shown in Table 2, the total phenolic content of the lotus leaf ethyl acetate soluble fraction was 418.19 ± 2.35mg / g fraction, indicating the highest total phenolic content among the lotus leaf fractions.
수율 : 연잎 추출물에서 각각의 용매 분획물의 퍼센트.Yield: percent of each solvent fraction in lotus leaf extract.
<실험 예 3> 연 부위 조추출물의 총 플라보노이드 성분의 정량Experimental Example 3 Determination of Total Flavonoid Components of Crude Extracts
연 부위 조추출물의 총 플라보노이드 성분의 정량은 Iqbal 등이 고안한 비색분석(colorimetric) 방법을 변형하여 측정하였다(Iqbal et al., J. Food Comp. Anal., 19, pp544-551, 2006). Determination of total flavonoid components of the soft spot crude extract was determined by modifying the colorimetric method devised by Iqbal et al. (Iqbal et al., J. Food Comp. Anal., 19, pp544-551, 2006).
0.1에서 2mg/ml 농도의 시료 1ml를 탈이온수 4ml로 희석하고, 5% NaNO2 0.3ml를 넣고 5분간 실온에 방치하였다.1 ml of the sample at a concentration of 0.1 to 2 mg / ml was diluted with 4 ml of deionized water, 0.3 ml of 5% NaNO 2 was added thereto, and left at room temperature for 5 minutes.
그리고 10% AlCl3을 0.3ml 넣고 6분간 방치한 후, 1M NaOH 2ml을 첨가한다. 총 부피가 2.4ml이 되도록 탈이온수를 넣고 혼합한 후, UV/Visible spectrophotometer를 이용하여 510nm에서 흡광도를 측정하였다. 모든 실험은 (+)-카테킨(catechin) (Sigma, St. Louis, MO, USA)을 CS로 사용하였으며, 각각의 결과는 건조 추출물 g당 (+)-카테킨의 당량으로 표시하였다[CE ((+)-catechin equivalent), mg/g of each extract or fraction].0.3 ml of 10% AlCl 3 was added and allowed to stand for 6 minutes, and then 2 ml of 1 M NaOH was added. Deionized water was added and mixed so that the total volume was 2.4 ml, and the absorbance was measured at 510 nm using a UV / Visible spectrophotometer. All experiments used (+)-catechin (Sigma, St. Louis, MO, USA) as CS, and each result was expressed in equivalents of (+)-catechin per gram of dry extract [CE (( +)-catechin equivalent), mg / g of each extract or fraction].
그 결과, 표 3에서 나타낸 바와 같이 연잎 조추출물에서 총 플라보노이드 성분함량이 125.61±1.14 mg/g extract로 연 부위 중 연잎에서 플라보노이드 성분의 함량이 가장 높은 것으로 나타났다.As a result, as shown in Table 3, the total flavonoid content of the crude extract of lotus leaf was 125.61 ± 1.14 mg / g extract, indicating that the highest content of flavonoid was found in the lotus leaf of the lotus leaf.
<실험 예 4> 연잎 분획물의 총 플라보노이드 성분의 정량Experimental Example 4 Determination of Total Flavonoid Components of Lotus Leaf Fractions
연잎 분획물의 총 플라보노이드 성분의 정량은 Iqbal 등이 고안한 비색분석 방법을 변형하여 측정하였다.Quantification of the total flavonoid components of the lotus leaf fraction was measured by modifying the colorimetric method devised by Iqbal et al.
0.1에서 2mg/ml 농도의 시료 1ml를 탈이온수 4ml로 희석하고, 5% NaNO2 0.3ml를 넣고 5분간 실온에 방치하였다.1 ml of the sample at a concentration of 0.1 to 2 mg / ml was diluted with 4 ml of deionized water, 0.3 ml of 5% NaNO 2 was added thereto, and left at room temperature for 5 minutes.
