Anti-Influenza Virus (H5N1) Activity Screening on the Phloroglucinols

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Article Anti-Inﬂuenza Virus (H5N1) Activity Screening on the Phloroglucinols from Rhizomes of Dryopteris crassirhizoma

Juan Wang 1, Yan-Tao Yan 1, Shen-Zhen Fu 1, Bing Peng 2, Lin-Lin Bao 3, Yan-Ling Zhang 1, Jing-Hong Hu 4, Zu-Ping Zeng 2, Dong-Hao Geng 5 and Zeng-Ping Gao 1,*

1 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China; [email protected] (J.W.); [email protected] (Y.-T.Y.); [email protected] (S.-Z.F.); [email protected] (Y.-L.Z.) 2 Beijing Institute of Traditional Chinese Medicine, Beijing Hospital of Traditional Chinese Medicine Afﬁliated to Capital Medical University, Beijing 100000, China; [email protected] (B.P.); [email protected] (Z.-P.Z.) 3 Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS&PUMC), Beijing 100730, China; [email protected] 4 School of Basic Medical Science, Beijing University of Chinese Medicine, Beijing 100029, China; [email protected] 5 College of Pharmacy, China Pharmaceutical University, Nanjing 211198, China; [email protected] * Correspondence: [email protected]; Tel.: +86-10-8473-8628

Academic Editor: Thomas J. Schmidt Received: 6 December 2016; Accepted: 2 March 2017; Published: 8 March 2017

Abstract: For screening the active phloroglucinols on influenza virus (H5N1) from Dryopteris crassirhizoma NaKai, a database was established including twenty-three phloroglucinols that had been isolated from Dryopteris crassirhizoma. Their inhibitory effect on the neuraminidase (NA) of influenza virus H5N1 was screened by molecular docking. As a result, three candidates were selected. The rhizomes of D. crassirhizoma were subjected to isolation and purification processes to obtain the inhibitor candidates. Thirteen phloroglucinols were obtained, including three selected candidates and two new phloroglucinols. The five phloroglucinols were investigated for their inhibitory activity on NA in vitro. The results showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory effects on NA with IC50 as 18.59 ± 4.53 and 29.57 ± 2.48 µM, respectively, and the other three phloroglucinols showed moderate inhibitory activity. Moreover, the anti-influenza virus (H5N1) activity and cytotoxicity of dryocrassin ABBA and filixic acid ABA were tested on Madin-Darby canine kidney (MDCK) cells with the cell counting kit-8 (CCK8) method. The results confirmed that dryocrassin ABBA exhibited an inhibitory activity with low cytotoxicity (TC50 > 400 µM) against influenza virus (H5N1) which will have to be investigated in further detail. In conclusion, phloroglucinols from D. crassirhizoma were shown to have anti-influenza virus activity, and especially dryocrassin ABBA, one of the phloroglucinols, may have the potential to control influenza virus (H5N1) infection.

Keywords: Dryopteris crassirhizoma; phloroglucinols; inﬂuenza virus H5N1; neuraminidase; dryocrassin ABBA

1. Introduction Highly pathogenic avian influenza virus (HPAIV), subtype H5N1, is of great concern for infecting and causing an inordinate amount of lethal disease in humans that could evolve into a pandemic risk [1]. Human H5N1 influenza infection was first recognized in 1997 when this virus infected

Molecules 2017, 22, 431; doi:10.3390/molecules22030431 www.mdpi.com/journal/molecules Molecules 2017, 22, 431 2 of 16

18 people in Hong Kong, causing six deaths [2,3]. According to the World Health Organization, a total of 665 people were infected with the H5N1 virus from 2003 to 2014, resulting in 392 deaths, with a mortality rate of 58.9%. Vaccine is an effective measure for preventing infection, but virus mutations are abrupt and rapid, so vaccines would be developed too late to be used for prevention during the more severe phases of recent pandemics. Amantadine, rimantadine, oseltamivir, and zanamivir have been approved by the United States Food and Drug Administration for the treatment of pandemic influenza. Studies have demonstrated that many of the avian influenza H5N1 viruses were resistant to both amantadine and rimantadine [4]. In addition, an H5N1 virus that was resistant to the drug oseltamivir has been isolated by Le et al. [5]. Therefore, much attention has been paid to Chinese herbs as potential sources for the discovery of new anti-influenza compounds with high efficacy and low toxicity. D. crassirhizoma (DC) is widely distributed in the northeast of China and rhizome has been traditionally used for treating various inflammatory [6–9] and infectious diseases [10,11]. Many studies demonstrated that phloroglucinols from DC had a wide range of pharmacological effects, such as antibacterial [12], anti-influenza virus [13], anti-tumor promoting [14,15], anti-reverse transcriptase [16], and antioxidant activities [17]. Furthermore, flavaspidic acid AB, one of the phloroglucinols from this plant, was able to inhibit replication of the porcine reproductive and respiratory syndrome virus (PRRSV) [18]. Our pilot study suggested that the extract fraction of this plant that was rich in phloroglucinols could reduce mortality and decrease lung index (dry lung-to-body weight ratio) of mice inoculated with avian influenza virus (H5N1). Dryocrassin ABBA was isolated from this fraction, which could protect mice infected with avian influenza virus via inhibiting inflammation and reducing viral loads [19]. Therefore, anti-influenza virus activity screening on phloroglucinols was carried out as a part of our program exploring the most active phloroglucinol from DC. In this report, virtual screening with molecular docking, isolation, structural characterization, and the activity on neuraminidase (NA) and influenza virus in vitro are described.

2. Results and Discussion

2.1. Molecular Docking Twenty-three phloroglucinols that had been isolated from DC were selected from the databases of CNKI and Web of Science (Table S1). To determine the probable binding conformations of the phloroglucinols with NA, Autodock Vina was used to dock them into the active site of NA (N1 Neuraminidase H274Y, PDB ID: 3CL0), isolated from H5N1-infected patients that have been reported to be oseltamivir-resistant [20]. The docking reliability was studied using the known X-ray structure of NA complexed with oseltamivir. As shown in Figure1, the low root mean-square deviation (RMSD) was 1.12 Å between the docked and the crystal conformation of oseltamivir, which indicated the high reliability of AutoDock Vina in reproducing the experimentally observed binding mode for the NA inhibitor. The results exhibited that the minimum binding energy of the twenty-three phloroglucinols ranged from –6.3 to –8.3 kcal/mol. The lowest binding energy of the protein-ligand complex was considered as the best [21]. Hence, the binding conformations of twenty-three phloroglucinols with a docking score ≤−8.0 were chosen. Finally, ten phloroglucinols were selected with the lowest docking energies of the protein-ligand complex (Table1). It has been reported that there was a ‘150-cavity’ adjacent to the active site of NA (PDB ID: 3CL0, H5N1), which was proven as an anti-influenza drug target to develop new NA inhibitors [22]. The binding modes of ten phloroglucinols with NA showed that trimeric and tetrameric phloroglucinols filixic acid ABA, ABB, and dryocrassin ABBA could dock into both the active site and the ‘150-cavity’ of NA. The other seven monomeric or dimeric phloroglucinols could only dock into the active site (Figure2A,B). Moreover, filixic acid ABA, ABB, and dryocrassin ABBA formed several hydrogen bonds and hydrophobic interactions with the key residues Arg118, Arg152, Arg292, Arg371, Glu119, and Glu227 in the active site and ‘150-cavity’ (Figure3A–C). Therefore, filixic acid ABA, filixic acid ABB, and dryocrassin ABBA may have the potential to inhibit NA of the H5N1 virus. Molecules 2017, 22, 431 3 of 16 Molecules 2017, 22, 431 3 of 16 Molecules 2017, 22, 431 3 of 16 Molecules 2017, 22, 431 3 of 16

