曾潔醇,楊 潤,黃 金,李艷平,高 路,汪偉光*
大薊內生真菌sp.DJ-1抗SARS-CoV-2 Mpro活性的化學成分研究
曾潔醇1,楊 潤1,黃 金1,李艷平2,高 路1,汪偉光1*
1. 云南民族大學 國家民委民族藥內生菌天然產物合成生物學重點實驗室,民族醫(yī)藥學院,云南 昆明 650031 2. 云南中醫(yī)藥大學中藥學院,云南 昆明 650500
研究大薊內生真菌sp. DJ-1中抑制新型冠狀病毒主蛋白酶(severe acute respiratory syndrome coronavirus 2 main protease,SARS-CoV-2 Mpro)活性的化學成分。運用新型冠狀病毒Mpro/3CLpro抑制劑篩選模型從30株菌株中篩選具有抑制活性的菌株和化學成分。采用正相硅膠、反相RP-18柱色譜和HPLC等技術對抑制率高的菌株的次生代謝產物進行分離純化,通過NMR、質譜等譜學技術解析化合物結構,利用熒光共振能量轉移法測定化合物對SARS-CoV-2 Mpro的抑制活性。在30株菌株中發(fā)現(xiàn)抑制活性良好的菌株sp. DJ-1,并從該菌的大米發(fā)酵提取物中分離到17個化合物,分別鑒定為德加隆內酯(1)、5-表細鏈格孢烯(2)、細鏈格孢烯(3)、4-表細鏈格孢烯(4)、3-表-二氫細鏈格孢烯A(5)、瓶霉酚(6)、1-脫氧紅藻內酯(7)、6-羥基-8-甲氧基-3a-甲基-3a,9b-二氫-3-呋喃并[3,2-]異色烯-2,5-二酮(8)、黃色籃狀菌酮(9)、鏈格孢毒素II(10)、二氫鏈格孢苝醇(11)、2-(2-羥基丙基)-5-甲基-7-羥基色酮(12)、7-羥基-2-羥甲基-5-甲基-4-色烯-4-酮(13)、三環(huán)鏈格孢霉烯6b(14)、三環(huán)鏈格孢霉烯D(15)、苯乙醇(16)和對羥基苯甲醛(17)?;衔?、9、11對SARS-CoV-2 Mpro的半數(shù)抑制濃度(median inhibition concentration,IC50)分別為42.77、25.85和24.26 μmol/L?;衔?~15和17均為首次從sp.屬中分離得到,化合物5、9、11對SARS-CoV-2 Mpro有較好的抑制作用。通過篩選活性菌株的策略,快速從真菌天然產物中挖掘抗新冠病毒活性的化合物,為抗新冠病毒藥物的研發(fā)提供了參考。
大薊;盾殼霉屬;內生真菌;次生代謝產物;抗病毒活性;新型冠狀病毒主蛋白酶;3-表-二氫細鏈格孢烯A;黃色籃狀菌酮;二氫鏈格孢苝醇
新型冠狀病毒肺炎(corona virus disease 2019,COVID-19)是一種由嚴重急性呼吸綜合征冠狀病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的急性呼吸道傳染病,其傳播速度快和致病率高,對全球衛(wèi)生和人口安全造成了嚴重的威脅。盡管一些抗病毒的藥物,如瑞德西韋、法匹拉韋、莫那匹拉韋等被各國批準用于作為COVID-19的治療藥物[1-5],但由于SARS-CoV-2的高突變性,這些治療藥物對于變異毒株的有效性面臨巨大的挑戰(zhàn)。因此,尋找和開發(fā)抗COVID-19有效藥物仍迫在眉睫。
研究表明,針對冠狀病毒中保守的靶點,研發(fā)抗冠狀病毒活性的抑制劑,可以提高藥物的有效性[6]。主蛋白酶(main protease,Mpro)是冠狀病毒的1個關鍵酶,在病毒復制和轉錄中發(fā)揮重要作用,且保守性高,在人體內沒有同源蛋白,所以Mpro是藥物設計和開發(fā)的理想靶點[7-8]。目前,已有文獻報道一些小分子藥物如奈瑪特韋、依布硒、tideglusib、disulfiram、carmofur、PX-12和紫草素等對SARS-CoV-2 Mpro有很好的抑制作用[8-10],同時在細胞檢測中也顯示了良好的抗病毒活性,這表明以SARS-CoV-2 Mpro作為靶點來篩選藥物是一種有效的策略。不僅如此,中藥在治療COVID-19方面發(fā)揮了巨大的作用,挽救了大量人民群眾的生命[11]。許多來源于傳統(tǒng)中藥的天然產物(銀杏酚酸、胡桃醌及其衍生物、4甲基黃芩素、新刺孢曲霉素A和甘草酸苷等)也紛紛被報道對SARS-CoV-2 Mpro有很強的抑制作用,均被認為是潛在的抗SARS-CoV-2藥物[12-18]。由此可見,從天然產物中發(fā)現(xiàn)具有抗SARS-CoV-2 Mpro活性的化合物,是獲得抗新冠病毒先導化合物的一種有效途徑。
