For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC50 values of 0.92 and 1.43 µg∙mL-1, respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC50 value of 82.4 µg∙mL-1. This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

Department of Applied Ecology Faculty of Environmental Sciences Czech University of Life Sciences Prague Kamýcká 129 165 21 Praha Suchdol Czech Republic

Department of Natural Drugs Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic

Zobrazit více v PubMed

Einsenberg R., Atanasiu D., Cairns T.M., Gallagher J.R., Krummenacher C., Cohen G.H. Herpes virus fusion and entry: A story with many characters. Viruses. 2012;4:800–832. doi: 10.3390/v4050800. PubMed DOI PMC

Gescher K., Hensel A., Hafezi W., Derksen A., Kühn J. Oligomeric proanthocyanidins from Rumex acetosa L inhibit the attachment of herpes simplex virus type-1. Antivir. Res. 2011;89:9–18. doi: 10.1016/j.antiviral.2010.10.007. PubMed DOI

Khan M.T., Alter A., Thompson K.D., Gambari R. Extracts and molecules from medicinal plants against herpes simplex viruses. Antivir. Res. 2005;67:107–119. doi: 10.1016/j.antiviral.2005.05.002. PubMed DOI

Keller M.J., Tujama A., Carlucci M.J., Herold B.C. Topical microbicides for prevention of genital herpes infection. J. Antimicrob. Chemother. 2005;55:420–423. doi: 10.1093/jac/dki056. PubMed DOI

Wang Z., Liu Q., Lu J., Fan P., Xie W., Qiu W., Wang F., Hu G., Zhang Y. Serine/Arginine-rich splicing factor 2 modulates herpes simplex virus type 1 replication via regulating viral gene transcriptional activity and pre-mRNA splicing. J. Biol. Chem. 2016;291:26377–26387. doi: 10.1074/jbc.M116.753046. PubMed DOI PMC

Hassan S.T.S., Berchová-Bímová K., Petráš J., Hassan K.T.S. Cucurbitacin B interacts synergistically with antibiotics against Staphylococcus aureus clinical isolates and exhibits antiviral activity against HSV-1. S. Afr. J. Bot. 2017;108:90–94. doi: 10.1016/j.sajb.2016.10.001. DOI

Hassan S.T.S., Masarčíková R., Berchová K. Bioactive natural products with anti-herpes simplex virus properties. J. Pharm. Pharmacol. 2015;67:1325–1336. doi: 10.1111/jphp.12436. PubMed DOI

Hassan S.T.S., Žemlička M. Plant-derived urease inhibitors as alternative chemotherapeutic agents. Arch. Pharm. 2016;349:507–522. doi: 10.1002/ardp.201500019. PubMed DOI

Konieczna I., Zarnowiec P., Kwinkowski M., Kolesinska B., Fraczyk J., Kaminski Z., Kaca W. Bacterial urease and its role in long-lasting human diseases. Curr. Protein Pept. 2012;13:789–806. doi: 10.2174/138920312804871094. PubMed DOI PMC

Follmer C.J. Ureases as a target for the treatment of gastric and urinary infections. Clin. Pathol. 2010;63:424–430. doi: 10.1136/jcp.2009.072595. PubMed DOI

Kosikowska P., Berlicki Ł. Urease inhibitors as potential drugs for gastric and urinary tract infections: A patent review. Expert Opin. Ther. Pat. 2011;21:945–957. doi: 10.1517/13543776.2011.574615. PubMed DOI

Mazzei L., Cianci M., Musiani F., Ciurli S. Inactivation of urease by 1,4-benzoquinone: Chemistry at the protein surface. Dalton Trans. 2016;45:5455–5459. doi: 10.1039/C6DT00652C. PubMed DOI

Mobley H.L., Island M.D., Hausinger R.P. Molecular biology of microbial ureases. Microbiol. Rev. 1995;59:451–480. PubMed PMC

Tanaka T., Kawase M., Tani S. Urease inhibitory activity of simple α, β-unsaturated ketones. Life Sci. 2003;73:2985–2990. doi: 10.1016/S0024-3205(03)00708-2. PubMed DOI

Zaborska W., Krajewska B., Olech Z. Heavy metal ions inhibition of jack bean urease: Potential for rapid contaminant probing. Enzyme Inhib. Med. Chem. 2004;19:65–69. doi: 10.1080/14756360310001650237. PubMed DOI

