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Mar. Drugs 2013, 11(11), 4628-4640; doi:10.3390/md11114628
Published: 21 November 2013
Abstract: Total lipids from the Brazilian brown seaweed Sargassum vulgare were extracted with chloroform/methanol 2:1 and 1:2 (v/v) at room temperature. After performing Folch partition of the crude lipid extract, the lipids recovered from the Folch lower layer were fractionated on a silica gel column eluted with chloroform, acetone and methanol. The fraction eluted with methanol, presented a strong orcinol-positive band characteristic of the presence of sulfatides when examined by TLC. This fraction was then purified by two successive silica gel column chromatography giving rise to fractions F4I86 and F4II90 that exhibited strong activity against herpes simplex virus type 1 and 2. The chemical structures present in both fractions were elucidated by ESI-MS and 1H/13C NMR analysis HSQC fingerprints based on their tandem–MS behavior as sulfoquinovosildiacylglycerols (SGDGs). The main SQDG present in both fractions and responsible for the anti-herpes activity observed was identified as 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol.
Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) are the most widely studied human herpes viruses  with an estimated 60%–95% of the adult population infected by at least one of them [1,2]. HSV-1 is generally related to oral–facial infections and encephalitis, whereas HSV-2 is responsible for genital infections, and can be transferred from infected mothers to neonates . Moreover, HSV infections are recognized as a risk factor for human immunodeficiency virus (HIV) infection . Efficient anti-herpes drugs already exist, but their extensive use can generate side effects and may also lead to the rise of drug-resistant virus strains [4,5]. Consequently, new types of anti-herpes compounds are urgently needed.
Marine organisms are a huge source of natural products with biological activities. Products of primary metabolism like amino acids, carbohydrates and proteins, are vital for maintaining life processes, while others such as alkaloids, phenolics, steroids, terpenoids, are secondary metabolites that have ecological, toxicological and pharmacological significance [6,7], encompassing bioactivities such as antiparasitic, antitumor, antimicrobial and antifoulant effects .
Recently, a great deal of interest has been expressed regarding compounds from seaweeds as potential antiviral agents . Polysaccharides (sulfated polysaccharides in particular), poliketides, terpenoids or peptides with anti-HSV activities have been isolated from these marine organisms [10,11,12,13]. Glycolipids represent a less studied class of antiviral secondary metabolites . Seaweeds synthesize three major types of glycolipids: monogalactosyldiacylglycerides (MGDG), digalactosyldiacylglycerides (DGDG), and sulfoquinovosyldiacylglycerides (SQDG) . SQDG has an important biological function in photosynthetic plant tissues , exhibits high biological activity , affects HIV  and neoplastic and inflammatory processes [17,19]. In a recent study, de Souza and coworkers  isolated SQDG from the red seaweed Osmundaria obtusiloba that exhibited potent anti-HSV-1 and HSV-2 activities. A SQDG with anti-HSV-1 activity was isolated from the microalga Spirula platensis . Wang and coworkers  highlighted the anti-HSV-2 activity of a SQDG isolated from the green seaweed Caulerpa racemosa. As SQDG is the main glycolipid found in brown seaweeds of the order Fucales , we have chosen the brown seaweed Sargassum vulgare as a model in order to isolate and test its glycolipids as potencial anti-HSV-1 and HSV-2 agents.
2. Results and Discussion
2.1. Lipid Fractionation
Total lipids from the brown seaweed Sargassum vulgare were successively extracted with chloroform/methanol 2:1 and 1:2 (v/v) at room temperature according to previous studies [14,23]. After filtration, the extracts were combined, concentrated in vacuo and the crude lipid extract was partitioned according to Folch and coworkers . The lower layer was evaporated and fractionated on silica gel column chromatography using chloroform, acetone, and methanol as solvents (Figure 1). Fractions were analyzed by TLC, developed with CHCl3:CH3OH:2M NH4OH (40:10:1 v/v/v) and the spots visualized with iodine and by spraying with orcinol/H2SO4 . The resulting fractions were combined in four fractions, F1, F2, F3 and F4 according to their TLC profiles. Thin-layer chromatography of F4 revealed an orcinol-positive band with chromatographic mobility corresponding to a sulfatide. This fraction was then chosen to carry out the purification protocol.
