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Metabolic Profiling of Melissa officinalis and Optimization of Separation of Its Metabolites

멜리사 오피시날리스의 대사체 프로파일링과 대사물의 분리공정

  • 김수아 (Kim, Sooah, 대학원 식품과학과 식품생물공학전공)

초록/요약

Various plant metabolites, which are the end products of cellular regulatory processes, are estimated to be over 100,000. The levels f plant metabolites are easily affected by the genetic and environmental changes. Therefore, the response of biological systems reflecting the physiological state and the specific biochemical processes in plants cannot be holistically elucidated by a single genotypes or phenotype. Therfore, the multivariate analysis of the metabolic data analyzed by using LC/MS, GC/MS, NMR and others are emerging technology to more systemically and holistically express the metabolic changes. In this study, lemom balm, Melissa officinalis, a perennial herb occurring throughout the east Asia, was selected as the target plant due to its health-benefiting effect mainly caused by its metabolites. Lemon balm is known as one of sources of antioxidants and polyphenolic acid since it has been used as a effective folk medicine for headache, digestion disorder, rheumatism and hypersensitivities. Accordingly, the number of reports on the medical effects of aqueous extracts of lemon balm, such as antioxidation, the moderation of Alzheimer`s disease, and the regulation of the central nervous system are rapidly increasing nowadays. We investigated the metabolite profiling and the identification of unkown metabolites in lemon balm treated with UV irradiation for different durations and with different concentrations of sucrose by GC/MS and LC/MS. The change of metabolite levels of lemon balm control and treated with various methods were statistically analyzed by a multivariate analysis, principal component analysis (PCA). The extracts of lemon balm treated with UV irradiation and sucrose was well discriminated by PCA. Through the PCA, phenotypic levels of several metabolites were found to be significantly affected by the various treatments such as UV irradiation and sucrose induction on Melisa officianalis. These data could be further used in the screening of cultivars overproducing high-value metabolites. Lemon balm is known to mainly possess rosmarinic acid (RA) and protocatechuic acid (PA) as polyphenolic acid. To investigate the biological activity of the polyphenolic acids in these plants, it is necessary to develop an effective system for extracting and purifying the polyphenolic acids with possible biological functions. Therefore, in this study, the extraction process with operating variable such as temperature, type and concentration of solvents and solid-to-solvent ratio was optimized by the response surface analysis methodology (RSM). RSM result presented that predicted maximal yield of RA extracted from lemon balm was 46.1 mg RA/g dry materials when lemon balm was extracted with 58.96% (v/v) aqueous methanol solution at 54.78ºC for 64.79 min. Also, the chromatographic purification of rosmarinic acid in the extract obtained from the solvent extraction was carried out by using adsorption chromatography resins such as Amberlite XADs and Sephadex LH-20 resins at a variety of conditions. The purity of RA increased from 12.66% to 38.83% when Sephadex LH-20 was used and elution was performed with 100% (v/v) methanol. This work is used as basic information for developing an effective purification strategy of rosmarinic acid from plant resources.

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초록/요약

본 연구에서 다양한 방법으로 레몬밤을 처리했을 때, 대사물질이 어떻게 변화되는지 GC/MS와 LC/MS를 이용하여 분석하고 PCA를 통하여 살펴보았다. 먼저, GC/MS 분석 결과를 살펴보면, UV를 시간 별로 처리한 레몬밤은 PCA에 의해서 control, UV를 1시간 처리한 것과 UV를 2시간 처리한 것들이 확연하게 구분되었다. 특히 control과 UV를 처리한 샘플 군은 PC1에 의해서 나뉘었으며, UV 1시간 처리한 샘플군과 2시간 처리한 샘플군은 PC2에 의해서 분리되는 것을 확인하였다. 특히 PC1에 영향을 미치는 대사물질은 glutamic acid, octadecanoic acid 등이며, oxalic acid, citric acid 등의 대사물질은 PC2에 영향을 미친다. Glutamic acid는 레몬밤에 UV처리를 하면 감소하지만, octadecanoic acid, citric acid는 증가하는 것을 보였다. sucrose를 농도별로 처리한 레몬밤의 결과를 살펴보면, 이것 역시 PCA의해서 sucrose를 농도별로 처리한 군끼리의 명백하게 나뉘는 것을 볼 수 있다. 특히, PC1은 이들을 나누는데 중요한 역할을 한다. PC1과 PC2의 score plot을 보면, 농도가 증가할수록 샘플군들이 시계반대방향으로 움직이는 모습을 보인다. 특히 PC1에 영향을 미치는 대사물질은 threonine, glutamic acid, fructose, citiric acid 등이다. Fructose와 citric acid는 농도가 증가 할수록 많이 생성되는 것을 볼 수 있다. 다음으로 LC/MS 분석결과를 보면, UV를 시간별로 처리한 레몬밤과 다양한 농도의 sucrose로 처리한 레몬밤 샘플군들은 PCA 의해서 확연하게 분리되었다. Malvidin-3-glucoside, petunidin-6-acetyl-3-glucoside, kaempferol-3-glucoside-7-rhamnoside, quercetin-3-glucoside-7-rhamnoside과 같은 anthocyanin 계통들이 분리에 영향을 미치는 중요한 metabolite로 확인되어졌다. 다양한 처리를 통해 우리가 원하는 대사물질을 많이 생성할 수 있음을 알 수 있었다. 본 연구는 또한 레몬밤으로부터 로즈마린 산의 최적 추출조건을 찾기 위해 반응표면분석법을 이용하였으며, 로즈마린산의 분리정제를 위해 Sephadex LH-20을 이용하였다. 로즈마린 산의 최적 추출 조건은 58.96% 메탄올을 이용하여 64.79분 동안 54.78°C에서 추출하는 것이다. 또한 로즈마린산을 레진에 흡착 시키고 탈착 시킬 때의 회수율을 살펴보면, 탈착 시 높은 농도의 용매를 사용할수록 회수율이 증가함을 알 수 있었다. 레몬밤으로부터 로즈마린 산의 분리정제를 위하여 Sephadex LH-20을 이용하였을 때의 순도가 38.83%로 이는 초기 12.66%에서 3.07배 높아진 것이다.

