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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 67  |  Page : 140-147  

Cytotoxicity, phagocytic activity, and leishmanicidal potential of extract standardized in geranylgeraniol obtained from the fruit of Pterodon emarginatus vogel


1 Faculty of Pharmacy, Natural Products Research Laboratory, Federal University of Goiás; Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goiás, Anápolis, Go, Brazil
2 Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goiás; Institute of Tropical Pathology and Public Health, Anápolis, Go, Brazil
3 Faculty of Pharmacy, Natural Products Research Laboratory, Federal University of Goiás, Anápolis, Go, Brazil
4 Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goiás; Faculty of Pharmacy Laboratory of Pharmacology and Cell Toxicology – Farmatec, Federal University of Goiás, Anápolis, Go, Brazil
5 Faculty of Pharmacy Laboratory of Pharmacology and Cell Toxicology – Farmatec, Federal University of Goiás, Anápolis, Go, Brazil
6 Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goiás; Pharmacy Course, University Unit of Exact and Technological Sciences, State University of Goiás, Anápolis, Go, Brazil
7 Replicon Research Center, Department of Biology, Pontifical Catholic University of Goiás, Goiânia, Brazil
8 Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goiás, Anápolis, Go, Brazil

Date of Submission29-May-2019
Date of Decision27-Jun-2019
Date of Web Publication11-Feb-2020

Correspondence Address:
Suzana Ferreira Alves
Pharmacy Course, School of Medical Sciences, Pharmaceuticals and Biomedical Sciences, Pontifical Catholic University of Goias
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_231_19

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   Abstract 


Aim: The propouse of this study was to available the cytotoxicity, phagocytic activity, and leishmanicidal potential of extract standardized from the fruits of Pterodon emarginatus Vogel. Background: P. emarginatus is a Brazilian medicinal plant with several constituents shown to have anti-inflammatory, antinociceptive, antimicrobial, and antiproliferative activities. Materials and Methods: The standardized extract in geranylgeraniol obtained from the fruit of P. emarginatus was investigated for clastogenic and cytotoxic effects, as well as phagocytic activity against Leishmania guyanensis. Results: A 24-h exposure of Allium cepa roots to standardized geranylgeraniol extract (9.77 mg/mL) promoted clastogenic effects, with a mitotic index (MI) of 10.33 ± 0.61. Extending the exposure time to 48 or 72 h significantly reduced the MI (P < 0.05). The IC50for geranylgeraniol in 3T3 and RAW 264.7 cells was found to be 15.5 mg/mL and 12.2 mg/mL, respectively. The standardized extract in geranylgeraniol exhibited high phagocytic and microbicidal activities in Leishmania, promoting a >60% increase in macrophage infection, with greater internalization and destruction of the parasite. In silico study results suggest that geranylgeraniol acts in Leishmania by interfering with the biosynthesis of sterols through steric hindrance of the active site of the enzyme CYP51. Conclusion: The standardized extract in geranylgeraniol may be useful as an antiproliferative agent, macrophage immunomodulator, and as a cytotoxic treatment against infectious agents.

Keywords: Biological safety, clastogenic effects, docking, geranylgeraniol, phagocytic activity, potential leishmanicide


How to cite this article:
Alves SF, Gomes CM, de Oliveira MG, de Andrade WM, Moreira LC, Borges LL, da Silva CC, de Souza GO, da Silva VB, Valadares MC, Freitas Bara MT. Cytotoxicity, phagocytic activity, and leishmanicidal potential of extract standardized in geranylgeraniol obtained from the fruit of Pterodon emarginatus vogel. Phcog Mag 2020;16:140-7

How to cite this URL:
Alves SF, Gomes CM, de Oliveira MG, de Andrade WM, Moreira LC, Borges LL, da Silva CC, de Souza GO, da Silva VB, Valadares MC, Freitas Bara MT. Cytotoxicity, phagocytic activity, and leishmanicidal potential of extract standardized in geranylgeraniol obtained from the fruit of Pterodon emarginatus vogel. Phcog Mag [serial online] 2020 [cited 2020 Jul 16];16:140-7. Available from: http://www.phcog.com/text.asp?2020/16/67/140/278003






