Pharmacological effects of Nephrolepis exaltata L. (fern) aqueous extract on an insect-based model (Nauphoeta cinerea)

In this work we used semi-isolated heart of the cockroach Nauphoeta cinerea for the investigation of the pharmacological effects of extracts (aqueous, 1:1, 1:2, 1:4 and 1:8) from Nephrolepis exaltata L. leaves, a popular ornamental fern considered to be safe. The use of insects in experimental studies has grown due to the easy handling, proliferation/growing assuring its rapid obtention, and absence of ethical issues. An aqueous extract 0.2 % was obtained after maceration of 1 g N. exaltata leaves powder with 20 mL of distilled water (1:20). Diluted extracts in water were obtained to have the following proportion 1:1, 1:2, 1:4 and 1:8. Experiments (n=4) consisted of 200 μL addition onto semi-isolated heart preparation of N. cinerea with concomitant heart beating counting. Aqueous, 1:1 and 1:2 extracts paralyzed completely the heart beatings of cockroachs (p<0.05 compared to saline control), but not 1:4 or 1:8, which showed only a slight decline (p>0.05 compared to saline control). A preliminary thin layer chromatography showed the presence of unidentifi ed terpenoid in aqueous extract of N. exaltata. These pharmacological fi ndings of N. exaltata can be exploited for future use as insecticide or as dose-dependently cholinergic agent. Research Article Pharmacological effects of Nephrolepis exaltata L. (fern) aqueous extract on an insect-based model (Nauphoeta cinerea) Danrley Mariel Egidio Sanchez1, Jocimar de Souza2, Yoko Oshima-Franco2 and Sandro Rostelato-Ferreira1* 1Biological Sciences Course, Health Sciences Institute, Paulista University (UNIP), Sorocaba, SP, Brazil 2Post-Graduate Program in Pharmaceutical Sciences, University of Sorocaba (UNISO), Sorocaba, SP, Brazil *Address for Correspondence: Sandro RostelatoFerreira, Biological Sciences Course, Health Sciences Institute, Paulista University (UNIP), Sorocaba, SP, Brazil, Email: sandrorostelato@yahoo.com.br Submitted: 16 March 2018 Approved: 26 March 2018 Published: 27 March 2018 Copyright: 2018 Sanchez DME, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The objectives of this work were: 1) to evaluate pharmacological effects of the aqueous extract of fern leaves from Nephrolepis exaltata on semi-isolated heart of cockroaches Nauphoeta cinerea, and 2) to ind bioactive compounds in N. exaltata aqueous extract using thin layer chromatography. Our hypothesis was based on the in luence of aqueous extract could have on cardiac beatings, since it is known that substances able in paralyzing the cardiac beatings could have insecticidal potential [11 ]. Research on cockroaches is becoming frequent by its easy maintenance, low cost, rapid reproduction and there are no restrictions on its use [12]. Besides, in recent years, organic agriculture has become an alternative to food consumption, since the problems that insecticides can cause to human health and the environment are already known [13]. However, the extract from Nephrolepis exaltata tested here showed a cardiotoxic effect, with a possible effect induced by a terpene, could be used as an insecticide.

Botanical material
The botanical material was commercially purchased from Companhia de Entrepostos e Armazéns Gerais de São Paulo (CEAGESP), in Sorocaba city, cleaned and weight (6.4 g), oven-air dried during 24 h at 40°C, and further milled to obtain 1.11 g of powder (percentage of incoming=17.15 %).

Extracts obtention
The extract was obtained by water maceration method [14]. For each 1 g plant powder was used 20 mL distilled water, put in contact during 48 h, resulting in a 0.2% aqueous extract. Dilutions of aqueous extract in water were also performed to have 1:1, 1:2, 1:4 and 1:8 extracts.

Biological material
Cockroaches (Nauphoeta cinerea) commercially purchased from Pet Shop Bicho & Cia, Sorocaba city, were put in an adequate recipient for its creation. The cockroaches had water and food (as fruits and vegetables) ad libitum.

