Genotoxic and cytotoxic activities of hexane extract in seeds from Platonia insignis

Platonia insignis Mart. is a vegetable of the Clusiaceae Lindl., family that is popularly known as “bacuri” in Brazil. It is widely used in folk medicine to treat human diarrheas and inflammatory diseases. It has antiparasitic activity, antioxidant, anticarcinogenic action, in addition to vasorelaxant potential. The present study investigates the leishmanicidal, cytotoxic and genotoxic activities in different biological systems, as well as the chemical constituents of hexane extract from P. insignis seeds. The analysis of the non-volatile constituents was analyzed by GC/MS. A total of sixteen main constituents were detected in the saponifiable material (fatty acid methyl esters, ethyl ester, alcohols and hydrocarbons). In unsaponifiable fraction, nineteen constituents were detected, including a series of nalkanes and prenylated xanthones gamma-mangostin as main component (70.23% of the total components). The results indicated that the seed hexane extract significantly inhibits the growth of promastigote forms of Leishmania amazonensis (IC50), displayed a significant toxicity against Artemia salina Leach and cytotoxicity and genotoxicity in V79 cell lines.


Introduction
Medicinal plants play a prominent role in the health system around the world, this involves the use of medicinal plants not only for the treatment of diseases, but also as a potential material for the maintenance of good health conditions. used in medicines to maintain physical, mental and social health (Devi et al., 2021;Garg et al., 2021). However, despite the therapeutic advantages of medicinal plants, some of their chemical constituents can be potentially toxic, mutagenic, carcinogenic or teratogenic (Doroftei et al., 2019). Studies have shown that many plants have considered toxicity (Campos et al., 2016;Matos et al., 2011). Therefore, it is important to screen medicinal plants for their cytogenotoxic potential, since genotoxicity is one of the main causes of the onset and development of many types of cancer (Schultz et al., 2021).
Platonia insignis Mart., (Clusiaceae) commonly known as "bacuri", is a native species of the Brazilian Amazonia region composed of pulp, shell and seeds (Monteiro et al., 1997). The fruits of P. insignis can be consumed raw or as juice, ice cream or jam. While the seeds from P. insignis is used to treat various skin diseases in both humans and animals, the seed decoction is used to treat human diarrhea and inflammatory diseases (Agra et al., 2007). The ethanolic extract demonstrated a strong protective effect on the central nervous system and garcinielliptone, isolated from the hexane extract seeds, an antioxidant action (Costa Júnior et al., 2010;Costa Júnior et al., 2011a).
The xanthones are the major secondary metabolites found in the seeds of the bacuri fruits (Costa Júnior et al., 2011b).
A broad range of biological activities of xanthones have been reported extensively, especially cytotoxicity on a variety of cancer cells, e.g. human breast cancer, colorectal cancer, hepatoma, leukaemia, and small cell lung cancer (Pedraza-Chaverri et al., 2008;Wang et al., 2011). These cytotoxicities were found to be associated with anti-proliferative and apoptotic effects.
There are no studies reporting the cytotoxicity of P. insignis seed extract. In view of the popular use and the absence of knowledge about the cytotoxic potential of P. insignis, the aim of this work was to identify the chemical constituents of P. insignis seeds hexane extract by GC/MS, and its toxic effects by employing three biological models: Leishmania amazonensis, Artemia salina Leach, and permanent mammalian fibroblast cell line derived from Chinese hamsters (V79 cells). The toxicity was determined in L. amazonensis (leishmanicidal activity) and A. salina. The V79 cells were employed to measure the cytotoxic effects by MTT assay and genotoxicity by using the standard comet assay.