Laghari H.A., Memona S., Nelofar A., Khan K.M., Yasmin A., Syed M.N., Aman A. A new flavanenol with urease-inhibition activity isolated from roots of manna plant camelthorn (Alhagi maurorum) J. Mol. Struct. 2010;965:65–67. doi: 10.1016/j.molstruc.2009.11.039. DOI

Paulo L., Oleastro M., Gallardo E., Queiroz J.A., Domingues F. Anti-Helicobacter pylori and urease inhibitory activities of resveratrol and red wine. Food Res. Int. 2011;44:964–969. doi: 10.1016/j.foodres.2011.02.017. DOI

Hassan S.T., Berchová K., Šudomová M. Antimicrobial, antiparasitic and anticancer properties of Hibiscus sabdariffa (L.) and its phytochemicals: In vitro and in vivo studies. Ceska Slov. Farm. 2016;65:10–14. PubMed

De Arruda A., Cardoso C.A., Vieira Mdo C., Arena A.C. Safety assessment of Hibiscus sabdariffa after maternal exposure on male reproductive parameters in rats. Drug Chem. Toxicol. 2016;39:22–27. doi: 10.3109/01480545.2014.1003938. PubMed DOI

Ademiluyi A.O., Oboh G. Aqueous extracts of Roselle (Hibiscus sabdariffa Linn.) varieties inhibit α-amylase and α-glucosidase activities in vitro. J. Med. Food. 2013;16:88–93. doi: 10.1089/jmf.2012.0004. PubMed DOI

Wang J., Cao X., Jiang H., Qi Y., Chin K. L., Yue Y. Antioxidant activity of leaf extracts from different Hibiscus sabdariffa accessions and simultaneous determination five major antioxidant compounds by LC-Q-TOF-MS. Molecules. 2014;19:21226–21238. doi: 10.3390/molecules191221226. PubMed DOI PMC

Da-Costa-Rocha I., Bonnlaender B., Sievers H., Pischel I., Heinrich M. Hibiscus sabdariffa L.—A phytochemical and pharmacological review. Food Chem. 2014;165:424–443. doi: 10.1016/j.foodchem.2014.05.002. PubMed DOI

Mo Z.-Z., Wang X.-F., Zhang X., Su J.-Y., Chen H.-M., Liu Y.-H., Zhang Z.-B., Xie J.-H., Su Z.-R. Andrographolide sodium bisulphite-induced inactivation of urease: Inhibitory potency, kinetics and mechanism. BMC Complement. Altern. Med. 2015;15:238. doi: 10.1186/s12906-015-0775-4. PubMed DOI PMC

Du N., Chen M., Liu Z., Sheng L., Xu H., Chen S. Kinetics and mechanism of jack bean urease inhibition by Hg2+ Chem. Cent. J. 2012;6:154. doi: 10.1186/1752-153X-6-154. PubMed DOI PMC

Krajewska B. Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme. J. Enzyme Inhib. Med. Chem. 2008;23:535–542. doi: 10.1080/14756360701743051. PubMed DOI

Wu G., Yuan Y., Hodge C.N. Determining appropriate substrate conversion for enzymatic assays in high-throughput screening. J. Biomol. Screen. 2003;8:694–700. doi: 10.1177/1087057103260050. PubMed DOI

Tanaka T., Kawase M., Tani S. Alpha-hydroxyketones as inhibitors of urease. Bioorg. Med. Chem. 2004;12:501–505. doi: 10.1016/j.bmc.2003.10.017. PubMed DOI

Firdous S., Ansari N.H., Fatima I., Malik A., Afza N., Iqbal L., Lateef M. Ophiamides A-B, new potent urease inhibitory sphingolipids from Heliotropium ophioglossum. Arch. Pharm. Res. 2012;35:1133–1137. doi: 10.1007/s12272-012-0702-x. PubMed DOI

Yang C.M., Cheng H.Y., Lin T.C., Chiang L.C., Lin C.C. The in vitro activity of geraniin and 1,3,4,6-tetra-O-galloyl-β-d-glucose isolated from Phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection. J. Ethnopharmacol. 2007;110:555–558. doi: 10.1016/j.jep.2006.09.039. PubMed DOI

Tseng T.H., Hsu J.D., Lo M.H., Chu C.Y., Chou F.P., Huang C.L., Wang C.J. Inhibitory effect of Hibiscus protocatechuic acid on tumor promotion in mouse skin. Cancer Lett. 1998;126:199–207. doi: 10.1016/S0304-3835(98)00010-X. PubMed DOI

