Anti-Phytomonas activity of the lyophilized residues obtained from the distillation of Lantana camara L. essential oil

Anti-Phytomonas activity of the lyophilized residues obtained from the distillation of Lantana camara L. essential oil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-Phytomonas activity of the lyophilized residues obtained from the distillation of Lantana camara L. essential oil Caroline Alves Soares, Tamíris Aparecida de Carvalho Santos, Luís Fernando de Andrade Nascimento, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4092579/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract On an industrial scale, the residues accumulated in essential oil distilleries can be compared to the volume of residues produced in a textile industry. Although these residues are discarded, they possess molecules with diverse biological activities, including their application in phytopathogen control. In this study, the chemical profile of the residue from the hydrodistillation of Lantana camara L. leaves was determined using High-Performance Liquid Chromatography (HPLC). Additionally, the effect of the residue on cells was assessed by determining plasma membrane integrity, levels of reactive oxygen species (ROS) production, and mitochondrial potential depolarization. The viability and cell density of Phytomonas serpens parasites significantly decreased after treatment with increasing concentrations of the lyophilized residue. RL038, the lyophilized residue from accession LAC-038, reduced cell viability by an average of 61.36%. ROS levels increased by approximately 2x and 3x at RL038 concentrations of 120 µg/mL and 180 µg/mL, respectively. It was observed that the same concentrations modified mitochondrial potential, reducing fluorescence by 44.6% and 46.8%, respectively. Analytical liquid chromatography of RL038 revealed the presence of 17 peaks subsequently classified as phenolic acids and flavonoids. RL038 from the hydrodistillation of Lantana camara L. leaves is a source of biologically active compounds with antiprotozoal potential. Residues from distillation Biological control Medicinal plants Mechanism of action Reactive oxygen species Mitochondrial membrane potential Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Essential oils are mixtures that are rich in aromatic compounds and they have several applications. They are currently used as ingredients in the production of beverages, food, fragrances, cosmetics, and drugs, in addition to being considered the primary tool of aromatherapy, a segment of medicine in which the treatment of patients occurs with the help of these oils (Shankar et al., 2021 ). Although there is a growing demand for these products (Alighiri et al., 2017 ), the yield of essential oil in many plant species is low. This results in a large volume of residues during the distillation process (Napoli et al., 2020 ). On an industrial scale, the waste that is accumulated in the distilleries of essential oils can be compared to the volume of waste that is produced by the textile industry (Gharred et al., 2020). The incorrect disposal of these materials can cause severe damage to the environment. The maximum use of raw materials, the reduction of energy expenditure and waste production, as well as their transformation into new products, is part of the biorefinery concept. In the essential oils industry, the residues are generally discarded. However, several useful compounds can be extracted from them in other production chains, such as rosmarinic acid, an important natural antioxidant that can be obtained from the liquid residue of the hydrodistillation process of the leaves of rosemary ( Rosmarinus officinalis L.) (Wollinger et al., 2016 ). This is together with the phenolic compounds that are extracted from the solid residue of the hydrodistillation of R. officinalis , which have a deterrent effect on pest insects (Sánchez-Vioque et al., 2015 ). Sankara et al. ( 2020 ) reported that the residues from the hydrodistillation of Eucalyptus camaldulensis could provide interesting bioactive compounds, as new anti-termite agents in plant protection. Mahmoudi et al. ( 2020 ) also reported that hydrodistilled basil leaves ( Ocimum basilicum ) have a good repellent activity against the stored grain pests, Tribolium castaneum . Lantana camara L. (Verbenaceae) is an aromatic species widely known throughout the world for its ornamental use. In traditional medicine, it is common to use its leaves, branches, and roots to treat cuts, fevers, headaches, palpitations, tetanus, rheumatism, malaria, ulcers, and so forth. (Gindri and Coelho, 2020 ). In the essential oil that is obtained from this species, several bioactive compounds are found, such as terpenoids, alkaloids, flavonoids, phenolic compounds, and glycosides. These compounds are to which various biological activities have been attributed, such as anticancer and antiangiogenic (Al-Hakeim et al., 2021 ), anti-inflammatory (Wu et al., 2020 ), anti-tuberculosis (Nirmal et al., 2020 ), as well as a sedative (Dougnon, Ito, 2020 ). This is in addition to antifungal (Bashir et al., 2019 ), herbicide (Qureshi, et al., 2019 ), nematicide (Borbodoli et al., 2021), insecticide (Ayalew, 2020 ), and trypanocide agents (Delgado-Altamirano et al., 2019 ). Trypanosomatids are microorganisms that are adapted to life in fluids that are rich in glucose, where most of them are parasites of animals, with the exception of the genus Phytomonas , whose representatives are plant parasites (Maslov et al., 2019 ). Some species cause serious diseases in large crops of economic interest. Phytomonas staheli , which causes acute lethal wilt in coconut palms ( Cocos nucifera ), and oil palm ( Elaeis guineensis ), which begins with the rotting of the leaves, and then progresses to the spear and the roots, as well as Phytomonas leptovasorum , a pathogen of necrosis in coffee (Arabica and Liberia) phloem (Jaskowska et al., 2015 ). These are some examples of the important plant pathogens, which are capable of offering an economic risk in tropical countries. Phytomonas serpens is not a pathogen of agricultural relevance, although it is an important biological model that helps in the understanding of pathogenic members belonging to the Trypanossomatidae family that affect plants and mammals. Due to its simplified biology, it helps in understanding the mechanisms of pathogenicity and the manifestations of diseases in plants and animals (Jaskowska et al., 2015 , Santos Junior et al., 2018). In a recent study, this current group demonstrated for the first time, the trypanocidal activity of the essential oil of L. camara on P. serpens (Pereira et al., 2022 ), demonstrating the potential of this product for the treatment of economically important diseases that are caused by other species of Phytomonas . However, the yield of L. camara essential oil is low (usually 0.2%), and by steam dragging, 500 kg of biomass yields about 200 mg of oil (Negi et al., 2019 ). On the other hand, the volume of waste generated by the oil hydrodistillation process is high and constitutes a promising source of bioactive molecules. Given the commercial importance of crops that are devastated by species of the genus Phytomonas , and the potential of the products in the metabolism of L. camara to control the proliferation of these phytopathogens, the present work evaluated the action of residues in the hydrodistillation of the leaves of L. camara on the viability of P. serpens in vitro . This was as well as their effects on the integrity of the plasma membrane, and the potential of the mitochondrial membrane on these phytopathogens. 2. Material and Methods 2.1 Plant material Four accessions of Lantana camara L., LAC-033, LAC-038, LAC-017, and LAC-025, from the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe, Brazil, were selected. The choice of the accessions was supported by previous works, in which the chemical characterization of the essential oils from the plants of the active germplasm bank was determined (Pereira et al., 2020 ). The accessions of the plant material were registered under the identification number A8CCB3B in the National System for the Management of Genetic Heritage and Traditional Knowledge (SisGen). The leaves were collected and subjected to hydrodistillation in a modified Clevenger apparatus, by using 75g samples of the dry leaves, with the addition of 1.5L of distilled water, for 140 minutes. The effluent, resulting from the decoction of the leaves, was filtered and separated into 200mL samples. Later, they were stored in an ultra-freezer at − 80°C (Liotop UFR30, Liobras, São Carlos, SP, Brazil) for 24 hours. They were then transferred to a freeze dryer (Liotop L101, Liobras, São Carlos, SP, Brazil), where they rested for five days at a temperature of − 54°C and a pressure of 79 µm Hg until the complete sublimation of the water that was contained in the samples. At the end of this process, the “Lyophilized Residues (LRs)” were obtained from each accession, hereinafter named LR038 (Accession LAC-038), LR017 (Accession LAC-017), LR025 (Accession LAC-025), and LR033 (Accession LAC-033). 2.2 Cell culture The Phytomonas serpens (9T) parasites were obtained from the Trypanosomatid Collection at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The promastigote forms were grown in a Schneider culture medium (Sigma-Aldrich®, St. Louis, Missouri, USA), which were supplemented with 10% fetal bovine serum (Sigma-Aldrich®, St. Louis, Missouri, USA) and 1% streptomycin at 26ºC in a BOD incubator. 2.3 Cell viability of Phytomonas serpens when exposed to the residues from the Lantana camara accessions The promastigotes were cultured at 26°C in a BOD incubator, and then 1x105 cells were deposited in 96-well microplates and treated for 48 hours, with the four lyophilized residues (LRs), in serial dilutions at concentrations of 100.0 to 1.5 µg/mL. The negative control consisted of the untreated cells, and for the positive control, the cells were treated with the antibiotic, Amphotericin B. After 48 hours of treatment, 50 µL of resazurin solution (200 µM) was added to each well, and the plates were incubated in the dark for 30 minutes. This particular dye is an indicator of cell viability, which when added to the culture, enters the cells and is reduced by the mitochondrial dehydrogenases to the resofurin of the viable cells while changing the color of the medium from blue to pink. The cell viability was measured by spectrofluorimetry at a wavelength of 555 nm excitation and 585 nm emission in a microplate reader (Synergy™ H1, BioTek Hybrid Technology, Winooski, Vermont, USA). From the cell viability values obtained, the IC 50 levels (the concentration that inhibits 50% of the cells) were calculated. The lyophilized residue (LR) from the accessions that presented the lowest IC 50 value was used in the subsequent tests. 2.4 Growth curve of Phytomonas serpens when treated with selected residue The promastigotes (1x10 5 cells/mL) were treated or not with the lyophilized residue (LR), which had a lower IC 50 at the concentrations of 6.25, 12.5, 25.0, 50.0, and 100 µg/mL, and then incubated in 12-well plates for 72 hours, in a BOD oven at 26ºC. The number of cells was evaluated at 24, 48, and 72 hours, by direct counting in a Neubauer chamber when under an optical microscope with a 10X lens. The results were used to construct the growth curve. 2.5 Effects of LR on the Phytomonas serpens cells The effects of LR on the integrity of the plasma membrane, on the potential of the mitochondrial membrane, and on the production of the reactive oxygen species, were evaluated for the P. serpens cells. In all of the evaluations, the promastigotes (5.10 5 cells/mL) were treated for 48 hours with the different concentrations of the selected lyophilized residue (LR). They were then used for the specific procedures for each assessment, as described below. 2.6 Plasma membrane integrity The treated cells were exposed to propidium iodide (40 µg/ml) for 15 minutes. The incorporation of propidium iodide, which was indicative of the loss of membrane integrity, was evaluated by flow cytometry when using an Attune® Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA). A total of 10,000 events were obtained and analyzed by using the Attune® Cytometer Software program. The incorporation of propidium iodide was also evaluated by fluorescence spectrometry when using the wavelengths of 488 nm excitation and 620 nm emission. As a negative control, the untreated cells were used, and the positive control was by the cells that were heated at 80°C for 10 minutes. 