Effects cytotoxic and genotoxic of Psittacanthus acinarius and Psittacanthus cordatus (mistletoe) on Allium cepa

This study assessed the cytotoxic and genotoxic potentials of extracts of Psittacanthus acinarius (Mart.) Mart. and Psittacanthus cordatus (Hoffmanns.) in the root cell cycle of Allium cepa L. Aqueous leaf extracts of P. acinarius and P. cordatus at three concentrations: 0.00, 5 and 20 mg/mL for 24 hours. Histological slides were prepared and mitotic indices (MI %) and chromosomal alteration indices (CAI %) were determined. Inhibitory effects of the aqueous extract of leaves of P. acinarius were observed in 46.33 and 46.00% at concentrations of 5 and 20 mg/mL, respectively, in relation to the control (62.83%), in addition to a higher chromosomal alteration index by 0.26% at a concentration of 20 mg/mL. In the aqueous extract of leaves of P. cordatus, the greatest inhibitory effects were 33.83 and 35.50% in the concentrations of 5 and 20 mg/mL, respectively, in relation to the control (88.16%) and the highest alteration index chromosomal (3.30%) at 5 mg/mL. The aqueous leaf extracts of P. acinarius and P. cordatus at concentration of 5 and 20 mg/mL inhibit MI %, reveal an irregular recovery or prevent it, and induce chromosomal alterations, suggesting cytotoxic and genotoxic effects on division of meristematic cells of A. cepa.


Introduction
The family Loranthaceae Juss. (Santalales), with 70 genera and 940 species [1] , has a great diversity of mistletoes distributed mainly in tropical areas, but also temperate regions [2] . Among the genera of this family, the Psittacanthus Mart. has approximately 120 species distributed from California (USA) to Argentina, extending through Jamaica and other Caribbean islands [3] . In Brazil, it is the most representative genus, being found in the Amazon, Atlantic Forest, Caatinga, Cerrado, and Pantanal [4] . Its flowers produce nectars enriched with biomolecules attractive to pollinators [5,6] . Besides presenting an ecological interaction, this genus has therapeutic potential, with description of plants used for diabetes and hypertension [7] , with anti-inflammatory [8] , antibacterial, and antioxidant activity [9] , vasodilator [10] and anti-hyperglycemic effect, and genotoxicity [11] .
The species Psittacanthus acinarius (Mart.) Mart. and Psittacanthus cordatus (Hoffmanns.) are among the plants of this genus. These species have been used popularly in the treatment of cancer [12] . Also, P. acinarius has medicinal uses for treating wounds, ulcers, and diabetes and disorders of the urinary, respiratory, cardiovascular, and reproductive systems [12] , while P. cordatus is used to treat eye infections [13] . Despite the medicinal importance of these species, few studies have described the possible toxic compounds synthesized by plants, being required studies that reveal the possible damage to human health.
The cytotoxic and genotoxic effects of plant extracts can be assessed in several cytogenetic assays, such as in the root tips of plant species [14] . Among these assays is the Allium cepa L. test, which allows the contact of the root of the test organism with the tested substance, allowing assessing different doses of the studied plant, showing possible cytotoxic and mutagenic potential [15] . Inhibition in the cell cycle, interruption in metaphases, induction of numerical and structural chromosomal alterations, and sister chromatid exchanges are among the alterations observed cytologically [15] . In addition to assessing various genetic parameters, it also allows verifying the mechanism of action of the substances tested in the DNA and stating that the action of a compound is cytotoxic, genotoxic, or carcinogenic [16] . Therefore, it is an important tool for such assessments [17][18][19] . Thus, this study aimed to assess the cytotoxic and genotoxic potentials of concentrations of aqueous leaf extract of P. acinarius (EAFPa) and P. cordatus (EAFPc) in the cell cycle of A. cepa.

Linking
This study is linked to the Mistletoes Project, under the responsibility of the Research Group Unemat/CNPq FLOBIO (Study of the Flora Bearing Bioactive Substances of Mato Grosso).

