Indian Journal of Traditional Knowledge Vol. 17 (2), April 2018, pp 255-262

In vitro anti-herpes simplex virus activity, antioxidant potential and total phenolic compounds of selected Iranian medicinal extracts

Mohammad-Taghi Moradi1, Ali Karimi1*, Somayeh Alidadi2 & Leila Hashemi3 1Medical Research Center; 2Cellular and molecular Research Center; 3Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Science, Shahrekord, Iran E-mails: [email protected], [email protected]

Received 11 September 2017, revised 17 January 2018

Drug resistant strains of herpes simplex virus-1 (HSV-1) have been increased recently. Essentially, medicinal plant-based new antiviral agents that are effective against HSV-1 infections are urgently required. Therefore, this research was conducted to evaluate in vitro anti HSV-1 activity of 25 plant extracts. In this study, the hydroalchoholic extracts of different parts of 25 medicinal plants belonging to 16 different families were prepared. Anti-HSV-1 activity was evaluated on Vero cell line using MTT (3-[4,5-dimethylthiazol–2-yl]-2,5-diphenyltetrazolium bromide) assay. The 50 % effective concentration (EC50) and 50 % cytotoxicity concentration (CC50) of the extract were determined using regression analysis. The inhibitory effect of the plant materials on adsorption and/or post-adsorption stages of HSV-1 replication cycle was determined. Antioxidant activity, total phenolic content, and total flavonoid content of the extracts were determined using 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay, Folin-Ciocalteu reagent, and aluminum chloride colorimetric method, respectively. Results showed that Pistacia atlantica Desf., Equisetum arvense L., Melissa officinalis L., Anthyllis vulneraria L., Punica granatum L., Syzygium aromaticum (L.) Merr. & L.M. Perry, Camellia sinensis (L.) Kuntze, and azarolus L., were active against HSV-1. There was a significant association of total phenolic contents (R = 0.773, p < 0.001) and the free radical scavenging property (R = -0.684, p < 0.01) with the antiviral activity of the extracts. Some of the Iranian plant extracts studied showed potent antiviral activity against HSV-1 and can be used to develop new and effective anti-HSV-1 agents.

Keywords: Antiviral activity, Herpes simplex virus, Antioxidant potential IPC Int. Cl.8: A61P31/12, C07K 14/035, A61K 39/245, C09K 15/00

Plants have the ability to synthesize a wide array of mild to severe life threatening infections such as compounds and have long been used as remedies, with encephalitis14. During the past two decades, the many of them being currently collected and examined mechanisms of replication and pathogenesis of HSV-1 to identify possible sources of herbal medicines1. and thus the potential antiviral targets in this virus have Some medicinal plant extracts have been recently used been thoroughly understood, leading to development of for treatment of a few diseases2 and some others antiviral compounds for this virus14. A category of have been evaluated to develop therapeutic agents with nucleoside analogues, acyclovir (ACV), is widely used promising results3-5. to treat HSV-1 infections15. However, a major problem Modern studies have shown that some of the with the use of ACV, is the development of drug medicinal plants with therapeutic application in resistant HSV strains, particularly in AIDS patients16. traditional medicine6-9 have antiviral effects. Many This type of drug resistance may occur after long-term screening efforts have been made to seek out antiviral treatment and is mainly due to mutations in the HSV-1 agents from natural sources10-13. thymidine kinase and/or DNA polymerase genes17. Persia ancient civilization has a long history in Therefore, antiviral, medicinal plant-based agents with identification of medicinal plants and treatment by active compounds that exhibiy different modes of these plants and Iranian scientists such as Avicenna action against viral infections are urgently needed. have tried to develop this science. Iran is endowed with rich and diverse local health Infections caused by HSV-1 represent a public tradition, which is matched with its rich and diverse health concern worldwide. These infections range from plant genetic source. Detailed investigations and —————— documentation of plants used in local healthcare *Corresponding author traditions and ethnopharmacological evaluation to 256 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 2, APRIL 2018

