(Luận văn thạc sĩ) nghiên cứu hoạt tính gây độc tế bào ung thư và kháng khuẩn của các hợp chất thứ cấp từ chủng vi nấm aspergillus niger IMBC NMTP01

BỘ V HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ Lê Ngọc Anh NGHIÊN CỨU HOẠT TÍNH GÂY ĐỘC TẾ BÀO UNG THƯ VÀ KHÁNG KHUẨN CỦA CÁC HỢP CHẤT THỨ CẤP TỪ CHỦNG VI NẤM Aspergillus niger IMBC-NMTP01 LUẬN VĂN THẠC SĨ SINH HỌC THỰC NGHIỆM Hà Nội - 2021 BỘ V HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ Lê Ngọc Anh NGHIÊN CỨU HOẠT TÍNH GÂY ĐỘC TẾ BÀO UNG THƯ VÀ KHÁNG KHUẨN CỦA CÁC HỢP CHẤT THỨ CẤP TỪ CHỦNG VI NẤM Aspergillus niger IMBC-NMTP01 Chuyên ngành : Sinh học thực nghiệm Mã số: 42 01 14 LUẬN VĂN THẠC SĨ NGÀNH SINH HỌC NGƯỜI HƯỚNG DẪN KHOA HỌC : TS Trần Hồng Quang TS Trần Thị Hồng Hạnh Hà Nội - 2021 i LỜI CAM ĐOAN Tôi xin cam đoan luận văn cơng trình nghiên cứu tơi dƣới hƣớng dẫn khoa học TS Trần Hồng Quang TS Trần Thị Hồng Hạnh Các số liệu, kết nghiên cứu đảm bảo trung thực khách quan Đồng thời, kết chƣa đƣợc công bố cơng trình khác Tác giả Lê Ngọc Anh ii LỜI CẢM ƠN Luận văn đƣợc hoàn thành Viện Hóa sinh biển, Viện Hàn lâm Khoa học Cơng nghệ Việt Nam Trong q trình nghiên cứu, nhận đƣợc nhiều giúp đỡ quý báu thầy cô, nhà khoa học, đồng nghiệp, gia đình bạn bè Tơi xin bày tỏ lời cảm ơn sâu sắc đến TS Trần Hồng Quang TS Trần Thị Hồng Hạnh – ngƣời thầy tận tâm hƣớng dẫn, dạy cho chun mơn, đồng thời động viên, khích lệ tạo điều kiện thuận lợi cho suốt thời gian thực luận văn nhƣ trình tơi làm việc Viện Hóa sinh biển Tơi xin cảm ơn lãnh đạo đồng nghiệp phòng Dƣợc liệu biển, Viện Hóa sinh biển, Viện Hàn lâm Khoa học Công nghệ Việt Nam tạo điều kiện giúp đỡ, truyền kinh nghiệm, đƣa lời khun bổ ích góp ý q báu suốt thời gian tơi làm việc phịng Tơi xin trân trọng cảm ơn tất thầy cô, cán Học viện Khoa học Công nghệ giảng dạy, cung cấp cho kiến thức giúp tơi hồn thành chƣơng trình đào tạo Cuối cùng, tơi xin bày tỏ lịng biết ơn tới tồn thể gia đình, ngƣời thân bạn bè quan tâm, khích lệ, động viên tơi q trình học tập nghiên cứu Luận văn đƣợc giúp đỡ mặt kinh phí thực khn khổ Đề tài trọng điểm cấp Viện Hàn lâm KHCNVN: ―Nghiên cứu nhận dạng chất độc độc tố có nấm mốc thực phẩm có thành phần (đậu tương, lạc, ngô, vừng) bảo quản không quy định, chất lượng‖ Mã số: TĐNDTP.06/19-21 Học viên Lê Ngọc Anh iii MỤC LỤC LỜI CAM ĐOAN i LỜI CẢM ƠN ii MỤC LỤC iii DANH MỤC KÝ HIỆU, CHỮ VIẾT TẮT vi DANH MỤC BẢNG .vii DANH MỤC HÌNH viii MỞ ĐẦU CHƢƠNG TỔNG QUAN 1.1 Vi nấm tầm quan trọng nghiên cứu hợp chất tự nhiên từ vi nấm .4 1.1.1 Giới thiệu chung vi nấm .4 1.1.2 Tầm quan trọng việc nghiên cứu hợp chất từ vi nấm 1.2 Giới thiệu chung Aspergillus niger 1.3 Tình hình nghiên cứu giới Aspergillus niger 11 1.3.1 Một số ứng dụng Aspergillus niger 11 1.3.2 Thành phần hóa học hoạt tính sinh học Aspergillus niger 12 1.3.2.1 Pyranones 13 1.3.2.2 Alkaloid 14 1.3.2.3 Cyclopeptide 16 1.3.2.4 Polyketide 17 1.3.2.5 Sterol 18 1.4 Tình hình nghiên cứu Aspergillus niger Việt Nam 18 CHƢƠNG NGUYÊN VẬT LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU 20 2.1 Nguyên liệu, hóa chất thiết bị nghiên cứu 20 2.1.1 Nguyên liệu hóa chất 20 2.1.2 Thiết bị nghiên cứu 21 iv 2.2 Phƣơng pháp nghiên cứu 21 2.2.1 Phƣơng pháp tạo dịch chiết nấm mốc 21 2.2.2 Phƣơng pháp phân lập hợp chất 21 2.2.3 Phƣơng pháp xác định cấu trúc hợp chất 22 2.2.4 Phƣơng pháp tính tốn phổ ECD 22 2.2.5 Phƣơng pháp đánh giá hoạt tính gây độc tế bào ung thƣ 22 2.2.6 Phƣơng pháp đánh giá ức chế sản sinh nitric oxide (NO) 23 2.2.6.1 Phƣơng pháp nuôi cấy tế bào in vitro 23 2.2.6.2 Phƣơng pháp MTT 24 2.2.6.3 Phƣơng pháp đánh giá khả ức chế sản sinh NO .24 2.2.7 Phƣơng pháp đánh giá hoạt tính kháng sinh 25 CHƢƠNG KẾT QUẢ VÀ THẢO LUẬN 26 3.1 Kết nhân sinh khối lƣợng lớn tạo cao chiết tổng chủng Aspergillus niger IMBC-NMTP01 26 3.2 Kết phân lập, làm sạch, tinh chế hợp chất từ chủng nấm mốc A niger IMBC-NMTP01 27 3.3 Kết xác định cấu trúc hóa học hợp chất 28 3.3.1 Hợp chất A1: epi-Aspergillusol (chất mới) 28 3.3.2 Hợp chất A3: Aspernigin (chất mới) 36 3.3.3 