Elsevier

Phytomedicine

Volume 23, Issue 12, 15 November 2016, Pages 1484-1493
Phytomedicine

Original article
Eremophila maculata—Isolation of a rare naturally-occurring lignan glycoside and the hepatoprotective activity of the leaf extract

https://doi.org/10.1016/j.phymed.2016.08.006Get rights and content

Abstract

Background

The Australian plant Eremophila maculata F. Muell (Scrophulariaceae) is cultivated worldwide as an ornamental plant.

Purpose

This study was designed to assess the antioxidant and hepatoprotective activities of a methanol extract from E. maculata leaves (EMM) both in vitro and in vivo (rats) experiments. Detailed phytochemical study was done on the extract followed by molecular docking experiments on TNF-α ascertain the efficacy of the isolated compounds.

Methods

The antiproliferative activity was evaluated in the human cancer cell lines A-495, PC3 and HepG2 cells using the SRB method. The antioxidant activity was evaluated in vitro using the DPPH• assay while the hepatoprotective properties were investigated by determining the amelioration of CCl4-induced cytotoxicity and oxidative stress in HepG2 cells. The activity was confirmed in vivo by studying tamoxifen-induced hepatotoxicity in rats. An in-depth phytochemical investigation of a methanol extract was performed using 1D and 2D NMR experiments. In silico molecular modeling studies of the isolated compounds on TNF-α (PDB ID 2AZ5) were carried out using Discovery Studio 2.5 software applying C-Docker protocol.

Results

The IC50 values of EMM were > 500 µg/ml for both PC3 and HepG2 cells indicating its safety. Similar to the standard drug silymarin, EMM could restore AST, ALT values; replenish GSH level, SOD activity and TAC in vitro. The hepatoprotective activity was confirmed in vivo in which the extract (20 mg/kg body weight) decreased ALT and AST levels by 45.23 and 45.79%, respectively as compared to the tamoxifen treated groups. Oxidative stress was reduced by lowering of thiobarbituric acid reactive substances by 28.57%. Additionally, hepatocyte inflammation was improved by reducing the pro-inflammatory mediator TNF-α by 54.29%. Phytochemical investigation resulted in the isolation of a rare naturally-occurring lignan glycoside, namely pinoresinol-4-O-[6″-O-(E)-feruloyl]-β-D-glucopyranoside for the first time from the Scrophulariaceae in addition to 12 known compounds.Pinoresinol-4-O-[6′'-O-(E)-feruloyl]-β-D-glucopyranoside was the strongest inhibitor of TNF-α as evidenced from its higher fitting scores comparable to lead compound.

Conclusions

These findings highlighted for the first time that EMM could be an interesting candidate as a safe, natural liver supplement for relieving of various hepatic disorders and counteracting the effect of many xenobiotics.

Introduction

Liver diseases, including hepatitis, cirrhosis, alcoholic liver disease and hepatocellular carcinoma have dramatically increased and are considered to be among the most widespread conditions that may lead to a loss of liver functions and ultimately death (Schiff et al., 2007).

Basically, the main activity of liver toxicants is the stimulation of lipid peroxidation together with other oxidative injuries; consequently, hepatoprotective agents exert their prominent effect by acting as antioxidants, immunomodulators, and anti-inflammatory agents in addition to directly counteracting liver cirrhosis (Chattopadhyay, 2003). Despite the enormous achievements in recent medicinal chemistry developments, no efficient synthetic therapeutic agents have been established to enhance hepatic function, exhibit liver protection or aid in the nourishment of hepatocytes (Adewusi and Afolayan, 2010).

Medicinal plants and drugs derived from them have been extensively used in almost all countries throughout history (Sewell and Rafieian-Kopaei 2014). Several of them were beneficial for the treatment of liver disorders without or with low toxicity or side effects (Heidarian and Rafieian-Kopaei, 2013). The activity of hepatoprotective plants has been related to certain secondary metabolites, such as alkaloids, coumarins, carotenoids, flavonoids, glycosides, lignans, xanthenes, lipids, monoterpenes, and organic acids (Oh et al., 2004, Yan et al., 2009).

