Jatropha curcas is a plant with a diverse spectrum of pharmacological properties. It contains mixtures of different chemical compounds that act individually or collectively to improve human health (Prasad et al., 2012). It is used for medicinal purposes because it has antifungal, antibacterial, antitumor, antiviral, anti-inflammatory  (Silva  Filho  et  al.,  2009)  and  antioxidant effects (Rocha, 2013; Ribeiro et al., 2020).

However, despite all the potentialities of the plant reported earlier, this work focuses on the antiviral property of J. curcas because first, ??public health is faced with numerous challenges imposed by viral diseases, such as HIV -AIDS, COVID-19, new variants of SARS-CoV-2 and  also  existing drugs are  no  longer  effective against HIV; second, Ferrão and Janeque (2019), in their work, found that the compounds present in the seeds of this plant species have multiple functions.

Furthermore, regarding studies that report that J. curcas can be used to treat HIV-AIDS, it is important to note that the experimental work carried out by Matsuse et al. (1999) and Dahake et al. (2013) showed promising results. The first group of authors that used the extracts from the stem bark of this plant observed that cytopathic effects induced by HIV were inhibited with low cytotoxicity, and the second group that analyzed the effect of the leaf extracts on HIV concluded that it is an excellent candidate for herbal medicine. However, there is no evidence on the use of this plant in the treatment of COVID-19.

Ferrão and Janeque (2019), in their work, used gas chromatography-mass spectrometry (GC-MS) to identify compounds present in the extracts of J. curcas seeds used for treating people in the municipal village of Massinga, Mozambique. Thus, based on the work of Ferrão and Janeque (2019) and the literary matrix that deals with substances with antiviral effect, the present work aims to trace compounds with anti-HIV and anti-SARS-CoV-2 potential in this plant structure.

The data obtained from the work of Ferrão and Janeque (2019) were used for the present study. The method adopted by these authors was GC-MS. Also, bibliographic consultations were carried out.

This research analyzed and discussed the results of Ferrão and Janeque (2019) obtained from the GC-MS analysis of J. curcas seed extracts. Ferrão and Janeque (2019) collected the material from Massinga district, Inhambane province, based on the procedure of Bessa et al. (2013), to reduce the degree of humidity. This process prevents microbial activity and hydrolysis. Therefore, the materials were left in the oven at a temperature of 40°C for 24 h. The J. curcas seeds underwent the process of Soxhlet extraction and maceration.

GC-MS analysis

GC-MS analysis was carried out at the Research and Extension Laboratory of the Department of Chemistry, Eduardo Mondlane University, Mozambique, as part of the work of Ferrão and Janeque (2019).

Sample preparation

According to Ferrão and Janeque (2019), about 80 mg of the plant was extracted in a 15 mL polypropylene tube, mixed with 1 mL of ethanol (99.9%) using a  vortex mixer at 2200 rpm for 5 min. Then, the mixture was centrifuged for 1 min and the supernatant was transferred to another polypropylene tube. The procedure was repeated two more times (to complete 3 extractions) and the supernatants were collected into the same polypropylene tube. Also, the  extract  was  subsequently  evaporated  to  dryness  on  a rotary evaporator at 65 to 67°C. It was recovered with 1 mL of methanol, filtered and injected into GC-MS.

GC-MS

J. curcas seed extract (2 ?L) was injected into a capillary column (HP 5MS IU: 30 m × 250 ?m × 0.25 ?m, -60 - 325°C) by splitless mode. Helium (99.9%) was used as a 1 mL-1 mobile phase. The analysis was performed in electron impact mode with ionization energy of 70 eV. The injection temperature was maintained at 250°C. The initial column temperature was 50°C (3 min), then it was raised to 280°C (3 min) at a rate of 10°C/min and finally to 300°C (10 min) at a rate of 20°C/min. The total analysis time was 40 min. Peak identification was performed by comparing the spectra of each eluted component with the spectra present in the NIST library database.

Articles reviewed

Articles, communications and papers available or not on online platforms (Google Scholar, Elsevier, Research Gate, Science Direct and Scielo) were consulted as the following. The works of Ferrão and Janeque (2019), Verma et al. (2015), Kaur et al. (2011), Abdelgadir and Van Staden (2013), Vincent et al. (2020), Fadilah et al. (2020), Sharmila et al. (2016), Kamiyama et al. (2013), Venkata et al. (2012), Hadi et al. (2016), Chinsembu (2019), Kaur et al. (2020), Bapia et al. (2021), Siwe-Noundou et al. (2019), Terefe et al. (2022), Silva Filho et al. (2009), Matsuse et al. (1999), Dahake et al. (2013), Padmashree et al. (2018b), Godara et al. (2019), and Beckstrom-Sternberg and Duke (1996).

The articles reviewed comprised two stages: the first concerned the articles that deals with the antiviral activity of J. curcas, and the second focused on the material that reports the compounds found in the extracts of this plant with anti-HIV-1 and SARS-Cov-2 indication, in order to  explain their forms of action.

