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Targeted isolation of diverse alkaloids from Alchornea rugosa, Persea americana, and Gymnema inodorum by dereplication methods using LC-MS/MS-based molecular networking : LC-MS/MS 기반 분자네트워킹의 선택적 표적화 방법을 이용한 Alchornea rugosa, Persea americana와 Gymnema inodorum의 다양한 alkaloids 표적분리 연구

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서울대학교 대학원
학위논문(박사) -- 서울대학교대학원 : 약학대학 약학과, 2022. 8. 오원근.
Natural products (NPs) that are small molecules derived from the environment by secondary metabolic pathways have played significant roles in drug discovery, ecology, and biotechnology. The conventional investigation of natural products in the early years such as grind and find or bio-guided isolation has successfully found important medicines including morphine (painkiller), aspirin (anti-inflammation), digitoxin (cardiac glycoside), silymarin (treatment of liver disease), quinine, artemisinin (antimalarial), and pilocarpine (treating glaucoma). Since the first plant-derived medicine was founded in 1803, the number of new natural products reported every year has risen dramatically. As many easily accessed natural products have been isolated for a long history, recent studies have faced a commonplace challenge – the rediscovery of known compounds. In this regard, analytical tools, especially mass spectrometry (MS), have recently been efficiently applied in the NP field for dereplication and rapid identification of new NPs in complex mixtures.
Remarkably, a recent technique using tandem MS/MS data sets called molecular networking (MN) has transited the traditional grind and find to the hypothesis-driven targeting isolation since its first introduction a decade ago. MN is the computer-based approach aiming to arrange MS/MS data, automatically compare it to available databases for the identification of specific metabolites, and give the best visualization of related compounds in the same group (cluster). This dereplication technique has been widely and successfully applied in NPs leading to the discovery of broad types of novel secondary metabolites that could be promising for research and development of new drugs.
Herein, the MN was successfully applied to target and isolate various types of alkaloids from different plant sources.

Part 1: Guanidine alkaloids from Alchornea rugosa and their glucose uptake and autophagy activities
Natural guanidines that are classified as alkaloids and as unusually modified peptides with guanidine functionalities are generally found in microorganisms and marine invertebrates but are rarely found in terrestrial plants and animals. The substantial hydrophilicity of guanidines makes them suitable for the development of drugs, some of which have been used clinically. It is noteworthy that although the natural guanidines in plants account for only a small number compared to other sources, plant guanidines have been frequently reported in some species belonging to the genus Alchornea including A. floribunda, A. hirtella, and A. cordifolia. Based on MS/MS-based molecular networking analysis, three pairs of novel configurationally semistable diastereomers featuring an unprecedented 1,6-dioxa-7,9-diazaspiro[4.5]dec-7-en-8-amine scaffold named rugonidines A–F (1–6), and nine new guanidine-fuse catechins named rugonines A–H (7–15) were isolated from Alchornea rugosa. Compounds 1−6 possess a 1,6-dioxa-7,9-diazaspiro[4.5]dec-7-en-8-aminefused catechin skeleton that features a naturally unique scaffold. Their structures were elucidated by NMR spectroscopy in combination with quantum-chemical calculations. The biological activities of 1−6 were tested in glucose uptake levels in differentiated 3T3-L1 adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe and compounds 1–3 showed a significant increase glucose uptake levels. Structures of 7–15 were determined by NMR, HRESIMS/MS, and CD data analysis. These guanidine-fused catechins were evaluated for their bioactivities in the autophagy modulation assay. The results indicated that compounds 10–13 had potential autophagy inhibition effects compared to the chloroquine which is known as autophagy inhibitor.

Part 2: Alkaloids from Persea americana and their SIRT1 activities
Sirtuin 1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)+-dependent deacetylase belonging to the mammalian sirtuins family that plays important roles in cellular and organismal processes, including metabolism and aging. Through deacetylation of various substrates, including histone proteins (acetylated histones H4K16 and H3K56) and non-histone targets (p53) to generate nicotinamide and the acetyl group, SIRT1 is involved in a broad range of physiological functions, including control of gene expression, metabolism, and aging. The function of SIRT1 in senescence, which leads to extending life span, ameliorates cellular senescence, and consequently prevents aging-related diseases, is mainly achieved by catalyzing the deacetylation of various downstream transcription factors.
Avocado seeds are the by-products of the food industry with rich bioactive compounds but the secondary metabolites have not been yet studied and applied. Herein, the phytochemicals in avocado seeds were dereplicated by using an LC-MS/MS-based molecular networking. A new finding in alkaloid compositions from avocado seeds was discovered allowing the isolation of seven new quinolone-alkaloids and two new benzoxazinone-alkaloids. Structures of isolated compounds were identified by NMR spectroscopy methods in combination with ECD calculations for absolute configuration elucidation. Later, the activities of isolated compounds were tested for their SIRT1 activities in HEK293 cells. The results showed that compound 1 has the most potent effect on SIRT1 activation with an elevated NAD+/NADH ratio and it could be a promising candidate for further investigation of anti-aging agents.

Part 3: Oleanane triterpenoids from Gymnema inodorum and their insulin mimetic activities
The molecular networking of n-hexane, EtOAc, and n-BuOH fractions of G. inodorum was generated and showed four main clusters including oleanane – triterpenoids (cluster 1), flavonoids (cluster 2), methyl anthranilate derivatives of oleanane triterpenoids (cluster 3), and benzoyl derivatives of oleanane triterpenoids (cluster 4). Seventeen compounds including 13 new oleananes (4–7), benzoyl derivatives of oleanane (3), and methyl anthranilate derivatives of oleanane triterpenoids (1, 2, 10–18), and four known compounds were isolated and their structures were identified by analysis of the NMR spectra. Compounds 1–9 were tested in an insulin-mimetic model to evaluate their biological activities. The results suggested that compounds 3, 5, 8, and 9 showed potential stimulatory effects on the uptake of 2-NBDG in 3T3-L1 adipocyte cells. Compounds 1, 8, 9, and 10–18 were evaluated for their effects on antimuscle atrophic activities. The results suggested that compound 12 showed the most potent effect on muscle atrophy at 2 µM. Later, the SAR was discussed to suggest the crucial point for antimuscle atrophy of oleanane triterpenoids from G. inodorum.
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