Bioactivity and Toxicity of Senna cana and Senna pendula Extracts

This work investigated the content of total polyphenolic compounds and flavonoids as well as their toxicity and larvicidal and acetylcholinesterase inhibitory activities. The antioxidant activities of two medicinal Senna species extracts (Senna cana and Senna pendula) were also investigated. The ethanol extract of the leaves of S. cana and the ethanol extract of the branches of S. pendula presented the best performance in the DPPH/FRAP and ABTS/ORAC assays, respectively. For the inhibition of acetylcholinesterase, the hexane extract of the flowers of S. pendula presented the lowest IC50 value among the ethanol extracts of the leaves of S. cana and showed the best performance in some assays. The hexane extract of the leaves of S. pendula and the hexane extract of the branches of S. cana were moderate to Artemia salina Leach. In the quantification of phenols and flavonoids, the ethanol extract of the leaves of S. cana presented the best results. The ethanol extracts of the leaves of S. cana were found to be rich in antioxidants, phenolic compounds, and flavonoids. These results indicate the antioxidant potential of the extracts of Senna species and can be responsible for some of the therapeutic uses of these plants.


Introduction
e genus Senna (Fabaceae) includes about 260 species, 200 of which occur in the Americas. Several of these occur in the Brazilian northeastern semiarid region, such as S. martiana and S. spectabilis var. excelsa, which are used in folk medicine to treat colds, as laxatives, and for antioxidant, cytotoxic, and acetylcholinesterase inhibitory activities [1].
Many of these species are reported in the literature to contain anthraquinone glycosides, responsible for laxative activity, and polyphenolic metabolites such as flavonoids, which are scientifically recognized as having considerable leishmanicidal and antioxidant activity, among other biological activities [1][2][3].
With the current spread of diseases caused by arboviruses transmitted through Aedes aegypti mosquitoes, such as dengue, zika, and chikungunya, the search for new sources of natural repellents extracted from plants is very important to control outbreaks [11]. erefore, the aim of this study was to evaluate the pharmacological potential of S. cana HS Irwin and Barneby and S. pendula HS Irwin and Barneby, native species of the Brazilian northeastern, through different methods of determining antioxidant activity, toxicity, larvicidal activity, acetylcholinesterase inhibition, and flavonoid content (Supplementary Materials (available here)).

Preparation of Plant Extracts.
e leaves and branches of Senna cana were collected in Mucugê, Bahia State (−12°57′ 7900″ S and −41°19′ 3600″ W, elevation of 400 m). e leaves, branches, and flowers of S. pendula were collected in Cratéus, Ceará State (−5°17′ 833″ S and −40°67′ 75″ W, elevation of 300 m) ( Figure 2). Both places are in northeastern Brazil. e botanical material was deposited in the Prisco Bezerra Herbarium of Universidad Federal of Ceará with respective identification numbers of 50297 and 54075. e plant material was dried, weighed, and macerated thoroughly in n-hexane for seven days at room temperature. Afterwards, the hexane extract was filtered and concentrated in a rotary evaporator, obtaining the hexane extracts. is process was repeated with ethanol to obtain the ethanol extracts. e residues were dried and stored at 27°C. e extracts obtained are shown in Table 1.

Chemical Screening.
e tests were carried out according to the method proposed by Matos [12], using freeze-dried extracts. e main secondary metabolite classes present in the extracts were identified by chemical reactions with specific reagents and formation of precipitates or color changes. Some of the chemical tests performed are described below.

Test for Detection of Xanthones and Flavonoids.
Solutions of the extracts were prepared at a concentration of 5 mg·mL 1 in methanol. Aliquots of 5 mL of this solution were removed and placed in test tubes for chemical testing. Magnesium strips and 4 drops of concentrated HCL were added to the tubes. e presence of flavonoids and xanthones was detected by the appearance and intensification of the red color in the solution.

Test for Detection of Tannins.
Solutions of the extracts were prepared in the same way as for the flavonoid test (5 mg·mL 1 in methanol). Aliquots of 5 mL of this solution were removed and placed in test tubes for chemical testing. Distilled water (5 mL) was added, and the solution was filtered to remove any solids, after which 5 drops of FeCl 3 were added to attain a concentration of 10%. e formation of blue color indicates the presence of hydrolyzable tannins and green color indicates the presence of condensed tannins.

