Development of natural resources for cardiovascular disease improvement from Pinus densiflora

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Pine trees (Pinus densiflora Sieb. et Zucc) belong to the family Pinaceae and are widely distributed around the world. In East-Asian countries such as Korea and China, Japan, various parts of pine trees, including the needles, cones, cortices, and pollen, are widely consumed as foods or dietary supplements to promote health and have shown a wide spectrum of biological and pharmacological actions such as anti-inflammatory, antibacterial, antiviral, antidepressant, antihyperternsion, and triglyceride decreasing effects. We have previously reported that the fermented pine needle extract (FPE) from these plants possesses fibrinolytic activity and antihypertension. Two fibrinolytic compounds, protocatechuic (PCA) and shikimic acids (SA), were isolated from the ethyl acetate- and n-butyl alcohol-soluble fractions of FPE, respectively. Fibrin clots lysis in compounds PCA and SA occurred completely in the pH 2-4 range.
These compounds strongly suppressed fibrin clots formation, which was determined by measuring turbidity. The SDS-PAGE pattern demonstrated that plasmin fibrinolysis was different from that of chemicals PCA and SA. Plasmin disassembles the Aα and Bβ chains of fibrin, but chemicals PCA and SA maintained the Aα, Bβ, and γ fibrin chains after fibrinolysis. Furthermore, we measured the changes in the quantities of compounds PCA and SA that were obtained from FPE. While the fermentation progressed, we determined that the quantity of compound PCA steadily increased, while the quantity of SA did not demonstrate significant changes. FPE is an excellent resource for chemicals PCA and SA and can provide inexpensive and sufficient quantities for industrial-scale extraction. The Angiotensin converting enzyme (ACE) is the key enzyme catalyzes angiotensin Ⅰ (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile) to angiotensin Ⅱ (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile) in renin-angiotensin system. Angiotensin Ⅰ produces aldosterone in combination with angiotensin Ⅱ receptor, which increases blood pressure through absorption of Na+. To investigate the antihypertensive compounds from Pinus densiflora Sieb. et Zucc (red pine) needles, water, ethyl acetate - and n-butyl alcohol-soluble fractions from red pine needles were screened for the inhibitory activity against ACE. The most potent ACE inhibitory activity was detected in the ethyl acetate alcohol-soluble fractions (5 mg/mL). After the purification of ACE inhibitor compounds with column chromatography, antihypertensive activity was determined by measuring Revers Phase-HPLC. We obtained two active compounds, abieta-8,11,13-trien-18-oic and 8(17),12,14-Labdatriene-19-oic acids. To measure
quantitative analysis of antihypertensive activity, we analyzed the quantities of hippuric acid using RP-HPLC. abieta-8,11,13-trien-18-oic acid (3 mM) and 8(17),12,14-Labdatriene-19-oic acid (3 mM) inhibited production hippuric acid in N-Hippuryl-His-Leu. Also, abieta-8,11,13-trien-18-oic acid (dehydroabietic acid) and 8(17),12,14-Labdatriene-19-oic acid (commnunic acid) regulated ACE and angiotensin Ⅱ receptor expression in endothelial cells. We determined that abieta-8,11,13-trien-18-oic and 8(17),12,14-Labdatriene-19-oic acids separated from the red pine needles are new ACE inhibitors.
| 솔잎은 소나무 과에 속하며 세계적으로 넓게 분포하고 있다. 한국, 중국, 일본 등 동아시아의 각 나라에서는 소나무의 잎, 열매, 껍지, 꽃가루 등 다양한 부위를 식용 또는 건강 증진을 위한 식사 보충제로 사용되고 있다. 건강 증진을 위한 솔잎의 효능은 항염증, 항균, 항바이러스, 항우울제, 항고혈압과 트리글리세리드 감소 효과 등 다양한 생리학적 및 약리학적 활성이 알려져 있다. 이전 연구에서 솔잎발효액은 fibirnolysis 및 antihypertension activity에 대해 연구되어졌다. 솔잎발효액에서 fibrinolysis 물질인 protocatehcuic acid과 shikimic acid은 에틸아세테이트와 부탄올 층에서 각각 분리되었다. protocatehcuic acid과 shikimic acid은 낮은 pH 범위에서(2 - 4) thrombus 분해가 완벽하게 발생되었다. fibrin 형성 억제 활성은 UV spectrophotometer를 이용하여 fibrin solution의 탁도 변화를 통해
확인하였고, protocatehcuic acid과 shikimic acid은 fibrin solution의 탁도가 증가되는 것을 억제하였고, 이는 protocatehcuic acid과 shikimic acid이 혈전 형성을 억제한다는 것이 확인되었다. Protocatehcuic acid과 shikimic acid의 혈전분해 방식을 확인하기 위해 SDS-PAGE를 이용하여 분석하였고, 분석결과 protocatehcuic acid과 shikimic acid의 fibrinolysis 방법은 plasmin과는 다르게 분해한다는 것을 확인하였다. Plasmin은 fibrin의 Aα, Bβ 단백질 밴드가 사라졌지만, protocatehcuic acid과 shikimic acid은 피브린의 Aα, Bβ, γ 단백질 밴드가 유지되었다. protocatehcuic acid과 shikimic acid의 양적 변화를 솔잎발효액에서 측정하였다. 발효과정 동안 protocatehcuic acid는 지속적으로 증가 하였지만, shikimic acid의 양은 특별한 변화가 없었다. 솔잎발효액은 protocatehcuic acid과 shikimic acid의 우수한 공급원이며 필요한 충분한 양을 값싸고 충분한 양을 공급할 수 있다. Renin angiotensin system에서 angiotensin converting enzyme은 angiotensin Ⅰ (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile)을 angiotensin Ⅱ(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile)로 바꾸는 중요한 단백질이다. Angiotensin Ⅱ은 angitensin Ⅱ receptor와 결합하여 aldosterone을 생산하고 Na+를 흡수하여 혈압을 상승시킨다. 적송의 잎에서 anhypertension 물질을 연구하기 위해서, 적송의 잎을 물, 에틸아세테이트, 부탄올 층으로 분리하여 angiotensin converting enzyme 억제 활성을 분석하였다. 가장 잠재적으로 angiotensin converting enzyme 억제 활성을 가지고 있는 것은 에틸아세테이트 층(5 mg/mL)에서 확인 되었다. 컬럼 크로마토그래피로 angiotensin converting enzyme 억제 물질의 정제 후, antihypertension activity은 역상 액체크로마토그래프를 이용하여 측정하였다. 두 개의 활성 물질을 분리 및 정제하였고, 구조분석 결과
angiotensin converting enzyme 억제 물질은 각각 abieta-8,11,13-trien-18-oic acid (dehydroabietic acid)와 8(17),12,14-Labdatriene-19-oic acid (communic acid)으로 확인되었다. Antihypertension 활성의 분석을 위해, 역상 액체크로마토그래프를 이용하여 hippuric acid의 양을 측정하였다. Abieta-8,11,13-trien-18-oic (3 mM)과 8(17),12,14-Labdatriene-19-oic acid (3 mM)은 N-Hippurly-His-Leu에서 hippuric acid 생산을 억제하였다. 또한, abieta-8,11,13-trien-18-oic acid와 8(17),12,14-Labdatriene-19-oic acid은 endothelial cell에서 angiotensin converting enzyme와 angiotensin Ⅱ receptor 발현을 조절하였다. 적송의 잎에서 분리된 두 개의 abieta-8,11,13-trien-18-oic acid (dehydroabietic acid)과 8(17),12,14-Labdatriene-19-oic acid (communic acid)는 새로운 angiotensin convering enzyme inhibitor임을 확인하였다.
Alternative Title
솔잎에서 심혈관 질환 개선용 천연물 소재 개발
Alternative Author(s)
Jaeyoung Park
일반대학원 생명과학
Awarded Date
Table Of Contents