Sample extraction
The fruits of P. insignis were collected from Barras, Piauí State, Brazil in March 2009. A voucher specimen was identified and deposited at the Herbarium of the Biology Department of the Federal University of Piauí, Brazil (Voucher No. ICN TEPB27164). The seeds were dried at 55ºC and powdered in a Soxhlet apparatus, the powder (848.2 g) was extracted with hexane (63%, w/w). The hexane extract (hexane. Ext.) was subjected to saponification followed by methylation reaction (Hartman & Lago, 1973) and a small portion (F. saponifiable and F.unsaponifiable) of the crude reaction was analyzed in GC/MS (Gas Chromatography coupled to the detector by Mass Spectrometry).

Gas chromatography/mass spectrometry (GC/MS) analysis
The saponifiable and unsaponifiable fractions from P. insignis seeds were analyzed by gas chromatography to determine the fatty acid composition. The fractions 10µl were analyzed using a Shimadzu GC-17A/MS QP5050A (GC/MS system) and a DB-5HT capillary column (30 m x 0.251 mm, 0.1 m film thickness). The experimental conditions used were: carrier gas; helium 1.7 mL/min; column inlet pressure = 107.8 kPa; column flow = 1.7 mL/min; linear velocity = 47.3 cm/sec; total flow = 24 mL/min; carrier flow = 24 mL/min; injector temperature = 280 o C; detector temperature = 300 o C; and column temperature = 80 (1 min) -300 o C at 10 o C/min (15 min). The ionization energy used for the mass spectrometer was 70 eV. The mass spectra were recorded from 40 -650 m/z. The quantity of all identified components was investigated by using a percent relative peak area. The tentative identification of the compounds was performed based on the comparison of their relative retention time and mass spectra with those of the WILEY229 library data of the GC/MS system. Spectra were considered coincident if the similarity index was higher than 90%.
Amphotericin B was used as a control. After 48 h, parasites were collected, fixed in an isotonic solution (10.5 g citric acid, 7.0 g NaCl, 5.0 mL formalin and 1,000 mL distilled water) and examined using light microscopy. The inhibitory effect of the fraction on cellular growth was estimated by cell counting using a Neubauer chamber. The concentration that inhibited 50% of the growth (IC50) was determined by regression linear analysis (Oliveira- Silva et al., 2008).

Toxicity against A. salina Leach.
The brine shrimp assay is a safe, practical, and economical method to determine the bioactivity of natural compounds.
The brine shrimp lethality bioassay was performed following the reported procedure (Meyer et al., 1982). The growth medium was prepared with filtered seawater in a small tank divided into two compartments. Shrimp eggs were added to the covered compartment, and a lamp was placed above the open side of the tank to attract hatched shrimps through perforations in the partition wall. After 48 h, the shrimps matured as nauplii (A. salina Leach.) and were ready for the assay. Stock solutions of the extracts of P. insignis seeds were prepared in dimethylsulfoxide (DMSO) and seawater and filtered (the final concentration of DMSO in the mixture never exceeded 0.2% v/v). Appropriate volumes of stock solutions were then added to the tubes containing seawater and 10 nauplii each. Four different extract concentrations were applied in triplicate to each tube. After 24 h of incubation under light, the numbers of dead and surviving brine shrimps were counted in each tube. The LC50 values were calculated from the graphics of drug concentration versus lethality percentage using a probit adjust scale.

V79 cell culture and treatment
Chinese hamster lung fibroblast cells (V79 cells) were cultured under standard conditions in DMEM supplemented with 10% heat-inactivated-FBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin and 100 g/mL streptomycin. Cells were kept in tissue-culture flasks at 37°C in a humidified atmosphere containing 5% CO2 in air and were harvested by treatment with 0.15% trypsin-0.08% EDTA in PBS. Cells (5 x 10 5 cells) were seeded in the medium and grown for one day prior to treatment with hexane extract. The hexane extract of P. insignis seeds was dissolved in DMSO and added to the FBS-free medium to give various concentrations (10, 25, 50 and 100 g/mL). The cells were treated for 2 h under standard conditions. The appropriate concentrations were obtained by dilution of stock solution in sterile distilled water and the final concentration of DMSO in the incubation mixture never exceeded 0.2% (v/v).