Occurrence of species Senna pendula
Crateús, Ceará, Brazil  control, di-tert-butylmethylphenol (BHT), were mixed, and the absorbance of the reaction was read at 515 nm. e test was performed in triplicate at various concentrations (mg·mL −1 ) [13]. Absorbance measurements were determined in a Spekol 1100 spectrophotometer.

Folin-Ciocalteu Method.
Total phenol content was determined by the spectrophotometric method using the Folin-Ciocalteu reagent and gallic acid as the reference standard [14]. Ethanol extracts (7.5 mg) were dissolved in methanol and transferred to a 25 mL volumetric flask, and the final volume was completed with methanol. An aliquot of 100 µL of the latter solution was shaken with 500 µL of the Folin-Ciocalteu reagent and 6 mL of distilled water for 1 min. After this time, 2 mL of 15% Na 2 CO 3 was added to the mixture and stirred for 30 s. Finally, the solution was diluted to 10 mL volume with distilled water. After 2 h of incubation, the absorbance of the samples was measured at 750 nm.

ABTS Radical Scavenging Assay.
is method was based on Re et al. [15]. It measures the antioxidant capacity based on the ability of the substances to inactivate the cation radical 2,2′-azinobis-(ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS°+). Solutions of the extracts were prepared at a concentration of 600 mg·L −1 . Aliquots of 10 µL, 20 µL, and 30 µL of these solutions were added to test tubes, and the volume in the first two cases was completed with distilled water to 30 µL (extract and water). In dark environment, 3 mL of each solution (radical ABTS°+ + ethanol P.A.) was added to a test tube, which already had absorbance preset to 0.70 in the absence of light. Readings were taken at 734 nm in a spectrophotometer six minutes after addition of the radical. e percentage inhibition of ABTS°+ was determined from the standard curve of trolox, and the results were expressed as TEAC (trolox equivalent antioxidant capacity) µmol·g −1 .

Potential Antioxidant FRAP (Ferric-Reducing Antioxidant Power).
is method is based on the capacity of metabolites to reduce Fe 3+ to Fe 2+ . When this occurs in the presence of 2,4,6-tripyridyl-S-triazine (TPTZ), the formation of Fe 3+ /TPTZ with blue staining of the Fe 2+ occurs. e extracts were prepared in methanol at different concentrations between 0.25 and 1.0 mg/mL. Ten microliters of the extracts was first incubated with 30 µL of bidistilled water and 300 µL of FRAP reagent, consisting of 25 mL of acetate buffer (300 mmol·L −1 sodium acetate, pH � 3.6), 2.5 mL of TPTZ (TPTZ in 10 mmol·L −1 and HCl 40 mmol·L −1 ), and 2.5 mL of FeCl 3 at 37°C, before measurement. A ferrous sulfate calibration curve (0.01-1.0 mmol·L −1 ) was used, and the results were expressed as (Fe 2+ mmol·L −1 )·L −1 . e reaction was measured at 595 nm in a universal microplate reader (ELx 800, BioTek Instruments, Winooski, Vermont, USA) after 5 min resting time. Using these regression curves, the EC values were calculated as the concentrations of the antioxidant (expressed as mg·mL −1 ) giving an absorbance equivalent to a 1 mM Fe(II) solution according to Pulido et al. [16].
From a stock solution of DMSO, trolox (10 mmol·L −1 ) was diluted in ORAC buffer solution to a concentration of 20 μmol·L −1 .
e ORAC buffer contains 75 mmol·L −1 of sodium hydrogen phosphate/hydrogen phosphate potassium with pH � 7.4. e decline of fluorescein was measured at 37°C at constant intervals of 2 minutes between measurements until completing 122 min using a CytoFluor 4000 fluorescence microplate reader (excitation wavelength at 530 nm was measured every minute for 25 min and emission wavelength at 585 nm was measured every minute for 30 min) (Perspective Biosystems, Minnesota, USA). e final ORAC results were calculated using a regression equation between the trolox concentrations and AUC, expressed as ORAC units, where 1 ORAC unit inhibits the decline produced by 1 μmol·L −1 of trolox.