Hợp chất A2: Pyrophen 45 3.3.4 Hợp chất A4: 2-(Hydroxyimino)-3-(4-hydroxyphenyl)propanoic acid .46 3.3.5 Hợp chất A5: Aspergillusol A 47 3.3.6 Hợp chất A6: Rubrofusarin B 48 3.3.7 Hợp chất A7: Nigerasperone A 50 3.3.8 Hợp chất A8: Fonsecin 51 3.3.9 Hợp chất A9: TMC-256C1 52 3.3.10 Hợp chất A10: Pyranonigrin A 53 v 3.3.11 Hợp chất A11: Orlandin 54 3.3.12 Hợp chất A12: Nigerasperone C 55 3.3.13 Hợp chất A13: Asperpyrone A 57 3.3.14 Hợp chất A14: 5-(Hydroxymethyl)-2-furancarboxylic acid 59 3.3.15 Tổng hợp hợp chất đƣợc phân lập 60 3.4 Kết đánh giá hoạt tính gây độc tế bào ung thƣ hợp chất 61 3.5 Kết đánh giá hoạt tính ức chế NO hợp chất 62 3.6 Kết đánh giá hoạt tính kháng khuẩn, kháng nấm hợp chất .64 KẾT LUẬN VÀ KIẾN NGHỊ 66 Kết luận 66 Kiến nghị 66 DANH MỤC CƠNG TRÌNH ĐÃ CƠNG BỐ 67 TÀI LIỆU THAM KHẢO 68 A niger 13 C-NMR 1 H- H COSY H-NMR CC CD CYA DMSO GC-MS Hep-G2 HL-60 HMBC HPLC HSQC IC50 KB MCF-7 MIC50 MS NOESY PDA TCA PDB SK-Mel-2 TLC SRB LNCaP tR vii DANH MỤC BẢNG Bảng 1.1 Một số hợp chất dùng dƣợc phẩm có nguồn gốc từ vi nấm 13 Bảng 3.1 Số liệu phổ H C NMR hợp chất A1 ……………………………31 13 13 Bảng 3.2 Số liệu phổ H C NMR hợp chất A3 38 Bảng 3.3 Số liệu phổ H C NMR hợp chất A2 45 13 Bảng 3.4 Số liệu phổ H C NMR hợp chất A4 46 13 Bảng 3.5 Số liệu phổ H C NMR hợp chất A5 48 13 Bảng 3.6 Số liệu phổ H C NMR hợp chất A6 49 13 Bảng 3.7 Số liệu phổ H C NMR hợp chất A7 50 13 Bảng 3.8 Số liệu phổ H C NMR hợp chất A8 51 13 Bảng 3.9 Số liệu phổ H C NMR hợp chất A9 52 13 Bảng 3.10 Số liệu phổ H C NMR hợp chất A10 53 13 Bảng 3.11 Số liệu phổ H C NMR hợp chất A11 54 13 Bảng 3.12 Số liệu phổ H C NMR hợp chất A12 56 13 Bảng 3.13 Số liệu phổ H C NMR hợp chất A13 58 13 Bảng 3.14 Số liệu phổ H C NMR hợp chất A14 59 Bảng 3.15 Hoạt tính gây độc tế bào ung thƣ hợp chất A1-A14 61 Bảng 3.16 Hoạt tính ức chế NO hợp chất A1-A14 63 viii DANH MỤC HÌNH Hình 1.1 Một số loài vi nấm phổ biến .5 Hình 1.2 Một số loại kháng sinh đƣợc sản xuất vi nấm Hình 1.3 Một số loài nấm thuộc chi Aspergillus .9 Hình 1.4 Sinh sản vơ tính A niger 10 Hình 1.5 A niger mơi trƣờng Czapek PDA 10 Hình 1.6 Bào tử A niger chụp kính hiển vi điện tử quét (Scanning Electron Microscope – SEM) 10 Hình 1.7 Các cụm gen sinh tổng hợp chất trao đổi thứ cấp 12 chủng A niger 13 Hình 1.8 Một số hợp chất pyranones từ A niger 14 Hình 1.9 Một số hợp chất alkaloids từ A niger 16 Hình 1.10 Một số hợp chất cyclopeptides từ A niger 17 Hình 1.11 Một số hợp chất polyketide từ A niger 17 Hình 1.12 Một số hợp chất sterol từ A niger 18 Hình 3.1 Lên men nhân sinh khối lƣợng lớn mẫu IMBC-NMTP01……………… 26 Hình 3.2 Ngâm chiết, siêu âm thu dịch chiết mẫu A niger IMBC-NMTP01 .26 Hình 3.3 Sơ đồ phân lập hợp chất từ chủng nấm mốc A niger IMBC-NMTP01 28 Hình 3.4 Cấu trúc tƣơng tác HMBC (→ COSY (—) hợp chất A1 29 Hình 3.5 Phổ ECD tính tốn lý thuyết thực nghiệm hợp chất A1 30 Hình 3.6 Phổ HRESIMS A1 31 Hình 3.7 Phổ H NMR (CDCl3, 500 MHz) A1 32 Hình 3.8 Phổ 13 C NMR (CDCl3, 125 MHz) A1 32 Hình 3.9 Phổ HSQC (CDCl3, 500 MHz) A1 33 Hình 3.10 Phổ HMBC (CDCl3, 500 MHz) A1 33 Hình 3.11 Phổ H NMR (CD3OD, 500 MHz) A1 34 Hình 3.12 Phổ 13 C NMR (CD3OD, 125 MHz) A1 34 Hình 3.13 Phổ HSQC (CD3OD, 500 MHz) A1 35 Hình 3.14 Phổ HMBC (CD3OD, 500 MHz) A1 35 Hình 3.15 Phổ 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Anti-Inflammatory and Cytoprotective Effects of TMC-256C1 from Marine-Derived Fungus Aspergillus sp SF-6354 via up-Regulation of Heme Oxygenase-1 in Murine Hippocampal and Microglial Cell Lines Int J Mol Sci., 17, pp 529 109 Natural Product Research Formerly Natural Product Letters ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20 Secondary metabolites from a peanutassociated fungus Aspergillus niger IMBCNMTP01 with cytotoxic, anti-inflammatory, and antimicrobial activities Tran Hong Quang , Nguyen Viet Phong , Le Ngoc Anh , Tran Thi Hong Hanh , Nguyen Xuan Cuong , Nguyen Thi Thanh Ngan , Nguyen Quang Trung , Nguyen Hoai Nam & Chau Van Minh To cite this article: Tran Hong Quang , Nguyen Viet Phong , Le Ngoc Anh , Tran Thi Hong Hanh , Nguyen Xuan Cuong , Nguyen Thi Thanh Ngan , Nguyen Quang Trung , Nguyen Hoai Nam & Chau Van Minh (2020): Secondary metabolites from a peanutassociated fungus Aspergillus niger IMBC-NMTP01 with cytotoxic, anti-inflammatory, and antimicrobial activities, Natural Product Research, DOI: 10.1080/14786419.2020.1868462 To link to this article: https://doi.org/10.1080/14786419.2020.1868462 View supplementary material Published online: 30 Dec 2020 Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1868462 Secondary metabolites from a peanut-associated fungus Aspergillus niger IMBC-NMTP01 with cytotoxic, antiinflammatory, and antimicrobial activities a a a Tran Hong Quang , Nguyen Viet Phong , Le Ngoc Anh , Tran Thi Hong a a b Hanh , Nguyen Xuan Cuong , Nguyen Thi Thanh Ngan , Nguyen Quang c a a Trung , Nguyen Hoai Nam and Chau Van Minh a Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), Hanoi Vietnam; bInstitute of Genome Research, VAST, Hanoi, Vietnam; c Center for Research and Technology Transfer, VAST, Hanoi, Vietnam ABSTRACT Chemical investigation of a peanut-associated fungal strain Aspergillus niger IMBC-NMTP01 resulted in isolation and identifica-tion of 14 secondary metabolites, including two new, epi-aspergil-lusol (1) and aspernigin (3), and 12 known compounds: pyrophen (2), 2(hydroxyimino)-3-(4-hydroxyphenyl)propanoic acid (4), aspergillusol A (5), rubrofusarin B (6), nigerasperone A (7), fonse-cin (8), TMC-256C1 (9), pyranonigrin A (10), orlandin (11), nigeras-perone C (12), asperpyrone A (13), and 5-(hydroxymethyl)-2-furancarboxylic acid (14) Compounds 9, 12-14 showed cytotox-icity toward all six human cancer cell lines, including HepG2, KB, HL-60, MCF-7, SK-Mel2, and LNCaP, with IC50 values ranging from 8.4 to 84.5 mM, compounds 3-5 were cytotoxic against five cancer cell lines except HepG2, whereas exhibited cytotoxicity toward HepG2, KB, and MCF-7 cells All of the compounds, except and 13, inhibited NO overproduction in LPSinduced RAW264.7 cells In addition, all of the compounds displayed antimicrobial effects against Enterococcus faecalis, whereas 13 compounds, except 10, significantly inhibited the growth of the yeast Candida albicans ARTICLE HISTORY Received 28 September 2020 Accepted 20 December 2020 KEYWORDS Peanut-associated fungus; Aspergillus niger; cytotoxic; anti-inflammatory; antimicrobial CONTACT Nguyen Hoai Nam namnguyenhoai@imbc.vast.vn; Tran Hong Quang quangtranhong@imbc.vast.vn Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1868462 2020 Informa UK Limited, trading as Taylor & Francis Group download by : skknchat@gmail.com T H QUANG ET AL Introduction Aspergillus niger is one the most popular fungi commonly found in soils, seeds, dried fruit, and nuts and as contaminant of food This fungus involved in biological deterioration of maize, peanuts, grapes and grape products, coffee, tea, beans, and spice (Palencia et al 2010) It has been used for many years as an industrial producer of extracellular enzymes and citric acid, which are considered Generally Recognized as Safe (GRAS) by the Food and Drug Administration of the United States (Schuster et al 2002) In addition to the wide applications in biotechnological industry, A niger has also been proven to be a rich source of secondary metabolites, including diketopipera-zine and other alkaloids (Nielsen et al 2009; Li et al 2015), pyrones (Sakurai et al 2002; Liu et al 2011; Li et al 2016), bicoumarins and malformins (Nielsen et al 2009), of which some compounds were shown to exhibit immunomodulatory (Sakurai et al 2002) and cytotoxic activities (Liu et al 2011; Li et al 2015; 2016) However, the chem-ical profile of the peanut-associated A niger has yet to be understood so far During the primary screening, the fungal strain A niger IMBC-NMTP01 isolated from a moldy peanut showed significant cytotoxic and antimicrobial effects, leading to further inves-tigation of the chemical constituents of this fungal strain In the present