컬럼 크로마토그래피를 이용한 포도 추출물로부터 레스베라트롤의 정제

Sewon Kim,Jaeho Pyee 한국약용작물학회 2016 한국약용작물학술대회 발표집 Vol.2016 No.10

Background : Resveratrol is the stillbenoid material made by the stress factors from the plants like grape, blueberry and Polygonum cuspidatum and it has useful pharmacological activity such as anti-cancer and longevity effect. Although grapevine contains less resveratrol compared to Polygonum cuspidatum, it is expected that a large amount of resveratrol can be obtained using by-products from the processing because grapes are one of the crops grown and processed considerably in south Korea. Methods and Results : As the result of the analysis of resveratrol content in grape extract using high-performance liquid chromatography (HPLC), it was showed that the amount of the resveratrol was 46.8~47.8 mg(about 4.7~4.8%) per 1 g of grape extract. The method used for purifying resveratrol from grape extract was a column chromatography and adsorption resin was used in packing the column. The content of resveratrol in the eluate obtained by column chromatography was analyzed by HPLC. Then the solid content obtained by freeze-drying was analyzed to get the purity and the collect rate of the resveratrol by measuring the weight. The result showed that the purity of the resveratrol in eluate obtained by column chromatography was measured to 15.2~18.1% and collect rate was up to 95%. Conclusion : From the above results, we have isolated resveratrol which is more pure than the conventional from grape extract and assumed that it is helpful to produce functional material at a low cost by using this isolation method.

A New Process for Mass Production of Resveratrol (I): Analysis of Resveratrol Content and the Expression Profile of a Gene Encoding Resveratrol Synthase in Various Tissues of Polygonum cuspidatum Sieb.Et Zucc

Kisuk Bae,Jaeho Pyee 한국산업식품공학회 2004 산업 식품공학 Vol.8 No.4

The roots of Polygonum cuspidatum have been used as herbal medicines in Asia and resveratrol is one of the main active chemicals of root extract. Although a great number of studies have reported its biological and pharmacological activities and purification, resveratrol biosynthesis has never been investigated at the biochemical or genetic level in P. cuspidatum. Hence, we tested resveratrol content in various tissues. Roots accumulated approximately 20 to 50-fold higher levels of resveratrol than other parts of P. cuspidatum plant. Resveratrol content in roots was increased by about 1.84-fold by irradiation with ultraviolet as compared to untreated roots. RT-PCR analysis also showed that PcSTSY, a gene encoding resveratrol synthase (STSY) was expressed at the highest level in roots compared to other tissues in P. cuspidatum plant. These results suggest that resveratrol synthesis is spatially regulated in this plant and that the biosynthesis is induced by irradiation with ultraviolet. In conclusion, this biochemical and genetic results will be applicable to development of foods and beverages containing a high level of resveratrol with beneficial effects on health. The roots of Polygonum cuspidatum have been used as herbal medicines in Asia and resveratrol is one of the main active chemicals of root extract. Although a great number of studies have reported its biological and pharmacological activities and purification, resveratrol biosynthesis has never been investigated at the biochemical or genetic level in P. cuspidatum. Hence, we tested resveratrol content in various tissues. Roots accumulated approximately 20 to 50-fold higher levels of resveratrol than other parts of P. cuspidatum plant. Resveratrol content in roots was increased by about 1.84-fold by irradiation with ultraviolet as compared to untreated roots. RT-PCR analysis also showed that PcSTSY, a gene encoding resveratrol synthase (STSY) was expressed at the highest level in roots compared to other tissues in P. cuspidatum plant. These results suggest that resveratrol synthesis is spatially regulated in this plant and that the biosynthesis is induced by irradiation with ultraviolet. In conclusion, this biochemical and genetic results will be applicable to development of foods and beverages containing a high level of resveratrol with beneficial effects on health.