verify their efficacy and safety can lead to the dissolved in 70 % ethylealcohole and kept at room development of invaluable herbal drugs or isolation temperature in an orbital shaker for 96 h. Next, the of compounds of therapeutic value. Therefore, this mixture was filtered and concentrated under research was conducted to examine 25 plant extracts approximately vacuum pressure and 40 °C in a for antiviral activity against HSV-1 in vitro. rotary evaporator. The extracts were kept in sterile bottles under refrigerated conditions until further Methodology use. The extracts were suspended at 37 °C in 10 % dimethylsulphoxide (DMSO) to yield a stock solution Plant collection and extraction of 10 mg/mL. This solution was filtered (Millipore® Different parts of 25 medicinal plants belonging to 0.22 μm) and stored (at 4 °C) until used. The small 16 different families (Table 1) that are traditionally percentage (up to 0.2 %) of DMSO present in the wells used for treatment of diseases were tested for their has no effect on the results of the experiments18. antiviral activity. The plant materials were provided from medicinal plants market in Shahrekord, a South Determination of free-radical scavenging activity west city of Iran. Then, both genus and species of the The free-radical scavenging activity was measured plants were identified and confirmed in the Herbarium by the 2, 2 diphenyl-1-picrylhydrazyl (DPPH) assay of Medical Plants Research Center of Shahrekord described by Moon & Terao, with some modification19. University of Medical Sciences. The plant materials Briefly, different amounts of the extracts and methanol were shadow dried, pulvarized, and then extracted were added to a solution of 0.3 mg/mL methanol using maceration method. The plant material was solution of DPPH to a final volume of 3.0 m.

Table 1 — Ethnobotanical data of selected medicinal plants NO (Family) Local name Voucher number Common name Parts used 1 Allium ampeloprasum L. (Amaryllidaceae) Tarrehkoei MPSKUMS-169 Wild leek Aerial parts 2 Allium jesdianum Boiss. (Amaryllidaceae) bosor MPSKUMS-204 - Leaves 3 Pistacia atlantica Desf. (Anacardiaceae) Baneh MPSKUMS-93 Atlas mastic tree Leaves 4 Peucedanum membranaceum Boiss (Apiaceae) Chowak MPSKUMS-57 Hog's fennel Aerial parts 5 Kelussia odoratissima Mozaff (Apiaceae) Kelos MPSKUMS-144 Wild celery Leaves 6 Heracleum persicum Desf. (Apiaceae) Golpar MPSKUMS-267 Golpar Fruits 7 Achilleam illefolium L. (Asteraceae) Boomadaran MPSKUMS-304 Yarrow Aerial parts 8 Calendula officinalis L. (Asteraceae) Hamyshebahar MPSKUMS-116 Marigold Aerial parts 9 Berberis integerrima Bunge (Berberidaceae) Zereshkekoohi MPSKUMS-512 Barberry root 10 Equisetum arvense L. (Equisetaceae) Domeasb MPSKUMS-516 Horsetail Aerial parts 11 Vaccinium arctostaphylos L. (Ericaceae) Ghareghat MPSKUMS-264 Whortleberry Fruits 12 Melissa officinalis L. Badranjbuoye MPSKUMS-107 lemon balm Aerial parts (Lamiaceae) 13 Stachys lavandulifolia Vahl (Lamiaceae) Chaeekoohi MPSKUMS-204 - flowers 14 Ziziphora clinopodioides Lam. (Lamiaceae) Kakooti MPSKUMS-253 - Aerial parts 15 Zataria multiflora Boiss. (Lamiaceae) Avishanshirazi MPSKUMS-443 - Aerial parts 16 Thymus daenensis Celak. (Lamiaceae) Avishandenaei MPSKUMS-248 thyme Aerial parts 17 Hyssopus officinalis L. (Lamiaceae) Zofa MPSKUMS-177 hyssop flowers 18 Trifolium campestre Schreb. (Leguminosae) Shabdarezard MPSKUMS-1001 Kidney vetch Leaves 19 Punica granatum L. Anar MPSKUMS-316 Pomegranate Peel fruits (Lythraceae) 20 Althaea officinalis L. Khatmi MPSKUMS-139 Marsh-mallow Flowers (Malvaceae) 21 Syzygium aromaticum (L.) Merr. & L.M. Perry Mikhak MPSKUMS-1002 Clove Flower buds (Myrtaceae) 22 Rheum ribes L. Rivas MPSKUMS-527 Currant-fruited Petiolules (Polygonaceae) rhubarb 23 Adiantum capillus-veneris L. (Pteridaceae) Parsiyavash MPSKUMS-131 maidenhair Leaves 24 Crataegus azarolus L. Zalzalak MPSKUMS-499 Hawthorn Leaves () 25 Camellia sinensis (L.) Kuntze (Theaceae) Chaee MPSKUMS-1003 Black tea Leaves MORADI et al.: ANTI-VIRAL ACTIVITY OF IRANIAN PLANT EXTRACTS 257