Eremophila maculata (Scrophulariaceae), known as Spotted Emu Bush or Native Fuchsia is native to Australia but grown in many countries of the world as an ornamental plant. Traditionally, many Eremophila species have gained popularity among Australians for curing respiratory, gastro-intestinal tract and skin infections (Singab et al., 2013). The genus is known for several beneficial biological activities, including anti-infective, immunomodulatory, anti-inflammatory as well as antiproliferative activities (Tomlinson and Palombo, 2005, Liu et al., 2006, Beattie et al., 2011). Moreover, many secondary metabolites belonging to different classes such as flavonoids, lignans, phenylpropanoids and terpenoids upon which the previously mentioned activities could rely, have been isolated from this genus. Antimicrobial and xanthine oxidase inhibitory activities of E. maculata were assumed to be caused by pinoresinol and its glucoside, prunasin, catalpol and two sesquiterpenes. Besides, the essential oils of its leaf, flower and stem were carefully analyzed, and their antimicrobial properties were assessed (Singab et al., 2013, Youssef et al., 2014).

In spite of the presence of flavonoids, lignans, phenylpropanoids that are known for being potent antioxidants neither the antioxidant nor the hepatoprotective activities of E. maculata have been studied. In this communication, we report on antioxidant and hepatoprotective activities of a methanol extract from E. maculata leaves both in vitro and in vivo (rats) experiments. Furthermore, the phytochemical composition of the methanol extract was studied in detail to identify its main secondary metabolites that may be responsible for the observed activities. Additionally, molecular docking experiments were carried out for the isolated compounds on TNF-α, a major inflammation mediator.

Section snippets

Plant material

Eremophila maculata (KerGawl.) F.Muell. leaves were obtained from a private botanical garden in Egypt (30 km on the Cairo-Alexandria road) between January to April 2011. The plant was kindly identified and authenticated morphologically by Mrs Terease Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture and El-Orman Botanical Garden, Giza, Egypt and further confirmed by Dr. Mohamed El-Gibaly, Department of Botany, National Research Centre (NRC), Giza, Egypt. Furthermore, the plant

Cytotoxic activity

EMM extract exhibited low to no cytotoxicity across all the tested tumor cell lines (PC3, A-495 and HepG2), being more cytotoxic in PC3 as compared to A549 and HepG2 cells (Table 2). These findings suggest that the extract can be safely used to conduct further in vitro and in vivo biological studies.

In vitro antioxidant and hepatoprotective activity

EMM exerted a moderate in vitro antioxidant activity as indicated by reduction of the DPPH radicals attaining an IC50 value of 320 µg/ml. Additionally, treatment of HepG2 cells with EMM at

Discussion

Oxidative stress can be defined as a distinct imbalance in the oxidative-antioxidative equilibrium that ultimately leads to cell injury (Rafieian-Kopaie and Baradaran 2013). Generally, a plethora of reactions can precede this serious imbalance causing disturbances and overproduction of reactive oxygen species (ROS) (Nasri and Rafieian-Kopaei, 2014). Notably, ROS mainly damage lipids, protein and DNA (causing mutations), which can affect gene expression, cellular adhesion, cellular metabolism

Conclusions

In this communication we could show for the first time that the methanol extract of E. maculata leaves displayed promising antioxidant and hepatoprotective activities in vitro and in vivo owing to the presence of non-toxic polyphenolics, mainly of lignans and phenylpropanoids. However, further in depth in vivo investigations on the isolated compounds are needed to confirm the claimed activities before further drug development.

Conflict of interest

The authors declare that they have no conflict of interest and confirm that this work receives no financial supports.

Acknowledgments

We would like to thank Mr. T. Timmermann and Mr. H. Rudi (Department of Pharmaceutical Chemistry, Heidelberg University) for their technical assistance in recording both NMR and MS spectra.

References (40)

  • E.A. Adewusi et al.

    A review of natural products with hepatoprotective activity

    J. Med. Plants Res

    (2010)
  • D.C. Ayres et al.

    Lignans: Chemical, Biological and Clinical Properties

    (1990)
  • A. Baradaran et al.

    Oxidative stress and hypertension: possibility of hypertension therapy with antioxidants

    J. Res. Med. Sci.

    (2014)
  • J. Buege et al.

    The thiobarbituric acid assay

    Methods Enzymol

    (1978)
  • M. Burits et al.

    Antioxidant activity of Nigella sativa essential oil

    Phytother. Res.

    (2000)
  • Y.-W. Chin et al.

    Lignans and other constituents of the fruits of Euterpe oleracea (Acai) with antioxidant and cytoprotective activities

    J. Agric. Food Chem

    (2008)
  • D.A. Daels-Rakotoarison et al.

    Neurosedative and antioxidant activities of phenylpropanoids from Ballota nigra

    Arzneimittelforschung

    (2000)
  • P.M. Dewick

    Medicinal Natural Products : A Biosynthetic Approach

    (2009)
  • S.H. El-Ahmady et al.