MTT assay
The MTT assay was performed according to Denizot &Lang (1986). After treatments, cells were briefly washed with PBS. A serum-free medium (0.15 mL) containing a yellow dye of a tetrazolium salt (MTT; 1 mg/mL) was then added and the mixture was incubated for 3 h at 37 °C. After incubation, the supernatant was removed. The residual purple formazan product was solubilized in 0.2 mL DMSO and stirred for 15 min. The absorbance of the product was measured at 570 nm. The absorbance of the negative control (solvent) was considered as corresponding to the viability of 100%, and the values of treated cells were calculated as a percentage of the negative control (solvent).

Comet assay V79 cell culture
An alkaline comet assay was performed as described by Hartmann & Speit (1997) and Tice et al. (2000) with minor changes. At the end of the treatment, the cells were washed with ice-cold PBS and trypsinized with 100 L of trypsin (0.15%).
Immediately thereafter, 20 L of the cell suspension was dissolved in 0.75% low-melting-point agarose (Hartmann et al., 2001) and spread on regular pre-coated agarose-point (1%) microscope slides. Cells were ice-cold lysed (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, pH 10.0, with freshly added 1% Triton X-100 and 10% DMSO) at 4 °C for at least 1 h in order to remove cellular proteins and membranes, with DNA remaining as "nucleoids." The slides were then placed in a horizontal electrophoresis box containing a freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH ~13.0) at 4 °C for 20 min to allow DNA unwinding. A 300 mA and 25 V (0.90 V/cm) electric current were applied for 20 min to perform DNA electrophoresis. All of the above steps were performed under yellow light or in the dark to prevent additional DNA damage.
The slides were then neutralized (0.4 M Tris, pH 7.5), stained with silver nitrate as described by Nadin et al. (2001), and analyzed using an optical microscope. One hundred cells (50 cells from each of two replicate slides of each organ or tissue) were selected and analyzed for DNA migration. When selecting cells, edges and cells around air bubbles were discarded (Tice et al., 2000). Cells were visually scored according to tail length into five classes: (1) class 0: undamaged with no tail; (2) class 1: tail shorter than the diameter of the head (nucleus); (3) class 2: tail as long as 1-2x the diameter of the head; (4) class 3: tail longer than 2x the diameter of the head; and (5) class 4: comets with no heads. Visual scoring of comets is considered a valid evaluation method in international guidelines and recommendations for the comet assay (Burlinson et al., 2007;Hartmann & Speit, 1997;Tice et al., 2000). The damage index (DI) is an arbitrary score calculated for cells in different damage classes that were visually scored by measuring the DNA migration length and the amount of DNA in the tail. DI was thus assigned to each comet according to its class and ranged from 0 (Completely undamaged: 100 cells×0) to 400 (With maximum damage: 100 cells×4). Damage frequency (DF), which is the proportion of cells presenting tails after electrophoresis, was also considered in our study. The DF (%) was calculated as the number of cells with tails versus those without (0-100%) (Hartmann & Speit, 1997).

Statistical analysis
All experiments were independently repeated at least three times, with triplicate samples for each treatment. The experimental results were expressed as mean and standard deviation (S.D.). Data were analyzed by one-way analysis of variance (ANOVA) and the means were compared using the Tukey test. A P value lower than 0.05 was considered to be statistically significant. Data analysis was performed with SPSS 10.0 for Windows and GraphPad Prism version 4.00, GraphPad Software (San Diego, USA).

In vitro leishmanicidal activity
The P. insignis seeds hexane extract strongly inhibited the growth of the promastigote form of L. amazonensis. After 48 h of incubation, the IC50 value for the hexane was 3.49µg/mL. The IC50 value for amphotericin B was 0.04 μg/mL (Table   2).

Toxicity against Artemia salina Leach.
According to McLaughlin (1991), compounds with IC50 < 1.000 ppm in the brine shrimp lethality assay are considered active and potentially cytotoxic against cell lines. The hexane. Ext. presented IC50 of 130.9 µg/mL and considered toxicity against brine shrimps ( Table 2). The control samples with solvents (seawater and DMSO) did not lead to significant brine shrimp mortality.