After standing for 15 min at RT, the absorbance was grown in Dulbecco's Modified Eagle's Medium read at 517 nm using UV–Vis pectrophotometer (DMEM; Gibco, USA) supplemented with 10 % of (UNICO 2100: USA). High absorbance of the reaction fetal bovine serum (FBS) (Gibco, USA), 100 µg/mL of mixture indicated low free radical scavenging activity. streptomycin, 100 UI/mL of penicillin, and 0.25 µg/mL Butylated hydroxytoluene (BHT) was used as positive of amphotericin B (Gibco, USA) at 37 °C with 5 % control. Inhibition of free radical by DPPH was CO2. The same medium containing 2 % PBS was used calculated according to the formula below: for cytotoxicity and antiviral assays. HSV-1 (HSV-1, KOS strain) was kindly provided by University of Antiradical activity (%) = (Acontrol – Asample)/Acontrol × 100. Tarbiat Modares, Tehran, Iran. Virus stock was

prepared by infection of confluent monolayer Vero The IC50 value, defined as the amount of antioxidant 2 necessary to decrease the initial DPPH concentration cells in 75 cm culture flasks using DMEM medium by 50 % and was calculated by using linear regression with 2 % FBS, at 37 °C with 5 % CO2. Virus titer was of plots of antiradical activity percentage against the determined by cytopathic effect (CPE) of HSV-1 on concentration of the studied compounds20. Experiments Vero cells and was expressed as the 50 % tissue culture were carried out in triplicate and results were presented infective dose (TCID 50)/mL. The infective titer of the stock solution was 106/4 TCID 50/mL. as the average of three independent trials. Screening of antiviral activity Determination of total phenolic content The antiviral activity of all plant extracts was The total phenolic content of all plant extracts was screened in 96-well culture plates by measuring the determined using Folin-Ciocalteu reagent21. Briefly, reduction of the viral CPE10. Confluent Vero cell the extracts at 1 mg/mL were dissolved in 60 % monolayers were infected with 100 TCID50/well of methanol and 0.1 ml of the diluted sample was added HSV-1 in the presence of 25, 50, and 100 μg/mL of to 0.5 mL of 10 % (v/v) Folin–Ciocalteu reagent and extract10. Infected cells in the absence of extract, as kept at room temperature for 3-8 min. Then, 0.4 mL of control virus, and mock-infected cells with and without 7.5 % (w/v) sodium carbonate solution was added to extract, as control cell, and cytotoxicity control were the mixture. After the reaction mixture was left in total included. Plates were incubated at 37 °C in a darkness for 30 min, its absorbance was read at 765 nm humidified atmosphere containing 5 % CO until viral using a UV–VIS spectrophotometer (UNICO 2100: 2 CPE in the control virus reached 90 %. The reduction USA). Total phenolic content was calculated using a of viral CPE was determined by measuring cell gallic acid calibration curve and results were presented viability by thetetrazolium salt MTT [3-(4, 5- as mg of gallic acid equivalent (GAE)/g of extract dimethylthiazol-2ol) 2, 5 diphenyl tetrazolium powder. bromide), (Sigma, USA)] colorimetric assay. The Determination of total flavonoid content absorbance was read at 570 nm in an enzyme-linked The total flavonoid content of all plant extracts was immunosorbent assay (ELISA) reader (STATA FAX measured as described previously22. Briefly, the 2100, USA). extracts at 1 mg/mL were dissolved in 60 % methanol Results of the screening were expressed as positive and 0.5 mL of diluted plant material was separately (+) (reduction of viral CPE higher than 50 % at all three mixed with 1.5 mL of methanol, 0.1 mL of 10 % (w/v) concentrations), negative (−) (without protection at all aluminum chloride, 0.1 mL of 1 M potassium acetate, three concentrations), (++/−) (reduction of viral CPE and 2.8 m of distilled water. After a 40 min incubation higher than 50 % at 100 and 50 μg/mL), and +/−− at room temperature, the absorbance of the reaction (reduction of viral CPE higher than 50 % only at 100 mixture was read at 415 nm using a UV–Vis μg/mL)10. spectrophotometer (UNICO 2100: USA). The results Cytotoxicity assay were expressed as mg of rutin equivalent/g of extract Before the investigation of anti-HSV-1 activity, the powder (mg RE/g) in comparison with the standard cytotoxic effect (CPE) of the studied compounds were curve, which was plotted in the same. determined. Briefly, Vero cells at a concentration of Cell and virus 10000 cells/well were seeded onto 96-well plates Vero (African green monkey kidney) cells were to a final volume of 100 µL/well. After incubation at procured from Pasteure Institute of Iran. The cells were 37 °C with 5% CO2 for 24 h, when the cell monolayer 258 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 2, APRIL 2018