    Chemical composition and anti-inflammatory activity of the essential oils of Psidium guajava fruits and leaves

    J. Essent. Oil Res.

    (2013)
  • M. Ellman

    A spectrophotometric method for determination of reduced glutathione in tissues

    Anal. Biochem.

    (1959)
  • Cited by (36)

    • Authentication and discrimination of green tea samples using UV–vis, FTIR and HPLC techniques coupled with chemometrics analysis

      2019, Journal of Pharmaceutical and Biomedical Analysis
      Citation Excerpt :

      After incubation in the dark for 30 min at room temperature, the absorbance was then measured against a blank at λmax = 520 nm using Awareness Technology ChroMate® Microplate Reader (Florida, USA) and compared to DPPH• control after background subtraction. The percent inhibition was calculated from three different experiments using the following equation [12]. Inhibition (%) = [Ac – As/Ac] × 100,Where, Ac: absorbance of control; As: absorbance of sample.

    • New flavonoid glycosides from two Astragalus species (Fabaceae) and validation of their antihyperglycaemic activity using molecular modelling and in vitro studies

      2018, Industrial Crops and Products
      Citation Excerpt :

      Docking was carried out using C-docker protocol applying both pH-based ionization and rule-based ionization methods. Docking was performed in a similar manner as previously described (Youssef et al., 2016, 2017; Ashour et al., 2017). However, the free binding energies were calculated in Kcal/mol using the following equation:ΔGbinding = Ecomplex − (EAG + E ligand)Where; ΔGbinding: The ligand–enzyme interaction binding energy, Ecomplex: The potential energy for the complex of AG bound with the ligand, Eprotein: The potential energy of AG alone and Eligand: The potential energy for the ligand alone.

    • Validation of the antihyperglycaemic and hepatoprotective activity of the flavonoid rich fraction of Brachychiton rupestris using in vivo experimental models and molecular modelling

      2018, Food and Chemical Toxicology
      Citation Excerpt :

      Although numerous synthetic drugs have been discovered for the management of diabetes mellitus and liver disorders, some of them resulted in a plethora of serious intolerable adverse effects and undesirable drug interactions (Youssef et al., 2017). Scientific community always turn to nature to mine its treasures and find bioactive molecules that are relatively safe, inexpensive and acceptable by a large category of people both in developing and developed countries in compare with the synthetic agents (Youssef et al., 2016). Family Malvaceae comprises nearly 200 genera with 2300 species of flowering plants.

    • Furofuran lignans from the Simpson Desert species Eremophila macdonnellii

      2018, Fitoterapia
      Citation Excerpt :

      The thriving diversity that has evolved to survive in extreme Australian arid landscapes has to-date only attracted meagre attention in terms of desert plant bioactive profiling and secondary metabolite discovery. Of those species that have been investigated thus far (e.g. Acacia victoriae [1–4], and Myoporum deserti [5,6]), the majority of investigations have focussed on the Eremophila family (i.e. E. neglecta [5,7,8], E. serrulata [9], E. mitchellii [10,11], E. microtheca [12], E. longifolia [13], E. duttonii [14–16], E. maculata [17,18], E. sturtii [19], E. alternifolia [20], E. latrobei [21], E. freelingii [22], E. bignoniifloru [23], E. cuneifolia [24], E. dalyunu [25], E. fruseri [26], E. gilesii [27], E. foliosissima [27], E. georgei [27], E. paisley [28]). Much of the inspiration behind these substantial efforts has been in the pursuit of evaluating biological activity as informed by Australian indigenous traditional medicine [7,9,10,12–14,16,18,19,20,22–29].

    • Phenolic compounds from Syzygium jambos (Myrtaceae) exhibit distinct antioxidant and hepatoprotective activities in vivo

      2018, Journal of Functional Foods
      Citation Excerpt :

      Pretreatment of animals with SJE significantly reduced the levels of the hepatic markers relative to the CCl4-challenged group; effects are similar to those of silymarin (Table 2). Similar activities were reported for other plant crude extracts (Abbas & Wink, 2014; Youssef et al., 2016). The pathogenesis and progression of liver diseases is often associated with oxidative stress, which is also pivotally involved in promoting drug-induced hepatotoxicity (Al-Sayed, El-Lakkany, Seif el-Din, Sabra, & Hammam, 2014).

    View all citing articles on Scopus
    View full text