Targeted classification, identification and metabolite profiling of triterpenoids in the genus Gymnema and Gynostemma by developing a building block strategy using UHPLC-QTOF/MS : UHPLC-QTOF/MS를 활용한 building block 전략 및 이를 활용한 Gymnema 및 Gynostemma 속 triterpenoids 대사체 프로파일링 연구
- Pham Ha Thanh Tung
- Oh, Won Keun
- Issue Date
- 서울대학교 대학원
- 학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2019. 8. Oh, Won Keun.
Gymnema sylvestre는 전통적으로 아유르베다 의학에서 당뇨치료에 사용되어왔다. 본 연구에서는 베트남과 인도의 Gymnema sylvestre을 형태학적으로 구분하였고 베트남의 Gymnema sylvestre에서 분리한 6개의 신규화합물 (1-6)의 조각이온 패턴을 화학성분 분석에 활용하였다. 본 식물의 분리 및 동정을 통하여 이미 알고 있는 조각이온의 패턴과 이 식물의 oleanane triterpenoid 의 생합성 과정을 토대로 building block 전략을 활용하여 각각의 피크를 동정하였다. 이 과정에서 소분획, mass 조각이온 패턴, relative mass defect filtering, reference와 비교 등의 방법이 적용되었다. 총 119개의 피크가 oleanane triterpenoid 계열로 분류되었으며 그 중 77개의 피크가 신규화합물임을 확인하였다.
천연물은 PTP1B억제를 통한 항당뇨 효과 후보물질의 훌륭한 원천이며 이에 따라 선별된 Korea Bioactive Natural Material Bank의 식물추출물들에 대한 스크리닝 작업을 실시하였으며 그 결과 Gymnema latifolium 70% 에탄올 추출물이 PTP1B 억제 효능을 보였다. 이는 현재 베트남에서 항당뇨 치료제로 Gymnema latifolium이 활용되고 있는 결과와도 일치하였다. 이에 본 연구는 14종의 신규 oleanane triterpene인 Gymlatinosides (1-14)와 6개의 기지물질을 분리 동정하였다. Gymlatinoside GL2와 GL3는 생리활성 결과 각각 IC50 값 28.66±2.57 과 19.83±0.40을 나타내어 PTP1B를 유의미하게 억제하였음을 확인하였다. 또한 building block 전략을 이용하여 54개의 신규 피크를 추정하였다.
Gynostemma longipes 에서는 8종의 신규 12,23-dione dammarane triterpenoid와 1종의 gypetonoside가 분리되었으며 building block 전략을 이용하여 32개의 피크가 신규물질로 추정됨을 확인하였다. 분리한 9종의 화합물에 대해서는 AMPK 활성을 검사하였으며 C2C12 근육세포에 7종의 화합물 (1, 3-8)을 처치하였을 경우 AMPK 활성을 증가시키는 것으로 확인하였다. 화합물 1의 경우 식물 건조량 대비 2.08%의 함량을 가지는 것으로 평가되어 향후 기능성식품으로의 발전 가능성을 제시하였다. 본 연구에서는 Gynostemma longipes의 AMPK 활성을 통한 근육세포 증식을 확인하였다.
The quality control of a medicinal plant requires a rapid, accurate, and comprehensive analysis of the chemical profile of the bioactive extract. Common HRMS-based compound annotations relied on the mass fragmentation analysis of detected peaks to identify the partial structure of the compounds before merging them together. Herein, we applied a universally accepted concept that most of the natural products of the same organic origin are generated by the same building blocks using several conservative biosynthetic processes. The detection of metabolites - both known and unknown - can be redefined by an inverse approach, in which, prebuilt natural products were predicted using key prefabricated LEGO-type building blocks and their biosynthetic construction rules. The conventional mass fragmentation pattern analysis and NMR experiments are for the confirmation of the existence of predicted compounds.
Furthermore, we proposed a new idea of multilayer metabolite profiling, which provides simple, distinct, and comprehensive chromatogram interface with not only original information about retention time of metabolites, but also considerable insight into structure formation as well as the chemical relationship among plant metabolites. In this study, the building block strategy was applied effectively to explore the triterpenoid composition of three medicinal plants including Gymnema sylvestre, Gymnema latifolium and Gynostemma longipes. Overall, we expect this new approach can be used, with much practicality, for massive structural characterization and for exploring the biosynthetic relationships among various compounds in medicinal plants.
Part 1: Discrimination of different geographic varieties of Gymnema sylvestre and development of a building block strategy to classification, identification and metabolite profiling of its oleanane triterpenoids
The major class of bioactive metabolites in Gymnema sylvestre, a popular Ayurevedic medicinal plant for the treatment of diabetes mellitus, is oleanane triterpenoids. In this study, a targeted, biosynthesis-inspired approach using UHPLC-qTOF/MS was implemented to elucidate the whole chemical profile of the plant for the standardization of the Vietnamese G. sylvestre variety. The known compounds reported in the literature were first analyzed to identify the building blocks of the biosynthetic intermediates and the construction rules for synthesizing oleanane triterpenoids in the plant. These blocks were recombined to build up a theoretical virtual library of all reasonable compounds consistent with the deduced construction rules. Various techniques, including microfractionation, relative mass defect filtering, multiple key ion analysis, mass fragmentation analysis, and comparison with standard references, were applied to determine the presence of these predicted compounds. Conventional isolation and structure elucidation of 6 of the new compounds were carried out to identify new building blocks and validate the assignments. Consequently, 119 peaks were quickly assigned to oleanane triterpenoid, and among them, 77 peaks are predicted to be new compounds based on their molecular formulas and mass fragmentation patterns. All the identified metabolites were then classified into different layers to analyze their logical relationships and construct a multilayered chemical profile of the oleanane triterpenoids.
Part 2: Oleanane triterpenoids from Gymnema latifolium and their PTP1B inhibitory activities
Natural products are promising sources of lead compounds that play significant roles in the discovery of new antidiabetic agents via the mechanism of PTP1B inhibition. In our ongoing research to find PTP1B inhibitors from natural products, hundreds of plants extracts available in Korea Bioactive Natural Material Bank has been screened against this biological target. An extract of the Gymnema latifolium Wall. ex. Wight showed considerable PTP1B inhibitory activity. This result was in good agreement with the use of this plant in Vietnam as an antidiabetic herbal medicine, similar with its taxonomical relative G. sylvestre. It is also well-known that most species belonging to the same genus possess similar chemical composition and thus exhibit similar biological activities. Further chemical investigation of this plant led to the isolation of 14 new oleanane triterpenes Gymlatinosides GL1-GL14 and 6 known oleanolic acid analogs. The structures of the new compounds were elucidated using diverse spectroscopic methods. Among them, compounds Gymlatinosides GL2 and GL3 showed significant PTP1b inhibitory effect. The building block strategy was also applied successfully to predict the structures of 54 new compounds which may have similar skeleton and biosynthesis construction with those isolates.
Part 3: 12,23-Dione dammarane triterpenes from Gynostemma longipes and their muscle cell proliferation activities via activation of the AMPK pathway.
The aging population is growing rapidly around the world. Sarcopenia, characterized by decreased muscle mass, strength, and function, is a common feature of the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid, and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids (1–3 and 5–9), as well as one known gypentonoside A from Gynostemma longipes. Application of the building blocks and their construction rules also led to the assignment of 26 possible new compounds. All 9 isolated compounds were tested for their AMPK activation activities, seven compounds (1 and 3–8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound Longipenoside A1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.