was confluent, the cell culture medium of cells was Antiviral activity (%) = (Atv–Acv)/(Acv–Acv) × 100 % aspirated and washed with PBS. Cells were incubated where A , A , and A are the absorbance of the with 100 µL/well of different concentrations of tv cv cv studied compounds on virus infected cells, the extracts (in triplicates) and incubated at 37 °C with absorbance of the virus control, and the absorbance of 5 % CO for further 3 days. Cell viability was 2 the cell control, respectively18. The procedure was examined by the ability of the cells to cleave the carried out in triplicate. The 50 % effective tetrazolium salt MTT by the mitochondrial enzyme concentration (EC ) was determined according to succinate dehydrogenase, which creates a blue 50 the inhibition-concentration curve of the extracts. formazan and the procedure was followed as described Selectivity index (SI), as a marker of antiviral activity, previously23. Briefly, the supernatants were removed was determined as the ratio of CC50 to EC50. from the wells and 50 μL of an MTT (Sigma, USA) solution (1mg/mL in PBS) was added to each well. The Time-of-addition assay plates were incubated at 37 °C for 4 h, and 100 μL of The time-of-addition effect of the positive extracts DMSO (sigma, USA) was added to the wells to was evaluated by a previously described method minor dissolve the MTT crystals. The plates were placed on a modifications10. To assess the effect of the positive shaker for 15 min and the absorbance was read on an extracts in the adsorption and post-adsorption of HSV-1 ELISA reader (STATA FAX 2100, USA) at 492 nm. replication, the virus was treated with one time EC90 of Data were expressed as the percentage of toxicity using the plant materials by three different manners in the the following formula: presence of the extracts; 1) only during the adsorption period (adsorption); 2) after adsorption and until the Toxicity (%) = [100– (At/As) × 100] % end of the experiment (post-adsorption); and 3) during Where At and At represent the absorbance of and after the adsorption (throughout) (Fig. 1). To carry the studied substance and the solvent control, out these experiments, 90 % confluent cells were 18 respectively . The CC50 was considered the cytotoxic chilled at 4 °C for 1 h followed by infection with concentration of the crude extracts measured by probit 100 µL/well of HSV-1 (100 TCID50) in the presence or regression model. absence of extracts and incubated at 4 °C for further 1 h allowing only the adsorption stage of the viral Antiviral assay particles to the cells (adsorption). Next, the supernatant Antiviral activity of the positive extracts were was removed, the cells were washed with PBS, the evaluated by inhibitory activity assay using MTT medium with or without the extracts was replaced with assay, as described previously18. Briefly, non-cytotoxic an equal volume of DMEM and 1% PBS, and then concentrations of extracts (under the CC value) were 50 incubated at 37 °C with 5 % CO for two days. Using used to test their ability to inhibit CPE of HSV-1 in 2 the MTT assay, cell viability and the percentage of tissue culture. One hundred µL of virus suspension viral inhibition was evaluated in comparison with the (100 TCID50) was added to confluent Vero cell control. Data were presented as the percentage of virus monolayer in a 96-well plate, and incubated at 37 °C inhibition in comparison with untreated control, as for about one hour to allow virus adsorption. mean ± SD (n = 3). Statistical analysis was carried out Thereafter, serial two fold dilutions prepared from using Kruskal-Wallis test. non-toxic doses of the crude extracts were added and tested in triplicate. The cells infected with the same concentration of virus but without addition of extract were considered positive control. The cells to which 1 % DMEM was add were considered negative or control cells. The plates were incubated at 37 °C in a humidified CO2 atmosphere for 3 days. DMSO at 0.1 % concentration and a solution of aciclovir (9-(2-hydroxyethoxymethyl) (Sigma, USA) were used as negative and positive controls, respectively. Cell viability was also determined using a previously 18 Fig. 1 — Pattern of extracts addition during adsorption and post- described MTT assay . Data were calculated as the adsorption stages of the virus10. White and black arrows indicate percentage of inhibition using the following formula: the absence and presence of extract, respectively MORADI et al.: ANTI-VIRAL ACTIVITY OF IRANIAN PLANT EXTRACTS 259