真菌來源的天然產物結構多樣,生物活性顯著,是藥物先導化合物的重要源泉[19-23]。目前有學者針對性地從真菌天然產物中尋找抗新冠病毒活性的化合物,如Pang等[24]從1株青霉屬菌株sp. SCSIO06868.中發(fā)現(xiàn)了1個tetramic acid衍生物對新冠病毒有抑制作用。本課題組在1株真菌sp.中發(fā)現(xiàn)了化合物曲霉狐扁枝衣酮D、M和R對SARS-CoV-2有抑制活性,且能減少SARS-CoV-2引起的炎癥反應[25]。為了從真菌天然產物中尋找更多活性較好的抗SARS-CoV-2藥物先導化合物,本課題組對30株植物內生真菌的大米發(fā)酵提取物進行了活性篩選,發(fā)現(xiàn)有多株菌株對SARS-CoV-2 Mpro有明顯的抑制作用。其中1株大薊Fisch. ex DC.內生真菌sp. DJ-1的組分在40 μg/mL質量濃度下對SARS-CoV-2 Mpro的抑制率達75.0%,提示該菌株代謝物中極有可能存在抗COVID-19活性較好的化合物。為了挖掘該菌株抑制SARS-CoV-2 Mpro活性的化學成分,進一步對sp. DJ-1進行擴大發(fā)酵。綜合運用現(xiàn)代色譜和光譜技術,從sp. DJ-1的大米發(fā)酵粗提物中分離鑒定了17個單體化合物,分別為德加隆內酯(djalonensone,1)、5-表細鏈格孢烯(5-epialtenuene,2)、細鏈格孢烯(altenuene,3)、4-表細鏈格孢烯(4-epialtenuene,4)、3-表-二氫細鏈格孢烯A(3-- dihydroaltenuene A,5)、瓶霉酚(phialophoriol,6)、1-脫氧紅藻內酯(1-deoxyrubralactone,7)、6-羥基-8-甲氧基-3a-甲基-3a,9b-二氫-3-呋喃并[3,2-]異色烯-2,5-二酮(6-hydroxy-8-methoxy-3a-methyl-3a,9b- dihydro-3-furo[3,2-c]isochromene-2,5-dione,8)、黃色籃狀菌酮(talaroflavone,9)、鏈格孢毒素II(altertoxin II,10)、二氫鏈格孢苝醇(dihydro- alterperylenol,11)、2-(2-羥基丙基)-5-甲基-7-羥基色酮[2-(2-hydroxypropyl)-5-methyl-7-hydroxy- chromone,12]、7-羥基-2-羥甲基-5-甲基-4-色烯-4-酮(7-hydroxy-2-hydroxymethyl-5-methyl-4-chromen- 4-one,13)、三環(huán)鏈格孢霉烯6b(tricycloalternarene 6b,14)、三環(huán)鏈格孢霉烯D(tricycloalternarene D,15)、苯乙醇(2-phenylethan-1-ol,16)和對羥基苯甲醛(4-hydroxybenzaldehyde,17)?;钚詼y定發(fā)現(xiàn),化合物5、9和11對SARS-CoV-2 Mpro有較好的抑制作用,其半數(shù)抑制濃度(median inhibition concentration,IC50)分別為42.77、25.85和24.26 μmol/L。
Avance III HD 600 MHz核磁共振波譜儀(瑞士Bruker公司),Acquity UPLC I-Class plus Xevo G2-XS Qtof高分辨液質聯(lián)用儀(美國Waters公司),AUTOPOL V plus高精度旋光儀(美國魯?shù)婪蚬荆琒pectraMax i3x多功能酶標儀(美國Molecular Devices公司),Aglient 1100高效液相色譜儀(美國Aglient公司),Venusil MP-C18柱(250 mm×10 mm,5 μm;美國Aglient公司),LDZX-40B型立式自動電熱蒸汽滅菌鍋(上海博訊實業(yè)有限公司醫(yī)療設備廠),SW-CJ-ZFD型無菌操作臺(蘇凈集團安泰公司),DHP-9082型電熱恒溫培養(yǎng)箱(上海一恒科學儀器有限公司),ZWY-2112B型恒溫培養(yǎng)振蕩器(上海智城分析儀器制造有限公司)。
正相色譜柱硅膠(200~300目,青島譜科公司)、2019-nCoV Mpro/3CLpro抑制劑篩選試劑盒(上海碧云天生物技術有限公司)、依布硒(Ebselen,上海碧云天生物技術有限公司)、大米(秋田小町)、馬鈴薯、葡萄糖、瓊脂、娃哈哈純凈水、乙腈(色譜純)、二甲基亞砜(DMSO)、二氯甲烷、甲醇、石油醚、醋酸乙酯(均為分析純)。
大薊植株采自云南省昆明市呈貢區(qū),經云南民族大學熊勇副教授鑒定為菊科薊屬植物薊Fisch. ex DC.。本實驗所用菌株從新鮮的大薊莖部分離得到,根據(jù)其菌落形態(tài)特征和ITS rDNA序列比對鑒定為盾殼霉屬sp.,登錄號(MH205926.1)。菌株培養(yǎng)在斜面并保存于云南民族大學國家民委民族藥內生菌天然產物合成生物學重點實驗室。