Total Flavonoid Contents.
e total flavonoid contents of the extracts were determined using the spectrophotometric method described in Quettier et al. [18], where quercetin was used as a standard, in a solution of aluminum chloride. Twenty milligrams of each extract was placed in a test tube, and methanol was added to complete the volume of 50 mL, producing a second extract. en, 5 mL of this second extract was removed and 0.5 mL of 2% AlCl 3 solution was added, after which the volume was completed to 10 ml with 5% acetic acid solution. Following incubation for 30 min, the absorbance of the reaction mixture was measured at λ max � 425 nm with a Femto 700 plus spectrophotometer. e calibration curve was plotted using concentrations of 5, 10, 25, 50, and 75 µg·mL −1 of quercetin.

Evaluation of Larvicidal Activity.
is assay was based on Cavalcanti et al. [22]. Solutions of 100, 250, 500, and 1000 µg·mL −1 of all extracts were prepared with water and DMSO. en, 25 Aedes aegypti larvae were added in stage 3.
e solution was left with the larvae for 24 hours; then, the dead larvae were counted and the LC 50 of each extract was calculated.

Statistical Analysis.
e relation between the phenolic compound and antioxidant variables was determined by linear regression using Excel and Origin 6.0 software.

Chemical Screening.
e chemical screening of the hexane extracts LHESC, BHESC, LHESP, BHESP, and FHESP was positive only for anthraquinones and triterpenes. However, for the ethanol extracts LEESC, BEESC, LEESP, BEESP, and FEESP, the presence of anthraquinones, flavonoids, triterpenes, tannins, and xanthones was confirmed. e results for the chemical screening of ethanol extracts are shown in Table 2.
ese metabolites are frequently found in Senna species [23].

Determination of Antioxidant Activity and Quantification of Flavonoids and Total Phenol Contents.
Antioxidant tests can be divided into two types: the direct method (ORAC), which is used to ascertain chemical kinetics, and indirect methods (DPPH, ABTS, and FRAP), which are mediated by electron transfer. Each method has its specificity. Some methods involve an acid medium, as is the case of the FRAP assay, while others are already in the basic medium, such as the ABTS assay, and other tests are carried out in a neutral medium, such as the test of total phenolic content. e pH of the medium can have an important effect on antioxidant capacity of the compounds [17].
In this work, we related the results to ethanol extracts because the hexane extracts were negative for flavonoids, which are recognized as having a significant antioxidant potential. Additionally, the hexane extracts were not compatible with all the antioxidant screening methods. e results obtained for the antioxidant tests are shown in Table 3.
e extracts that showed the best performance against DPPH/FRAP were the ethanol extract of the leaves of S. cana According to the literature, direct methods (in this case, ORAC) are more suitable for the evaluation of antioxidant activity, especially those based on the controlled chain Biochemistry Research International reaction model, because in general they are more sensitive. e best practice is always to make a comparison between the data obtained by both methods (direct and indirect) in order to obtain greater analytical safety [17,24].

Correlation between the Antioxidant Assays.
According to the statistical analysis and a comparison of correlation between the 4 tests, the strongest correlation (Table 4) among the antioxidant capacity assays was observed between the DPPH and the FRAP assays ( Figure 3) (r � 0.8908; n � 5) and the ABTS and ORAC assays (Figure 4) (r � 0.8353; n � 5).
In the spectrophotometric quantification of the total flavonoid content (Table 5), the ethanol extract of the leaves of S. cana (LEESC) presented the best results (228.9 mg·g −1 ), followed by the ethanol extract of the leaves of S. pendula (LEESP), which presented a value of 221.1 mg·g −1 .
Regarding the total phenolic content, the leaf extract of S. cana (LEESC) also presented the best result (724.5 mg EAG·g −1 extract), which confirms that this extract, because it contains a high phenolic content, automatically has a high content of antioxidants, since phenolic compounds are excellent natural antioxidants. It is noteworthy that the extracts that presented the best results had more satisfactory performance than the standards used (BHT and rutin), even though rutin is a flavonoid, which is a natural antioxidant.
is suggests that the synergism of the compounds present in extracts may have influenced this excellent result. Phenolic compounds have been reported in the literature to have good antioxidant activity [25,26].
Like in the antioxidant tests, the branch extract of S. cana (BEESC) showed the lowest phenolic content and the second lowest total flavonoid content. ese results partly explain the weak performance of this extract in the antioxidant trials because of the lower content of phenolic compounds and flavonoids in this extract [7,9].
Another factor that can be considered for the good performance of the leaf extracts of S. cana, both in relation to the different antioxidant tests and the total phenolic content, may be the high content of flavonoids present in the extract, considering that the extract was prepared in an organic solvent, which increases the solubility of these flavonoids in the solution. While ORAC and Folin-Ciocalteu tests are not     suitable for measuring liposoluble antioxidants, ABTS can measure the activity of both water-soluble antioxidants and liposoluble ones and DPPH in turn is soluble only in organic solvents [27,28]. When comparing the value obtained with the values reported in the literature, the TEAC value found for the ethanol extract of the leaves of Senna alata was 125 μmol trolox·g −1 (31.29 mg trolox·g −1 ), a value well below the values obtained for all extracts studied in this work [29]. According to Liczano [7], the aqueous extract of the leaves of S. reticulata presented an ORAC value of 226.6 μmol trolox·g −1 and a TEAC value of 34.04 μmol trolox·g −1 . Both these results are lower than those found for all extracts presented here. Based on the data observed, the extracts of S. cana and S. pendula had excellent performance in the different antioxidant trials and contain high concentrations of phenolic compounds and flavonoids in their composition.
Also in comparison with the values in the literature, Mak et al. [30] reported that the ethanol extract of the flowers of S. bicapsularis L. contained total flavonoids and phenols of 12.93 mg quercetin·g −1 extract and 262.23 mg EAG·g −1 extract, respectively. In contrast, the extracts of S. cana and S. pendula studied contained much higher total flavonoids and phenols, and the lowest value found for the flavonoid content was for the ethanol extract of the branches of S. pendula, which presented a value of 87.04 mg of quercetin·g −1 extract. For the total phenol content, the lowest value was found for the ethanol extract of the branches of S. cana, which presented a content of 473.7 mg EAG·g −1 extract.