The F4 fraction was first treated with activated charcoal in order to remove the pigments, and was then purified on a silica gel column, which was sequentially eluted with chloroform/methanol with increasing concentrations of methanol (95:5, 90:10, 80:20, 50:50, v/v) and finally with 100% methanol, providing ninety-five sub-fractions. These fractions were pooled according to their TLC profiles, resulting in twelve final fractions: F4I1, F4I5, F4I7, F4I11, F4I23, F4I35, F4I41, F4I63, F4I69, F4I86, F4I90 and F4I95.
Fraction F4I90 was further purified on a silica gel column, which was sequentially eluted with chloroform/methanol with increasing concentrations of methanol (90:10, 80:20 v/v) and finally with 100% methanol, providing hundred fifty-one sub-fractions. These fractions were pooled according to their TLC profiles, resulting in seven final fractions: F4II1, F4II13, F4II63, F4II70, F4II90, F4II121, and F4II148.
Fractions F4I86 and F4II90, which TLC profiles indicate the presence of SQDGs, were then analyzed using ESI-MS and NMR, and their antiviral activity was tested against HSV-1 and HSV-2.
2.2. Mass Spectrometry of Sulfolipids
The spectrum obtained in negative MS1 from fraction F4I86 exhibited six deprotonated ions with m/z 766, 794, 808, 820, 836 and 892 [M − H]− compatible with sulfoquinovosyldiacylglycerol structures.
In order to confirm the structures, the ions at m/z 766, 794, 808, 820, 836 and 892 were fragmented by the second stage tandem-MS. Each ion gave fragments at m/z 225, 165, 153, 95 and 81 characteristic of the 6-deoxy-6-sulfono-hexosyl residue of the SQDG (Figure 2).
The ion at m/z 793.9 was the most abundant and gave fragments at m/z 537.5 (M − C16:0 from the sn-2 position), 536.9 (M–C16:0 from the sn-1 position), 224.7, 164.8, 152.8, 95.2 and 81.1, as indicated in the fragmentation pathway, consistent with a SQDG structure, esterified by two palmitic acids (C16:0) (Figure 2). The structure was confirmed comparing our data to the fragmentation pathway already described by Zianni and coworkers  for a similar SQDG isolated in a lipid extract from spinach leaves.
The fragmentation pathway of the six deprotonated ions with m/z at 765.7, 793.6, 807.4, 819.5, 835.9 and 891.9 [M − H]−, is compatible with sulfoquinovosyldiacylglycerol structures represented in Table 1 and Figure 3.
|Table 1. Identification of sulfoquinovosyldiacylglycerides (SQDGs) present in fractions F4I86 and F4II90.|
|Fraction||Compound||R1/R2||[M − H]− m/z||[M − R1]− m/z||[M − R2]− m/z|
The spectrum obtained from fraction F4II90 presents the same ions that fraction F4I86, with the exception of the ions at m/z = 819.5 and m/z = 891.9 that correspond to SQDG structures esterified by palmitic and oleic acids, and by palmitic and tricosanoic acids respectively.
2.3. NMR Spectroscopy of Sulfolipids
The structure of the main sulfoglycolipid present in fraction F4I86 and F4II90 was confirmed by 1H and 13C NMR analysis, based on HSQC fingerprints. The anomeric region (H1/C1 Qui) contained a single signal at δ 4.78/99.3, consistent with α-quinovopyranosyl group. Moreover, 1H/13C-HSBC signals at δ 3.25, 2.990/53.5 were observed (Figure 4). The presence of doublets of CH2 signals in a high-field region is characteristic of S-substituted C-6, typical of 6-sulfo-α-quinovopyranosyl unit [14,26,27].
These results and those obtained from mass spectrometry allowed us to identify the main SQDG from fractions F4I86 and F4II90 as 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol.
3. Experimental Section
3.1. Biological Material
Thalli of S. vulgare were collected by free diving in the shallow subtidal zone from Ilha de Itacuruçá, a large nearshore island inside Sepetiba Bay (Mangaratiba district, Rio de Janeiro State, Southeastern Brazil—22°56′ S, 43°52′ W). After collection, specimens of S. vulgare were immediately transferred to the laboratory in isothermic boxes filled with local seawater, where they were gently washed in seawater, sorted, and carefully cleaned from associated biota. Thalli were then freeze-dried and ground to a fine powder before performing extraction.
