Juniperus Communis L

Abstract

Juniperus communis L. is commonly used in various applications, from pharmaceutical to food industry and from cosmetics to beverage industry. Nevertheless, the main utilisation of juniper since ancient time is as a medicinal plant. The aim of the study is to present, for the first time in the literature, the potential uses of a hydroalcoholic extract obtained from wild-growing Romanian native juniper berries, and to evaluate its antioxidant, antifungal and anti-inflammatory effects. The hydroalcoholic extract was obtained from wild-growing Juniperus communis L. (voucher specimens nos. 40003 and 40004 – BUAG Herbarium), and consisted of 20 g of shade-dried berries in 1:1 mixture water-ethanol (100:100 mL). The extract was characterised by UV-Vis spectrometry, GC-MS and phytochemical assays. The antioxidant potential was evaluated using the DPPH assay and the antifungal effect was studied on two fungal lines (Aspergillus niger and Penicillium hirsutum). The anti-inflammatory effect was evaluated in two inflammation experimental models (dextran and kaolin) by plethysmometry. The analytical characterisation revealed the chemical composition, as well as the total terpenoids, flavonoids and phenolics content of the extract. The in vitro studies prove that the extract has good antioxidant and antifungal potential. The in vivo evaluation of the extract (prepared as a microemulsion) shows a significant anti-inflammatory effect in the tested experimental models. The results obtained from the in vivo and in vitro evaluations recommend the Juniperus communis L. hydroalcoholic extract as a promising biologically-active material.

Keywords: Juniperus communis L.; extract; microemulsion; antioxidant; antifungal; anti-inflammatory.

1. Introduction

Natural products (extracts or essential oils) obtained from various plants are complex mixtures, containing hundreds of organic compounds that are usually used as food, beverages flavouring and aroma agents (Soukand et al., 2015). Medicinal plants are considered a very important source of new compounds for drug development (Lantto et al., 2009; Samaradivakara et al., 2016). These natural products are currently promoted as anticancer, anti-diabetic, antibacterial, antiviral, and antioxidant agents, and in various other medical applications (Lantto et al., 2009; Barros et al., 2010; Kritsidima et al., 2010; Ortan et al., 2015; Samaradivakara et al., 2016; Sutan et al., 2016) . Most researchers assign the therapeutic activity of natural products to their polyphenolic compounds (Soobrattee et al., 2005).

The Juniperus L. genus consists of 67 species and 34 varieties (Adams, 2008). The most common juniper species in Central and Southeast Europe is Juniperus communis L., which can be identified based on macroscopic and microscopic differences compared to other species of juniper (Bercu et al., 2009; Lakusic and Lakusic, 2011).

Juniperus communis L. is an evergreen tree growing in many regions in Eurasia, North Africa and North America. Its usable parts (berries – Juniperi fructus and needles – Juniperi foliage) contain an essential oil with a characteristic flavour. The essential oil and extracts obtained from juniper can be used as a diuretic, in gastrointestinal diseases, renal, genital, pulmonary and rheumatic disorders, in pharmaceutical and food industries, perfumery or in cosmetics (Bojor, 2003).

Recent papers studied the use of juniper extracts natural products (essential oil or extracts) mainly as antioxidants (Hoferl et al., 2014) and antimicrobial agents (Pepeljnjak et al., 2005; Gordien et al., 2009). Their hypoglycemic and hypolipidemic effects and cytotoxic activity were also investigated (Ju et al., 2008).

However, when dealing with natural extracts it is important to consider that their composition strongly depends on various ecological factors, the time period, weather conditions and area of harvesting, cultivation technology, biological value of the cultivar, and, last but not least, the extraction and processing techniques (Shanjania et al., 2010; Sultana et al., 2009; Kurti et al., 2015; Fierascu et al., 2015). Thus, it is possible that different researchers will obtain different results for the same plant species.

The present paper describes the preparation, chemical characterisation, and the assessment of antioxidant, antifungal and anti-inflammatory properties of a hydroalcoholic extract obtained from the fruits of Romanian native Juniperus communis L.

2. Material and methods

2.1 Plant material and extraction techniques

Wild-growing Juniperus communis L. was identified in Dobresti area, Pitesti hills (N 44°57’48”, E 25°6’58”, 450 meters above sea level) in 2014. Voucher specimens nos. 40003 and 40004 were deposited in BUAG Herbarium, Bucharest for future reference.

Fruits were carefully collected in order to choose only the ripe ones, as fruits in all stages of a multi-annual ripening cycle (which covers a period of approx. 18 months) are usually found on the same plant (Farjon, 2005).