將30株內生真菌用PDA培養(yǎng)基活化后分別接種到PDB培養(yǎng)基中,在28 ℃、200 r/min的條件下培養(yǎng)3 d;將種子液以10%的接種量接種到大米培養(yǎng)基中,在28 ℃組培室靜置培養(yǎng)30 d后,在無菌條件下,分別取出20 g大米發(fā)酵物,用醋酸乙酯超聲萃取2次,每次用100 mL醋酸乙酯,將2次萃取的醋酸乙酯減壓濃縮得到粗提組分。將此粗提組分溶解于DMSO中,終質量濃度為40 μg/mL;利用2019-nCoV Mpro/3CLpro抑制劑篩選試劑盒進行SARS-CoV-2 Mpro活性實驗,通過酶標儀讀取相應的熒光值,計算分析抑制效果,以此篩選出對SARS-CoV-2 Mpro抑制效果最好的活性菌株。
菌株sp. DJ-1用PDA平板活化,將長滿菌落的平板切成1 mm×1 mm×1 mm的小塊,以5%的接種量加入到10瓶PDB培養(yǎng)基中,每瓶體積為200 mL,在28 ℃、200 r/min條件下培養(yǎng)3 d,此10瓶作為大量發(fā)酵的種子液。配制100瓶大米培養(yǎng)基(大米80 g、珍珠巖10 g、100 mL純凈水),高溫滅菌備用。將10瓶種子液以10%的接種量加入到100瓶大米培養(yǎng)基中,于28 ℃組培室靜置培養(yǎng)30 d。
采用醋酸乙酯對菌株sp. DJ-1的大米發(fā)酵物超聲提取,每次20 L,提取4次。將提取液減壓濃縮,得到78 g總浸膏;經正相硅膠柱色譜,二氯甲烷-甲醇(1∶0→1∶1)梯度洗脫,合并相同流分得到9個組分(Fr. 1~9)。Fr. 2(7.5 g)經正相硅膠柱色譜,石油醚-醋酸乙酯(30∶1→0∶1)洗脫,得到5個亞組分(Fr. 2-1~2-5)。Fr. 2-3經半制備HPLC(乙腈-水47∶53)得到化合物16(3.1 mg);Fr. 2-5經半制備HPLC(乙腈-水58∶42)得到化合物7(6.1 mg)和8(33 mg)。Fr. 4(6.0 g)經正相硅膠柱色譜,石油醚-醋酸乙酯(10∶1→1∶1)洗脫,得到7個亞組分,其中Fr. 4-2經半制備HPLC(乙腈-水38∶62)得到化合物17(14.2 mg);Fr. 4-5經半制備HPLC(乙腈-水67∶33)得到化合物1(26.1 mg)、6(3.4 mg)、10(31.0 mg)和15(10.5 mg)。Fr. 6(5.8 g)經正相硅膠柱色譜,石油醚-醋酸乙酯(5∶1→0∶1)洗脫,得到6個亞組分,其中Fr. 6-4經半制備HPLC(乙腈-水52∶48)得到化合物11(6.0 mg);Fr. 6-5經半制備HPLC(乙腈-水45∶55)得到化合物2(3.3 mg)、3(30.3 mg)和9(4.2 mg)。Fr. 7(1.1 g)經RP-18柱色譜并結合半制備HPLC純化,得到化合物4(7.4 mg)、5(4.0 mg)、12(7.6 mg)、13(2.0 mg)和14(2.5 mg)。
在熒光共振能量轉移的基礎上,通過2019-nCoV Mpro/3CLpro抑制劑篩選試劑盒(增強型)對SARS-CoV-2 Mpro進行抑制實驗。將所有待測化合物溶解于DMSO中,備用。設空白組、100%酶活性組、陽性對照組和給藥組。在黑色的96孔板中,各組分別加入90 μL的Assay Buffer,100%酶活性組、陽性對照組和給藥組加入1 μL SARS-CoV-2 Mpro、空白組補加1 μL Assay Buffer,空白組和100%酶活性組各加入5 μL DMSO,陽性對照組和給藥組分別加入5 μL的陽性藥物依布硒(Ebselen)和待測化合物溶液,混勻。在37 ℃恒溫避光孵育30 min后,轉移至冰上冷卻10 min,再快速加入4 μL底物,混勻。然后,在SpectraMax i3x多功能酶標儀上讀取相對熒光單元(relative fluorescence unit,RFU),激發(fā)波長為325 nm,發(fā)射波長為393 nm。按照公式計算各化合物對SARS-CoV-2 Mpro的抑制率。
抑制率=(RFU100%酶活性-RFU樣品)/(RFU100%酶活性-RFU空白)
采用2019-nCoV-Mpro/3CLpro抑制劑篩選模型對30株菌株的大米發(fā)酵組分進行活性篩選,結果見表1。根據(jù)結果可以判斷出在這30株菌株中,有9株菌的抑制率≥60.0%,其中菌株sp. DJ-1的抑制效果是最好的,抑制率為75.0%。