study, we report the isolation and structural elucidation of 14 secondary metabolites, including two new compounds, epi-aspergillusol (1) and 2,3,4-trihydroxybutyl 2-(hydroxyimino)-3-(4hydroxyphenyl)propanoate (3) from the EtOAc extract of the fungal strain A niger IMBCNMTP01 Furthermore, cytotoxic, anti-inflammatory, and antimicrobial effects of the isolated compounds were also evaluated To the best of our knowledge, this is the first time to evaluate the cytotoxicity of and 11, the anti-inflammatory effects of 2, 4, 5, 7, 10, and 12, and the antimicrobial effects of and Results and discussion The EtOAc extract of A niger IMBC-NMTP01 was subjected to the multiple chromatographic steps to give 14 compounds, including two new secondary metabolites (1 and 3) The structures of the 12 known compounds were identified as: pyrophen (2) (Varoglu and Crews 2000), 2-(hydroxyimino)-3-(4-hydroxyphenyl)propanoic acid (4) (Korwar et al 2016), aspergillusol A (5) (Ingavat et al 2009), rubrofusarin B (6) (Shaaban et al 2012), nigerasperone A (7) (Zhang et al 2007), fonsecin (8) (Priestap 1986), TMC-256C1 (9) (Sakurai et al 2002), pyranonigrin A (10) (Schlingmann et al 2007), orlandin (11) (Huttel€ and Muller€ 2007), nigerasperone C (12) (Zhang et al 2007), asperpyrone A (13) (Akiyama et al 2003), and 5-(hydroxymethyl)-2-furancarboxylic acid (14) (Klemke et al 2004) by the 1D and 2D NMR and MS spectroscopic analyses in comparison with those of the reported compounds (Figure 1) Compound was isolated as a white, amorphous powder Its molecular formula, ỵ C14H14O4 was deduced by a protonated molecular ion at m/z 247.0963 [M ỵ H] (calcd ỵ for C14H15O4 , 247.0965) in the HRESITOFMS, implying eight degrees of unsaturation The H NMR spectrum (in CDCl3) of showed signals of five aromatic protons at d H 7.22 (d, J ¼ 8.0 Hz, H-9 and H-13), 7.32 (t, J ¼ 8.0 Hz, H-10 and H-12), and 7.26 (t, J ¼ 8.0 Hz, H-11), characterizing the presence of a mono-substituted benzene ring The H NMR spectrum additionally exhibited signals of two olefinic protons at d H 5.44 (d, download by : skknchat@gmail.com NATURAL PRODUCT RESEARCH Figure Chemical structures of compounds 1-14 from A niger IMBC-NMTP01 J ¼ 2.5 Hz, H-2) and 6.05 (dd, J ¼ 1.0, 2.5 Hz, H-4), one oxymethine proton at d H 4.60 (dd, J ¼ 4.0, 8.0 Hz, H-6), two germinal-coupled protons at d H 3.26 (dd, J ¼ 4.0, 14.0 Hz, Ha-7) and 2.93 (dd, J ¼ 8.0, 14.0 Hz, Hb-7), and a methoxy group at d H 3.84 (s, 31 OCH3) The observed H NMR data in combination with analysis of the 13 C NMR and HSQC spectra (in CDCl3) revealed the presence of a 3-methoxy-1-oxo-1H-pyran-5-yl moiety, as evidenced by carbon signals of one carbonyl carbon at d C 164.2 (C-1), four sp car-bons at dC 88.2 (C-2), 171.3 (C-3), 99.0 (C-4), and 165.2 (C-5) (Zhang et al 2010) The location of the methoxy group at C-3 was confirmed by an HMBC correlation from its proton (dH 3.84) to C-3 (Figure S3) In addition, a COSY interaction between H-6 and H2-7, together with HMBC correlations from H 2-7 to C-8, C-9, and C-13 allowed to rec-ognize the occurrence of a 1-hydroxy-2-phenylethyl moiety (Figure S3) The planar structure of was finally established as 5-(6-hydroxy-7-phenylethyl)-3-methoxy-1Hpyran-1-one by HMBC cross-peaks observed from H-6 to C-4 and C-5 Comparison of 13 the H and C NMR data of with those of aspergillusol resulted in an excellent agreement, revealing that the planar structures of both compounds are identical (Zhang et al 2010) However, the optical rotation values observed for and aspergillu-sol were shown to be opposite in direction [1: ẵa 25 D ẳ 61.0 (c ẳ 0.5, CHCl3) vs asper-gillusol: 25 D ẵa ẳ ỵ35 (c ẳ 0.4, CHCl 3)] (Zhang et al 2010), suggesting that is an enantiomer of aspergillusol Therefore, the absolute configuration of was further confirmed using time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculation by Gaussian 16 W program (Frisch et al 2016) Firstly, an analysis of conformations was implemented using MMFF94 force field for