2.7 Mitochondrial membrane potential After the treatments, the cells were washed twice in sterile phosphate buffered saline (PBS) (3000 rpm / 10 minutes / 4°C) and were resuspended in 100 µL of PBS. The cells that were treated with LR were exposed to 10 µg/mL of Rhodamine 123 (Rh123, Sigma-Aldrich, St. Louis, Missouri, USA) in the absence of light for 15 minutes. After the treatment period, 400 µL of PBS was added, and the changes in the mitochondrial membrane potential were assessed by flow cytometry by using an Attune® Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA). A total of 10,000 events were obtained and studied when using the Attune® Cytometer Software program. The cells that were not treated with LR were used as a negative control, and the cells that were treated with 50 µM of 4-chlorophenylhydrazine carbonyl cyanide (CCCP) were used as a positive control. The variation index (VI) was determined by the equation (MTC-MUC)/MUC, in which MTC corresponded to the Rh123 fluorescence median of the cells that were treated with the LR, while MUC was the median fluorescence of the untreated cells. 2.8 Reactive oxygen species After the treatments, a volume containing 1,107 promastigotes was washed twice in sterile PBS (3000 rpm / 10 minutes / 4°C) and later resuspended in 100 µL of PBS, and then transferred to 96-well microplates. The cells that were treated with LR were exposed to 20 µM 2,7-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in the absence of light for 30 minutes. The fluorescence produced by the formation of 2,7-dichlorofluorescein (DCF), as a result of the interaction with H 2 DCFDA with the produced ROS, was measured by spectrofluorimetry at wavelengths of 492 nm excitation and 527 nm emission when using a Synergy™ H1 microplate reader (BioTek Hybrid Technology, Winooski, Vermont, USA). As a negative control, the untreated cells were used, and as a positive control, the cells that were treated with 750 µM of hydrogen peroxide (H 2 O 2 ) for 30 minutes were used. 2.9 Qualitative chemical profile of the lyophilized residue (LR) The chromatographic exploration to obtain the qualitative chemical profile of the lyophilized residue from the accessions of the L. camara collection that displayed a greater antiprotozoal activity was performed by a Shimadzu Prominence network-ready system for analytical liquid chromatography (Kyoto, Japan). This consisted of a CBM-20A communication module, a DGU-20A3 degasser, an LC-20AT binary pump system, a SIL-20AHT automatic injector, a spectrophotometric detector with an SPD-M20A diode array, and a CTO-20A column oven. The analyzed samples were prepared by dissolving 10 mg of LR in 1 mL of ultrapure water (10 mg/mL), then strained through a 0.45 µm syringe filter, and transferred to the analysis channel. For the separation of the chemical compounds that were present in the samples, a C18 stationary phase chromatographic column (250 x 4.6mm, 5µm, 100 A, Kinetex, Phenomenex, Torrance, California, USA) was used. This was connected to a guard column of the same stationary phase, with exploratory elution gradients of 5 to 100% B for 60 min, 100 to 100% B for 10 min, 100 to 5% B for 5 min, as well as with 40 minutes of conditioning the column to 5% B. The sample injection volume was 25 µL and the column temperature was 30°C. The mobile phase was formed by a binary mixture of methanol (B) and an aqueous solution of formic acid 0.5% (v/v) (A) at a flow rate of 1mL/minute. The wavelength used for the detection of the chemical compounds was 254 nm. 2.10 Data analysis The data was subjected to an Analysis of Variance (ANOVA), while using Tukey as a post-test, with the aid of the GraphPad Prism 7.01 program. The results were expressed as mean ± standard error, and the differences concerning the untreated controls were considered significant when P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). All of the experiments were carried out in triplicate. 3. Results 3.1 Cell viability of Phytomonas serpens when exposed to the residues from the Lantana camara accessions Initially, a screening of the antiprotozoal activities of the lyophilized residues (LR) of the four L. camara accessions on the cell viability of P. serpens was carried out. The parasite cell viability decreased as the LR concentrations increased (Fig. 1 A). The IC 50 values that were obtained from the four LRs of L. camara were 13.68 µg/mL (LR038), 17.50 µg/ml (LR017), 35.16 µg/ml (LR025), and 43.69 µg/ml (LR033) (Fig. 1 B). Although the four LRs showed good inhibition of the parasite viability, LR038 was selected for further experiments because of its lower IC 50 . 3.2 Growth curve of Phytomonas serpens when treated with selected residue LR038 affected the proliferation of P. serpens at all of the concentrations evaluated, and the concentrations of 50 µg/ml and 100 µg/m of LR038 markedly reduced the cell density of the promastigote forms (Fig. 2 A). After 24 hours, the concentration of 50µg/mL reduced 47% of the cell growth and the concentration of 100µg/mL inhibited 59%. After 48 hours, the same concentrations reduced 52% and 76% of the growth of promastigotes, respectively. After 72 hours, there was a 65% reduction in the cell density at the 25 µg/mL concentration of LR038, and an 84% and 95% inhibition was observed at the 50 µg/mL and 100 µg/mL concentrations, respectively (Fig. 2 B). 3.3 Evaluation of the cell membrane integrity of the Phytomonas serpens promastigotes To determine the integrity of the membrane, a high molecular weight DNA intercalant that did not penetrate the cell with an intact membrane was used with the fluorescent probe, propidium iodide (PI). The flow cytometry and spectrofluorimetric analysis demonstrated that LR038 at the concentrations evaluated did not affect the integrity of the plasma membrane (data not shown). 3.4 Evaluation of the reactive oxygen species production of the Phytomonas serpens promastigotes The production of the reactive oxygen species in the P. serpens promastigotes that were treated with 60, 120, and 180 µl/mL of LR038 was evaluated by using the fluorescent probe, H 2 DCFDA. It was observed that at the two highest concentrations, there was a significant increase in the accumulation of the ROS when compared with the untreated cells (P < 0.05, P < 0.01) (Fig. 3 ). The accumulation of the ROS was approximately 2x and 3x at the concentrations of 120 µl/mL and 180 µl/mL, respectively. 3.5 Evaluation of the mitochondrial potential of the Phytomonas serpens promastigotes The P. serpens promastigotes that were treated with 60, 120, and 180 µg/mL of LR038 were stained with Rhodamine 123 and evaluated by flow cytometry, to verify the effects of the lyophilized residue. The cells that were treated with 120 and 180 µl/ml of LR038 showed a reduction in the mitochondrial potential when compared with the untreated cells. The Rhodamine 123 total fluorescence histogram (Fig. 4 C) and the index of the variation results (Fig. 4 A) showed a reduction in the fluorescence at concentrations of 120µl/mL and 180µl/mL, respectively. These two concentrations reduced the fluorescence by 44.6% (IV= -0.446) and 46.8% (IV= -0.468), respectively. These reductions did not differ statistically from the reduction of fluorescence in the positive control, 56.0% (IV= -0.560). These results were confirmed by the statistical analysis of the median of fluorescence, which was obtained and compared with the negative control (P < 0.05) (Fig. 4 B). 3.6 Qualitative chemical profile of the residue. The qualitative chemical profile of LR038 was determined by analytical liquid chromatography (Fig. 5 A). The presence of 17 peaks and their respective ultraviolet spectra were observed, which were used to identify the classes of the secondary metabolites that were present in LR038. The results of this qualitative analysis indicated the presence of phenolic acids and flavonoids (Fig. 5 B). Discussion The global essential oils market had a turnover of 18.62 billion US dollars in 2020, and strong growth is estimated for the next few years, reaching a value of 33.26 billion US dollars by 2027, with estimated sales of 473 .31 thousand tons of essential oils (Grand View Research, 2020). Although there is a great demand for these products, the essential oil yield from many plant species is almost always low (Alighiri et al., 2017 ). In contrast, millions of tons of waste are generated during production and they are disposed of into the environment, in some cases, without treatment (Peshev, 2020 ). Although these residues are discarded, they have many biologically active molecules, which exhibit diverse biological activities. The residue from the hydrodistillation of Dittrichia graveolens , with a high content of flavonoids, presented antioxidant, antibacterial, and anti-inflammatory activities, with the advantage of not being cytotoxic to the fibroblasts (Gharred et al., 2021 ). The genus Phytomonas , belonging to the Trypanosomatidae family, has pathogenic members, which are parasites that cause diseases in plants and humans. Some species of this genus cause severe infections in large crops, such as acute lethal wilt in oil palm and coconut trees ( Phytomonas staheli ), necrosis in coffee phloem ( Phytomonas leptovasorum ), and root bubbling in cassava ( Phytomonas françai ) (Jaskowska et al., 2015 ). Despite not causing a severe systemic disease, P. serpens can compromise the commercial value of tomato plants, due to the appearance of yellow spots on the parasitized fruits (Schwelm et al., 2018 ). However, this species constitutes an important biological model, as it presents natural characteristics similar to other pathogenic members belonging to the Trypanosomatidae family. In addition, unlike other pathogenic species, P. serpens is readily isolated and cultivated in the laboratory, enabling in vitro studies in the most diverse areas. Studies have proven that compounds from medicinal plants have important molecules, with antimicrobial activities on the promastigote forms of P. serpens . Piper crassinervium and P. amalago leaf extracts have inhibited the P. serpens proliferation in vitro, with low to moderate cytotoxicity in mammalian cells (Cancini et al., 2020 ). Silva et al. ( 2019 ) reported that the use of the essential oil of Varronia curassavica inhibited the growth of P. serpens , causing a loss of the cell membrane integrity. Pereira et al. ( 2022 ) showed an inhibitory effect of L. camara essential oil on the promastigote forms of this same species. Thus, this study aimed to evaluate the effect of residual water from the hydrodistillation process of L. camara leaves, as a source of natural compounds for the control of protozoa of the genus Phytomonas . In this work, it was possible to demonstrate the antiprotozoal actions of the residues from the production chain of essential oils from four accessions of L. camara on P. serpens . The results have shown that the four samples of the lyophilized residues evaluated showed antiprotozoal activities at low concentrations, with the IC 50 value ranging from 13.68 to 43.69 µg/mL. Although the four LRs showed a promising inhibition of parasite viability, LR038 was selected for having a lower IC 50 value. From the qualitative chemical profile of the LR038 sample, two classes of natural products were detected, the phenolic acids and flavonoids, which can be attributed to the detected trypanocidal activities. Although the molecules from these classes have known antiprotozoal activities, there are no reports on the activity of these molecules against P. serpens . Caffeic acid, belonging to the phenolic acid class, is a good representative to demonstrate the antiprotozoal capacity of this group of compounds. Silva Bortoleti et al. ( 2019 ) reported that caffeic acid could inhibit the proliferation of the Leishmania amazonensis promastigotes, and cause changes in the cell morphology through the loss of plasma membrane integrity, as well as with the accumulation of the levels of the reactive oxygen species, thus increasing the oxidative response. Flavonoids also have antiprotozoal activities, like quercetin and myricetin, which have shown a trypanocidal activity on Trypanosoma brucei , the etymological agent of African human trypanosomiasis (Larit et al., 2021 ). Since it was shown that LR038 markedly decreased the viability of P. serpens at low concentrations, the effects of this residue on the cell membrane integrity, on the intracellular levels of the reactive oxygen species, and on the mitochondrial membrane potential of the P. serpens promastigotes, were determined. One of the possible mechanisms of action of natural products on cell viability includes their effects on the integrity of the plasma membrane. The evaluation that was based on the permeability of propidium iodide showed that the treatment with LR038 did not alter the integrity of the plasma membrane of the P. serpens promastigotes. This might be due to the low liposolubility of LR038, as molecules typically require that the hydrophobic groups interact directly with the membrane (Jesus et al., 2021 ). The hydroxyl (polar) groups that were present in the flavonoids and phenolic acids likely entered the cell through controlled endocytosis. The biological activities of flavonoids are mainly related to their antioxidant effects. However, several studies have also demonstrated the pro-oxidant action of many molecules of