key: cord-0981012-cigb807w
authors: Ticona L, Apaza; P, Bermejo; JA, Guerra; MJ, Abad; M, Beltrán; R, Martín–Lázaro; J, Alcamí; LM, Bedoya
title: 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
date: 2020-08-03
journal: J Ethnopharmacol
DOI: 10.1016/j.jep.2020.113163
sha: 821c98490e4064743f1b3cc9f6111b6a9946a171
doc_id: 981012
cord_uid: cigb807w

ETHNO–PHARMACOLOGICAL RELEVANCE: The 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 STUDY: The 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 METHODS: Antiviral 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. RESULTS: Ethanolic and aqueous extracts of Artemisia campestris subsp. glutinosa (Besser) Batt. subsp. glutinosa displayed anti–HIV activity in vitro, although ethanolic extract was more powerful (IC(50) 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 IC(50) 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. CONCLUSION: We 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.

Viral infections can be treated by means of pharmacological interventions, although a cure is not always achieved. Hepatitis C virus (HCV) infections are successfully treated from a pharmacological point of view, clearing the virus from the human body. However, other viral infections as herpesvirus (HSV) or human papilloma virus (HPV) can be treated but not cured with drug therapy alone, due to the establishment of viral reservoirs (Massanella and Richman, 2016) . In this context, human immunodeficiency virus type 1 (HIV-1) infection is effectively controlled by antiretroviral therapy (ART), although it is unable to eliminate the infection. The main reason of viral persistence is the presence of latent viral reservoirs, mainly in infected resting CD4+ T cells, which are not susceptible to ART. New approaches are under study to eliminate these reservoirs, such as the "shock and kill" strategy (Deeks, 2012) , but none of them has shown effectiveness in clinical trials yet. Furthermore, only 24.5 million of the 37.9 million people infected with HIV were on ART in 2019 (UNAIDS, AIDS info accessed April 2020), and it seems unlikely that an effective vaccine will be obtained in coming years.

Artemisia genus comprises several species and some are among the most used plants in traditional Chinese medicine for the treatment of infectious diseases produced by fungi, bacteria and viruses (Abad, M. J. et al., 2012; Bora & Sharma, 2011; Pandey & Singh, 2017; Taleghani et al., 2020; Tan et al., 1998) . The best-known compound isolated from an Artemisia species (A. annua) is an endoperoxide sesquiterpene lactone sesquiterpene, artemisinin, an antimalarial drug used in artemisinin combination therapies (ACT) which is active against the intraerythrocytic forms of Plasmodium falciparum by preventing the detoxification of hemodigestion products (Talman et al., 2019) . Novel derivatives have been tested for their activity against malaria or other infectious diseases (for a review see Efferth, 2018; Liu et al., 2019; Taleghani et al., 2020) .

Other species of Artemisia genus, known as wormwoods, have been used traditionally as medicinal plants (Guibourt, 1862) . The pharmacological importance of Artemisia campestris, known as field wormwood, was revised by Al-Sanafi in 2015 (Al-Sanafi, 2015) . Traditionally, A. campestris was used as antiseptic (Redwood, 1857) , and for the treatment of pleurisy, a pathological condition caused by viral or bacterial infections (Breverton, 2011) . Moreover, North American indigenous people use A. campestris extracts to treat the common cold, caused by rhinovirus or coronavirus (Palmer 1975) . However, neither the mechanism of action nor the compounds responsible for this activity were described. In the Mediterranean region, this species is commonly used as an anthelmintic drug , as well as for the treatment of cutaneous, respiratory and digestive disorders. Several reviews on taxonomical aspects, cytogeography, biological activities and bioactive compounds of A. campestris have been published (Dib et al., 2017a; Dib & El Alaoui-Faris, 2019; Dib et al., 2017b; Dib et al., 2017c , Aniya et al., 2000 ; Jabri et al., 2018; Kadi et al., 2019; Memmi et al., 2007) which describe how the aqueous extracts and essential oils, rich in flavonoids and terpenes, are responsible for the biological activity. Antioxidant and anti-inflammatory activities have been evaluated through several in vitro and in vivo methods by measuring glutathione levels, superoxide dismutase activity, protein oxidation and nitric oxide products and in carrageenan-induced rat paw edema (Ghlissi et al., 2016; Sefi et al., 2010; Sefi et al., 2012) , identifying some of the targets of this species in human pathologies.

In this paper we have studied the potential activity of A. campestris in HIV-1 replication in vitro. Hence, the antiviral activity against HIV-1 of A. campestris extracts, the isolation of the active compounds, two terpenes and four flavonoids, and the identification of their targets in the HIV replication cycle are reported in this study. 