both of R download by : skknchat@gmail.com T H QUANG ET AL and 6S isomers by Spartan’18 program Optimization of the selected conformers was carried out using B3LYP/6-31ỵỵG(d,p) basic set and the conductor-like polarizable continuum model (CPCM) in methanol (O’Boyle et al 2011) The ECD calculation was subsequently performed using TDDFT at the same theory level Eventually, simulation of the calculated ECD spectra was performed by overlapping Gaussian functions for each transition using SpecDis 1.71 (Bruhn et al 2013; 2017) As shown in Figure S4, the calculated ECD spectrum for 6S isomer was shown to be in a good agreement with the experimental data, whereas the calculated ECD spectrum for R configuration exhibited the opposite direction, suggesting that the absolute configuration of C-6 of is S Consequently, the structure of was identified as shown in Figure 1, named epi-aspergillusol Compound was obtained as a white, amorphous powder Its molecular formula was determined to be C13H17NO7 by a chlorinated molecular ion at m/z 334.0692 [M ỵ Cl] (calcd for C13H17NClO7 , 334.0699) and a deprotonated ion at m/z 298.0917 [M-H]1 (calcd for C13H16NO7 , 298.0932) in the HRESITOFMS, along with an analysis of the H 13 and C NMR data In the H NMR spectrum (in DMSO-d6), signals of an 1,40 disubstituted benzene ring were observed at d H 7.03 (br d, J ¼ 8.5 Hz, H-5 and H-9 ) 0 and 6.66 (br d, J ¼ 8.5 Hz, H-6 and H-8 ) The H NMR spectrum further exhibited two pairs of germinal coupling protons at dH 4.30 (dd, J ¼ 3.0, 11.5 Hz, Ha-1), 4.10 (dd, J ¼ 7.0, 11.5 Hz, Hb-1), 3.55 (dd, J ¼ 3.5, 11.0 Hz, Ha-4), and 3.39 (dd, J ¼ 5.5, 11.0 Hz, Hb-4), two oxymethine protons at dH 3.62 (m, H-2) and 3.40 (m, H-3), and a singlet of a methylene group at dH 3.72 (H2-30) Analysis of 13C NMR and HSQC spectra (in DMSO-d6) pointed out signals of one carbonyl carbon at d C 163.9 (C-1 ), seven sp2 car0 bons, including six signals of the para-disubstituted benzene ring [d C 126.5 (C-4 ), 129.7 0 0 (C-5 and C-9 ), 115.0 (C-6 and C-8 ), and 155.7 (C-7 )] and one non-protonated 0 carbon bearing nitrogen at dC 150.1 (C-2 ), and one methylene group at dC 29.2 (C-3 ) 0 In the HMBC spectrum, the proton signal at d H 3.72 showed correlations with C-1 , C-2 , 0 C-5 , and C-6 , revealing the presence of a tyrosine oxime unit in the molecule (Figure 13 S3) (Ingavat et al 2009; Korwar et al 2016) The C NMR additionally showed signals characteristic of a butane-1,2,3,4-tetraol partial structure at d C 67.1 (C-1), 69.3 (C-2), 72.1 (C-3), and 62.9 (C-4) This was supported by HMBC correlations from the oxymethylene proton signals at d H 4.30/4.10 (H2-1) and dH 3.55/3.39 (H2-4) to C-2 and C-3, along with COSY correlations between H 2-1/H-2/H-3/H2-4 (Figure S3) The connection of the butane-1,2,3,4-tetraol and tyrosine oxime moieties through an ester linkage was deduced unambiguously by an HMBC correlation from H 2-1 to C-1 Comparison of 13 the H and C NMR data of with those of aspergillusol A resulted in a good agreement, suggesting that both compounds have the similar structures, except for the lack of a 4-hydroxyphenylpyruvic acid oxime unit in compared with the symmet-ric structure of aspergillusol A (Ingavat et al 2009) The relative configuration of was suggested based on its 1H and 13C NMR data in comparison with those of the reported 0 analogs The carbon chemical shifts of C-2 (dC 150.1) and C-3 (dC 29.2) posi-tions of 0 were closely consistent with those of aspergillusol A [d C 150.5 (C-2 ) and 29.6 (C-3 )] (Ingavat et al 2009) and 2-(hydroxyimino)-phenylpropanoic acid [d C 150.13 (C-2 ) and 29.85 (C-3 )] (Korwar et al 2016) as well as the co-existent analog, 2-(hydrox-yimino)-3(4-hydroxyphenyl)propanoic acid (4), suggesting that the oxime functional download by : skknchat@gmail.com NATURAL PRODUCT RESEARCH 13 group of possesses the Z geometry Similarly, the H and C NMR data of the butane-1,2,3,4-tetraol moiety of were in a good agreement with those of (ỵ)-erythrin and (ỵ)-montagnetol (Basset et al 2010; Mallavadhani et al 2018), suggesting that both hydroxy groups at C-2 and C-3 have the erythro relationship This was supported by the small coupling value (J ¼ 3.0 Hz) observed between H-2 and H-3 in comparison with those reported for both threo and erythro acyclic vicinal diol groups (Xu et al 2017) Consequently, the structure of was established as 2,3,4-trihydroxybutyl 2(hydroxyimino)-3-(4-hydroxyphenyl)propanoate, namely aspernigin All the isolated compounds were evaluated for cytotoxicity toward six human carcinoma cell lines, including HepG2, KB, HL-60, MCF-7, SK-Mel-2, and LNCaP using the sulforhodamine B (SRB) assay (Monks et al 1991; Quang et al 2020) As the result, among the naphtho-c-pyrones (6-9), compound was shown to have the strongest cytotoxicity toward all the cell lines, with the IC 50 values ranging from 9.1 to 23.5 mM, while was cytotoxic against only HepG2 and MCF-7 cells, with IC 50 values of 77.1 and 68.3 mM, respectively (Table S1) Both of the dimeric naphthopyrones (12 and 13) and the furancarboxylic acid derivative (14) exhibited moderate cytotoxicity against all the carcinoma cell lines, with IC50 values ranging from 47.7 to 84.5 mM Of the two phenethyl-a-pyrones (1 and 2), compound displayed moderate cytotoxicity toward HepG2, KB, and MCF-7 cell lines, with IC50 values of 52.0, 91.2, and 65.0 mM, respectively, while its analog (2), with an acetamide functional group at C-6, was noncytotoxic at the tested concentrations Moderate cytotoxicity of the three hydroxyimine metabo-lites (3-5) were observed against five cancer cell lines, except HepG2, with IC 50 values in a range of 58.7 to 88.9 mM Compound 11 was weekly cytotoxic toward MCF-7 and SKMel-2 cells, with IC50 values of 91.5 and 83.8 mM, respectively In vitro anti-inflammatory effects of the isolated compounds were evaluated through inhibition of NO overproduction in LPS-stimulated RAW264.7 cells The results revealed that, except and 13, all of the tested compounds inhibited NO production in a dose-dependent manner, with IC50 values ranging from 2.1 to 84.4 mM (Table S1) Notably, the NO inhibitory effect of TMC-256C1 (9) (IC50 ¼ 2.1 mM) was more potent than that of the positive control, LNMMA (IC50 ¼ 7.6 mM) Nigerasperone A (7) showed a significant NO inhibitory effect (IC 50 ¼ 7.4 mM) which was comparable to that of L-NMMA Interestingly, the NO inhibitory effect of rubrofusarin B (6) (IC50 ¼ 84.4 mM) which possesses a methyl group at C-3 was decreased dramatically compared with that of its hydroxylated analog (7) Compounds 1, 4, 10, 12, and 14 significantly suppressed NO production, with IC 50 val-ues ranging from 14.5 to 24.0 mM, whereas compounds 3, 5, and 11 exhibited moderate inhibitory effects, with IC 50 values in a range of 41.3 to 56.0 mM As fungal metabolites have been considered as a promising source of antibiotics, we examined antimicrobial actions of the isolated compounds against a number of pathogens, including Gram-positive bacteria (Enterococcus faecalis ATCC299212, Staphylococcus aureus ATCC25923, and Bacillus cereus ATCC13245), Gram-negative bac-teria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Salmonella enterica ATCC13076), and a yeast strain Candida albicans ATCC 24433 The result indi-cated that all the compounds significantly inhibit the growth of the Gram-positive bacterium E faecalis, with MIC values of 32 or 64 mM, however no inhibition was observed toward the remaining the five strains of experimental bacteria (Table S2) In download by : skknchat@gmail.com T H QUANG ET AL addition, all of the compounds, except 10, suppressed the growth of the yeast Candida albicans, with MIC values of 64 or 128 mM Experimental 3.1 Fungal material The fungal strain Aspergillus niger IMBC-NMTP01 was isolated from a mouldy peanut col-lected from a market in Hanoi, Vietnam in 2019 In brief, the peanuts were left for fungi growing naturally for seven days The fungal spores were collected, mixed with the medium