this class of natural products. A good example is quercetin, which is capable of inducing the reactive oxygen species in human embryonic stem cells (hESCs), thus triggering selective cell deaths (Kim et al., 2017 ). Another representative of the flavonoid class with an oxidizing action is cynaroside, which inhibits the growth of the Leishmania donovani promastigotes, by inducing the production of ROS (Tabrez et al., 2021 ). The ROS levels in the P. serpens promastigotes that were treated with LR038 were determined by using H 2 DCFDA, a non-fluorescent probe that when permeating the cell, is converted into dichlorodihydrofluorescein (DCFH) by the action of the esterases. In the presence of free radicals, such as ROS, DCFH is oxidized and converted to 2,7-dichlorofluorescein (DCF), a highly fluorescent product. The results obtained revealed that an intracellular increase in the ROS was accompanied by an increase in the concentration of LR038. Notably, the levels of ROS that were detected in the parasites that were treated with 120 and 180 ug/mL of the residues were approximately 2x and 3x higher than in the untreated parasites. Reactive oxygen species result from cellular metabolism, and their levels are regulated by the presence of an endogenous antioxidant complex that when affected, can lead to an intracellular accumulation of ROS. The consequent oxidative stress generated might cause metabolic changes, including a loss of the mitochondrial membrane potential (Fonseca-Silva, et al., 2015 , Mittler, 2017 ). In this study, reductions of 44.6% and 46.8% in the Rhodamine-generated fluorescence were observed in the P. serpens promastigotes that were treated with 120 and 180 µg/mL of LR038, respectively. This reduction in the fluorescence demonstrated a decrease in the mitochondrial membrane potential that was generated by LR038, which might be related to the increase in ROS that was observed in the cells that were treated with this residue. Since trypanosomatids have a single mitochondrion, the proper functioning of this organelle is essential for the survival of these organisms, hence constituting an important target for the construction of drugs (Menna-Barreto and Castro, 2014 , Fidalgo and Gille, 2011 ). A study by Bombaça et al. pointed out that the mitochondrial dysfunction and the production of ROS were the main determinants of the antiprotozoal activity of the naphthoimidazoles in Trypanosoma cruzi (Bombaça et al., 2019 ). Similarly, the apigenin flavonoid has been shown to exert antileishmanicidal activity by altering the membrane potential, as the result of an increase in the production of ROS (Fonseca-Silva et al., 2015 ). When considering the results that were obtained here, the antiprotozoal activities that were presented by the residues of the hydrodistillation of the essential oil of L. camara were due to its ability to induce oxidative stress. This was evidenced by the increase in the ROS levels and the functional alterations in the mitochondria, which interfered with the production of energy that compromised the parasite's survival. Conclusions The residues from the hydrodistillation of the L. camara leaves can serve as a source of biologically active compounds, phenolic acids, and flavonoids, with an antiprotozoal potential. The highest concentrations tested promoted changes in the potential of the mitochondrial membrane and caused the accumulation of the reactive oxygen species. This demonstrated the effectiveness of the residues that were generated during the hydrodistillation of L. camara leaves to obtain the essential oils. Declarations 6.1. Ethical Approval Not applicable. 6.2. Consent to Participate Not applicable. 6.3. Consent to Publish Not applicable. 6.4. Authors Contributions Caroline Alves Soares: Conceptualization, Data curation, Methodology, Roles/Writing - original draft. Tamíris Aparecida de Carvalho Santos: Conceptualization, Methodology. Luís Fernando de Andrade Nascimento: Conceptualization, Methodology. Raphael Amancio de Jesus: Conceptualization, Methodology. Arie Fitzgerald Blank: Conceptualization; Writing - Review & Editing; Funding acquisition. Ricardo Scher: Conceptualization, Methodology. Valéria Regina de Souza Moraes: Conceptualization, Methodology. Roberta Pereira Miranda Fernandes: Conceptualization, Methodology, Writing - Review & Editing. Maria de Fátima Arrigoni-Blank: Conceptualization, Writing - Review & Editing; Supervision. 6.5. Funding This study was funded, in part, by Brazil’s Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação de Apoio à Pesquisa e a Inovação Tecnológica do Estado de Sergipe (Fapitec/SE), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001), and the Financiadora de Estudos e Projetos (FINEP). 6.6. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6.3. Author contributions Caroline Alves Soares: Conceptualization, Data curation, Methodology, Roles/Writing - original draft. Tamíris Aparecida de Carvalho Santos: Conceptualization, Methodology. Luís Fernando de Andrade Nascimento: Conceptualization, Methodology. Raphael Amancio de Jesus: Conceptualization, Methodology. Arie Fitzgerald Blank: Conceptualization; Writing - Review & Editing; Funding acquisition. Ricardo Scher: Conceptualization, Methodology. Valéria Regina de Souza Moraes: Conceptualization, Methodology. Roberta Pereira Miranda Fernandes: Conceptualization, Methodology, Writing - Review & Editing. Maria de Fátima Arrigoni-Blank: Conceptualization, Writing - Review & Editing; Supervision. 7. Acknowledgments The authors would like to thank the Trypanosomatids Collection of the Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil for providing P. serpens isolate (strain 9T). References Al-Hakeim HK, Al-Zabibah, RS, Alzihari HF, Almensoori AK, Al-Zubaidi HA, Hassan LEAH (2021) Anticancer and antiangiogenic activities of alkaloids isolated from Lantana camara by adsorption on the magnetic nanoparticles. Karbala Int. J. Mod. Sci 7(1), e11. https://doi.org/10.33640/2405-609X.2577 Alighiri D, Eden WT, Supardi KI, Purwinarko A (2017) Potential development of essential oil production of Central Java, Indonesia. J. Phys.: Conf. Ser 824, 12-21. https://doi.org/10.1088/1742-6596/824/1/012021 Ayalew AA (2020) Insecticidal activity of the Lantana camara extract oil on controlling maize grain weevils. Toxicology Research and Application 4, 1-10. https://doi.org/10.1177/2397847320906491 Bashir S, Jabeen K, Iqbal S, Javed S, Naeem A (2019) Lantana camara : phytochemical analysis and antifungal prospective. Planta Daninha 37, e019193526. http://dx.doi.org/10.1590/s0100-83582019370100137 Bombaça ACS, Viana PG, Santos AC, Silva TL, Rodrigues ABM, Guimarães ACR, Menna-Barreto RF (2019) Mitochondrial disfunction and ROS production are essential for anti- Trypanosoma cruzi activity of β-lapachone-derived naphthoimidazoles. Free Radic. Biol. Med 130, 408-418. https://doi.org/10.1016/j.freeradbiomed.2018.11.012 Bordoloi K, Bhagawati B, Baruah AM, Neog PP, Kurulkar U (2021) Biochemical mechanism of Lantana camara leaf extracts in the management of Meloidogyne incognita on tomato. J. Pharmacogn. Phytochem 10(1), 2828-2834. https://doi.org/10.22271/phyto.2021.v10.i1an.13789 Cancini NDS, Beraldo-Borrazzo J, Menezes JLD, Cortez DAG, Valdez RH, Garcia FP, Abreu Filho BAD (2020) Activity of Piperaceae extracts and fractions in the control of Phytomonas serpens. Cienc. Rural 50(10), e20200343. https://doi.org/10.1590/0103-8478cr20200343 Delgado-Altamirano R, López-Palma RI, Monzote L, Delgado-Domínguez J, Becker I, Rivero-Cruz JF, Rojas-Molina A (2019) Chemical constituents with leishmanicidal activity from a pink-yellow cultivar of Lantana camara var. aculeata (L.) collected in Central Mexico. Int. J. Mol. Sci 20(4), e872. https://doi.org/10.3390/ijms20040872 Dougnon G, Ito M (2020) Sedative effects of the essential oil from the leaves of Lantana camara occurring in the Republic of Benin via inhalation in mice. J. Nat. Med 74(1), 159-169. https://doi.org/10.1007/s11418-019-01358-9 Fidalgo LM, Gille L (2011) Mitochondria and trypanosomatids: targets and drugs. Pharm. Res 28(11), e2758. https://doi.org/10.1007/s11095-011-0586-3 Fonseca-Silva F, Canto-Cavalheiro MM, Menna-Barreto RFS, Almeida-Amaral EE (2015) Effect of apigenin on Leishmania amazonensis is associated with reactive oxygen species production followed by mitochondrial dysfunction. J. Nat. Prod 78(4), 880-884. https://doi.org/10.1021/acs.jnatprod.5b00011 Gharred N, Baaka N, Bettache N, Hamdi A, Dbeibia A, Dhaouadi H, Dridi-Dhaouadi S (2021) Wastewater to Ecological dyeing process and bioactive compounds resources: a case study of Dittrichia graveolens hydrodistillation aqueous residue. Waste Biomass Valorization 12, 5065-5077. https://doi.org/10.1007/s12649-021-01375-4 Gindri DM, Coelho CMM (2020) Metabolitos aleloquímicos de Lantana camara L.: potencial para o desenvolvimento de bioherbicida - revisão. Revista Técnico-Científica do CREA-PR 24, e7. Accessed: 22/02/21 Grand View Research, 2020 Essential oils market size, share & trends analysis report by application (food & beverages, spa & relaxation), by product (orange, peppermint), by sales channel, and segment forecasts, 2020-2027. Accessed: 22/02/21 Jaskowska E, Butler C, Preston G, Kelly S (2015) Phytomonas: trypanosomatids adapted to plant environments. PLoS Pathog 11(1), e1004484. https://doi.org/10.1371/journal.ppat.1004484 Jesus JA, Laurenti MD, Antonangelo L, Faria CS, Lago JHG, Passero LFD (2021) Related pentacyclic triterpenes have immunomodulatory activity in chronic experimental visceral leishmaniasis. J. Immunol. Res 2021, e6671287. https://doi.org/10.1155/2021/6671287 Kim SY, Jeong HC, Hong SK, Lee MO, Cho SJ, Cha HJ (2017) Quercetin-induced ROS production triggers mitochondrial cell death of human embryonic stem cells. Oncotarget 8(39), 64964-64973. https://doi.org/10.18632/oncotarget.11070 Larit F, Elokely KM, Nael MA, Benyahia S, León F, Cutler SJ, Ghoneim MM (2021) Proposed mechanism for the antitrypanosomal activity of quercetin and myricetin isolated from Hypericum afrum Lam.: phytochemistry, in vitro testing and modeling studies. Molecules 26(4), e1009. https://doi.org/10.3390/molecules26041009 Mahmoudi H, Marzouki M, M'rabet Y, Mezni M, Ouazzou AA, Hosni K (2020) Enzyme pretreatment improves the recovery of bioactive phytochemicals from sweet basil ( Ocimum basilicum L.) leaves and their hydrodistilled residue by-products, and potentiates their biological activities. Arab. J. Chem 13(8), 6451-6460. https://doi.org/10.1016/j.arabjc.2020.06.003 Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V (2019) Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy, and evolution. Parasitology 146(1), 1-27. https://doi.org/10.1017/S0031182018000951 Menna-Barreto RFS, Castro SL (2014) The double-edged sword in pathogenic Trypanosomatids: the pivotal role of mitochondria in oxidative stress and bioenergetics. Biomed Res. Int 2014 e614014. https://doi.org/10.1155/2014/614014 Mittler R (2017) ROS are good. Trends Plant Sci 22(1), 11-19. https://doi.org/10.1016/j.tplants.2016.08.002 Napoli E, Dattilo S, Ruberto G (2020) Hydrodistillation of Trachelospermum jasminoides Lindl. flowers. An analysis of essential oil, hydrolate, and polyphenols content of the process wastes. J. Essent. Oil Res 32(6), 556-561. https://doi.org/10.1080/10412905.2020.1808538 Negi GC, Sharma S, Vishvakarma SC, Samant SS, Maikhuri RK, Prasad RC, Palni LM (2019) Ecologia e uso de Lantana camara na Índia. The Bot. Rev 85(2), 109-130. https://doi.org/10.1007/s12229-019-09209-8 Nirmal CR, Ebenezer RS, Kannan P, Balasubramanian M, Thirunavukkarasu I, Mondal R, Dusthackeer A (2020) Anti-tuberculosis activity of bioactive compounds from Lantana camara L., Euphorbia hirta L., Mukia maderaspatana (L.) M. Roem, and Abutilon indicum (L.). Eur. J. Integr. Med 35, 101-105. https://doi.org/10.1016/j.eujim.2020.101105 Pereira KLG, Nizio DAC, Lima PCN, Fernandes RPM, Arrigoni-Blank MF, Sá Filho JCF, Nascimento LFA, SouzaVT (2022) Seasonal variance in the chemical composition of essential oils from Lantana camara accessions and their tripanocida do óleo essencial sobre Phytomonas serpens . Bol. Latinoam. Caribe Plantas Med. Aromat 21(4) Pereira KLG, Nogueira PCL, Arrigoni-Blank MF, Nizio DAC, Silva DC, Pinto JAO, Blank AF (2020) Chemical diversity of essential oils of Lantana camara L. native populations. J. Essent. Oil Res 32(1), 32-47. https://doi.org/10.1080/10412905.2019.1661879 Peshev D (2020) Theoretical assessment of the use of nanofiltration for fractionation of waste aqueous fractions from the essential oil industry. Bulg. Chem. Commun 52(4), 533-542. https://doi.org/10.34049/bcc.52.4.MP05 Qureshi H, Anwar T, Ali Q, Haider MZ, Habib N, Fatima S, Waseem M, Bibi Y, Arshad M, Adkins SW (2019) Isolation of the natural herbicidal compound from Lantana camara . Int. J. Environ. Anal. Chem 101(5), 631-638. https://doi.org/10.1080/03067319.2019.1670822 Sánchez-Vioque R, Izquierdo-Melero ME, Polissiou M, Astraka K, Tarantilis PA, Herraiz-Peñalver