   Introduction Top


Pterodon emarginatus Vogel (family Fabaceae), popularly known as “sucupira-branca” or “faveiro,” is found in the Cerrado region of Brazil. Having proven anti-inflammatory and analgesic properties,[1],[2],[3],[4],[5],[6] its oil resin and fruit extract are used in folk medicine for the treatment of rheumatism, sore throats, and respiratory disorders (bronchitis and tonsillitis). Studies have ascribed cercaricide, antimicrobial and anti-inflammatory,[7],[8],[9] analgesic,[10] antinociceptive,[8],[11],[12],[13] and anticancer properties [14],[15],[16] to the oil and fruit extracts of P. emarginatus.

The compounds involved in the antinociceptive and anticancer activities were isolated from Pterodon genus and identified as geranylgeraniol, 6α-acetoxy, 7α-hydroxylvouacapan, 6α,7α-di-hydroxy-vouacapan-17α-oate methyl ester, and its isomers 6α-hydroxy-7α-acetoxy-vouacapan-17α-oate methyl ester, and 6α-acetoxy-7α-hydroxy-vouacapan-17α-oate methyl ester. [11,17] Geranylgeraniol (3, 7, 11, 15-tetramethyl- 2, 6, 10, 14-hexadecatraen-ol) [Figure 1] is an acyclic diterpene that has shown antinociceptive activity in capsaicin and glutamate animal models, and the likely pharmacological mechanisms involved have been described.[18],[19] In addition, geranylgeraniol inhibits the proliferation of the amastigote form of the parasite Trypanosoma cruzi at concentrations that did not affect cell viability in the mammalian host.[20]
Figure 1: Chemical structure of geranylgeraniol (acyclic diterpene)

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In vitro studies of the essential oil extracted from the fruit of P. emarginatus have investigated its cytotoxic and antiproliferative activities, as well as its activity against the protozoan Leishmania.[21],[22] However, these studies were not performed using standardized extracts of geranylgeraniol. Proper evaluation of the toxic properties of plant extracts is important for safety considerations since exposure to complex chemicals can have adverse effects in humans.[23] Thus, toxicity studies of the diverse bioactivity of compounds found in the Pterodon genus are needed.

In a study of the potential clastogenic effects of oil extracted from the seeds of Pterodon pubescens, Dias et al.[24] reported no chromosomal abnormalities. Sabino et al.[25] also observed no mutagenic or toxic effects of this seed oil in the cells of humans or other animals. Similar toxicity studies of the standardized geranylgeraniol extracts of P. emarginatus fruit are needed. Accordingly, this study aims to assess the cytotoxic potential of standardized extracts of geranylgeraniol in 3T3-A31 fibroblasts and RAW macrophage cells. In addition, the mutagenic and clastogenic effects of geranylgeraniol are investigated in meristematic cells of Allium cepa and its possible biological sites of action are predicted in silico.


   Materials and Methods Top


Cell culture

BALB/c 3T3-A31 fibroblasts were kindly donated by Dr. Mari Cleide Sogayar from the Chemistry Institute, São Paulo University, Brazil. These cells were cultured in Dulbecco's Modified Eagle Medium® medium containing inactivated fetal bovine serum (FBS) (10%), glutamine (1%), erythromycin, and streptomycin at 37°C in a humidified atmosphere containing 5% CO2 in the air. RAW 264.7 cells were donated by Dr. Milton Adriano Pelli Oliveira from the Institute of Tropical Pathology and Public Health, Federal University of Goiás (UFG), Brazil. These cells were grown in RPMI-1640 (Sigma Chemical Co., St Louis, MO, USA) supplemented with 10% FBS (Cripion, São Paulo, Brazil). Cells were inactivated by incubation at 56°C for 30 min in RPMI containing 2 mM L-glutamine (Sigma Chemical Co.), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma-Aldrich), and 2 mM Hepes (Sigma-Aldrich). Cells (2 × 105) were grown in 2 mL of RPMI in a well of 6-well culture plates (Costar, Nova York, USA) in a 37°C incubator at 5% CO2.

