ABSTRACT

Objective

Most research on the therapeutic effects of wild pistachio species has focused on bacteria and fungi. This study investigated the flavonoid and phenolic contents of different leaves and hulls of Pistacia atlantica(P. atlantica) subsp. Kurdica (P. atlantica subsp. Kurdica) extracts and their effect on herpes simplex virus 1 (HSV1).

Methods

In this study, aqueous, ethanolic, and methanolic extracts of the leaf and hull of P. atlantica subsp. Kurdica were prepared by the percolation method. The total phenolic and flavonoid contents in different extracts were measured by UV/Vis spectrophotometry using the Folin-Ciocâlteu reagent and the colorimetric method of aluminum chloride, respectively. On African green monkey kidney cells (VERO), the ethanolic extract of P. atlantica subsp. Kurdica was tested for toxicity. Furthermore, 50% cytotoxic and inhibitory concentrations (CC₅₀ and IC₅₀) were identified.

Results

Compared with aqueous and methanolic extracts, the ethanolic extract of the leaf and hull of P. atlantica subsp. Kurdica had the highest phenolic and flavonoid concentrations. In addition, our study showed that CC₅₀ values were 661.81 and 795.21 μg/mL. Also, IC₅₀ values were 97.51 and 110.82 μg/mL for leaf and hull extracts, respectively. The selectivity index for the ethanolic leaf and hull extracts were 6.79 and 7.18, respectively.

Conclusion

Our results showed that P. atlantica subsp. Kurdica had an anti-HSV1 effect in a dose-dependent manner but at a higher dose than acyclovir. Ethanolic extract of P. atlantica subsp. Kurdica is probably a suitable herbal medicine with anti-herpetic effects.

INTRODUCTION

Herpes simplex virus 1 (HSV1) is a highly prevalent and contagious virus that can appear as permanent and latent infections with frequent outbreaks of oral lesions (1). Although there is currently no medication for the virus, antiviral drugs are frequently used to suppress and minimize outbreaks. Chemical antiviral drugs have caused the occurrence of allergies, side effects, and drug resistance, and finding new and effective treatments for the prevention and treatment of HSV outbreaks has been considered (2-5). It has been reported that approximately 65-80% of the world’s population uses traditional medicine and medicinal plants as complementary medicine (6).

Pistacia atlantica (P. atlantica) subsp. Kurdica (P. atlantica subsp. Kurdica) is one of the subspecies of wild pistachio P. atlantica in the west (Zagros mountains) and less in the central and eastern regions of Iran (7, 8). The leaves and fruits of this tree in folk medicine have many properties, including anti-atherogenic, blood sugar-lowering, liver protective, cell-protective, anti-genotoxic, anti-inflammatory, anti-ulcer, antipyretic, anti-fungal, anti-bacterial, anti-parasitic, Anti-mutagenic, antioxidant, and anti-cancer activity, as well as stimulant and diuretic (3, 9, 10). These pharmacological properties have been used in many diseases therapies (7, 10).

Most studies have been conducted on the effects of this plant on bacteria and fungi (10-12), but there are very few reports on its antiviral effects (13). The current study determined the antiviral effects of P. atlantica subsp. Kurdica hull and leaf extracts on HSV1.

MATERIALS AND METHODS

Plant Collection and Extraction

Hulls and leaves of P. atlantica subsp. Kurdica were obtained from Ilam Province, Iran. Under sterile conditions, the samples were dried for 10 days in the shade. After that, a grinding machine ground them. Extraction was performed by the percolation method using different solvents such as ethanol, methanol 70%, and water. In brief, 50 g of plant powder was poured into the percolator and soaked in various solvents for 72 h. Then, the solid part was separated from the solvent part using Whatman paper with 11 μm pore sizes. The obtained extracts were then concentrated using a rotary evaporator under vacuum at a temperature of 40 °C. The extracts were kept in small sterile containers at a temperature of 4 °C in a dark place for further tests.