D, Santana-Méridas O (2015) Comparative chemistry and biological properties of the solid residues from hydrodistillation of Spanish populations of Rosmarinus officinalis L. Grasas Aceites 66(2), e079. https://doi.org/10.3989/gya.1060142 Sankara A, Ouédraogo JCW, Pignolet L, Thévenon MF, Bonzi-Coulibaly Y (2020) Chemical profiles and anti-termite activity of hydrodistillation residues from three aromatic plants acclimated in Burkina Faso. J. Agric. Sci 12(8), 245-256. https://doi.org/10.5539/jas.v12n8p245 Santos Júnior ADCM, Ricart CAO, Pontes AH, Fontes W, Souza ARD, Castro MS, Lima BD (2018) Proteome analysis of Phytomonas serpens , a phytoparasite of medical interest. PloS One 13(10), e0204818. https://doi.org/10.1371/journal.pone.0204818 Schwelm A, Badstöber J, Bulman S, Desoignies N, Etemadi M, Falloon RE, Neuhauser S (2018) Not in your usual Top 10: protists that infect plants and algae. Mol. Plant Pathol 19(4), 1029-1044. https://Doi.Org/10.1111/Mpp.12580 Shankar S, Prasad S, Owaiz M, Yadav S, Manhas S, Yaqoob M (2021) Essential oils, components, and their applications: a review. Plant Arch 21(1), 2027-2033. https://doi.org/10.51470/PLANTARCHIVES.2021.v21.S1.331 Silva Bortoleti BT, Tomiotto-Pellissier F, Gonçalves MD, Miranda-Sapla MM, Assolini JP, Carloto AC, Pavanelli WR (2019) Caffeic acid has antipromastigote activity by the apoptosis-like process, and anti-amastigote by TNF-α/ROS/NO production and decreased of iron availability. Phytomedicine 57, 262-270. https://doi.org/10.1016/j.phymed.2018.12.035 Silva KP, Carvalho Santos TA, Moutinho BL, Silva RS, Pinto VS, Blank AF, Fernandes RPM (2019) Using Varronia curassavica (Cordiaceae) essential oil for the biocontrol of Phytomonas serpens . Ind. Crops Prod 139, e111523. https://doi.org/10.1016/j.indcrop.2019.111523 Tabrez S, Rahman F, Ali R, Alouffi AS, Akand SK, Alshehri BM, Rub A (2021) Cynaroside inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response. Biosci. Rep 41(1), eBSR20203857. https://doi.org/10.1042/BSR20203857 Wollinger A, Perrin E, Chahboun J, Jeannot V, Touraud D, Kunz W (2016) Antioxidant activity of hydro distillation water residues from Rosmarinus officinalis L. leaves determined by DPPH assays. C. R. Chim 19(6), 754-765. https://doi.org/10.1016/j.crci.2015.12.014 Wu P, Song Z, Wang X, Li Y, Cui J, Tuerhongc M, Da-Qing J, Abudukeremuc M, Leed D, Xu J, Guo Y (2020) Bioactive triterpenoids from Lantana camara showing anti-inflammatory activities in vitro and in vivo. Bioorg. Chem 101, e104004. https://doi.org/10.1016/j.bioorg.2020.104004 Supplementary Files GraphicalAbstract.jpg HighlightsLantanacamara.docx Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Major Revision 25 Aug, 2024 Reviewers agreed at journal 13 Apr, 2024 Reviewers invited by journal 30 Mar, 2024 Editor invited by journal 21 Mar, 2024 Editor assigned by journal 19 Mar, 2024 First submitted to journal 14 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4092579","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285600216,"identity":"8b118f4e-e3a3-4558-8ad2-cef6f7005174","order_by":0,"name":"Caroline Alves Soares","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"Alves","lastName":"Soares","suffix":""},{"id":285600217,"identity":"25490fa7-79c9-4945-9513-d66eca80ed29","order_by":1,"name":"Tamíris Aparecida de Carvalho Santos","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Tamíris","middleName":"Aparecida de Carvalho","lastName":"Santos","suffix":""},{"id":285600218,"identity":"2b394577-5250-41fc-838b-d5cd0f3bb52b","order_by":2,"name":"Luís Fernando de Andrade Nascimento","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Luís","middleName":"Fernando de Andrade","lastName":"Nascimento","suffix":""},{"id":285600219,"identity":"97a5b3b8-bfc6-4342-96e7-18de517397e3","order_by":3,"name":"Raphael Amancio de Jesus","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Raphael","middleName":"Amancio","lastName":"de Jesus","suffix":""},{"id":285600220,"identity":"6b2ce25f-369c-4e9f-a51c-e882ffc375c9","order_by":4,"name":"Arie Fitzgerald Blank","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Arie","middleName":"Fitzgerald","lastName":"Blank","suffix":""},{"id":285600221,"identity":"bc57d4d5-5244-49c0-8784-76992132e024","order_by":5,"name":"Ricardo Scher","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Scher","suffix":""},{"id":285600222,"identity":"cea0e29c-7ef6-444f-8e54-d0041b4ffefe","order_by":6,"name":"Valéria Regina de Souza Moraes","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Valéria","middleName":"Regina de Souza","lastName":"Moraes","suffix":""},{"id":285600223,"identity":"4dbcdcaf-08d1-4705-a5ad-a8b74e65ad42","order_by":7,"name":"Maria de Fátima Arrigoni-Blank","email":"","orcid":"","institution":"Universidade Federal de Sergipe","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"de Fátima","lastName":"Arrigoni-Blank","suffix":""},{"id":285600224,"identity":"e96fe76d-0bd8-42f1-bfbe-e91d2919ee1a","order_by":8,"name":"Roberta Pereira Miranda Fernandes","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1467-3946","institution":"Universidade Federal de Sergipe","correspondingAuthor":true,"prefix":"","firstName":"Roberta","middleName":"Pereira Miranda","lastName":"Fernandes","suffix":""}],"badges":[],"createdAt":"2024-03-13 12:35:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4092579/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4092579/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-35142-4","type":"published","date":"2024-09-26T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53993811,"identity":"4245405b-1016-4e57-b9d6-1b03c5caabc5","added_by":"auto","created_at":"2024-04-03 06:42:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":281215,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the lyophilized residue from four \u003cem\u003eLantana camara\u003c/em\u003e L. accessions on the promastigote forms of \u003cem\u003ePhytomonas serpens\u003c/em\u003e. The parasites (1x10\u003csup\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sup\u003e cells/ml) were treated with 1.5; 3.0; 6.0; 12.0; 25.0; 50.0, and 100 µg/mL for 48 hours, and the viability was evaluated by the resazurin colorimetric test. (a) Cell viability. (b) Values (IC\u003csub\u003e50\u003c/sub\u003e) of the lyophilized residue. The data is expressed as the mean ± standard error of three independent experiments.\u003c/p\u003e","description":"","filename":"Fig1.EPS.png","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/cb21b5baabe6a0800db970fe.png"},{"id":53993809,"identity":"262de80b-2850-4bcd-990b-a3a74cb651eb","added_by":"auto","created_at":"2024-04-03 06:42:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":223442,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the lyophilized residue from the \u003cem\u003eLantana camara\u003c/em\u003e L. accession LAC-038, named LR038, on the growth of the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes. The parasites (1x10\u003csup\u003e5\u003c/sup\u003e cells/ml) were treated with 6.25; 12.5; 25.0; 50.0, and 100 µg/mL of the lyophilized residue for 72 hours, with direct counting performed every 24 hours. (a) Cell growth curve. (b) Percentage of inhibition. Control equal to the untreated cells. The data is expressed as the mean ± standard error of three independent experiments.\u003c/p\u003e","description":"","filename":"Fig2.EPS.png","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/0847602c04dd719639e57b7b.png"},{"id":53993810,"identity":"439d0e11-c2da-45ef-8c3a-92070f66c437","added_by":"auto","created_at":"2024-04-03 06:42:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174045,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the lyophilized residue from the \u003cem\u003eLantana camara\u003c/em\u003e accession LAC-038, named LR038, on the production of the reactive oxygen species in the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes. The parasites (5,105 cells/ml) were treated with 60, 120, and 180µg/ml of the lyophilized residue for 48 hours. The EROS levels were assessed spectrofluorimetrically when using the H2DCFDA probe. The untreated cells represent the negative control (CN), and the cells incubated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (750 µM, 30 min) correspond to the positive control (CP). The fluorescence data was relativized with the negative control. The results were expressed as the mean ± standard error of three independent experiments. The data was submitted to the Analysis of Variance (ANOVA) and the Tukey test, (*) P\u0026lt;0.05, (**) P\u0026lt;0.01, (***) P\u0026lt;0.001, while (ns) was not significant.\u003c/p\u003e","description":"","filename":"Fig3.EPS.png","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/ffaf73c19e9be017126e68b1.png"},{"id":53993814,"identity":"4f405592-99da-4ec4-960e-93bdd81d411b","added_by":"auto","created_at":"2024-04-03 06:42:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":630178,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the lyophilized residue from the \u003cem\u003eLantana camara\u003c/em\u003e accession LAC-038, named LR038, on the mitochondrial potential of the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes. The parasites (5,105 cells/mL) were treated with 60, 120, and 180µg/mL of the lyophilized residue for 48 hours and stained with Rhodamine 123, and evaluated by flow cytometry. The untreated cells represent the negative control (CN), and the cells incubated with CCCP correspond to the positive control (CP). (a) Representative histogram of the mitochondrial membrane potential. (b) The medians of fluorescence relative to the negative control. (c) The variation index results were expressed as a percentage of the relative fluorescence ± standard error. (*) Indicates the statistical difference to the untreated control (P\u0026lt;0.05), while (ns) was not significant.\u003c/p\u003e","description":"","filename":"Fig4.EPS.png","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/a93b131f0813462f84370209.png"},{"id":53993816,"identity":"b4aaaca7-f6db-44af-90b5-d6493185787f","added_by":"auto","created_at":"2024-04-03 06:42:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4131182,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of the \u003cem\u003eLantana camara\u003c/em\u003e L compound classes. (a) Chemical profile of the lyophilized residue from the accession LAC-038, named LR038. Phenolic Acids (Peak 1 - 6), Flavonoids (Peak 11 - 17), not clear (Peak 7 -10). (b) The classes of the compounds that were detected in the lyophilized residue of the accession LAC-038 (-) Absence (+) Presence.\u003c/p\u003e","description":"","filename":"Fig5.TIF.png","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/338fbf2e1840e3db8cfd7915.png"},{"id":65627297,"identity":"110786da-bb00-4e74-b820-0a05e6e30a04","added_by":"auto","created_at":"2024-09-30 16:14:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9307467,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/e0449c4b-3e37-45e7-a7a7-7f62714975b3.pdf"},{"id":53993812,"identity":"e0f0e7bd-d1b4-4943-8612-c099ae68f1a3","added_by":"auto","created_at":"2024-04-03 06:42:33","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":305874,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/6f36ea948a42f67e65e8264a.jpg"},{"id":53993813,"identity":"5ff82aa1-3773-407b-a541-16e6c9a6f46b","added_by":"auto","created_at":"2024-04-03 06:42:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14686,"visible":true,"origin":"","legend":"","description":"","filename":"HighlightsLantanacamara.docx","url":"https://assets-eu.researchsquare.com/files/rs-4092579/v1/c6644145c482768b1f673b78.docx"}],"financialInterests":"","formattedTitle":"Anti-Phytomonas activity of the lyophilized residues obtained from the distillation of Lantana camara L. essential oil","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEssential oils are mixtures that are rich in aromatic compounds and they have several applications. They are currently used as ingredients in the production of beverages, food, fragrances, cosmetics, and drugs, in addition to being considered the primary tool of aromatherapy, a segment of medicine in which the treatment of patients occurs with the help of these oils (Shankar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although there is a growing demand for these products (Alighiri et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the yield of essential oil in many plant species is low. This results in a large volume of residues during the distillation process (Napoli et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On an industrial scale, the waste that is accumulated in the distilleries of essential oils can be compared to the volume of waste that is produced by the textile industry (Gharred et al., 2020). The incorrect disposal of these materials can cause severe damage to the environment.