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 GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VN HỌC VIỆN KHOA HỌC VÀ CƠNG 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 GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VN 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 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 cơng bố cơng trình khác Tác giả Lê Ngọc Anh ii LỜI CẢM ƠN Luận văn đƣợc hồn thành Viện Hóa sinh biển, Viện Hàn lâm Khoa học Công nghệ Việt Nam Trong q trình nghiên cứu, tơi nhận đƣợc nhiều giúp đỡ quý báu thầy cơ, nhà khoa học, đồng nghiệp, gia đình bạn bè Tôi 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 chuyên môn, đồng thời động viên, khích lệ tạo điều kiện thuận lợi cho suốt thời gian thực luận văn nhƣ q 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 Cơng nghệ Việt Nam tạo điều kiện giúp đỡ, truyền kinh nghiệm, đƣa lời khuyên 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 tơi 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 khuôn 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, ngơ, 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 1.1.1 Giới thiệu chung vi nấm 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 Đà CÔNG BỐ 67 TÀI LIỆU THAM KHẢO 68 vi DANH MỤC KÝ HIỆU, CHỮ VIẾT TẮT A niger Aspergillus niger 13 Phổ cộng hƣởng từ hạt nhân carbon C-NMR H-1H COSY Phổ tƣơng tác proton-proton H-NMR Phổ cộng hƣởng từ hạt nhân proton CC Cột sắc ký CD Circular dichroism Spectroscopy – Phổ lƣỡng sắc tròn CYA Czapek Yeast Agar DMSO Dimethylsulfoside GC-MS Gas chromatography – mass spectrometry – Sắc ký khí – khối phổ Hep-G2 Tế bào ung thƣ biểu mô gan HL-60 Tế bào ung thƣ máu ngƣời HMBC Phổ tƣơng tác di hạt nhân qua nhiều liên kết HPLC Sắc ký lỏng hiệu cao HSQC Phổ tƣơng tác dị hạt nhân qua liên kết IC50 Nồng độ ức chế 50% KB Tế bào ung thƣ biểu mô ngƣời MCF-7 Tế bào ung thƣ vú ngƣời MIC50 Nồng độ ức chế tối thiểu 50% MS Phổ khối lƣợng NOESY Phổ NOESY PDA Potato Dextrose Agar TCA Trichloro acetic acid PDB Potato Dextrose Broth SK-Mel-2 Tế bào ung thƣ da ngƣời TLC Thin layer chromatography – Sắc ký lớp mỏng SRB Sulforhodamine B LNCaP Tế bào ung thƣ tiền liệt tuyến tR Thời gian lƣu vii DANH MỤC BẢNG Bảng 1.1 Một số hợp chất dùng dƣợc phẩm có nguồn gốc từ vi nấm Bảng 3.1 Số liệu phổ 1H 13C NMR hợp chất A1 ……………………………31 Bảng 3.2 Số liệu phổ 1H 13C NMR hợp chất A3 38 Bảng 3.3 Số liệu phổ 1H 13C NMR hợp chất A2 45 Bảng 3.4 Số liệu phổ 1H 13C NMR hợp chất A4 46 Bảng 3.5 Số liệu phổ 1H 13C NMR hợp chất A5 48 Bảng 3.6 Số liệu phổ 1H 13C NMR hợp chất A6 49 Bảng 3.7 Số liệu phổ 1H 13C NMR hợp chất A7 50 Bảng 3.8 Số liệu phổ 1H 13C NMR hợp chất A8 51 Bảng 3.9 Số liệu phổ 1H 13C NMR hợp chất A9 52 Bảng 3.10 Số liệu phổ 1H 13C NMR hợp chất A10 53 Bảng 3.11 Số liệu phổ 1H 13C NMR hợp chất A11 54 Bảng 3.12 Số liệu phổ 1H 13C NMR hợp chất A12 56 Bảng 3.13 Số liệu phổ 1H 13C NMR hợp chất A13 58 Bảng 3.14 Số liệu phổ 1H 13C 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 lồi vi nấm phổ biến Hình 1.2 Một số loại kháng sinh đƣợc sản xuất vi nấm Hình 1.3 Một số lồi nấm thuộc chi Aspergillus 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 1H NMR (CDCl3, 500 MHz) A1 32 Hình 3.8 Phổ 13C 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 1H NMR (CD3OD, 500 MHz) A1 34 Hình 3.12 Phổ 13C 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 COSY (CD3OD, 500 MHz) A1 36 Hình 3.16 Cấu trúc hóa học tƣơng tác HMBC (), COSY (—) hợp chất A3 36 Hình 3.17 Phổ HRESIMS A3 38 Hình 3.18 Phổ 1H NMR (DMSO-d6, 