Plant material and standardized geranylgeraniol extract

The fruit of P. emarginatus was collected in the city of Campestre in the state of Goiás, Brazil (S 16° 46' 01“; W 49° 42' 06”; 612 m). A voucher specimen (UFG-41714) was deposited at the UFG herbarium. The crushed fruits were subjected to filtration using 95% alcohol as the solvent. After obtaining the crude alcoholic extract, it was concentrated in a rotary evaporator (Buchi R-220 SE), under reduced pressure (30 rpm, 40°C, −600 bar). The concentrated extract was standardized with respect to geranylgeraniol using the standard method for quantification by high-performance liquid chromatography (HPLC) using a Waters model HPLC Alliance with separation module e2695, photodiode array detector (PDA) 2998, and data-processing system Enpower 2.0. Chromatographic separations were conducted using the Zorbax Eclipse column XDB C18(5 mm, 250 × 4.6 mm, Agilent) at a wavelength of 210 nm, elution mode gradient, with the mobile phase consisting of acetonitrile acidified with phosphoric acid 0.2% (v/v) (A), methanol (B), and phosphoric acid 0.5% (v/v) in water (C) [Table 1]. The mobile phase flow rate was 1 mL/min, injection volume was 10 mL, and the column heater was maintained at a temperature of 30°C. The external standard of geranylgeraniol (purity ≥98.5%, Sigma-Aldrich) and the concentrated extract were diluted with methanol and filtered through a Millex filter (Millipore, Billerica, MA, USA).
Table 1: Elution model gradient system

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Mutagenicity and clastogenicity test

A. cepa bulbs were exposed to geranylgeraniol at concentrations of 3.24, 6.48, 9.77, 12.95, and 16.19 mg/mL for 24, 48, or 72 h. Control tests were carried out using Milli-Q water. After reaching 1.5 cm in length, the geranylgeraniol-exposed A. cepa roots were collected and fixed in 3:1 (v/v) ethanol/glacial acetic acid (Carnoy's solution). Microscope slides were prepared by carefully squashing the material, as described by Matsumoto et al.[26] Meristems previously fixed in Carnoy's solution were washed with distilled water in three baths of 5 min each. The material was then hydrolyzed in HCl 1 M at 60°C for 11 min, followed by three baths of 5 min in distilled water. The slides were stained with acetic carmine (2%) and later covered with coverslips. One thousand cells were observed by optical microscopy, noting those that had undergone any type of change. The mitotic index (MI) was calculated according to:



We also calculated the average number of changes at each concentration for exposures of 24, 48, and 72 h. Disorders of mitosis, including c-metaphases, chromosome bridges, sticky and lagging chromosomes, “budding” nuclei, nucleoli partly outside nuclei, micronuclei, and binucleated cells, were also investigated using previously described methods.[27],[28] Statistical analysis was performed using ANOVA followed by posttests (for group comparisons, Tukey's test) to a significance level of 5% (P < 0.05) using the Statistical Analysis System software version 7.0. (SAS Institute Inc., North Carolina, USA).

Neutral red uptake assay

The viability of 3T3 cells (3 × 104 cells/mL) after treatment with nine concentrations of standardized extract in geranylgeraniol (0.39–100 μg/mL) for 48 h was evaluated by measuring the uptake of neutral red by lysosomes in viable cells. Cell suspensions (3 × 104 cells/mL) were distributed in 96-well plates (TPP, Trasadingen, Switzerland) and incubated at 37°C at 5% CO2 for 24 h. Next, the cells were exposed to 50 mL of the standardized extract at a concentration of 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, or 100 mg/mL and incubated for 48 h under the same conditions. The adhered cells were exposed to a 250 mL of a solution containing 5% FBS (Cultilab), 1% neutral red dye at 0.3% (Sigma-Aldrich), and culture medium DMEM-1640 (Sigma, St. Louis, MO, USA) for 3 h at 37°C in an oven with CO2 at 5%. The solution containing neutral red was removed, and the cells were washed with 250 mL of PBS at 37°C, resuspended in 100 mL of a solution of 1% glacial acetic acid (Vetec, Rio de Janeiro, RJ, Brazil), 50% ethanol (Vetec, Rio de Janeiro, RJ, Brazil), and 49% water, and incubated for 20 min. The absorbance of each sample at 540 nm was determined using an ELISA plate reader (Start fax 2100, Awareness Technology, Dusseldorf, Germany).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay

This assay was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction test as previously described by Mosmann [29] and modified by Mota et al.[30] Briefly, the RAW 264.7 cells (1 × 106 cells/mL) were plated in triplicate in 96-well plates in RPMI-1640 medium supplemented with 10% FBS and treated with one of 9 concentrations of standardized geranylgeraniol extract (0.39–100 μg/mL). After 48 h of incubation, 10 mL of MTT (5 mg/mL) was added to each well and the plates were incubated for an additional 3 h. The blue MTT formed a precipitate, which was then dissolved in Dimethyl sulfoxide solution (DMSO). The absorbance was measured using a spectrophotometer (Start fax 2100, Awareness Technology, Dusseldorf, Germany) at 550 nm. Cell viability is expressed as a percentage of the control, and IC50 values (concentration that inhibited cell growth in 50% relative to untreated group) were obtained.

Phagocytic activity

The RAW 264.7 macrophages cells were counted in a Neubauer chamber, plated at 1 × 106 cells per well on glass coverslips in 24-well culture plates, and infected with 5 × 105 metacyclic forms of Leishmania guyanensis (M4147) and also counted in a Neubauer chamber. The negative control contained cells, the parasite and RPMI; the positive control contained cells, parasite, 1 ng/mL interferon-g (IFN-g) and 20 ng/mL lipopolysaccharide (LPS). Test 1 (T1) contained cells, the parasite and 10 mL of the standardized extract (6.25 mg/mL) in RPMI and test 2 (T2) contained cells, the parasite, 1 ng/mL IFN-g, 20 ng/mL LPS and 10 mL of the standardized extract (6.25 mg/mL) in RPMI. After exposure, the plates were incubated at 37°C at 5% CO2 for 1 h or 48 h. After incubation, the coverslips were removed from the wells and immediately stained using Kit Instant Prov (Newprov, Pinhais, PR, Brazil) and then fixed on microscope slides. Under optical microscopy (magnification, ×1000), 300 cells were observed and the number of parasites in the infected cells was counted. The phagocytic activity was determined as the percentage of infected cells versus the average number of parasites per infected cell.

Prediction of biological activity spectra

Bioactivity prediction based on the two-dimensional (2D) structure of geranylgeraniol was performed using PASS [31] and SwissTargetPrediction [32] tools, which operate based on the assumption that bioactive compounds with similar structures may similar biological activity. The top-ranked activities indicated by each tool were selected for analysis.

Molecular docking

Docking simulations using geranylgeraniol and Leishmania infantum sterol 14-alpha demethylase (CYP51), originally complexed with fluconazole (PDB code: 3L4D), were performed using the DockThor server.[33] The calculations were performed inside a sphere with a 5 Š radius centered at the N2 atom of the fluconazole-complexed ligand. The advanced options were maintained as default. The docked orientations were clustered according to the root-mean-square deviation for the analysis.