UV spectra were recorded on a Shimadzu 160A spectrophotometer. IR spectra were acquired with a Perkin-Elmer FT-1725X spectrophotometer. 1H NMR and 13C NMR spectra were measured on a BRUCKER Advance DRX-700 and Advance DRX-500 spectrometer, using TMS as internal standard. EIMS were obtained on a Hewlett Packard 5930 spectrometer.

The following adsorbents were used for purification: gel filtration chromatography, Carlo Erba (SDS ®) (40/60 μm; 20/45 μm); analytical thin layer chromatography (TLC) Al, Merck Si gel 60 F254. TLC chromatograms were visualized under UV light at 254 and 366 nm and/or sprayed with Naturstoffreagents-PEG.

Known compounds were isolated and identified by comparing their spectral data with those in the literature.

The air-dried aerial parts of Artemisia campestris subsp. glutinosa (Besser) Batt.

(1.0 kg) were repeatedly extracted at room temperature with ethanol (126 g) and water (146 g 

The plasmid pNL4.3-luc was generated by cloning the luciferase gene in the (Oberlin et al., 1996) . The 3-enh-κB-ConA-luc plasmid carries a luciferase gene under the control of three synthetic copies of the κB consensus of the immunoglobulin k-chain promoter cloned into the BamHI site located upstream from the conalbumin transcription start site (Arenzana-Seisdedos et al., 1993 Cell viability was measured in treated mock-infected cells in parallel under the same conditions as in the antiviral assay by the CellTiter Glo (Promega) assay system.

Cell viability is expressed as percentage of viable cells compared to a non-treated (DMSO same concentration as compound) control (100%). CC 50 was calculated with a non-linear regression formula using Prism v8.0 (GraphPad Software).

To evaluate viral entry we have compared MT-2 infections performed with recombinant wild type HIV (NL4.3-Ren) (100.000 RLUs per well or 20 ng p24 per well) and a VSV-pseudotyped recombinant HIV (HIV-VSV) (100.000 RLUs per well or 20 ng p24 per well), which enter the target cells through a receptor independent mechanism. The methodology is similar to the antiviral activity evaluation. Infections were performed with both viruses (HIV and VSV-HIV) in parallel and, after 48 hours, RLUs obtained on a luminometer. Enfuvirtide was used as a reference control of viral entry inhibition. IC 50 was calculated with a non-linear regression, dose response formula, and ANOVA analysis was performed to calculate the significant differences (p value) between HIV IC 50 and VSV-HIV IC 50 , both using Prism v8.0 (GraphPad Software).

MT-2 cells were maintained in culture without stimuli and collected prior to the assay in RPMI without serum and antibiotics. MT-2 cells were then suspended in 350 µL of RPMI without supplements and electroporated using an Easyject plus Electroporator (Equibio) at 260V, 1500 mF and maximum resistance with 1µg/10 6 cells of a luciferase plasmid under the control of the whole genome of HIV-1 (pNL4.3-Luc), NF-κB (3-enh-κB-ConA-luc) or Sp1 (pSp1-Luc). Afterwards, MT-2 cells were seeded in 24 well microplates and treated with different concentrations of compounds, using PMA as a reference control of HIV-1 transactivation, and left in culture in complete RPMI at 37°C. 48 hours later, cultures were lysed with luciferase buffer (luciferase assay system buffer, Promega) and luciferase activity (RLUs) measured in a luminometer (Berthold Detection Systems).

Total glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined in THP-1 cells extracts following the Hissin and Hill method (Hissin and Hilf, 1976) . Briefly, cell culture was lysed with phosphate-EDTA (0.1 M sodium phosphate and 0.005 M EDTA) buffer (pH=8) at 100 mg/mL concentration, adding 10 μL/mL of HClO 4 (60 %). Then, cell homogenates were spun at 10.000 rpm (6000g) for 10 min at 4°C and supernatants were maintained at 4°C until reduced glutathione (GSH) and oxidized glutathione ( RI=GSH/(GSH+GSSG). Results are expressed as EC 50 or concentration that produces 50% of the maximum antioxidant effect, being the maximum 1 (100% of glutathione is in its reduced form). Furthermore, we have also calculated the E max reached by each compound to evaluate the antioxidant efficacy. Both parameters were calculated using Prism v8.0 (GraphPad Software) using non-linear regression, dose-response curves.