Determination of the Total Phenolic Content

The total phenolic content in samples of different hull and leaf extracts was measured by UV-Vis spectrophotometer using Folin-Ciocâlteu reagent (FCR). Briefly, 1 mg/mL of the various hull and leaf extracts were added to 0.5 mL of FCR and kept for 7 min at room temperature. Then, 0.4 mL of 7.5% sodium carbonate solution was added. After 1 h, the absorbance of different samples was read using a spectrophotometer at 765 nm. Gallic acid was used as a standard to draw the calibration curve. The total phenolic content was reported on the basis of mg of gallic acid equivalent/g of extract powder.

Determination of the Total Flavonoid Content

The aluminum chloride colorimetric assay was used to determine the total flavonoid content in the samples. Thus, 1 mg/mL of each extract was added to 0.1 mL of 10% aluminum chloride and 0.1 mL of 1 M potassium acetate. The solutions were placed at room temperature for 30 min. The total flavonoid content was measured on the basis of mg RE/g of extract powder using the absorbance of a spectrophotometer at 415 nm.

Cells and Viruses

African green monkey kidney (VERO) cells and HSV1 were obtained from the Traditional Medicine Research Institute, Isfahan, Iran. VERO cells were cultured in Eagle’s minimum essential medium enriched with 10% newborn calf serum, 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin sulfate (Gibco).

Cytotoxicity Assay

The cytotoxic effects of different extracts were determined by a photometric technique using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (14). In each well of the 96-well plate, 180 microliters of cell suspension (VERO cells) equal to 3x10⁴ µL cells were added. The extracts were added to the wells at different concentrations from 50 to 1000 µg/mL. The culture medium containing 0.5% dimethyl sulfoxide (DMSO) without plant extract was considered a negative control, whereas the antiviral drug acyclovir was considered a positive control. The plate was incubated for 48 h in an incubator with 5% CO₂ at 37 °C. Then 20 µL of MTT solution was added to the wells and incubated for 2 h. The 100 µL of DMSO was poured into the wells and mixed well to dissolve the formazan crystals. The absorbance was determined at 560 nm using an ELISA reader. The VERO cell survival percentage was estimated using the following equation, and for the positive and negative control groups, 100 was considered. A concentration of the tested compounds that reduces the cell survival rate by half was considered IC₅₀.

Antiviral Activity

A plaque reduction test on VERO cells was used to determine the anti-HSV1 activity of the hull and leaf extracts. In each well of a 24-well plate, 400x10³ µL of VERO cells were incubated in 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) containing 3% inactivated FBS for 24 h. Then one µL of thawed viral suspension was added to each well. After 10 min, 20 µL of different concentrations of extracts were added to each well along with 2 mL of preheated DMEM medium with methylcellulose. In the negative and positive control groups, 20 µL DMSO diluted with deionized water and 20 µL acyclovir were added to the wells. The plates were incubated for 48 h and stained. Plaques were counted in each well, and the inhibition percentage of each extract concentration was determined using the following equation:

Statistical Analysis

ANOVA was used to analyze the data using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA), and Tukey’s multiple comparison tests were used to compare means. Differences with a p˂0.05 were considered significant.

RESULTS

The phenolic and flavonoid contents of the ethanolic, methanolic, and aqueous extracts of the leaf and hull are shown in Figure 1. The present results showed that the phenolic and flavonoid contents were the highest in the ethanolic extract and the lowest in the aqueous extract.

Antiviral activity indices of ethanolic extracts of the leaves and hulls of P. atlantica subsp. Kurdica are shown in Table 1. The ethanolic extract of the hull had higher indices than the leaf. The antiviral activity of each extract was expressed as a selectivity index (SI), which was determined to be 7.18 and 6.79 for hull and leaf extracts, respectively (Table 1).

