Elsevier

Journal of Chromatography B

Volumes 1068–1069, 15 November 2017, Pages 335-342
Journal of Chromatography B

UPLC–PDA–ESI–qTOF-MS profiling and potent anti-HSV-II activity of Eucalyptus sideroxylon leaves

https://doi.org/10.1016/j.jchromb.2017.10.065Get rights and content

Highlights

  • UPLC–MS analysis allowed the identification of 70 metabolites in E. sideroxylon (Myrtaceae) leaves

  • Phloroglucinol-rich extract (PGRE) was found to be non-cytotoxic (CC50 is 0.808 mg/mL).

  • Potent antiviral activity was observed against HSV-II (87.65% inhibition).

  • PGRE decrease HSV-II replication and attachment on VERO cells.

  • Weak antiviral activity against HSV-I (8.88%) and no activity against CoxB4 and hepatitis A viruses were reported.

Abstract

Eucalyptus is one of the most important and highly exploited genus in family Myrtaceae. An UPLC/PDA/ESI-qTOF-MS method was adopted to identify Eucalyptus sideroxylon Cunn. ex Woolls leaves phytoconstituents. Cytotoxicity of E. sideroxylon leaves phloroglucinol-rich extract (PGRE) on VERO cells was determined. The antiviral effect of PGRE against hepatitis A (HAV), herpes simplex type 1 (HSV-I), herpes simplex type 2 (HSV-II), coxsackie (CoxB4), and adenoviruses was in vitro evaluated using MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). UPLC–MS analysis allowed the identification of 70 metabolites including: 26 triterpenes, 13 phloroglucinols, 8 fatty acids, 5 flavonoids, 5 oleuropeic acid glucosides, 3 gallic acid derivatives, and 10 miscellaneous. Twenty four metabolites identified in the leaves of E. sideroxylon and four in the genus Eucalyptus are reported herein for the first time. PGRE was found to be non-cytotoxic; the concentration that reduced the cell viability by 50% (CC50) was 0.808 mg/mL. Maximum non-toxic concentration (MNTC) of PGRE on Vero cells was 0.312 mg/mL. The best antiviral activity was observed against HSV-II. Its mechanism was through decreasing the viral replication (IC50 189.36 μg/mL, 87.65% inhibition) and attachment on Vero cells (IC50 199.34 μg/mL, 83.13% inhibition) rather than virucidal effect (IC50 293.1 μg/mL, 50.68% inhibition). This study provides a complete map for E. sideroxylon leaves composition. It also suggests the plant as a source of new antiviral agents.

Introduction

Eucalyptus is a diverse genus of Myrtaceae flowering trees and shrubs. It comprises more than 700 species that are widely distributed and native to Australia. Eucalyptus leaves accumulate a very large number of secondary metabolites. Phloroglucinol-terpene adducts, phloroglucinol dimers, flavonoids, tannins, triterpenes, and glucose esters of oleuropeic acid are among the most famous Eucalyptus metabolites. In addition, leaves of Eucalyptus species were reported to possess several biological activities [1], [2], [3], [4]. Eucalyptus sideroxylon Cunn. ex Woolls known as red-iron bark, iron wood, and mugga [5], [6]. The leaves are used in the production of cineole based eucalyptus oil [7]. Many Eucalyptus species essential oils and metabolites are well known antiviral agents [8], [9], [10], [11], [12], [13]. One of the major challenges is the in vivo practical use of essential oils. Thus we focused the antiviral potency evaluation on the total extract of E. sideroxylon leaves not its essential oil.

The outbreaks of several viruses worldwide in last decades drove the author’s interest towards the evaluation of E. sideroxylon antiviral activity. Herpes simplex virus (HSV) type I and II were among the selected viruses to be screened; they cause common infections worldwide [14]. HSV-I and HSV-II are the cause of most genital herpes [15]. Coxsackie B virus was also selected, it is linked to insulin dependent diabetes mellitus a disease with a high distribution all over the world [16]. Adenovirus has a well-documented role in obesity [17], which in turn is a direct cause of several illness. In addition, hepatitis A virus (HAV), infects liver cells and impair liver function[18]. In spite of the presence of several reports [19], [20], [21] concerning the leaves of E. sideroxylon; nothing is known about its antiviral potential. Also, the leaves secondary metabolites are not yet well explored. Thus, we focused this study on analysis of its leaves secondary metabolites to present a complete image for its constituents using UPLC/PDA/ESI-qTOF-MS method.

Section snippets

Plant material

Eucalyptus sideroxylon Cunn. ex Woolls leaves were collected, in April 2013, from El-Kobba palace, Cairo. Identity of the plant was authenticated by the Royal Botanic Gardens Herbarium, Kew, Richmond, Surrey, United Kingdom. A voucher specimen (No. 27.04.16.1) was deposited in the museum of Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

High resolution UPLC–MS analysis

Dried leaves were ground, extracted with methanol and analyzed using reversed phase ultra performance liquid chromatography, coupled to

High resolution UPLC–MS analysis

The base peak chromatogram is presented in Fig. 1. Retention times, observed molecular weight and fragment ions for each metabolite, and their identities are presented in Table 1.

The E. sideroxylon leaf UPLC-ESI–MS chromatogram (Fig. 1) was characterized by two main regions; the first one with retention time 30 till 299 s with peaks mainly due to flavonoids and oleuropeic acid derivatives and the second region starts from 300 till 700 s for triterpenes, phloroglucinols and fatty acids. A total of

Discussion

Acylphloroglucinols are phenolic compounds biosynthesized by intramolecular Claisen-type acylation between atoms C-1 and C-6 of the corresponding acetogenin. A wide range of acylphloroglucinols are biosynthesized in Eucalyptus species. The most common acylpholoroglucinols in the genus are the typical diformyl monomeric phloroglucinols, dimeric acylphloroglucinols, phloroglucinol glycosides, acylphloroglucinol-monoterpene adducts, and phloroglucinol-sesquiterpene adducts [26]. E. sideroxylon

Conclusion

The UPLC–MS fingerprint of E. sideroxylon Cunn. ex Woolls leaves provides the first metabolome based comprehensive profile of different metabolites of the plant and provides chemically based evidence for its identification among other Eucalyptus species. Its efficacy against HSV-II and adenovirus will be greatly facilitated by further clinical studies. Its combination with other naturally discovered antiviral agents may decrease the risk of development of viral strains resistance to drugs.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgment

The authors are deeply thankful to Prof. Dr. Mohammed Farag, Prof. in Faculty of Pharmacy, Cairo University, for his help in the UPLC analysis.

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