Non-linear regression was used to calculate IC 50 , EC 50 and CC 50 . One-way ANOVA statistical analysis (post-test Bonferroni multiple comparison test, * p<0.05; **p<0.01) was performed to evaluate the significant differences among values.

Antioxidant and transcriptional activity correlation analysis was performed using the non-parametric Spearman analysis and the r coefficient was calculated. All the analyses were performed using Prism v8.0 (GraphPad Software).

Artemisia campestris subsp. glutinosa (Besser) Batt. was extensively extracted with ethanol and water. The obtained extracts were evaporated and submitted to chemical complexity (figures S1, S2 and S3) and anti-HIV and cytotoxic activity evaluation ( DCM-EE, selected as the most active fraction, was then subjected to repeated column chromatography obtaining 18 fractions (I-XVIII) which were submitted again to antiviral activity evaluation (Table 2) . Again, we found that several fractions showed anti-HIV activity, but some of them displayed lower IC 50 values. Further purification of the most active fractions, 10-1954; Aponte et al., 2010; Goldsby and Burke, 1987; Herz et al., 1981; Saeed et al., 2015; Villagomez et al., 2013) . Canrenone is a steroidal triterpene lactone never isolated before from any plant species although its structure is described elsewhere (Cashman & Peña 1989 ).

On the other hand, flavonoids are almost ubiquitous compounds, also found in Artemisia spp. In fact, cardamonin was first identified in A. absinthium L. (Hatziieremia et al., 2006) and acerosin was isolated from Artemisia afra Jacq. ex Willd (Wollenweber & Mann, 1989) . However, 6,2',4'-trimethoxyflavone and xanthimicrol were never isolated from Artemisia spp.

The chemical structure of artemisinin, a sesquiterpene lactone, and other related antimalarial compounds isolated from A. annua, is related to damsin, although the former is a cyclohexane lactone rather than a cyclopentane lactone (Abad et al., 2014) .

Moreover, the α-methylene-γ-lactone fragment characteristic of sesquiterpene lactones has been previously identified as responsible for the anti-inflammatory and anti-tumor activities (Chaturvedi, 2011) due to its interaction with specific biological nucleophiles, such as the cysteine sulfhydryl groups of target proteins (Hwang et al., 2006) .

Furthermore, there are references in the literature indicating that sesquiterpene lactones with a α-methylene-γ-lactone fragment inhibits influenza A virus replication (Zhang et al., 2018) .

On the other side, canrenone only presents the γ-lactone fragment. If we compare it to damsin, the main differences are the absence of the methylidene group in the lactonic ring and the presence of a steroidal fragment (cyclopentane perhydro phenanthrene). This has two main implications. First, the absence of the methylidene group (exocyclic bond) prevents the conjugation with the carbonyl function of the lactonic ring, reducing its binding capacity and causing a reduction in its biological activity (Gach et al., 2015) . Second, the steric effect caused by the steroidal ring of canrenone can cause a decrease in its activity by preventing the compound binding (Cheng & Yuan, 2006) . Interestingly, canrenone is thought to be the main metabolite of the aldosterone antagonist spironolactone, and both structures are terpene steroids. In fact, canrenone is widely known as a diuretic due to its antagonism of mineralocorticoids receptors in the kidney (potassium canreronate) (Armanini et al., 2014) .

Regarding the four flavonoids isolated, they are widely described in the literature but all of them share different rates of hydroxyl groups methylation as a structural characteristic, with cardamonin as the less methoxylated and trymethoxyflavone, with no free hydroxyl groups, as the most methoxylated flavonoid. As a matter of fact, their antiviral activity could be related to the hydroxylation rate of the rings (Ahmad et al., 2015) . For example, flavonols were reported to be more active in comparison to flavones against the herpes simplex type I virus (Dillard & German, 2000) . Moreover, the mild selective anti-HIV activity of myricetin relative to quercetin as a result of the presence of one additional hydroxyl group at the 5' position (Mahmood et al., 1993) .

Although 6,2',4'-trimethoxyflavone, acerosin and xanthomicrol have the same number of methoxy groups they have a different number of hydroxyl groups: acerosin 3, xanthomicrol 2 and trimethoxyflavone 0. These observations suggest potential differences in antiviral and antioxidant activities. Regarding cardamonin, previous literature reported the anti-HIV activity of a related chalcone, 2-methoxy-3-methyl-4,6dihydroxy-5-(3'-hydroxy) cinnamoylbenzaldehyde (Wu et al., 2003) . However, the structural differences between both chalcones could lead to a diverse biological activity, since cardamonin lacks the hydroxyl group in the C position.