3.2. Extraction and Fractionation of Lipids
The powder obtained from S. vulgare freeze-dried specimens was successively extracted at room temperature with chloroform/methanol 2:1 and 1:2 (v/v). After filtration, the extracts were combined, dried and the crude lipid extract was partitioned according to Folch and coworkers . The lipids recovered from the Folch lower phase were fractionated on a silica gel column, which was eluted with chloroform, acetone and methanol, giving rise to fractions F1–F4. Fraction F4, eluted with methanol and enriched in sulfatides was further purified on a silica gel column, which was sequentially eluted with chloroform/methanol with increasing concentrations of methanol (95:5, 90:10, 80:20, 50:50 v/v) and finally 100% methanol. The resulting fractions were combined in twelve final fractions. Fraction F4I86 was guarded for further analyses and fraction F4I90, eluted with 80/20 chloroform/methanol, was further purified on a second silica gel column yielding a purified sulfolipid fraction, F4II90.
All the fractions were analyzed by TLC developed with CHCl3:CH3OH: 2 M NH4OH (40:10:1 v/v) and the spots visualized with iodine and by spraying with orcinol/H2SO4 .
3.3. Mass Spectrometry
The samples were prepared in MeOH at 1 mg/mL, then diluted to 0.1 mg/mL in MeOH-H2O (7:3, v/v) and direct infused into ESI source, at a flow rate of 10 μL/min, following the protocol described by de Souza and coworkers . The MS analysis was carried out in an electrospray ionization mass spectrometry (ESI-MS), model Quattro-LC (Waters) with a triple-quadrupole mass analyzer, operating at atmospheric pressure ionization (API), assisted by a syringe pump (Model KDS-100-CE, KD Scientific, Holliston, MA, USA) for sample infusion. Nitrogen was used as nebulizing and desolvation gas and the ionization energies were 50 V on the cone and 2 kV on the capillary, operating in the negative ionization mode. The second stage tandem-MS was obtained by collision induced dissociation mass spectrometry (CID-MS) using argon as collision gas and collision energies ranging between 35 and 60 eV.
3.4. Nuclear Magnetic Resonance
NMR analyses were performed on a Bruker Avance III 400 MHZ spectrometer with a 5 mm inversed gradient probe. The samples were dissolved in deuterated chloroform and methanol (1:1, v/v) at 20 mg/mL. Two-dimensional homo- and heteronuclear 1H/13C correlation experiments (HSQC) were developed. The chemical shifts (δ = ppm) were obtained on the basis of tetramethylsilane shifts (δ13C = 0; δ1H = 0) .
3.5. Cells and Viruses
Vero cells (African green monkey kidney) were grown in Eagle’s minimum essential medium (Eagle-MEM) and supplemented with 10% (v/v) fetal bovine serum, glutamine (2 mM), garamycin (50 μg/mL), fungizone (amphotericin B) (2.5 μg/mL), NaHCO3 (0.25%) and HEPES (10 mM). HSV-1 and HSV-2 were isolated from a typical lip and genital lesion respectively, in the Virology Department of the Federal University of Rio de Janeiro (UFRJ), Brazil. Viruses were typed by polymerase chain reaction (PCR) using specific primers for identification [14,43].
3.6. Cytotoxicity Assay
The cytotoxicity of glycolipids was performed by incubating triplicate Vero cell (African green monkey kidney cell) line monolayers cultivated in 96-well microplates with two-fold serial dilutions (200–3.1 μg/mL) of the SQDG fractions for 48 h at 37 °C in a 5% CO2 atmosphere. Cellular viability was evaluated by the neutral red dye-uptake method . The 50% cytotoxic concentration (CC50) was defined as the SQDG concentration, which caused a 50% reduction in the number of viable cells.
Antiviral SQDGs were isolated and characterized for the first time in Sargassum vulgare from Brazil. Other studies already highlighted antifouling, anticoagulant, antithrombotic, antioxidant and anti-inflammatory activities from S. vulgare extracts and isolated compounds. Our results reinforce the potential of S. vulgare as a source of natural products with biotechnological applications. Future studies will be necessary to understand more precisely the mechanism of action of SQDGs and to fully determine the potential applications of these seaweed compounds.