List of Figures ⅰ

List of Tables ⅳ

Abbreviation ⅴ

Abstract in Korean 1

Abstract in English 4

Introduction 7

Chapter 1. Screening of compunds with biological activity on fibrinolysis from fermented Pinus densiflora extract 15

1.1 Introduction 15

1.2 Material and Methods 19

1.2.1 General experimental procedures 19
1.2.2 Plant material 20

1.2.3 Fermented pine needle extract preparation 20

1.2.4 Extraction and isolation 21

1.2.5 Fibrinolysis activity assay in fibrin and fibrinogen solution 21

1.2.6 Quantitative analysis of protocatechuic and shikimic acids 22

1.3 Results and discussion 24

1.3.1 Isolation and purification of fibrinolysis compounds 24

1.3.2 Fibrinolysis activity of protocatechuic acid and shikimic acid 28

1.3.3 Fibrin and fibrinogen lysis pattern of protocatechuic acid and shimic acid 35

1.3.4 Antithrombotic and bleeding time effects of protocatechuic and shikimic acids in mouse 39
Chapter 2. Screening of compounds with biological activity on angiotensin converting enzyme inhibition form Pinus densiflora 52

2.1 Introduction 52

2.2 Material and Methods 54

2.2.1 General experimental procedures 54
2.2.2 Plant material 54

2.2.3 Extraction and isolation 55

2.2.4 Measurement of antihypertensive activity using an angiotensin converting enzyme spectrophotometer assay 56

2.2.5 Cell culture 57

2.2.6 Cell treatments and cytotoxicity 57

2.2.7. Total RNA isolation and cDNA synthesis 58

2.2.8. Reverse transciption PCR 59
2.3 Results and discussion 60

2.3.1 Isolation and purification of antihypertension compounds 60

2.3.2 Angiotensin converting enzyme inhibitory activity of dehydroabietic acid and communic acid 66

2.3.3 Expression of angiotensin Ⅱ receptor, type 1 and endothelial nitric oxide synthase 3 (eNOS) in mouse endothelial cells 69

2.3.4 Cytotoxicity determination of dehydroabietic acid and communic acid 73

Chapter 3. The construction of angiotensin converting enzyme gene 76

3.1 Introduction 76

3.2 Material and Methods 77

3.2.1 RNA preparation and cDNA synthesis 77

3.2.2 Constructions for structure study of ACE and inhibitor binding assay 79
3.2.1 DNA sequence analysis 79

3.3 Results and discussion 80

3.3.1 Cloning of angiotensin converting enzyme gene 80

3.3.2 Confirm transformation on DH 5α 81

Reference 93

감사의 글 106
박재영. (2014). Development of natural resources for cardiovascular disease improvement from Pinus densiflora.
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