그리고 10% AlCl3을 0.3ml 넣고 6분간 방치한 후, 1M NaOH 2ml을 첨가한다. 0.3 ml of 10% AlCl 3 was added and allowed to stand for 6 minutes, and then 2 ml of 1 M NaOH was added.
총 부피가 2.4ml이 되도록 탈이온수를 넣고 혼합한 후, UV/Visible spectrophotometer를 이용하여 510nm에서 흡광도를 측정하였다.Deionized water was added and mixed so that the total volume was 2.4 ml, and the absorbance was measured at 510 nm using a UV / Visible spectrophotometer.
모든 실험은 (+)-카테킨을 CS로 사용하였으며, 각각의 결과는 건조 추출물 g당 (+)-카테킨의 당량으로 표시하였다.All experiments used (+)-catechin as CS and each result was expressed as equivalent of (+)-catechin per gram of dry extract.
그 결과, 표 4에서 나타낸 바와 같이 연잎 에틸아세테이트 가용성 분획물의 총 플라보노이드 성분함량이 342.66±2.05 mg/g fraction로 연잎 분획물들 중에서 연잎 에틸아세테이트 가용성 분획물이 총 플라보노이드 성분의 함량이 가장 높은 것으로 나타났다.As a result, as shown in Table 4, the total flavonoid component content of the lotus leaf ethyl acetate soluble fraction was 342.66 ± 2.05 mg / g fraction, indicating that the lotus leaf ethyl acetate soluble fraction had the highest total flavonoid content.
<실험 예 5> 연 부위 조추출물의 알도즈 환원효소(AR) 억제활성Experimental Example 5 Aldose Reductase (AR) Inhibitory Activity of Crude Extracts
연 부위 조추출물의 랫드 렌즈 알도즈 환원효소 억제활성을 측정하기 위한 효소원의 조제는 Hayman and Kinoshite(Hayman S. and Kinoshite I. H., J. Biol. Chem., 240, pp877-882, 1965)가 사용한 방법을 수정하여 실시하였다.Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of the soft extract of soft region was used by Hayman and Kinoshite (Hayman S. and Kinoshite IH, J. Biol. Chem., 240, pp877-882, 1965). The method was modified.
흰쥐의 안구에서 수정체를 적출하고, 그 습중량에 따라 일정량(수정체 1개당 sodium phosphate buffer(pH 6.2) 0.5ml 첨가)의 sodium phosphate buffer(pH 6.2)를 가하여 균질화(homogenization)하였다.The lens was isolated from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.
이를 4℃에서 10,000g로 20분간 원심 분리한 후, 그 상등액을 취하여 효소원으로 사용하였다.After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.
1.5ml 석영 큐벳에 potassium phosphate buffer(pH 7.0) 621μl, 효소 90μl, 조효소인 NADPH(1.6 mM) 90μl, DMSO에 녹인 측정시료 9μl를 넣고, 마지막으로 기질인 DL-glyceraldehyde(50 mM)을 90μl을 넣어 총 반응액이 900μl가 되도록 하여 340nm에서 4분간 spectrophotometer를 측정하여 NADPH의 흡광도 감소율을 측정하였다.Put 621μl potassium phosphate buffer (pH 7.0), 90μl enzyme, 90μl coenzyme NADPH (1.6 mM), 9μl sample dissolved in DMSO, and finally add 90μl substrate DL-glyceraldehyde (50 mM) to 1.5ml quartz cuvette. The total reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.
대조군은 시료 대신에 DMSO를 첨가하여 측정하고, 양성 대조군은 퀘르세틴, 켐프페롤(quercetin, kaempferol: Sigma, St. Louis, MO, USA)을 이용하여 농도별로 측정하였다.The control group was measured by adding DMSO instead of the sample, and the positive control group was measured for each concentration using quercetin and camphorol (quercetin, kaempferol: Sigma, St. Louis, MO, USA).