This work was supported by a research grant from FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. Processo E26/111.823/2012). B.A.P.G., R.C.P., G.L.S. and E.B.B. are CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) research fellows. The authors also thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), the Fundação Araucária, and the Centers of NMR and MS from the Federal University of Paraná (UFPR).
Conflicts of Interest
The authors declare no conflict of interest.
- Vo, T.; Ngo, D.; Ta, Q.V.; Kim, S. Marine organisms as a therapeutic source against herpes simplex virus infection. Eur. J. Pharm. Sci. 2011, 44, 11–20, doi:10.1016/j.ejps.2011.07.005.
- Brady, R.C.; Bernstein, D.I. Treatment of herpes simplex virus infections. Antiviral. Res. 2004, 61, 73–81, doi:10.1016/j.antiviral.2003.09.006.
- Celum, C.L. The interaction between herpes simplex virus and human immunodeficiency virus. Herpes 2004, 11, 36A–45A.
- Bacon, T.H.; Levin, M.J.; Leary, J.J.; Sarisky, R.T.; Sutton, D. Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clin. Microbiol. Rev. 2003, 16, 114–128, doi:10.1128/CMR.16.1.114-128.2003.
- Morfin, F.; Thouvenot, D. Herpes simplex virus resistance to antiviral drugs. J. Clin. Virol. 2003, 26, 29–37, doi:10.1016/S1386-6532(02)00263-9.
- Maschek, J.A.; Baker, B.J. The chemistry of algal secondary metabolism. In Algal Chemical Ecology; Amsler, C.D., Ed.; Springer-Verlag: Berlin, Germany, 2008; pp. 1–24.
- Chakraborty, S.; Ghosh, U. Oceans: A store house of drugs—a review. J. Pharm. Res. 2010, 3, 1293–1296.
- Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2012, 29, 144–222, doi:10.1039/c2np00090c.
- Kim, S.K.; Karadeniz, F. Anti-HIV activity of extracts and compounds from marine algae. Adv. Food. Nutr. Res. 2011, 64, 255–265, doi:10.1016/B978-0-12-387669-0.00020-X.
- Saha, S.; Navid, M.H.; Bandyopadhyay, S.S.; Schnitzler, P.; Ray, B. Sulfated polysaccharides from Laminaria angustata: Structural features and in vitro antiviral activities. Carbohydr. Polym. 2012, 87, 123–130, doi:10.1016/j.carbpol.2011.07.026.
- Bandyopadhyay, S.S.; Navid, M.H.; Ghosh, T.; Schnitzler, P.; Ray, B. Structural features and in vitro antiviral activities of sulfated polysaccharides from Sphacelaria indica. Phytochemistry 2011, 72, 276–283, doi:10.1016/j.phytochem.2010.11.006.
- Cardozo, F.T.; Camelini, C.M.; Mascarello, A.; Rossi, M.J.; Nunes, R.J.; Barardi, C.R.; de Mendonça, M.M.; Simões, C.M. Antiherpetic activity of a sulfated polysaccharide from Agaricus brasiliensis mycelia. Antivir. Res. 2011, 92, 108–114, doi:10.1016/j.antiviral.2011.07.009.
- Adhikari, U.; Mateu, C.G.; Chattopadhyay, K.; Pujol, C.A.; Damonte, E.B.; Ray, B. Structure and antiviral activity of sulfated fucans from Stoechospermum marginatum. Phytochemistry 2006, 67, 2474–2482, doi:10.1016/j.phytochem.2006.05.024.
- de Souza, L.M.; Sassaki, G.L.; Romanos, M.T.; Barreto-Bergter, E. Structural characterization and anti-HSV-1 and HSV-2 activity of glycolipids from the marine algae Osmundaria obtusiloba isolated from Southeastern Brazilian coast. Mar. Drugs 2012, 10, 918–931, doi:10.3390/md10040918.
- Kind, T.; Meissen, J.K.; Yang, D.W.; Nocito, F.; Vaniya, A.; Cheng, Y.S.; VanderGheynst, J.S.; Fiehn, O. Qualitative analysis of algal secretions with multiple mass spectrometric platforms. J. Chromagraph. A 2012, 1244, 139–147.
