Comparative anti-herpes simplex virus type 1 and chemical profiling of Thymus capitatus and Artemisia herba-alba collected from North Africa

Abstract Herpes simplex virus (HSV) can infect millions of people worldwide causing mild to life-threating infections. The current study demonstrates the first comparative anti-HSV type 1 activity and phytochemical investigation of Artemisia herba-alba and Thymus capitatus collected from Egypt and Libya. Liquid chromatography/mass spectrometry (LC/MS) analysis allowed the identification of 56 and 38 compounds in the Egyptian and Libyan Artemisia herba-alba ethanolic extracts, respectively, in addition to 46 and 50 compounds in the Egyptian and Libyan Thymus capitatus ethanolic extracts, respectively. Gas chromatography/mass spectrometry (GC/MS) analysis of their corresponding essential oils revealed the presence of 15, 17, 17 and 8 compounds in Egyptian and Libyan Artemisia herba-alba and Thymus capitatus, respectively. The major chemical classes of the identified compounds were phenolic acids, flavonoids and oxygenated monoterpenes. Evaluation of the anti-HSV1 activities of the studied extracts showed that the Egyptian Thymus capitatus ethanolic extracts were the most potent extract with more than 200-fold reduction in the viral PFU. Graphical Abstract


Introduction
Herpes simplex virus (HSV) is a human pathogen causing billions of lifelong infections worldwide (James et al. 2020).HSV infections can cause mild symptoms, however, they can be associated with high morbidity and mortality (Madavaraju et al. 2021).HSV can be divided into two serotypes: HSV-1 and HSV-2 (Juan et al. 2022).It is estimated that 80% of humans worldwide are infected with HSV-1 with roughly 40% recurrent infections (Reusser 2001).Medicinal plants are considered one of the most promising sources of new antiviral drugs (Hassan et al. 2015).Artemisia herba-alba, family Asteraceae and Thymus capitatus, family Lamiaceae are widely distributed in the world, especially in the Mediterranean basin regions (Amor et al. 2019).They are rich in diverse secondary metabolites as essential oils, flavonoids, lactones and phenolics (Amkiss et al. 2021).They are among the most extensively used medicinal plants in North Africa traditional medicine mainly for their antihyperglycemic, anti-inflammatory and potent antimicrobial properties for gastrointestinal and respiratory tract illnesses (Amor et al. 2019).In the present work, we are reporting the first comparative phytochemical and anti-HSV-1 evaluation of A. herba-alba and T. capitatus collected from North Africa namely from Egypt and Libya.Analysis and identification of the chemical composition were achieved using LC/MS and GC/MS.In addition, the anti-HSV-1 activities of the extracts of the two studied species were correlated to the identified chemical constituents.

Results and discussion
2.1.Comparative chemical profiling of A. herba-alba samples using LC/MS and GC/MS LC/MS analysis was performed on the ethanolic extracts of A. herba-alba specimens collected from Egypt and Libya.Identification of detected compounds was achieved as previously reported (Mustafa et al. 2020) relying on the retention time and fragmentation of the detected compounds in comparison to an in-house authentic library.LC/MS revealed the presence of 56 compounds in the A. herba-alba Egyptian samples and 38 compounds in the Libyan samples.The chemical composition of the studied Egyptian and Libyan species are listed in Tables S1 and S2 (Figures S1 and S2).Despite the qualitative and quantitative differences in the identified compounds of the Egyptian and Libyan species, di-O-caffeoylquinic acids, artemisinic acid, a-ketoglutaric acid and scopoline represented the most abundant secondary metabolites in both species.
GC/MS analysis of the extracted essential oil samples revealed the presence of 15 and 17 compounds in the Egyptian and Libyan A. herba-alba essential oils, respectively (Tables S3 and S4 and Figures S3 and S4).The main components in both essential oil samples were vanillyl alcohol, a-thujone and b-thujone.Unlike the Egyptian A. herbaalba, the essential oil of the Libyan plant contains a significant amount of camphor, and it lacks 2-propenoic acid, 3-phenyl-, ethyl ester.

Comparative chemical profiling of T. capitatus samples using LC/MS and GC/MS
LC/MS analysis of the T. capitatus revealed the presence of 46 and 50 compounds in the studied Egyptian and Libyan samples, respectively (Tables S5 and S6 and Figures  S5 and S6).Both extracts shared several major compounds as thymol, paraben, ferulic acid, cinnamic acid, apigenin, p-hydroxybenzoic and terpinen-4-ol.However, the Egyptian sample contained higher amounts of epicatechin while the Libyan showed higher concentrations of a-terpinolene and b-ocimene in addition to the unique occurrence of gallic acid.
GC/MS showed the presence of 17 and 8 compounds in the Egyptian and Libyan T. capitatus essential oils, respectively (Tables S7 and S8 and Figures S7 and S8).Although thymol represented the major constituent in both essential oils, it was more abundant in the Libyan samples (89.38%) compared to the Egyptian samples (62.9%).

Anti HSV-1 activity
The anti-HSV1 activities of studied extracts were investigated by two models.The first was achieved through preincubating the tested compounds with viruses before being added to host cells (co-infection model), while the second model was performed by pre-treatment of host cells with the studied extracts before adding viruses (pre-treatment model).The obtained results, summarized in Table S9, indicated the potent antiviral effects of the total ethanolic extracts of the Egyptian and Libyan T. capitatus samples against HSV1 virus in the pre-treatment model reducing viral PFU from 9 Â 10 5 to 0.54 Â 10 5 and 0.5 Â 10 5 , respectively via preventing viral invasion.On the other side, the Egyptian A. herba-alba and T. capitatus ethanolic extracts showed potent effects in the co-infection model by reducing viral PFU from 9 Â 10 5 to 0.04 Â 10 5 for both extracts, through either direct viricidal effects or binding to viral particles.The detected anti-HSV1 activities might be attributed to the high content of monoterpenes as thymol, 1,8-cineole, terpinen-4-ol and alpha-terpineol in the studied ethanolic extracts.These findings support the previously reported potent anti-HSV-1 activity of these compounds (Astani et al. 2010).It is clear from the obtained results that the Egyptian T. capitatus ethanolic extract showed the highest activity against HSV1 in both co-infection and pre-treatment models.

Experimental
This part is described in the Supplementary Material.

Conclusion
The current study demonstrated the first comparative phytochemical and anti-HSV-1 study of A. herba-alba and T. capitatus samples collected from North Africa (Egypt and Libya).LC/MS revealed the variation in the secondary metabolites in the Egyptian and Libyan plants with a maximum of 56 compounds in the Egyptian Artemisia herba-alba and 50 compounds in the Libyan Thymus capitatus.While GC/MS identified from 8 to 17 volatile components in the studied extracts.This was reflected on the anti HSV-1 activities of the studied extracts.In general, ethanolic extracts were found to be more potent than essential oil of the tested samples.T. capitatus showed higher anti HSV-1 activities compared to A. herba-alba samples.Libyan and Egyptian T. capitatus as well as Egyptian A. herba-alba ethanolic extracts showed promising anti HSV-1 profile with more than 200-fold reduction in the viral PFU.The obtained results supported the wide folk medicine use of the studied plants as potent antimicrobial agents in North Africa.