랫드 렌즈 알도즈 환원효소 억제활성 실험 %는 하기의 수학식 1로 구하였고, 선회귀방정식을 사용하여 통계처리 하였다. % Of rat lens aldose reductase inhibitory activity was calculated by Equation 1 below and statistically processed using a regression equation.
그 결과, 표 5에서 나타낸 바와 같이 연자육을 제외한 연근, 연잎, 연심, 연수는 IC50 값이 8.8±0.12, 34.3±0.16, 36.0±0.72 및 48.3±0.86μg/ml로 높은 알도즈 환원효소 억제활성이 나타냈다.As a result, the table as shown in 5, except for yeonjayuk lotus root, lotus leaf, yeonsim, stations, IC 50 value of 8.8 ± 0.12, 34.3 ± 0.16, 36.0 ± 0.72 and 48.3 ± 0.86μg / Al higher dose reductase inhibitory activity in ml Showed this.
<실험 예 6> 연잎 조추출물 및 분획물의 알도즈 환원효소(AR) 억제활성Experimental Example 6 Aldose Reductase (AR) Inhibitory Activity of Crude Extracts and Fractions of Lotus Leaf
연잎 조추출물 및 각 용매 분획물의 랫드 렌즈 알도즈 환원효소 억제활성을 측정하기 위한 효소원의 조제는 Hayman and Kinoshite가 사용한 방법을 수정하여 실시하였다.Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of the crude leaf extract and each solvent fraction was carried out by modifying the method used by Hayman and Kinoshite.
흰쥐의 안구에서 수정체를 적출하고, 그 습중량에 따라 일정량(수정체 1개당 sodium phosphate buffer(pH 6.2) 0.5ml 첨가)의 sodium phosphate buffer(pH 6.2)를 가하여 균질화하였다.The lens was extracted from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.
이를 4℃에서 10,000g로 20분간 원심 분리한 후, 그 상등액을 취하여 효소원으로 사용하였다.After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.
1.5 ml 석영 큐벳에 potassium phosphate buffer(pH 7.0) 621μl, 효소 90μl, 조효소인 NADPH(1.6mM) 90μl, DMSO에 녹인 측정시료 9μl를 넣고, 마지막으로 기질인 DL-glyceraldehyde(50mM)을 90μl을 넣어 총 반응액이 900μl가 되도록 하여 340nm에서 4분간 spectrophotometer를 측정하여 NADPH의 흡광도 감소율을 측정하였다.Into a 1.5 ml quartz cuvette add 621 μl potassium phosphate buffer (pH 7.0), 90 μl enzyme, 90 μl coenzyme NADPH (1.6 mM), 9 μl of sample dissolved in DMSO, and finally add 90 μl of DL-glyceraldehyde (50 mM) substrate. The reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.
대조군은 시료 대신에 DMSO를 첨가하여 측정하고, 양성 대조군은 퀘르세틴, 켐프페롤을 이용하여 농도별로 측정하였다.The control group was measured by adding DMSO instead of the sample, and the positive control group was measured by concentration using quercetin and camphorol.
랫드 렌즈 알도즈 환원효소 억제활성 실험 %는 하기의 수학식 2로 구하였고, 선회귀방정식을 사용하여 통계처리 하였다. % Of rat lens aldose reductase inhibitory activity was calculated by
그 결과, 표 6에서 나타낸 바와 같이 연잎 에틸아세테이트 가용성 분획물과 부탄올 가용성 분획물의 IC50 값이 2.40±0.11, 3.26±0.10 μg/ml로 대조 화합물인 퀘르세틴(IC50 값이 1.92±0.03μg/ml)과 캠프페롤(IC50 값이 2.47±0.10μg/ml)과 비교할만한 강한 알도즈 환원효소 억제활성이 나타났다.As a result, as described lotus leaf ethyl acetate soluble fraction and the butanol-soluble fraction of the IC 50 value of 2.40 ± 0.11, 3.26 ± 0.10 μg / ml in the control compound, quercetin, as shown in Table 6 (the IC 50 value of 1.92 ± 0.03μg / ml) Strong aldose reductase inhibitory activity was shown to be comparable with that of caffeferol (IC 50 value of 2.47 ± 0.10 μg / ml).
