Cytotoxicity of isolated compounds from Picrasma crenata (Vell.) Engl. in animal tumor cell (HTC)

Abstract The study aim was to evaluate the cytotoxic activity, using the MTT test [3-(4,5-Dimethilthiazol-2-yl)-2,5-diphenil tetrazolium bromide], from the crude extract of Picrasma crenata (Pau Tenente) and its isolated compounds, quassin and parain, in culture of rat liver tumor cells (HTC). The test was carried out exposing the cells for 24, 48 and 72 hours to concentrations of 5, 10, 50, 100, 200, 300, 400, 500 and 1000 μg of crude extract of Pau Tenente/mL of culture medium and 1, 5, 10, 15, 20, 40, 60, 80 and 100 μg of quassin or parain compounds/mL of culture medium. The absorbances averages results obtained showed that the crude extract did not present cytotoxicity for the HTC cells in all the concentrations and evaluated times. For quassin, the concentrations of 80 and 100 μg/mL were cytotoxic, after 72 hours of treatment. For parain, the concentrations of 1, 5, 20, 40, 60, 80 and 100 µg/mL, in 72 hours, were cytotoxic, revealing a new activity for this compound. Thus, the results demonstrate a first indication of the cytotoxic activity of compounds quassin and parain, adding an important social and economic value to them, and may have application in future research and in pharmaceutical industry.


Introduction
Medicinal plants are made up of various substances that can be used in therapeutic and alternative medicine, and the recognition of their efficiency and cultural influence has increased their use (Lapa et al., 2004;Bach et al., 2014;Gomes et al., 2014;Vendruscolo et al., 2022).Mainly due to the growing increase in the incidence of cancer and mortality, stimulating the search for new less invasive treatments, since the results are of great interest to the The crude extract of Pau Tenente (lyophilized) was dissolved in DMEM culture medium in the following concentrations : 5, 10, 50, 100, 200, 300, 400, 500 and 1000 μg/mL.
The concentrations tested were chosen in order to perform an evaluation screening, from low to high doses, as proposed by Meerloo et al. (2011), Vendruscolo et al. (2022) and Santos et al. (2023).

Cytotoxicity test
The ,5-Dimethilthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cytotoxicity activity test followed the protocol suggested by Mosmann (1983).96-well cell culture plates were used, 2.0x10 4 HTC cells/well were seeded.After 24 hours the culture medium was discarded and 100 μL of new medium was added for the groups: negative control (CO-) (culture medium); positive control (CO+) (cytotoxic agent methyl methane sulfonate (MMS -500 μM); solvent control (CS) (25 μL of DMSO/mL of culture medium for quassin and 20 μL of DMSO/mL of culture medium for paraína), and treatments with different concentrations of crude extract of Pau Tenente and quassin and parain compounds.
The cells were incubated for 24, 48 and 72 hours and, after this time, the culture medium was replaced by 100 μL of serum-free medium, plus MTT (0.167 mg/mL).The plates were incubated for another 4 hours and, afterwards, the medium containing MTT was discarded and 100 μL of DMSO was added to the wells, for dilution of the formed formazan crystals.
The reading was performed in a micro plate reader at 492 nm and the data were expressed by means of the absorbances obtained in the three biological repetitions.The statistical analysis was performed with the data of the average absorbances, by one-way analysis of variance (ANOVA), followed by the Dunnet tests (α=0.05,p<0.05, n=3), to compare the absorbances negative control means with the absorbances of each treated group, at each treatment time, and Tukey (α=0.05,p<0.05, n=3), to compare the average absorbances between the concentrations of each treatment, at each time, between each concentration within the different times tested and between the same concentrations, at the same times, for quassin and parain, by the Action Stat Program.
The percentage values of cell viability (VC) were estimated by Equation 1.
Where: VC: Cell viability [%]; A T : Absorbance of treatment; A CO-: Absorbance of the negative control.
Considering that medicinal plants have innumerable biologically active chemical constituents, the aim of the present study was to investigate the action of Pau Tenente and its isolated compounds quassin and parain as cytotoxic agents against liver tumor cells of Rattus norvegicus (HTC).