Figure 1. Co-crystallized (green) and redocked (pink) oseltamivir. The reliability of molecular docking Figure 1. Co-crystallized(green) (green) and and redocked redocked (pink) (pink) oseltamivir. oseltamivir. The The reliability reliability of molecular of molecular docking docking was Figurewas validated 1. Co-crystallized using the known (green) X-ray and structureredocked of (pink) neuraminidase oseltamivir. (NA) The complexed reliability of with molecular oseltamivir. docking The validatedwas validated using using the known the known X-ray X-ray structure structure of neuraminidase of neuraminidase (NA) (NA) complexed complexed with oseltamivir. with oseltamivir.The result The wasresult validated demonstrated using the that known the low X-ray root structure mean-square of ne uraminidasedeviation (RMSD) (NA) complexed was 1.12 Å with between oseltamivir. the docked The demonstratedresult demonstrated that the that low the root low mean-square root mean-square deviation deviation (RMSD) (RMSD) was 1.12was Å1.12 between Å between the docked the docked and resultand the demonstrated crystal conformation that the lowof oseltamivir, root mean-square which indicated deviation the (RMSD) high reliability was 1.12 ofÅ AutoDockbetween the Vina. docked theand crystalthe crystal conformation conformation of oseltamivir, of oseltamivir, which which indicated indicated the the high high reliability reliability of AutoDockof AutoDock Vina. Vina. and the crystal conformation of oseltamivir, which indicated the high reliability of AutoDock Vina.

Figure 2. Binding modes of trimeric, tetrameric phloroglucinols (A) and monomeric, dimer Figure 2. Binding modes of trimeric, tetrameric phloroglucinols (A) and monomeric, dimer Figurephloroglucinols 2.2. BindingBinding (B) inmodes modes the active of of trimeric, trimeric,site and ‘150-cavity’tetrameric tetrameric ofphloroglucinols NA. ( A)) and monomeric,monomeric, dimer phloroglucinols (B) in the active site and ‘150-cavity’ of NA. phloroglucinols (B)) inin thethe activeactive sitesite andand ‘150-cavity’‘150-cavity’ ofof NA.NA.

Figure 3. Cont.

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Figure 3. Binding modes of dryocrassin ABBA ( A); filixic ﬁlixic acid ABA ( B) and filixic ﬁlixic acid ABB (C) with key residues of NA.

TableTable 1. Vina energy scores of the screened ten phloroglucinolsphloroglucinols boundbound toto NA.NA. Compound MF MW Affinity (Kcal/mol) Compound MF MW Affinity (Kcal/mol) Dryofragin C28H38O5 454 −8.0 Dryofragin C H O 454 −8.0 Albaspidin AA 28 C2138H245 O8 404 −8.0 Albaspidin AA C21H24O8 404 −8.0 Albaspidin AB C23H28O8 432 −8.1 Albaspidin AB C23H28O8 432 −8.1 22 26 8 AlbaspidinAlbaspidin AP AP C22 CH26HO8 O 418418 −−8.08.0 FlavaspidicFlavaspidic acid acid AB AB C22 CH2226HO268 O8 418418 −−8.28.2 − FlavaspidicFlavaspidic acid acid PB PB C23 CH2328HO288 O8 432432 −8.18.1 Norflavaspidic acid AB C21H24O8 404 −8.0 Norflavaspidic acid AB C21H24O8 404 −8.0 Filixic acid ABA C32H36O12 612 −8.0 Filixic acid ABA C32H36O12 612 −8.0 Filixic acid ABB C32H36O12 640 −8.1 DryocrassinFilixic acid ABBA ABB C43 CH3248HO3616O12 820640 −−8.38.1 Dryocrassin ABBA C43H48O16 820 −8.3 2.2. Isolation and Structure Characterization 2.2. Isolation and Structure Characterization Compound 7 was obtained as a yellowish amorphous powder which showed a positive response (pinkCompound spot) to fast 7 bluewas obtained BB salt (4-benzoylamino-2,5-diethoxy-benzenediazonium as a yellowish amorphous powder which showed a salt) positive spray response reagent on(pink a silica spot)gel to fast Thin blue Layer BB chromatographysalt (4-benzoylamino-2,5-diethoxy-benzenediazonium (TLC) plate. The characteristic UV absorptions salt) spray werereagent at 242on a and silica 286 gel nm. Thin The Layer absorption chromatography bands in the (TLC) IR spectrumplate. The showed characteristic the presence UV absorptions of hydroxyl were group(s) at 242 (3216and 286 cm nm.−1), The carbonyl absorption group(s) bands (1612 in the cm −IR1), spectr phenylum group(s) showed (1395,the presence 1445, 1556 of hydroxyl cm−1) and group(s) alkyl group(s)(3216 cm− (29611), carbonyl cm−1 ).group(s) The quasi-molecular (1612 cm−1), phenyl ion in group(s) negative (1395, high 1445, resolution 1556 cm electrospray−1) and alkyl ionization group(s) mass(2961 spectroscopycm−1). The quasi-molecular (HRESIMS) of 7 ionat m in/z negative833.3004 [Mhigh− H]resolution− (calcd forelectrospray 833.3015) correspondedionization mass to − thespectroscopy molecular (HRESIMS) formula of Cof44 7H 50atO m16/z. The833.3004 characteristic [M − H] resonances (calcd for for833.3015) hydroxyl corresponded protons (δH to10.14, the molecular formula of C44H50O16. The characteristic resonances for hydroxyl protons (δH 10.14, 11.75, 11.75, 16.09, 18.54, etc.), a number of aromatic quaternary carbons (Table2), and methylenes at δH/C 3.57/17.416.09, 18.54, (C-7,7 etc.),0)) a number and 3.85/17.1 of aromatic (C-14) quaternary in the 1H- carbons and 13C-NMR (Table 2), spectra and methylenes suggested at the δH/C tetrameric 3.57/17.4 phloroglucinol(C-7,7′)) and 3.85/17.1 skeleton (C-14) [23 ,in24 ].the13 1H-C-NMR and 13C-NMR showed spectra 25 signals, suggested which the suggested tetrameric that phloroglucinol structure of 13 compoundskeleton [23,24].7 had symmetry.C-NMR showed The resonances 25 signals, at whichδH 3.85 suggested (2H, H-14) that are structure characteristic of compound for methylenes 7 had H 0 0 situatedsymmetry. between The resonances two phloroglucinol at δ 3.85 (2H, units H-14) (ring are B/B characteristic)); δH 3.57 (4H, for methylenes H-7,7 )) are situated characteristic between for methylenestwo phloroglucinol situated betweenunits (ring hexadioldienone B/B′)); δH 3.57 (ring (4H, A/A H-7,70)) and′)) phloroglucinol are characteristic (ring B/Bfor 0methylenes)) units [23]. 0 0 0 Thesituated HMBC between correlations hexadioldienone (Figure4) of(ring H-7,7 A/A)(′))δ Hand3.57) phloroglucinol with C-1,1 )( (ringδC 188.1, B/B′)) 188.0), units C-5,5[23]. The)(δ CHMBC172.8, 0 H 0 C 0 C 0 172.5),correlations C-6,6 (Figure)(δH 111.2, 4) of H-7,7 111.1),′) ( C-8,8δ 3.57))(δ withC 105.6, C-1,1 105.3),′) (δ 188.1, C-9,9 188.0),)(δC 160.5), C-5,5′) and (δ 172.8, C-13,13 172.5),)(δC C-6,6158.7)′) confirmed(δH 111.2, 111.1), the positions C-8,8′) ( ofδC methylenes105.6, 105.3), between C-9,9′) ( theδC 160.5), ringsA and and C-13,13 B, and′) A(δ0)C and158.7) B 0).confirmed The HMBC the 0 0 0 correlationspositions of methylenes of H-14 (δC between3.85) with the C-12,12 rings A)( andδC 106.7), B, and C-13,13A′) and )(B′δ).C The158.7), HMBC and correlations C-11,11 )(δ Cof159.7) H-14 confirmed(δC 3.85) with the C-12,12 position′) of(δC methylene 106.7), C-13,13 between′) (δC the 158.7), rings and B and C-11,11 B0). NMR′) (δC 159.7) spectral confirmed data (Table the2 position) of this compoundof methylene resembled between thatthe rings of dryocrassin B and B′). ABBA NMR [spectral25,26], suggesting data (Table they 2) of have this thecompound same skeleton. resembled that of dryocrassin ABBA [25,26], suggesting they have the same skeleton.