500 MHz) A3 39 Hình 3.19 Phổ 13C NMR (DMSO-d6, 125 MHz) A3 39 Hình 3.20 Phổ HSQC (DMSO-d6, 500 MHz) A3 40 73 60 Zhou X., Fang W., Tan S., Lin X., Xun T., Yang B., Liu S., Liu Y., 2016, Aspernigrins with Anti-HIV-1 Activities from the marine-derived Fungus Aspergillus niger SCSIO Jcsw6F30, Bioorg Med Chem Lett., 26(2), pp 361–365 61 Nielsen K.F., Mogensen J.M., Johansen M., Larsen T.O., Frisvad J.C., 2009, Review of Secondary Metabolites and Mycotoxins from the Aspergillus niger Group, Anal Bioanal Chem., 395(5), pp 1225–1242 62 Sørensen L.M., Lametsch R., Andersen M.R., Nielsen P.V., Frisvad J.C., 2009, Proteome Analysis of Aspergillus niger: Lactate Added in StarchContaining Medium Can Increase Production of the Mycotoxin Fumonisin B2 by Modifying Acetyl-CoA Metabolism, BMC Microbiol., 9, pp 255 63 Li X., Pan L., Wang B., Pan L., 2019, The histone deacetylases HosA and HdaA affect the phenotype and transcriptomic and metabolic profiles of Aspergillus niger, Toxins, 11(9), pp 520 64 Zabala A.O., Xu W., Chooi Y.H., Tang Y., 2012, Characterization of a Silent Azaphilone Gene Cluster from Aspergillus niger ATCC 1015 Reveals a Hydroxylation-Mediated Pyran-Ring Formation, Chem Biol 19(8), pp 1049– 1059 65 Chiang Y.M., Meyer K.M., Praseuth M., Baker S.E., Bruno K.S., Wang C.C.C., 2011, Characterization of a Polyketide Synthase in Aspergillus niger Whose Product Is a Precursor for Both Dihydroxynaphthalene (DHN) Melanin and Naphtho-γ-Pyrone, Fungal Genet Biol., 48(4), pp 430–437 66 Li S., Yong-Hao Y., Xiao-Ting W., Hai-Liang Z., Chen X., Yong-Cun S., Hai L., Ren-Xiang T., 2006, Structure and Total Synthesis of Aspernigerin: A Novel Cytotoxic Endophyte Metabolite, Chem Eur J., 12(16), pp 4393–4396 67 Li X.B., Li Y.L., Zhou J.C., Yuan H.Q., Wang X.N., Lou H.X., 2015, A New Diketopiperazine Heterodimer from an Endophytic Fungus Aspergillus niger, J Asian Nat Prod Res., 17(2), pp 182–187 68 Varoglu M., Corbett T.H., Valeriote F.A., Crews P., 1997, Asperazine, a Selective Cytotoxic Alkaloid from a Sponge-Derived Culture of Aspergillus niger, J Org Chem., 62(21), pp 7078–7079 69 Varoglu M., Crews P., 2000, Biosynthetically diverse compounds from a saltwater culture of sponge-derived Aspergillus niger, J Nat Prod., 63(1), pp 41–43 74 70 Suda S., Curtis R.W., 1966, Antibiotic Properties of Malformin, Appl Microbiol, 14(3), pp 475–476 71 Liu D., Li X.M., Li C.S., Wang B.G., 2013, Nigerasterols A and B, Antiproliferative Sterols from the Mangrove-Derived Endophytic Fungus MA132, Hca, 96(6), pp 1055–1061 72 Wang J., Jiang Z., Lam W., Gullen E.A., Yu Z., Wei Y., Wang L., Zeiss C., Beck A., Cheng E.C., 2015, Study of malformin C, a fungal source cyclic pentapeptide, as an anti-cancer drug, PLoS One, 10, pp 1–19 73 Rao K.C.S., Divakar S., Babu K.N., Rao A.G.A., Karanth N.G., Sattur A.P., 2002, Nigerloxin, a novel inhibitor of aldose reductase and lipoxygenase with free radical scavenging activity from Aspergillus niger CFR-W-105, J Antibiot., 55(9), pp 789–793 74 Santhakumaran I., Kesavan S.S., Arumugam G., 2019, Asperyellone pretreatment protects HaCaT cells from UVB irradiation induced oxidative damages: Assessment under in vitro and in vivo conditions and at molecular llevel, J Cel Biochem, 120(6), pp 10715–10725 75 Ayer W.A., Muir D.J., Chakravarty P., 1996, Phenolic and other metabolites f Phellinus Pini, a fungus pathogenic to pine, Phytochemistry, 42(5), pp 1321– 1324 76 Kodukula K., Arcuri M., Cutrone J.Q., Hugill R.M., Lowe S.E., Pirnik D.M., Shu Y.Z., Fernandes P.B., Seethala