\u003c/p\u003e \u003cp\u003eThe maximum use of raw materials, the reduction of energy expenditure and waste production, as well as their transformation into new products, is part of the biorefinery concept. In the essential oils industry, the residues are generally discarded. However, several useful compounds can be extracted from them in other production chains, such as rosmarinic acid, an important natural antioxidant that can be obtained from the liquid residue of the hydrodistillation process of the leaves of rosemary (\u003cem\u003eRosmarinus officinalis\u003c/em\u003e L.) (Wollinger et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This is together with the phenolic compounds that are extracted from the solid residue of the hydrodistillation of \u003cem\u003eR. officinalis\u003c/em\u003e, which have a deterrent effect on pest insects (S\u0026aacute;nchez-Vioque et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sankara et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that the residues from the hydrodistillation of \u003cem\u003eEucalyptus camaldulensis\u003c/em\u003e could provide interesting bioactive compounds, as new anti-termite agents in plant protection. Mahmoudi et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also reported that hydrodistilled basil leaves (\u003cem\u003eOcimum basilicum\u003c/em\u003e) have a good repellent activity against the stored grain pests, \u003cem\u003eTribolium castaneum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLantana camara\u003c/em\u003e L. (Verbenaceae) is an aromatic species widely known throughout the world for its ornamental use. In traditional medicine, it is common to use its leaves, branches, and roots to treat cuts, fevers, headaches, palpitations, tetanus, rheumatism, malaria, ulcers, and so forth. (Gindri and Coelho, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the essential oil that is obtained from this species, several bioactive compounds are found, such as terpenoids, alkaloids, flavonoids, phenolic compounds, and glycosides. These compounds are to which various biological activities have been attributed, such as anticancer and antiangiogenic (Al-Hakeim et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), anti-inflammatory (Wu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), anti-tuberculosis (Nirmal et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as a sedative (Dougnon, Ito, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This is in addition to antifungal (Bashir et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), herbicide (Qureshi, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), nematicide (Borbodoli et al., 2021), insecticide (Ayalew, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and trypanocide agents (Delgado-Altamirano et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTrypanosomatids are microorganisms that are adapted to life in fluids that are rich in glucose, where most of them are parasites of animals, with the exception of the genus \u003cem\u003ePhytomonas\u003c/em\u003e, whose representatives are plant parasites (Maslov et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Some species cause serious diseases in large crops of economic interest. \u003cem\u003ePhytomonas staheli\u003c/em\u003e, which causes acute lethal wilt in coconut palms (\u003cem\u003eCocos nucifera\u003c/em\u003e), and oil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e), which begins with the rotting of the leaves, and then progresses to the spear and the roots, as well as \u003cem\u003ePhytomonas leptovasorum\u003c/em\u003e, a pathogen of necrosis in coffee (Arabica and Liberia) phloem (Jaskowska et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These are some examples of the important plant pathogens, which are capable of offering an economic risk in tropical countries.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhytomonas serpens\u003c/em\u003e is not a pathogen of agricultural relevance, although it is an important biological model that helps in the understanding of pathogenic members belonging to the Trypanossomatidae family that affect plants and mammals. Due to its simplified biology, it helps in understanding the mechanisms of pathogenicity and the manifestations of diseases in plants and animals (Jaskowska et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Santos Junior et al., 2018).\u003c/p\u003e \u003cp\u003eIn a recent study, this current group demonstrated for the first time, the trypanocidal activity of the essential oil of \u003cem\u003eL. camara\u003c/em\u003e on \u003cem\u003eP. serpens\u003c/em\u003e (Pereira et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), demonstrating the potential of this product for the treatment of economically important diseases that are caused by other species of \u003cem\u003ePhytomonas\u003c/em\u003e. However, the yield of \u003cem\u003eL. camara\u003c/em\u003e essential oil is low (usually 0.2%), and by steam dragging, 500 kg of biomass yields about 200 mg of oil (Negi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the other hand, the volume of waste generated by the oil hydrodistillation process is high and constitutes a promising source of bioactive molecules.\u003c/p\u003e \u003cp\u003eGiven the commercial importance of crops that are devastated by species of the genus \u003cem\u003ePhytomonas\u003c/em\u003e, and the potential of the products in the metabolism of \u003cem\u003eL. camara\u003c/em\u003e to control the proliferation of these phytopathogens, the present work evaluated the action of residues in the hydrodistillation of the leaves of \u003cem\u003eL. camara\u003c/em\u003e on the viability of \u003cem\u003eP. serpens in vitro\u003c/em\u003e. This was as well as their effects on the integrity of the plasma membrane, and the potential of the mitochondrial membrane on these phytopathogens.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material\u003c/h2\u003e \u003cp\u003eFour accessions of \u003cem\u003eLantana camara\u003c/em\u003e L., LAC-033, LAC-038, LAC-017, and LAC-025, from the Active Germplasm Bank of Medicinal and Aromatic Plants of the Federal University of Sergipe, Brazil, were selected. The choice of the accessions was supported by previous works, in which the chemical characterization of the essential oils from the plants of the active germplasm bank was determined (Pereira et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The accessions of the plant material were registered under the identification number A8CCB3B in the National System for the Management of Genetic Heritage and Traditional Knowledge (SisGen).\u003c/p\u003e \u003cp\u003eThe leaves were collected and subjected to hydrodistillation in a modified Clevenger apparatus, by using 75g samples of the dry leaves, with the addition of 1.5L of distilled water, for 140 minutes. The effluent, resulting from the decoction of the leaves, was filtered and separated into 200mL samples. Later, they were stored in an ultra-freezer at \u0026minus;\u0026thinsp;80\u0026deg;C (Liotop UFR30, Liobras, S\u0026atilde;o Carlos, SP, Brazil) for 24 hours. They were then transferred to a freeze dryer (Liotop L101, Liobras, S\u0026atilde;o Carlos, SP, Brazil), where they rested for five days at a temperature of \u0026minus;\u0026thinsp;54\u0026deg;C and a pressure of 79 \u0026micro;m Hg until the complete sublimation of the water that was contained in the samples. At the end of this process, the \u0026ldquo;Lyophilized Residues (LRs)\u0026rdquo; were obtained from each accession, hereinafter named LR038 (Accession LAC-038), LR017 (Accession LAC-017), LR025 (Accession LAC-025), and LR033 (Accession LAC-033).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell culture\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePhytomonas serpens\u003c/em\u003e (9T) parasites were obtained from the Trypanosomatid Collection at the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. The promastigote forms were grown in a Schneider culture medium (Sigma-Aldrich\u0026reg;, St. Louis, Missouri, USA), which were supplemented with 10% fetal bovine serum (Sigma-Aldrich\u0026reg;, St. Louis, Missouri, USA) and 1% streptomycin at 26\u0026ordm;C in a BOD incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell viability of \u003cem\u003ePhytomonas serpens\u003c/em\u003e when exposed to the residues from the \u003cem\u003eLantana camara\u003c/em\u003e accessions\u003c/h2\u003e \u003cp\u003eThe promastigotes were cultured at 26\u0026deg;C in a BOD incubator, and then 1x105 cells were deposited in 96-well microplates and treated for 48 hours, with the four lyophilized residues (LRs), in serial dilutions at concentrations of 100.0 to 1.5 \u0026micro;g/mL. The negative control consisted of the untreated cells, and for the positive control, the cells were treated with the antibiotic, Amphotericin B. After 48 hours of treatment, 50 \u0026micro;L of resazurin solution (200 \u0026micro;M) was added to each well, and the plates were incubated in the dark for 30 minutes. This particular dye is an indicator of cell viability, which when added to the culture, enters the cells and is reduced by the mitochondrial dehydrogenases to the resofurin of the viable cells while changing the color of the medium from blue to pink. The cell viability was measured by spectrofluorimetry at a wavelength of 555 nm excitation and 585 nm emission in a microplate reader (Synergy\u0026trade; H1, BioTek Hybrid Technology, Winooski, Vermont, USA). From the cell viability values obtained, the IC\u003csub\u003e50\u003c/sub\u003e levels (the concentration that inhibits 50% of the cells) were calculated. The lyophilized residue (LR) from the accessions that presented the lowest IC\u003csub\u003e50\u003c/sub\u003e value was used in the subsequent tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Growth curve of \u003cem\u003ePhytomonas serpens\u003c/em\u003e when treated with selected residue\u003c/h2\u003e \u003cp\u003eThe promastigotes (1x10\u003csup\u003e5\u003c/sup\u003e cells/mL) were treated or not with the lyophilized residue (LR), which had a lower IC\u003csub\u003e50\u003c/sub\u003e at the concentrations of 6.25, 12.5, 25.0, 50.0, and 100 \u0026micro;g/mL, and then incubated in 12-well plates for 72 hours, in a BOD oven at 26\u0026ordm;C. The number of cells was evaluated at 24, 48, and 72 hours, by direct counting in a Neubauer chamber when under an optical microscope with a 10X lens. The results were used to construct the growth curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Effects of LR on the \u003cem\u003ePhytomonas serpens\u003c/em\u003e cells\u003c/h2\u003e \u003cp\u003eThe effects of LR on the integrity of the plasma membrane, on the potential of the mitochondrial membrane, and on the production of the reactive oxygen species, were evaluated for the \u003cem\u003eP. serpens\u003c/em\u003e cells. In all of the evaluations, the promastigotes (5.10\u003csup\u003e5\u003c/sup\u003e cells/mL) were treated for 48 hours with the different concentrations of the selected lyophilized residue (LR). They were then used for the specific procedures for each assessment, as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Plasma membrane integrity\u003c/h2\u003e \u003cp\u003eThe treated cells were exposed to propidium iodide (40 \u0026micro;g/ml) for 15 minutes. The incorporation of propidium iodide, which was indicative of the loss of membrane integrity, was evaluated by flow cytometry when using an Attune\u0026reg; Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA). A total of 10,000 events were obtained and analyzed by using the Attune\u0026reg; Cytometer Software program. The incorporation of propidium iodide was also evaluated by fluorescence spectrometry when using the wavelengths of 488 nm excitation and 620 nm emission. As a negative control, the untreated cells were used, and the positive control was by the cells that were heated at 80\u0026deg;C for 10 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eAfter the treatments, the cells were washed twice in sterile phosphate buffered saline (PBS) (3000 rpm / 10 minutes / 4\u0026deg;C) and were resuspended in 100 \u0026micro;L of PBS. The cells that were treated with LR were exposed to 10 \u0026micro;g/mL of Rhodamine 123 (Rh123, Sigma-Aldrich, St. Louis, Missouri, USA) in the absence of light for 15 minutes. After the treatment period, 400 \u0026micro;L of PBS was added, and the changes in the mitochondrial membrane potential were assessed by flow cytometry by using an Attune\u0026reg; Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA).\u003c/p\u003e \u003cp\u003eA total of 10,000 events were obtained and studied when using the Attune\u0026reg; Cytometer Software program. The cells that were not treated with LR were used as a negative control, and the cells that were treated with 50 \u0026micro;M of 4-chlorophenylhydrazine carbonyl cyanide (CCCP) were used as a positive control. The variation index (VI) was determined by the equation (MTC-MUC)/MUC, in which MTC corresponded to the Rh123 fluorescence median of the cells that were treated with the LR, while MUC was the median fluorescence of the untreated cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Reactive oxygen species\u003c/h2\u003e \u003cp\u003eAfter the treatments, a volume containing 1,107 promastigotes was washed twice in sterile PBS (3000 rpm / 10 minutes / 4\u0026deg;C) and later resuspended in 100 \u0026micro;L of PBS, and then transferred to 96-well microplates. The cells that were treated with LR were exposed to 20 \u0026micro;M 2,7-dichlorodihydrofluorescein diacetate (H\u003csub\u003e2\u003c/sub\u003eDCFDA) in the absence of light for 30 minutes. The fluorescence produced by the formation of 2,7-dichlorofluorescein (DCF), as a result of the interaction with H\u003csub\u003e2\u003c/sub\u003eDCFDA with the produced ROS, was measured by spectrofluorimetry at wavelengths of 492 nm excitation and 527 nm emission when using a Synergy\u0026trade; H1 microplate reader (BioTek Hybrid Technology, Winooski, Vermont, USA). As a negative control, the untreated cells were used, and as a positive control, the cells that were treated with 750 \u0026micro;M of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) for 30 minutes were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Qualitative chemical profile of the lyophilized residue (LR)\u003c/h2\u003e \u003cp\u003eThe chromatographic exploration to obtain the qualitative chemical profile of the lyophilized residue from the accessions of the \u003cem\u003eL. camara\u003c/em\u003e collection that displayed a greater antiprotozoal activity was performed by a Shimadzu Prominence network-ready system for analytical liquid chromatography (Kyoto, Japan). This consisted of a CBM-20A communication module, a DGU-20A3 degasser, an LC-20AT binary pump system, a SIL-20AHT automatic injector, a spectrophotometric detector with an SPD-M20A diode array, and a CTO-20A column oven.\u003c/p\u003e \u003cp\u003eThe analyzed samples were prepared by dissolving 10 mg of LR in 1 mL of ultrapure water (10 mg/mL), then strained through a 0.45 \u0026micro;m syringe filter, and transferred to the analysis channel. For the separation of the chemical compounds that were present in the samples, a C18 stationary phase chromatographic column (250 x 4.6mm, 5\u0026micro;m, 100 A, Kinetex, Phenomenex, Torrance, California, USA) was used. This was connected to a guard column of the same stationary phase, with exploratory elution gradients of 5 to 100% B for 60 min, 100 to 100% B for 10 min, 100 to 5% B for 5 min, as well as with 40 minutes of conditioning the column to 5% B. The sample injection volume was 25 \u0026micro;L and the column temperature was 30\u0026deg;C. The mobile phase was formed by a binary mixture of methanol (B) and an aqueous solution of formic acid 0.5% (v/v) (A) at a flow rate of 1mL/minute. The wavelength used for the detection of the chemical compounds was 254 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Data analysis\u003c/h2\u003e \u003cp\u003eThe data was subjected to an Analysis of Variance (ANOVA), while using Tukey as a post-test, with the aid of the GraphPad Prism 7.01 program. The results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, and the differences concerning the untreated controls were considered significant when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***). All of the experiments were carried out in triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Cell viability of \u003cem\u003ePhytomonas serpens\u003c/em\u003e when exposed to the residues from the \u003cem\u003eLantana camara\u003c/em\u003e accessions\u003c/h2\u003e \u003cp\u003eInitially, a screening of the antiprotozoal activities of the lyophilized residues (LR) of the four \u003cem\u003eL. camara\u003c/em\u003e accessions on the cell viability of \u003cem\u003eP. serpens\u003c/em\u003e was carried out. The parasite cell viability decreased as the LR concentrations increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The IC\u003csub\u003e50\u003c/sub\u003e values that were obtained from the four LRs of \u003cem\u003eL. camara\u003c/em\u003e were 13.68 \u0026micro;g/mL (LR038), 17.50 \u0026micro;g/ml (LR017), 35.16 \u0026micro;g/ml (LR025), and 43.69 \u0026micro;g/ml (LR033) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Although the four LRs showed good inhibition of the parasite viability, LR038 was selected for further experiments because of its lower IC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Growth curve of \u003cem\u003ePhytomonas serpens\u003c/em\u003e when treated with selected residue\u003c/h2\u003e \u003cp\u003eLR038 affected the proliferation of \u003cem\u003eP. serpens\u003c/em\u003e at all of the concentrations evaluated, and the concentrations of 50 \u0026micro;g/ml and 100 \u0026micro;g/m of LR038 markedly reduced the cell density of the promastigote forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After 24 hours, the concentration of 50\u0026micro;g/mL reduced 47% of the cell growth and the concentration of 100\u0026micro;g/mL inhibited 59%. After 48 hours, the same concentrations reduced 52% and 76% of the growth of promastigotes, respectively. After 72 hours, there was a 65% reduction in the cell density at the 25 \u0026micro;g/mL concentration of LR038, and an 84% and 95% inhibition was observed at the 50 \u0026micro;g/mL and 100 \u0026micro;g/mL concentrations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Evaluation of the cell membrane integrity of the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes\u003c/h2\u003e \u003cp\u003eTo determine the integrity of the membrane, a high molecular weight DNA intercalant that did not penetrate the cell with an intact membrane was used with the fluorescent probe, propidium iodide (PI). The flow cytometry and spectrofluorimetric analysis demonstrated that LR038 at the concentrations evaluated did not affect the integrity of the plasma membrane (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Evaluation of the reactive oxygen species production of the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes\u003c/h2\u003e \u003cp\u003eThe production of the reactive oxygen species in the \u003cem\u003eP. serpens\u003c/em\u003e promastigotes that were treated with 60, 120, and 180 \u0026micro;l/mL of LR038 was evaluated by using the fluorescent probe, H\u003csub\u003e2\u003c/sub\u003eDCFDA. It was observed that at the two highest concentrations, there was a significant increase in the accumulation of the ROS when compared with the untreated cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The accumulation of the ROS was approximately 2x and 3x at the concentrations of 120 \u0026micro;l/mL and 180 \u0026micro;l/mL, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Evaluation of the mitochondrial potential of the \u003cem\u003ePhytomonas serpens\u003c/em\u003e promastigotes\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eP. serpens\u003c/em\u003e promastigotes that were treated with 60, 120, and 180 \u0026micro;g/mL of LR038 were stained with Rhodamine 123 and evaluated by flow cytometry, to verify the effects of the lyophilized residue. The cells that were treated with 120 and 180 \u0026micro;l/ml of LR038 showed a reduction in the mitochondrial potential when compared with the untreated cells. The Rhodamine 123 total fluorescence histogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and the index of the variation results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) showed a reduction in the fluorescence at concentrations of 120\u0026micro;l/mL and 180\u0026micro;l/mL, respectively. These two concentrations reduced the fluorescence by 44.6% (IV= -0.446) and 46.8% (IV= -0.468), respectively. These reductions did not differ statistically from the reduction of fluorescence in the positive control, 56.0% (IV= -0.560). These results were confirmed by the statistical analysis of the median of fluorescence, which was obtained and compared with the negative control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Qualitative chemical profile of the residue.\u003c/h2\u003e \u003cp\u003eThe qualitative chemical profile of LR038 was determined by analytical liquid chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The presence of 17 peaks and their respective ultraviolet spectra were observed, which were used to identify the classes of the secondary metabolites that were present in LR038. The results of this qualitative analysis indicated the presence of phenolic acids and flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global essential oils market had a turnover of 18.62\u0026nbsp;billion US dollars in 2020, and strong growth is estimated for the next few years, reaching a value of 33.26\u0026nbsp;billion US dollars by 2027, with estimated sales of 473 .31 thousand tons of essential oils (Grand View Research, 2020). Although there is a great demand for these products, the essential oil yield from many plant species is almost always low (Alighiri et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, millions of tons of waste are generated during production and they are disposed of into the environment, in some cases, without treatment (Peshev, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although these residues are discarded, they have many biologically active molecules, which exhibit diverse biological activities. The residue from the hydrodistillation of \u003cem\u003eDittrichia graveolens\u003c/em\u003e, with a high content of flavonoids, presented antioxidant, antibacterial, and anti-inflammatory activities, with the advantage of not being cytotoxic to the fibroblasts (Gharred et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003ePhytomonas\u003c/em\u003e, belonging to the Trypanosomatidae family, has pathogenic members, which are parasites that cause diseases in plants and humans. Some species of this genus cause severe infections in large crops, such as acute lethal wilt in oil palm and coconut trees (\u003cem\u003ePhytomonas staheli\u003c/em\u003e), necrosis in coffee phloem (\u003cem\u003ePhytomonas leptovasorum\u003c/em\u003e), and root bubbling in cassava (\u003cem\u003ePhytomonas fran\u0026ccedil;ai\u003c/em\u003e) (Jaskowska et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite not causing a severe systemic disease, \u003cem\u003eP. serpens\u003c/em\u003e can compromise the commercial value of tomato plants, due to the appearance of yellow spots on the parasitized fruits (Schwelm et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, this species constitutes an important biological model, as it presents natural characteristics similar to other pathogenic members belonging to the Trypanosomatidae family. In addition, unlike other pathogenic species, \u003cem\u003eP. serpens\u003c/em\u003e is readily isolated and cultivated in the laboratory, enabling in vitro studies in the most diverse areas.\u003c/p\u003e \u003cp\u003eStudies have proven that compounds from medicinal plants have important molecules, with antimicrobial activities on the promastigote forms of \u003cem\u003eP. serpens\u003c/em\u003e. \u003cem\u003ePiper crassinervium\u003c/em\u003e and \u003cem\u003eP. amalago\u003c/em\u003e leaf extracts have inhibited the \u003cem\u003eP. serpens\u003c/em\u003e proliferation in vitro, with low to moderate cytotoxicity in mammalian cells (Cancini et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Silva et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that the use of the essential oil of \u003cem\u003eVarronia curassavica\u003c/em\u003e inhibited the growth of \u003cem\u003eP. serpens\u003c/em\u003e, causing a loss of the cell membrane integrity. Pereira et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed an inhibitory effect of \u003cem\u003eL. camara\u003c/em\u003e essential oil on the promastigote forms of this same species. Thus, this study aimed to evaluate the effect of residual water from the hydrodistillation process of \u003cem\u003eL. camara\u003c/em\u003e leaves, as a source of natural compounds for the control of protozoa of the genus \u003cem\u003ePhytomonas\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this work, it was possible to demonstrate the antiprotozoal actions of the residues from the production chain of essential oils from four accessions of \u003cem\u003eL. camara\u003c/em\u003e on \u003cem\u003eP. serpens\u003c/em\u003e. The results have shown that the four samples of the lyophilized residues evaluated showed antiprotozoal activities at low concentrations, with the IC\u003csub\u003e50\u003c/sub\u003e value ranging from 13.68 to 43.69 \u0026micro;g/mL. Although the four LRs showed a promising inhibition of parasite viability, LR038 was selected for having a lower IC\u003csub\u003e50\u003c/sub\u003e value.