As the previous screening of extracts and fractions showed, the presence of structurally different compounds with anti-HIV-1 activity in A. campestris EE would yield puzzling results in the EE target identification, due to the concurrent interference with several different targets. Therefore, we have tried to study the mechanism of action of the isolated compounds.

We Table 3 . IC 50 s and CC 50 s calculated for the isolated compounds from Artemisia campestris in the HIV infection assay. MT-2 cells were infected with either recombinant wild type HIV (NL4.3-Ren. 100.000 RLUs or 20 ng p24/well) or VSV pseudotyped recombinant wild type HIV (VSV-HIV. 100.000 RLUs or 20 ng p24/well)) and treated with serial dilutions of damsin, canrenone, cardamonin, acerosin, trimethoxyflavone or xanthomicrol. The HIV-1 fusion inhibitor enfuvirtide was used as control. Results were obtained using the renillaluciferase assay system (Promega) following the manufacturer instructions after 48 hours in culture, measuring the RLUs in a luminometer. 100% was the RLUs obtained by untreated controls. Cell viability was determined in parallel in mock infected cells using CellTiter Glo reagent following the manufacturer instructions (Promega). IC 50 , CC 50 and p values were calculated using Prism v8.0 (GraphPad Software) using non-linear regression, dose-response curves, and one-way Anova analysis. IC 50 : Inhibitory Concentration 50%. CC 50 : Cytotoxic Concentration 50%. CI95%: Confidence interval 95%.

Sesquiterpene lactones have previously shown anti-HIV activity (Mohammed et al., 2014; Zhang et al., 2005) , although their mechanism of action remains elusive, while isolated flavonoids have shown previously mixed results. Regarding flavonoids, we did not find literature about the antiviral activity of acerosin, xanthomicrol and 6,2',4'-trimethoxyflavone, although other trimethoxyflavones, as 5,6,7trimethoxyflavone, showed activity against poliovirus and herpesvirus (Hayashi et al., 1997) or 4',5-dihydroxy-3,3',7-trimethoxyflavone, with selective activity against picornavirus (Ishitsuka et al., 1982) . Moreover, four different hydroxymethoxyflavones were found to inhibit HIV-1 replication, although their mechanism of inhibition was not determined (Kongkum et al., 2012) . Cardamonin, a chalcone commonly isolated from different plants, has proven to have antifungal (Lopez et al., 2011) , antibacterial (Aderogba et al., 2012) and anti-HIV protease (Tewtrakul et al., 2003) activities.

Additionally, cardamonin inhibited dengue-2 virus NS4 protease (Kiat et al., 2006) and

was also found to inhibit NF-κB activation in HUVEC cells, although it did not alter the grow of Hantaan virus (HTNV) infection (Yu et al., 2014) . Taking into consideration the diverse hydroxylation rate among flavonoids, differences were also found in their biological activity. Therefore, as it was hypothesized before, a higher hydroxylation rate would result in a higher activity. This hypothesis could not be confirmed with the chalcone. However, cardamonin lacks the hydroxyl group in the C position present in other bioactive chalcones (Wu et al, 2003) , which could be the reason for its low anti-HIV activity.

The multi-infectious activity of these compounds and the uses traditionally reported by plant extracts in several diseases suggest a common target in the inhibition of microorganisms. Since every microorganism encompasses its own and different proteins, we hypothesized here that this target, or targets, could be cellular processes suggesting that HIV-1 entry is impaired by the treatment with both flavonoids.

HIV-1 is a retrovirus that integrates its genetic material into the host cell genome.

Cellular transcriptional activity is then responsible for the viral RNA expression as well as for the completion of a successful viral replication cycle. Viral transcription is controlled by long terminal repeat (LTR) sequences, viral promoter regions present in the proviral form of the integrated provirus, which show binding sites in response to several stimuli. One of the most important viral transcription activators is the transcription factor NF-κB, although others are also involved, such as Sp1 or NFAT.

To evaluate the effect on viral transcription, we first used a plasmid encoding a luciferase reporter gene whose expression depends on the whole HIV genome (NL4.3-Luc). This plasmid was transfected in MT-2 cells treated or not with serial concentrations of isolated compounds. This allowed us to easily measure the effect of compounds specifically on HIV transcription, without any potential pre-transcription target interference ( Figure 3 ). Figure 3 and Table 4 . IC 50 s calculated for the isolated compounds from Artemisia campestris. in the viral transcription assay. MT-2 cells were transfected with a luciferase plasmid (pNL.43-Luc.1 ng/10 6 cells) under the control of the whole genome of the wild type HIV and treated with serial dilutions of damsin, canrenone, cardamonin, acerosin, trimethoxyflavone and xanthomicrol. Results were obtained using the luciferase assay system following the manufacturer instructions after 48 hours in culture measuring the RLUs in a luminometer. 100% was the RLUs obtained by untreated controls. IC 50 was calculated using Prism v8.0 (GraphPad Software) using nonlinear regression, dose-response curves. CI95%: Confidence interval 95%.