R., 1995, BMS-192548, a tetracyclic binding inhibitor of neuropeptide Y receptors, from Aspergillus niger WB2346 I taxonomy, fermentation, isolation and biological activity, J Antibiot 48(10), pp 1055–1059 77 Shu Y.Z., Cutrone J.Q., Klohr S.E., Huang S., 1995, BMS-192548, a tetracyclic binding inhibitor of neuropeptide Y receptors, from Aspergillus niger WB2346 II physico-chemical properties and structural characterization, J Antibiot, 48(10), pp 1060–1065 78 Li Y., Chooi Y.H., Sheng Y., Valentine J.S., Tang Y., 2011, Comparative characterization of fungal anthracenone and naphthacenedione biosynthetic pathways reveals an α-Hydroxylation-Dependent claisen-like cyclization catalyzed by a dimanganese thioesterase, J Am Chem Soc., 133(39), pp 15773–15785 75 79 Barton D.H.R., Bruun T., 1951, A new sterol from a strain of Aspergillus niger, J Chem Soc., pp 2728 80 Ano Y., Ikado K., Shindo K., Koizumi H., Fujiwara D., 2017, Identification of 14-dehydroergosterol as a novel anti-inflammatory compound inducing tolerogenic dendritic cells, Sci Rep., 81 Zhang Y., Li X.M., Proksch P., Wang B.G., 2007a, Ergosterimide, a new natural diels-alder adduct of a steroid and maleimide in the fungus Aspergillus niger, Steroids, 72(9-10), pp 723–727 82 Huỳnh Ngọc Oanh, Trần Ngọc Hùng, 2008, Thu nhận enzyme pectinase từ Aspergillus niger phƣơng pháp lọc gel lọc màng, Tạp chí Phát triển Khoa học Cơng nghệ, tập 11, số 83 Lê Thị Thu Trang, 2011, Nghiên cứu khả tổng hợp pectinesterase polygalacturonase A niger, luận văn thạc sĩ, Đại học Đà Nẵng 84 Trần Thanh Trúc, 2013, Phân lập tuyển chọn dòng Aspergillus niger sinh pectin methylesterase hoạt tính cao, luận án tiến sĩ, Trƣờng Đại học Cần Thơ 85 Frisch M.J., Trucks G.W., Schlegel H.B., Scuseria G.E., Robb M.A., Cheeseman J.R., Scalmani G., Barone V., Petersson G.A., Nakatsuji H., 2016, Gaussian 16 Rev C.01 Gaussian, Inc, Wallingford, CT 86 O'Boyle N.M., Vandermeersch T., Flynn C.J., Maguire A.R., Hutchison G.R., 2011, Confab-Systematic generation of diverse low-energy conformers, J Cheminform, 3(1), pp 1-9 87 Bruhn T., Schaumlöffel A., Hemberger Y., Pescitelli G., 2017, SpecDis version 1.71, Berlin, Germany https:/specdis-softwarejimdocom 88 Monks A., Scudiero D., Skehan P., Shoemaker R., Paull K., Vistica D., Hose C., Langley J., Cronise P., Vaigro-Wolff A., Gray-Goodrich M., Campbell H., Mayo J., Boyd M., 1991, Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines, Journal of the National Cancer Institute, 83, pp 757-766 89 Scudiero D.A., Shoemaker R.H., Paull K.D., Monks A., Tierney S., Nofziger T.H., Currens M.J., Seniff D., Boyd M.R., 1988, Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines, Cancer Research, 48, pp 4827-4833 76 90 Titheradge M.A., 1998, The enzymatic measurement of nitrate and nitrite, Methods in Molecular Biology, 100, pp 83-91 91 Andrews J.M., 2001, Determination of minimum inhibitory concentrations, J Antimicrob Chemother.,48, pp 5-16 92 Thi Q.V., Tran V.H., Maia H.D., Le C.V., Hong Mle T., Murphy B.T., Chau V.M., Pham V.C., 2000, Antimicrobial Metabolites from a Marine-Derived Actinomycete in Vietnam's East Sea, Nat Prod Commun., 11, pp 49-51 93 Zhang Y., Li X.M., Feng Y., Wang B.G., 2010, Phenethyl-α-pyrone derivatives and cyclodipeptides from a marine algous endophytic fungus Aspergillus niger EN–13, Nat Prod Res, 24(11), pp 1036-1043 94 Bruhn T., Schaumlöffel A., Hemberger Y., Bringmann G., 2013, SpecDis: Quantifying the comparison of calculated and experimental electronic circular dichroism