Brine shrimp cytotoxic activity of methanolic extracts of Juniperus communis L . berries

Methanolic extracts from Juniperus communis L. berries collected from five different localities in the Republic of Macedonia were evaluated for their cytotoxicity by Brine shrimp lethality assay. The obtained cytotoxic activity is descending as follows: Pelister (128 μg/ mL) > Jakupica (221 μg/mL) > Prilep (662 μg/mL) > Demir Hisar (863 μg/mL) > Makedonski Brod (969 μg/mL). Berries collected from mountain areas (Pelister and Jakupica) demonstrated prominent cytotoxic effects, while berries collected from localities near urban areas exhibited lower cytotoxicity. Variations in their bioactivity are probably due to their complex phytochemical composition, which may vary with different ecological and geographical conditions.


Introduction
Juniperus communis L. (common juniper) is an evergreen shrub belonging to the family Cupresaceae and is widely distributed throughout the Northern Hemisphere, from cool temperate to polar regions.Juniper trees are adapted for optimal growth on a wide range of soils and soil pH, and are drought tolerant (Lim, 2012).Common juniper shrubs grow natively in all parts of the Republic of Macedonia (Micevski, 1998).Ripe berries and needles have long been used for the treatment of gastrointestinal conditions, dyspepsia, flatulence, egzema (Charles, 2013).Indigenous people from the East were using the juniper berries as herbal tea to treat urinary tract infections and as tonic against flu, muscle aches and kidney problems, while the needles were mainly used as remedy for acute and chronic cystitis, albuminuria, renal suppression, amennorhoea (Bais et al., 2014).Despite the wide usage in traditional medicine, the biological activity of the Juniperus communis berries still remains insufficiently examined.Previous studies on the biological activity of the berries demonstrated their antibacterial, fungicidal (Pepeljnjak et al., 2005), antioxidant (Höferl et al., 2014), anticholinesterase (Orhan et al., 2011) and antiinflamatory (Han and Parker, 2017) activity.However, limited data are reported on their cytotoxic properties.Therefore, the aim of this study was in vivo determination of the cytotoxic potential of Juniperus communis L. berries collected from several localities of the Republic of Macedonia.

Plant materials
The plant material was harvested in the year 2010 and 2011 from five different localities: Pelister (JCP), Jakupica

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(JCJ), Prilep (JCPr), Demir Hisar (JCH) and Makedonski Brod (JCB).Dried plant material was milled to fine powder and stored in airtight containers until use.

Preparation of extracts
Plant samples were extracted with methanol in ultrasonic bath.Obtained filtrates were evaporated until dry and later freeze dried and preserved at -18 °C in a dark place until use.Lyophilizates were reconstituted with DMSO (dimethyl sulfoxide).

Brine shrimp lethality assay
The cytotoxic potential of the extracts was evaluated by Brine Shrimp Lethality Assay in vivo (Meyer et al., 1982) based on the number of dead Artemia salina nauplii after 24 hours exposure to the plant samples.K 2 Cr 2 O 7 (potassium dichromate) was used as a positive control in a concentration range from 0.01 to 10 mg/mL.Final results were expressed as LC 50 values using probit regression analysis (Finney, 1952).LC 50 values represent the concentration of each extract needed to elicit mortality in 50% of the tested population of brine shrimps.
The trend of mortality rate for the Artemia nauplii after their 24-hour exposure to each sample and to the positive control are demonstrated on Figure 1.A notable increase in toxicity was observed for the samples collected from Pelister (40% to 100%) and Jakupica (50% to 100%) at concentrations 1 mg/mL and 3 mg/mL, and for the positive control K 2 Cr 2 O 7 (100% mortality rate at 1 mg/mL).Moreover, a maximal mortality rate of 100% at the highest concentration (10 mg/mL) was observed for all samples except for the berries collected from Makedonski Brod.
J. communis berries collected from the localities of Pelister and Jakupica were classified as moderately toxic, while samples collected from locations near the cities of Prilep, Demir Hisar and Makedonski Brod were classified as extracts with low toxicity.The amount of certain bioactive compounds present in the berries may significantly vary as a result of differences in temperature, soil chemistry, soil acidity (Ložienė and Labokas, 2012), light quality (Artemkina et al., 2016), rainfall levels, season (Peñuelas et al., 2002) and altitude of the habitat (Martz et al., 2009).Ecological conditions of plant growth may significantly change the phytochemical composition of the plants upon transition from the base to the top of the mountains (Artemkina et al., 2016).In the current research, a moderate cytotoxicity was obtained for the samples collected from mountain areas (Pelister and Jakupica), while the berries collected from the localities near urban area with an altitude range from 550 m to 650 m (Prilep, Demir Hisar and Makedonski Brod) demonstrated low cytotoxicity.The variations in their cytotoxic activity are probably related to their complex phytochemical composition.This observation is supported by previous research on the variability in concentration of bioactive compounds present in Juniperus berries as a result of different latitude and altitude of the