Molecules 2017, 22, 431 5 of 16

Table 2. 1H- and 13C-NMR spectroscopic data for compounds 7 and 8 in CDCl3.

Compound 7 Compound 8 Molecules 2017, 22, 431Position 5 of 16 δH δC δH δC 1 (1′) 188.1 (188.0) 188.0 1 13 Table2 2. (2H-′) and C-NMR spectroscopic108.2 (108.1) data for compounds 7 and 108.28 in CDCl 3. 3 (3′) 18.54 (OH) 199.2 (198.5) 18.52 (OH) 199.3 4 (4′) Compound44.5 (44.3) 7 Compound 44.5 8 Position 5 (5′) 9.71 (OH)δH 172.8 (172.5)δC 9.80 (OH)δH 172.8 δC 1 (16 0(6)′) 111.2 188.1 (111.1) (188.0) 111.2 188.0 0 2 (27 (7)′) 3.57 s 108.217.4 (108.1) 3.57 s 17.4 108.2 3 (30) 18.54 (OH) 199.2 (198.5) 18.52 (OH) 199.3 8 (8′) 105.6 (105.3) 105.7 4 (40) 44.5 (44.3) 44.5 5 (59 0(9)′) 16.09 9.71 (OH) (OH) 160.5 172.8 (172.5)16.10 9.80 (OH) (OH) 160.6 172.8 610 (6 0(10)′) 158.9 111.2 (111.1) 158.9 111.2 0 711 (7 (11)′) 11.75 3.57(OH) s 159.7 17.4 11.73 (OH) 3.57 s 159.7 17.4 8 (80) 105.6 (105.3) 105.7 912 (9 0(12)′) 16.09 (OH)106.7 160.5 16.10 (OH)106.7 160.6 1013 (10 (130)′) 10.14 (OH) 158.7 158.9 10.12 (OH) 158.7 158.9 11 (11140) 11.753.85 s (OH) 17.1 159.7 3.87 11.73 s (OH) 17.1 159.7 0 12 (1215) 203.6 106.7 203.5 106.7 13 (130) 10.14 (OH) 158.7 10.12 (OH) 158.7 1415′ 3.85 s207.5 17.1 3.87 s203.5 17.1 1516 2.75 s 29.7 203.6 2.77 s 29.4 203.5 0 1516′ 3.21 m 34.9207.5 2.77 s 29.4 203.5 1716 (17′) 1.43 2.75 s s 25.3 29.7 1.46 2.77 s s 25.3 29.4 160 3.21 m 34.9 2.77 s 29.4 1718 (17 (180)′) 1.53 1.43 s s 24.1 25.3 1.57s 1.46 s 24.1 25.3 1819 (18 (190)′) 1.53 s208.2 (208.1) 24.1 1.57s 208.3 24.1 0 1920 (19 (20)′) 3.21 m 208.246.0 (208.1) 3.22 m 46.1 208.3 20 (200) 3.21 m 46.0 3.22 m 46.1 2121 (21 (210)′) 1.73 1.73 m m 18.0 18.0(17.8) (17.8) 1.76 1.76 m m 17.9 17.9 2222 (22 (220)′) 1.01 1.01 t t 14.0 14.0 1.03 1.03 t t 14.0 14.0 0 2323′ 1.191.19 t t 8.4 8.4

FigureFigure 4. StructureStructure of of compound compound 77 and its HMBC correlations.

Compared to dryocrassin ABBA, the additional resonances at δH 1.19 (H-23′)) and δC 8.4 (C-23′)) Compared to dryocrassin ABBA, the additional resonances at δ 1.19 (H-230)) and δ 8.4 (C-230)) indicated that compound 7 should be dryocrassin ABBP. Although Hthe positions of the acetylC group indicated that compound 7 should be dryocrassin ABBP. Although the positions of the acetyl group at C-2 in ring A, propionyl group at C-2′) in ring A′), and the two butyryl units at C-10,10′) were not at C-2 in ring A, propionyl group at C-20) in ring A0), and the two butyryl units at C-10,100) were not supported by the HMBC spectrum, they were confirmed by the mass fragments at m/z 625, m/z 417, supported by the HMBC spectrum, they were conﬁrmed by the mass fragments at m/z 625, m/z 417, m/z 403, m/z 207, m/z 195 observed in the ESIMS (Scheme 1). After complete assignment of 1H-, 13C-NMR m/z 403, m/z 207, m/z 195 observed in the ESIMS (Scheme1). After complete assignment of 1H-, 13 C-NMR and DEPT data by the HSQC and HMBC spectra, compound 7 was elucidated as dryocrassin ABBP. It was different from the known dryocrassin ABBA, possessing a propionyl group at C-20) instead of an acetyl group. Molecules 2017, 22, 431 6 of 16 and DEPT data by the HSQC and HMBC spectra, compound 7 was elucidated as dryocrassin ABBP. ItMolecules was different2017, 22, 431 from the known dryocrassin ABBA, possessing a propionyl group at C-2′) instead6 of of 16 an acetyl group.

