Evaluation of the in vitro schistosomicidal, leishmanicidal, and trypanocidal activities of the capsaicin metabolite, Capsicum frutescens, and Capsicum baccatum extracts and of their analysis of the main constituents by HPLC/UV and CG/MS

Abstract Neglected tropical diseases are significant causes of death and temporary or permanent disability for millions living in developing countries. Unfortunately, there is no effective treatment for these diseases. Thus, this work aimed to conduct a chemical analysis using HPLC/UV and GC/MS to identify the major constituents of the hydroalcoholic extracts of Capsicum frutescens and Capsicum baccatum fruits, evaluating these extracts and their constituents’ schistosomicidal, leishmanicidal and trypanocidal activities. The results obtained for the extracts of C. frutescens are better when compared to those obtained for C. baccatum, which can be related to the different concentrations of capsaicin (1) present in the extracts. The lysis of trypomastigote forms results for capsaicin (1) led to a significant value of IC50 = 6.23 µM. Thus, the results point to capsaicin (1) as a possible active constituent in these extracts. Graphical abstract


Introduction
The capsicum genus comprises a diverse group of bell peppers.It consists of about 31 species (Batiha et al. 2020), with Capsicum annuum, Capsicum chinense, Capsicum frutescens and Capsicum baccatum being the five domesticated species (Parry et al. 2021).Chili peppers are used as food additives in the composition of medicines (Abou-Elkhair et al. 2018).The capsaicinoids stand out among the several chemical constituents of the capsicum genus (Batiha et al. 2020).Different studies show that capsaicinoids, particularly capsaicin (1), have numerous biological activities (Nadi et al. 2020).However, there is still a vast field to be explored in this area, especially on neglected tropical diseases such as Leishmaniasis, Schistosomiasis and trypanosomiasis.
Schistosomiasis is an ancient disease caused by the parasite Schistosoma mansoni; 700 million people are infected worldwide.An estimated 200,000 people die each year (Verjee 2019).Praziquantel is the drug used in the treatment of Schistosomiasis.Due to the possible development of tolerance or resistance to praziquantel, research and production of new medicines to prevent and cure, Schistosomiasis is warranted (Mishra et al. 2019).
Similarly, Chagas disease is probably as old as man's presence in the Americas.American trypanosomiasis, caused by the protozoan parasite Trypanosoma cruzi, affects about 10 million people worldwide (Mishra et al. 2019), causing approximately 14,000 deaths per year and another 100 million deaths.One of the first drugs used to treat the disease is benznidazole (BNZ), which has the disadvantage of partial efficacy and high toxicity (Bustamante et al. 2020).Leishmaniasis is a parasitic disease that affects about 12 million people worldwide (Ikeogu et al. 2020;Gupta et al. 2022).An estimated 350 million people are at risk for the disease in 88 countries.About 90% of all cases of visceral Leishmaniasis occur in Bangladesh, Brazil, Ethiopia, India, Nepal, and Sudan (Gupta et al. 2022).Thus, this work aimed to conduct a chemical study using HPLC and GC/MS to identify the majority constituents of the hydroalcoholic extracts of the fruit of Capsicum frutescens (EHCF) and Capsicum baccatum (EHCB) and schistosomicidal, leishmanicidal and trypanocidal evaluate in vitro of extracts and their major constituents.

Extracts chemistry composition
The chemical profile (Figure S1) of EHCF and EHCB extracts is well described in the literature (Antonio et al. 2018).Still, in this present study, we sought to correlate the extract's biological activities with its chemical constituents, mainly the presence of capsaicin.The presence of capsaicin (1) in the extracts was determined by comparing an analytical standard's retention time and its UV spectrum with the peaks in the extract by HPLC/UV (Figure S2).
The comparative analysis of the chromatograms showed capsaicin (1) in EHCF extract as the principal constituent.However, when comparing the analytical standard with the EHCB extract, there is a similarity in retention times (rt), but the UV spectrum differs from the capsaicin standard.This fact may lead to the conclusion that the main constituent in the EHCB extract is not capsaicin.Therefore, to resolve such doubts and aim at greater detail of the chemical constitution of these extracts, it was decided to analyze them using the GC/MS technique (Figures S3 and S4).
As for EHCB extract, the mass spectra analysis obtained for the major compounds allows identifying the substances as being squalene (4) (rt = 61.93min, IS = 93%), capsaicin (1) (rt = 62.86 min, IS = 96%), dihydrocapsaicin (2) (rt = 63.438min, IS = 91%) and tocopherol (3) (rt = 76.33 min, IS = 93%).Thus, this result evidences the presence of capsaicin (1) in EHCB extract, which allowed us to conclude that in HPLC analysis, the signals of capsaicin (1) and squalene (4) were superimposed, causing the UV spectrum not to reflect the spectrum of a pure compound but of a mixture.In GC analysis, the signals of the two compounds were well resolved, allowing the conclusion of the presence of these two substances in the extract.

