Evaluation of the ethanolic extract of Myconia albicas (Old Cinnamon) in the alternative model of anxiety in zebrafish

Myconia albicans is the most abundant genus of the melastomaceous botanical family. In Brazil, it is known as "Canela de Velho", and is used mainly in inflammatory processes. Due to the scarcity of scientific works that report its pharmacological effect, the objective of this study was to perform its chemical characterization, toxicity, and the possible effect on the nervous system, using the experimental model Zebrafish (Dario regio). The organic extract of ethyl acetate (EtOAc) was elaborated through the dry leaves of the cinnamon tree of old. The EtOAc sample was submitted to preliminary phytochemical screening and showed the presence of phenols, flavanoids of the flavones type, flavonols and xanthones, triterpenoids, and alkaloids. Then, flavonoid compounds (369.58±0.074mgEQ/g) and total phenols (81.48±0.005mgEAG/g) were determined. EtOAc was evaluated for its toxicity, with Artemia salina, which showed low toxicity in all the studied doses. In the pharmacological action tests, the doses of 3, 10, or 30 mg/mL were used; 20 μL; oral (v.o.) or intraperitoneal (i.p.). The locomotor activity was evaluated in the open field test, which showed a decrease in the fish. And the anxiolytic activity, in the light & dark preference test, showed that EtOAc at 30 mg/mL, when administered intraperitoneally (IP), has an anxiolytic effect similar to Diazepam IP Research, Society and Development, v. 11, n. 1, e50811125048, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.25048 2 control. Thus the EtOAC of M. albicans showed the presence of total phenols and several flavonoids, with low toxicity, in which the highest dose presented anxiolytic effect in Zebrafish.


Introduction
Anxiety is the most common psychiatric disorder. WHO data show a worldwide prevalence of this disease in the order of 3.6%. In the American continent, it reaches 5.6% and in Brazil it is present in 9.3% of the population, with the highest number of cases among countries in the world (Ferreira et al., 2017;Garakani et al., 2020;Mufford et al., 2021) Classic pharmacological therapy in the treatment of anxiety is based on the use of traditional anxiolytic/hypnotic agents, such as benzodiazepines, which have numerous undesirable side effects Penninx et al., 2021;Reis et al., 2017). In 2006, the National Policy of Integrative and Complementary Practices (PICS) and the National Policy of Medicinal Plants and Phytotherapies were implemented in the Brazilian Unified Health System (Brasil. Ministério da Saúde, 2004Figueredo et al., 2014).
The Myconia albicans, populously known as "Old Cinnamon or Canela de Velho", is a species of the Brazilian cerrado and is the most abundant genus of the botanical family melastomatacea, with 4,300 species. The leaves of "Canela de Velho" are widely used in popular medicine, being sold in fairs and stores of natural products and used to treat gastric Research, Society andDevelopment, v. 11, n. 1, e50811125048, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.25048 3 disorders, rheumatoid arthritis and bacterial infections (Celotto et al., 2003;De Oliveira Viana Arruda et al., 2008;Lima et al., 2020;Vasconcelos et al., 2006). Zebrafish (Danio rario Hamilton 1822) is a teleosteal freshwater fish (2.0 cm long and weighing around 1.5g), and is considered an ideal alternative model for human disease research. Zebrafish (or Paulistinha do Brasil) has an environment of 70% genetic homology with human beings, besides other advantages such as low cost, easy handling and maintenance, little space for maintenance, fast embryonic development, and its behavior can be easily observed and quantified (Canedo & Rocha, 2021;Maximino et al., 2010;Mesquita et al., 2017;Sborgi et al., 2021;Stewart et al., 2013).
As the tea from the leaves of Miconia albicans has been widely used by the Brazilian population because of its antiinflammatory effects, and the anxiety presents in its mechanisms inflammatory components, this study aimed to evaluate the pharmacological potential of the ethanolic extract extracted from the leaves of Miconia albicans, commercialized in the city of Fortaleza, in the treatment of anxiety in zebrafish models.

Preparation of extracts
Samples of the ethanolic organic extract of ethyl acetate from the old cinnamon (EtOAc) were extracted from the dried leaves acquired commercially (Chás da Amazônia, Lot: 138) in a natural products store in the city of Fortaleza. The acquisition of EtOAc followed the parameters of Matos' methodology (Lorenzi & Matos, 2002), since, for the author, "every 70 grams of cinnamon leaves samples of old people, three liters of EtOAc solvent are used".

Phytochemical profile
EtOAc was subjected to a preliminary phytochemical screening in order to identify secondary metabolites. The compounds present in the samples were shown as present (+) or absent (-) (Lorenzi & Matos, 2002).

Total phenolic compounds and content of total flavonoids
The phenolic compounds were determined using the Folin-Ciocalteu method, in which the reference parameter is gallic acid (Slinkard & Singleton, 1977).
The characterization of the total flavonoid content was done according to Sousa et al. (Sousa et al., 2015) using the aluminium method. The absorbances were read by the spectrophotometer at 415nm, and all tests were done in triplicate. The results were displayed as mean ± standard deviation (SD), and the correlation coefficient defined using Excel Software (Lemos et al., 2011).

In vitro toxicity
We used 10 nauplii in larval state of Artemia salina, conditioned in the microplate 96 wells with the ethanolic extract in the following concentrations: 100, 500 and 1000 µg/mL (Meyer, Ferrigni, & Putnam, 1982). After 24 hours, CL50 (lethal concentration for 50% of the nauplii) was calculated, being the toxic potentials classified as non-toxic/toxic.

Open-field Test
The locomotor activity was evaluated according to Magalhães et al.(Magalhães et al., 2017) After 1 hour of treatment time, the animals were placed in Petri dishes (130mm diameter), previously marked in four quadrants and immersed in 60mL of water from the aquarium. The locomotor analysis was based on the number of crossings between the marked quadrants.