HIV-integrated provirus depends on several cellular and viral factors to initiate transcription, being the viral protein Tat and the cellular transcription factor NF-kB by far the most significant ones. Sp1 is another important transcriptional factor with consensus sequences in the long terminal repeats (LTR) of HIV. We, therefore, evaluated the effect of isolated compounds on NF-kβ and Sp1 activity through transfection of luciferase plasmids in MT-2 cells under the control of both transcription factors (Figure 4) .

To this purpose, we selected one active concentration of each compound and compared the effect on transcription factors with the effect on viral transcription to assure that selected concentrations were also active in HIV transcription. As shown in Figure 4 , damsin (4 µM) completely inhibits HIV transcription and NF-κB and Sp1 activity (p values <0.0001, 0.0036 and <0.0001, respectively) when compared to the untreated control. Canrenone was slightly less active, although it showed a more powerful activity as a NF-κB inhibitor than as a Sp1 inhibitor (p values <0.0001, 0.0066 and 0.0002, respectively). Thus, the main target of damsin, and to a lesser extend canrenone, is NF-κB, and to a lesser extent Sp1. Regarding flavonoids, cardamonin was not tested since it was not active in viral transcription (figure 3). When fixed concentrations of 20 µM were used, we found that the weak effect of acerosin in viral transcription was confirmed with the lack of activity in the inhibition of transcription factors. Moreover, acerosin was able to induce the activation of Sp1 and for that reason, theoretically, it could activate viral replication. However, acerosin showed first an IC 50 in the inhibition of viral infection of 2.7 µM, ruling out a potential pro-replicative effect. Besides, acerosin was one of the compounds that inhibited viral entry. Thus, this inhibitory activity could be responsible of the HIV infection inhibition, although its weak activity on viral transcription could aid to the final effect. The effect of xanthomicrol, although active as HIV transcriptional inhibitor, was only mediated through a weak inhibition of NF-κB with no effect on Sp1. This effect was expected, since xanthomicrol is the least powerful transcription inhibitor, excepting cardamonin, and showed a slight activity in viral entry. Finally, the most potent flavonoid, trimethoxyflavone confirmed its activity by the almost complete inhibition of HIV transcription and both transcription factors, NF-κB and Sp1 (p values <0.0001, 0.0044 and <0.0001, respectively).

Although we show here that NF-κB could be involved in the anti-HIV effect of some flavonoids, especially the trimethoxyflavone, the mechanism of inhibition of viral infection is not well understood. Some authors have linked antioxidant activity to NF-κB inhibition (Xiao et al., 2006) which could lead to HIV-1 inhibition. The involvement of oxidation in T-cell activation was first described in the 1980s by several groups, including the inhibition of PMA induced proliferation by antioxidants (Novogrodsky et al., 1982) . Reactive oxygen species (ROS) play a dual role in lymphocytes, the main target of HIV-1. They are needed for their activation but also promote apoptosis and cell death. Lymphocyte activation is crucial for the establishment of HIV infection and can be induced through several stimuli. In fact, T cell receptor (TCR) triggered by antigens activates an intracellular signalling cascade leading to transcriptional activation, including NF-κB activation. Moreover, oxidative stimuli in the cytosol are associated with activation and nuclear translocation of NF-κB, while reductive activation by thioredoxin is required for NF-κB binding to DNA (Chandrasekaran & Taylor, 2008) . In this process, ROS play an important role, either produced by T cells themselves or by the exposure to exogenous oxidants (Benhar et al., 2016) . Besides, NF-κB transcription factor can be activated by several stimuli including infection but also inflammation and oxidative stress (Pahl, 1999) .

In this sense, low reduced glutathione (GSH) levels in HIV+ patients were found to induce oxidative stress and increase viral replication (Schreck et al., 1991) .

Furthermore, the decrease of GSH levels seems to be related to HIV-1 LTR sensitization through the enhanced DNA binding ability of NF-κB (Yamaguchi et al., 2002) . Actually, antioxidants and low GSH levels could promote viral latency and thus, the maintenance of HIV-1 reservoirs in T cells (Kalebic et al., 1991) . However, some controversy has been found, since the role of oxidation in T cell activation varies when cell or environmental conditions change (Simeoni & Bogeski 2015) .