Scheme 1. ElectrosprayElectrospray Ionization Mass Spectr Spectroscopyoscopy (ESIMS) fragments of compound 7.

Compound 13 was obtained as an off-white powder which showed positive response (pink spot) Compound 13 was obtained as an off-white powder which showed positive response (pink spot) to fast-blue BB salt spray reagent on a silica gel TLC plate. The characteristic UV absorptions were at to fast-blue BB salt spray reagent on a silica gel TLC plate. The characteristic UV absorptions were at 242 and 281 nm. The absorption bands in the IR spectrum showed the presence of hydroxyl group(s) 242 and 281 nm. The absorption bands in the IR spectrum showed the presence of hydroxyl group(s) (3243 cm−1), carbonyl group(s) (1611 cm−1), phenyl group(s) (1392, 1446 cm−1), and alkyl groups (2963 cm−1). (3243 cm−1), carbonyl group(s) (1611 cm−1), phenyl group(s) (1392, 1446 cm−1), and alkyl groups The quasi-molecular ion in the negative HRESIMS of 13 at m/z 611.2125 [M − H]− (calcd for 611.2129) (2963 cm−1). The quasi-molecular ion in the negative HRESIMS of 13 at m/z 611.2125 [M − H]− (calcd corresponded to the molecular formula of C32H36O12. The characteristic resonances for hydroxyl for 611.2129) corresponded to the molecular formula of C32H36O12. The characteristic resonances protons (δH 11.65, 13.01, 16.01, 18.45, etc.), a number of aromatic quaternary carbons, and methylenes at for hydroxyl protons (δH 11.65, 13.01, 16.01, 18.45, etc.), a number of aromatic quaternary carbons, 1 13 δH/C 3.52/17.9 (C-7) and 3.80/17.1 (C-22) in the H-, C-NMR, and DEPT1 spectra13 suggested the trimeric and methylenes at δH/C 3.52/17.9 (C-7) and 3.80/17.1 (C-22) in the H-, C-NMR, and DEPT spectra phloroglucinol skeleton [23,24]. The resonances at δH 3.52 (2H, H-7) and δH 3.80 (2H, H-22) are suggested the trimeric phloroglucinol skeleton [23,24]. The resonances at δH 3.52 (2H, H-7) and δH characteristic for methylenes when situated between hexadioldienone (ring A) and phloroglucinol 3.80 (2H, H-22) are characteristic for methylenes when situated between hexadioldienone (ring A) unit (ring B), and two phloroglucinol units (rings B and C), respectively [23]. The HMBC correlations and phloroglucinol unit (ring B), and two phloroglucinol units (rings B and C), respectively [23]. (Figure 5) of H-7 (δH 3.52) with C-6 (δC 111.1), C-1 (δC 188.0), C-5 (δC 172.6), C-13 (δC 158.9), C-14 (δC 105.7), The HMBC correlations (Figure5) of H-7 ( δH 3.52) with C-6 (δC 111.1), C-1 (δC 188.0), C-5 (δC 172.6), and C-15 (δC 160.5) and those of H-22 (δH 3.80) with C-15 (δC 160.5), C-16 (δC 108.5), C-17 (δC 160.7), C-13 (δC 158.9), C-14 (δC 105.7), and C-15 (δC 160.5) and those of H-22 (δH 3.80) with C-15 (δC 160.5), C-24 (δC 159.6), C-23 (δC 106.7), and C-28 (δC 158.6) confirmed the sandwich positions of methylenes C-16 (δC 108.5), C-17 (δC 160.7), C-24 (δC 159.6), C-23 (δC 106.7), and C-28 (δC 158.6) conﬁrmed the between the rings A, B, and C. The NMR spectral data (Table 3) of this compound resembled that of sandwich positions of methylenes between the rings A, B, and C. The NMR spectral data (Table3) of trisflavaspidic acid ABB [27], except for one methyl group absence at C-27 on ring C. this compound resembled that of trisﬂavaspidic acid ABB [27], except for one methyl group absence at C-27 on ring C.

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Figure 5. Structure of compound 13 and its HMBC correlations.

Table 3.1 1H- and13 13C-NMR spectroscopic data for compounds 13 and trisflavaspidic acid ABB in CDCl3. Table 3. H- and C-NMR spectroscopic data for compounds 13 and trisflavaspidic acid ABB in CDCl3. Compound 13 Trisflavaspidic Acid ABB Position Compound 13 Trisﬂavaspidic Acid ABB Position δH δC δH δC 1 δH 188.0 δC δH 188.2δC 2 1 108.2 188.0 108.5 188.2 3 2 18.45 (OH) 199.1 108.2 18.34 (OH) 199.3 108.5 3 18.45 (OH) 199.1 18.34 (OH) 199.3 4 44.4 44.7 4 44.4 44.7 5 5 18.46 (OH) 18.46 (OH) 172.6 172.6 18.34 18.34 (OH) (OH) 172.7 172.7 6 6 111.1 111.1 111.4 111.4 7 7 3.52 s 3.52 s 17.9 17.9 3.46 s 3.46 s 17.4 17.4 8 8 203.5 203.5 203.7 203.7 9 2.74 s 29.7 2.68 s 29.6 9 10 2.74 s 1.43 s 29.7 25.2 2.68 s 1.37 s 29.6 25.5 10 11 1.43 s 1.55 s 25.2 24.1 1.37 s 1.47 s 25.5 24.4 11 12 1.55 s 24.1 106.8 1.47 s 24.4106.4 12 13 16.01 (OH) 106.8 159.6 16.01 (OH) 106.4 159.8 14 106.7 107.0 13 16.01 (OH) 159.6 16.01 (OH) 159.8 15 9.55 s 160.5 9.60 s 160.7 14 16 106.7 108.5 107.0 108.6 15 17 9.55 s 16.00 (OH) 160.5 160.7 9.60 15.97 s (OH) 160.7 160.2 16 18 108.5 208.0 108.6 208.2 17 19 16.00 (OH) 3.05 t 160.7 46.0 15.97 (OH) 3.08 t 160.2 46.2 20 1.73 m 18.0 1.66 m 18.4 18 21 1.01 m 208.0 14.0 0.94 m 208.2 14.2 19 22 3.05 t 3.80 s 46.0 17.1 3.08 3.76t s 46.2 17.0 20 23 1.73 m 18.0 105.7 1.66 m 18.4105.9 21 24 1.01 m 13.01 s 14.0 158.9 0.94 m 12.94 s 14.2159.0 25 105.3 105.6 22 3.80 s 17.1 3.76 s 17.0 26 11.65 s 159.5 11.59 s 159.1 23 27 5.90 s 105.7 96.6 105.9 102.0 24 28 13.01 s 10.09 (OH) 158.9 158.6 12.94 10.05 s (OH) 159.0 158.9 25 29 105.3 206.7 105.6 207.1 26 30 11.65 s 3.15 t 159.5 45.6 11.59 3.08s t 159.1 46.0 31 1.73 m 18.0 1.66 m 18.1 27 325.90 s 1.01 m 96.6 14.0 0.94 m 102.0 14.1 28 33 10.09 (OH) 158.6 10.05 (OH) 2.03 158.9 7.7 29 206.7 207.1 30 3.15 t 45.6 3.08 t 46.0 The acetyl group at C-2 was supported by the HMBC correlations of H-9 (δH 2.74) with C-2 31 1.73 m 18.0 1.66 m 18.1 (δC 108.2). Therefore, the two butyryl units should be located at C-12 and C-25, which were 32 1.01 m 14.0 0.94 m 14.1 conﬁrmed by mass fragments at m/z 403, m/z 207, and m/z 195 observed in the ESIMS (Scheme2). 33 2.03 7.7