Semi-isolated heart of Nauphoeta cinerea preparation
The cockroaches were immobilized using entomological pinheads. The lateral margins of abdomen were cut along on each side [15] and the cuticle removed using an ophtalmic scissor to expose the visceral organs which were carefully keep out using a tweezer, in order to view the vascular system. After, the insect was put under a 30X magnifying glass (Carl Zeiss, Germany) aiming to visualize the cardiac beatings. The mean beatings/minute in the irst 5 minutes were considered as zero time. After the extract addition (200 μL of each), the beatings were accounted in intervals of 5, 15, and 30 minutes [16]. Experimental groups (n=4) were randomly distributed in: saline control, pure extract, extract diluted in water 1:1, 1:2, 1:4 and 1:8. The obtained results were compared to pharmacological controls as acetylcholine chloride (110 μM, MW 181.66, Sigma-Aldrich) and adrenaline (110 μM, MW 183.20, Sigma-Aldrich), at the same 200 μL.

Statistical analysis
Results were expressed as the mean ± standard error mean (±SEM). The Student's t-test or repeated measures ANOVA were used for statistical comparison of the data.
The signi i cance level was set at 5%. Figure 1 expresses the percentage of heart beating (Y-axis) in function of counting in intervals of 5, 15 and 30 minutes (X-axis) of classic pharmacological drugs as adrenaline and acetylcholine in comparison to saline. Notice the effects sympathomimetic (increase of heart beatings) and parasympathomimetic (decrease of heart beatings) in semi-isolated heart of N. cinerea, induced by adrenaline and acetylcholine, respectively. Adrenaline is the active sympathomimetic hormone from the adrenal medulla. It stimulates both the alpha-and beta-adrenergic systems, consequently stimulates the heart [20]. Acetylcholine is a neurotransmitter found at neuromuscular junctions, autonomic ganglia, parasympathetic effector junctions, a subset of sympathetic effector junctions, and at many sites in the central nervous system 21]. At heart, acetylcholine acts causing bradycardia due to blockade of postjunctional muscarinic receptors, whereas the slight recovery seen at 15 and 30 minutes can express the vagal stimulation [22]. Figure 2 shows the pharmacological effects of extracts in different concentrations. The heart beating blocker effect seen with aqueous and diluted 1:1 and 1:2 extracts can be exploited for insecticidal use of the fern N. exaltata, which is among the most popular ornamental ferns [23]. Diluted 1:4 N. exaltata extract did exhibit similar pro ile of acetylcholine, which means that the blocker effect is a dose-dependent pharmacological event. On the other words, other than insecticidal use can be ventured to N. exaltata, for example, as a cholinergic agonist. However, the future pharmacodynamic of N. exaltata must be studied. Figure 3 shows the chromatographical pro ile of N. exaltata aqueous extract face to two systems of mobile phases and three chromogenic agents, which matched to each other, resulted in the phytogroup identi ication. Note that N. exaltata exhibits the same chromatographical pro ile of terpenes ( Figure 3A, 3B) but does not as lavonoids ( Figure 3E).  both 110 μM, n=4), in comparison to SALINE CONTROL (n=4), on the exposed preparation, with sympatho-and parasympatho-mimetic effects, respectively. * p<0.05 compared with saline control.

Results and Discussion
Terpenes are a diverse group of more than 30,000 lipid-soluble compounds. Terpenoids are classi ied according to the number of isoprene units they contain. Thus, 1 unit, hemiterpene; 2 units, monoterpenes; 3 units, sesquiterpenes; 4 units, diterpenes; 5 units, sesterpenes; 6 units, triterpenes; and 8 units, tetraterpenes [24]. Terpenes exhibit a range of toxicity which confer antimicrobial properties and a wide range of effects within the insect central nervous system, but very low toxicity to mammals [25]. Phytochemical studies must be carried out in order to identify the terpene structure in aqueous extract of N. exaltata.

Conclusion
Concluding, aqueous extract of fern Nephrolepis exaltata is cardiotoxic to cokroaches Nauphoeta cinerea on a dose-dependent manner. An unidenti ied terpene present in N. exaltata could contribute to this effect.