Total phenolic content and antioxidant and anticholinesterase activities of medicinal plants from the State’s Cocó Park (Fortaleza-CE, Brazil)

The State’s Cocó Park in the city of Fortaleza-CE present mainly a mangrove flora and include landscape and medicinal plants. The aim of this study is determining the total phenol content, antioxidant activity against the free radical DPPH and the inhibition of the enzyme acetylcholinesterase (AChE) in ethanolic extracts of 30 medicinal plants and thus assess which plants have potential against Alzheimer's Disease. The plants rich in phenolic compounds with amounts ranging from 297.46 ± 26.94 to 599.30 ± 17.08 mg GAE/g plant extract, which showed greater antioxidant activities (with IC50 against DPPH radical from 3.44 ± 0.16 to 3.73 ± 0.12 μg mL) and higher acetylcholinesterase inhibiting power (IC50 < 20 μg mL) were Anacardium occidentale, Ceiba pentandraLaguncularia racemosa, Mangifera indica, Myracrodrum urundeuva and Terminalia catappa. Then, these species and their constituents are recommended for more specific studies related to Alzheimer ́s Disease.


Introduction
The park bordering the Cocó river occupies an environmental conservation area containing 1,571.29 hectares, making it the largest natural park in an urban area in North/Northeast Brazil and the fourth in Latin America. Consisting mainly of mangroves, the park is a haven for the city's fauna. Being considered a kind of natural nursery for mollusks, crustaceans, fish, reptiles, birds and mammals, mainly Callithrix jacchus (Sousa & Santos, 2016)). The State's Cocó Park besides offering leisure and tourist attractions, gives the opportunity for the elaboration of scientific researches.
Oxidative stress is an imbalance of free radicals and antioxidants in the body, which occurs naturally and plays a role in the aging process and it is related to many diseases such as cardiovascular diseases, acute and chronic kidney disease, neurodegenerative diseases, macular degeneration, biliary diseases, and cancer. There is a close relationship between oxidative stress, inflammation, and aging (Liguori et al., 2018). Oxidative stress is involved in the development of several human pathologies, such as hypertension, atherosclerosis, asthma, cancer, rheumatoid arthritis, cataracts, diabetes mellitus and degenerative diseases (sclerosis multiplies, Parkinson's disease and Alzheimer's disease) (Phaniendra et al, 2015). There is a lower incidence of degenerative diseases in populations that use diets composed of cereals, fruits, vegetables and natural foods, which are rich in antioxidant compounds, the most common being found are phenolic acids, flavonoids, vitamin C, vitamin E, selenium and carotenoids (Falco et al., 2016). Several studies have linked plant antioxidants with AChE inhibition and thus opened up several treatment options for AD (Akram & Nawaz, 2017;Penido et al, 2017).
Drugs that have AChE inhibition as a mechanism of action are called anticholinesterase or indirectly cholinergics.
AChE when blocked is unable to hydrolyze ACh, thus, this neurotransmitter tends to remain active for a longer period in the synaptic cleft, a fact that increases cholinergic transmission. Drugs that block AChE in the peripheral nervous system (PNS), such as neostigmine, are used in atonic constipation, intestinal atony, urinary retention, myasthenia gravis and as an antagonist for myorelaxants. If the AChE inhibitor has an action in the central nervous system (CNS), such as Rivastigmine, it is useful in the treatment of dementia associated with Alzheimer's and Parkinson's diseases (Araujo et al., 2016).
Medicinal plants are relatively safe compared to synthetic drugs, according to the World Health Organization (WHO) it is estimated that about 80% of people worldwide depend on herbal medicines. These drugs are even used to treat neurological disorders associated with AChE regulation (Patel et al, 2018). Thus, this study aims to determine the total phenolic content, antioxidant activity and the inhibition of the enzyme acetylcholinesterase of medicinal plants present in the State's Cocó Park in Fortaleza, Ceará and elaborate a bibliographic survey on the activities reported for plants, to try to correlate with the activities determined in order to discover new herbal agents with potential to be used in the treatment of AD.

Plant extracts
The collection of plant material was carried out at State's Cocó Park in Fortaleza-CE in 2018 and the samples were from specific parts of each plant species according to popular use. All samples were placed in plastic bags and transported to the Laboratory of Chemistry of Natural Products at the State University of Ceará (UECE) where exsiccates were prepared, following the botanical criteria of drying and cataloging (Matos, 1988), then they were deposited in the Prisco Bezerra herbarium of the Federal University of Ceará (UFC) with exsiccate numbers shown in Table 1.  Research, Society andDevelopment, v. 10, n. 5, e7510514493, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i5.14493 To obtain the ethanol extracts, 50 grams of dry sample (oven at 60 ºC) were added to glass vessels containing 200 mL of ethyl alcohol (96%). The samples were left for 10 days, then the resulting solution was filtered and concentrated on a rotary evaporator under pressure at 60 °C to evaporate the ethanol, then placed in a water bath to dry completely.