Statistical analysis Equisetum arvense L. and Punica granatum L. (6.5, All experiences were carried out in triplicate. The 6.8, and 7.7 μg/mL, respectively) when compared to two-sided Spearman's correlation test was used to the other studied extracts. The results were expressed investigate the correlation of the total phenolic content with reference to BHT with IC50 of 25.41±1.89 μg/mL. and the free radical scavenging property with the Our results showed that among the 25 medicinal antiviral activity score. The EC50 and CC50 values were plant extract, Punica granatum L. (282.9), Camellia calculated using dose-response analyses and related sinensis (L.) Kuntze (341.8), and Pistacia atlantica models with probit procedure using SPSS. p value < Desf. (353.4) extracts had the highest total phenolic 0.05 was considered significance level. content. Pearson’s correlation coefficient showed a significant relationship between the total phenolic Results content and the free radical scavenging property of the

DPPH radical scavenging activity and total phenolic studied extract (R = -0.592, p < 0.01). The highest and flavonoid content flavonoid content (57.2 mg/mL) was obtained for Adiantum capillus-veneris L. (Table 2). To standardize the crude extracts, their DPPH radical scavenging activity and their total phenolic and Antiviral activity flavonoid content were measured. DPPH radical Eight of the 25 plant extracts, at a nontoxic scavenging activity showed that the highest IC50 was concentration to Vero cell line, were found to have obtained for Pistacia atlantica Desf., followed by antiviral activity against HSV-1 (Tables 1 & 2). The

Table 2 — DPPH-radical scavenging activity (IC50), total phenolic and flavonoid content, and screening of antiviral activity of plant extracts against HSV-1 DPPH-radical Total phenolics Flavonoid content Anti HSV1 NO Plant name Scavenging activity (mg GAE/g)b (mgRUT/g)c Activity a IC50 (μg/mL) 1 Pistacia atlantica Desf. 6.5 353.4 33.5 ++/- 2 Peucedanum membranaceum Boiss 288.5 38.4 5.7 - 3 Kelussia odoratissima Mozaff. 79.75 104.8 38 - 4 Heracleum persicum Desf. 418.4 75.4 42.9 - 5 Achillea millefolium L. 60.7 73.4 21.8 - 6 Calendula officinalis L. 96.1 60.2 17.3 - 7 Equisetum arvense L. 6.8 262.1 27.85 + 8 Trifolium campestre Schreb. 327.5 22.3 12 - 9 Melissa officinalis L. 16.8 227.6 12.5 +/-- 10 Anthylis vulneravia L. 26.8 146.75 49.7 +/-- 11 Allium ampeloprasum L. 364.2 47.7 7.2 - 12 Allium jesdianum Boiss. 305.6 40.2 6.1 - 13 Punica granatum L. 7.7 282.9 57.6 ++/- 14 Althaea officinalis L. >500 16.3 0 - 15 Syzygium aromaticum 8.4 225.6 29.3 ++/- 16 Rheum ribes L. 17.36 251.8 53 - 17 Camellia sinensis (L.) Kuntze 8 341.8 21.1 ++/- 18 Crataegus azarolus L. 231 214.2 28.6 ++/- 19 Stachys lavandulifolia Vahl 66.44 67.7 28.6 - 20 Berberis integerrima Bunge 163.2 94 0 - 21 Ziziphora clinopodioides Lam. 31.8 159.4 47.4 - 22 Zataria multiflora Boiss. 228.8 194.4 56.8 - 23 Thymus daenensis Celak. 51.9 108.4 40.3 - 24 Adiantum capillus-veneris L. 235.9 107.2 57.2 - 25 Hyssopus officinalis L. 663.9 55.61 14.5 - a b The results are expressed with reference to butylated hydroxytoluene, a reference standard with IC50 of 25.41±1.89 μg/mL. mg of gallic acid equivalent/g of extract powder. c mg of rutin equivalent/g of extract powder. The antiviral activity was determined by the reduction of viral cytopathic effect (CPE) assays; (+) positive: Reduction of viral CPE higher than 50% at all three concentrations; (++/−): Reduction of viral CPE higher than 50% at 100 and 50 μg/mL; (+/−−) positive/negative: Reduction of viral CPE only at 100 μg/mL; (−) negative: without protection at 25, 50, and 100 μg/mL. 260 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 2, APRIL 2018