특히 연잎 에틸아세테이트 가용성 분획물은 부탄올 가용성 분획물 보다 더 강력한 알도즈 환원효소 억제활성이 나타났다.In particular, lotus leaf ethyl acetate soluble fraction showed stronger aldose reductase inhibitory activity than butanol soluble fraction.
<실험 예 7> 연잎에서 분리된 플라보노이드 성분의 HPLC 정량분석Experimental Example 7 HPLC Quantitative Analysis of Flavonoid Components Isolated from Lotus Leaf
연잎의 에틸아세테이트 가용성 분획물을 키에셀 겔, RP-18 gel을 이용한 반복적인 column chromatography를 수행하여 5개의 플라보노이드 화합물 즉, 퀘르세틴, 퀘르세틴 3-글루코사이드, 퀘르세틴 3-글루크로나이드, 퀘르세틴 3-갈락토사이드, 퀘르세틴 3-류티노사이드를 분리하였다.Ethyl acetate soluble fraction of lotus leaf was subjected to repeated column chromatography using kiesel gel and RP-18 gel to give five flavonoid compounds: quercetin, quercetin 3-glucoside, quercetin 3-gluronide, quercetin 3-galactoside Quercetin 3-leutinoside was isolated.
연잎 에틸아세테이트 가용성 분획물과 부탄올 가용성 분획물에서 활성성분의 정량분석을 위해서 HPLC 분석을 실시하였다.HPLC analysis was performed for quantitative analysis of the active ingredient in the lotus leaf ethyl acetate soluble fraction and butanol soluble fraction.
Chromatography는 Dionex HPLC system Dionex Co., Germering, Germany)으로 on-line vacuum degasser가 포함된 quatenary gradient pump (Model P680A), 자동 sample injector(Model ASI-100), 4-channel multi UV-Vis detector Model 170 U)를 사용하였다.Chromatography is a Dionex HPLC system Dionex Co., Germering, Germany), a quatenary gradient pump (Model P680A) with an on-line vacuum degasser, an automatic sample injector (Model ASI-100), and a 4-channel multi UV-Vis detector Model 170 U) was used.
연잎 추출물과 분획물로부터 플라보노이드 성분의 분석은 guard column(2.0×40 mm, 3μm, Phenomenex, Torrance, CA, USA)과 C-18 column(2.0×150 mm, 3μm, Phenomenex, Torrance, CA, USA)를 이용하여 정량분석을 수행하였다.Analysis of flavonoid components from lotus leaf extracts and fractions was performed using guard columns (2.0 × 40 mm, 3μm, Phenomenex, Torrance, CA, USA) and C-18 columns (2.0 × 150 mm, 3μm, Phenomenex, Torrance, CA, USA). Quantitative analysis was performed.
HPLC에서 사용된 이동상에서 이동상 A는 0.1% 포름산(formic acid)이 함유된 탈이온수, 이동상 B는 0.1% 포름산이 함유된 아세토나이트릴(acetonitrile)을 각각 사용하였다.In the mobile phase used in HPLC, mobile phase A used deionized water containing 0.1% formic acid, and mobile phase B used acetonitrile containing 0.1% formic acid, respectively.
이동상의 gradient 조건은 0분에서 10분까지는 10% B, 10분에서 40분까지는 10→20% B, 40분에서 42분까지는 20% B, 42분에서 45분까지는 60% B, 45분에서 60분까지는 10% B를 사용하였고, 유속은 1분당 0.2ml이며, injection volume은 1ml의 메탄올에 시료 2mg을 녹인 용액을 5μl injection 하였다.Gradient condition of mobile phase is 10% B from 0 to 10 minutes, 10 → 20% B from 10 to 40 minutes, 20% B from 40 to 42 minutes, 60% B from 42 to 45 minutes, from 45 minutes Up to 60 minutes, 10% B was used, the flow rate was 0.2 ml per minute, and the injection volume was 5 μl injection of a solution of 2 mg of sample dissolved in 1 ml of methanol.