   Results and Discussion Top


Standardized extract in geranylgeraniol

The alcoholic extract of the fruit of P. emarginatus yielded a total solid content of 32.39 g/100 g of extract. Thus, after quantification of geranylgeraniol by HPLC [Figure 2], the average content of geranylgeraniol in the extract was determined to be 5.55% (w/w). The content of geranylgeraniol in the standardized extract is 1.80 g/100 g of extract. Menna-Barreto et al.[20] determined that geranylgeraniol is the major component of the oil of P. pubescens. Isolation and characterization studies of the oil and extracts from fruits of species of the genus Pterodon have identified geranylgeraniol as a component of relevance for genus identity and monitoring.[11],[17],[19] The question of dosage represents a major obstacle to ensure the safety and efficacy of medicinal herbs, even in the case of plants with confirmed biological activities. A small dose may not have any effect, whereas a higher dose may be dangerous to the organism.[34] Thus, a standardized extract allows for greater biological safety and obtaining an analytical quality profile of the pharmacological activities reported for the vegetal extract.
Figure 2: Two-dimensional chromatographic profile at 210 nm. (a) Geranylgeraniol standard (Sigma-Aldrich), with integration, at 19.65 min. (b) Alcoholic extract marker geranylgeraniol identified and standardized, with integration, at 19.73 min. High-performance liquid chromatography-photodiode array (m, 210 nm); column, C18Zorbax (250 mm × 4.6 mm × 5 mm). Mobile phase in a gradient system; flow rate, 1.0 mL/min; and column temperature, 30°C. Empower 2.0 software

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Allium cepa test

Data on the frequency of metaphase and anaphase abnormalities in 1000 interphase cells and the MI for each concentration of geranylgeraniol extract tested are shown in [Figure 3]. In the five concentrations tested, no micronuclei were observed, indicating the absence of mutagenicity.
Figure 3: The mean percentage and standard deviation of the mitotic index for the control group (Milli-Q water) and the groups treated with standardized geranylgeraniol extract at five concentrations (mg/mL) for different times (24, 48, and 72 h). ANOVA factorial followed by posttest of grouping (Tukey's test) to a significance level of 5% (P < 0.05). *Compared to control (P < 0.05)

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The interaction of concentration and time exerts a significant influence on the number of abnormalities observed during metaphase and anaphase (P < 0.05), as observed at concentrations of 9.77 mg/mL and 16.19 mg/mL, for which there were significant differences in MI between exposure times at the same concentration [Figure 3].

After exposure to standardized extract in geranylgeraniol, meristematic cells of the roots were observed for chromosomal abnormalities such as chromosomal fragments, chromosomal bridges, delayed chromosomes, and polyploidy [Figure 4].
Figure 4: Photomicrograph of chromosomal abnormalities observed after analysis of Allium cepa meristematic cells exposed to standardized extracts of geranylgeraniol at 24, 48, and 72 h. Chromosome fragments (a), chromosomal bridges (b), straggler chromosomes (c), and polyploid cells (d). Images obtained in optical photomicroscope (objective, 100×)

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The cytogenetic changes observed suggest that bioactive components of the extract interact with proteins that control the mitotic spindle, resulting in delays in chromosomal migration. The appearance of micronuclei is the consequence of chromosomal breakdown, demonstrating disturbances of the mitotic process [35],[36] that did not occur after treatment with standardized extracts. The MI and replication index are indicators of cell proliferation.[37] The MI differenced significantly between exposure times for cells treated with 9.77 mg/mL and 16.19 mg/mL standardized geranylgeraniol extracts (P < 0.05) [Figure 3]. The MI for both concentrations was higher after treatment for 24 h than that after 48 or 72 h; at 16.19 mg/mL, the MI after treatment for 72 h was lower than that after 24 or 48 h.

The MI increased in a concentration-dependent manner [Table 2], showing that the standardized geranylgeraniol extract induces cell proliferation with 24 h of exposure. However, after 48 and 72 h of exposure, the MI decreased significantly (P < 0.05), indicating that the time of exposure may influence the antiproliferative activity of A. cepa, as observed by Peron et al.[38] using extracts of Hypericum perforatum. According to Lloret et al.,[17] geranylgeraniol is a precursor of vouacapanic derivatives, among them 6α-hydroxy-7α-acetoxyvouacapan, which plays an important role in inhibiting cell growth in cell lines such as cancer PC-03. In this same study, the concentration of geranylgeraniol was inversely proportional to the content of vouacapans, which may explain the lag in the antiproliferative effect observed in our study after 48 and 72 h, as indicated by the significant decrease in MI.
Table 2: Frequency of clastogenic events expressed as the mitotic index in metaphase and anaphase in 1000 cells at interphase at 24, 48, and 72 h