Table 3 —Assessment of cytotoxicity and anti-herpes simplex virus activity of plant extracts by MTT assay

a b c d Plant name CC50 (μg/mL) EC50 (μg/mL) EC90 (μg/mL) SI Equisetum arvense L. 574.4 23.9 35.4 24 Melissa officinalis L. 631.5 93.7 135 6.7 Camellia sinensis (L.) Kuntze 216.9 42.1 70 5.15 Anthyllis vulneraria L. >1400 118 190 >11.9 Syzygium aromaticum 375 40.11 83 9.34 Punica granatum L. 401.9 37.7 89 10.6 Pistacia atlantica Desf. 529 90.6 142 5.8 Crataegus azarolus L. 969.68 97.24 129.3 9.97 Aciclovir* 177.5 1.3 2 136.5 total phenolic content (R = 0.773, p < 0.001) and the free radical scavenging property of the plant extracts (R = -0.684, p < 0.01) were significantly associated with their antiviral activity score.

Cytotoxicity and antiviral assay The antiviral activity of the extracts against HSV-1 was investigated in vitro using the viral CPE reduction assay. Based on the results (Table 2), the extracts of Pistacia atlantica Desf., Equisetum arvense L., Melissa officinalis L., Anthyllis vulneraria L., Punica granatum L., Syzygium aromaticum, Camellia sinensis (L.) Kuntze, and Crataegus azarolus L. exhibited Fig. 2 — The effect of extracts on the adsorption and post-adsorption strong activity against HSV-1. Most of these extracts of herpes simplex virus 1 (HSV-1) to Vero cells. had partial activity at low concentrations studied. The results showed that Equisetum arvense L. and Anthyllis added between the adsorption and post-adsorption vulneraria L. extracts had the highest SIs against HSV- stages of HSV-1 replication cycle (p > 0.05). 1 (24 and > 11.9, respectively) (Table 3). The increase of viral inhibition observed in the post-adsorption stage could be due to the inhibitory Characterization of antiviral activity effect of Pistacia atlantica Desf., Equisetum arvense To determine whether the inhibitory effect of the L., Melissa officinalis L., Anthyllis vulneraria L., and plant extracts occur during the adsorption or post- Crataegus azarolus L. extracts on the replication adsorption stages of HSV-1 replication cycle, different stages of HSV-1 in Vero cells (Fig. 2). experiments were carried out with one time IC90 of these plant materials (Fig. 1). Our results showed that Discussion Pistacia atlantica Desf., Equisetum arvense L., The initial screening of plants for their active natural Melissa officinalis L., Anthyllis vulneraria L., and products typically begins by using crude aqueous or Crataegus azarolus L. extracts inhibited HSV-1 alcohol extraction and then will be followed by various replication in post-adsorption stage (p < 0.01) similar organic extraction methods. These naturally occurring to aciclovir (as positive control). There was no products are often obtained through primary ethanol or significance difference in the percentage of post- methanol extraction24. In this study, we investigated 25 adsorbtion viral inhibition when Pistacia atlantica plant extracts for their antiviral activity against HSV-1 Desf., Equisetum arvense L., Melissa officinalis L., using ethanol as crude extraction solvent. To the best Anthyllis vulneraria L., and Crataegus azarolus L. of our knowledge, the preliminary data of these plants’ extracts and aciclovir were added throughout all antiviral activity based on in vitro studies are not experiments. There was also no significant difference available. Eight of the 25 extracts at 100 μg/mL in the percentage of viral inhibition of Punica showed potential antiviral activity against HSV-1. granatum L., Syzygium aromaticum, and Camellia Based on these results, further qualitative and sinensis (L.) Kuntze extracts when the extracts were quantitative analyses of these extracts were carried out MORADI et al.: ANTI-VIRAL ACTIVITY OF IRANIAN PLANT EXTRACTS 261