The Juniperus communis L. extract used for the study was obtained from ground shade-dried fruits (approx. 20 g.) in 1:1 mixture water-ethanol (100:100 mL). This method was previously described by our group as appropriate for obtaining valuable hydroalcoholic extracts (Fierascu et al., 2015; Ortan et al., 2015). The analytic grade ethanol used for all experiments was purchased from Merck KGaA (Germany), while the bidistilled water was prepared in our laboratory using a GFL 2102 water still.

2.2 Analytical characterisation methods

In order to evaluate its chemical composition, the extract was characterized by modern analytical techniques, Gas chromatography–mass spectrometry and UV-Vis spectrometry

An UV-Vis spectrometer Unicam Helios α Thermo Orion was used to acquire scans from 200 to 900 nm, at a resolution of 1 nm, with 1 nm slit width and automatic scan rate. Results were processed using specialized data analysis software (Origin Pro 8.0). The Extraction Factor (EF) of the extract was determined from the absorption values (A λmax) multiplied with the dilution factor (DF) (Bunghez et al., 2013; Fierascu et al., 2015).

A Varian 3800 gas chromatograph coupled to a Varian 2000 mass spectrometer (GC–MS) was used to analyze the natural products. An analytical column FactorFour WCOT fused silica column (stationary phase: VF- 624 ms; column length: 30 m; inside diameter: 0.25 mm; film thickness: 1.40 mm) supplied by Varian Inc. was used for the separation of analytes. The GC–MS conditions were as follows: column temperature, programmed from 50°C (held for 1 min) to 280°C (held for 10 min) at a rate of 6°C min−1; injector temperature, 200°C; injection mode, splitmode (20); helium carrier gas flow rate, 1.0 mL min−1; MS transfer temperature, 280°C; ion source temperature, 250°C; ionization mode, electron impact; ionization energy, 70 eV; mass scan range, m/z 50–650. Output files were analyzed using Varian MS workstation version 6 and the NIST98 Mass Spectral Database. Before injection, the extract was first evaporated using a rotary evaporator and then diluted using a non-polar solvent of choice (hexane, 1 g / 10 mL of solvent).

For the study of the microemulsion formulations, conductibility studies were performed using a Corning 441 conductivity meter (Corning, NY, USA). The refractive index was determined at 25ºC using a digital Abbe refractrometer. The mean diameter of the droplets and the Zeta potential of the microemulsion were measured using a Mastersizer 2000 (Malvern, UK) particle size analyser.

2.3. Phytochemical Analyses

Phytochemical quantification procedures were applied for the determination of total terpenoids (Misra and Dey, 2012), total flavonoids (Singleton and Rossi, 1965) and total phenolics content (Maurya and Singh, 2010) of the extract. The procedures were described in detail in our previous papers (Fierascu et al., 2015; Ortan et al., 2015). Results are presented as standard equivalents per gram of dried extract.

2.4. Antioxidant assay

The antioxidant activity was determined using the DPPH assay.

For the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, 0.5 mL of sample was mixed with 1 mL of 0.02 mg/mL DPPH solution (Sigma Aldrich, USA). After a 30-minute incubation period, solutions were analyzed by reading the absorbance at 517 nm on the UV-Vis spectrophotometer. Bidistilled water was used for the blank sample.

The antioxidant activity percentage (AA%) was calculated using the formula:

(1)

where Acontrol is the absorbance of the DPPH solution without sample, and Asample is the absorbance of the extract mixed with 0.02 mg/mL DPPH solution (Fierascu et al., 2015).

The half maximal effective concentration (EC50) was calculated using specialized data analysis software (Origin Pro 8.0) (Chen et al., 2013).

2.5 Determination of antifungal effect

The antifungal activity was evaluated using the disc diffusion or Kirby-Bauer method (Bauer et al., 1966; Soare et al., 2012; Jorgensen and Turnidge, 2015). The antifungal activity was tested against two relevant fungal strains, Aspergillus niger (ATCC 15475) and Penicillium hirsutum (ATCC 52323). The stock culture was maintained at 4°C.

The fungal strains were cultivated onto potato-dextrose agar (PDA) from Sigma-Aldrich, with the following composition: agar, 15 g/L, dextrose, 20 g/L and potato extract, 4 g/L.