Flavonoids are widely known antioxidants and for this reason, this activity could be related to the viral transcription inhibition. Therefore, we have also tried to evaluate the antioxidant effect of all the isolated compounds, including terpenes and flavonoids, to assess if the effect on the cellular redox state is related to the transcriptional effect. Table 5 . EC 50 calculated for the isolated compounds from Artemisia campestris. in the glutathione assay. Total, reduced (GSH) and oxidized glutathione (GSSG) levels were determined in THP-1 cells extracts. Fluorimetric measurement of the interaction of thiols with OPT (o-Phtaldehyde) in the presence of serial dilutions of damsin, canrenone, cardamonin, acerosin, trimethoxyflavone, xanthomicrol and resveratrol (control) were compared to a standard curve produced by measuring increasing concentrations of GSH. Fluorescence was measured at λexc=350 nm and λem= 420 nm. The redox index (RI), a parameter that indicates the antioxidant status of the tissue, was expressed as follows: RI=GSH/(GSH+GSSG). Results are expressed as EC 50 (concentration that produce the 50% of the maximum antioxidant effect), being the maximum 1 (all of the glutathione is in the reduced form). Besides, we have also calculated the E max reached by each compound to evaluate antioxidant efficacy (E max ). Both parameters were calculated using Prism v8.0 (GraphPad Software) using non-linear regression, dose-response curves.

All the flavonoids showed antioxidant activity and, as expected, terpenes did not. The most effective antioxidants were acerosin and xanthomicrol (E max 0.95 and 0.93, respectively). The high methoxylation rate and the lack of hydroxyl groups of trimethoxiflavone could explain its lower efficacy as antioxidant (E max 0.74), although it was still higher than terpenes, damsin and canrenone. EC 50 was very similar among all the flavonoids, with trimethoxyflavone as the most potent antioxidant (EC 50 3.79 µM).

It could be hypothesized that a high methoxylation rate could increase cell penetrability and due to this fact, a lower flavonoid concentration would be needed. On the other hand, the lack of hydroxyl groups could diminish its antioxidant activity once inside.

In summary, acerosin and xanthomicrol were the most effective antioxidants and trimethoxiflavone the most powerful one. Although it is difficult to correlate these results with the viral transcriptional inhibition, we found that trimethoxyflavone was the most powerful HIV-transcription inhibitor and one of the most powerful antioxidants, although its efficacy was lower than the efficacy of acerosin or xanthomicrol.

Cardamonin was the less powerful antioxidant but it showed a good antioxidant efficacy. However, cardamonin did not show any transcriptional activity. Acerosin and xanthomicrol were medium transcriptional inhibitors, and showed the highest antioxidant effect, although with slightly lower concentrations than trimethoyflavone.

To analyse the potential correlation between the antioxidant effect and the transcriptional inhibition of the isolated compounds we have performed a Spearman correlation analysis, and the r value has been calculated for EC 50 transcription (EC 50 trans), EC 50 antioxidant (EC 50 ox) and antioxidant efficacy (E max ) for all the isolated compounds, including terpenes (figure 6A), and only for the flavonoids with proven antioxidant activity ( figure 6B) .

Surprisingly, we could not find correlation between transcriptional and antioxidant activities (IC 50 trans and IC 50 ox), even when the flavonoids alone were analysed. Moreover, we found a negative correlation even if terpenes were included or not (Spearman r of -0.67 and -0.40, respectively). However, when we compare the effectiveness as antioxidants (E max ) with the transcriptional activity (EC 50 trans), a positive r was obtained, even when terpenes were included (Spearman r of 0.60 and 0.40, respectively). However, in this last case, the correlation is inverse. A higher Emax indicates a higher efficacy as antioxidant and it correlates with a higher IC 50 which indicates less potency as transcriptional inhibitor. Consequently, the correlation is, in fact, negative. Therefore, the more antioxidant the activity is, the less transcriptional.

This rules out a potential involvement of the antioxidant activity in the transcriptional inhibition achieved with these compounds. Despite this fact, the presence of flavonoids with preferred transcriptional activity, as the trimethoxyflavone, and flavonoids with preferred antioxidant activity, as acerosin, highlights the multitarget activity of A. campestris composition. However, more research would be needed to address this issue.

