백장미 꽃잎 페놀화합물의 추출 조건 최적화 및 근아세포 보호 효과 평가 = Optimization of Extraction Conditions for Phenolic Compounds from White Rose Petals and Evaluation of Their Protective Effects in C2C12 Myoblasts

국문 초록 (Abstract)

우리 사회가 경제적으로 발전하고 고령화됨에 따라, 노화 및 관련 질병에 관한 연구 또한 증가하고 있다. 근육의 감소는 노화 증상 중 가장 눈에 띄게 나타나는 현상으로, 나이가 들어감에 따라 자연스럽게 발생한다. 선행 연구에 따르면, 백장미(Rosa spp.) 꽃잎 추출물은 강력한 항산화 능력과 항염증 효과뿐만 아니라 추출물에 다량 함유된 gallic acid(3,4,5-trihydroxybenzoic acid)가 근아세포(C2C12)의 분화와 증식을 촉진한다고 보고 되었다. 따라서 백장미 꽃잎 유래의 추출물이 근아세포에 대한 잠재적 항노화 효능이 있을 것으로 기대된다. 그러나 백장미 꽃잎에서의 경제적 추출 조건 및 효능을 극대화 할 수 있는 추출방법, 추출물에 함유된 생리활성 성분을 분석하는 연구는 현재까지 매우 미비한 실정이다. 특히, 근아세포내에서 항산화, 항염증, 근육관련 단백질의 발현에 대한 연구는 아직까지 수행되지 않았다. 따라서 본 연구의 목적은, 백장미 꽃잎에서 페놀화합물의 추출조건을 최적화하고, 생리활성 성분을 분리 및 동정하며, 산화 및 염증이 유도된 쥐 C2C12 근아세포에서 추출물의 세포 보호효과를 평가하는 것이다.
백장미 꽃잎으로부터 자유라디칼(DPPH)에 대한 높은 항산화 효능이 있는 추출물의 추출조건을 최적화하기 위해 반응표면 분석법(Response surface methodology)을 사용하여, 에탄올 농도(X1), 추출 온도(X2) 및 추출 시간(X3)을 세 가지 독립 변수로 설정하고 종속변수의 효과를 예측했다. 항산화 활성을 갖는 페놀화합물의 추출조건 최적화 연구결과, 에탄올 농도 42 %(X1), 추출 시간 80분(X3) 및 추출 온도 75 ℃(X2)의 독립변수 조건을 확인 할 수 있었다. 반면 플라보노이드 화합물의 최적 추출조건은 에탄올 농도 41 %(X1), 추출 시간 119 분(X3) 및 추출 온도 75 ℃(X2) 였다. 이러한 추출조건 아래에서, 페놀화합물 및 플라보노이드 화합물의 예측된 함량은 각각 243.5 mg GAE/g 및 19.93 mg CE/g이었다.
백장미 꽃잎에서 잘 알려지지 않은 생리활성 성분을 분리 및 동정하기 위해, 극성이 다른 유기용매로 분획을 실시하고, 분취용 고속액체크로마토그래피(Preparative high-performance liquid chromatography, Prep HPLC)로 정제하여 DPPH 라디칼 소거능, 산화질소(NO) 생성 억제 활성을 평가하였다. 분석 결과, 에틸아세테이트(EA) 분획물이 가장 뛰어난 DPPH 라디칼 소거능과 산화질소(NO) 생성 억제 활성을 보였고, 에탄올(EtOH) 추출물 또한 우수한 생리활성을 나타냈다. 에틸아세테이트(EA) 분획물과 에탄올(EtOH) 추출물에서 각각 분리, 정제된 2가지 화합물(F4, F11)의 구조를 핵자기공명기(1H-NMR, 13C-NMR) 및 질량분석기(Gas chromatography–mass spectrometry, GC-MS)로 분석하여 확인한 결과, 에탄올(EtOH) 추출물에서 분리, 정제된 화합물(F4)은 methyl gallate (methyl 3,4,5-trihydroxy benzoate)이었고, 에틸아세테이트(EA)에서 분리, 정제된 화합물(F11)은 gallic acid(3,4,5-tri-hydroxybenzoic acid)로 확인되었다.