UV 파장은 340nm에서 분석하였다. 다섯 개의 상대적인 양은 하기의 수학식 3으로 구하였다. UV wavelength was analyzed at 340 nm. Five relative amounts were obtained from
그 결과, 도 1, 도 2 및 표 8에 나타낸 바와 같이 연잎 에틸아세테이트 가용성 분획물에서 퀘르세틴 3-글루코사이드가 32.1%, 퀘르세틴 3-글루크로나이드 30.4%로 이들 화합물이 주성분임을 확인하였고, 퀘르세틴 3-갈락토사이드는 4.3%, 퀘르세틴은 2.3%, 퀘르세틴 3-루티노사이드가 0.3%로 미량이 존재함을 확인하였다.As a result, as shown in Fig. 1, Fig. 2 and Table 8, it was confirmed that these compounds were the main component of the quercetin 3-glucoside in the lotus leaf ethyl acetate soluble fraction, 32.1% and quercetin 3-gluronide 30.4%, and the quercetin 3-gal Lactobacillus was found to be 4.3%, quercetin 2.3%, and quercetin 3-lutinoside 0.3%.
그리고 연잎 부탄올 가용성 분획물에서는 퀘르세틴 3-글루크로나이드가 38.8%를 차지하여 주성분임을 확인할 수 있었다. In the lotus leaf butanol soluble fraction, quercetin 3-gluronide accounted for 38.8%, confirming that it was the main component.
이때, 상기 도 1에서의 (a)는 연잎 부탄올 가용성 분획물의 HPLC chromatograms를 나타내고 있고, (b)는 연잎 아세테이트 가용성 분획물의 HPLC chromatograms를 나타내고 있다. 또한 도 1의 그래프에서 표시된 1 내지 5는 각각 '1 : 퀘르세틴 3-루티노사이드, 2 : 퀘르세틴 3-갈락토사이드, 3 : 퀘르세틴 3-글루코사이드, 4 : 퀘르세틴 3-글루크로나이드, 5 : 퀘르세틴'을 나타내고 있으며, '*'는 'unknown peak'을 나타내고 있다.In this case, (a) in FIG. 1 shows HPLC chromatograms of lotus leaf butanol soluble fraction, and (b) shows HPLC chromatograms of lotus leaf acetate soluble fraction. In addition, 1 to 5 shown in the graph of Fig. 1 is' 1: quercetin 3-lutinoside, 2: quercetin 3-galactoside, 3: quercetin 3-glucoside, 4: quercetin 3-gluronide, 5: quercetin ',' And '*' represents 'unknown peak'.
<실험 예 8> 연잎에서 분리된 플라보노이드 성분의 알도즈 환원효소 (AR) 억제활성Experimental Example 8 Aldose Reductase (AR) Inhibitory Activity of Flavonoid Components Isolated from Lotus Leaf
연잎의 에틸아세테이트 가용성 분획물에서 분리된 플라보노이드 성분의 랫드 렌즈 알도즈 환원효소 억제활성을 측정하기 위한 효소원의 조제는 Hayman and Kinoshite가 사용한 방법을 수정하여 실시하였다.Preparation of enzyme source for measuring rat lens aldose reductase inhibitory activity of flavonoid component isolated from ethyl acetate soluble fraction of lotus leaf was carried out by modifying the method used by Hayman and Kinoshite.
흰쥐의 안구에서 수정체를 적출하고, 그 습중량에 따라 일정량(수정체 1개당 sodium phosphate buffer(pH 6.2) 0.5ml 첨가)의 sodium phosphate buffer(pH 6.2)를 가하여 균질화하였다.The lens was extracted from the eye of the rat, and homogenized by adding a certain amount (0.5 ml of sodium phosphate buffer (pH 6.2) per lens) according to the wet weight.