at widely various concentrations. In order to confirm Acknowledgment that the inhibitory effect of the extracts was only due to This work was financially supported by Shahrekord their anti HSV-1 activity and not due to overlapping University of Medical Sciences, Shahrekord, Iran drug toxicity, the cytotoxicity assay was carried out to (Grant No.:1701). Authors are thankful to the Director measure cell viability in presence of the drug alone. of Medical Plants Research Center and to the Research Based on our results, the total phenolic content and and Technology Deputy of this university. the antiviral activity of the plant extracts were References associated. Thus, the antiviral activity of these extracts 1 Naithani R, Huma LC, Holland LE, Shukla D, McCormick could be attributed to their phenolic compounds. DL, Mehta RG, et al., Antiviral activity of phytochemicals: a Phenolic compounds have been shown to have comprehensive review, Mini Rev Med Chem, 8(11) (2008) antibacterial and anti-viral activities6,25-27. 1106-1133. PubMed PMID: 18855727. To investigate the antiviral mechanism of the extract 2 Rafieian-Kopaei M & Sewell RD, The history and ups and against HSV-1, we also carried out additional experiments downs of herbal medicines usage, J Herbmed Pharmacol, 3(1) including during the adsorption (adsorption), after (2014) 1-3. 3 Karimi A, Mohammadi-Kamalabadi M, Rafieian-Kopaei M, adsorption until the end of the experiment (post- Amjad L & Salimzadeh L, Determination of antioxidant adsorption), and during and after the adsorption activity, phenolic contents and antiviral potential of methanol (throughout). Our results showed that Pistacia atlantica extract of Euphorbia spinidens Bornm (Euphorbiaceae), Trop Desf., Equisetum arvense L., Melissa officinalis L., J Pharm Res, 15(4) (2016) 759-764. Anthyllis vulneraria L., and Crataegus azarolus L. extracts 4 Moradi MT, Gatreh-Samani K, Farrokhi E, Rafieian-Koupaei M inhibited HSV-1 replication in post-adsorption stage. & Karimi A, The effects of purslane (Portulaca oleracea L.) on serum level of lipids, lipoproteins and paraoxanase It could be argued that these extracts have strong 1(PON1) activity in hypercholesterolemia patients, Life Sci J, antiviral activities against this virus because of their 9(4) (2012) 5548-5552. ability to inhibit the viral cycle, particularly during the 5 Moradi MT, Karimi A & Alidadi S, In vitro antiproliferative post-adsorption period. Therefore, further understanding and apoptosis-inducing activities of crude ethyle alcohole of the action mechanism of and the bioassay guided extract of Quercus brantii L. acorn and subsequent fractions, Chin J Nat Med, 14(3) (2016) 196-202. fractionation of this extract seem valuable. Further 6 Asadi-Samani M, Moradi MT, Bahmani M & Shahrani M, characterization of this extract probably leads to Antiviral medicinal plants of Iran: A review of ethnobotanical development of a potential specific anti-HSV-1 agent. evidence, Int J PharmTech Res, 9(5) (2016) 427-434. Our data also indicated that HSV-1 replication 7 Karimi A, Moradi MT, Alidadi S & Hashemi L, Anti-adenovirus was significantly impaired only if the Punica activity, antioxidant potential, and phenolic content of black granatum L., Syzygium aromaticum, and Camellia tea (Camellia sinensis Kuntze) extract, J Comple Integr Med, sinensis (L.) Kuntze extracts were added at the time 13(4) (2016) 357-363. PubMed PMID: 27567600. 8 Moradi M-T, Rafieian-Kopaei M & Karimi A, A review study of adsorption. Therefore, these results suggest that on the effect of Iranian herbal medicines against in vitro these extracts may exert their antiviral activity by replication of herpes simplex virus, Avicenna J Phytomed, inactivating viral particles at high concentrations and 6(5) (2016) 506–515. probably by interfering with the adsorption of the virus 9 Moradi MT, Karimi A, Rafieian-Kopaei M & Fotouhi F, to the cells. In vitro antiviral effects of seed extract and its total alkaloids against Influenza virus, Microbial Pathogen, Conclusion 110 (2017) 42-49. Based on our results, the Iranian medicinal plant 10 Visintini Jaime MF, Redko F, Muschietti LV, Campos RH, Martino VS & Cavallaro LV, In vitro antiviral activity of plant extracts investigated in this study including Pistacia extracts from Asteraceae medicinal plants, Virol J, 10 (2013) atlantica Desf., Equisetum arvense L., Melissa 245. PubMed PMID: 23890410. officinalis L., Anthyllis vulneraria L. and Crataegus 11 Vijayan P, Raghu C, Ashok G, Dhanaraj SA & Suresh B, azarolus L. extracts inhibit HSV-1 replication during Antiviral activity of medicinal plants of Nilgiris, Indian J Med the post-adsorption. Therefore, further research is needed Res, 120(1) (2004) 24-29. PubMed PMID: 15299228. to elucidate the active constituents of these extracts to 12 Bhandary MJ & Chandrashekar KR, Herbal therapy for herpes be used as sources of novel anti-HSV-1 agents. in the ethno-medicine of Coastal Karnataka, Indian J Tradit Knowle, 10(3) (2011) 528-532.