In summary, aqueous and ethanolic extracts of Artemisia campestris subsp.

glutinosa (Besser) Batt. displayed anti-HIV activity, with EE as the most powerful inhibitor. Two terpenes and four flavonoids were isolated from this active extract: a pseudoguainolide sesquiterpene lactone, damsin, a steroid triterpene, canrenone, a chalcone, cardamonin, and three methoxylated flavones, acerosin, 6,2',4'trimethoxyflavone and xanthomicrol. All the isolated compounds showed anti-HIV replication activity in vitro with IC 50 s in the medium nanomolar to low-medium micromolar range. Acerosin and xanthomicrol inhibit viral entry and all of them, excepting cardamonin, showed activity in HIV transcription, mainly though NF-κB inhibition. Antioxidant activity was showed exclusively by flavonoids, although a negative correlation with transcriptional activity was found. However, the antioxidant activity would probably add other benefits to the treatment of the AIDS pathology.

In this work, we have shown the anti-HIV-1 activity of A. campestris subsp. glutinosa (Besser) Batt. and its components, through the inhibition of multiple 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 several infections. Besides, multitarget activity is receiving increased attention in the field of drug discovery and the presence of several compounds with dual and different mechanisms of action could be of interest in pathologies like HIV-1 infection, treated with a mixture of drugs directed at different targets, since it could aid in overcoming drug resistances and simplify drug therapy.

The authors declare no conflict of interest. 

The writing assistance of Ms Brooke-Turner and Ms Luisa López is gratefully acknowledged. We would like to thank Dr. Molina for collecting the plant samples.

This project was supported by the Spanish Agency for International Cooperation and Development (AECID) (D/020523/08) and the Universidad Complutense de Madrid UCM-Santander (PR87/19-22685). This work was partially supported by Instituto de Salud Carlos III and co-funded by European Regional Development Fund (ERDF) "A way to build Europe" (projects AIDS Research Network RD16CIII/0002/0001 and RD16CIII/0002/0001 to JA). The cell culture was then lysed and relative luminescence units (RLUs) were measured in a luminometer. Cell viability was evaluated in mock infected cells in parallel using the CellTiter Glo reagent (Promega). Results were analyzed using Prism v8.0 (GraphPad Software), non-linear regression and dose response curves. IC 50 : inhibitory concentration 50%. CC 50 : cytotoxic concentration 50%. CI95%: confidence interval 95%. cardamonin, acerosin, trimethoxyflavone and xanthomicrol were compared to a standard curve produced by measuring increasing concentrations of GSH. Fluorescence was measured at λexc=350 nm and λem= 420 nm. The redox index (RI), a parameter that indicates the antioxidant status of the tissue, was expressed as follows:

RI=GSH/(GSH+GSSG). Results are expressed as EC 50 or concentration that produce the 50% of the maximum antioxidant effect, being the maximum 1 (maximum of the glutathione is in the reduced form or GSH). Besides, we have also calculated the E max reached by each compound to evaluate the antioxidant efficacy (E max ). Both parameters were calculated using Prism v8.0 (GraphPad Software) using non-linear regression, dose-response curves. 

The artemisia L. Genus: a review of bioactive essential oils

Chapter 2 -The Artemisia L

Pharmacological Potentials of Artemisinin and Related Sesquiterpene Lactones: Recent Advances and Trends

The chemistry of Ambrosia maritima L

Hydrogenation, oxidation, and dehydrogenation of ambrosin and damsin

Isolation of antioxidant constituents from Combretum apiculatum subsp. apiculatum

Therapeutic potential of flavonoids and their mechanism of action against microbial and viral infections-A review

The pharmacological importance of Artemisia campestris-a review

Antioxidant and hepatoprotective actions of the medicinal herb Artemisia campestris from the Okinawa Islands

Cytotoxic and antiinfective sesquiterpenes present in Plagiochila disticha (Plagiochilaceae) and Ambrosia peruviana (Asteraceae)

Phosphatidylcholine hydrolysis activates NF-kappa B and increases human immunodeficiency virus replication in human monocytes and T lymphocytes

Aldosterone receptor blockers spironolactone and canrenone: two multivalent drugs

Dual targeting of the thioredoxin and glutathione systems in cancer and HIV

Breverton´s Complete Herbal, based on Culpeper´s the english physician and complete herbal of 1653

CCR5 receptor antagonism inhibits hepatitis C virus (HCV) replication in vitro

The genus Artemisia: a comprehensive review

Canrenone formation via general-base-catalyzed elimination of 7alpha-(methylthio)spironolactone S-oxide