Preparation of extracts
The leaves (500 g of each species) were dehydrated in an air-circulation oven at 45°C for seven days and ground in a mill (IKA ® , model M20). The extracts were prepared with 150 g of the crushed material and 750 mL of distilled water at ambient temperature for 60 minutes, being subsequently filtered on ordinary filter cordatus (mistletoe) on Allium cepa Silva, Destacio, Paula, Rodrigues et al paper. This process was repeated three times. The extract was then taken to an air-circulation oven at 45°C for seven days until the water evaporated, obtaining the concentrated aqueous extract.

Model system
Onions (Allium cepa L.; 2n=16) obtained commercially were subjected to rooting in 50 mL of distilled water and maintained in a germination chamber of the biochemical oxygen demand (BOD) type at 25°C and under constant white light for 72 hours. The roots reached an average length of 2 cm and were submitted to concentrations of aqueous extract for each species, that is, D1=5 mg/mL and D2=20 mg/mL, including the negative control (NC, D0=0 mg/mL), which remained exclusively in distilled water. The experimental unit  1). The roots (still in the onions) were subjected to diluted doses in 50 mL of distilled water for 24 hours, whose environment was qualified as treatment (Ti).
After this period, 50% of the roots were collected and the remaining roots were subjected to distilled water without the concentrations for 24 hours to assess the recovery of the mitosis process, which was qualified as the recovery environment (Ri). The roots collected in the process of submission to treatments and the recovery process were fixed in Carnoy solution (95% ethanol + glacial acetic acid, 3:1 v/v), and stored in a refrigerator for further analysis.

Cytogenetic analysis
Root ends were washed in distilled water for four minutes, hydrolyzed in 5N HCL for five minutes, and washed again in distilled water for four minutes. The root ends were transferred to histological slides and sectioned with cutting blades. Subsequently, a drop of Giemsa dye (5%) was superimposed on the coverslip, the tissue was crushed, the excess dye was removed using a filter paper [20] , and photographed under an optical microscope (400x magnification). A total of 3000 cells were identified in each treatment, and normal The data were subjected to analysis of variance and the means compared by the Tukey (for treatments) and Student tests (for submission environment) at the 5% significance level, using the statistical program SISVAR ® for analysis [21] . The compared means were considered different when the conclusive error was

Cytotoxicity effect
An inhibitory effect (P < 0.05) of the mitotic index (MI %) was found in roots of A. cepa for doses of aqueous leaf extracts of P. acinarius (EAFPa) and P. cordatus (EAFPc) in the exposure environments (Ti) and with irregular partial recovery in the recovery environment (Ri) (

Genotoxicity effect
The aqueous leaf extracts of P. acinarius and P. cordatus at the doses D1 (5 mg/mL) and D2 (20 mg/mL) under submission (Ti) and recovery (Ri) presented chromosomal alterations (TABLE 3). However, no chromosomal alterations were observed in the cell cycle of roots of A. cepa exposed only to distilled water, that is, the zero dose (D0) or control (NC) (   [19] , phenolic compounds (rosmarinic acid) [22] , the leaf extract of Parkinsonia aculeata L. [23] , and the aqueous extract of Grewia lasiocarpa [24] . SD -standard deviation. Means followed by the same lowercase letter in the column and uppercase letter in the row do not differ from each other by the Tukey test (for treatments) and Student t-test (for submission environment) at the significance level of 5% probability. The reviewed literature showed no studies revealing the chemical constituents present in the studied plants.
However, several chemical constituents of the secondary metabolism were found in plants of the genus Psittacanthus, which may be related to the inhibition of the cell cycle division. The presence of flavonoids, coumarins, and hydrolyzable tannins was observed in the phytochemical analysis of aqueous and hydroalcoholic leaf extracts P. plagiophyllus Eichl. [25] . Also, found gallic acid, flavonoids, and non-protein amino acid in the soluble fraction of methanol from the aqueous extract of P. calyculatus (DC.) G. Don [26] . Identified