In vitro safety assessment of the strawberry tree (Arbutus unedo L.) water leaf extract and arbutin in human peripheral blood lymphocytes

Abstract

Strawberry tree (Arbutus unedo L.) leaves have long been used in the traditional medicine of the Mediterranean region. One of their most bioactive constituents is the glycoside arbutin, whose presence makes A. unedo suitable as a potential substitute for bearberry [Arctostaphylos uva ursi (L.) Spreng] leaves, an herbal preparation widely used for treating urinary tract infections. The safety and biocompatibility of strawberry tree water leaf extract have not yet been documented well. This study estimated arbutin content in strawberry tree water leaf extract (STE) using high performance liquid chromatography. Furthermore, we performed an in vitro safety assessment of the 24 h exposure to three presumably non-toxic concentrations of standardized STE and arbutin in human peripheral blood lymphocytes using the apoptosis/necrosis assay, the alkaline comet assay, and the cytokinesis-block micronucleus cytome assay. The STE was also tested for total antioxidant capacity and lipid peroxidation. At a concentration corresponding to the maximum allowable daily intake of arbutin, the tested extract was not cytotoxic, had a negligible potential for causing primary DNA damage and even hindered micronuclei formation in lymphocytes. It also showed a valuable antioxidant capacity, and did not exert marked lipid peroxidation. These promising results represent a solid frame for further development of STE-based herbal preparations. Although arbutin generally had a low DNA damaging potential, the slowing down of lymphocyte proliferation observed after 24 h of exposure points to a cytostatic effect, which merits further research.

Keywords: Apoptosis; Arbutus unedo L.; Cytokinesis-block micronucleus “cytome” assay; Oxidative stress; Primary DNA damage.

Fig. 1

Strawberry tree (Arbutus unedo L.), a small evergreen tree or shrub native to the Mediterranean

Fig. 2

HPLC–DAD chromatogram of the Arbutus unedo L. water leaf extract. The extract contained 10.7 mg arbutin per g of lyophilisate

Fig. 3

Results of the quantitative fluorescent assay for simultaneous identification of viable, apoptotic and necrotic cells in peripheral blood lymphocytes treated with standardized strawberry tree (STE) water leaf extract and arbutin (ARB) in vitro for 24 h and in the negative (NC) and positive control (PC) samples. Mean values ± SD of three independent evaluations are shown (3 × 100 cells per sample per each experimental point were analysed under epifluorescence microscope, magnification ×400). Positive control—lymphocytes treated for 24 h in vitro with bleomycin at 1.25 µg/mL. Statistical significance of data was evaluated using χ² test. The level of statistical significance was set at P < 0.05. The abbreviations next to the means indicate from which groups the relevant group differs with statistical significance: *versus all other samples; nc—versus negative control; e1—versus sample treated with standardized STE which contained 11.4 µg/mL of arbutin; e2—versus sample treated with standardized STE which contained 200 µg/mL of arbutin; a1—versus sample treated with 11.4 µg/mL of arbutin; a2—versus sample treated with 200 µg/mL of arbutin

Fig. 4

Photomicrographs of lymphocyte nuclei observed under epifluorescence microscope after dual ethidium bromide/acridine orange (EtBr/AO) staining. a viable cell in the negative control; b dead cell in the sample treated with 400 µg/mL of standardized strawberry tree water leaf extract for 24 h; c early apoptosis in the sample treated with 11.4 µg/mL of standardized strawberry tree water leaf extract for 24 h; d late apoptosis in the positive control sample, treated with 1.25 µg/mL of bleomycin for 24 h. Magnification ×1000

Fig. 5

Primary DNA damage in peripheral blood lymphocytes treated with standardized strawberry tree water leaf extract (STE) and arbutin (ARB) in vitro for 24 h and in the negative (NC) and positive control (PC) sample as determined by alkaline comet assay. For each sample, duplicate slides were prepared and two hundred independent comet measurements per sample per experimental point were performed. As indicators of DNA damage, tail length (a) and tail intensity (b) were chosen. Lymphocytes treated ex vivo with 100 μM hydrogen peroxide (H₂O₂) applied for 10 min on ice were used as positive control. Results are shown as the mean value/median, and the range of the measured values (min–max). Statistical significance of data was evaluated using descriptive statistics, ANOVA with post hoc Scheffé’s test (intra-group comparisons). The level of statistical significance was set at P < 0.05. The abbreviations above whiskers indicate which groups differ with statistical significance; from negative control (nc), or all other groups (*)

Fig. 6

Photomicrographs of lymphocyte nuclei observed under an epifluorescence microscope after the alkaline comet assay, and captured using a black and white camera coupled with image analysis software (Comet Assay IV™, Instem-Perceptive Instruments Ltd., Bury Saint Edmunds, UK). a negative control; b sample treated with 200 µg/mL of arbutin for 24 h; c sample treated with 11.4 µg/mL of standardized strawberry tree water leaf extract for 24 h. d positive control (100 μM hydrogen peroxide, 10 min). Stained with ethidium bromide. Magnification ×200

Fig. 7

Photomicrographs of lymphocyte nuclei observed under a light microscope after the cytochalasin B-blocked micronucleus assay. Binucleated lymphocytes observed in the sample treated with 400 µg/mL of standardized strawberry tree water leaf extract for 24 h: a one micronucleus (arrow), b two micronuclei (arrows). Binucleated lymphocytes observed in the sample treated with 400 µg/mL of arbutin for 24 h: c nuclear bud (arrow), d nucleoplasmic bridge (arrow). Binucleated lymphocytes observed in the positive control sample treated with 1.25 µg/mL of bleomycin for 24 h: e late apoptosis, f necrotic cell with a vacuolated cytoplasm (arrows). Stained with Giemsa dye. Magnification ×1000

Fig. 8

Photomicrographs of lymphocyte nuclei observed under a light microscope after the cytochalasin B-blocked micronucleus assay in the negative control sample. The number of nuclei varied depending on the number of nuclear divisions occurring since cytochalasin B addition, which was used to determine the nuclear division index. a mononucleated lymphocyte (M1); b binucleated lymphocyte (M2); c lymphocytes with three nuclei (M3); d lymphocyte with four nuclei (M4). c and d are also called multinucleated lymphocytes. Stained with Giemsa dye. Magnification ×1000

Fig. 9

Effect of standardized strawberry tree water leaf extract (STE) on the extent of lipid peroxidation in lymphocytes treated in vitro for 24 h at three concentrations and in the negative control sample. Results are expressed as means ± SD of four independent measurements. The level of statistical significance was set at P < 0.05. No statistically significant differences were observed

Fig. 10

Effect of standardized strawberry tree water leaf extract (STE) on the total antioxidant capacity in lymphocytes treated in vitro for 24 h at three concentrations and in the negative control sample. Results are expressed as means ± SD of four independent measurements. The level of statistical significance was set at P < 0.05. *Significantly different versus negative control