[논문]비자나무 추출물의 항당뇨 활성물질의 특성 연구

김지원; 김동섭; 이화신; 박보배; 유선녕; 황유림; 김상헌; 안순철

doi:10.5352/jls.2022.32.1.1

초록
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비자나무는 한반도 남부지역과 제주도에 자생하고 있으며 식용으로 이용이 가능하며, 전통적으로 구충 빛 변비 예방의 목적으로 사용되어 왔었다. 그러나 항산화 활성에 대한 연구는 보고되어 왔지만 항당뇨 활성에 대한 연구는 제대로 이루어지지 않고 있다. 새로운 당뇨 치료제로서 천연물은 독성의 염려가 낮고 매우 오랫동안 사용되어 왔기 때문에 약물에 대한 안전성의 장점이 있다. 식용 및 한방 재료로 사용되고 있는 비자나무는 지금까지 살충 및 항균성에 대해 보고되어 왔으나 항당뇨에 관한 연구는 이루어진 바가 없다. 따라서 본 연구에서는 비자나무 추출물의 항당뇨 효과를 조사하고 그 특성을 조사하였다. 먼저, 비자나무의 과육과 종자를 메탄올로 각각 추출한 후, 항당뇨 활성과 관련된 α-glucosidase와 protein tyrosine phosphatase 1B (PTP1B)에 대한 저해활성을 포함한 다양한 생리활성을 조사하였다. 그 결과, 비자나무의 종자 추출물에서 항당뇨활성을 나타내는 α-glucosidase와 PTP1B 효소에 대한 억제 효능이 높게 나타났으며, 특히 과육에 비해 종자 추출물이 각각 14.5배, 4.3배 높은 저해활성을 보였다. 또한 비자나무의 과육 추출물의 pH와 열 안정성 테스트를 수행한 결과로, 활성 물질은 산, 알카리 조건과 고온의 조건에서 안정한 저해활성을 보였다. 비자 과육의 메탄올 추출물을 한외 여과(ultrafiltration)한 결과, 항당뇨 활성물질은 분자량이 300 kDa 이상에 해당하였고 Diaion HP-20 수지에 대한 흡착능을 조사한 결과, 50-100% 메탄올 조건에서 항당뇨 활성물질이 용출되었다. 따라서 비자나무 종자의 메탄올 추출물로부터 buthanol 추출, 한외 여과, Diaion HP-20 컬럼 크로마토그래피를 통해 비자나무의 항당뇨 활성 물질의 분리, 정제를 시도하였다. 따라서 비자나무 종자 추출물의 인슐린 저항성에 대한 개선 효과에 대한 추가 연구를 통해 천연물 유래 당뇨병 예방 및 치료용 조성물로서의 가능성을 보여주었다.

Abstract ▼ AI-Helper

Natural products have gained increasing attention due to their advantage of long-term safety and low toxicity for a very long time. Torreya nucifera is widespread in southern Korea and Jeju Island and its seeds are commonly used as edible food. Oriental ingredients have often been reported for their...

Natural products have gained increasing attention due to their advantage of long-term safety and low toxicity for a very long time. Torreya nucifera is widespread in southern Korea and Jeju Island and its seeds are commonly used as edible food. Oriental ingredients have often been reported for their insecticidal, antioxidant and antibacterial properties, but there have not yet been any studies on their antidiabetic effect. In this study, we investigated several biological activities of T. nucifera pericarp (TNP) and seeds (TNS) extracts and proceeded to characterize the antidiabetic compounds of TNS. The initial results suggested that TNS extract at 15 and 10 ㎍/ml concentration has inhibitory effects on α-glucosidase and protein tyrosine phosphatase 1B, that is 14.5 and 4.35 times higher than TNP, respectively. Thus, the stronger antidiabetic TNS was selected for the subsequent experiments to characterize its active compounds. Ultrafiltration was used to determine the apparent molecular weight of the active compounds, showing 300 kDa or more. Finally the mixture was then partially purified using Diaion HP-20 column chromatography by eluting with 50~100% methanol. Therefore we concluded that the active compounds of TNS have potential as therapeutic agents in functional food or supplemental treatment to improve diabetic diseases.