Cell line
The cells derived from Rattus norvegicus (HTC) hepatoma, obtained from the Cell Bank of Rio de Janeiro-RJ-Brazil, were grown in 25 cm 2 culture flasks, containing 10 ml of DMEM culture medium (Dulbecco's Modified Eagle's Medium), supplemented with 10% fetal bovine serum and incubated in a BOD oven at 37 ºC.

Adjusting polynomials from data
The polynomials adjusted by the empirical equations, which described the behavior of the HTC cells for each tested compound, were constructed from the experimentally obtained data and were valid only within the concentration and time ranges employed.The coefficients of each equation were obtained using the Scilab 6.0.1 software, using the "coeff" command, with a 95% confidence interval.

Crude extract
Statistical analysis of the cytotoxicity activity data of HTC cells treated for 24, 48 and 72 hours with the crude ethanol extract (80%) of Pau Tenente (Table 1) show that no concentration of this extract was statistically different from the control negative, in none of the evaluation times (24, 48 and 72 hours), indicating absence of cytotoxic effect.The cell viability of the groups treated with the extract was even greater than 99.80% (24 hours), 102.10% (48 hours) and 65.86% (72 hours).The lowest values of cell viability were at 72 hours, where the lowest concentration (5 μg/mL) showed mean absorbance statistically lower than the absorbances of the highest concentrations (500 and 1000 μg/mL).
These results corroborate those found by Novello et al. (2008), who also confirmed low toxic activity of the hydro alcoholic extract of P. crenata in vivo tests.In this experiment, the masses of the body, lungs, liver and heart of rats treated intravenously with 2500 and 5000 mg of the extract/kg remained unchanged.The study by Toma et al. (2002) also demonstrated absence of signs of toxicity and/or death of mice before oral administration (5000 mg/kg) and intraperitoneal (1000 mg/kg) of 70% ethanolic, 100% ethanol, dichloromethane and hexane extracts of Q. amara, a plant that has properties similar to the one studied in the present study (P.crenata) and which, in turn, did not show a cytotoxic effect to HTC cells with the use of crude ethanolic extract (80%).
However, in general, a slight proliferation of HTC cells can be seen in 48 hours, evidenced by their high cell viability at this time, and also by the fact that their average absorbances that were statistically higher and different from those obtained in times of 24 hours (concentrations of 10, 50, 100, 200, 300, 500 and 1000 μg/mL) and 72 hours (5, 10, 50 μg/mL), by the Tukey test.Maranhão et al. (2014), found similar results evaluating the aqueous extract of Simarouba amara Aublet, a medicinal plant also belonging to the Simaroubaceae family, where all concentrations of the extract induced proliferation of hepatocytes from treated rats, possibly due to their inhibitory action on development of free radicals by catechins, explained by the recovery of antioxidant enzymes and the decrease in lipid peroxidation.
However, it is worth noting that the ethanolic extracts of Picrasma quassinoides, a species of Picrasma native to Asia, showed cytotoxic effect for SiHa (cervical cancer) (Gong et al., 2020) and HepG2 G12V cells (liver cancer) (Xie et al., 2020), indicating an effect dependent on the species, soil and/or climate in which these species grow.From the adjusted curve of the data obtained for the crude extract of Pau Tenente (Figure 1), it was possible to obtain the polynomial of Equation 2. Its coefficients (a, b and c) (Table 2) with lower and upper limits of 95% of confidence were obtained by the Scilab 6.0.1 software, using the "coeff" command.When the limits of the coefficients do not include zero as a result, it means that the data has been well adjusted, since the null solution (0,0,0) has no physical meaning, nor can there be a saddle point (point where the slope of the surface is null).
This adjustment was defined by the coefficient of determination (R 2 ), which must be the closest to a unit to be the best possible.In this specific case, R 2 =0.8895 was reached, which shows that 88.95% of the results are explained by the factors studied, indicating a good relationship between the experimental data and the curve established by the empirical equation found.This result is excellent considering that experimental data do not always follow a pattern, as in the case of Thomas et al. (2006), who prove a good quality of the curve obtained by means of a larger one R 2 .And, from these data, it is possible to perceive a greater dependence on absorbance, that is, on cell viability, with the treatment time than with the concentration of the crude extract.