V79 cells cytotoxicity measured by an MTT assay
The MTT assay was used for evaluation of the cytotoxicity of the P. insignis seeds hexane extract in V79 cells and was shown in Figure 1. At low tested concentrations, the hexane extract did not induce significant cytotoxic effects. At concentrations up to 100 µg/mL the hexane extract was cytotoxic with a significant decrease in survival. Values shown are the mean and standard deviation at least three determinations. ***Significant difference as compared to the negative control (solvent; DMSO 0.2%) at P < 0.001/One-way ANOVA Tukey's multiple comparison test. Source: Authors.

V79 cell genotoxicity by comet assay
The induction of genotoxicity by P. insignis in V79 cell lines using an alkaline version of the comet assay is shown in Table 3. The hexane extract clearly resulted in a significant increase in DI and DF values, as compared to the values obtained for the control groups, at concentrations of up to 25 g/mL. In addition, this increase in damage score occurred in a doserelated manner.

Discussion
The saponifiable portion of P. insignis seeds (bacuri fat) is yellowish, solid and rich in triacylglycerols and fatty acids. Hilditch and Pathak (1949) determined the composition of bacuri fat; they found that its main acidic components were palmitic (55%) and oleic (32%) acids, with smaller proportions of stearic (6%) and palmitoleic (3%) acids and traces of myristic, arachidic, and linoleic acids. Our results, shown in Table 1, differ from previously reported data (Bentes et al., 1986;Hilditch & Pathak, 1949). In our study, palmitic (25.31%) and oleic (27.59%) acids also contributed significantly to the composition of the oil, although both studies revealed palmitic and oleic acid as the main constituents.
As shown in Table 1, oleic and palmitic acids are the most abundant fatty acids present in hexane extract. In addition, ester and hydrocarbon residues are freed from unsaponifiable matter. Despite a total molar content of 35% of unsaturated acids, the fat contained over 20% of trisaturated glycerides (largely tripalmitin), which is different from the majority of seed fats.
The antimicrobial, trypanocidal and antitumoral activities of natural products are mainly associated with prenylated compounds, especially in the case of benzophenones. It has been suggested that the biological activity of a compound can be increased by increasing the number of attached prenyl residues (Pereira et al., 2010). In addition, benzophenones isolated from plants were found to display a high antioxidant activity and protect cells from oxidative stress and the formation of reactive oxygen species (ROS) that are involved in inflammatory processes (Acuna et al., 2009).
Leishmaniasis is a public health problem in developing countries, where 350 millions people are at risk of infection.
Systematic studies in different parts of the world searching for the anti-protozoal activity of medicinal plants have been reported (Braga et al., 2007). In the present study, P. insignis seeds showed a potent inhibitory effect against the promastigote form ( Table 2). The results described in this study provide an additional contribution to the development of new compounds with potential anti-leishmanial properties. Nevertheless, further studies are required to characterize the active component of P.
insignis against Leishmania, using highly purified compounds. A systematic in vitro evaluation of an extract obtained from the latex of Moronobea coccinea Aubl. (Clusiaceae) exhibited a strong anti-plasmodial activity against Plasmodium falciparum that was attributed to the presence of polycyclic polyprenylated acylphloroglucinols (Marti et al., 2009).
The use of plants in therapies is a worldwide phenomenon. Currently, drugs derived from plants are being investigated for the possible presence of cytotoxic, mutagenic and genotoxic substances, as well as other biological activities. The detection and evaluation of the cytotoxic, mutagenic and carcinogenic effects of plant compounds are of fundamental importance to minimize the possible risks of these agents. Many plants occasionally consumed by the human population can have toxic effects (Varanda et al., 2002).