Schistosomicidal evaluation in vitro
As shown in Table S1, the capsaicin (1) at concentrations between 12.5 and 50 µM did not affect adult S. mansoni worms.At 100 µM, it caused a decrease in the motor activity of 25% of the parasites, and this same effect was observed throughout the evaluation period.On the other hand, at the concentration of 200 µM, 100% of the parasites showed minimal motor activity during the 24-h and 120-h periods, respectively.At the same concentration, it was also observed that capsaicin (1) causes partial alteration of the integument of the parasites.
The EHCF and EHCB extracts had no effect at 12.5 and 25 µg/mL.On the other hand, the EHCF extract at a 100 µg/mL concentration had a lethal impact on 25% of the worms and a minimal reduction in motor activity in 75% of them in all cultivation periods.The EHCF extract at 100 µg/mL caused partial alteration of the parasite's integument.Furthermore, at 50 µg/mL, a decrease in motor activity was observed in 50% of the worms in 120 h of culture.Regarding the EHCB extract, it did not show any lethal effect.However, EHCB extract caused a decrease in the motor activity of more than 50% of the worms at concentrations of 50 and 100 µg/mL in 120 h of culture (Table S1).
According to the literature, male parasites are more susceptible than female worms in resistance studies to praziquantel (PZQ) and certain plant extracts (Pica-Mattoccia and Cioli 2004).However, other studies have shown that female parasites were more susceptible to the action of some substances than male worms (De Araújo et al. 2007;De Oliveira Penido et al. 2008).However, there was no difference between male and female worms in our studies.Furthermore, it is essential to report that the effect on the integument of S. mansoni caused by capsaicin and EHCF extract was similar to those observed in studies with other natural products and with PZQ (Moraes et al. 2011).
In the negative controls (RPMI 1640 medium and RPMI 1640 medium + 0.2% DMSO), the parasites remained viable without integumentary changes throughout the analysis period.On the other hand, PZQ (12.5 µM or µg/mL) used as a positive control caused the death of 100% of the parasites and integumentary alteration within 24 h.

Leishmanicidal evaluation in vitro
Table S2 shows the results of leishmanicidal activity expressed in the percentage of lysis at different concentrations of EHCF and EHCB extracts.The EHCF extract was inactive at the concentrations evaluated, while the EHCB extract showed moderated activity.The differences in the results of leishmanicidal activity must be related to the chemical profile presented by the extracts and the differences in the concentrations of capsaicinoids, mainly capsaicin (1).Previous studies have shown that the percentage of capsaicin (1) in EHCF and EHCB extracts is different, which can modulate biological activities.Nakagawa and Hiura (2006) state that capsaicin and several related components known as capsaicinoids are produced as secondary metabolites by peppers of the genus Capsicum.Probably as barriers against herbivores, classifying the different species in terms of capsaicin (1) content, highlighting C. frutescens (chili peppers) with 0.89%, C. baccatum (pepper) with 0.48%, C. annuum (garden pepper) with 0.20%, and the different peppers (C.annuum) with 0 .0% of said compound (Takikawua et al. 2002;Ozguven and Yaldiz 2011).Table S3 shows the evaluation results of the leishmanicidal activity of capsaicin (1).The results show that this substance is related to EHCF extract's activity

Trypanocidal evaluation in vitro
According to the empirical process of obtaining drugs, an effective drug emerges every 10,000 to 50,000 attempts in the pharmaceutical industry.The lack of new drugs is almost complete in the case of some parasitic diseases, such as Chagas disease and Leishmaniasis.Under these conditions, new alternatives for the specific treatment of Chagas disease have been the drugs on the market that fortuitously can act against T. cruzi or drugs that are still in the process of scientific investigation (Gupta et al. 2022).
The analysis of the results obtained from the in vitro evaluation against the Y strain of T. cruzi (Table S3) indicates that the EHCF and EHCB extracts evaluated did not show significant trypanocidal activity since the IC 50 values obtained were 119.5 µg/ mL and 278.6 µg/mL, respectively.Likewise, the best values of the percentage of parasite lysis were obtained at the highest concentrations, that is, 200 µg/mL for EHCF extract (52.3%) and 100 µg/mL for EHCB extract (42 .6%),demonstrating that the results obtained for the extracts were not significant.However, it can be noted that the results obtained for EHCF extract are better when compared to those obtained for EHCB extract, which may also be related to the different concentrations of capsaicin (1) present in the extracts (Table S4).

Conclusion
The EHCF extract predominantly consists of capsaicin (1) and dihydrocapsaicin (2).In contrast, in EHCB extract, squalene (4) occurs as the predominant compound.EHCF and EHCB extracts were evaluated against three in vitro models for neglected endemic diseases: Schistosomiasis, Leishmaniasis, and Chagas disease.Thus, this is the first results report approaching these assays for C. frutescens and C. baccatum extracts.The results point to capsaicin (1) as a possible active constituent in the extracts responsible for biological activities.The results obtained for C. frutescens extracts are better when compared to those obtained for C. baccatum, which may also be related to the different concentrations of capsaicin (1) present in the extracts.