- Packter, N.M. Lipids in plants and microbes: By J L Harwood and N J Russel. pp 162. George Allen & Unwin, London. 1984. Biochem. Educ. 1985, 13, 94–94, doi:10.1016/0307-4412(85)90058-5.
- Morimoto, T.; Murakami, N.; Nagatsu, A.; Sakakibara, J. Studies on glycolipids VII. Isolation of two new sulfoquinovosyl diacylglycerols from green alga Chlorella vulgaris. Chem. Pharm. Bull. 1993, 41, 1545–1548, doi:10.1248/cpb.41.1545.
- Gustafson, K.R.; Cardellina, J.H.; Fuller, R.W.; Weislow, O.S.; Kiser, R.F.; Snader, K.M.; Patterson, G.M.L.; Boyd, M.R. AIDS-antiviral sulfolipids from cyanobacteria (Blue-Green Algae). J. Natl. Cancer Inst. 1989, 81, 1254–1258, doi:10.1093/jnci/81.16.1254.
- Kikuchi, H.; Tsukitani, Y.; Manda, T.; Fujii, T.; Nakanishi, H.; Kobayashi, M.; Kitagawa, I. Marine Natural Products. X. Pharmacologically active glycolipids from the Okinawan marine sponge Phyllospongia foliascens (PALLAS). Chem. Pharm. Bull. 1982, 30, 3544–3547, doi:10.1248/cpb.30.3544.
- Chirasuwan, N.; Chaiklahan, R.; Kittakoop, P.; Chanasattru, W.; Ruengjitchatchawalya, M.; Tanticharoen, M.; Bunnag, B. Anti HSV-1 activity of sulphoquinovosyl diacylglycerol isolated from Spirulina platensis. Scienceasia 2009, 35, 137–141.
- Wang, H.; Li, Y.L.; Shen, W.Z.; Rui, W.; Ma, X.J.; Cen, Y.Z. Antiviral activity of a sulfoquinovosyldiacylglycerol (SQDG) compound isolated from the green alga Caulerpla racemosa. Bot. Mar. 2007, 50, 185–190.
- Khotimchenko, S.V. Distribution of glyceroglycolipids in marine algae and grasses. Chem. Nat. Compd. 2002, 38, 223–229, doi:10.1023/A:1020471709232.
- Barreto-Bergter, E.; Sassaki, G.L.; Souza, L.M. Structural analysis of fungal cerebrosides. Front. Microbiol. 2011, 2, 1–11.
- Folch, J.; Lees, M.; Sloane-Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957, 226, 497–509.
- Zianni, R.; Bianco, G.; Lelario, F.; Losito, I.; Palmisano, F.; Cataldi, T.R.I. Fatty acid neutral losses observed in tandem mass spectrometry with collision-induced dissociation allows regiochemical assignment of sulfoquinovosyl-diacylglycerols. J. Mass Spectrom. 2013, 48, 205–215.
- Sassaki, G.L.; Gorin, P.A.J.; Tischer, C.A.; Iacomini, M. Sulfonoglycolipids from the lichenized basidiomycete Dictyonema glabratum: Isolation, NMR, and ESI-MS approaches. Glycobiology 2001, 11, 345–351, doi:10.1093/glycob/11.4.345.
- Souza, L.M.; Iacomini, M.; Gorin, P.A.J.; Sari, R.S.; Haddad, M.A.; Sassaki, G.L. Glyco- and sphingophosphonolipids from the medusa Phyllorhiza punctata: NMR and ESI-MS/MS fingerprints. Chem. Phys. Lipids 2007, 145, 85–96, doi:10.1016/j.chemphyslip.2006.11.001.
- Tsai, C.J.; Pan, B.S. Identification of sulfoglycolipid bioactivities and characteristic fatty acids of marine macroalgae. J. Agric. Food Chem. 2012, 60, 8404–8410, doi:10.1021/jf302241d.
- Al-Fadhli, A.; Wahidulla, S.; D’Souza, L. Glycolipids from the red alga Chondria armata (Kutz.) Okamura. Glycobiology 2006, 16, 902–915, doi:10.1093/glycob/cwl018.