Molecules 2017, 22, 431 8 of 16 Molecules 2017, 22, 431 8 of 16 Molecules 2017, 22, 431 8 of 16 The acetyl group at C-2 was supported by the HMBC correlations of H-9 (δH 2.74) with C-2 (δC 108.2). The acetyl group at C-2 was supported by the HMBC correlations of H-9 (δH 2.74) with C-2 (δC 108.2). Therefore, the two butyryl units should be located at C-12 and C-25, which were confirmed by mass Therefore, the two butyryl units should be located at C-12 and C-25, which were confirmed by mass fragmentsAfter comparing at m/z 403, the m NMR/z 207, resonances and m/z 195 with observed those in of the desaspidins ESIMS (Scheme [28] and 2). After trisflavaspidic comparing acid the NMR ABB, fragments at m/z 403, m/z 207, and m/z 195 observed in the ESIMS (Scheme 2). After comparing the NMR resonancescompound with13 was those elucidated of desaspidins as nortrisflavaspidic [28] and trisflavaspidic acid ABB. acid ABB, It was compound different 13 from was the elucidated known resonances with those of desaspidins [28] and trisflavaspidic acid ABB, compound 13 was elucidated astrisflavaspidic nortrisflavaspidic acid acid ABB ABB. which It was did different not have fr aom methyl the known group trisflavaspidic (C-33) at C-27 acid but ABB had which a hydrogen did not as nortrisflavaspidic acid ABB. It was different from the known trisflavaspidic acid ABB which did not haveatom a on methyl ring C. group (C-33) at C-27 but had a hydrogen atom on ring C. have a methyl group (C-33) at C-27 but had a hydrogen atom on ring C.

Scheme 2. ESIMS fragments of compound 13. SchemeScheme 2.2. ESIMS fragments ofof compoundcompound 1313.. The eleven known compounds (Figure 6) were elucidated as albaspidin AP (1) [28,29], albaspidin TheThe eleveneleven knownknown compoundscompounds (Figure(Figure6 6)) were were elucidated elucidated as as albaspidin albaspidin AP AP ( (11)[) [28,29],28,29], albaspidinalbaspidin AB (2) [30], albaspidin PB (3) [28,29], albaspidin AA (4) [31], albaspidin PP (5) [28,29], filixic acid ABA ABAB (2)[) [30],30], albaspidin albaspidin PB PB (3 ()3 )[[28,29],28,29 ],albaspidin albaspidin AA AA (4) ([31],4)[31 albaspidin], albaspidin PP ( PP5) [28,29], (5)[28 ,filixic29], filixic acid ABA acid (6) [32], dryocrassin ABBA (8) [25,26], filixic acid ABB (9) [33], filixic acid ABP (10) [29,31], norflavaspidic ABA(6) [32], (6 )[dryocrassin32], dryocrassin ABBA (8 ABBA) [25,26], (8)[ filixic25,26 acid], filixic ABB ( acid9) [33], ABB filixic (9)[ acid33], ABP filixic (10) acid [29,31], ABP norflavaspidic (10)[29,31], acid AB (11) [34], and flavaspidic acid AB (12) [35] by using similar spectroscopic methods as norflavaspidicacid AB (11) [34], acid and AB (flavaspidic11)[34], and acid flavaspidic AB (12) acid[35] ABby using (12)[ 35similar] by using spectroscopic similar spectroscopic methods as described for Compounds 7 and 13. Their spectroscopic data were compared with those reported in methodsdescribed as for described Compounds for Compounds 7 and 13. Their7 and spectroscopic13. Their spectroscopic data were compared data were with compared those reported with those in the literature. Compound 2 was isolated from DC for the first time. reportedthe literature. in the Compound literature. Compound2 was isolated2 was from isolated DC for from the first DC fortime. the first time.

Figure 6. Cont. FigureFigure 6.6. Cont.Cont.

Molecules 2017, 22, 431 9 of 16 Molecules 2017, 22, 431 9 of 16

FigureFigure 6. 6. StructuresStructures of of the the 11 11 known known phloroglucinols phloroglucinols isolated isolated from from DryopterisDryopteris crassirhizoma crassirhizoma (DC).(DC).

2.3. Validation of Molecular Docking by NA Inhibition Assay 2.3. Validation of Molecular Docking by NA Inhibition Assay After virtual screening of all phloroglucinols isolated from DC via molecular docking, three of After virtual screening of all phloroglucinols isolated from DC via molecular docking, three of them were screened as candidates. The three candidates and two new compounds were evaluated them were screened as candidates. The three candidates and two new compounds were evaluated for their NA inhibition activity, and oseltamivir was used as a positive control. The purified protein for their NA inhibition activity, and oseltamivir was used as a positive control. The purified protein of NA (N1 Neuraminidase H274Y) from A/Anhui/1/2005 (H5N1) was used in the test. The results of NA (N1 Neuraminidase H274Y) from A/Anhui/1/2005 (H5N1) was used in the test. The results showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against NA with showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against NA with IC50 values of 18.59 ± 4.53 and 29.57 ± 2.48 μM (Table 4), respectively. The other compounds showed IC50 values of 18.59 ± 4.53 and 29.57 ± 2.48 µM (Table4), respectively. The other compounds showed moderate activity in the NA inhibition assay. However, whether the two phloroglucinols could moderate activity in the NA inhibition assay. However, whether the two phloroglucinols could inhibit inhibit the influenza H5N1 virus directly or not is unknown, and the molecular mechanism still needs the influenza H5N1 virus directly or not is unknown, and the molecular mechanism still needs to be to be clarified by further investigation. clarified by further investigation.