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Neutral red uptake assay

A decline was observed in the viability of 3T3 basal cells after treatment with all nine concentrations of standardized geranylgeraniol extract (0.39–100 μg/mL) for 48 h in a concentration-dependent manner. The IC50 value was 15.5 μg/mL [Figure 5].
Figure 5: Cytotoxic effects of standardized geranylgeraniol extract in 3T3 basal cells. The cells (3 × 104 cells/mL) were treated with nine concentrations of geranylgeraniol (0.39–100 μg/mL) for 48 h, and cell viability was evaluated by neutral red uptake assay. Results represent the mean ± standard deviation of three independent experiments using six replicates

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The neutral red assay is a biological safety assay that evaluates cell viability after exposure to chemicals capable of causing cytotoxicity. The geranylgeraniol compound treats an acyclic diterpene precursor of other vouacapan compounds that were evaluated for cytotoxicity in 3T3 cells, giving the following results: 6α-acetoxy-7β-hydroxyvouacapan, IC50, 34.33 μg/mL; 6α,7β-dihydroxyvouacapan-17β-oate methyl ester, IC50, 22.83 μg/mL; and 6α,7β-dihydroxyvouacapan-17β-methylene-ol, IC50, 23.55 μg/mL; and exhibited pronounced cytotoxic activity (antitumor/antiproliferative activity) in the following cancer cells in vitro: UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-3 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia), and NCI-ADR/RES (ovarian expressing multidrug-resistant phenotype).[15] Although the geranylgeraniol compound has a low IC50, suggesting cytotoxicity, its antinociceptive, anti-inflammatory, and antitumor properties already observed in oils and extracts of various species of the genus Pterodon[9],[12],[14],[16],[18],[19] also make it promising for treating numerous pathological conditions. The oil extracted from the fruit of P. emarginatus, which is known to have diterpene compounds such as geranylgeraniol, demonstrated cytotoxic activity against cancer cell lines (line C6) (IC50, 24.9 μg/mL) but had no effect on peripheral blood mononuclear cells (PBMCs), which demonstrated a viability of 94 ± 1.5%, 93 ± 3.5%, and 95 ± 3.3%, after 12, 24, and 48 h of treatment at 100 μg/mL, respectively.[22]

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay

A concentration-dependent decline in the viability of RAW 264.7 cells was observed after treatment with nine concentrations of standardized geranylgeraniol extract (0.39–100 μg/mL) for 48 h. The IC50 value was 12.2 μg/mL [Figure 6].
Figure 6: Cytotoxic effects of standardized geranylgeraniol extract in RAW 264.7 cells (macrophages). The cells (3 × 104 cells/mL) were treated with nine concentrations of extract (0.39–100 μg/mL) for 48 h and the cell viability evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Results are presented as the mean ± standard deviation of three independent experiments using six replicates

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The viability of RAW murine cells (macrophages) as assessed by MTT assay was dose-dependent, with an IC50 of 12.2 μg/mL. This IC50 was higher than that observed by Sabino et al.[25] for human PBMC treated with oil extracted from P. pubescens after 24 (2 μg/mL) and 48 (1 μg/mL) h. These authors did not consider the material evaluated as cytotoxic for PBMC. This result is consistent with that present study, as no dead cells were observed after treatment with standardized extract (6.25 μg/mL). Determination of the geranylgeraniol IC50 in RAW 264.7 cells was essential for evaluating phagocytic activity, clarifying the optimal concentration for verifying the microbicidal potential of the standardized extract geranylgeraniol. We determined this concentration to be 6.25 μg/mL against L. guyanensis, an important parasite of murine macrophages. Our study showed that geranylgeraniol has a high phagocytic capacity and microbicidal potential against the experimental model of L. guyanensis, corroborating the findings of Menna-Barreto et al.[20] using a similar infection model in which geranylgeraniol inhibited the proliferation of intracellular amastigote forms of T. cruzi. The treatment of macrophages infected with the amastigote forms of T. cruzi with 2 and 6 μg/mL of geranylgeraniol showed a decrease in the dose and time dependence of infection and a decrease in the number of internalized parasites. The mechanism underlying the geranylgeraniol-induced induction of phagocytosis involves the rupture of the mitochondrial membrane in trypomastigotes and epimastigotes.