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목차

CHAPTER 1 Introduction = 1
1.1 Response surface methodology = 2
1.2 Metabolomics = 3
1.3 Data pretreatment methods = 6
1.4 Principal component analysis (PCA) = 10
References = 11
CHAPTER 2 Metabolic profiling by GC/MS for Melissa officinalis under sucrose stress = 13
2.1 Introduction = 15
2.2 Materials and Methods = 16
2.2.1 Chemicals = 16
2.2.2 Plant materials = 16
2.2.3 Analysis by GC/MS = 16
2.2.4 Data pretreatment and statistical analysis = 17
2.3 Results and Discussion = 18
2.3.1 Metabolic profiling of lemon balm treated with sucrose by GC/MS = 18
2.3.2 Score and loading plots of lemon balm treated with sucrose = 19
2.3.3 Metabolite change accroding to concentration of sucrose = 25
2.4 Conclusion = 28
References = 29
CHAPTER 3 Metabolic profiling by GC/MS for Melissa officinalis treated by UV-trradiation = 31
3.1 Introduction = 33
3.2 Materials and Methods = 34
3.2.1 Chemicals = 34
3.2.2 Extraction = 34
3.2.3 GC/MS analysis = 35
3.2.4 Statistical analysis = 35
3.3 Results and Discussion = 36
3.3.1 GC/MS analysis of lemon balm treated with UV = 36
3.3.2 Score and loading plots of control and lemon balm treated with UV irradiation = 37
3.3.3 Metabolite change though UV irradiation = 43
3.4 Conclusion = 45
References = 46
CHAPTER 4 Metabolic Profiling by LC/MS for Melissa officinalis Under Sucrose Stress = 49
4.1 Introduction = 51
4.2 Materials and Methods = 51
4.2.1 Sample preparation = 51
4.2.2 LC/MS analysis = 52
4.2.3 Statistical analysis = 53
4.3 Results and Discussion = 53
4.3.1 LC/MS analysis of lemon balm treated with sucrose = 53
4.3.2 PCA analysis = 54
4.4 Conclusion = 60
References = 61
CHAPTER 5 Metabolic Profiling by LC/MS for Melissa officinalis Treated by UV Irradiation = 62
5.1 Introduction = 64
5.2 Materials and Methods = 65
5.2.1 Sample preparation = 65
5.2.2 LC/MS analysis = 65
5.2.3 Statistical analysis = 66
5.3 Results and Discussion = 66
5.3.1 LC/MS analysis of lemon balm treated with UV irradiation and recovered for different durations = 66
5.3.2 Effect of UV strength = 67
5.3.3 Effect of UV irradiation time = 75
5.3.4 Effect of recovery time = 82
5.4 Conclusion = 89
References = 90
CHAPTER 6 Extraction and Purification of Rosmarinic Acid from Melisssa officinalis = 91
6.1 Introduction = 93
6.2 Materials and Methods = 94
6.2.1 Materials = 94
6.2.2 Extraction of lemon balm = 94
6.2.3 HPLC analysis of RA = 95
6.2.4 Response surface methodology and data analysis = 95
6.2.5 Elution profile of RA on Sephadex LH-20 = 98
6.2.6 Isolation and purification of RA from lemon balm = 98
6.3 Results and Discussion = 99
6.3.1 Optimization of extraction of RA from lemon balm using response surface methodology (RSM) = 99
6.3.2 Elution profile of RA on Sephadex LH-20 = 106
6.3.3 Isolation and purification of RA from lemon balm = 108
6.4 Conclusion = 110
References = 111
CHAPTER 7 Conclusions = 114
Conclusions = 115
SUMMARY IN KOREAN = 117

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