13 Goswami D, Mukherjee PK, Kar A, Ojha D, Roy S & Conflict of interest Chattopadhyay D, Screening of ethnomedicinal plants of The authors declare that they have no conflicts of diverse culture for antiviral potentials, Indian J Tradit Knowle, interest to disclose. 15(3) (2016) 474-481. 262 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 2, APRIL 2018

14 Koelle DM & Corey L, Herpes simplex: insights on 22 Asgari S, Setorki M, Rafieian-kopaei M, Heidarian E, pathogenesis and possible vaccines, Annu Rev Med, 59 (2008) Shahinfard N, Ansari R, et al., Postprandial hypolipidemic and 381-395. PubMed PMID: 18186706. hypoglycemic effects of Allium hertifolium and Sesamum 15 Villarreal EC, Current and potential therapies for the treatment indicum on hypercholesterolemic rabbits, Afr J Pharm of herpes-virus infections, Prog Drug Res, 60 (2003) 263-307. Pharmacol, 6(15) (2012) 1131-1135. PubMed PMID: 12790345. 23 Goswami D, Mukherjee PK, Kar A, Ojha D, Roy S & 16 Bush Larry M, Talledo-Thais K, Fernandez Alyn Casal- & Chattopadhyay D, Screening of ethnomedicinal plants of Perez Maria T, Resistant Herpes Simplex Virus Infection and diverse culture for antiviral potentials, Indian J Tradit Knowle, HIV: A Potential diagnostic and Therapeutic Dilemma, Lab 15(3) (2016) 474-481. Med, 42(8) (2011) 452-457. 24 Vilegas JHY, de Marchi E & Lancas FM, Extraction of low- 17 Schmidt S, Bohn-Wippert K, Schlattmann P, Zell R & polarity compounds (with emphasis on coumarin and Sauerbrei A, Sequence Analysis of Herpes Simplex Virus 1 kaurenoic acid) from Mikania glomerata (‘guaco’) leaves, Thymidine Kinase and DNA Polymerase Genes from over 300 Phytochem Anal, 8(5) (1997) 266-270. Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral 25 Karimi A, Rafieian-Kopaei M, Moradi M-T & Alidadi S, Resistance, Antimicrob Agents Chemother, 59(8) (2015) Anti–Herpes Simplex Virus Type-1 Activity and Phenolic 4938-45. PubMed PMID: 26055375. Content of Crude Ethanol Extract and Four Corresponding 18 Jadhav P, Kapoor N, Thomas B, Lal H & Kshirsagar N, Fractions of Quercus brantii L Acorn, J Evid Based Comple Antiviral potential of selected Indian medicinal (ayurvedic) Altern Med, 22(3) (2017) 455-461. PubMed PMID: plants against herpes simplex virus 1 and 2, N Am J Med Sci, 27899436. 4(12) (2012) 641-647. PubMed PMID: 23272307. 26 Moradi M-T, Karimi A, Lorigooini Z, Pourgheysar B, Alidadi 19 Moon J-H & Terao J, Antioxidant Activity of Caffeic Acid S & Hashemi L, In vitro anti influenza virus activity, and Dihydrocaffeic Acid in Lard and Human Low-Density antioxidant potential and total phenolic content of twelve Lipoprotein, J Agric Food Chem, 46(12) (1998) 5062-5065. Iranian medicinal plants, Marmara Pharm J, 21(4) (2017) 20 Nahak G & Sahu RK, In vitro antioxidative acitivity of 843-851. Azadirachta indica and Melia azedarach leaves by DPPH 27 Karimi A, Rafieian-Kopaei M, Moradi MT & Alidadi S, Anti– scavenging assay, Nat Sci, 8(4) (2010) 22-28. Herpes Simplex Virus Type-1 Activity and Phenolic Content 21 Ainsworth EA & Gillespie KM, Estimation of total phenolic of Crude Ethanol Extract and Four Corresponding Fractions content and other oxidation substrates in plant tissues using of Quercus brantii (L.) Acorn, J Evid-Based Comple Altern Folin–Ciocalteu reagent, Nat Protoc, 2(4) (2007) 875-877. Med, 22(3) (2017) 455-461.