백장미 꽃잎 추출물(WRPE)은 강력한 항산화 및 항염증 효과를 갖는 것으로 알려져 있지만, 생리활성 특성은 추출방법 및 적용된 추출조건의 다양한 매개변수에 영향을 받는다. 백장미 꽃잎 에탄올 추출물(WRPE-EtOH)은 고온가압 추출물(WRPE-HTHP) 및 효소분해 추출물(WRPE-enzyme)과 비교하였을 때, 가장 효과적인 항산화 (DPPH소거능, TBARS생성억제)효과 및 항염증 활성(NO 생성억제, SOD2와 iNOS 발현 억제)을 나타내었다. 골격근량과 강도를 감소시키는 근육 손실의 주요 원인은 반응성 산소종(reactive oxygen species, ROS)에 의한 세포 내인성 산화스트레스 및 사이토카인(cytokine)에 의해 유발된 염증으로 알려져있다. 그러므로, 내인성 산화스트레스 및 염증으로부터 WRPE-EtOH가 C2C12근아세포의 생존력, 지질산화 및 근손실 지표 단백질의 생성 억제 효과가 있는지 확인하기 위해MTT검정과 TBARS(Thiobarbituric acid reactive substances) 분석, 근육파괴단백질인MuRF-1(muscle RING-finger protein-1) 및 Atrogin-1의 발현 정도를 평가하였다. WRPE-EtOH는 세포독성(cytotoxicity) 없이 종양괴사인자(tumor necrosis factor alpha, TNF-α)에 의한 세포사멸을 농도 의존적으로 감소시켰다. 유사하게, 반응성 산소종(reactive oxygen species, ROS)에 의한 세포 손상 또한 추출물의 농도가 증가함에 따라 유의적으로 회복되었다. 이러한 결과는 WRPE-EtOH가 항산화 및 항염증 활성과 관련된 TNF-α 및 ROS에 의한 손상에 대해 보호 효과가 있음을 뒷받침한다. 또한, 유비퀴틴리가제인 MuRF-1 및 Atrogin-1 단백질의 발현은 추출물 처리 후 현저하게 감소하였는데, 이는WRPE-EtOH가 항산화 및 항염증 기작을 통해 근손실 억제 효과가 있음을 시사한다.
이번 연구를 통해 페놀화합물의 추출 특성과 최적 조건이 확인되었고, 페놀화합물을 함유한 추출 수율은 이전 연구(15%)보다 약2.5 배 높은 40%로 증가되었다. 또한 그 동안 명확하게 보고되지 않았던 백장미 꽃잎 추출물의 주요 생리 활성 성분으로 gallic acid와 methyl gallate를 핵자기공명기 및 질량분석기로 최초로 확인하였다. 더불어 WRPE-EtOH는 C2C12근아세포 내에서 강력한 항산화 및 항염증 활성 기작을 통해 MuRF-1 및 Atrogin-1 단백질 발현을 억제함으로써 잠재적인 근손실 억제 물질임을 시사하였다. 결과적으로 뛰어난 항산화 활성과 근아세포에서 항노화 효과를 가진 백장미 추출물은 향후 건강기능식품 시장의 활성화와 공중보건 개선에 기여할 것으로 예상한다.

다국어 초록 (Multilingual Abstract)

Recently, researches on aging and aging-related diseases are increasing, as our society has been developed economically and has dominant aging population. As symptoms of aging, muscle loss is the most visible phenomena and it is expressed naturally with aging. Based on previous researches, white rose petal extracts (WRPE) were known to contain large amount of gallic acid that induces myoblast differentiation and promotes proliferation. Therefore, it was expected that the extracts have anti-aging potentials in muscular fiber due to their ability to exert potent antioxidative and anti-inflammatory effects. However, researches on the bioactive components and economical extraction conditions of white rose petals were very lacking. In addition, studies have not been conducted to assess their activities to inhibit inflammation and muscle-related protein degradation in cells. Therefore, the purposes of this research were to optimize the extraction conditions of phenolic compounds from white rose petals, to identify the compounds having biological activities in the extracts, and to evaluate their protective effects in mouse myoblasts (C2C12).