Sterile PDA plates were prepared by pouring the sterilized media into sterile Petri dishes under aseptic conditions. The test organism (1 mL) was spread on the plates. Wells were made using a sterile Durham tube of 6 mm diameter, and were inoculated with 50 μL of hydroalcoholic extract. Each plate also carried a blank well containing solvent (ethanol:H2O = 1:1) alone to serve as a negative control. Miconazole nitrate (Sigma-Aldrich) solution (30 µg/mL) was used as positive control. All plates containing fungal strains were incubated at 37°C for 84 hours.

The antifungal activity of the hydroalcoholic extract against the microorganism species was determined by measuring the sizes of inhibition zone (IZ, mm) as clear, distinct zones of inhibition surrounding the agar wells. Values < in diameter were considered as not active against microorganisms. The percent inhibition of the target fungi was calculated according to the formula:

(2)

where IZ – inhibition zone diameter and NC – negative control.

All data were expressed as the mean ± standard deviation (SD) by measuring three independent replicates. Standard deviation was calculated as the square root of variance using STDEV function in Excel 2010.

2.6 Microemulsion preparation

Due to the general low bioavailability of the polyphenolic compounds, the extract was formulated as a microemulsion and tested for anti-inflammatory action (Rios-Hoyo et al., 2014; Mahmood et al., 2015).

Equilibrium solubility experiments were carried out in order to select the appropriate oil, surfactant, and cosurfactant constituents of the microemulsion. An excess amount of extract was added to 5 mL of oil, surfactant or co-surfactant, and the resulting mixture was stirred for 30 seconds at 2500 rpm on an IKA Genius 3 vortex mixer (IKA Werke GmbH & Co. KG, Staufen, Germany). The mixture was then shaken (250 rpm) at room temperature for 24 h on a IKA HS 260 orbital shaker (IKA Werke GmbH & Co. KG, Staufen, Germany), followed by centrifugation for 10 min at 12,000 rpm on a Hettich Mikro 220R centrifuge (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). The supernatant was filtered through a 0.45 μm Teflon® filter, and UV spectra were recorded after suitable dilution. Based on preliminary solubility experiments, oleic acid, Tween 80 and propylene glycol were selected as the oil phase, surfactant, and as cosurfactant, respectively.

The behaviour of the multi-component microemulsion system was studied by constructing pseudo-ternary phase diagrams. The ratio of surfactant to co-surfactant was fixed at 1:1 based on their weights. Oleic acid was mixed with the surfactant: co-surfactant mixture at ratios of 1:9, 2:8, 3:7, 4:6, 5:5. 6:4, 7:3, 8:2, 9:1, and distilled water was added to the mixture in increments of 100 μL by micropipette under vigorous shaking. In order to reach equilibrium prior to further evaluation, the resulting samples were maintained at 25°C for 24 h. The mixtures were then visually assessed and samples that remained homogeneous and visually transparent were selected as microemulsions. The same procedure was applied when preparing microemulsions containing Juniperus communis L. extract, with the extract being dispersed into the surfactant: cosurfactant mixture.

2.7 In vivo determination of the anti-inflammatory effect

Male Wistar rats weighing 212 ± 45g from the University of Medicine and Pharmacy, Bucharest animal facility (rodent farm) were used for the in vivo studies. The rats were housed in plastic cages in an air-conditioned room and fed on granulated food with free access to water. Temperature and relative humidity were continuously monitored with a thermohygrometer; the temperature was kept between 20 and 22 C, and the relative humidity was generally maintained at 35-45 %.

Inflammation was evaluated in two inflammation experimental models by plethysmometry (Ugo Basile 7140 Plethysmometer), as previously reported (Badea et al., 2014). Inflammation was induced by intraplantar administration of 0.2 mL inflammatory agent (0.6% solution of dextran and 10 % aqueous suspension of kaolin, respectively) into the rat’s inferior right paw. The anti-inflammatory effect was compared with a negative control group (untreated rats), a positive control group (rats treated with the microemulsions vehicle) and a reference substance (diclofenac) group.

A number of 64 male Wistar rats were randomly distributed in 8 groups of 8 animals. For both inflammatory agents (dextran and kaolin), the negative control rats were administered by gavage with distilled water, 1 mL/100 g body weight (b.w.); the positive control rats were treated by gavage with the microemulsions vehicle, 1 mL/100 g b.w., the reference rats were treated by gavage with 1 % solution sodium diclofenac, 100 mg/kg b.w., and the experimental group was treated by gavage with 1 mL/100 g b.w. of microemulsions containing juniper extract. After the administration of substances, all animals were anesthetized intraperitoneally with 13 % solution of urethane, 130 mg/kg b.w. and the initial paw volume was measured. Paw edemas was further evaluated at 1, 2, 3, 4, 5 and 24 hours after the inflammatory agent administration. The research was conducted in accordance with the European Community guidelines (86/609/).