Phagocytic activity

[Figure 7] shows the number of parasites in all cells counted for each treatment (a), the percentage of infected macrophages (b), the phagocytic activity of the macrophages (c), and the relationship of parasites by infected macrophages (d). During the 24 or 48 h of the experiment, endocytosis of parasites should be observed. However, this did not occur in this study because RAW 264.7 cells are known to release reactive oxygen and nitrogen during this time period, killing the parasite. In light of this observation, we evaluated the phagocytic and microbicidal activity during the 1st hour after treatment and compared the results to those of positive controls treated with INF-γ and LPS. We observed that treatment with the standardized geranylgeraniol extract inhibited the action of INF-g and LPS, as shown in [Figure 7].
Figure 7: Index of phagocytosis and microbicidal activity (%) of mononuclear phagocytes (RAW 264.7 cells) against Leishmania guyanensis exposed to the standardized geranylgeraniol extract for 1 h. CT, negative control; γ + lipopolysaccharide, positive control containing cells, parasite, 1 ng/mL interferon-γ, and 20 ng/mL lipopolysaccharide; E + G, test 1 (T1) containing cells, parasite, 10 mL standardized extract (6.25 mg/mL); γ + lipopolysaccharide + (E + G), test 2 (T2) containing cells, parasite, 1 ng/mL interferon-γ, 20 ng/mL lipopolysaccharide, and 10 mL standardized extract (6.25 mg/mL). (a) Phagocity activity; (b) % infection macrophages; (c ) Phagocity Index; (d) Number de parasites infective por macrophages. The data are presented as mean standard deviation of three experiments performed in triplicate were compared for significance by Student's test or ANOVA followed by Bonferroni test using the GraphPad Prism software 5.0 (Inc. San Diego, CA, USA). P < 0.05 was considered significant

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Because of the similarity between the model used in the present study and that of Menna-Barreto et al.[20] including the geranylgeraniol concentration and the use of parasites of the same family, it is likely that the pronounced phagocytic activity observed in RAW 264.7 cells is related to the treatment with geranylgeraniol. The 1-h period for evaluation of phagocytic activity was considered adequate since it corresponds to the macrophage activation period observed by Dias et al.[39] who showed that the maximum activation of macrophages in the peritoneal cavity of Brycon amazonicus inoculated with Saccharomyces cerevisiae yeast occurred during a 2-h study period, allowing determination of the phagocytic index.

Cardoso et al.[40] observed that the ethanolic extract from the seeds of P. pubescens inhibits nitric oxide (NO) production in vivo, suggesting that it affects macrophage function. Because NO is one of the main microbicidal agents produced by macrophages, this observation suggests that the extract would promote a decrease in phagocytic activity, a prediction not supported by the findings of our present study. However, a study reporting that NO synthase inhibition in macrophages contributes to anti-inflammatory and immunosuppressive actions [41] is consistent with findings of studies of the anti-inflammatory activity of P. emarginatus extracts and oils.[4],[8],[9] Several compounds of vegetal origin exhibit immunomodulatory activities,[42],[43] including the phagocytosis of bacteria by mononuclear phagocytes, a microbicidal activity stimulated by immunomodulatory agents.[44] Immunomodulatory compounds capable of modifying immune system responses are considered alternative treatments for infections.

Biological activity prediction in silico and docking of geranylgeraniol

The PASS and SwissTargetPrediction tools were used to predict the bioactivity of geranylgeraniol. This analysis revealed that it may have anti-inflammatory and antineoplastic activities, regulate lipid metabolism, and interfere with enzymes involved in sterol biosynthesis. The inhibition of farnesyltranstransferase and 2,3-oxidosqualene-lanosterol cyclase was suggested by the PASS tool, and the inhibition of squalene monooxygenase was suggested by the SwissTargetPrediction tool.