To investigate the optimal conditions to extract anti-oxidative compounds from white rose petals, response surface methodology based on a central composite design was used by comparing the effects of three independent variables: ethanol concentration (X1), extraction temperature (X2), and extraction time (X3). The estimated optimal conditions to obtain phenolic compounds with antioxidant activities were as follows: ethanol concentration of 42 % (X1), extraction time of 80 min (X3), and extraction temperature of 75 ℃ (X2). Meanwhile, the estimated optimal conditions to obtain flavonoid compounds with antioxidant activities were an ethanol concentration of 41 % (X1), extraction time of 119 min (X3), and an extraction temperature of 75 ℃ (X2). Under these conditions, predicted response values for the phenolic and flavonoid contents were 243.5 mg gallic acid equivalent (GAE)/g dry mass and 19.93 mg catechin equivalent (CE)/g dry mass, respectively.
Bioactive compounds in Rosa spp. with white petals were not well known, and thus isolation and identification of those compounds were necessary. Therefore, chromatographic techniques, solvent fractionation, and evaluations of biological activities, such as antioxidant capacity and nitric oxide scavenging activity, were applied to obtain the solvent extracts and their fractions. As result, ethyl acetate (EA) fractions from white rose petals showed the highest antioxidant capacity and nitric oxide scavenging activity. Ethanol (EtOH) extracts also showed high biological activities in all parts. The structure of the purified compound was determined by spectroscopic methods including ultraviolet, proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), and mass spectrometry (MS). As results, two phenolic compounds (F4, F11) were isolated and identified from the secondary fractionation of EA fractions and EtOH extracts. Based on data obtained from 1H-NMR and 13C-NMR with molecular weight (MW) of MS, the two compounds isolated from EtOH extracts and EA fractions were determined as methyl gallate (methyl 3,4,5-trihydroxy benzoate) and gallic acid (3,4,5-trihydroxybenzoic acid), respectively.
WRPE have potent antioxidant and anti-inflammatory activities, meanwhile, the biological properties are known to be influenced by various parameters of employed extraction techniques. It was confirmed WRPE-EtOH is the extracts with the most effective antioxidant and anti-inflammatory activities compared to WRPE-high temperatures-high pressures (HTHP) or WRPE-enzyme. Both endogenous oxidative stress by reactive oxygen species (ROS) and inflammation induced by cytokines have been accepted as the major causes of muscle loss that include loss of skeletal muscle mass and strength. Therefore, the protective effects of WRPE-EtOH were examined in mouse myoblast (C2C12) by using various methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, thiobarbituric acid reactive substances (TBARS) levels, viability of C2C12 myotubes, and expression levels of muscle RING-finger protein-1 (MuRF-1) and Atrogin-1 protein. According to the result of evaluating the antioxidant, anti-inflammatory and protective effects of WRPE-EtOH in C2C12 myoblasts, WRPE-EtOH decreased tumor necrosis factor-α (TNF-α) triggered muscle atrophy without cytotoxicity in a dose-dependent manner. Similarly, ROS-induced cell damage was recovered significantly with increasing concentrations of WRPE-EtOH. Also, WRPE-EtOH decreased TNF-α-induced oxidative damage. These results support that WRPE-EtOH has protective effect against the damages by TNF-α and ROS, which is related to its antioxidant and anti-inflammatory activity. The total protein content was increased in a dose-dependent manner of WRPE-EtOH treatment. In addition, the expression of ubiquitin ligases MuRF-1 and Atrogin-1 proteins significantly decreased after WRPE-EtOH treatment. These results suggest that the ubiquitin ligase inhibition effect of WRPE-EtOH is associated with its protective effect in C2C12 cells.
Taken together, in this study, the optimum extraction conditions of white rose petals for polyphenol compounds and flavonoids were determined; the yield of extracting solid contents containing polyphenols was increased to 40%, which was 2.5 times higher compared to 15% of previous studies. In addition, the main biologically active compounds of WRPE were newly identified as gallic acid and methyl gallate. Furthermore, the WRPE-EtOH was effective in protecting C2C12 cells from muscle loss with its strong antioxidative and anti-inflammatory activity. The results of this study demonstrate that WRPE are strong antioxidative and anti-aging substance to be used as helath promoting materials in the future.