Table 4. Inhibitory effects of the five phloroglucinols on influenza virus (H5N1) NA. Table 4. Inhibitory effects of the five phloroglucinols on influenza virus (H5N1) NA. H5N1 (A/Anhui/1/2005) Compounds H5N1 (A/Anhui/1/2005) Compounds IC50 (μM) Dryocrassin ABBA 18.59IC50 (±µ 4.53M) DryocrassinFilixic acid ABBA ABA 29.57 18.59 ±± 2.484.53 FilixicFilixic acid acid ABA ABB 96.58 29.57 ±± 7.672.48 ± FilixicDryocrassin acid ABB ABBP 48.90 96.58 ± 2.917.67 Dryocrassin ABBP 48.90 ± 2.91 NortrisflavaspidicNortrisflavaspidic acid acid ABB ABB 51.49 51.49 ±± 6.846.84 OseltamivirOseltamivir (nM) (nM) 4.62 4.62 ± 0.770.77

2.4.2.4. Anti-Influenza Anti-Influenza Virus Virus H5N1 H5N1 Activity Activity In In Vitro Vitro DryocrassinDryocrassin ABBA ABBA and and filixic filixic acid acid ABA ABA were were eval evaluateduated for for their antiviral activity against against the the H5N1(A/Vietnam/1203/2004)H5N1(A/Vietnam/1203/2004) strain strain on on MDCK MDCK cells cells via via CCK8 CCK8 and and CPE CPE assays assays in in a a biosafety biosafety BSL-3 BSL-3 laboratory,laboratory, and oseltamivir was was used as a positive control. The antiviral assays demonstrated that that dryocrassin ABBA exhibited anti-influenza virus activity with an EC50 value of 16.55 μM (Figure 7), dryocrassin ABBA exhibited anti-influenza virus activity with an EC50 value of 16.55 µM (Figure7), which was was weaker than than oseltamivir. oseltamivir. The The inhibition inhibition rate rate of of filixic filixic acid acid ABA ABA was was less less than than 50% 50% at at its its maximalmaximal non-cytotoxic non-cytotoxic concentrations (100 μµM).

Molecules 2017, 22, 431 10 of 16 MoleculesMolecules 2017 2017, ,22 22, ,431 431 1010 of of 16 16

FigureFigure 7. 7. Inhibitory InhibitoryInhibitory effect effect effect of of of dryocrassin dryocrassin dryocrassin ABBA ABBA ABBA on on on H5 H5 H5N1N1N1 virus virus in in Madin-Darby Madin-Darby canine canine kidney kidney (MDCK) (MDCK) cells.cells. Oseltamivir Oseltamivir was was used used as as a a positive positive control. control. Culture Culture medium medium in in 96-well 96-well plates platesplates was waswas removed removedremoved and and cellscells were were washed washed with with PBS. PBS. Then, Then, 100 100 μ µμLL sample samplesample solution solutionsolution at at different differentdifferent concentrations concentrations and and 100 100 μ μµLL of of virus at 100 TCID50 were added. After incubation at 37 °C ◦in 5% CO2 for 72 h, the antiviral activity was virusvirus at at 100 100 TCID TCID5050 werewere added. added. After After incubation incubation at 37 at °C 37 inC 5% in CO 5%2 CO for2 72for h, 72 the h, antiviral the antiviral activity activity was investigatedwasinvestigated investigated by by cell cell by counting counting cell counting kit-8 kit-8 (CCK8) (CCK8) kit-8 (CCK8) and and cytopathic cytopathic and cytopathic effect effect (CPE) effect(CPE) reduction (CPE)reduction reduction assay. assay. assay.

2.5.2.5. Cellular Cellular Toxicity Toxicity of ofof Dryocrassin DryocrassinDryocrassin ABBA ABBAABBA WeWe evaluated evaluatedevaluated the thethe cytotoxicity cytotoxicitycytotoxicity of of dryocrassin dryocrassin ABBA ABBA by by CCK8 CCK8 assay. assay. MDCK MDCK cells cells were were incubated incubated withwith media media in in in the the the absence absence absence or or or presence presence presence of of ofdryocrassin dryocrassin dryocrassin ABBA ABBA ABBA for for for72 72 h. 72h. The The h. Theresult result result of of the the of cytotoxicity thecytotoxicity cytotoxicity test test showed that the 50% toxicity concentration (TC50) of dryocrassin ABBA was greater than 400 μM showedtest showed that thatthe 50% the 50% toxicity toxicity concentration concentration (TC (TC50) 50of) dryocrassin of dryocrassin ABBA ABBA was was greater greater than than 400 400 μµMM (Figure(Figure(Figure 88), 8),), whichwhich which suggestedsuggested suggested thatthat that thisthis this compoundcompound compound mightmight might bebe be consideredconsidered considered asas as aa a potentialpotential potential therapeutic therapeutic agent agent in in thethe treatment treatment of of of H5N1 H5N1 H5N1 infection, infection, infection, but but but further further further studies studies studies are are are still still still needed needed needed to to validate to validate validate its its efficacy its efficacy efﬁcacy and and and toxicity. toxicity. toxicity.

FigureFigure 8. 8. The The toxicity toxicitytoxicity of of dryocrassin dryocrassindryocrassin ABBA ABBA on on MDCK MDCK cells. cells. Culture Culture medium medium in in 96-well 96-well plates plates were were removedremoved and and cells cells were were washed washedwashed with with PBS. PBS. Then, Then,Then, 100 100 μµμLL dryocrassin dryocrassindryocrassin ABBA ABBA solution solution at at different different concentrations were added. After incubation at 37 ◦°C in 5% CO2 for 72 h, the relative cell viability was concentrationsconcentrations were were added. added. After After incubation incubation at at 37 37 °CC in in 5% 5% CO CO22 forfor 72 72 h, h, the the relative relative cell cell viability viability was was investigatedinvestigated by byby CCK8 CCK8CCK8 assay. assay.assay.

3.3. Materials Materials and and Methods Methods

3.1.3.1. Plant Plant Material Material RhizomeRhizome of of D. D. crassirhizoma crassirhizoma was was collected collected in in Shuangyashan Shuangyashan (Heilongjiang (Heilongjiang province province of of China) China) in in JulyJuly 2014 2014 and and authenticated authenticated by by Professor Professor Chun-Sheng Chun-Sheng Liu Liu at at the the School School of of Chinese Chinese Materia Materia Medica, Medica, BeijingBeijing University University of of Chinese Chinese Medicine, Medicine, where where the thethe specimen specimenspecimen (GZDCRA02) (GZDCRA02)(GZDCRA02) has hashas been beenbeen deposited. deposited.deposited.