Light-Dark Preference Test
For the evaluation of the anxiolytic activity, the Clear & Dark Test was used according to the methodology proposed by Gebauer et al (Gebauer et al., 2011). The administration of the drugs occurred 60 minutes before each test. The animals were then positioned in the light zone in a glass tank (height: 7.0cm; width: 9.0cm; length: 18.0cm; black side: 9.0cm and white side: 8.0cm) with 3cm of water, and evaluated within 5 minutes the following items: I) Latency time: interval of the first passage to the dark compartment; II) Time in the light: total time to stay in the light zone; III) Number of crossings: Total number of crossings made between the light and dark zones. Table 1 shows the yield of Ethyl Acetate extract from the Old Cinnamon after freeze-drying, in which, for each 70g of raw material, 3L of solvent with a yield of 0.5314g was used.

Determination of total phenolic compounds
The concentration of phenolic compounds was reached after the standard gallic acid curve, which varied significantly (p<0.05). (Table 3)

Determination of the total flavonoid content
The flavonoid content of EtOAc was measured using the aluminum method, using quercetin. The total flavonoid concentration was significant (p<0.001), from 0.61 to 369.58 mg of EQuer/g of extract. (Table 3)

Toxicity to Artemia salina
The lethality test before Artemia salina did not cause mortality of 50% of the nauplii, confirming no toxicity in the sample, as illustrated in Table 4.

Open-field test (Locomotor behavior)
The tests of locomotor activity with EtOAc via oral route in doses 10 and 30 mg/mL; 20 µL showed compromise in the locomotor system of the fish, since it reduced significantly (p<0.05 or p<0.001 vs. Naive) the locomotor activity of zebrafish (AL = 35.9 or 56.7%) (Figure 1).

Discussion
Myconia represents one of the largest genera of the Melastomatacea family and with approximately 1000 species.
Studies show that this genus has several biological activities such as analgesic, antimicrobial, fungicidal and trypanocidal effects Li et al., 2001;Pieroni et al., 2011;Torrão et al., 2012;Lemos Vasconcelos et al., 2003) The The phenolic compounds, because they have an antioxidant effect, are able to neutralize the activity of free radicals present in the body, and these are directly related to several chronic-degenerative diseases such as diabetes, cancer, inflammatory processes and cardiovascular diseases (Bessa et al., 2014), in addition to diseases of the central nervous system (Lajolo, 2009).
Artemia salina is a filtering crustacean that feeds basically on bacteria, unicellular algae, small protozoa and debris dissolved in the medium. A. salina is a kind of microcrustacean of the Anostraca order, used in this work as a bioindicator of toxicity. Studies prove the toxic action of several natural substances to this crustacean (Barroso Rios, 1995;Lagarto Parra et al., 2001;Martins et al., 2014;Scalco & Munhoz, 2016;Widdows, 2009). Its use is easy to manipulate, besides having low economic cost (Nascimenlo et al., 2008). It was found that EtOAc has no toxic effect on Artemia salina, with no mortality of 50% of the animals. The study of Scalco (Scalco & Munhoz, 2016) tested the toxicity of the methanolic extract of M. albicans in different concentrations, also using the Artemia salina for 24 hours. The results confirm the low toxicity of M. albicans. The toxicity tests are elaborated with the objectives of evaluating or predicting the toxic effects on biological systems and dimensioning the relative toxicity of the substances (Forbes & Forbes, 1994).
For decades, animal models have been used in neuropsychopharmacology for pre-clinical studies, seeking to understand the pathophysiology and new drugs for the treatment of mental disorders (Nunes & Hallak, 2014 M. albicans extract, at doses of 10 and 30 mg/mL, showed decreased locomotor activity in the open field model in zebrafish. Through the low motor activity and the reduction of the exploratory activity of the animal in relation to the new environment, a possible action of the extract in the central nervous system is signaled, interpreted as a possible anxiolytic effect (Taylor et al., 2010).
To evaluate this anxiolytic effect of EtOAc, the light & dark preference test was used, in which the Zebrafish presents a behavior called scottotaxis which refers to the preference for dark environments. This fish species tends to remain about 80% of the time in the darker environment (Maximino et al., 2010(Maximino et al., , 2007. Zebrafish treated with the highest dose of M. albicans extract had an anxiolytic effect when compared to control and Diazepam. Both showed a very similar length of stay in the clear zone. Thus, the fish that received the EtOAc stayed 80.8% of the time in the lighter zone, and the animals that received the Diazepam stayed for 81.5% of the time in the clear, against less than 4% in relation to the DMSO control and 1.8% of the naive. This effect can be justified by the large amount of phenolic and flavonoid compounds found in M. albicans, since these bioactive compounds have the ability to overcome the Blood-Brain Barrier, promoting a protective effect on the Central Nervous System through its antioxidant action and neuronal modulation (Lajolo, 2009). According to some authors, flavonoids have high affinity as binders of GABA A receptors in the Central Nervous System (Fernández et al., 2006;Hanrahan et al., 2011;Wasowski & Marder, 2012), leading to the anxiolytic effect observed in zebrafish treated with ethanolic extracts of M. albicans.

Conclusion
The ethanolic extract of ethyl acetate from Myconia albicans (Old Cinnamon) presented, in its composition, several phenols and flavonoids with a good yield and low toxicity effect for Artemia salina. In Zebrafish models, M. albicans extract showed that the dose of 30mg/mL, both in the oral and intraperitoneal routes, presents an anxiolytic effect. However, further studies are needed in order to evaluate the main active compounds in Myconia albicans extract, in order to elucidate the anxiolytic action mechanisms present in this plant.