Pinosylvin at a high concentration induces AMPK-mediated autophagy for preventing necrosis in bovine aortic endothelial cells

Park, Jinsun,Pyee, Jaeho,Park, Heonyong Canadian Science Publishing 2014 Canadian journal of physiology and pharmacology Vol.92 No.12

<P> Pinosylvin is a known functional compound of the Pinus species. Pinosylvin at low concentrations (∼pmol/L) was reported to promote cell proliferation in endothelial cells. However, this study found that pinosylvin at a high concentration (100 μmol/L) induces cell death in bovine aortic endothelial cells. Therefore, we examined how pinosylvin was associated with apoptosis, autophagy, and necrosis. Pinosylvin at a high concentration appeared to promote caspase-3 activation, nuclear condensation, and the “flip-flop” of phosphatidylserine, indicating that pinosylvin induces apoptosis. However, based on flow cytometry data obtained from double-staining with annexin V and propidium iodide, pinosylvin was shown to inhibit necrosis, a postapoptotic process. Pinosylvin induced LC3 conversion from LC3-I to LC3-II and p62 degradation, which are important indicators of autophagy. In addition, AMP-activated protein kinase (AMPK) appeared to be activated by pinosylvin, and an AMPK inhibitor was markedly shown to reduce the LC3 conversion. The inhibitory effect of an AMPK inhibitor was reversed by pinosylvin. These results suggest that pinosylvin induces autophagy via AMPK activation. Further, necrosis was found to be promoted by an autophagy inhibitor and then restored by pinosylvin, while the caspase-3 inhibitor had no effect on necrosis. These findings indicate that pinosylvin-induced autophagy blocks necrotic progress in endothelial cells. </P>

Purification of Resveratrol in Grape Extract using Column Chromatography

Sewon Kim(김세원),Jaeho Pyee(피재호) 한국약용작물학회 2016 한국약용작물학회 학술대회논문집 Vol.2016 No.2

A new method for immobilization of lipid-binding proteins to the glass slides

Jaeyoung Shin,Minho Cho,June Won Hyun,Jaeho Pyee,Heonyong Park 한국물리학회 2006 Current Applied Physics Vol.6 No.2

Previously, we constructed nickel-coated glass slides to immobilize histidine-tagged proteins [Bull. Korean Chem. Soc. 23 (2002)17241728]. Here, we further investigated whether the nickel-coated glass slide could be utilized for assaying molecular interactionbetween lipid and immobilized protein. Proteins interact with various lipid molecules in biological systems. More importantly, thisinteraction is responsible for a broad spectrum of physiological functions. Accordingly, to more eciently analyze various types ofthe high-throughput analytical purpose, we immobilized an FITC (uorescein isothiocyanate)-tagged lipid-binding protein tonickel-coated plates, incubated the plates with various lipid molecules and then measured uorescence intensities to analyze its bind-ing anity. Our systems seemed to be reliable for the application to the high-throughput system consisted of lipid-binding proteins.

Regeneration of Grape(Vitis labruscana cv. Kyoho) by Shoot-Tip Culture)

Park, Hye-Jeong,Lee, Ho-Rim,Pyee, Jaeho,Cha, Hyeon-Cheol 한국식물학회 2001 Journal of Plant Biology Vol.44 No.4

We investigated the optimal levels of growth regulators, culture media, and pH on callus growth and organogenesis of in-vitro cultured 'Kyoho' grapes. Calli were induced by culturing leaf blades on an MS basal medium supplemented with 1㎎/L BA and 0.01㎎/L 2,4-D. In addition, calli originating from the exocarp and mesocarp of grape fruits developed on MS media supplemented with 0.1㎎/L IAA, NAA, or 2,4-D, or with 0.2㎎/L BA. In testing the potential for plant regeneration from shoot tips on various media, we found that the Nitsch medium, with 1㎎/L BA, was optimal for caulogenesis. The type of shoot development depended on the pH of the medium, with vigorous multiple-shoot development occurring at pH 6.0, and single shoots forming at pH 5.0. Finally, we were able to obtain rooted seedlings from the regenerated shoots that had been cultured on 1/4-strength Nitsch medium supplemented with 0.03㎎/L NAA.

Extract from <i>Acanthopanax senticosus</i> prevents LPS-induced monocytic cell adhesion via suppression of LFA-1 and Mac-1

Kim, Hyun Jeong,McLean, Danielle,Pyee, Jaeho,Kim, Jongmin,Park, Heonyong Canadian Science Publishing 2014 Canadian journal of physiology and pharmacology Vol.92 No.4