Molecules 2017, 22, 431 11 of 16

3.2. General Apparatus and Chemicals Column chromatography was performed on 200–300 mesh silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China). All puriﬁcations were monitored by TLC using commercially available glass plates pre-coated with silica gel 60 G (E. Merck Co., Billerica, Germany) and LC-6AD pHPLC (Shimadzu, Kyoto, Japan) with a UV SPD-20A detector using a reversed-phase C18 column (5 µm, 21.2 × 150 mm; Phenomenex Inc., Torrance, CA, USA). All the silica gel and GF254 plates were treated with citrate buffer (pH 2.0). The analytical HPLC was performed using an Agilent LC1100 (Agilent Technologies, Santa Clara, CA, USA) equipped with a MWD detector using a reversed-phase C18 column (5 µm, 4.6 × 250 mm; Phenomenex Inc., Torrance, CA, USA). 1H- and 13C-NMR data were all obtained on an Advance DRX-500 spectrometer (Bruker, Rheinstetten, Germany) in CDCl3 with TMS as an internal standard. HRESIMS was performed on a QTOF-MS (AB SCIEX, Framingham, MA, USA). Anti-inﬂuenza virus H5N1 activity was assayed with a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CAUSA).

3.3. Viruses, Cells and Reagents The purified protein of NA (N1 Neuraminidase H274Y) from A/Anhui/1/2005 (H5N1) was purchased from Sino Biological Inc. (Beijing, China). Influenza virus strain A/Vietnam/1203/2004 8.5 (10 TCID50/mL, H5N1) and Madin-Darby canine kidney (MDCK) cells were provided by the Institute of Laboratory Animal Science, CAMS&PUMC (Beijing, China). The experiments were performed at a BSL-3 laboratory in the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College. Viruses were propagated on MDCK cells and incubated in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.01% antibiotic-antimycotic solution (100 U/mL penicillin, and 100 µg/mL streptomycin) at 37 ◦C. Antibiotic-antimycotic solution, trypsin-EDTA, FBS, and DMEM were supplied by GIBCO (USA). Oseltamivir phosphate capsules were obtained from Roche Pharmaceuticals Ltd. (Shanghai, China). The stock solution of oseltamivir was prepared as follows: The capsule content powder (50 mg) was dissolved in 50 mL distilled water by ultrasonication and was centrifuged. 4 mL 20% NaOH was added to 32 mL of the supernatant and stirred at room temperature for 6 h. Then, the pH value of the solution was adjusted to 6.5 with glacial acetic acid [36]. Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Japan). MES and 20-(4-Methylumbelliferyl)-α-N-acetylneuraminic (4-MUNANA) were purchased from Sigma (USA). Stock solutions of the compounds were made in dimethyl sulfoxide (DMSO) and subsequently diluted in DMEM. The final DMSO concentration was a maximum of 0.1%, which had no effect on the cell cultures, and 0.1% DMSO was also added to all the no-drug control samples.

3.4. Molecular Docking Molecular docking is a well-recognized method for finding the optimal conformation and orientation of a ligand when binding to a receptor and has been extensively validated over the years [37–39]. In this research, AutoDock 4.2 and AutoDock Vina were used to evaluate the binding affinity of the phloroglucinols isolated from DC with NA (PDB ID 3CL0) of influenza virus H5N1. AutoDock Tools-1.5.6 was used to prepare the receptor by removing the bound ligand (oseltamivir) and crystallographic water molecules from the active site of the protein, and by adding all polar hydrogen atoms and the charges to the macromolecule. Gasteiger charges were calculated for each atom of the receptor molecule in AutoDock 4.2. The grid map was centered at the active site pocket of the protein by running the Auto Grid file. We used grid maps with a grid box size of 72 × 92 × 64 with a grid-point spacing of 0.375 Å. During docking, center grid parameters were specified for the x, y, and z axis as −31.409, −56.287, and 8.115, respectively. Docking calculations were performed by Autodock Vina. The default settings for docking in Vina were used except for num_modes (20) and exhaustiveness (100). The empirical scoring function and the iterated local search for global Molecules 2017, 22, 431 12 of 16 optimization of Vina were employed here to achieve a significantly improved calculation speed and better efficiency compared with AutoDock 4.2 [40]. After extracting the binding ligand oseltamivir, the X-ray structure of NA was used for redocking with oseltamivir and the score was calculated to check the accuracy of the AutoDock Vina program. Twenty-three phloroglucinols were prepared to dock into the active site of NA. The highest scored conformation was selected as the potential bioactive conformation. Additionally, the schematic plan of ligand interactions showing hydrogen bonds and hydrophobic interactions were visualized by LigPlot+ and PyMOL 1.5.0.4.

3.5. Extraction and Isolation The dried rhizome (15 kg) of DC was refluxed with 90% EtOH three times (2, 2, and 1 h, respectively). The combined extracts were evaporated under reduced pressure at 35 ◦C to yield a dark brown syrupy residue (2.92 kg), which was suspended in H2O (3 L), and then filtered. The filtrate was sequentially partitioned with petroleum ether (Pet), chloroform, and EtOAc, respectively, to yield 436 g Pet extract, 119 g chloroform extract, 111 g EtOAc extract, 1046 g water extract, and 202 g precipitate. The precipitation fraction was subjected to column chromatography (CC) on a silica gel column (54.0 cm × 9.4 cm, 200–300 mesh), and gradient eluted with n-hexane/chloroform (8:1–1:1) to give sixteen fractions (A1–P1). The fraction K1 was divided into seven parts (A2–G2) by a silica gel column (54.0 cm × 5.0 cm, 200–300 mesh), and gradient eluted with n-hexane/chloroform (3:1–1:1). A portion of D2 (0.87 g) was subjected to a silica gel column (45.0 cm × 1.2 cm, 200–300 mesh) with n-hexane/chloroform (16:1) to obtain 3 sub-fractions (d1, d2, d3). Fraction d1 was purified by preparative High Performance Liquid Chromatography (pHPLC) using MeOH/0.1%TFA (94:6) as the eluent to afford compound 1 (51 mg), compound 2 (5.3 mg), and compound 3 (3.1 mg). Fraction d2 was applied to pTLC [silica gel 60 G (4.5 g/plate), glass plates (15.0 cm × 15.0 cm)], developed with n-hexane/chloroform (1:4), and visualized under UV at 365 nm, then further purified by pHPLC with MeOH/0.1%TFA (93:7) to afford compound 4 (25.1 mg). Compound 5 (10.7 mg) was obtained from part d3 by pTLC, developed with n-hexane/acetone (25:1) and visualized under UV at 365 nm, and purified by pHPLC with MeOH/0.1% TFA (94:6). A portion of G2 was separated by pTLC, developed with n-hexane/acetone (15:1), and further purified by pHPLC with MeOH/0.1% TFA (99:1) to afford compound 6 (96.6 mg). Compound 7 (18 mg), compound 8 (896 mg), and compound 9 (15.6 mg) were obtained from the precipitate by pTLC, developed with n-hexane/chloroform/methanol (30:15:1) and n-hexane/acetone (20:1), and visualized under UV at 365 nm. The chloroform fraction (119 g) was subjected to a silica gel column (72.5 cm × 10 cm, 200–300 mesh), gradient eluted with n-hexane/acetone (32:1–1:1) to give seven fractions, GZ-1–GZ-7. The fraction GZ-2 (2.72 g) was separated by pTLC, developed with n-hexane/acetone (64:1) and recrystallized to give compound 10 (17 mg). A portion of GZ-4 (6.0 g) was subjected to a medium voltage silica gel column (46.0 cm × 4.9 cm, 200–300 mesh), and gradient eluted with Pet/acetone (50:1-1:1) to obtain 4 sub-fractions (GZ-4-1–GZ-4-4). The GZ-4-2 was separated by pHPLC with MeOH/0.1% TFA (95:5) to give compound 11 (25.3 mg). Compound 12 (13.4 mg) and compound 13 (15.3 mg) were obtained from GZ-5 (26.0 g) by a medium voltage silica gel column (46.0 cm × 7.0 cm, 200–300 mesh), gradient eluted with n-hexane/acetone/ethylacetate (60:1:1–3:1:1) and prepared with pTLC (Pet/ethylacetate = 3:1; n-hexane/acetone = 1:1). Compound (7): yellowish amorphous powder, UV (CHCl3) λmax: 242, 286 nm; IR (FT-IR) νmax 3216, 2961, 1612, 1556, 1445, 1395, 1323, 1289, 1194, 1161, 898, 610 cm−1; for 1H- and 13C-NMR data in − CDCl3, see Table2; HRESIMS m/z 833.3004 [M − H] (Calcd. for C44H49O16, 833.3015). Compound (13): off-white powder, UV (CHCl3) λmax 242, 281 nm; IR (FT-IR) νmax 3243, 2963, −1 1 13 1611, 1446, 1392, 1223, 1150, 896, 822, 609 cm ; for H- and C-NMR data in CDCl3, see Table3; − HRESIMS m/z 611.2125 [M − H] (Calcd. for C32H35O12, 611.2129). Molecules 2017, 22, 431 13 of 16

