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. 2014 Mar;31(1):29-36.
doi: 10.5152/balkanmedj.2014.8377. Epub 2014 Mar 1.

Assessment of eyebright (euphrasia officinalis L.) extract activity in relation to human corneal cells using in vitro tests

Roman Paduch  1 , Anna Woźniak  2 , Piotr Niedziela  3 , Robert Rejdak  2
Affiliations

Assessment of eyebright (euphrasia officinalis L.) extract activity in relation to human corneal cells using in vitro tests

Roman Paduch et al. Balkan Med J. 2014 Mar.

Abstract

Background: Euphrasia officinalis L. is an herb traditionally used in folk medicine, mainly in the treatment of eye disorders.

Aims: The present study analyzed the activity of three extracts of E. officinalis L. (ethanol, ethyl acetate and heptane) on cultured human corneal epithelial cells (10.014 pRSV-T).

Study design: In vitro study.

Methods: Toxicity, free radical scavenging activity and the immunomodulatory effects of the extracts were tested using the thiazolyl blue tetrazolium bromide (MTT) or Neutral Red, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ELISA tests, respectively. Moreover, nitric oxide levels and cytoskeleton architecture were analyzed after corneal cell incubation with the plant extracts.

Results: We show that the biological effect depended on both the concentration and the extraction solvent used. Heptane extracts, distinct from those in ethanol and ethyl acetate, were toxic to 10.014 pRSV-T cells at low concentrations (25 μg/mL) and did not demonstrate free radical scavenging effects. All tested extracts decreased pro-inflammatory cytokine expression (IL-1β, IL-6 and TNF-α) and also anti-inflammatory IL-10 expression by human corneal cells when the extracts were added to the cell culture medium for 24 h.

Conclusion: In conclusion, we show that the promising effects of the application of E. officinalis L. preparations as a supplementary therapy for eye disorders are associated with the ethanol and ethyl acetate extracts, not the heptane extract.

Keywords: Cytotoxicity; Euphrasia officinalis extracts; human corneal cells; immunomodulation; reactive oxygen species scavenging effect.

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Figures

FIG. 1. a, b.
FIG. 1. a, b.
The effect of 24 h treatment of 10.014 pRSV-T with methanol, ethyl acetate and heptane extracts of E. officinalis L. The Neutral Red assay (NR) (a) and MTT assay (b). The results are presented as a percentage of the control, arbitrarily set to 100%. The figure shows an average of three independent experiments
FIG. 2.
FIG. 2.
Nitric oxide (NO) secretion in culture of 10.014 pRSV-T human normal corneal cells during 24 h of incubation with the methanol, ethyl acetate and heptane extracts of E. officinalis L. Two concentrations of the extracts were used: 8 μg/mL and 20 μg/mL. Analysis was performed using the Griess method. Columns and bars show the mean±standard deviation (n=3). *p≤0.01 - culture of corneal cells treated with plant extracts compared to the non-treated culture control
FIG. 3. a–d.
FIG. 3. a–d.
IL-1β (a), IL-6 (b), TNF-α (c) and IL-10 (d) secretion as assessed by ELISA in the culture of 10.014 pRSV-T human normal corneal cells after 24 h of incubation with the methanol, ethyl acetate and heptane extracts of E. officinalis L. Two concentrations of the extracts were used: 8 μg/mL and 20 μg/mL. *p≤0.01 - culture of corneal cells treated with plant extracts compared to the non-treated culture control
FIG. 4.
FIG. 4.
Cytoskeleton organization and reciprocal, direct interactions among human normal corneal cells (10.014 pRSV-T). Control sample. Fluorescent staining of cells using TRITC-phalloidin dye. Magnification 400×. Bar 50 μm
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