Quassin
The data in Table 3 shows the results of the cytotoxicity activity test performed with the compound quassin.Statistical analysis indicates that no concentration of quassin, at the evaluation times of 24 and 48 hours, had a cytotoxic effect on the liver tumor cells of rats.In fact, cell viability was greater than 92.96% (24 hours) and 83.58% (48 hours).It is also possible to notice an increase in the absorbance of time from 24 to 48 hours for all concentrations tested, being statistically significant for the lowest concentrations (1, 10, 15, 20 and 60 μg/mL), indicating a proliferation of HTC cells within 48 hours, as well as that observed for the crude ethanolic extract.
The absence of cytotoxicity to quassin against cells derived from cervical cancer (HeLa) was also demonstrated by Fukamiya et al. (2005).Almeida et al. (2007), after 48 hours of treatment, also confirmed that quassin was one of the least efficient against cancer, testing it for the inhibitory effect against the activation of the Epstein-Barr virus antigen (EBV-EA), induced by 12-O-tetradecanoylforbol-13-acetate (TPA), in Raji cells.And, Xu et al. (2016), also showed that none of the quassinoid compounds, isolated from the 95% ethanolic extract of the Picrasma quassioides stems, showed cytotoxic activity.
At 72 hours, the highest concentrations (80 and 100 μg/mL) were statistically different from the negative control and cytotoxic to HTC cells, with cell viability of 60.76% (100 μg/mL) and 61.89% (80 μg/mL) (Table 3).This may have occurred because quassin, in high concentrations and treatment time, may have acted as a cytotoxic or antiproliferative agent, as shown by Mata-Greenwood et al. (2002), who assessed the cytotoxicity of the quassinoid brusatol identified the interruption of the cell cycle of tumor cells in phase G1.
This effect can already be seen in the time of 48 hours, where the comparison between the different concentrations tested, in the same evaluation time, already indicated a difference between the lowest concentrations (1, 5, 10, 20 and 60 μg/mL) and the higher concentrations (80 and 100 μg/mL), which showed lower average absorbances and, consequently, lower cell viability of HTC cells.Within 72 hours, this effect was maintained, with the lowest concentrations (1, 5, 10, 15, 20 and 40 μg/mL) also showing absorbances statistically different from the highest concentrations (60, 80 and 100 μg/mL).
From the adjusted curve of the data obtained for quassin (Figure 2), it was possible to obtain the polynomial of Equation 3, and the values of the coefficients of the generated equation are in Table 4.In this case, the R 2 was 0,8908, which is again an optimal value, as 89.08% of the results are explained by the factors studied.In this case, one can perceive, again, a greater dependence on absorbance (cell survival) over time than with concentration.Means followed by the same lowercase letter do not differ statistically from each other, when comparing the concentrations at the same evaluation time, and upper case, within the same concentration at the different evaluation times, using the Tukey test (p<0.05).These data can be confirmed by the statistical analysis of the cytotoxicity test (Table 3), since only for higher concentrations and times the absorbance declines with more intensity, demonstrating the cellular unfeasibility and the existing cytotoxicity.

