Essential Oil of Cynophalla flexuosa and its Cytotoxicity, Antioxidant, and Anti-Acetylcholinesterase Effect

Published in Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2021, pp. 480–482.

The interest in glucosinolate (GLS) hydrolysis products has grown due to their preventive potential against diseases by inducing a variety of physiological functions, including antioxidant activity, enzymatic regulation, apoptosis and cell cycle control, thus inhibiting the development of various types of cancer [[1]]. These are biologically inactive compounds which, when undergoing enzymatic hydrolysis by β-thioglucosidase (myrosinase), produce isothiocyanates, nitriles, and thiocyanates, which act in the defense of plants against herbivores and pathogens [[2]]. As these are found expressively in the Capparaceae family, the objective of the present study is to analyze the chemical composition and to evaluate the toxicological, anticholinesterase, and antioxidant potential of the essential oil of Cynophalla flexuosa (L.) J.Presl, the search for active substances of which offers a way to study the chemical-biological contribution of the oil.

Table 1 shows the compounds identified by GC/MS, with isothiocyanates and nitriles representing 39.4%. These compounds are identified in the essential oils of foods such as mustard, which act as natural preservatives due to their antimicrobial and antioxidant activities [[4]].

TABLE 1. Composition of the Essential Oils of Cynophalla flexuosa

C. flexuosa oil exerted high toxicity against Artemia salina, with an LC50 of 97.54 μg/mL. Nitriles present in the volatile oil of Bunias erucago flowers are related to their cytotoxic activity against human bladder cancer cell lines, with IC50 ranging from 7.8–418.4 μg/mL [[5]].

Studies point to the superior toxicity of isothiocyanates, being 1000 times more potent than nitriles when tested in human liver cancer cell line, but equally genotoxic. The cytotoxic potential of nitriles is directly related to their structure, as compounds that contain sulfur, aromatic rings, or possess longer chains have higher toxicity [[6]]. Analysis of the toxicity mechanism of glucosinolate derivatives of isothiocyanate compounds show that blockade of the G2/M phase of the cell cycle causes cell death via necrosis, and nitriles promote necrosis and apoptosis, which gives these compounds chemoprotective properties [[7]].

The essential oil showed strong inhibition of acetylcholinesterase with IC50 of 7.9 μg/mL when compared to the control with physostigmine, which has a value of 1.2 μg/mL. This activity is probably related to the presence of isothiocyanates. Burcul et al. [[8]] tested 11 isothiocyanate derivatives and concluded that those of aromatic origin have higher inhibitory activity of AChE and BChE enzymes. Inhibition of these enzymes is the main form of treatment against Alzheimer′s disease. Due to the many side effects that conventional drugs have, there is a need to find safe and less aggressive ways to treat this disorder.

For all oil concentrations tested, relevant capture percentages were obtained, ranging from 87.1 to 91.3%, showing better percentages for the first two concentrations compared with those obtained for ascorbic acid, with an IC50 of 183.2 μg/mL, only two times higher compared to the 62.7 μg/mL positive control. Other species rich in isothiocyanates show a moderate ability to capture the ABTS radical, such as the methanolic extract of Capparis spinosa L. fruits, which showed elimination activity ranging from 23.1 to 44.9% [[9]]. On the other hand, the ethanolic extract of Moringa oleifera L. leaves showed an IC50 of 34.7 μg/mL, which is higher than the one obtained in this study [[10]].

Isothiocyanates are known to be promising compounds in the prevention and treatment of neurodegenerative disorders such as Alzheimer′s, Parkinson′s, and Huntington's disease, among others, as they act as an antioxidant by activating the Nrf2/ARE pathway that regulates several genes involved in the protection against neurodegenerative conditions, where oxidative stress plays a fundamental role in its development [[11]].

The essential oil showed significant activity in the protection of deoxyribose, showing different capacity for each system (FeSO4 < H2O2 < H2O2 + FeSO4). The best protection was observed in the H2O2 + FeSO4 system, with percentages ranging from 36.7 to 89.7%. This result is relevant because these compounds participate in the Fenton reaction, forming the hydroxyl radical (·OH), considered the most reactive among ROS. It can affect nitrogenous bases, denature proteins by oxidation of sulfhydryl groups and disulfide bridges, as well as cause damage to carbohydrates and remove hydrogen atoms from methylene groups of polyunsaturated fatty acids, initiating the lipidic peroxidation process [[12]].

Evaluation of the antioxidant activity with Lepidium meyenii tubers found that in addition to the ability to eliminate free radicals, the aqueous extract also has the property of protecting cells against oxidative stress, where 1–3 mg/mL of the extract showed protection from deoxyribose 57–74%; these positive results were attributed to the presence of polyphenols and isothiocyanates [[13]]. Furthermore, these compounds are known to have indirect antioxidant characteristics, such as preventing any increase in ROS production, thus decreasing the activity of cisplatin-induced oxidative enzymes and avoiding renal damage in the body [[11]].