Total phenol content
The determination of the total phenol content was carried out using the Folin-Ciocalteu method described by Sousa et al. (2007). For each extract, 7.5 mg was dissolved in 10 mL of P.A. methanol (99.8%) using an ultrasonic bath, then transferred quantitatively to a 25 mL volumetric flask and the final volume was made up with methanol. A 100 μL aliquot of this solution was transferred to a 10 mL volumetric flask with 500 μL of the Folin-Ciocalteu reagent and stirred for 30 seconds, 6 mL of distilled water and 2 mL of Na2CO3 (15%) were added, stirring the mixture for more 60 seconds and the final volume was filled with distilled H₂O, the solution was kept at rest for 2 hours in a dark place. The white reagent was conducted under the same conditions. All determinations were made in triplicate. The same procedure was used to prepare the calibration standard curve (y = 0,127x + 0,011, R 2 = 0,995) obtained with 0 a 4 μg. mL -1 gallic acid solutions. The absorbances from the several concentrations were obtained in the UV-Vis (Genesys 10S UV-Vis Thermo Scientic) at 750 nm. The results were determined by interpolation of the data with the gallic acid calibration standard equation and expressed in terms of mg GAE/g sample extract.

Determination of antioxidant activity
The determination of antioxidant capacity was carried out according to the free radical DPPH (2,2 diphenyl-1-pricrylhydrazil) methodology proposed by Yepez et al. (2002), with some modifications. A 6.5x10 -5 mol L -1 DPPH methanolic solution was prepared by diluting 1.3 mg of radical in 50 mL of methanol PA (Neon 99.8%) in a volumetric flask. Then, the DPPH solution was read and corrected to have a wavelength between 0.600 and 0.700 nm. The extracts were initially solubilized to prepare a stock solution with a concentration of 10,000 ppm (15 mg of the extract in 1.5 mL of methanol), afterwards, the stock solution was diluted in the respective concentrations of 5,000, 1,000, 500, 100, 50, 10 and 5 ppm. After dilutions, 1.9 mL of the 6.5x10 -5 mol L -1 DPPH solution and 0.1 mL of the sample solution from each dilution were placed in test tubes to react. For the positive control, quercetin was used in the same concentrations and the negative control consisted of 0.1 mL of methanol with 1.9 mL of the DPPH solution. The test was maintained in the absence of light for 30 minutes and then the reading was performed on the UV-Vis spectrophotometer (Genesys 10S UV-Vis Thermo Scientic), at a wavelength of 515 nm. All procedures were performed in triplicate. The percentage of DPPH free radical inhibition by extracts at different concentrations was calculated by expressing the scavenging index percentage (IV%): SI% = (AbsDPPH -AbsAmostra /AbsDPPH) x 100.
The effective concentration to inhibit 50% of the free radical DPPH (CE50) was obtained with the aid of the Excel 2019 software, using the sample concentration values and the scavenging index (SI%). Dispersion graphs were generated whose linear equations were used to obtain the values of the mean and standard deviation. For comparison purposes, a calibration curve was constructed with different percentages of the quercetin flavonoid, which has high antioxidant activity in the DPPH free radical to calculate the IC50 of the extracts.

Statistical analysis
The statistical analysis was performed using the Graph Pad Prism v5.01 program, where the data were submitted to the one-way test of variance analysis (ANOVA) to determine the statistical differences followed by the multiple comparison between pairs by the Tukey test, considering significant values at P <0.05. To analyze the correlation between the data, Pearson's correlation coefficient was used in the Microsoft Excel 2019 software, which measures the degree of linear correlation between two quantitative variables.