이를 4℃에서 10,000g로 20분간 원심 분리한 후, 그 상등액을 취하여 효소원으로 사용하였다.After centrifugation at 10,000 g for 20 minutes at 4 ℃, the supernatant was taken and used as the enzyme source.
1.5ml 석영 큐벳에 potassium phosphate buffer(pH 7.0) 621μl, 효소 90μl, 조효소인 NADPH(1.6mM) 90 μl, DMSO에 녹인 측정시료 9μl를 넣고, 마지막으로 기질인 DL-glyceraldehyde(50mM)을 90μl을 넣어 총 반응액이 900μl가 되도록 하여 340nm에서 4분간 spectrophotometer를 측정하여 NADPH의 흡광도 감소율을 측정하였다.Put 621μl potassium phosphate buffer (pH 7.0), 90μl enzyme, 90μl coenzyme NADPH (1.6mM), 9μl sample dissolved in DMSO, and finally add 90μl substrate DL-glyceraldehyde (50mM) to 1.5ml quartz cuvette. The total reaction solution was 900 μl and the absorbance reduction rate of NADPH was measured by measuring a spectrophotometer at 340 nm for 4 minutes.
대조군은 시료 대신에 DMSO를 첨가하여 측정하여 농도별로 측정하였다.The control group was measured by concentration by adding DMSO instead of the sample.
랫드 렌즈 알도즈 환원효소 억제활성 실험 %는 하기의 수학식 4로 구하였고, 선회귀방정식을 사용하여 통계처리 하였다. % Of rat lens aldose reductase inhibitory activity was calculated by Equation 4 below and statistically processed using a regression equation.
그 결과, 표 8에서 나타낸 바와 같이 연잎 에틸아세테이트 가용성 분획물로 분리된 플라보노이드 성분 중에서 퀘르세틴-3-루티노사이드가 IC50 값이 2.49±0.04μM로 가장 강력한 알도즈 환원효소 억제활성이 나타났고, 그 다음으로 퀘르세틴(IC50 값이 5.54±0.15μM), 퀘르세틴-3-글루크로나이드(IC50 값이 13.59±0.06μM), 퀘르세틴-3-갈락토사이드(IC50 값이 19.30±0.00μM), 퀘르세틴-3-글루코사이드(IC50 값이 20.83±0.51μM) 순으로 알도즈 환원효소 억제활성이 나타났다.As a result, as shown in Table 8, among the flavonoid components separated from the lotus leaf ethyl acetate soluble fraction, quercetin-3-rutinoside showed the strongest aldose reductase inhibitory activity with an IC 50 value of 2.49 ± 0.04 μM. Next, quercetin (IC 50 value is 5.54 ± 0.15 μM), quercetin-3-gluronide (IC 50 value is 13.59 ± 0.06 μM), quercetin-3-galactoside (IC 50 value is 19.30 ± 0.00 μM), Quercetin-3-glucoside (IC 50 value of 20.83 ± 0.51 μM) showed aldose reductase inhibitory activity.
<실험 예 9> 연 부위 조추출물의 최종당화산물 (AGEs) 생성 억제활성Experimental Example 9 Inhibitory Activity on the Production of Final Glycation Products (AGEs)
연 부위 조추출물의 최종당화산물 생성 억제활성의 측정은 Vinson(Vinson J. A., J. Nutr. Biochem., 7, pp659-663, 1996)이 사용한 방법을 수정하여 실시하였다.Determination of the final glycation end product production inhibitory activity of the crude extract was performed by modifying the method used by Vinson (Vinson J. A., J. Nutr. Biochem., 7, pp659-663, 1996).