<P> A crude extract from Acanthopanax senticosus (AS) has drawn increased attention because of its potentially beneficial activities, including anti-fatigue, anti-stress, anti-gastric-ulcer, and immunoenhancing effects. We previously reported that AS crude extract exerts anti-inflammatory activity through blockade of monocytic adhesion to endothelial cells. However, the underlying mechanisms remained unknown, and so this study was designed to investigate the pathways involved. It was confirmed that AS extract inhibited lipopolysaccharide (LPS)-induced adhesion of monocytes to endothelial cells, and we found that whole extract was superior to eleutheroside E, a principal functional component of AS. A series of PCR experiments revealed that AS extract inhibited LPS-induced expression of genes encoding lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) in THP-1 cells. Consistently, protein levels and cell surface expression of LFA-1 and Mac-1 were noticeably reduced upon treatment with AS extract. This inhibitory effect was mediated by the suppression of LPS-induced degradation of IκB-α, a known inhibitor of nuclear factor-κB (NF-κB). In conclusion, AS extract exerts anti-inflammatory activity via the suppression of LFA-1 and Mac-1, lending itself as a potential therapeutic galenical for the prevention and treatment of various inflammatory diseases. </P>

A new method for immobilization of lipid-binding proteins to the glass slides

Shin, Jaeyoung,Cho, Minho,Hyun, June Won,Pyee, Jaeho,Park, Heonyong Elsevier 2006 Current Applied Physics Vol.6 No.2

<P><B>Abstract</B></P><P>Previously, we constructed nickel-coated glass slides to immobilize histidine-tagged proteins [Bull. Korean Chem. Soc. 23 (2002) 1724–1728]. Here, we further investigated whether the nickel-coated glass slide could be utilized for assaying molecular interaction between lipid and immobilized protein. Proteins interact with various lipid molecules in biological systems. More importantly, this interaction is responsible for a broad spectrum of physiological functions. Accordingly, to more efficiently analyze various types of interactions and understand the complicated biological systems, it is essential to develop a high-throughput analytical device. For the high-throughput analytical purpose, we immobilized an FITC (fluorescein isothiocyanate)-tagged lipid-binding protein to nickel-coated plates, incubated the plates with various lipid molecules and then measured fluorescence intensities to analyze its binding affinity. Our systems seemed to be reliable for the application to the high-throughput system consisted of lipid-binding proteins.</P>

담배 형질전환체를 이용한 포도 UDP-glucose flavonoid glucosyl transferase (UFGT) 유전자의 기능 분석

Ji-Yeon Park(박지연),Sung-chool Park(박성출),Jaeho Pyee(피재호) 한국생명과학회 2010 생명과학회지 Vol.20 No.2

페놀화합물인 안토시아닌은 과일, 꽃잎 등 식물 조직에서 청 혹은 적색을 나타내는 색소이다. 포도의 경우, 안토시아닌은 적포도 품종의 과피에만 축적되는데, 이것은 안토시아닌 생합성에 관여하는 효소 중에서 UDP-glucose: flavonoid 3-O-glucosyltransferase(UFGT)를 암호화하는 ufgt 유전자에 의해 조절된다고 보고된 바 있다. ufgt 유전자에 의해 안토시아닌의 축적 양상이 변하는지를 검증하기 위해, 포도로부터 ufgt cDNA를 분리한 다음, 각각 sense와 antisense 방향으로 발현하는 벡터를 제작하고, 이를 야생형 담배에 도입하였다. 담배 형질전환체를 선발하여 RT-PCR을 수행한 결과, 포도 ufgt 유전자가 sense 방향으로 들어간 snese 형질전환체에서는 야생형 담배에 비해 ufgt 전사체의 양이 증가하였고, antisense 방향으로 들어간 antisense 형질전환체에서는 전사체의 양이 도리어 감소하였다. 한편, 담배 형질전환체의 꽃을 분석한 결과, sense 형질전환체의 안토시아닌 함량은 야생형담배에 비해 최고 44% 증가하였고, antisense 형질전환체에서는 최고 88% 감소하였다. 또한 sense 형질전환체의 꽃 색깔은 야생형 담배에 비해 더 진해졌다. 이러한 결과는 외부로부터 도입된 포도 ufgt 유전자의 발현으로 ufgt 전사체의 양이 증가하면 담배 내 안토시아닌의 함량이 높아지고 꽃 색깔이 진해진다는 것을 시사한다. Anthocyanin, a phenolic compound, is a pigment that shows blue or red color in the fruit, petal and other tissues. It is an important factor in grape berry skin pigment and accumulates only in the skin. This skin-specific accumulation of anthocyanin has been reported to be regulated by the ufgt gene which encodes UDP-glucose: flavonoid 3-O-glucosyltransferase that participates in the biosynthesis of anthocyanin. The ufgt gene is expressed only in berry skin, while the other genes involved in the biosynthetic pathway are expressed in both skin and flesh tissues. In order to determine whether anthocyanin accumulation is primarily regulated by compartment of UFGT, a ufgt cDNA clone was isolated from grape berry, its open reading frame was ligated in pBI121 vector in either a sense or an antisense orientation under the control of the CaMV35S promoter and the recombinant constructs were incorporated into tobacco plants. Several transgenic lines were selected and characterized to determine the level of expression of the grapevine ufgt transcript and endogenous homologs of tobacco. Compared to the wild-type, the amount of anthocyanins in sense transgenic plants increased by 44%, while the amount of anthocyanins in antisense transgenic plants decreased by 88%. In addition, the color of flowers became intense in the sense transgenic plants. These results suggest that over-expression or repression of the ufgt gene affected the accumulation of anthocyanin in flowers of tobacco.