The statistical analyses were performed using Origin 8.0 Pro. The evolution of paw edema was calculated using the formula

(3)

where V0 is the initial paw volume and Vxh is the paw volume at each time measurement. The anti-inflammatory effect was calculated as the difference between the evolutions of paw edema of the treated groups and the negative control group or the reference group. Results are expressed as mean ± standard deviation. The applied parametrical tests (t test, one way ANOVA) have a 90 % confidence interval and statistical differences were considered for p value < 0.05.

3. Results and discussions

The UV-Vis analyses of the extract (Figure 1) identifies peaks at wavelengths specific to phenolic acids (220-280 nm) and to flavonoids and quinones (290-420 nm) (Bunghez et al., 2013). No peaks were observed for the specific wavelengths of chlorophylls (around 605 and 665 nm). Table 1 presents the specific absorption values for the plant extract, as well the extraction efficiency (EF factor) calculated according to the formula described in Section 2.2.

The extraction efficiency strongly depends on solvent polarity and the polarity of the plant compounds. In our case, the solvent used for the extraction (ethanol/water) proved to be very efficient in extracting phenolic compounds, as indicated by the high values of EF220-280nm. The EF290-420 nm values specific to flavonoids and quinones were relatively low (EF=11.28 and 59.94, respectively), while the absence of values for the EF600-670nm indicates no extraction of chlorophylls.

GC-MS characterization of the juniper extract identifies 57 compounds. The GC-MS chromatogram is shown in Figure 2, while the identified components (based on comparison of the GC-MS spectra and RI with those of internal NIST library) are summarized in Table 2.

Phytochemical evaluations of the crude extract were performed by spectrophotometric methods as described in Section 2.3. Standard curves for linalool [y=0.0016x+00168, R² = 0.993], gallic acid [y=0.01122x+0.00804, R² = 0.9979] and rutin [y=0.0067x-0.0401, R²= 0.996] were used to quantify the content of total terpenoids (13.44 ± 0.14 mg LE/g extract), total phenolics (19.23 ± 1.32 mg GAE/g extract) and total flavonoids (5109.603 ± 21.47 µg RE/g extract), respectively.

The results of the phytochemical assays reveal that the analyzed samples have comparable amounts of polyphenols to other studies of European native juniper. Kurti et al. (2015) reported values ranging from 4.7 to 5.83 mg/g dry plant weight (no standard reported) for ethanol extracts obtained from 20 localities in the Republic of Macedonia, while Miceli et al. (2009) reported values of 17 to about 60 mg GAE/g extract for methanol extracts of Juniperus communis L. var. communis and Juniperus communis L. var. saxatilis Pall. The high mono-terpenoid content could be an indicator of very good antioxidant pontential (Misra and Dey, 2012). However, the terpenoid content varies strongly with the time of harvesting, growing area and other factors (Owens et al., 1998), therefore making it difficult to compare samples of different origins. The total flavonoids content is also comparable with the results of Kurti et al. (2015) and Miceli et al. (2009), who obtained values ranging from 2 up to 49 mg/g.

The antioxidant activity of the extract was evaluated following the DPPH radical scavenging assay, as described in Section 2.4.

The antioxidant potential of the crude extract was found to be 81.63±0.38% (measured by the DPPH method). The calculated EC50 (half maximal effective concentration) (1.42 ± 0.11 mg/mL) reveals a good antioxidant activity of the tested extract. The IC50 value is comparable with data reported in the literature (for example, Miceli et al. (2009) obtained values ranging from 0.63 to 1.84 mg/mL).

The antifungal activity of the natural products was investigated as described, using miconazole nitrate and solvent (ethanol:H2O=1:1) as positive and negative controls, respectively. Figure 3 shows images of the plates containing the positive and negative controls, used for fungicidal activity testing against Aspergillus niger ATCC 15475 and Penicillium hirsutum ATCC 52323. The diameters of inhibition zones (in millimetres) against test strains are shown in Figure 4.

The extract strongly affected the growth of all target fungi. The strong antifungal activity of the extract is probably correlated with the high content of phenolic acids, flavonoids and terpenoids. The juniper extract showed a very good antifungal potential compared with the available literature data (Sokovic et al., 2004; El-Sawi et al., 2007; Glisic et al., 2007).

In vitro pharmacological effects of polyphenolic compounds are numerous (Rios-Hoyo et al., 2014; Farzaneh and Carvalho, 2015), but the in vivo success appears to be limited by their poor bioavailability (Mahmood et al., 2015). Therefore, the juniper extract was formulated as a microemulsion and tested for its anti-inflammatory action.