AGE 반응 시약은 세균 증식을 막기 위해서 0.02% sodiumazide가 첨가된 50mM sodium phosphate buffer(pH 7.4)에 10mg/ml의 bovine serum albumin(Sigma, St. Louis, MO, USA)과 0.2M fructose와 0.2 M glucose를 넣어 만든다.AGE reaction reagents were used in 50 mM sodium phosphate buffer (pH 7.4) with 0.02% sodiumazide (10 mg / ml bovine serum albumin (Sigma, St. Louis, MO, USA)), 0.2 M fructose and 0.2 M glucose Make it put
시료는 10% DMSO에 녹인 후, AGE 반응 시약 950μl에 반응시료 50μl를 넣은 후, 37℃에서 7 일간 배양한다.The sample was dissolved in 10% DMSO, 50 μl of the reaction sample was added to 950 μl of the AGE reaction reagent, and then incubated at 37 ° C. for 7 days.
배양 후, 반응 생성물의 형광강도를 spectrofluorometric detector로 excitation과 emission 파장을 350nm과 450nm에서 측정한다.After incubation, the fluorescence intensity of the reaction product was measured with a spectrofluorometric detector at excitation and emission wavelengths of 350 nm and 450 nm.
최종당화산물 생성 억제활성 실험 %는 하기의 수학식 5로 구하였고, 선회귀방정식을 사용하여 통계처리 하였다. % Of the final glycation product inhibitory activity was calculated by Equation 5 below and statistically processed using a regression equation.
그 결과, 표 9에서 나타낸 바와 같이 연 부위 중 연잎과 연수는 IC50 값이 110.5±2.50과 125.5±7.80μg/ml로 양성 대조군인 아미노구아니딘(IC50 값 = 84.9±0.51μg/ml)과 비교할만한 강력한 최종당화생성물 생성 억제활성이 나타났다.As a result, as shown in Table 9, the lotus leaf and soft water in the soft parts had IC 50 values of 110.5 ± 2.50 and 125.5 ± 7.80μg / ml, compared with aminoguanidine (IC 50 value = 84.9 ± 0.51μg / ml) as a positive control. There was a potent inhibitory activity on the production of end glycosylated products.
<실험 예 10> 연잎 조추출물 및 분획물의 최종당화산물 (AGEs) 생성 억제활성Experimental Example 10 Inhibitory Activity of Crude Extracts and Fractions of Final Glycation Products (AGEs)
연잎 조추출물 및 분획물의 최종당화산물 생성 억제활성의 측정은 Vinson이 사용한 방법을 수정하여 실시하였다.The crude glycoside extract and fractions of the final glycation product production inhibition activity was measured by modifying the method used by Vinson.
AGE 반응 시약은 세균 증식을 막기 위해서 0.02% sodiumazide가 첨가된 50mM sodium phosphate buffer(pH 7.4)에 10mg/ml의 bovine serum albumin과 0.2M fructose와 0.2M glucose를 넣어 만든다.The AGE reaction reagent is prepared by adding 10 mg / ml bovine serum albumin, 0.2 M fructose and 0.2 M glucose in 50 mM sodium phosphate buffer (pH 7.4) containing 0.02% sodiumazide to prevent bacterial growth.
시료는 10% DMSO에 녹인 후, AGE 반응 시약 950μl에 반응시료 50μl를 넣은 후, 37℃에서 7일간 배양한다.The sample is dissolved in 10% DMSO, 50 μl of the reaction sample is added to 950 μl of the AGE reaction reagent, and then incubated at 37 ° C. for 7 days.
배양 후, 반응 생성물의 형광강도를 spectrofluorometric detector로 excitation과 emission 파장을 350nm과 450nm에서 측정한다.After incubation, the fluorescence intensity of the reaction product was measured with a spectrofluorometric detector at excitation and emission wavelengths of 350 nm and 450 nm.
최종당화산물 생성 억제활성 실험 %는 하기의 수학식 6으로 구하였고, 선회귀방정식을 사용하여 통계처리 하였다. % Of the final glycation product inhibitory activity was calculated by Equation 6 below and statistically processed using a regression equation.