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표/그림 (6)

그림 Fig. 1. Evaluation of biological activities of Torreya nucifera extract. (A) α-glucosidase inhibitory activity. The positive control was acarbose. (B) PTP1B inhibitory activity. The positive control was ursolic acid. (C) Tyrosinase inhibitory activity. The positive control was kojic acid. (D) DPPH free radical scavenging activity. The positive control was ascorbic acid. (E) ACE inhibitory activity. The positive control was captopril. (F) XO inhibitory activity. The positive control was allopurinol. Each experiment was performed in duplicate or triplicate. TNP, methanol extract of Torreya nucifera pericarp; TNS, methanol extract of Torreya nucifera seeds; PTP1B, Protein tyrosine phosphatase 1B; ACE, Angiotensin-converting enzyme; XO, xanthine oxidase. Data were presented as mean ± SD (n=3 in each group, *p<0.05, **p<0.01, ***p<0.001 compared to control).
$Fig. 2. Extraction conditions of active compounds from Torreya nucifera seeds (TNS). (A) Extraction with hot water or methanol. To confirm the maximum extraction conditions for the active substance of TNS, methanol extract of remaining precipitates (HWME-1) after heating at 100℃ for 10 minutes (HWE-1), methanol extract of remaining precipitates (HWME-2) after heating at 121℃ for 15 minutes (HWE-2), and methanol extract (ME) at room temperature (RT) were prepared and investigated to observe α-glucosidase inhibition activity. (B) Organic solvent extraction. Each sample was fractionated into butanol layer and water layer and their inhibitory activities were determined on α-glucosidase. Data were presented as mean ± SD (n=3 in each group, *p<0.05, **p<0.01, ***p<0.001 compared to control).$ 그림 Fig. 2. Extraction conditions of active compounds from Torreya nucifera seeds (TNS). (A) Extraction with hot water or methanol. To confirm the maximum extraction conditions for the active substance of TNS, methanol extract of remaining precipitates (HWME-1) after heating at 100℃ for 10 minutes (HWE-1), methanol extract of remaining precipitates (HWME-2) after heating at 121℃ for 15 minutes (HWE-2), and methanol extract (ME) at room temperature (RT) were prepared and investigated to observe α-glucosidase inhibition activity. (B) Organic solvent extraction. Each sample was fractionated into butanol layer and water layer and their inhibitory activities were determined on α-glucosidase. Data were presented as mean ± SD (n=3 in each group, *p<0.05, **p<0.01, ***p<0.001 compared to control).
$Fig. 3. Molecular weight fractionation of active compounds from Torreya nucifera seeds (TNS) extract. After ultrafiltration of TNS extract using 300 kDa molecular weight cut-off membrane, their fractions were assayed to observe α- glucosidase inhibition activity. Data were presented as mean ± SD (n=3 in each group, **p<0.01, ***p<0.001 compared to control).$ 그림 Fig. 3. Molecular weight fractionation of active compounds from Torreya nucifera seeds (TNS) extract. After ultrafiltration of TNS extract using 300 kDa molecular weight cut-off membrane, their fractions were assayed to observe α- glucosidase inhibition activity. Data were presented as mean ± SD (n=3 in each group, **p<0.01, ***p<0.001 compared to control).
그림 Fig. 4. Heat and pH stability of active compounds from Torreya nucifera seeds. (A) pH stability. (B) Heat stability. TNS extracts were adjusted to pH 2, 7, and 10 and heated at 100℃ for 10 minutes and 121℃ for 15 minutes, respectively. Each extract was assayed to observe remaining α-glucosidase inhibition activity. Data were presented as mean ± SD (n=3 in each group, **p<0.01, ***p<0.001 compared to control).
$Fig. 5. Preliminary adsorption experiment on Diaion HP-20 resin of the Torreya nucifera seeds (TNS) extract. (A) Adsorption of TNS extract at pH 2. (B) Adsorption of TNS extract at pH 7. Passing fractions from Diaion HP-20 were collected in 2 ml of each fraction (fr.#1~fr.#2). In 20% methanol (MeOH), 50% MeOH, and 100% MeOH elution, each 2 ml was collected into 4 fractions (fr.#1~fr.#4). The main bar showed the α-glucosidase inhibitory ability and the line showed the total solid (mg) of each fraction.$ 그림 Fig. 5. Preliminary adsorption experiment on Diaion HP-20 resin of the Torreya nucifera seeds (TNS) extract. (A) Adsorption of TNS extract at pH 2. (B) Adsorption of TNS extract at pH 7. Passing fractions from Diaion HP-20 were collected in 2 ml of each fraction (fr.#1~fr.#2). In 20% methanol (MeOH), 50% MeOH, and 100% MeOH elution, each 2 ml was collected into 4 fractions (fr.#1~fr.#4). The main bar showed the α-glucosidase inhibitory ability and the line showed the total solid (mg) of each fraction.
$Fig. 6. Partial purification of active compounds from Torreya nucifera seeds (TNS) extract. (A) Schematic diagram of partial purification from TNS extract. (B) Inhibitory activity of partial purified fraction on α-glucosidase. (C) Inhibitory activity of partial purified fraction on protein tyrosine phosphatase 1B (PTP1B). Data were presented as mean ± SD (n=3 in each group, *p<0.05, ***p<0.001 compared with DW control).$ 그림 Fig. 6. Partial purification of active compounds from Torreya nucifera seeds (TNS) extract. (A) Schematic diagram of partial purification from TNS extract. (B) Inhibitory activity of partial purified fraction on α-glucosidase. (C) Inhibitory activity of partial purified fraction on protein tyrosine phosphatase 1B (PTP1B). Data were presented as mean ± SD (n=3 in each group, *p<0.05, ***p<0.001 compared with DW control).