\u003c/p\u003e \u003cp\u003eFrom the qualitative chemical profile of the LR038 sample, two classes of natural products were detected, the phenolic acids and flavonoids, which can be attributed to the detected trypanocidal activities. Although the molecules from these classes have known antiprotozoal activities, there are no reports on the activity of these molecules against \u003cem\u003eP. serpens\u003c/em\u003e. Caffeic acid, belonging to the phenolic acid class, is a good representative to demonstrate the antiprotozoal capacity of this group of compounds. Silva Bortoleti et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that caffeic acid could inhibit the proliferation of the \u003cem\u003eLeishmania amazonensis\u003c/em\u003e promastigotes, and cause changes in the cell morphology through the loss of plasma membrane integrity, as well as with the accumulation of the levels of the reactive oxygen species, thus increasing the oxidative response. Flavonoids also have antiprotozoal activities, like quercetin and myricetin, which have shown a trypanocidal activity on \u003cem\u003eTrypanosoma brucei\u003c/em\u003e, the etymological agent of African human trypanosomiasis (Larit et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince it was shown that LR038 markedly decreased the viability of \u003cem\u003eP. serpens\u003c/em\u003e at low concentrations, the effects of this residue on the cell membrane integrity, on the intracellular levels of the reactive oxygen species, and on the mitochondrial membrane potential of the \u003cem\u003eP. serpens\u003c/em\u003e promastigotes, were determined.\u003c/p\u003e \u003cp\u003eOne of the possible mechanisms of action of natural products on cell viability includes their effects on the integrity of the plasma membrane. The evaluation that was based on the permeability of propidium iodide showed that the treatment with LR038 did not alter the integrity of the plasma membrane of the \u003cem\u003eP. serpens\u003c/em\u003e promastigotes. This might be due to the low liposolubility of LR038, as molecules typically require that the hydrophobic groups interact directly with the membrane (Jesus et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The hydroxyl (polar) groups that were present in the flavonoids and phenolic acids likely entered the cell through controlled endocytosis.\u003c/p\u003e \u003cp\u003eThe biological activities of flavonoids are mainly related to their antioxidant effects. However, several studies have also demonstrated the pro-oxidant action of many molecules of this class of natural products. A good example is quercetin, which is capable of inducing the reactive oxygen species in human embryonic stem cells (hESCs), thus triggering selective cell deaths (Kim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Another representative of the flavonoid class with an oxidizing action is cynaroside, which inhibits the growth of the \u003cem\u003eLeishmania donovani\u003c/em\u003e promastigotes, by inducing the production of ROS (Tabrez et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ROS levels in the \u003cem\u003eP. serpens\u003c/em\u003e promastigotes that were treated with LR038 were determined by using H\u003csub\u003e2\u003c/sub\u003eDCFDA, a non-fluorescent probe that when permeating the cell, is converted into dichlorodihydrofluorescein (DCFH) by the action of the esterases. In the presence of free radicals, such as ROS, DCFH is oxidized and converted to 2,7-dichlorofluorescein (DCF), a highly fluorescent product. The results obtained revealed that an intracellular increase in the ROS was accompanied by an increase in the concentration of LR038. Notably, the levels of ROS that were detected in the parasites that were treated with 120 and 180 ug/mL of the residues were approximately 2x and 3x higher than in the untreated parasites.\u003c/p\u003e \u003cp\u003eReactive oxygen species result from cellular metabolism, and their levels are regulated by the presence of an endogenous antioxidant complex that when affected, can lead to an intracellular accumulation of ROS. The consequent oxidative stress generated might cause metabolic changes, including a loss of the mitochondrial membrane potential (Fonseca-Silva, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Mittler, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, reductions of 44.6% and 46.8% in the Rhodamine-generated fluorescence were observed in the \u003cem\u003eP. serpens\u003c/em\u003e promastigotes that were treated with 120 and 180 \u0026micro;g/mL of LR038, respectively. This reduction in the fluorescence demonstrated a decrease in the mitochondrial membrane potential that was generated by LR038, which might be related to the increase in ROS that was observed in the cells that were treated with this residue.\u003c/p\u003e \u003cp\u003eSince trypanosomatids have a single mitochondrion, the proper functioning of this organelle is essential for the survival of these organisms, hence constituting an important target for the construction of drugs (Menna-Barreto and Castro, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Fidalgo and Gille, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A study by Bomba\u0026ccedil;a et al. pointed out that the mitochondrial dysfunction and the production of ROS were the main determinants of the antiprotozoal activity of the naphthoimidazoles in \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e (Bomba\u0026ccedil;a et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, the apigenin flavonoid has been shown to exert antileishmanicidal activity by altering the membrane potential, as the result of an increase in the production of ROS (Fonseca-Silva et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen considering the results that were obtained here, the antiprotozoal activities that were presented by the residues of the hydrodistillation of the essential oil of \u003cem\u003eL. camara\u003c/em\u003e were due to its ability to induce oxidative stress. This was evidenced by the increase in the ROS levels and the functional alterations in the mitochondria, which interfered with the production of energy that compromised the parasite's survival.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe residues from the hydrodistillation of the \u003cem\u003eL. camara\u003c/em\u003e leaves can serve as a source of biologically active compounds, phenolic acids, and flavonoids, with an antiprotozoal potential. The highest concentrations tested promoted changes in the potential of the mitochondrial membrane and caused the accumulation of the reactive oxygen species. This demonstrated the effectiveness of the residues that were generated during the hydrodistillation of \u003cem\u003eL. camara\u003c/em\u003e leaves to obtain the essential oils.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6.1. Ethical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2. Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3. Consent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.4. Authors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaroline Alves Soares:\u003c/strong\u003e Conceptualization, Data curation, Methodology, Roles/Writing - original draft.\u0026nbsp;\u003cstrong\u003eTam\u0026iacute;ris Aparecida de Carvalho Santos:\u003c/strong\u003e Conceptualization, Methodology.\u0026nbsp;\u003cstrong\u003eLu\u0026iacute;s Fernando de Andrade Nascimento:\u003c/strong\u003e Conceptualization, Methodology.\u0026nbsp;\u003cstrong\u003eRaphael Amancio de Jesus:\u003c/strong\u003e Conceptualization, Methodology.\u0026nbsp;\u003cstrong\u003eArie Fitzgerald Blank:\u003c/strong\u003e Conceptualization; Writing - Review \u0026amp; Editing; Funding acquisition.\u0026nbsp;\u003cstrong\u003eRicardo Scher:\u003c/strong\u003e Conceptualization, Methodology.\u0026nbsp;\u003cstrong\u003eVal\u0026eacute;ria Regina de Souza Moraes:\u003c/strong\u003e Conceptualization, Methodology.\u0026nbsp;\u003cstrong\u003eRoberta Pereira Miranda Fernandes:\u003c/strong\u003e Conceptualization, Methodology, Writing - Review \u0026amp; Editing.\u0026nbsp;\u003cstrong\u003eMaria de F\u0026aacute;tima Arrigoni-Blank:\u003c/strong\u003e Conceptualization, Writing - Review \u0026amp; Editing; Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.5. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded, in part, by Brazil\u0026rsquo;s Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), the Funda\u0026ccedil;\u0026atilde;o de Apoio \u0026agrave; Pesquisa e a Inova\u0026ccedil;\u0026atilde;o Tecnol\u0026oacute;gica do Estado de Sergipe (Fapitec/SE), the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES, Finance Code 001), and the Financiadora de Estudos e Projetos (FINEP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6.6. Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6.3. Author contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaroline Alves Soares:\u003c/strong\u003e Conceptualization, Data curation, Methodology, Roles/Writing - original draft. \u003cstrong\u003eTam\u0026iacute;ris Aparecida de Carvalho Santos:\u003c/strong\u003e Conceptualization, Methodology. \u003cstrong\u003eLu\u0026iacute;s Fernando de Andrade Nascimento:\u003c/strong\u003e Conceptualization, Methodology. \u003cstrong\u003eRaphael Amancio de Jesus:\u003c/strong\u003e Conceptualization, Methodology. \u003cstrong\u003eArie Fitzgerald Blank:\u003c/strong\u003e Conceptualization; Writing - Review \u0026amp; Editing; Funding acquisition. \u003cstrong\u003eRicardo Scher:\u003c/strong\u003e Conceptualization, Methodology. \u003cstrong\u003eVal\u0026eacute;ria Regina de Souza Moraes:\u003c/strong\u003e Conceptualization, Methodology. \u003cstrong\u003eRoberta Pereira Miranda Fernandes:\u003c/strong\u003e Conceptualization, Methodology, Writing - Review \u0026amp; Editing. \u003cstrong\u003eMaria de F\u0026aacute;tima Arrigoni-Blank:\u003c/strong\u003e Conceptualization, Writing - Review \u0026amp; Editing; Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e7. Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Trypanosomatids Collection of the Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro, Brazil for providing \u003cem\u003eP. serpens\u003c/em\u003e isolate (strain 9T).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Hakeim HK, Al-Zabibah, RS, Alzihari HF, Almensoori AK, Al-Zubaidi HA, Hassan LEAH (2021) Anticancer and antiangiogenic activities of alkaloids isolated from \u003cem\u003eLantana camara\u003c/em\u003e by adsorption on the magnetic nanoparticles. Karbala Int. J. Mod. Sci 7(1), e11. https://doi.org/10.33640/2405-609X.2577\u003c/li\u003e\n\u003cli\u003eAlighiri D, Eden WT, Supardi KI, Purwinarko A (2017) Potential development of essential oil production of Central Java, Indonesia. J. Phys.: Conf. Ser 824, 12-21. https://doi.org/10.1088/1742-6596/824/1/012021\u003c/li\u003e\n\u003cli\u003eAyalew AA (2020) Insecticidal activity of the \u003cem\u003eLantana camara\u003c/em\u003e extract oil on controlling maize grain weevils. Toxicology Research and Application 4, 1-10. https://doi.org/10.1177/2397847320906491\u003c/li\u003e\n\u003cli\u003eBashir S, Jabeen K, Iqbal S, Javed S, Naeem A (2019) \u003cem\u003eLantana camara\u003c/em\u003e: phytochemical analysis and antifungal prospective. Planta Daninha 37, e019193526. http://dx.doi.org/10.1590/s0100-83582019370100137\u003c/li\u003e\n\u003cli\u003eBomba\u0026ccedil;a ACS, Viana PG, Santos AC, Silva TL, Rodrigues ABM, Guimar\u0026atilde;es ACR, Menna-Barreto RF (2019) Mitochondrial disfunction and ROS production are essential for anti-\u003cem\u003eTrypanosoma cruzi\u003c/em\u003e activity of \u0026beta;-lapachone-derived naphthoimidazoles. Free Radic. Biol. Med 130, 408-418. https://doi.org/10.1016/j.freeradbiomed.2018.11.012\u003c/li\u003e\n\u003cli\u003eBordoloi K, Bhagawati B, Baruah AM, Neog PP, Kurulkar U (2021) Biochemical mechanism of \u003cem\u003eLantana camara\u003c/em\u003e leaf extracts in the management of \u003cem\u003eMeloidogyne incognita\u003c/em\u003e on tomato. J. Pharmacogn. Phytochem 10(1), 2828-2834. https://doi.org/10.22271/phyto.2021.v10.i1an.13789\u003c/li\u003e\n\u003cli\u003eCancini NDS, Beraldo-Borrazzo J, Menezes JLD, Cortez DAG, Valdez RH, Garcia FP, Abreu Filho BAD (2020) Activity of Piperaceae extracts and fractions in the control of \u003cem\u003ePhytomonas serpens.