表1 30株內生真菌的來源和SARS-CoV-2 Mpro抑制活性
“–”表示無抑制作用
“–”means no inhibition activity
化合物1:白色粉末。HR-ESI-MS/273.075 8 [M+H]+(計算值C15H13O5,273.075 7),確定分子式為C15H12O5。1H-NMR (600 MHz, CD3COCD3): 11.98 (1H, s, 3-OH), 9.27 (1H, s, 4-OH), 7.31 (1H, d,= 2.2 Hz, H-6), 6.80 (1H, m, H-5), 6.71 (1H, d,= 2.7 Hz, H-3), 6.58 (1H, d,= 2.2 Hz, H-4), 3.98 (3H, s, 5-OCH3), 2.80 (3H, s, 6-CH3);13C-NMR (150 MHz, CD3COCD3): 167.6 (C-5), 166.1 (C-7), 166.0 (C-3), 159.4 (C-4), 154.1 (C-2), 139.7 (C-6), 139.1 (C-1), 118.4 (C-5), 110.6 (C-1), 104.6 (C-6), 102.7 (C-3), 99.9 (C-4), 99.8 (C-2), 56.3 (5-OMe), 25.6 (6-Me)。以上數(shù)據(jù)與文獻報道的數(shù)據(jù)基本一致[26],故鑒定化合物1為德加隆內酯。
化合物7:白色粉末。HR-ESI-MS/261.075 8 [M+H]+(計算值C14H13O5,261.075 7),確定它的分子式為C14H12O5。1H-NMR (600 MHz, CDCl3): 11.32 (1H, s, 6-OH), 6.68 (2H, t,= 1.7 Hz, H-7, 9), 3.93 (3H, s, 8-OCH3), 3.43 (1H, td,= 6.8, 1.3 Hz, H-1), 2.94 (1H, dd,= 19.0, 6.5 Hz, H-2α), 2.30 (1H, dd,= 18.9, 1.3 Hz, H-2β), 1.45 (3H, d,= 7.0 Hz, 1-CH3);13C-NMR (150 MHz, CDCl3): 195.4 (C-3), 167.0 (C-8), 165.4 (C-6), 165.0 (C-5), 148.3 (C-3a), 144.8 (C-10a), 134.6 (C-9a), 103.4 (C-7), 103.2 (C-9), 100.9 (C-5a), 56.2 (8-OCH3), 42.9 (C-2), 28.5 (C-1), 21.1 (CH3-1)。以上數(shù)據(jù)與文獻報道基本一致[31],故鑒定化合物7為1-脫氧紅藻內酯。
化合物9:黃色固體。HR-ESI-MS/277.071 6 [M+H]+(計算值C14H13O6,277.070 6),確定它的分子式為C14H12O6。1H-NMR (600 MHz, CDCl3): 6.49 (1H, d,= 1.8 Hz, H-4), 6.33 (1H, d,= 1.6 Hz, H-6), 6.07 (1H, d,= 1.8 Hz, H-3), 4.82 (1H, s, H-5), 3.82 (3H, s, 5-OCH3), 1.92 (3H, d,= 1.5 Hz, 2-CH3);13C-NMR (150 MHz, CDCl3): 199.9 (C-4), 170.6 (C-2), 170.4 (C-1), 167.8 (C-5), 158.2 (C-7), 147.1 (C-3a), 130.0 (C-3), 104.5 (C-7a), 102.2 (C-4), 101.4 (C-6), 93.7 (C-3), 78.7 (C-5), 56.3 (5-OCH3), 13.9 (2-CH3)。以上數(shù)據(jù)與文獻報道基本一致[31],因此鑒定化合物9為黃色籃狀菌酮。