매향 딸기로부터 anthocyanin 합성 유전자의 분리 및 과실발달 과정에서의 발현 분석

배기석(Kisuk Bae),길준영(Joon Yeong Kihl),피재호(Jaeho Pyee) 한국생명과학회 2008 생명과학회지 Vol.18 No.2

‘매향’ 딸기의 안토시아닌 생합성은 개화 후 26일째 시작되어 과실의 성숙기 동안 계속된다. 딸기로부터 안토시아닌의 생합성에 관여하는 주요 유전자를 분리하였다. 각각의 유전자에 대해, 다양한 식물체의 유사 유전자의 염기서열을 비교하여 PCR (polymerase chain reaciton) primer를 제작하였다. 숙기의 딸기에서 분리된 total RNA로부터 합성된 cDNA와각 primer를 이용하여 RT (reverse transcriptase)-PCR을 수행하였다. 각 cDNA clone의 염기서열을 작성하여 분석한 결과, 이들은 안토시아닌 생합성에 관여하는 phenylalanine ammonialyase (PAL), 4-cummarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidine synthase (ANS) 그리고 UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) 효소에 해당되었다. Northern blot 분석 결과, 이들 유전자는 과실 발달 과정에서 시기적으로 조절되었다. 특히 PAL을 제외한 모든 유전자는 과실에서만 주로 발현되었다. PAL, DFR 그리고 ANS 유전자는 과실 초기 발달 단계인 개화 후 10일에 검출된 후 감소하다가, 22일에 다시 증가하기 시작하여 34일에 최대가 되었다. 한편, 다른 유전자들은 초기에는 발현되지 않다가, 안토시아닌이 축적되기 시작하는 개화 후 22~30일에 처음으로 검출되었다. 본 연구를 통해, 딸기 과실 발달과정에서 안토시아닌 생합성 과정에 관여하는 여러 유전자가 과실 숙기에 함께 조절되는 현상을 알 수 있다. 이러한 연구 결과는 안토시아닌 합성과정을 제어하는 조절 유전자가 존재한다는 것을 시사한다. 그리고 딸기의 안토시아닌 생합성 유전자의 발현 패턴을 크게 두 가지로 나눌 수 있는 것으로 보아, 딸기의 안토시아닌 생합성에는 적어도 두 가지 서로 다른 조절 기작이 관여하여 색소 발달 과정을 제어할 것으로 보인다. Anthocyanin synthesis in strawberry (Fragaria x ananassa cv Maehyang) begins approximately 26 days postflowering and continued throughout fruit ripening. A set of cDNA clones encoding the anthocyanin biosynthetic enzymes were isolated from strawberry. A pair of primers were designed for polymerase chain reaction (PCR) through the comparison of the nucleotide sequences of homologous genes from diverse plants. Reverse transcriptase-PCRs were performed using cDNA synthesized from ripe fruit total RNA and the primers corresponding to each gene. Eight genes of the anthocyanin pathway were cloned and confirmed by sequencing to code for phenylalanine ammonia lyase (PAL), 4-cummarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidine synthase (ANS), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT). Northern analyses showed that the corresponding genes were differentially ex-pressed during the fruit development process. All genes except PAL were predominantly expressed in fruit. Expression of PAL, DFR and ANS was detected 10 days postflowering at the early stage of fruit development, declined for a while and sharply increased 22 days postflowering then showed a peak 34 days postflowering. The other genes, however, were not expressed up to 22 or 30 days post-flowering when the initial fruit ripening events occur at the time of initiation of anthocyanin accumulation. The onset of anthocyanin synthesis in ripening strawberry coincides with a coordinated induction of the anthocyanin pathway genes, suggesting the involvement of regulatory genes. We pro-pose that at least two different regulatory mechanisms play a role in the biosynthesis of anthocyanin during color development of strawberry.