and transferred to a potato dextrose agar (PDA) plate which was then incubated at 25 C (Figure S1) After sub-culturing the colony several times, the final pure isolate was obtained and preserved at 80 C The fungal strain was identified based on an analysis of its internal transcribed spacers (ITS) rDNA sequence (GenBank accession number MW000457.1) (Figure S2) A voucher specimen was deposited at the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam 3.2 Fermentation, extraction, and isolation The fungal strain was cultured on a PDA plate at 25 C for days The seed culture of the fungus was collected and mixed with liquid medium to make a spore suspension The spores were inoculated individually into 80 L Erlenmeyer flasks, each contain-ing 150 mL of PDA culture medium After 30 days of static fermentation at 25 C, the combined growing media and mycelia of the fungus were extracted by maceration with EtOAc (3 times x 16 L) to provide an organic phase After concentrating under reduced pressure, a residue (5.2 g) of the fungal extract was subjected to fractionation over reversedphase (RP) C18, eluting with a stepwise gradient of MeOH in H 2O from 20:80 to 100:0 (v/v) to give six fractions F1 – F6 F1 was separated by Sephadex LH-20 column chromatography (CC), using MeOH-H2O (4:1, v/v) as eluent to afford 14 (18 mg) F.2 was washed repeatedly with MeOH and filtered through a filter paper to obtain a white powder (10, 11 mg) and three subfractions (F2.1-F2.3) F2.1 was sepa-rated by silica gel CC, eluting with CH2Cl2-MeOH (7:1, v/v) and further purified by RP C 18 prep HPLC, using an isocratic elution of 11% CH3CN in water (containing 0.1% HCOOH) to give (2.2 mg, tR ¼ 28.2 min) F2.2 was introduced to a silica gel CC, using CH 2Cl2-MeOH (7:1, v/v) as eluent to provide (3 mg) F3 was introduced to a Sephadex LH-20 CC, eluting with MeOH-H2O (4:1, v/v) to yield six subfraction (F1.1-F1.6) F3.1 was chromatographed over silica gel, using CH 2Cl2-MeOH (8:1, v/v) as a mobile phase to give (5 mg) F3.2 was separated by a silica gel CC, using n-hexane-EtOAc (1:2.5, v/v) as a mobile phase to obtain (13 mg) F.3.3 was chromatographed over silica gel, eluting with n-hexane-EtOAc (1:1, v/v) to afford (2.4 mg) and a sub-fraction which was further purified by RP C18 prep HPLC, using 28% CH3CN in water (containing 0.1% HCOOH) as eluent to release 11 (2 mg, t R ¼ 28.3 min) From subfrac-tion F3.5, compound (6 mg) was isolated by silica gel CC, eluting with CH 2Cl2-EtOAc (4:1, v/v) F4 was separated over Sephadex LH-20, using MeOH-H 2O (4:1, v/v) as eluent to provide five subfractions (F4.1-F4.5) F4.2 was chromatographed over silica gel, elut-ing with CH2Cl2-EtOAc (3.5:1, v/v) and further purified by RP C18 prep HPLC, using an download by : skknchat@gmail.com NATURAL PRODUCT RESEARCH isocratic elution of 36% CH3CN in H2O (containing 0.1% HCOOH) to give 12 (4 mg, tR ¼ 17.0 min) F4.3 was separated by silica gel CC, eluting with CH 2Cl2-acetone (8:1, v/v) to afford (16 mg) and a subfraction which was further purified through a RP C18 prep HPLC, using 33% of CH3CN in H2O (containing 0.1% HCOOH) as eluent to give (2.3 mg, tR ¼ 28.7 min) F4.4 was introduced to RP C 18 CC, eluting with CH3CN-H2O (1:1, v/v) to provide subfractions F4.4.1-F4.4.4 F4.4.1 was purified by RP C18 prep HPLC, using an isocratic elution of 47% CH 3CN in H2O (containing 0.1% HCOOH) to give (4 mg, t R ¼ 31.8 min) Similarly, compound 13 (5 mg, t R ¼ 30.6 min) was obtained from the subfraction F4.4.4 by RP C 18 prep HPLC, using 45% CH3CN in H2O (containing 0.1% HCOOH) as a mobile phase epi-Aspergillusol (1): white, HRESITOFMS: m/z 247.0963 [M ỵ H] (kmax): 2.13 (283), ỵ0.97 (234), dH 5.44 (d, J ẳ 2.5 Hz, H-2), 6.05 (dd, J ¼ 1.0, 2.5 Hz, H-4), 4.60 (dd, J ¼ 4.0, 8.0 Hz, H-6), 3.26 (dd, J ¼ 4.0, 14.0 Hz, Ha-7), 2.93 (dd, J ¼ 8.0, 14.0 Hz, Hb-7), 7.22 (d, J ¼ 8.0 Hz, H-9 and H-13), 7.32 (t, J ¼ 8.0 Hz, H-10 and H-12), 7.26 (t, J ¼ 8.0 Hz, H-11), and 3.84 (s, 3-OCH 3); 13 C NMR (CDCl3, 125 MHz): dC 