spectra, Chirality, 25(4), pp 243-249 95 Ingavat N., Dobereiner J., Wiyakrutta S., Mahidol C., Ruchirawat S., Kittakoop P., 2009, Aspergillusol A, an α-glucosidase inhibitor from the marine-derived fungus Aspergillus aculeatus, J Nat Prod, 72(11), pp 2049-2052 96 Korwar S., Morris B.L., Parikh H.I., Coover R.A., Doughty T.W., Love I.M., Hilbert B.J., Royer W.E., Kellogg G.E., Grossman S.R., 2016, Design, synthesis, and biological evaluation of substrate-competitive inhibitors of Cterminal Binding Protein (CtBP), Bioorg Med Chem, 24(12), pp 2707-2715 97 Basset J.F., Leslie C., Hamprecht D., White A.J., Barrett A.G., 2010, Studies on the resorcylates: biomimetic total syntheses of (+)-montagnetol and (+)-erythrin, Tetrahedron Lett, 51(5), pp 783-785 98 Mallavadhani U.V., Boddu R., Rathod B.B., Reddy Setty P., 2018, Stereoselective synthesis of the lichen metabolite,(+) montagnetol and its congeners as antimicrobial agents, Synth Commun, 48(23), pp 2992-2999 99 Xu K., Yang P.F., Yang Y.N., Feng Z.M., Jiang J.S., Zhang P.C., 2017, Direct assignment of the Threo and Erythro configurations in polyacetylene glycosides by 1H NMR spectroscopy, Org Lett, 19(3), pp 686-689 100 Varoglu M, Crews P., 2000, Biosynthetically Diverse Compounds from a Saltwater Culture of Sponge-Derived Aspergillus niger, J Nat Prod., 63(1), pp 41-43 77 101 Shaaban M., Shaaban K.A., Abdel-Aziz M.S., 2012, Seven naphtho-γ- pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties, Org Med Chem Lett, 2(1), pp 102 Priestap H.A., 1986, 13C NMR spectroscopy of naphtho-γ-pyrones, Magn Reson Chem., 24(10), pp 875-878 103 Schlingmann G., Taniguchi T., He H., Bigelis R., Yang H.Y., Koehn F.E., Carter G.T., Berova N., 2007, Reassessing the Structure of Pyranonigrin, J Nat Prod, 70(7), pp 1180-1187 104 Hüttel W., Müller M., 2007, Regio‐and Stereoselective Intermolecular Oxidative Phenol Coupling in Kotanin Biosynthesis by Aspergillus niger, ChemBioChem 8(5), pp 521-529 105 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), pp 136-139 106 Ying Y.M., Shan W.G., Liu W.H., Zhan Z.J., 2013, Studies on the metabolites of a fungal endophyte Penicillium sp HS-5 from Huperzia serrata, Asian J Chem., 25, pp 1208 107 Klemke C., Kehraus S., Wright A.D., König G.M., 2004, New Secondary Metabolites from the Marine Endophytic Fungus Apiospora montagnei, J Nat Prod, 67(6), pp 1058-1063 108 Xu K., Guo C., Shi D., Meng J., Tian H., Guo S., 2019, Discovery of Natural Dimeric Naphthopyrones as Potential Cytotoxic Agents Through ROSMediated Apoptotic Pathway, Marine Drugs, 17(4), pp 207 109 Kim D.C., Cho K.H., Ko W., Yoon C.S., Sohn J.H., Yim J.H., Kim Y.C., Oh H., 2016, 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 Natural Product Research Formerly Natural Product Letters ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20 Secondary metabolites from a peanut-associated fungus Aspergillus niger IMBC-NMTP01 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 peanut-associated fungus Aspergillus�niger IMBCNMTP01 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 Tran Hong Quanga, Nguyen Viet Phonga, Le Ngoc Anha, Tran Thi Hong Hanha, Nguyen Xuan Cuonga, Nguyen Thi Thanh Nganb, Nguyen Quang Trungc, Nguyen Hoai Nama and Chau Van Minha a Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), Hanoi Vietnam; bInstitute of Genome Research, VAST, Hanoi, Vietnam; cCenter for Research and Technology Transfer, VAST, Hanoi, Vietnam ABSTRACT ARTICLE HISTORY Chemical investigation of a peanut-associated fungal strain