Plant Material and Obtaining Essential Oil. The pods of C. flexuosa were collected in 2019, in Jardim, Ceara, Brazil. A sample of the botanical material was deposited and identified in the Hebario Caririense Dardano de Andrade Lima of the Universidade Regional do Cariri under registration number 13257. For the extraction of the essential oil, the pods (3.80 kg) were previously dried in an oven at 55°C. The essential oil was obtained by hydrodistillation using a Clevenger apparatus for a period of 2 h. After this step, anhydrous Na2SO4 was added, yielding 0.0076%.

GC/MS Analysis. The C. flexuosa essential oil was analyzed using a Shimadzu GC-MS QP2010 series fitted with a fused silica Rtx-5MS (30 m × 0.25 mm I.D.; 0.25 m film thickness) capillary column and temperature programmed as follows: 60–240°C at 3°C/min, then to 280°C at 10°C/min, ending with 10 min at 280°C. The carrier gas was He at a flow rate of 1.5 mL/min, and the split mode had a ratio of 1:50. The injection port was set at 220°C. Significant quadrupole MS operating parameters are as follows: interface temperature 240°C; electron impact ionization at 70 eV with scan mass range of 40–350 m/z at a sampling rate of 1.0 scan/s; injected volume 1 μL of 5 μg/mL solution in dichloromethane. Constituents were identified by computer search using digital libraries of mass spectral data (NIST 08) and by comparison with authentic mass spectra [[14]].

Toxicity Assay. Acute toxicity in Artemia salina was evaluated by the method described by Meyer et al. [[15]] with modifications. The final sample concentrations dissolved in saline water were 25, 50, 100, 250, 500, and 1000 μg/mL. A negative control was used to demonstrate that dissolution conditions were inactive. Potassium chromate (26 mM) was used as a positive control. All tests were done in triplicate and repeated three times. Results are expressed as percentages of larvae killed after 24 h. Nonlinear regression analysis was used to determine the LC50.

Acetylcholinesterase Bioassay. The acetylcholinesterase inhibition assay was determined using the method described by Ellman et al. [[16]]. The following solutions were added to 96-well plates: 25 μL acetylcholine iodide (15 mM), 125 μL 5,5′-Dithiobis 2-nitrobenzoic acid in Tris/HCl solution (50 nM, pH = 8, 0.1 M NaCl and 0.02 M MgCl2·6H2O (Ellman′s reagent), 50 μL Tris/HCl solution containing bovine serum albumin, and 25 μL essential oil at a final concentration of 2 mg/mL. The absorbance was measured for 30 s at 405 nm; then 25 μL of the acetylcholinesterase enzyme was added and the absorbance read every minute for 25 min. Absorbance values for the oil were extinct, and the percentage of acetylcholinesterase inhibition was calculated by comparing the reaction rates (substrate hydrolysis) of the samples in relation to the blank. The standard used as control was physostigmine, and the tests were performed in triplicate.

ABTS ·+ Free Radical Capture. Radical capture was measured by the method proposed by Rufino et al. [[17]]. Initially the ABTS·+ solution was obtained by reacting the ABTS solution (7 mM) with the sodium persulfate solution (140 mM), which remained for 16 h in the dark. The concentrations evaluated ranged from 25 to 1000 μg/mL. In a dark environment, a 30 μL aliquot of each concentration was transferred to 3.0 mL ABTS·+ radical vials. The reading was performed using a spectrophotometer at 734 nm after 6 min of reaction of the mixture. Pure ABTS·+ radical was used as negative control, methyl alcohol as blank, and ascorbic acid as positive control.

Oxidative Degradation of 2-Deoxyribose (2-DR). The method used was adapted by Puntel et al. [[18]]. The ethanolic solution of the essential oil was prepared at concentrations of 500, 250, 100, and 50 μg/mL. For the test, 80 μL potassium phosphate buffer (0.5 M), 120 μL deoxyribose (20 mM), 80 μL H2O2, 80 μL FeSO4 (1 mM), and 340 μL distilled water were added to 100 μL of each concentration. For the blank, the same mixture was prepared in the absence of deoxyribose, H2O2, and FeSO4. All samples were incubated for 30 min at 37°C. After this time, 750 μL trichloroacetic acid (2.8%) and thiobarbituric acid (0.6%) were added, and the whole re-incubated for 20 min in a heated bath at 100°C. The reading was performed in a spectrophotometer at 412 nm.

Statistical Analysis. Values were expressed as means ± S.E.M. (n = 3). For all tests, after normalization of data, a nonlinear regression curve test was performed using the statistical program GraphPad Prism version 7.0 to obtain IC50 and LC50 values. With the obtained values, ANOVA and Tukey′s test for multiple comparison between pairs were performed, and values of P < 0.05 were considered significant.