Ethanolic extract of Artemisia campestris subsp. glutinosa (Besser) Batt. inhibits HIV–1 replication in vitro through the activity of terpenes and flavonoids on viral entry and NF–κB pathway

Abstract

Ethno–pharmacological relevanceThe genus Artemisia spp. is well known for its anti–infectious properties and its high content in anti–infectious compounds, like the well–known sweet wormwood (Artemisia annua L.). Another Artemisia species, Artemisia campestris subsp. glutinosa (Besser) Batt., field wormwood, has been traditionally used as medicinal plant in the Mediterranean region. Aim of the studyThe aim of this study is to investigate the anti–HIV activity of field wormwood, to identify the compounds responsible for this activity and their structure and mechanism of action. Materials and methodsAntiviral activity of isolated compounds and extracts was evaluated in HIV–1 infections of lymphoblastoid cells. We also evaluated the mechanism of action of isolated compounds. Viral entry was studied comparing the inhibitory effect of isolated compounds on wild type HIV–1 and VSV pseudotyped HIV–1. To assess the viral transcriptional effect, plasmids encoding luciferase reporter genes under the control of the whole genome of HIV–1 or NF–κB or Sp1 transcription factors were transfected in the presence of the compounds under evaluation. Finally, antioxidant activity was assessed by quantitation of reduced and total glutathione in treated cell cultures. ResultsEthanolic and aqueous extracts of Artemisia campestris subsp. glutinosa (Besser) Batt. subsp. glutinosa displayed anti–HIV activity in vitro, although ethanolic extract was more powerful (IC50 14.62 μg/mL). Bio–guided ethanolic extract fractionation leads to the isolation and characterization of two terpenes, damsin and canrenone, and four flavonoids, 6, 2′, 4′–trimethoxyflavone, acerosin, cardamonin and xanthomicrol. All the isolated compounds inhibited HIV–1 replication in vitro with IC50 values between the middle nanomolar and the low micromolar range. Their anti–HIV mechanism of action is due to the bloking of viral entry and/or transcription inhibition, without correlation with the antioxidant activity, through interference with the cellular transcription factors NF–κB and Sp1, which are targets that are not currently reached by antiretroviral therapy. ConclusionWe describe here the anti–HIV activity of field wormwood, Artemisia campestris subsp. glutinosa (Besser) Batt., and the isolation and study of the mechanism of action of two terpenes and four flavonoids, responsible, at least in part, for its activity, through the inhibition of two different cellular targets affecting the HIV replication cycle. The activity of these compounds in cellular targets could explain why plant extracts can be used in the treatment of different diseases. Besides, the presence of several compounds with dual and different mechanisms of action could prove useful in the treatment of HIV–1 infection, since it could aid to overcome drug resistances and simplify drug therapy. This work is a further step in understanding the anti–infectious activity of wormwood species and their use in treating infectious diseases.