CC₅₀ leaf: y=-0.2027x + 184.15,

CC₅₀ hull: y=-0.1627x + 179.38,

IC₅₀ leaf: y=1.5445x - 100.61,

IC₅₀ hull: y=1.8442x - 154.37.

The survival rate of VERO cells at concentrations of 400 to 900 μg/mL for leaf and 400 to 1100 μg/mL for hull showed that the cell survival rate decreased with increasing dose (Figure 2). Ethanolic extracts of the leaves and hulls of P. atlantica subsp. Kurdica at the concentration of 400-500 and 400 μg/mL, respectively, caused the maximum survival rate, and at concentrations of 900 and 1100 μg/mL, respectively, the survival rate was equal to zero.

The anti-herpes activity of the ethanolic extracts of the leaf and hull has been shown in comparison with that of acyclovir (Figure 3). The results of this study stated that the concentration range of anti-herpes activity of leaf and hull extracts was from 60 to 130 and 80 to 140 μg/mL, respectively, which increased with the dose of this antiviral activity. In the positive control group (acyclovir), this range was 0.04 to 0.4 μg/mL.

DISCUSSION

According to reports, many traditional medicinal plants and herbs have potent antiviral activity (15). Some antiviral constituents include various structural classes such as flavonoids, tannins, alkaloids, terpenes, coumarins, proteins, peptides, polysaccharides, lignans, naphtho- and anthraquinones (16, 17). The antiviral activity, and the antimicrobial and antifungal activities of pistachio, is related to phenolic and flavonoid compounds (18-20). The main phenolic compounds of the Pistacia genus include caffeic acid, catechin, eryodictiol-7-O-glucoside, gallic acid, protocatechuic acid, ellagic acid, ursolic acid, chlorogenic acid, ferulic acid, juglone, synaptic acid, and vanillic acid (13, 18). However, there have been few studies on the antiviral effect of wild pistachio species (13). Our findings confirm the anti-HSV1 effect of P. atlantica subsp. Kurdica. Chlorogenic acid and caffeic acid have antiviral effects (21).

The antibacterial, antifungal, and antiviral effects of various sections of cultivated Vera pistachios were investigated by Ozçelik et al. (22). Their results showed that these extracts have significant antibacterial and antifungal effects. They also showed high antiviral effects against HSV and parainfluenza virus; therefore, its seed and kernel extracts showed higher antiviral effects than other plant organs (22). In addition, the antiviral effects of 75 plant species have been investigated against herpes virus, Sindbis virus, and poliovirus. Owing to its phenolic compounds, Pistacia lentiscus extract was found to have the greatest antiviral action against the herpes virus (23).

Our study showed that CC₅₀ values were 661.81 and 795.21 μg/mL. Also, IC₅₀ values were 97.51 and 110.82 μg/mL for leaf and hull extracts, respectively. The SI for the ethanolic leaf and hull extracts were 6.79 and 7.18, respectively. In line with our study, Karimi et al. (13) showed anti-adenovirus effects of ethanolic extract and n-butanol fraction of P. atlantica leaf. They stated that CC₅₀, IC₅₀, and SI on Hep-2 cells were 434.7, 16.37 μg/mL, and 26.5.

Musarra-Pizzo et al. (18) demonstrated that polyphenol-rich extracts of natural Pistacia vera L. shelled at 0.4, 0.6, and 0.8 mg/mL concentration reduced the expression of the viral proteins ICP8, UL42, and US11, and finally lead to minimize of viral DNA synthesis in VERO cells. They reported values of CC₅₀, IC₅₀, and SI equal to 1.2, 0.4, mg/mL, and 3. These differences may be caused by the pistachio species or the tested cells.

CONCLUSION

The results of this study showed that the ethanol extract of wild pistachio leaves and hull both have similar anti-HSV1 effects. These extracts are comparable to acyclovir at a higher dose. Further clinical research is required to confirm the therapeutic effects of anti-HSV1.