목차 (Table of Contents)

• CHAPTER Ⅰ: Literature review 18
• 1. Introduction 18
• 1.1 Rose 18
• 1.2 Uses of rose 19
• 1.3 Phytochemicals in roses 21
• 1.4 Biological activity of roses 23
• 1.5 Sarcopenia 25
• 1.7 Research necessity 29
• 1.8 Research objectives 31
• CHAPTER Ⅱ: Extraction conditions for phenolic compounds with antioxidant activities from white rose petals 32
• 1. Introduction 32
• 2. Materials and Methods 35
• 2.1 Plant material and chemicals 35
• 2.2 Extraction procedure 36
• 2.2.1 Ethanol extraction 36
• 2.2.2 High temperatures-high pressures (HTHP) extraction 37
• 2.2.3 Enzymatic digestion extraction 37
• 2.2.4 Hot water extraction 38
• 2.3 Measurement of total phenolic contents (TPC) and total flavanoid contents (TFC) 38
• 2.4 Determination of total antioxidant activity (TAA) 39
• 2.5 Statistical analysis 40
• 2.6 Central composite design (CCD) 40
• 3. Results and Discussion 43
• 3.1 Determination of an efficient method for phenolic compounds 43
• 3.2 Determination of the experimental ranges 45
• 3.3 Statistical analysis and model fitting 51
• 3.4 Extraction yields 53
• 3.5 Total phenolic contents (TPC) 58
• 3.6 Total flavanoid contents (TFC) 59
• 3.7 Total antioxidant activity (TAA) 60
• 3.8 Optimization and verification 61
• 4. Conclusion 63
• CHAPTER Ⅲ : Identification of bioactive compounds from white rose petal extracts 64
• 1. Introduction 64
• 2. Materials and Methods 66
• 2.1 Plant material and chemicals 66
• 2.2 Ethanol extraction 67
• 2.3 Fractionation and isolation 69
• 2.4 GC-MS and NMR 70
• 2.5 Measurement of total phenolic contents (TPC) 71
• 2.6 DPPH-scavenging assay 72
• 2.7 Protein degradation assay 72
• 2.8 NO-scavenging assay 73
• 2.9 Statistical analysis 74
• 3. Results 75
• 3.1 Biological activities of EtOH extracts and solvent fractions 75
• 3.1.1 Total phenolic contents 75
• 3.1.2 Reactive oxygen scavenging activities 77
• 3.1.3 Anti-inflammatory activity 80
• 3.2 Isolation and identification 82
• 3.2.1 Isolation and identification of EtOH extracts 82
• 3.2.2 Isolation and identification of EA fractions 86
• 3.3 Biological functions of identified polyphenols 90
• 4. Discussion 92
• 5. Conclusion 95
• CHAPTER Ⅳ: Protective effects of white rose petal extracts in C2C12 myoblasts 96
• 1. Introduction 96
• 2. Materials and Methods 99
• 2.1 Plant material and chemicals 99
• 2.2 Extraction procedure 100
• 2.2.1 Ethanol extraction 100
• 2.2.2 High temperatures-high pressures (HTHP) extraction 100
• 2.2.3 Enzymatic digestion extraction 101
• 2.3 Culture condition and differentiation 101
• 2.4 Cytotoxicity assay 102
• 2.5 DPPH-scavenging assay 103
• 2.6 Protein degradation assay 104
• 2.7 Lipid peroxidation assay 104
• 2.8 NO-scavenging assay 105
• 2.9 Gene expression of superoxide dismutase 2 and inducible nitric oxide synthase 106
• 2.10 Lipid peroxidation assay in C2C12 myoblasts cells 109
• 2.11 Total protein assay 109
• 2.12 Western blotting 110
• 2.13 Statistical analysis 111
• 3. Results 112
• 3.1 Antioxidants activities of white rose petal extracts (WRPE) 112
• 3.1.1 DPPH-scavenging activity 112
• 3.1.2 Protective effects against protein degradation 114
• 3.1.3 Lipid peroxidation inhibition 116
• 3.2 Anti-inflammatory activities of white rose petal extracts (WRPE) 118
• 3.2.1 NO-scavenging activity 118
• 3.2.2 Gene expression of antioxidant and inflammatory enzymes 120
• 3.3 Non-cytotoxicity of WRPE-EtOH in C2C12 cell 122
• 3.4 Protective effect of WRPE-EtOH against TNF-α-induced cell death 124
• 3.5 Antioxidant activity of WRPE-EtOH in C2C12 cells 126
• 3.6 Alleviation of oxidative damages through WRPE-EtOH treatment 128
• 3.7 Attenuation of the MuRF-1 and Atrogin-1 protein expression in WRPE-EtOH-treated cells 130
• 4. Discussion 133
• 5. Conclusion 139
• REFERENCES 141
• SUMMARY IN KOREAN 164