化合物11:紅色粉末。HR-ESI-MS/351.088 3 [M-H]?(計算值C20H15O6,351.087 4),確定它的分子式為C20H16O6。1H-NMR (600 MHz, DMSO-6): 12.70 (1H, s, 3-OH), 12.29 (1H, s, 10-OH), 8.03 (1H, d,= 8.8 Hz, H-1), 7.97 (1H, d,= 8.7 Hz, H-12), 7.01 (1H, d,= 8.7 Hz, H-2), 6.91 (1H, d,= 8.6 Hz, H-11), 5.36 (1H, s, 7-OH), 5.26 (1H, s, 6a-OH), 4.54~4.43 (1H, m, H-7), 3.05 (1H, dd,= 17.3, 14.4 Hz, H-6b), 3.02 (1H, m, H-5ax), 2.98 (1H, m, H-5eq), 2.92 (1H, d,= 8.3 Hz, H-8ax) 2.83 (1H, dd,= 15.8, 4.6 Hz, H-8eq), 2.59~2.50 (1H, m, H-6ax), 2.27 (1H, td,= 14.3, 3.9 Hz, H-6eq);13C- NMR (150 MHz, DMSO-6): 206.1 (C-4), 204.2 (C-9), 161.0 (C-3), 160.4 (C-10), 140.7 (C-9b), 138.4 (C-12c), 132.9 (C-1), 132.6 (C-12), 124.8 (C-12a), 123.5 (C-12b), 117.9 (C-2), 116.5 (C-9a), 115.6 (C-11), 113.8 (C-3a), 68.0 (C-6a), 64.7 (C-7), 51.4 (C-6b), 47.5 (C-8), 34.8 (C-6), 33.5 (C-5)。以上數(shù)據(jù)與文獻報道基本一致[34],故鑒定化合物11為二氫鏈格孢苝醇。
化合物12:白色粉末。HR-ESI-MS/235.096 7 [M+H]+(計算值C13H15O4,235.096 4),確定它的分子式為C13H14O4。1H-NMR (600 MHz, CD3OD): 6.62 (1H, d,= 2.5 Hz, H-6), 6.60 (1H, dd,= 2.4, 1.1 Hz, H-8), 6.03 (1H, s, H-3), 4.16 (1H, m, H-2), 2.68 (3H, s, 5-CH3), 2.63 (2H, m, H-1), 1.25 (3H, d,= 6.3 Hz, 2-CH3);13C-NMR (150 MHz, CD3OD): 182.1 (C-4), 167.2 (C-2), 163.7 (C-7), 161.7 (C-9), 143.7 (C-5), 118.4 (C-3), 115.8 (C-10), 112.6 (C-6), 101.9 (C-8), 66.5 (C-2), 44.4 (C-1), 23.7 (2-CH3), 23.3 (CH3-5)。以上數(shù)據(jù)與文獻報道基本一致[35],故鑒定化合物12為2-(2-羥基丙基)-5-甲基-7-羥基色酮。
化合物13:白色粉末。HR-ESI-MS/207.065 3 [M+H]+(計算值C11H11O4,207.065 1),確定它的分子式為C11H10O4。1H-NMR (600 MHz, CD3OD): 6.66~6.62 (2H, m, H-6, 8), 6.22 (1H, d,= 1.1 Hz, H-3), 4.43 (2H, d,= 1.0 Hz, 2-CH2OH), 2.71 (3H, s, 5-CH3);13C-NMR (150 MHz, CD3OD): 182.1 (C-4), 168.7 (C-2), 163.9 (C-7), 161.3 (C-8a), 143.8 (C-5), 118.4 (C-6), 115.8 (C-4a), 109.1 (C-3), 101.8 (C-8), 61.3 (2-CH2OH), 23.2 (5-CH3)。以上數(shù)據(jù)與文獻報道基本一致[36],故鑒定化合物13為7-羥基-2-羥甲基- 5-甲基-4-色烯-4-酮。
化合物16:無色油狀物。HR-ESI-MS/145.062 6 [M+Na]+(計算值C8H10ONa,145.062 3),確定它的分子式為C8H10O。1H-NMR (600 MHz, CDCl3): 7.35~7.29 (2H, m, H-4), 7.24 (3H, m, H-2, 3, 5, 6), 3.87 (2H, td,= 6.6, 1.4 Hz, H-8), 2.88 (2H, dd,= 7.2, 5.9 Hz, H-7);13C-NMR (150 MHz, CDCl3): 138.6 (C-1), 129.2 (C-3, 5), 128.7 (C-2, 6), 126.6 (C-4), 63.9 (C-8), 39.3 (C-7)。以上數(shù)據(jù)與文獻報道的數(shù)據(jù)基本一致[39],故鑒定化合物16為苯乙醇。