Docking calculations for geranylgeraniol within the Leishmania CYP51 active site revealed that 20% of the orientations obtained were capable of interacting directly with the heme group through hydrogen bonds between the hydroxyl group of geranylgeraniol and the heme propionate moiety. Another hydrogen bond was observed with TYR102. In addition, van der Waals interactions were observed between geranylgeraniol and Val 212, Met 459, Phe 104, Phe 109, Tyr 115, and Thr 294 [Figure 8].
Figure 8: (a) Docking 3D model of geranylgeraniol (carbon atoms in yellow) within the Leishmania CYP51 active site, highlighting the main intermolecular interactions with the heme group and amino acid residues. (b) Two-dimensional diagram of interaction between geranylgeraniol and the Leishmania CYP51 heme group and amino acid residues

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The hexane and butanol fractions obtained from oil extracted from P. emarginatus fruit exhibited activity against promastigote forms of Leishmania amazonensis (hexane fraction IC50, 50.06 mg/mL; butanol fraction IC50, 46.65 mg/mL). These fractions had no activity against Leishmania chagasi.[21] This observation suggests that further studies to evaluate the leishmanicidal potential of standardized derivatives and substances isolated P. emarginatus extracts would be worthwhile.

Because the sterol biosynthesis pathway is vital to Leishmania, it is an important source of antimicrobial targets.[45] Inhibition of farnesyltranstransferase, a target of bisphosphonates, may interfere with the production of substrates for enzymes catalyzing the first committed step in sterol biosynthesis. Another target in this pathway is squalene monooxygenase, a known target of the allylamines. Squalene monooxygenase participates in the conversion of the 30-carbon chain squalene to a tetracyclic sterol skeleton.[45],[46]

Geranylgeraniol may also be an inhibitor of 2,3-oxidosqualene-lanosterol cyclase, which catalyzes the cyclization of 2,3-oxidosqualene to lanosterol. This enzyme has been confirmed as a therapeutic target against Trypanosomatidae.[46] Thus, the findings obtained in our bioactivity prediction analysis indicate that geranylgeraniol might interfere with enzymes involved in the Leishmania sterol biosynthesis pathway.

Sterol 14-alpha demethylase (CYP51) is a key enzyme of the sterol biosynthesis pathway. This enzyme catalyzes the removal of the C-14 alpha-methyl group from sterol precursors, a reaction essential for membrane biogenesis.[47] This enzyme was selected for molecular docking simulations to determine whether geranylgeraniol also can interact with this Leishmania target.

The 3D structure of L. infantum CYP51 is 95% similar to four Leishmania species, qualifying it as a suitable molecule for predictive docking in other Leishmania species. Docking simulations revealed that geranylgeraniol is accommodated by the CYP51 active site and interacts with it both electrostatically and hydrophobically. The hydrogen bonding between geranylgeraniol and the enzyme active site is of interest because the formation of the fluconazole/CYP51 complex is reported to involve hydrogen bonding.[47]

In addition, the hydrophobic pocket in the active site offers a favorable binding region for the geranylgeraniol alkyl chain. These hydrophobic interactions may be important for the binding affinity since the natural substrate of this enzyme is also a lipophilic molecule.[47],[48]


   Conclusion Top


That standardized geranylgeraniol extract increases cell division in A. cepa radicles after 24 h of exposure; further exposure results in antiproliferative effects. The preliminary cytotoxicity study revealed a low IC50 for geranylgeraniol in 3T3 and RAW 264.7 cells and cytotoxic effects in the parasite L. guyanensis. This result explains the pronounced phagocytic and microbicidal potential in treated cells, suggesting that geranylgeraniol has an important immunomodulatory activity in macrophages. In silico findings support the hypothesis that the effects of geranylgeraniol on Leishmania involve interference with enzymes of the sterol biosynthesis pathway. The present study indicates that the standardized geranylgeraniol extract from P. emarginatus might be useful as an immunomodulator and microbicide against Leishmania species.

Acknowledgements

We are thankful to all the sponsors of this research: Goiás State Research Support Foundation; Institute of Tropical Pathology and Public Health at the Federal University of Goiás; Replicon Research Center of the Pontifical Catholic University of Goiás; and Cellular Pharmacology and Toxicology Laboratory - Farmatec, Federal University of Goiás.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
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