- Fusetani, N.; Hashimoto, Y. Structures of two water-soluble hemolysins isolated from green alga Ulva pertus. Agric. Biol. Chem. 1975, 39, 2021–2025, doi:10.1271/bbb1961.39.2021.
- Araki, S.; Sakurai, T.; Oohusa, T.; Kayama, M.; Sato, N. Characterization of sulphonoquinovosyl diacylglycerol from marine red alga. Plant Cell Physiol. 1989, 30, 775–781.
- Son, W.B. Glycolipids from Gracilaria verrucosa. Phytochemistry 1990, 29, 307–309, doi:10.1016/0031-9422(90)89057-G.
- Siddantha, A.K.; Ramvat, B.K.; Chauvan, V.D.; Achari, B.; Dutta, P.K.; Pakrashi, S.C. Sulphoglycolipid from the green alga Enteromorpha flexuosa (Wulf). J. Agric. Bot. Mar. 1991, 34, 365–367.
- Logvinov, S.V.; Denisenko, V.A.; Dmitrenok, P.S.; Moiseenko, O.P. Sulfoquinovosyldiacylglycerins from Scaphechinus mirabilis. Chem. Nat. Compd. 2012, 48, 175–179, doi:10.1007/s10600-012-0198-0.
- Harada, H.; Yamashita, U.; Kurihara, H.; Fukushi, E.; Kawabata, J.; Kamei, Y. Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. Anticancer Res. 2002, 22, 2587–2590.
- Kabara, J.J.; Swieczkowski, D.M.; Truant, J.P.; Conley, A.J.; Truant, J.P. Fatty-acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 1972, 2, 23–28, doi:10.1128/AAC.2.1.23.
- Santoyo, S.; Jaime, L.; Plaza, M.; Herrero, M.; Rodriguez-Meizoso, I.; Ibañez, E.; Reglero, G. Antiviral compounds obtained from microalgae commonly used as carotenoid sources. J. Appl. Phycol. 2012, 24, 731–741, doi:10.1007/s10811-011-9692-1.
- Lee, D.Y.; Lin, X.; Paskaleva, E.E.; Liu, Y.; Puttamadappa, S.S.; Thornber, C.; Drake, J.R.; Habulin, M.; Shekhtman, A.; Canki, M. Palmitic acid is a novel CD4 fusion inhibitor that blocks HIV entry and infection. AIDS Res. Hum. Retrovir. 2009, 25, 1231–1241, doi:10.1089/aid.2009.0019.
- Spear, P.G. Herpes simplex virus: Receptors and ligands for cell entry. Cell Microbiol. 2004, 6, 401–410, doi:10.1111/j.1462-5822.2004.00389.x.
- Wang, W.; Wang, S.X.; Guan, H.S. The antiviral activities and mechanisms of marine polysaccharides: An Overview. Mar. Drugs 2012, 10, 2795–2816, doi:10.3390/md10122795.
- Neyts, J.; Snoeck, R.; Schols, D.; Balzarini, J.; Esko, J.D.; Van Schepdael, A.; De Clercq, E. Sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate. Virology 1992, 189, 48–58, doi:10.1016/0042-6822(92)90680-N.
- Skipski, V.P. Thin layer chromatography of neutral glycolipids. Methods Enzymol. 1975, 35, 396–425, doi:10.1016/0076-6879(75)35178-1.
- Markoulatos, P.; Georgopoulou, A.; Siafakas, N.; Plakokefalos, E.; Tzanakaki, G.; Kourea-Kremastinou, J. Laboratory diagnosis of common herpesvirus infections of the central nervous system by a multiplex PCR assay. J. Clin. Microbiol. 2001, 39, 4426–4432, doi:10.1128/JCM.39.12.4426-4432.2001.
- Borenfreund, E.; Puerner, J.A. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 1985, 24, 119–124, doi:10.1016/0378-4274(85)90046-3.
- Reed, L.J.; Muench, H. A simple method of estimating 50 per cent end-points. Am. J. Hyg. 1938, 27, 493–497.
- Nishimura, T.; Toku, K.; Fukuyasu, H. Antiviral compounds. XII. Antiviral activity aminohydrazones of alkoxyphenyl substituted carbonyl compounds against influenza virus in eggs and mice. Kitasato Arch. Exp. Med. 1977, 50, 39–46.
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