Aspergillus niger IMBC-NMTP01 resulted in isolation and identification of 14 secondary metabolites, including two new, epi-aspergillusol (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), fonsecin (8), TMC-256C1 (9), pyranonigrin A (10), orlandin (11), nigerasperone C (12), asperpyrone A (13), and 5-(hydroxymethyl)-2furancarboxylic acid (14) Compounds 9, 12-14 showed cytotoxicity 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 LPS-induced 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 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 ß 2020 Informa UK Limited, trading as Taylor & Francis Group 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 diketopiperazine 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 chemical 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 investigation 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-(4-hydroxyphenyl)propanoate (3) from the EtOAc extract of the fungal strain A niger IMBC-NMTP01 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 €ttel and Mu €ller 2007), nigerasperone C (12) (Zhang et al 2007), 2007), orlandin (11) (Hu 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, C14H14O4 was deduced by a protonated molecular ion at m/z 247.0963 [M ỵ H]ỵ (calcd for C14H15O4ỵ, 247.0965) in the HRESITOFMS, implying eight degrees of unsaturation The 1H NMR spectrum (in CDCl3) of showed signals of five aromatic protons at dH 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 dH 5.44 (d, 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 dH 4.60 (dd, J ¼ 4.0, 8.0 Hz, H-6), two germinal-coupled protons at dH 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 dH 3.84 (s, 3-OCH3) The observed 1H NMR data in combination with analysis of the 13C 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 dC 164.2 (C-1), four sp2 carbons 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 H2-7 to C-8, C-9, and C-13 allowed to recognize 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 the 1H and 13C 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 aspergillusol were shown to be opposite in direction [1: ẵa25 D ẳ 61.0 (c ẳ 0.5, CHCl3) vs asper25 gillusol: ẵaD ẳ ỵ35 (c ¼ 0.4, CHCl3)] (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 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]- (calcd for C13H16NO7-, 298.0932) in the HRESITOFMS, along with an analysis of the 1H and 13C NMR data In the 1H NMR spectrum (in DMSO-d6), signals of an 1,4disubstituted benzene ring were observed at dH 7.03 (br d, J ¼ 8.5 Hz, H-50 and H-90 ) and 6.66 (br d, J ¼ 8.5 Hz, H-60 and H-80 ) The 1H 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 dC 163.9 (C-10 ), seven sp2 carbons, including six signals of the para-disubstituted benzene ring [dC 126.5 (C-40 ), 129.7 (C-50 and C-90 ), 115.0 (C-60 and C-80 ), and 155.7 (C-70 )] and one non-protonated carbon bearing nitrogen at dC 150.1 (C-20 ), and one methylene group at dC 29.2 (C-30 ) In the HMBC spectrum, the proton signal at dH 3.72 showed correlations with C-10 , C20 , C-50 , and C-60 , revealing the presence of a tyrosine oxime unit in the molecule (Figure S3) (Ingavat et al 2009; Korwar et al 2016) The 13C NMR additionally showed signals characteristic of a butane-1,2,3,4-tetraol partial structure at dC 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 dH 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 H2-1/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 H2-1 to C-10 Comparison of the 1H and 13C 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 