Correlation between Total Phenolic Content and Antioxidant Capacity.
e best correlation (Table 6), that is, positive correlation, between total phenolic compounds and antioxidant capacity was obtained in the ABTS assay (r � 0.595982; n � 5) followed by the ORAC assay (r � 0.576957; n � 5).

Correlation between Total Flavonoid Content and Antioxidant Capacity.
e best correlation (Table 7) between total flavonoid and antioxidant capacity was obtained in the ABTS assay (r � 0.29654; n � 5) followed by the ORAC assay (r � 0.28349; n � 5).

Evaluation of Antiacetylcholinesterase Activity.
Plants are promising sources of new drugs, including some used to treat Alzheimer's disease (AD), such as galantamine. e drugs available at present are effective only in the early stages of AD, whose period is very short. erefore, it is important to search for new drugs of natural origin that inhibit the enzyme acetylcholinesterase (AchE), both in the early stages and advanced stages of AD [31,32].
Phenols and flavonoids are important natural products that inhibit acetylcholinesterase and thus restore the level of acetylcholine, which is essential for brain function [33]. Since the Senna extracts contained significant concentrations of phenolic compounds and flavonoids, the evaluation of antiacetylcholinesterase activity was carried out to confirm this aspect.
To date, no studies have been published evaluating the anticholinesterase activity of extracts of Senna species. ere are only two reports involving alkaloids isolated from Senna multijuga who had this activity [34,35]. e results obtained for the inhibition of cholinesterase are shown in Table 8. Only the ethanol extracts of the branches and leaves of S. cana showed no activity in this qualitative test. e hexane extract of the bark of S. pendula was the only one that presented significant activity. e hexane extracts of S. cana and the hexane and ethanol extracts of S. pendula presented inhibitory activity and can be considered for future studies against Alzheimer's disease, neurodegenerative diseases, and dysfunction of the cholinergic system. rough quantitative analysis of anticholinesterase activity, it was possible to verify an IC 50 value different from the value obtained for the eserine standard. e extract that presented the best IC 50 value was FHESP, in agreement with the result obtained in the qualitative test, where this extract also presented the best performance.
Another observation is related to the ethanol extracts of S. cana, which in the qualitative test had a negative result, while in the quantitative test, they presented satisfactory results. e  LEESC extract presented the third best performance in the quantitative test. is fact may be related to the high content of phenolic compounds and flavonoids found in this extract. Regarding antioxidant activity, this same extract performed best in the DPPH and FRAP assays and the second best in the ABTS and ORAC assays. ese results corroborate the finding of Penido et al. [33] that the higher the content of phenolic compounds and flavonoids, the better the performance against inhibition of acetylcholinesterase.