164.2 (C-1), 88.2 (C-2), 171.3 (C-3), 99.0 (C-4), 165.2 (C-5), 71.3 (C-6), 41.6 (C-7), 136.2 (C-8), 129.5 (C-9 and C-13), 128.8 (C-10 and C-12), 127.1 (C-11), and 56.0 (31 OCH3) H NMR (CD3OD, 500 MHz): dH 5.56 (d, J ¼ 2.0 Hz, H-2), 6.09 (dd, J ¼ 1.0, 2.0 Hz, H-4), 4.58 (dd, J ¼ 5.0, 7.5 Hz, H-6), 3.15 (dd, J ¼ 5.5, 13.5 Hz, Ha-7), 2.97 (dd, J ¼ 8.0, 13.5 Hz, Hb-7), 7.23 (d, J ¼ 8.0 Hz, H-9 J ¼ 8.0 Hz, H-11), and 3.84 (s, 3-OCH3); 2), 173.6 (C-3), 100.6 (C-4), 167.8 (C-5), 72.5 (C-6), 42.3 (C-7), 138.5 (C-8), 130.5 (C-9 and C13), 129.3 (C-10 and C-12), 127.6 (C-11), and 56.9 (3-OCH 3) Aspernigin (3): white, HRESITOFMS: m/z 334.0692H]- (calcd for C13H16NO7 , 298.0932) ECD (MeOH) mdeg (kmax): no significant Cotton effect was observed H NMR (DMSO-d6, 500 MHz): dH 4.30 (dd, J ¼ 3.0, 11.5 Hz, Ha-1), 4.10 (dd, J ¼ 7.0, 11.5 Hz, Hb-1), 3.62 (td, J ¼ 3.0, 7.0 Hz, H-2), 3.40 (m, H-3), 3.55 (dd, J ¼ 3.5, 11.0 Hz, Ha0 0 4), 3.39 (dd, J ¼ 5.5, 11.0 Hz, Hb-4), 3.72 (s, H 2-3 ), 7.03 (br d, J ¼ 8.5 Hz, H-5 and H-9 ), and 0 6.66 (br d, J ¼ 8.5 Hz, H-6 and H-8 ); 13 C NMR (DMSO-d6, 125 MHz): dC 67.1 (C-1), 69.3 (C-2), 0 0 72.1 (C-3), 62.9 (C-4), 163.9 (C-1 ), 150.1 (C-2 ), 29.2 (C-3 ), 126.5 (C-4 ), 129.7 (C-5 and 0 C-9 ), 115.0 (C-6 J ¼ 3.0, 11.5 Hz, Ha-1), 4.26 (dd, J ¼ 7.0, H-3), 3.77 (dd, J d, J ¼ 8.5 Hz, H-5 0 0 125 MHz): dC 68.4 (C-1), 71.1 (C-2), 73.4 (C-3), 64.5 (C-4), 165.4 (C-1 ), 152.2 (C-2 ), 30.3 (C-3 ), 0 0 0 128.5 (C-4 ), 131.1 (C-5 and C-9 ), 116.2 (C-6 and C-8 ), and 156.9 (C-7 ) Conclusion Chemical investigation of the EtOAc extract of the peanut-associated fungus A niger IMBC-NMTP01 resulted in the isolation and structural identification of 14 secondary metabolites, including two new compounds, epi-aspergillusol (1) and aspernigin (3) Compounds 9, 12-14 were shown to have cytotoxicity toward all HepG2, KB, HL-60, download by : skknchat@gmail.com T H QUANG ET AL MCF-7, SK-Mel2, and LNCaP carcinoma cells, compounds 3-5 were cytotoxic toward five cancer cell lines except HepG2, while possessed cytotoxicity toward HepG2, KB, and MCF-7 All of the compounds, except and 13, inhibited NO overproduction in LPS-induced RAW264.7 cells, in which the inhibitory actions of and were more potent than the positive control Furthermore, all the compounds displayed notably antimicrobial effects against the Gram-positive bacterium E faecalis, whereas 13 com-pounds, except 10, significantly inhibited the growth of the yeast C albicans Our results contribute to unraveling the chemical profile of the peanut-derived A niger and provide a scientific rationale for further studies of this fungal strain and its metab-olites for the therapeutic uses Acknowledgements The authors are thankful to the Institute of Chemistry, VAST for measurement of the NMR spec-tra; Prof Do Thi Thao, Institute of Biotechnology, VAST for the cytotoxicity and NO inhibitory assays; Dr Vu Thi Quyen, Institute of Marine Biochemistry, VAST for antimicrobial assay; and Dr Bui Huu Tai, Institute of Marine Biochemistry, VAST for measurement of the HRESITOFMS and ECD spectra Disclosure statement No potential conflict of interest was reported by the authors Funding This work was financially supported by Vietnam Academy of Science and Technology under - grant number TDNDTP 06/19-21 References Akiyama K, Teraguchi S, Hamasaki Y, Mori M, Tatsumi K, Ohnishi K, Hayashi H 2003 New dimeric naphthopyrones from Aspergillus niger J Nat Prod 66(1):136–139 Basset J-F, Leslie C, Hamprecht D, White AJ, Barrett AG 2010 Studies on the resorcylates: biomimetic total syntheses of (ỵ)-montagnetol and (ỵ)-erythrin Tetrahedron Lett 51(5):783–785 Bruhn T, Schaumloffel€ A, Hemberger Y, Bringmann G 2013 SpecDis: Quantifying the comparison of calculated and experimental electronic circular dichroism spectra Chirality 25(4):243–249 Bruhn T, Schaumloffel€ A, Hemberger Y, 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62 3.6 Kết đánh giá hoạt tính kháng khuẩn, kháng nấm hợp chất .64 KẾT LUẬN VÀ KIẾN NGHỊ