symmetric 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 analogs The carbon chemical shifts of C-20 (dC 150.1) and C-30 (dC 29.2) positions of were closely consistent with those of aspergillusol A [dC 150.5 (C-20 ) and 29.6 (C-30 )] (Ingavat et al 2009) and 2-(hydroxyimino)-phenylpropanoic acid [dC 150.13 (C-20 ) and 29.85 (C-30 )] (Korwar et al 2016) as well as the co-existent analog, 2-(hydroxyimino)-3-(4-hydroxyphenyl)propanoic acid (4), suggesting that the oxime functional NATURAL PRODUCT RESEARCH group of possesses the Z geometry Similarly, the 1H and 13C 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 IC50 values ranging from 9.1 to 23.5 mM, while was cytotoxic against only HepG2 and MCF-7 cells, with IC50 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 metabolites (3-5) were observed against five cancer cell lines, except HepG2, with IC50 values in a range of 58.7 to 88.9 mM Compound 11 was weekly cytotoxic toward MCF-7 and SK-Mel-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 dosedependent 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, L-NMMA (IC50 ¼ 7.6 mM) Nigerasperone A (7) showed a significant NO inhibitory effect (IC50 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 IC50 values ranging from 14.5 to 24.0 mM, whereas compounds 3, 5, and 11 exhibited moderate inhibitory effects, with IC50 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 bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Salmonella enterica ATCC13076), and a yeast strain Candida albicans ATCC 24433 The result indicated 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 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 collected 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 À80  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 containing 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 reversed-phase (RP) C18, eluting with a stepwise gradient of MeOH in H2O 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 separated by silica gel CC, eluting with CH2Cl2-MeOH (7:1, v/v) and further purified by RP C18 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 CH2Cl2-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 (F1.1F1.6) F3.1 was chromatographed over silica gel, using CH2Cl2-MeOH (8:1, v/v) as a mobile phase to give (5 mg) F3.2 was separated by a silica gel CC, using n-hexaneEtOAc (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 subfraction 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, tR ¼ 28.3 min) From subfraction F3.5, compound (6 mg) was isolated by silica gel CC, eluting with CH2Cl2-EtOAc (4:1, v/v) F4 was separated over Sephadex LH-20, using MeOH-H2O (4:1, v/v) as eluent to provide five subfractions (F4.1-F4.5) F4.2 was chromatographed over silica gel, eluting with CH2Cl2-EtOAc (3.5:1, v/v) and further purified by RP C18 prep HPLC, using an 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 CH2Cl2-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 C18 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% CH3CN in H2O (containing 0.1% HCOOH) to give (4 mg, tR ¼ 31.8 min) Similarly, compound 13 (5 mg, tR ¼ 30.6 min) was obtained from the subfraction F4.4.4 by RP C18 prep HPLC, using 45% CH3CN in H2O (containing 0.1% HCOOH) as a mobile phase epi-Aspergillusol (1): white, amorphous powder ẵa25 D ẳ 61.0 (c ẳ 0.5, CHCl3) HRESITOFMS: m/z 247.0963 [M ỵ H]ỵ (calcd for C14H15O4ỵ, 247.0965) ECD (MeOH) mdeg (kmax): 2.13 (283), ỵ0.97 (234), 2.67 (219), ỵ2.09 (205) nm 