\u003c/em\u003e Cienc. Rural 50(10), e20200343. https://doi.org/10.1590/0103-8478cr20200343\u003c/li\u003e\n\u003cli\u003eDelgado-Altamirano R, L\u0026oacute;pez-Palma RI, Monzote L, Delgado-Dom\u0026iacute;nguez J, Becker I, Rivero-Cruz JF, Rojas-Molina A (2019) Chemical constituents with leishmanicidal activity from a pink-yellow cultivar of \u003cem\u003eLantana camara \u003c/em\u003evar. \u003cem\u003eaculeata \u003c/em\u003e(L.) collected in Central Mexico. Int. J. Mol. Sci 20(4), e872. https://doi.org/10.3390/ijms20040872\u003c/li\u003e\n\u003cli\u003eDougnon G, Ito M (2020) Sedative effects of the essential oil from the leaves of \u003cem\u003eLantana camara\u003c/em\u003e occurring in the Republic of Benin via inhalation in mice. J. Nat. Med 74(1), 159-169. https://doi.org/10.1007/s11418-019-01358-9\u003c/li\u003e\n\u003cli\u003eFidalgo LM, Gille L (2011) Mitochondria and trypanosomatids: targets and drugs. Pharm. Res 28(11), e2758. https://doi.org/10.1007/s11095-011-0586-3 \u003c/li\u003e\n\u003cli\u003eFonseca-Silva F, Canto-Cavalheiro MM, Menna-Barreto RFS, Almeida-Amaral EE (2015) Effect of apigenin on \u003cem\u003eLeishmania amazonensis\u003c/em\u003e is associated with reactive oxygen species production followed by mitochondrial dysfunction. J. Nat. Prod 78(4), 880-884. https://doi.org/10.1021/acs.jnatprod.5b00011 \u003c/li\u003e\n\u003cli\u003eGharred N, Baaka N, Bettache N, Hamdi A, Dbeibia A, Dhaouadi H, Dridi-Dhaouadi S (2021) Wastewater to Ecological dyeing process and bioactive compounds resources: a case study of \u003cem\u003eDittrichia graveolens\u003c/em\u003e hydrodistillation aqueous residue. Waste Biomass Valorization 12, 5065-5077. https://doi.org/10.1007/s12649-021-01375-4\u003c/li\u003e\n\u003cli\u003eGindri DM, Coelho CMM (2020) Metabolitos aleloqu\u0026iacute;micos de \u003cem\u003eLantana camara\u003c/em\u003e L.: potencial para o desenvolvimento de bioherbicida - revis\u0026atilde;o. Revista T\u0026eacute;cnico-Cient\u0026iacute;fica do CREA-PR 24, e7. Accessed: 22/02/21 \u0026lt;http://creaprw16.creapr.org.br/revista/Sistema/index.php/revista/article/view/595\u0026gt;\u003c/li\u003e\n\u003cli\u003eGrand View Research, 2020\u003cstrong\u003e \u003c/strong\u003eEssential oils market size, share \u0026amp; trends analysis report by application (food \u0026amp; beverages, spa \u0026amp; relaxation), by product (orange, peppermint), by sales channel, and segment forecasts, 2020-2027. Accessed: 22/02/21\u003cstrong\u003e \u0026lt;\u003c/strong\u003ehttps://www.grandviewresearch.com/industry-analysis/essential-oils-market\u0026gt;\u003c/li\u003e\n\u003cli\u003eJaskowska E, Butler C, Preston G, Kelly S (2015) Phytomonas: trypanosomatids adapted to plant environments. PLoS Pathog 11(1), e1004484. https://doi.org/10.1371/journal.ppat.1004484 \u003c/li\u003e\n\u003cli\u003eJesus JA, Laurenti MD, Antonangelo L, Faria CS, Lago JHG, Passero LFD (2021) Related pentacyclic triterpenes have immunomodulatory activity in chronic experimental visceral leishmaniasis. J. Immunol. Res 2021, e6671287. https://doi.org/10.1155/2021/6671287\u003c/li\u003e\n\u003cli\u003eKim SY, Jeong HC, Hong SK, Lee MO, Cho SJ, Cha HJ (2017) Quercetin-induced ROS production triggers mitochondrial cell death of human embryonic stem cells. Oncotarget 8(39), 64964-64973. https://doi.org/10.18632/oncotarget.11070 \u003c/li\u003e\n\u003cli\u003eLarit F, Elokely KM, Nael MA, Benyahia S, Le\u0026oacute;n F, Cutler SJ, Ghoneim MM (2021) Proposed mechanism for the antitrypanosomal activity of quercetin and myricetin isolated from \u003cem\u003eHypericum afrum\u003c/em\u003e Lam.: phytochemistry, \u003cem\u003ein vitro\u003c/em\u003e testing and modeling studies. Molecules 26(4), e1009. https://doi.org/10.3390/molecules26041009\u003c/li\u003e\n\u003cli\u003eMahmoudi H, Marzouki M, M\u0026apos;rabet Y, Mezni M, Ouazzou AA, Hosni K (2020) Enzyme pretreatment improves the recovery of bioactive phytochemicals from sweet basil (\u003cem\u003eOcimum basilicum\u003c/em\u003e L.) leaves and their hydrodistilled residue by-products, and potentiates their biological activities. Arab. J. Chem 13(8), 6451-6460. https://doi.org/10.1016/j.arabjc.2020.06.003\u003c/li\u003e\n\u003cli\u003eMaslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Luke\u0026scaron; J, Yurchenko V (2019) Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy, and evolution. Parasitology 146(1), 1-27. https://doi.org/10.1017/S0031182018000951\u003c/li\u003e\n\u003cli\u003eMenna-Barreto RFS, Castro SL (2014) The double-edged sword in pathogenic Trypanosomatids: the pivotal role of mitochondria in oxidative stress and bioenergetics. Biomed Res. Int 2014 e614014. https://doi.org/10.1155/2014/614014\u003c/li\u003e\n\u003cli\u003eMittler R (2017) ROS are good. Trends Plant Sci\u003cstrong\u003e \u003c/strong\u003e22(1), 11-19. https://doi.org/10.1016/j.tplants.2016.08.002\u003c/li\u003e\n\u003cli\u003eNapoli E, Dattilo S, Ruberto G (2020) Hydrodistillation of \u003cem\u003eTrachelospermum jasminoides\u003c/em\u003e Lindl. flowers. An analysis of essential oil, hydrolate, and polyphenols content of the process wastes. J. Essent. Oil Res 32(6), 556-561. https://doi.org/10.1080/10412905.2020.1808538\u003c/li\u003e\n\u003cli\u003eNegi GC, Sharma S, Vishvakarma SC, Samant SS, Maikhuri RK, Prasad RC, Palni LM (2019) Ecologia e uso de \u003cem\u003eLantana camara\u003c/em\u003e na \u0026Iacute;ndia. The Bot. Rev\u003cstrong\u003e \u003c/strong\u003e85(2), 109-130. https://doi.org/10.1007/s12229-019-09209-8\u003c/li\u003e\n\u003cli\u003eNirmal CR, Ebenezer RS, Kannan P, Balasubramanian M, Thirunavukkarasu I, Mondal R, Dusthackeer A (2020) Anti-tuberculosis activity of bioactive compounds from \u003cem\u003eLantana camara\u003c/em\u003e L., \u003cem\u003eEuphorbia hirta\u003c/em\u003e L., \u003cem\u003eMukia maderaspatana\u003c/em\u003e (L.) M. Roem, and \u003cem\u003eAbutilon indicum\u003c/em\u003e (L.). Eur. J. Integr. Med\u003cstrong\u003e \u003c/strong\u003e35, 101-105. https://doi.org/10.1016/j.eujim.2020.101105\u003c/li\u003e\n\u003cli\u003ePereira KLG, Nizio DAC, Lima PCN, Fernandes RPM, Arrigoni-Blank MF, S\u0026aacute; Filho JCF, Nascimento LFA, SouzaVT (2022) Seasonal variance in the chemical composition of essential oils from \u003cem\u003eLantana camara\u003c/em\u003e accessions and their tripanocida do \u0026oacute;leo essencial sobre \u003cem\u003ePhytomonas serpens\u003c/em\u003e. Bol. Latinoam. Caribe Plantas Med. Aromat 21(4)\u003c/li\u003e\n\u003cli\u003ePereira KLG, Nogueira PCL, Arrigoni-Blank MF, Nizio DAC, Silva DC, Pinto JAO, Blank AF (2020) Chemical diversity of essential oils of \u003cem\u003eLantana camara\u003c/em\u003e L. native populations. J. Essent. Oil Res 32(1), 32-47. https://doi.org/10.1080/10412905.2019.1661879\u003c/li\u003e\n\u003cli\u003ePeshev D (2020) Theoretical assessment of the use of nanofiltration for fractionation of waste aqueous fractions from the essential oil industry. Bulg. Chem. Commun 52(4), 533-542. https://doi.org/10.34049/bcc.52.4.MP05 \u003c/li\u003e\n\u003cli\u003eQureshi H, Anwar T, Ali Q, Haider MZ, Habib N, Fatima S, Waseem M, Bibi Y, Arshad M, Adkins SW (2019) Isolation of the natural herbicidal compound from \u003cem\u003eLantana camara\u003c/em\u003e. Int. J. Environ. Anal. Chem\u003cstrong\u003e \u003c/strong\u003e101(5), 631-638. https://doi.org/10.1080/03067319.2019.1670822\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Vioque R, Izquierdo-Melero ME, Polissiou M, Astraka K, Tarantilis PA, Herraiz-Pe\u0026ntilde;alver D, Santana-M\u0026eacute;ridas O (2015) Comparative chemistry and biological properties of the solid residues from hydrodistillation of Spanish populations of \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L. Grasas Aceites 66(2), e079. https://doi.org/10.3989/gya.1060142\u003c/li\u003e\n\u003cli\u003eSankara A, Ou\u0026eacute;draogo JCW, Pignolet L, Th\u0026eacute;venon MF, Bonzi-Coulibaly Y (2020) Chemical profiles and anti-termite activity of hydrodistillation residues from three aromatic plants acclimated in Burkina Faso. J. Agric. Sci 12(8), 245-256. https://doi.org/10.5539/jas.v12n8p245\u003c/li\u003e\n\u003cli\u003eSantos J\u0026uacute;nior ADCM, Ricart CAO, Pontes AH, Fontes W, Souza ARD, Castro MS, Lima BD (2018) Proteome analysis of \u003cem\u003ePhytomonas serpens\u003c/em\u003e, a phytoparasite of medical interest. PloS One 13(10), e0204818. https://doi.org/10.1371/journal.pone.0204818\u003c/li\u003e\n\u003cli\u003eSchwelm A, Badst\u0026ouml;ber J, Bulman S, Desoignies N, Etemadi M, Falloon RE, Neuhauser S (2018) Not in your usual Top 10: protists that infect plants and algae. Mol. Plant Pathol 19(4), 1029-1044. https://Doi.Org/10.1111/Mpp.12580\u003c/li\u003e\n\u003cli\u003eShankar S, Prasad S, Owaiz M, Yadav S, Manhas S, Yaqoob M (2021) Essential oils, components, and their applications: a review. Plant Arch\u003cstrong\u003e \u003c/strong\u003e21(1), 2027-2033. https://doi.org/10.51470/PLANTARCHIVES.2021.v21.S1.331\u003c/li\u003e\n\u003cli\u003eSilva Bortoleti BT, Tomiotto-Pellissier F, Gon\u0026ccedil;alves MD, Miranda-Sapla MM, Assolini JP, Carloto AC, Pavanelli WR (2019) Caffeic acid has antipromastigote activity by the apoptosis-like process, and anti-amastigote by TNF-\u0026alpha;/ROS/NO production and decreased of iron availability. Phytomedicine 57, 262-270. https://doi.org/10.1016/j.phymed.2018.12.035\u003c/li\u003e\n\u003cli\u003eSilva KP, Carvalho Santos TA, Moutinho BL, Silva RS, Pinto VS, Blank AF, Fernandes RPM (2019) Using \u003cem\u003eVarronia curassavica\u003c/em\u003e (Cordiaceae) essential oil for the biocontrol of \u003cem\u003ePhytomonas serpens\u003c/em\u003e. Ind. Crops Prod 139, e111523. https://doi.org/10.1016/j.indcrop.2019.111523\u003c/li\u003e\n\u003cli\u003eTabrez S, Rahman F, Ali R, Alouffi AS, Akand SK, Alshehri BM, Rub A (2021) Cynaroside inhibits \u003cem\u003eLeishmania donovani\u003c/em\u003e UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response. Biosci. Rep 41(1), eBSR20203857. https://doi.org/10.1042/BSR20203857\u003c/li\u003e\n\u003cli\u003eWollinger A, Perrin E, Chahboun J, Jeannot V, Touraud D, Kunz W (2016) Antioxidant activity of hydro distillation water residues from \u003cem\u003eRosmarinus officinalis\u003c/em\u003e L. leaves determined by DPPH assays. C. R. Chim 19(6), 754-765. https://doi.org/10.1016/j.crci.2015.12.014\u003c/li\u003e\n\u003cli\u003eWu P, Song Z, Wang X, Li Y, Cui J, Tuerhongc M, Da-Qing J, Abudukeremuc M, Leed D, Xu J, Guo Y (2020) Bioactive triterpenoids from \u003cem\u003eLantana camara\u003c/em\u003e showing anti-inflammatory activities in vitro and in vivo. Bioorg. Chem 101, e104004. https://doi.org/10.1016/j.bioorg.2020.104004 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Residues from distillation, Biological control, Medicinal plants, Mechanism of action, Reactive oxygen species, Mitochondrial membrane potential","lastPublishedDoi":"10.21203/rs.3.rs-4092579/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4092579/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOn an industrial scale, the residues accumulated in essential oil distilleries can be compared to the volume of residues produced in a textile industry. Although these residues are discarded, they possess molecules with diverse biological activities, including their application in phytopathogen control. In this study, the chemical profile of the residue from the hydrodistillation of \u003cem\u003eLantana camara\u003c/em\u003e L. leaves was determined using High-Performance Liquid Chromatography (HPLC). Additionally, the effect of the residue on cells was assessed by determining plasma membrane integrity, levels of reactive oxygen species (ROS) production, and mitochondrial potential depolarization. The viability and cell density of \u003cem\u003ePhytomonas serpens\u003c/em\u003e parasites significantly decreased after treatment with increasing concentrations of the lyophilized residue. RL038, the lyophilized residue from accession LAC-038, reduced cell viability by an average of 61.36%. ROS levels increased by approximately 2x and 3x at RL038 concentrations of 120 \u0026micro;g/mL and 180 \u0026micro;g/mL, respectively. It was observed that the same concentrations modified mitochondrial potential, reducing fluorescence by 44.6% and 46.8%, respectively. Analytical liquid chromatography of RL038 revealed the presence of 17 peaks subsequently classified as phenolic acids and flavonoids. RL038 from the hydrodistillation of \u003cem\u003eLantana camara\u003c/em\u003e L. leaves is a source of biologically active compounds with antiprotozoal potential.\u003c/p\u003e","manuscriptTitle":"Anti-Phytomonas activity of the lyophilized residues obtained from the distillation of Lantana camara L. essential oil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 06:42:29","doi":"10.21203/rs.3.rs-4092579/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-08-25T05:05:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-13T07:59:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-30T09:11:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-03-21T17:27:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-19T04:23:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-03-14T12:01:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"568b8376-bd05-4f0e-b477-4614db1296c5","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:04:49+00:00","versionOfRecord":{"articleIdentity":"rs-4092579","link":"https://doi.org/10.1007/s11356-024-35142-4","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-09-26 15:57:48","publishedOnDateReadable":"September 26th, 2024"},"versionCreatedAt":"2024-04-03 06:42:29","video":"","vorDoi":"10.1007/s11356-024-35142-4","vorDoiUrl":"https://doi.org/10.1007/s11356-024-35142-4","workflowStages":[]},"version":"v1","identity":"rs-4092579","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4092579","identity":"rs-4092579","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}