化合物17:淺黃色粉末。HR-ESI-MS/123.043 4 [M+H]+(計算值C7H7O2,123.044 0),確定它的分子式為C7H6O2。1H-NMR (600 MHz, CD3OD): 9.75 (1H, s, H-7), 7.79 (2H, d,= 8.0 Hz, H-2, 6), 6.93 (2H, d,= 8.0 Hz, H-3, 5);13C-NMR (150 MHz, CD3OD): 193.0 (C-7), 165.7 (C-4), 133.6 (C-2, 6), 130.3 (C-1), 117.1 (C-3, 5)。以上數(shù)據(jù)與文獻報道的數(shù)據(jù)基本一致[40],故鑒定化合物17為對羥基苯甲醛。
以依布硒(1 μmol/L)為陽性對照,所有化合物在40 μmol/L的濃度下對SARS-CoV-2 Mpro抑制活性如圖1所示??梢园l(fā)現(xiàn)化合物1、4、6、13、16和17對SARS-CoV-2 Mpro基本無抑制作用(抑制率<10%),化合物2、3、7、8、10、12、14和15有一定抑制作用(抑制率20%~45%),而化合物5、9、11有較好的抑制作用(抑制率>48%),對化合物5、9和11進一步進行濃度梯度測試,得到IC50值分別為42.77、25.85和24.26 μmol/L。
圖1 化合物1~17對SARS-CoV-2 Mpro的抑制作用
本實驗通過聚焦大量內生真菌菌株抗SARS-CoV-2活性篩選,導向發(fā)現(xiàn)大薊內生真菌sp. DJ-1對SARS-CoV-2 Mpro有較好的抑制作用,并進一步對該菌抗SARS-CoV-2活性的化學成分進行研究。從sp. DJ-1菌株的大米發(fā)酵提取物中分離鑒定了17個化合物,其中化合物1~15和17均為首次從屬菌株中分離得到。通過活性實驗發(fā)現(xiàn)了該菌次生代謝產物中的主要活性成分是化合物5、9和11,IC50值分別為42.77、25.85和24.26 μmol/L。此外,本研究發(fā)現(xiàn)化合物11是鏈格孢毒素類化合物,曾被報道在2.2 μmol/L時能完全抑制HIV-1的復制[33],這意味著化合物11可能具有廣譜抗病毒活性。本研究表明,通過大量內生真菌菌株的篩選并結合活性跟蹤導向分離策略,可以提高抗COVID-19活性化合物發(fā)現(xiàn)的效率。此外,也為植物內生真菌的應用提供了參考。
利益沖突 所有作者均聲明不存在利益沖突
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Chemical constituents with potential anti-SARS-CoV-2 Mproactivity from endophytic fungussp. DJ-1 of
ZENG Jie-chun1, YANG Run1, HUANG Jin1, LI Yan-ping2, GAO Lu1, WANG Wei-guang1
1. Key Laboratory of Natural Products Synthetic Biology of Ethnic Medicinal Endophytes, State Ethnic Affairs Commission, School of Ethnic Medicine, Yunnan Minzu University, Kunming 650031, China 2. College of Traditional of Chinese Medicine, Yunnan University of Chinese Medicine, Kunming 650500, China
To study the chemical constituents with severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 Mpro) inhibitory activity fromsp. DJ-1, an endophytic fungus isolated from.The strains and chemical constituents both with inhibitory activity were screened from thirty endophytic fungi by using 2019-nCoV Mpro/3CLproinhibitor screening model. The secondary metabolites of the strain with high inhibition rate were separated and purified by normal phase silica