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habitat.The proportion of limonene, sabinene, 3-carene, myrcene, β-pinene and terpinolene, the most abundant compounds in the juniper essential oil was significantly higher in species collected from higher altitudes compared to lower habitats, as it was demonstrated by Martz et al. (2009).The proportion of a-pinene was affected insignificantly by the geographical factors (Ložienė and Labokas, 2012).Taking into account their complex phytochemical composition, it is highly probable that the bioactive profile of the examined species is due to the synergic action of several phytochemical components with cytotoxic potential.Additionally, LC 50 values obtained for the most potent samples in the current research were similar to obtained values for the cytotoxic potential against several in vitro cell lines demonstrated in previous studies on the bioactivity of Juniperus communis berries collected from different localities.In the study of Ghaly et al. (2016), treatment with methanolic extracts from the berries resulted in a significant cytotoxic activity against human breast cancer cells MCF7 and the human colon cancer cells HCT-116.The aquaeous extract of leaf and berries from Juniperus communis L. var.saxatilis Pall.demonstrated cytotoxic and antitumor activity against CRPC (Castration-resistant prostate cancer) cells in the study of Eryilmaz et al. (2017).The anticancer activity of Juniperus communis berries is also possible via induction of cell cycle arrest which has an important role in cancer prevention.This type of inhibitory activity was observed for isolated compounds from methanolic extract of J. communis berries against the cell cycle progression in p53-null human cancer CaLu-6 cells in the study of Marino et al. (2011).
Furthermore, our results are in conjuction with previously reported studies on other species from the Juniperus genus.Methanolic extracts from Juniperus drupacea Labill.berries demonstrated significant cytotoxic potential against Artemia larvae (LC 50 489.47± 27.8 μg/ mL) and a significant decrease in HepG2 cells viability (5-10 μg/mL) (Miceli et al., 2011).Moreover, similar results were also obtained for the hydroalcoholic extract of Juniperus sabina berries against HeLa and MDA-MB-468 cells in the study of Jafarian-Dehkordi et al. (2004).

Conclusion
Overall, Juniperus communis berries were identified as samples with a certain cytotoxic potential in vivo, and the obtained results suggest that the amount of bioactive compounds could be correlated with the altitude of their habitat.Berries collected from Pelister and Jakupica demonstrated moderate cytotoxicity, while the berries collected from localities near Prilep, Demir Hisar and Makedonski Brod were characterized with low cytotoxic

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activity.Obtained results in the current research emphasize the need of more intensive natural product screening for new bioactive compounds with cytotoxic properties.Therefore, further studies would be needed to clarify the mechanism of cytotoxicity and to identify the responsible bioactive compounds present in berries from Juniperus communis L.

Table 1 .
Cytotoxic potential of methanolic extracts of Juniperus communis berries collected from 5 different localities and a positive control according the Brine Shrimp Lethality Assay