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대상 데이터

Torreya nucifera seeds were purchased from a market of Namhae region in Korea. Methanol (MeOH), n-butanol (BuOH), and ethyl acetate (EtOAc) as solvents were purchased from SK Chemicals (Ulsan, Korea) and Diaion HP-20 was purchased from the Mitsubishi Kasei Co.

데이터처리

Unless otherwise stated, data were expressed as the mean ± SD. Statistical comparisons were used by ANOVA. Results were statistically significant at p<0.

이론/모형

The remaining biological activities of TNS ME under different pH and temperature were examined by α-glucosidase assay.

성능/효과

As a result, fraction of molecular weight of 300 kDa or higher from TNS ME was most on α-glucosidase inhibitory activity (Fig
As a result, methanol (MeOH) was more efficient to extract active compounds than hot water at high temperature, based on the observation of higher α-glucosidase activity in the MeOH extraction than HWE of T
In this study, active substances included in TNS were likely to be therapeutic agents that can improve diabetes, because its seeds showed significant α-glucosidase inhibitory activity (Fig
Taken together, it seemed that TNS has stronger antidiabetic activity on the base of the results from α-glucosi- dase and PTP1B inhibitory activities
Therefore MeOH extraction from TNS at room temperature was chosen as a best extraction process to yield active compounds with α-glucosidase inhibitory activity
1D). These results suggested that TNS contains more antioxidant substances than TNP. Captopril is a synthetic angiotensin-con- verting enzyme (ACE) inhibitor, which is associated with oxidative stress and hypertension.

후속연구

Finally the most active MW≥300 kDa fraction of TNS extract was used for further experiments to characterize its active compounds.
nucifera seeds could treat type 2 diabetes by improving the insulin resistance signaling by regulating α- glucosidase and PTP1B activities. Therefore, by analyzing their antidiabetic activity in additional in vivo experiments using animal models in the future, active substances of TNS can be an appropriate functional food and supplemental treatment in T2DM in various experimental aspects.

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비자나무 추출물의 항당뇨 활성물질의 특성 연구
Characterization of Antidiabetic Compounds from Extract of Torreya nucifera 원문보기

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비자나무 추출물의 항당뇨 활성물질의 특성 연구 Characterization of Antidiabetic Compounds from Extract of Torreya nucifera 원문보기

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비자나무 추출물의 항당뇨 활성물질의 특성 연구
Characterization of Antidiabetic Compounds from Extract of Torreya nucifera 원문보기

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