In order to evaluate the optimum microemulsion composition, different component ratios from the microemulsion were selected for further characterization. The microemulsion with the highest stability and lowest mean droplet size was selected and used in the experiments. The selected formulation contained 5% Juniperus communis L. extract, 9.29 % oleic acid, 57.14 % Tween 80: PG 1:1 (w/w) mixture and 28.57% water.

The droplet size was 134±7 nm, and was not significantly affected by incorporation of the extract when compared to the droplet size of microemulsion alone. Also, no significant diameter change was found after 3 months of storage at 25°C. Zeta potential was observed to be – 39.3±3.7 mV, also a good indicator of a stable formulation. The high conductivity (163±3.4 µS/cm) revealed the o/w structure of the microemulsion. The refractive index varied between 1.32 and 1.37 over three months, showing that the prepared microemulsion remained transparent and clear even after long-term storage. The pH ranged between 4.9 and 5.1 in this time interval.

The anti-inflammatory effect in the two inflammation experimental models used (acute-dextran and subacute-kaolin) (Srinivasan et al., 2001) was studied by the plethysmometry method, using two control groups (untreated rats and rats treated with the microemulsion vehicle) and a reference substance (diclofenac).

The results of the plethysmometric measurements were statistically analysed in order to obtain paw edema evolution for the groups with dextran- and kaolin-induced inflammation, respectively. These data are summarized in Tables 3 and 4 and Figures 5, 6, 7 and 8.

For the dextran-induced inflammation model, the paw edema volume was significantly higher compared to the initial values (p<0.05) for all animal groups, without return to baseline at the end of the observation period.

For the dextran-induced inflammation model, the global paw edema evolution process for the groups treated with microemulsion was different from the negative control group and the group treated with diclofenac (ANOVA, p<0.05). The microemulsion vehicle influenced the global paw edema evolution process (ANOVA, p<0.05), but the process was similar to the negative control group at the measurement times (t test, p>0.05).

The juniper-containing microemulsion presented a similar paw edema evolution to the group treated with diclofenac at all the measurement times (t test, p>0.05)

For the kaolin-induced inflammation model, the paw edema volume was significantly higher compared to the initial values (p<0.05) for all the animal groups, without return to baseline at the end of the observation period.

For the kaolin-induced inflammation model, the global paw edema evolution process for the groups treated with juniper-containing microemulsion was different from the negative control group and the group treated with diclofenac (ANOVA, p<0.05). The microemulsion vehicle did not influence the global paw edema evolution process, the process being similar to the negative control group (ANOVA, p>0.05). The microemulsion had a similar paw edema evolution after 24 hours with the group treated with diclofenac (t test, p>0.05). The animals treated with the juniper-containing microemulsion exerted a maximum anti-inflammatory effect in the first 4 hours of the experiment.

The results presented here establish the anti-inflammatory action of the juniper extract administered as microemulsion in both models, with increased activity when compared to kaolin subacute inflammation induced model. However, the mechanism of action is uncertain, and the active chemical compounds responsible for the anti-inflammatory activity of the extract remain to be elucidated. The literature data proposes a synergistic action of multiple compounds, and does not attribute the biological effects (antioxidant, antifungal, or anti-inflammatory) to a single compound (Xie et al., 2008; Picerno et al., 2011; Meshram et al., 2015; Pereira Uliana et al., 2015;). Many authors propose correlations between phenol/flavonoid content and the anti-inflammatory action (Deliorman et al., 2007; Saeed et al., 2010; Deng et al., 2011).

Our results indicate the possibility of developing the extract into a potent, lower-cost and safer therapeutic agent, compared with currently used synthesised agents.

4. Conclusions

In the present work we report the analytical characterization of Romanian native Juniperus communis L. berries extract, revealing 57 compounds. The hydroalcoholic extract revealed a very important in vitro antifungal activity on the studied fungal lines, which leads to the conclusion that it can be used as a natural antifungal agent for the treatment of several infectious diseases.

The microemulsion containing the juniper extract exerted anti-inflammatory effects in the tested experimental models. The microemulsion vehicle did not interfere with the extract effect. The anti-inflammatory effect obtained after the administration of juniper extract as microemulsion may recommend this formulation for further studies as dietary factors in pathologies with inflammatory component.

The results obtained from the in vivo and in vitro evaluations recommend the Juniperus communis L. hydroalcoholic extract as a promising biologically-active material.