Screening of medicinal plants from El Salvador for anti-viral activity against Herpes simplex 1

Abstract Herpes simplex virus 1 is one of the most prevalent pathogens worldwide. Resistant strains to current anti-viral treatment have been reported, requiring the search for novel anti-virals. Using a qPCR method to assess anti-herpetic activity from natural products, we analyzed 72 plant extracts from El Salvador and identified eighteen methanolic extracts with anti-viral activity of ≥ 75%. Anti-herpetic activity has not been previously reported in fourteen of the plants (Euphorbia lancifolia, Piper tuberculatum, Cordia alliodora, Tecoma stans, Taraxacum officinale, Hamelia patens, Witheringia solanacea, Emilia fosbergii, Gnaphalium viscosum, Citrus aurantium, Ambrosia peruviana, Carica papaya, Solanum hazenii and Melothria pendula). Four extracts were from species with previously reported anti-herpetic activity (Plantago major, Psidium guajava, Sida acuta and Bursera simaruba). These extracts effective anti-viral concentrations (EC50) were between 203 and 6.31 µg/mL, while the selectivity indexes (SI) were between 55.91 and 2.57. Euphorbia lancifolia showed the most effective anti-viral activity (EC50 = 6.31 µg/mL, SI = 51.82). Graphical Abstract


Introduction
Herpes simplex virus 1 (HSV-1) is one of the most common viral pathogens worldwide, with a prevalence of 67% in 2012 (Looker et al. 2015). The standard treatment for HSV-1 infection is acyclovir and its derivatives. However, resistant strains have been identified, mainly in immunosuppressed patients. Therefore, sources of new anti-virals are necessary to treat this disease.
In traditional medicine, medicinal plants are widely used to treat many pathologies, some of which have been reported to have potent anti-viral activity (Li et al. 2018). Salvadoran traditional medicine is mainly based on the use of medicinal plants. It is considered a syncretic medicine since it combines the ancient heritage of the Indigenous peoples (Nahuat-Pipiles, Lencas, and Cacaoperas) who still inhabit this Central American territory with European and Arabic contributions that the Spanish introduced (Ramos 2004).
Recently, our group reported a qPCR method for evaluating anti-viral activity against HSV-1 ( Gabaglio et al. 2019). This work evaluated the anti-viral activity of 72 methanolic extracts of medicinal plants from El Salvador using this method. A 50% cytotoxic concentration (CC 50 ), a 50% anti-viral effective concentration (EC 50 ), and the selectivity index (SI) of the best candidates were also analyzed.
Initially, we analyzed the anti-viral activity and cytotoxicity of the 72 methanolic extracts at 125 mg/mL. We observed that 60 extracts exhibited a cytotoxicity of lower than 10%, and of these, 14 showed an anti-viral activity of >75% (Table S1). Since a cytotoxicity of higher than 10% could affect anti-viral activity, we analyzed the remaining 12 extracts at 62.5 mg/mL. Four extracts showed an anti-viral activity of >75% at this concentration and a cytotoxicity of <10% (Table S2).
This analysis identified 18 plant extracts with anti-viral activity >75% (Tables S1 and  S2). We analyzed the CC 50 , EC 50 , and SI of these extracts. Table S3 summarizes the results of these analyses, as well as the plants' vernacular names and popular uses (Ayala 1994;Ram ırez et al. 2002). A wide range of results with SI from 55.91 to 2.57 was observed.
Of the 18 extracts, four species (22%) had previously been reported to exhibit antiherpetic activity: Plantago major (Chiang et al. 2002) Euphorbia lancifolia showed the highest SI of all the analyzed plant extracts. Although there have been no previous reports of this plant's anti-viral activity, there have been reports of the anti-herpetic activity of other species of this genus. Triterpenes and steroids were isolated, with anti-viral activity against HSV-1 and -2 from E. segetalis and E. denticulata species (Madureira et al. 2003;Shamsabadipour et al. 2013).
The methanolic extract of Citrus aurantium showed an EC 50 of 37.15 mg/mL and an SI of 9.97. It was found that the Citrus sinensis alkaloid Citrusinine-I inhibited HSV-1 at EC 50 of 0.56 mg/mL and HSV-2 at 0.74 mg/mL with SI of > 17.8 and 13.5, respectively (Yamamoto et al. 1989).
Cordia alliodora showed an EC 50 of 15.02 mg/mL and an SI of 47.83. Previous studies have found that the hydroalcoholic extract of Cordia americana, with an SI of 55, exhibited similar behavior (Moura-Costa et al. 2012). In addition, the chloroform fraction of Cordia salicifolia exhibited virucidal action against HSV-1 with an SI of 261 (Toshimitsuhayashi et al. 1990).
Previous studies have noted anti-viral activity in members of the genus Solanum. In a previous screening of Paraguayan plants, we identified the anti-viral activity of Solanum americanum , Solanum comptum C. V. Morton, and Solanum sisymbriifolium Lam with an SI between 208.66 and 118.41 (Gabaglio et al. 2019). However, the SI of S. hazenii (SI ¼ 3.23) was lower and thus more comparable to the extract of Solanum paniculatum (SI ¼ 1.4) (Valadares et al. 2009). Several glycosylated steroids isolated from the genus Solanum have shown activity against HSV-1 (Ikeda et al. 2000).

Experimental
Experimental information along with tables, are available online as supplementary material.

Conclusion
We determined that 18 plant extracts show high anti-viral activity and can therefore be sources for isolating new compounds against HSV-1. Additionally, we found that the qPCR method proved effective for screening natural products. This work establishes the bases for the search for new compounds with anti-viral activity from the flora of El Salvador.