The extract recovered with 1 mL of methanol, filtered and injected into GC-MS showed the presence of n-Propyl acetate, ethanethioic acid, S-(dihydro-2,5-dioxo-3-furanyl) ester,  thymine, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, d-Glycero-d-ido-heptose, 5 hydroxymethylfurfural, ethanol, 2-(2-butoxyethoxy)-,acetate, l-Pyrrolid-2-one, N-carboxyhydrazide, methyl 6-oxoheptanoate, 2,4,7,9-tetramethyl-5-decyn-4,7-diol,  hexadecanoic acid, methyl ester, melezitose, n-Hexadecanoic acid, 9,12-Octadecadienoic acid (Z,Z)-, methyl ester, 6-octadecenoic acid, methyl ester, (Z)-, 9,12-Octadecadienoic acid (Z,Z)-, cis-Vaccenic acid, oleic acid, octadecanoic acid, hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, glycerol 1-palmitate, 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester, 9-octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester; squalene, campesterol, stigmasterol, and gamma-sitosterol.

Table 1 show the retention times and areas of these compounds and the respective biological activities reported in the consulted bibliography.

Verma et al. (2015), Kamiyama et al. (2013), Venkata et al. (2012), and Hadi et al. (2016) report anti-HIV activity of stigmasterol. Vincent et al. (2020) and Fadilah et al. (2020) verified from their molecular modeling studies, in which computational method was used for the rational planning of bioactive compounds (Sant'Anna, 2009) that there is a possibility that gamma-sitosterol acts as an anti-SARS-CoV-2.

Figures 1 and 2  present  the  chromatograms  resulting from the GC-MS analysis of stigmasterol and gamma-sitosterol. Figures 1 and 2 show the retention times and peak areas corresponding to stigmasterol and gamma-sitosterol. The retention time and peak area of the first compound was 32.705 min and 4648026.21, respectively, and the second was 33.47 min and 23863798.04. In the research on antiviral compounds, attention is paid to those that interfere both in the mechanism of infection and in the process of viral multiplication, preventing its continuity (Kaur et al., 2020).

In this regard, Chinsembu (2019) and Kaur et al. (2020) explain how interference occurs in the HIV viral cycle, while Vincent et al. (2020) do the same for SARS-CoV-2.

Stigmasterol and HIV-1

Verma et al. (2015), Padmashree et al. (2018b), Godara et al. (2019), and Beckstrom-Sternberg and Duke (1996) linked stigmasterol to antiviral activity. Terefe et al. (2022) evaluated the anti-HIV action of some compounds and found the following results for stigmasterol: IC50 (μg/mL) 0.14 ± 0.04, EmaxAV (%) 76.77 ± 23.24 and SI 86.5. In another line of investigation, Kamiyama et al. (2013) found that the test compound does not affect the transduction efficiency of the HIV-1 vector during the infection phase within the viral cycle.

Regarding the group of substances with anti-HIV-1 reverse transcriptase properties in some plant species, Chinsembu (2019) mentioned stigmasterol. In describing these substances, this author stated that they have a structure that is similar in form to nucleoside analogues, and appear to be close homologs that share structural and functional similarities with non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Sluis-Cremer (2018), cited by Chinsembu (2019), defines NNRTI as "small molecules that bind to the HIV-1 reverse transcriptase site distinct from the enzyme DNA polymerase active site and block the reverse transcription of HIV-1 through an allosteric mechanism of action". Therefore, if stigmasterol shows structural and functional similarities with non-nucleoside reverse transcriptase inhibitors, it can be assumed that it has the same mechanism of action.

Still on natural reverse transcriptase inhibitors, Chinsembu (2019) highlights the pleiotropic effect and its pharmacological benefits. Supporting his point of view, this author states that, during infection, pleiotropic inhibitors help to fight opportunistic diseases due to the diverse spectrum of action they can present. In the case of stigmasterol, several actions that may be beneficial during infection and disease have been reported, namely: anti-hepatotoxic, anti-inflammatory, anti-osteoarthritis, antioxidant, hypocholesterolemic and antitumor.

In another aspect, Kaur et al. (2020), when discussing the mechanism of action of anti-HIV phytochemicals, refer to the role of antioxidants in inhibiting HIV-1 transcription. Arguing, these authors advance that many reactive oxygen species are produced due to reduced levels of antioxidant enzymes, which, in addition to causing DNA damage, can stimulate the nuclear factor kappa B (NF-κB factor) that helps in the transcription of HIV, thus promoting its replication. In the same context, Bapia et al. (2021), when studying the isolation of stigmasterol from Kra Don (Careya arborea Roxb.) and the bioactivity of its crude  extracts  against  free  radicals and human immunodeficiency virus, referred to several authors who reinforced the perspective presented by Kaur et al. (2020). Among them are Eldeen et al. (2011), Kapewangolo et al. (2013), Valle et al. (2013) and Allard et al. (1998). In this order of ideas, it can be understood that as an antioxidant, stigmasterol has the ability to act by reducing reactive oxygen species and contributing to the reduction of conditions for viral replication. However, it is assumed that this hypothesis must be proven in experimental work.