Evaluation of Toxicity against Artemia salina.
According to Silva et al. [36], the determination of the toxicity of plant samples against Artemia salina L. allows the evaluation of toxicity involving only one parameter: life or death. erefore, this model is considered as a preliminary form of testing, with low cost and easy handling, to identify bioactive compounds.
e absence of cytotoxicity of the extracts tested against A. salina is an indicator that the plant can be well tolerated by the biological system.
In the in vitro evaluation of the toxicity of the samples against A. salina, only the BHESC and LHESP extracts were able to cause 50% mortality of the larvae of A. salina, and they also presented low toxicity, with LC 50 of 790.94 and 746.35 μg·mL −1 , respectively. All values are reported in Table 9.
ese results confirm the toxic action for only these extracts, since only the samples with LC 50 less than 1000 μg·mL −1 are considered to be toxic according to the classification described by Meyer [21]. ese results corroborate the possible use of the other extracts tested, as reported by Simões and De Almeida [37]. If a sample is not shown to be toxic to A. salina, its effects will also be the same in humans. e toxicity results of the BHESC and LHESP extracts are also valuable, since samples that are toxic to A. salina can contain bioactive compounds with antitumor, antimalarial, trypanosomicidal, and insecticidal potential [38], possible antiplasmodic activity [39], and antimicrobial effect [40].
Parra et al. [41], when testing the aqueous extract of the leaves of Senna alata, obtained LC 50 of 7.74 μg·mL −1 , much lower than what is considered to be below toxicity. In other words, this aqueous extract showed high toxicity, unlike the ethanol extracts of S. cana and S. pendula shown in this work.

Evaluation of Larvicidal Activity.
e results of the larvicidal activity were analyzed and interpreted according to LC 50 values. Due to the low solubility of the hexane extracts in DMSO, only the ethanol extracts of each part of the two Senna species were used. According to the literature, for results to be considered good, the sample should have LC 50 below 100 ppm. As seen in Table 10, all the results found for S. cana and S. pendula were well above 100 ppm, meaning that these plants are not considered promising sources of substances with relevant larvicidal activity [22].
Edwin et al. [42] investigated the larvicidal activity of the ethanol and aqueous extracts of leaves and stems of Senna alata: for the aqueous extract, the values were 0.840 (% w/v) for the leaves and 0.935 (% w/v) for the stems, while for the ethanol extract of the leaves, the value was 0.791 (% w/v) and for the stems, it was 0.923 (% w/v). Unlike the extracts of S. cana and S. pendula, those of S. alata [43] and S. occidentalis    [23] presented excellent larvicidal activity. When comparing the chemical composition of these species of Senna, there is a very peculiar difference, which may explain this great difference in toxicity against Aedes aegypti: S. alata was found to contain cassiaindoline, a dimeric indole alkaloid, a compound that has not been identified in S. cana and S. pendula.

Conclusions
e chemical study of the Senna cana and Senna pendula ethanol extracts revealed the presence of anthraquinones, flavonoids, tannins, triterpenes, and xanthones, while hexane extracts presented positive results for anthraquinones and triterpenes. e ethanol extract of the leaves of S. cana (LEESC) performed the best in all the antioxidant tests, surpassing the standard values, meaning that this species is a promising source of antioxidants, possibly because of the presence of polyphenolic compounds, especially flavonoids, anthraquinones, and tannins, as confirmed by the quantitative analyses.
e ethanol extract of the branches of Senna cana (BEESC) presented the worst performance in all the antioxidant trials, the lowest phenolic content, and the second lowest total flavonoid content. ese results confirm that the lower the content of phenolic compounds and flavonoids, the worse the antioxidant capacity of the extract.
is study determined that the hexane extracts presented the best results for acetylcholinesterase activity, which can be attributed to the presence of triterpenes and/or anthraquinones, but the ethanolic extracts of the leaves of S. cana (LEESC) presented a satisfactory activity according to the quantitative tests, confirming that the higher the content of phenolic compounds and flavonoids, the better the performance regarding inhibition of acetylcholinesterase.
Most of the extracts investigated showed no toxicity against Artemia salina. As stated before, if a sample does not show toxicity to A. salina, this means that its effects will also be the same in humans. None of the extracts presented relevant larvicidal activity. e results obtained in this work indicate that these species are sources of substances with promising pharmacological activities, with emphasis on antioxidant activity, mainly in extracts from the leaves of Senna cana.

Conflicts of Interest
e authors declare that they have no conflicts of interest.