gel column chromatography, reversed-phase RP-18 column chromatography, and HPLC. Their structures were identified by NMR, MS, and optical rotation data. And the compounds’ anti-SARS-CoV-2 Mproactivities were determined by fluorescence resonance energy transfer method.Thesp. DJ-1 strain with good inhibitory activity was screened from thirty endophytic fungi. Seventeen compounds were isolated from the rice fermentation extract ofsp. DJ-1, and identified as djalonensone (1), 5-epialtenuene (2), altenuene (3), 4-epialtenuene (4), 3--dihydroaltenuene A (5), phialophoriol (6), 1-deoxyrubralactone (7), 6-hydroxy-8-methoxy-3a-methyl-3a,9b-dihydro-3- furo[3,2-]isochromene-2,5-dione (8), talaroflavone (9), altertoxin II (10), dihydroalterperylenol (11), 2-(2-hydroxypropyl)-5- methyl-7-hydroxychromone (12), 7-hydroxy-2-hydroxymethyl-5-methyl-4-chromen-4-one (13), tricycloalternarene 6b (14), tricycloalternarene D (15), 2-phenylethan-1-ol (16) and 4-hydroxybenzaldehyde (17). Compounds 5, 9 and 11 exhibited potent inhibitory effects on SARS-CoV-2 Mprowith IC50values of 42.77, 25.85 and 24.26 μmol/L, respectively.Compounds 1—15 and 17 have been isolated from the extracts ofsp. genus for the first time. Compounds 5, 9 and 11 exhibit potent inhibitory effects on SARS-CoV-2 Mpro. The research concludes that the method of screening active strains is helpful for us to quickly excavate active compounds from natural fungal products, moreover provides a basis for the research and development of drugs for COVID-19.
Fisch. ex DC.;sp.; endophytic fungus; secondary metabolites; antiviral activity; SARS-CoV-2 Mpro; 3--dihydroaltenuene A; talaroflavone; dihydroalterperylenol
R284.1
A
0253 - 2670(2023)10 - 3071 - 09
10.7501/j.issn.0253-2670.2023.10.004
2023-01-24
國家自然科學基金項目(31960095);國家自然科學基金項目(82160670);云南省應用基礎項目(202101AS070022,202201AT070228);云南省萬人計劃-青年拔尖人才(W.-G. Wang)
曾潔醇(1997—),女,碩士研究生,研究方向為天然產物化學。E-mail: mzengjiechun@163.com
汪偉光(1984—),男,研究員,碩士生導師,研究方向為天然藥物化學。E-mail: wwg@live.cn
[責任編輯 王文倩]