1H NMR (CDCl3, 500 MHz): dH 5.44 (d, J ¼ 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 H13), 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-OCH3); 13C 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 (C6), 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 (3-OCH3) 1H 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 and H-13), 7.27 (t, J ¼ 8.0 Hz, H-10 and H-12), 7.21 (t, J ¼ 8.0 Hz, H-11), and 3.84 (s, 3-OCH3); 13C NMR (CD3OD, 125 MHz): dC 167.0 (C-1), 88.7 (C2), 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-OCH3) Aspernigin (3): white, amorphous powder ½aŠ25 ẳ ỵ17.0 (c ẳ 0.08, MeOH) D HRESITOFMS: m/z 334.0692 [M ỵ Cl]- (calcd for C13H17NClO7-, 334.0699), m/z 298.0917 [MH]- (calcd for C13H16NO7-, 298.0932) ECD (MeOH) mdeg (kmax): no significant Cotton effect was observed 1H 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, Ha-4), 3.39 (dd, J ¼ 5.5, 11.0 Hz, Hb-4), 3.72 (s, H2-30 ), 7.03 (br d, J ¼ 8.5 Hz, H-50 and H-90 ), and 6.66 (br d, J ¼ 8.5 Hz, H-60 and H-80 ); 13C NMR (DMSO-d6, 125 MHz): dC 67.1 (C-1), 69.3 (C-2), 72.1 (C-3), 62.9 (C-4), 163.9 (C-10 ), 150.1 (C-20 ), 29.2 (C-30 ), 126.5 (C-40 ), 129.7 (C-50 and C-90 ), 115.0 (C-60 and C-80 ), and 155.7 (C-70 ) 1H NMR (CD3OD, 500 MHz): dH 4.45 (dd, J ¼ 3.0, 11.5 Hz, Ha-1), 4.26 (dd, J ¼ 7.0, 11.5 Hz, Hb-1), 3.82 (td, J ¼ 3.0, 7.0 Hz, H-2), 3.59 (m, H-3), 3.77 (dd, J ¼ 3.5, 11.0 Hz, Ha-4), 3.64 (dd, J ¼ 5.5, 11.0 Hz, Hb-4), 3.87 (s, H2-30 ), 7.13 (br d, J ¼ 8.5 Hz, H-50 and H-90 ), and 6.70 (br d, J ¼ 8.5 Hz, H-60 and H-80 ); 13C NMR (CD3OD, 125 MHz): dC 68.4 (C-1), 71.1 (C-2), 73.4 (C-3), 64.5 (C-4), 165.4 (C-10 ), 152.2 (C-20 ), 30.3 (C-30 ), 128.5 (C-40 ), 131.1 (C-50 and C-90 ), 116.2 (C-60 and C-80 ), and 156.9 (C-70 ) 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, 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 compounds, 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 metabolites for the therapeutic uses Acknowledgements The authors are thankful to the Institute of Chemistry, VAST for measurement of the NMR spectra; 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 - NDTP.06/19-21 grant number TD 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 €ffel A, Hemberger Y, Bringmann G 2013 SpecDis: Quantifying the comparison Bruhn T, Schaumlo of calculated and experimental electronic circular dichroism spectra Chirality 25(4):243–249 €ffel A, Hemberger Y, Pescitelli G 2017 SpecDis version 1.71, Berlin, Germany Bruhn T, Schaumlo https:/specdis-softwarejimdocom Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakatsuji H, et al 2016 Gaussian 16 Rev C.01 Wallingford, CT: Gaussian, Inc H€ uttel W, M€ uller M 2007 Regio- and stereoselective intermolecular oxidative phenol coupling in kotanin biosynthesis by Aspergillus niger Chembiochem 8(5):521–529 Ingavat N, Dobereiner J, Wiyakrutta S, Mahidol C, Ruchirawat S, Kittakoop P 2009 Aspergillusol A, an alpha-glucosidase inhibitor from the marine-derived fungus Aspergillus aculeatus J Nat Prod 72(11):2049–2052 €nig GM 2004 New secondary metabolites from the marine Klemke C, Kehraus S, Wright AD, Ko endophytic fungus Apiospora montagnei J Nat Prod 67(6):1058–1063 NATURAL PRODUCT RESEARCH Korwar S, Morris BL, Parikh HI, Coover RA, Doughty TW, Love IM, Hilbert BJ, Royer WE, Kellogg GE, Grossman SR, et al 2016 Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP) Bioorg Med Chem 24(12):2707–2715 Li D-H, Han T, Guan L-P, Bai J, Zhao N, Li Z-L, Wu X, Hua H-M 2016 New naphthopyrones from marine-derived