Molecular modeling of the oxidized form of nuclear factor-kappa B suggests a mechanism for redox regulation of DNA binding and transcriptional activation

Sesquiterpene lactones: structural diversity and their biological activities

Quantitative study of electrostatic and steric effects on physicochemical property and biological activity

HIV: Shock and kill

Artemisia campestris L.: Ethnomedicinal, phytochemical and pharmacological review

Artemisia campestris L.: review on taxonomical aspects, cytogeography, biological activities and bioactive compounds

Chemical composition, vasorelaxant, antioxidant and antiplatelet effects of essential oil of Artemisia campestris L. from Oriental Morocco

Antihypertensive and vasorelaxant effects of aqueous extract of Artemisia campestris L. from Eastern Morocco

Phytochemicals: nutraceuticals and human health

Beyond malaria: The inhibition of viruses by artemisinin-type compounds

The role of oxidative stress in anticancer activity of sesquiterpene lactones

A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1

Antioxidant, antibacterial, anti-inflammatory and wound healing effects of Artemisia campestris aqueous extract in rat

Sesquiterpene lactones and a sesquiterpene diol from jamaican ambrosia peruviana

Historia natural de las drogas simples

The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages

Antiviral activity of 5,6,7-trimethoxyflavone and its potentiation of the antiherpes activity of acyclovir

Damsinic acid and ambrosanolides from vegetative ambrosia hispida

A fluorometric method for determination of oxidized and reduced glutathione in tissues

The CCR5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction

Synthesis and anti-viral activity of a series of sesquiterpene lactones and analogues in the subgenomic HCV replicon system

Antipicornavirus flavone Ro 09-0179

Protective effects of Artemisia campestris extract against gastric acid reflux-induced esophageal mucosa injuries

Synergistic antinociceptive activity of combined aqueous extracts of Artemisia campestris and Artemisia herbaalba in several acute pain models

Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine

inhibitor as novel acute graft versus host disease prophylaxis in children and young adults undergoing allogeneic stem cell transplant: results of the phase II study

Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease

DNA topoisomerase IIalpha inhibitory and anti-HIV-1 flavones from leaves and twigs of Gardenia carinata

Biological Activities of Artemisinin Derivatives Beyond Malaria

Detection of antifungal compounds in Polygonum ferrugineum Wedd. extracts by bioassay-guided fractionation. Some evidences of their mode of action

Inhibition of HIV infection by flavanoids

Measuring the latent reservoir in vivo

Use of medicinal plants against scorpionic and ophidian venoms

Isolation and anti-HIV-1 activity of a new sesquiterpene lactone from Calocephalus brownii F. Muell

Hydroxyl radical scavengers inhibit lymphocyte mitogenesis

The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1

Activators and target genes of Rel/NF-kappaB transcription factors

The Genus Artemisia

Supplement to the Pharmacopoeia third ed

Cytotoxicity of the Sesquiterpene Lactones Neoambrosin and Damsin from Ambrosia maritima Against Multidrug-Resistant Cancer Cells

Reactive Oxygen Intermediates as Apparently Widely Used Messengers in the Activation of the NF-kappa B Transcription Factor and HIV-1

Mitigating effects of antioxidant properties of Artemisia campestris leaf extract on hyperlipidemia, advanced glycation end products and oxidative stress in alloxan-induced diabetic rats

Artemisia campestris leaf extract alleviates early diabetic nephropathy in rats by inhibiting protein oxidation and nitric oxide end products

Redox regulation of T-cell receptor signaling

Artemisia: a promising plant for the treatment of cancer

Artemisinin Bioactivity and Resistance in Malaria Parasites

Biologically active substances from the genus Artemisia

HIV-1 protease inhibitory substances from the rhizomes of Boesenbergia pandurata Holtt

Multiple anticancer effects of damsin and coronopilin isolated from Ambrosia arborescens on cell cultures

Exudate flavonoids in three essential oil plants from the Ciskei (South Africa)

Anti-AIDS Agents 54. A Potent Anti-HIV Chalcone and Flavonoids from Genus Desmos

Activity of the dietary antioxidant ergothioneine in a virus gene-based assay for inhibitors of HIV transcription

Azidothymidine causes functional and structural destruction of mitochondria, glutathione deficiency and HIV-1 promoter sensitization

Involvement of the Akt/NF-kappaB pathways in the HTNV-mediated increase of IL-6

Sesquiterpenes and butenolides, natural anti-HIV constituents from Litsea verticillata

Antiviral Activity of the Sesquiterpene Lactones from Centipeda minima against Influenza A Virus in vitro