The MTT assay was used to obtain information on cytotoxicity of P. insignis seeds hexane extract in V79 cells. A significant decrease in survival was observed at concentrations of up to 100 µg/mL (Figure 1). This information is important for the evaluation of the safety of this compound for possible pharmacological applications and for the study of the mechanisms underlying its anti-proliferative effects.
A literature review from a chemical and biological point of view for the genus Calophyllum (Clusiaceae) reveals a cytotoxic activity against several cell lines due to the presence of coumarins, xanthones, flavonoids, and triterpenes (Cechinel Filho et al., 2009). Among the eight Calophyllum species found on the American continent, Calophyllum brasiliense is the most widely distributed. Chemical analysis of this species revealed the presence of xanthones having chemo-preventive properties and anti-fungal activity. A cytological study showed that coumarins from C. brasiliense reduced the survival of BMK cells (baby mouse kidney cells) by inducing apoptosis and, to a lesser degree, necrosis. Several xanthones, coumarins, terpenoids and phenolic compounds were isolated from C. brasiliense with remarkable activities, including anti-HIV, antibacterial, trypanocidal, and anti-cancer against KM-12 (colon adenocarcinome), U-251 (gliome), PC-3 (prostate), and K-562 and HL-60 (leukemias) cell lines (Mesquita et al., 2009).
The evaluation of genotoxic damage caused by plant compounds together with a cytotoxicity determination is important to minimize the possible risks of these agents, especially when they are part of a long-term treatment (Cardozo et al., 2006).
The alkaline, single-cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues (Moøller et al., 2010). In this study, we showed that the hexane extract of P. insignis seeds induced genotoxicity in the V79 cell line in a dose-related manner ( Table 3). The alkaline comet assay (pH > 13) can detect single and double-stranded breaks, incomplete repair sites, alkali labile sites, and possibly both DNA-protein and DNA-DNA cross-links in almost any eukaryotic cell population that can be obtained as a single cell suspension (Burlinson et al., 2007). The genotoxic effects observed in our study with this assay can be explained by the results of the chemical constituents detected in the extracts.
The seeds from P. insignis are rich in free fatty acids, as shown in Table 1. Palmitic, oleic, linoleic, α-linolenic, and stearic acids were also identified in P. insignis seeds. Saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Palmitate and stearate, but not linoleate, oleate and palmitoylmethyl esters, induced growth inhibition and increased apoptosis in RINm5F cells following a 24-h exposure. However, linoleic acid protected against palmitic-acid-induced apoptotic and necrotic cell death (Beeharry et al., 2004). As also shown in Table 1, the amount of saturated fatty acids in P. insignis and the percentage of oleic and palmitic acids were high relative to those of other compounds. Oleic acid is more steatogenic, but less apoptotic, than palmitic acid in hepatocyte cell cultures (Ricchi et al., 2009). A promising study by Souza et al. (2017) using stem bark extract showed the inhibitory values of the EtOH-Ext, Hex-F, and Lupeol inhibited the growth of L. Amazonenses promastigote forms at IC50 of 174.24, 45.23, and 39.06 g/mL, respectively, as well as L. amazonenses axenic amastigote forms at IC50 of 40.58, 35.87, and 44.10 g/mL, respectively. The mean cytotoxic concentrations for macrophages (CC50) were higher than those for amastigotes (341.95, 71.65, and 144.0 g/mL, resp.), indicating a selective cytotoxicity towards the parasite rather thanthe macrophages. Interestingly, all treatments promoted antileishmanial effect against macrophage-internalized amastigotes atconcentrations lower than CC50.

Conclusion
In conclusion, the present findings indicate that hexane extract of P. insignis seeds are toxic against A. salina , present leishmanicidal activity on promastigote forms, and are cytotoxic and genotoxic in V79 mammalian cell lines. All these activities seem to be related, at least in part, to the chemical composition that includes the presence of saturated fatty acids and xanthone (gamma-mangostin). Further studies are needed to identify the molecular mechanism underlying hexane extract of P.
insignis seeds induced-cytotoxicity in different biological models.