fungus Aspergillus niger 2HL-M-8 and their in vitro antiproliferative activity Nat Prod Res 30(10):1116–1122 Li X-B, Li Y-L, Zhou J-C, Yuan H-Q, Wang X-N, Lou H-X 2015 A new diketopiperazine heterodimer from an endophytic fungus Aspergillus niger J Asian Nat Prod Res 17(2):182–187 Liu D, Li X-M, Meng L, Li C-S, Gao S-S, Shang Z, Proksch P, Huang C-G, Wang B-G 2011 Nigerapyrones A-H, a-pyrone derivatives from the marine mangrove-derived endophytic fungus Aspergillus niger MA-132 J Nat Prod 74(8):1787–1791 Mallavadhani UV, Boddu R, Rathod BB, Reddy Setty P 2018 Stereoselective synthesis of the lichen metabolite,(ỵ) montagnetol and its congeners as antimicrobial agents Synth Commun 48(23):2992–2999 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A 1991 Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines J Natl Cancer Inst 83(11):757–766 Nielsen KF, Mogensen JM, Johansen M, Larsen TO, Frisvad JC 2009 Review of secondary metabolites and mycotoxins from the Aspergillus niger group Anal Bioanal Chem 395(5):1225–1242 O’Boyle NM, Vandermeersch T, Flynn CJ, Maguire AR, Hutchison GR 2011 Confab-systematic generation of diverse low-energy conformers J Cheminform 3(1):8– Palencia ER, Hinton DM, Bacon CW 2010 The black Aspergillus species of maize and peanuts and their potential for mycotoxin production Toxins (Basel)) 2(4):399–416 Priestap HA 1986 13C NMR spectroscopy of naphtho-c-pyrones Magn Reson Chem 24(10): 875–878 Quang TH, Phong NV, Hanh TTH, Cuong NX, Ngan NTT, Oh H, Nam NH, Minh CV 2020 Cytotoxic and immunomodulatory phenol derivatives from a marine sponge-derived fungus Ascomycota sp VK12 Nat Prod Res doi:10.1080/14786419.2020.1786829 Sakurai M, Kohno J, Yamamoto K, Okuda T, Nishio M, Kawano K, Ohnuki T 2002 TMC-256A1 and C1, new inhibitors of IL-4 signal transduction produced by Aspergillus niger var niger TC 1629 J Antibiot (Tokyo)) 55(8):685–692 Schlingmann G, Taniguchi T, He H, Bigelis R, Yang HY, Koehn FE, Carter GT, Berova N 2007 Reassessing the Structure of Pyranonigrin J Nat Prod 70(7):1180–1187 Schuster E, Dunn-Coleman N, Frisvad J, Van Dijck P 2002 On the safety of Aspergillus niger-a review Appl Microbiol Biotechnol 59(4-5):426–435 Shaaban M, Shaaban KA, Abdel-Aziz MS 2012 Seven naphtho-c-pyrones from the marinederived fungus Alternaria alternata: structure elucidation and biological properties Org Med Chem Lett 2(1):6 Varoglu M, Crews P 2000 Biosynthetically diverse compounds from a saltwater culture of sponge-derived Aspergillus niger J Nat Prod 63(1):41–43 Xu K, Yang P-F, Yang Y-N, Feng Z-M, Jiang J-S, Zhang P-C 2017 Direct assignment of the Threo and Erythro configurations in polyacetylene glycosides by 1H NMR spectroscopy Org Lett 19(3):686–689 Zhang Y, Li X-M, Feng Y, Wang B-G 2010 Phenethyl-alpha-pyrone derivatives and cyclodipeptides from a marine algous endophytic fungus Aspergillus niger EN-13 Nat Prod Res 24(11): 1036–1043 Zhang Y, Li X-M, Wang B-G 2007 Nigerasperones A $ C, new monomeric and dimeric naphthoc-pyrones from a marine Alga-derived endophytic fungus Aspergillus niger EN-13 J Antibiot 60(3):204–210 ... DỤC VÀ ĐÀO TẠO VI? ??N HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VN HỌC VI? ??N KHOA HỌC VÀ CƠNG 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... bệnh nhiễm khuẩn ung thƣ Trên sở đó, chúng tơi lựa chọn đề tài : « Nghiên cứu phân lập số hợp chất có hoạt tính gây độc tế bào ung thƣ kháng khuẩn từ chủng vi nấm Aspergillus niger IMBC- NMTP01? ?... 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