In an experimental work, Siwe-Noundou et al. (2019) tested all compounds isolated from the crude methanolic extract of Alchornea cordifolia as anti-HIV integrase (anti-HIV IN) and found activity against HIV-1 IN, including for stigma sterol (IC50=20, 5 μg/ml).

Gamma-sitosterol and SARS-CoV-2

Shortly after Covid-19 was considered a pandemic by the World Health Organization (WHO), in 2020, studies around SARS-CoV-2 gained interest. According to Vincent et al. (2020), in the structure of this virus, the S proteins (spike) responsible for the viral invasion of the host cell stand out. This is as a result of the affinity they have with the receptor of the target cell (the angiotensin 2 converting enzyme ACE2), and the proteases Mpro (3C-Like also called 3CLpro) responsible for replication, viral transcription and protein cleavage. This inhibits the processes for which it is responsible and PLpro (enzyme of protein cleavage and suppression of the innate immune response of the host).

Based on the knowledge we have about the structure of SARS-CoV-2, research is being carried out in search of compounds that inhibit the replication or maturation processes of this virus, or those that act by blocking its binding to the receptors of target human cells. Subsequently, Gamma-sitosterol, a compound with antiviral properties (Venkata et al., 2012; Verma et al., 2015) ended up being studied by Vincent et al. (2020) and Fadilah et al. (2020).

Vincent et al. (2020), through molecular docking, evaluated the ability of Kabasura kudineer phytochemicals to bind to one of the main proteases of this virus (3CLpro), inhibiting the processes it is responsible for. In this study, these authors found that gamma-sitosterol is part of the group of compounds that showed affinities for 3CLpro with binding energy of -81.94 kcal/mol, which comprises the interaction of van der Waal and hydrogen. This finding shows that gamma-sitosterol can act as an inhibitor of 3CLpro and, because of this, J. curcas seed extracts can be candidates for in vitro assays.

In turn, using the molecular docking method, Fadilah et al. (2020) evaluated the phytochemicals of Psidium guajava, having also observed that gamma-sitosterol has affinities to the proteases 3 CLpro,  PLpro, Spike proteins  (S or surface binding proteins) and ACE2 (angiotensin-converting enzyme 2, cell receptor hostess). These data reinforce the evidence found by Vincent et al. (2020).

From the aforementioned, it can be inferred that, by competitively binding the S protein of the virus or ACE2, gamma-sitosterol prevents the interaction between them, blocking the fusion and consequently the viral entry. In another aspect, by binding the proteases Mpro (3CLpro) and PLpro, this compound interferes with the functions of these enzymes during viral replication, inhibiting the process. In fact, in this regard, Khan et al. (2021) explain the mechanism of action of different substances on the coronavirus, stating that a drug candidate may be useful if it inhibits viral entry, replication, or even provokes an immune response to produce Type I IFN against SARS-CoV-2.

Toxicity

Khan et al. (2021) advance that safety aspects should always be taken into account, despite the perception that herbal medicines are completely safe and free from any side effects.

The imperative to consider the safety aspects in relation to these drugs, despite having the perception mentioned earlier, is reinforced by Sharmila et al. (2016), Abdelgadir and Van Staden (2013), and Alamery and Algaraawi (2020) who indicated glycerol 1-palmitate, stigmasterol and gamma-sitosterol as cytotoxic compounds. In addition to these compounds, the plant under analysis has others such as Phorbol Ester and Curcin, considered the most toxic phytochemicals found in its seeds (Devappa et al., 2010a cited by Abdelgadir and Van Staden, 2013). These facts make this part of the plant potentially toxic, thus requiring a toxicity assessment.

GC-MS analysis of J. curcas seed extracts showed the presence of three compounds with antiviral reference in the literature, namely, glycerol-1-palmitate, stigmasterol and gamma-sitosterol. Stigmasterol and gamma-sitosterol were associated with anti-HIV-1 and anti-SARS-COV-2 activity, respectively. Glycerol-1-palmitate was not discussed in this study, due to insufficient information on its mode of action. As an anti-HIV-1, stigmasterol can act on the enzymes HIV-1TR and HIV-1 IN. Gamma-sitosterol is associated with the inhibition of the main proteases of SARS-VOC-2. The results of the present research provide horizons for the next level of research, in vitro assays, as they support the hypothesis that the seed exhibits antiviral properties against HIV-AIDS and COVID-19. The presence of cytotoxic in J. curcas seed extracts suggests that, in  case  the  extracts are proven to be effective in in vitro studies, their toxicity should also be evaluated.

The authors have not declared any conflict of interests.

The authors thank the National Research Fund of Mozambique for funding and the Laboratory of the Department of Chemistry at the University Eduardo Mondlane for their partnership in carrying out the analyses.