Tseng T.H., Kao T.W., Chu C.Y., Chou F.P., Lin W.L., Wang C.J. Induction of apoptosis by hibiscus protocatechuic acid in human leukemia cells via reduction of retinoblastoma (RB) phosphorylation and Bcl-2 expression. Biochem. Pharmacol. 2000;60:307–315. doi: 10.1016/S0006-2952(00)00322-1. PubMed DOI

Charlton A.J., Baxter N.J., Khan M.L., Moir A.J., Haslam E., Davies A.P., Williamson M.P. Polyphenol/peptide binding and precipitation. J. Agric. Food Chem. 2002;50:1593–1601. doi: 10.1021/jf010897z. PubMed DOI

Friend D.R. Pharmaceutical development of microbicide drug products. Pharm. Dev. Technol. 2010;15:562–581. doi: 10.3109/10837450903369879. PubMed DOI

Benini S., Rypniewski W.R., Wilson K.S., Mangani S., Ciurli S. Molecular details of urease inhibition by boric acid: Insights into the catalytic mechanism. J. Am. Chem. Soc. 2004;126:3714–3715. doi: 10.1021/ja049618p. PubMed DOI

De Oliveira A., Adams S.D., Lee L.H., Murray S.R., Hsu S.D., Hammond J.R., Dickinson D., Chen P., Chu T.C. Inhibition of herpes simplex virus 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate. Food Chem. Toxicol. 2013;52:207–215. doi: 10.1016/j.fct.2012.11.006. PubMed DOI PMC

Hassan S.T.S., Šudomová M. The development of urease inhibitors: What opportunities exist for better treatment of Helicobacter pylori infection in children? Children. 2017;4:2. doi: 10.3390/children4010002. PubMed DOI PMC

Nyam K.L., Leao S.Y., Tan C.P., Long K. 2014. Functional properties of roselle (Hibiscus sabdariffa L.) seed and its application as bakery product. J. Food Sci. Technol. 2014;51:3830–3837. doi: 10.1007/s13197-012-0902-x. PubMed DOI PMC

Carvalho D.O., Curto A.F., Guido L.F. Determination of phenolic content in different barley varieties and corresponding malts by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry. Antioxidants. 2015;4:563–576. doi: 10.3390/antiox4030563. PubMed DOI PMC

ISO 14502–1: Determination of Substances Characteristic of Green and Black Tea—Part 1: Content of Total Polyphenols in Tea—Colorimetric Method Using Folin-Ciocalteu Reagent. International Organization for Standardization; Geneva, Switzerland: 2005.

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. PubMed DOI PMC

Reed L.J., Muench H. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 1938;27:493–497.

Walker W.E., Waisbren B.A., Martins R.R., Batayias G.E. A method for determining sensitivities of antiviral drugs in vitro for possible use as clinical consultation. Am. J. Clin. Pathol. 1971;56:687–692. doi: 10.1093/ajcp/56.6.687. PubMed DOI

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. PubMed DOI

Nishimura T., Toku H., Fukuyasu H. Antiviral compounds. XII. Antiviral activity of amidinohydrazones of alkoxyphenyl-substituted carbonyl compounds against influenza virus in eggs and in mice. Kitasato Arch. Exp. Med. 1977;50:39–46. PubMed

Insights into Antiviral Properties and Molecular Mechanisms of Non-Flavonoid Polyphenols against Human Herpesviruses

Flavonoids Target Human Herpesviruses That Infect the Nervous System: Mechanisms of Action and Therapeutic Insights

Nutraceutical Curcumin with Promising Protection against Herpesvirus Infections and Their Associated Inflammation: Mechanisms and Pathways

Brassicasterol with Dual Anti-Infective Properties against HSV-1 and Mycobacterium tuberculosis, and Cardiovascular Protective Effect: Nonclinical In Vitro and In Silico Assessments

Natural Products-Derived Chemicals: Breaking Barriers to Novel Anti-HSV Drug Development

A Multi-Biochemical and In Silico Study on Anti-Enzymatic Actions of Pyroglutamic Acid against PDE-5, ACE, and Urease Using Various Analytical Techniques: Unexplored Pharmacological Properties and Cytotoxicity Evaluation

Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties

In Vitro Study of Multi-Therapeutic Properties of Thymus bovei Benth. Essential Oil and Its Main Component for Promoting Their Use in Clinical Practice

Anti-Infectivity against Herpes Simplex Virus and Selected Microbes and Anti-Inflammatory Activities of Compounds Isolated from Eucalyptus globulus Labill

Biological Evaluation and Molecular Docking of Protocatechuic Acid from Hibiscus sabdariffa L. as a Potent Urease Inhibitor by an ESI-MS Based Method