3.6. NA Inhibition Assay The NA inhibition assay was conducted using purified protein NA (1 U/mL) from the influenza virus strain A/Anhui/1/2005 H5N1. The inhibition activity of the three candidates and two new compounds on NA was assayed by quantifying the fluorescent product resulting from the cleavage of the substrate 4-MUNANA. The reported method was adopted with some modifications [41]. The reaction mixture consisted of the tested phlorolucinols, NA and 4-MUNANA in 33.3 mM MES ◦ buffer (containing 4 mM CaCl2, pH 6.5) in a 96-well plate. After incubation for 30 min at 37 C, the reaction was terminated with 100 µL 0.15 M glycine buffer (pH 10.3) and the fluorescence of the mixture was recorded for the excitation wavelength of 355 nm and emission wavelength of 460 nm [42]. The inhibition ratio was obtained using the equation: Inhibition activity (%) = (FNA − FSample)/(FNA − FSubstrate) × 100%, where FNA is the fluorescence of the influenza virus NA control (NA, buffer, and substrate), FSubstrate is the fluorescence of the substrate control (buffer and substrate), and FSample is the fluorescence of the tested samples (NA, sample solution, and substrate). Subsequently, the 50% inhibitory concentration (IC50) was determined by extrapolation of the results from various doses tested using a linear equation. The experiment was repeated three times with a similar finding each time. The three independent measurements were collected to determine the mean and SD values.

3.7. Anti-Inﬂuenza Virus H5N1 Activity in Vitro Assays of antiviral activity were performed using the CCK8 and CPE methods. Oseltamivir was used as a positive control. The CCK8 and CPE assays were performed in a biosafety BSL-3 laboratory as follows. MDCK cells were grown in a 96-well culture plate (104 cells/well) for 24 h. Then, the medium was removed and washed with PBS, and 100 µL sample solution at different concentrations ◦ and 100 µL virus at 100 TCID50 were inoculated onto MDCK cells for 72 h at 37 C under 5% CO2. After 3 days, we observed the cytopathic effect by a Fluorescence Inversion Microscope System and recorded the results, and then the solution was removed and the cells were washed with PBS, and then 190 µL DMEM and 10 µL CCK8 were added to each well. The plates were incubated in the dark for 3 h at 37 ◦C. The absorbance was read at 450/630 nm using a microplate reader. All tests were performed in quadruplicate. The inhibition ratio was determined as follows: inhibition activity (%) = (ODsample – ODvirus)/(ODcellular control − ODvirus) × 100%, where ODsample is the optical density of the tested sample at a certain concentration, ODvirus is the optical density of the inﬂuenza virus control, and ODcellular control is the optical density of normal cells. The 50% inhibition concentration (IC50) was determined by linear extrapolation of the results from various doses tested.

3.8. Cytotoxicity Assay The CCK8 method was used to measure the cytotoxicity of the tested sample. MDCK cells were grown in a 96-well plate 200 µL (104 cells/well) for 24 h. The culture supernatant was replaced with maintenance medium containing the tested samples at various concentrations. After 72 h incubation, we removed the culture supernatant and added 190 µL DMEM and 10 µL CCK8 to each well. The plates were incubated at 37 ◦C for 3 h. The ﬂuorescence intensity was recorded with a microplate reader at 450/630 nm. All tests were performed in quadruplicate. The 50% toxic concentration (TC50) was calculated by regression analysis.

4. Conclusions This is the first comprehensive investigation of anti-influenza virus H5N1 activity on all phloroglucinols from the rhizome of D. crassirhizoma by molecular docking. Experimental results indicate that dryocrassin ABBA may have the potential to be used against influenza virus H5N1 infection which will have to be verified in further detailed studies. Two new phloroglucinols were obtained and they also have moderate inhibitory activity on NA. Molecules 2017, 22, 431 14 of 16

Supplementary Materials: Structures of the twenty-three phloroglucinols, Table S1; UV, IR, 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC, and HRESIMS spectra are available as supporting data, Figures S1–S16. Supplementary materials are available online. Acknowledgments: The authors are grateful to Professor Chun-Sheng Liu, Beijing University of Chinese Medicine for authentication of the plant material. This work was supported by grants from the National Natural Science Foundation of China (No. 31270400). Author Contributions: Zeng-Ping Gao conceived and designed the experiments; Juan Wang, Shen-Zhen Fu, and Bing Peng performed the extraction and isolation of DC; Yan-Tao Yan and Lin-Lin Bao performed the biological assay and data analysis; Yan-Ling Zhang and Dong-Hao Geng performed the molecular docking; Zu-Ping Zeng and Jing-Hong Hu contributed reagents/materials/analysis tools; Juan Wang wrote the paper. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–13 are available from the authors.

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