그 결과, 표 10에서 나타낸 바와 같이 연잎 분획물들 중에서 에틸아세테이트 가용성 분획물은 IC50 값이 28.18±0.63μg/ml로 가장 강력한 최종 당화생성물 생성 억제활성이 나타났고, 양성 대조군인 아미노구아니딘(IC50 값 = 45.51±0.69μg/ml) 보다 더 강력한 억제효과가 나타났다.As a result, as shown in Table 10, the ethyl acetate soluble fraction of the lotus leaf fractions showed the strongest inhibitory activity of the final glycation product formation with an IC 50 value of 28.18 ± 0.63 μg / ml, and aminoguanidine (IC 50 value). = 45.51 ± 0.69 μg / ml).
본 발명은 이상에서 살펴본 바와 같이 바람직한 실시 예를 들어 도시하고 설명하였으나, 상기한 실시 예에 한정되지 아니하며 본 발명의 정신을 벗어나지 않는 범위 내에서 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 의해 다양한 변경과 수정이 가능할 것이다.Although the present invention has been shown and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.
도 1은 연잎 부탄올 가용성 분획물 및 연잎 아세테이트 가용성 분획물의 HPLC chromatograms를 나타내는 그래프이다.1 is a graph showing HPLC chromatograms of lotus leaf butanol soluble fraction and lotus leaf acetate soluble fraction.
도 2는 연잎 디클로로메탄 가용성 분획물, 연잎 에틸아세테이트 가용성 분획물 및 연잎 부탄올 가용성 분획물에서의 퀘르세틴 3-루티노사이드, 퀘르세틴 3-갈락토사이드, 퀘르세틴 3-글루코사이드, 퀘르세틴 3-글루크로나이드 및 퀘르세틴의 함유량을 보여 주는 그래프이다.Figure 2 Contents of quercetin 3-lutinoside, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-gluronide and quercetin in the lotus leaf dichloromethane soluble fraction, the lotus leaf ethyl acetate soluble fraction and the lotus leaf butanol soluble fraction This graph shows
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KR100978849B1 (en) * | 2010-01-22 | 2010-08-30 | 대상 주식회사 | Chlorella suspension for sea feed comprising extract of lotus leaf |
CN102827220A (en) * | 2012-08-24 | 2012-12-19 | 湖州市食品药品检验所 | Method for separating rutin, hyperoside, isoquercitrin and quercetin from lotus leaves |
KR20160141963A (en) | 2015-06-02 | 2016-12-12 | 안병준 | Edible plant mucilages as carrier for plant extracts containing flavonoid compounds and a composition comprising thereof |
JP2017514913A (en) * | 2014-05-05 | 2017-06-08 | 南京睿▲鷹▼▲潤▼▲澤▼生物医▲薬▼科技有限公司Nanjing Ruiying Runze Biopharmaceutical Technology Co., Inc. | A composition containing a fat lowering active ingredient of Chinese medicine hair chicory |
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KR100978849B1 (en) * | 2010-01-22 | 2010-08-30 | 대상 주식회사 | Chlorella suspension for sea feed comprising extract of lotus leaf |
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WO2011090343A3 (en) * | 2010-01-22 | 2011-12-15 | 대상 주식회사 | Chlorella suspension for aquaculture feed containing folium nelumbinis extract |
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JP2017514913A (en) * | 2014-05-05 | 2017-06-08 | 南京睿▲鷹▼▲潤▼▲澤▼生物医▲薬▼科技有限公司Nanjing Ruiying Runze Biopharmaceutical Technology Co., Inc. | A composition containing a fat lowering active ingredient of Chinese medicine hair chicory |
KR20160141963A (en) | 2015-06-02 | 2016-12-12 | 안병준 | Edible plant mucilages as carrier for plant extracts containing flavonoid compounds and a composition comprising thereof |
WO2019088381A1 (en) * | 2017-10-31 | 2019-05-09 | 경희대학교 산학협력단 | Composition for preventing, ameliorating or treating obesity and metabolic diseases, comprising complex extract from peach blossom and lotus leaf |
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