Parain
The data in Table 5 shows the results of the parain cytotoxicity activity test.Statistical analysis indicates that no concentration of parain was statistically different from the negative control in the evaluation times of 24 and 48 hours, with no cytotoxic effect, similar to that found with crude extract and quassin.Cell viability in these cases was greater than 96.84% (24 hours) and 94.39% (48 hours).It can also be noted an increase in absorbance from 24 to 48 hours, statistically significant (p≤0.05), for concentrations of 5, 10, 15 and 20 μg/mL, meaning a proliferation behavior of HTC cells within 48 hours, similar to that observed with the crude extract and the quassin compound.
At 72 hours, the concentrations of 1, 5, 20, 40, 60, 80 and 100 μg/mL of parain were statistically different from the negative control, promoting the mortality of HTC tumor cells, with cell viabilities below 72.74% and reaching 53.40% (100 μg/mL) (Table 5).Only concentrations of 10 and 15 μg/mL were not cytotoxic at 72 hours, as well as being statistically different from the concentration of 100 μg/mL.The presence of a ketocarbonyl group in the in the C ring of isoparain resulted in low cell viability for SH-SY5Y cells co-treated with this compound and H 2 O 2 , by the MTT method (Zhao et al., 2019).Therefore, the chemical structures of the quassinoids isolated from P. crenata can influence their biological activities.
These results are relevant, because Guo et al. (2005) already highlighted the importance of the search for new natural sources of quassinoids as anticancer drugs.The cytotoxic activity found in the present study, with parain, may have been due to the inhibition of NADH oxidase of cell membranes, an effect proven by the glaucarubolone quassinoid in HeLa cells (Morré et al., 1998).
From the adjusted curve of the data obtained for parain (Figure 3), it was possible to obtain the polynomial of Equation 4, and the coefficients of the equation are plotted in Table 6.In this case, the R 2 was 0,7633, which is also a value well, considering that experimental data does not always follow a pattern perfectly and that biological systems are very sensitive.The similarity between Equations 3 and 4 is observed, but the values of the coefficients change.This means that time has a greater influence on absorbance (cell viability), since the longer the time, the lower the absorbance and, consequently, the lower the viability of these cells.Means followed by the same lowercase letter do not differ statistically from each other, when comparing the concentrations at the same evaluation time, and upper case, within the same concentration at the different evaluation times, using the Tukey test (p<0.05).
Table 6.Coefficients of the fitted curve equation for the experiment with parain from Pau Tenente.In addition, this equation and its coefficients corroborate with the statistical analyzes, which show cytotoxicity of parain from the lowest concentrations tested, indicating that the inducing effect of apoptosis or cell death occurs due to the presence of the compound, regardless of the concentration tested, but influenced by exposure time.

Conclusions
Based on the experimental results, we highlight the isolated compound parain, which under experimental conditions, showed cytotoxic activity against the tested HTC cells, with a good extraction yield (0.132%), when compared to quassin (0.046%), and potential for future research and applications in the pharmaceutical industry.Although the highest extraction yield was from the crude ethanolic extract (4.5%), it did not show any cytotoxic activity.Thus, the results prove the cytotoxic activity of quassin and parain, being of interest to the general population as important information, since data and activities in the literature are scarce.
of the tested compound (crude extract of Pau Tenente); t: Treatment time.

Figure 2 .
Figure 2. Adjusted curve for absorbance versus concentration versus time data, with quassin isolated from Pau Tenente.

Figure 3 .
Figure 3. Adjusted curve for absorbance versus concentration versus time data, with parain isolated from Pau Tenente.

Table 1 .
Percentage of viability of HTC cells (VC) treated with different concentrations of the crude extract of Pau Tenente incubated for 24, 48 and 72 hours.
CO-: Negative Control; CO+: Positive Control.*: Result statistically different from the negative control (Dunnet test, p <0.05).Averages followed by the same lowercase letter do not differ statistically from each other, when comparing concentrations at the same time of assessment, and upper case, within the same concentration at different times of assessment (Tukey Test, p <0.05).

Table 2 .
Coefficients of the fitted curve equation for the experiment with the crude extract of Pau Tenente.
Figure 1.Adjusted curve for absorbance versus concentration versus time data, with crude Pau Tenente extract.

Table 3 .
Viability percentages of HTC cells (VC) treated with different concentrations of quassin, incubated for 24, 48 and 72 hours.

Table 4 .
Coefficients of the fitted curve equation for the experiment with quassin from Pau Tenente.

Table 5 .
Percentage of viability of HTC cells (VC) treated with different concentrations of parain, incubated for 24, 48 and 72 hours.