Results and Discussion
In the State's Cocó Park, 30 species of medicinal plants were collected and the plant part was chosen according to the popular use. About the families the most prevalent was Fabaceae (8), followed by Anacardiaceae (5), Rubiaceae (2) and Combretaceae (2). The activities of the plants are displayed in Table 2. The most common use of plants is for gastrointestinal diseases as diarrhea and dysentery (12/30), following the treatment of dermatitis, wounds (healing) and mycosis (9/30); for inflammation and infections -7 plants; respiratory tract diseases such as bronchitis, asthma and cough -6 plants; diabetes -5 plants and for cancer or anti-tumor Annona glabra, Cordia oncocalyx and Moringa oleifera. An ethnobotanical survey with High School students about medicinal plants in Maranguape-Ceará showed that the most cited therapeutic indications were related to diseases of the respiratory, digestive and circulatory systems (Castro et al, 2021). In another study 22 plants from Caxias city of Maranhão State, cited by the population, being mainly indicated for poor digestion, insomnia, hypertension, cough, flu and inflammation (Silva et al, 2021). Then, these informations confirm that the medicinal plants present in Cocó Park are representatives of the popular use of plants for medicinal purposes. In general, these diseases can be related to oxidative stress and to the action of the acetylcholinesterase enzyme, then phenolic compounds can act in several pathogens and strengthen the immune system, contributing to treat Alzheimer´s disease.  Research, Society and Development, v. 10, n. 5, e7510514493, 2021 (CC BY 4. Cholinergic stimulation by administration of acetylcholinesterase inhibitors, enhances intestinal antimicrobial activity and prevents systemic dissemination of pathogenic bacteria, and the mechanism is a crucial pathway between neural and immune systems that acts at the mucosal interface to protect the host against invading pathogens (Al- Barazie et al., 2018).
Phenolic compounds including flavonoids are well-known antioxidants and presenting also many other important bioactivities for human health, curing and preventing many diseases, such as antibacterial, anti-cancer, cardioprotective, immunostimulant and anti-inflammatory and skin protective effect from UV radiation (Tungmunnithum et al., 2018).
The antioxidant and anticholinesterase activities of plant extracts may reveal the potential for the treatment of Alzheimer's Disease (AD). Bigueti, Lellis and Dias (2018) demonstrated that the intake of antioxidant substances contributed to the reduction of the disease incidence, and can be used as an alternative therapy for the treatment of the disease.
Previous studies (Achkar et al., 2014;Silva et al., 2010) have shown that the total phenol content above 100 µg mL -1 is already considered a high value in plant extracts, and in the ethanolic extracts of thirty plants from Cocó Park, 21 entered in this relation. Taking into account the best results in the present work, it is observed that ten species demonstrated antioxidant activity correlating linearly with the phenol content: A. occidentale, C. pentandra, H. stigonocarpa, L. racemosa, L. férrea, M. indica, M. tenuiflora, M. urundeuva, S. mombim, T. cattapa, whose total phenol content ranges from 297.46 ± 26.94 µg mL -1 (S. mombim) to 599.30 ± 17.08 µg mL -1 (M. tenuiflora) -with antioxidant activities with IC50 against DPPH radical ranging from 3.44 ± 0.16 to 3.73 ± 0.12 µg mL -1 , respectively (Table 3). Such a result is expected, since phenolic compounds have a high antioxidant capacity. Therefore, many medicinal plants present in Cocó Park are sources of antioxidant compounds. The results obtained in the inhibition of AChE activity were compared to that of the alkaloid physostigmine, which was the first discovered natural inhibitor. Santos et al. (2018), determined the anticholinesterase activity of extracts and fractions from 54 plants and classified the action according to the IC50 values as: high potency (IC50 <20 μg mL -1 ); moderate power (20 <IC50 <200 μg mL -1 ) and low power (200 <IC50 <1000 μg mL -1 ). Evaluating the results obtained in the AChE inhibition test, it is observed that 15 plants presented results below 20 μg mL -1 , therefore with a high inhibition power, they are: A. occidentale, A. glabra, C. pulcherrima, C. pentandra, C. pyramidale, C. tapia, H. impetiginosus, L. racemosa, M. indica, M. urundeuva, S. cumini, T. esculenta, T. guianensis, T. catappa and Z. joazeiro. In the correlation of the total phenol content with AChE enzyme inhibiting action, considering the 15 best results, but only six species that presented IC50 between 11 and 14 μg mL -1 correlate linearly with the levels of phenols: C. pulcherrima, M. indica, M. urundeuva, S. cumini, T. guianensis and T. catappa.
A weak correlation was obtained between the total phenolic content and AChE inhibition activity and these results are corroborated by studies by Barbosa Filho et al. (2006), who showed many other types of compounds exerting this activity.
They report that out of 260 chemical compounds studied with AChE inhibiting action, only 51 were phenolic compounds (18 coumarins, 14 flavonoids, 13 benzenoids, 3 stilbenes, 2 lignans and 1 quinoid), however alkaloids were the majority class with 139 compounds. Another study reported by Santos et al. (2018) on the anti-acetylcholinesterase activity of plant species extracts, it was concluded that out of 54 plant species belonging to 29 different families, 36 compounds were identified, of which 16 showed potent inhibition, being superior to galantamine (1 terpene, 2 coumarins and 13 alkaloids) and another 20 compounds with lower potency (phenolic and flavonoid acids). Observing that the phenolic compounds did not show prominent AChE inhibition results, and the alkaloids represent the class with the highest potency in the evaluation.
Similarly, previous work with plants from living pharmacies, Morais et al. (2013) deduced that there was no linear correlation between AChE enzyme inhibition and the mean inhibition concentration of the DPPH radical -CE50 from the extracts of the tested plants.
Other studies have also shown that the mechanism of action of AChEIs is not limited to their effects on the neuron-toneuron transmission involving acetylcholine but extends to their protective effects of a cell against free radical toxicity as well as increased production of antioxidants. A diet rich in polyphenols and polyunsaturated fatty acids helps boost the production of the brain's stem cells -neurogenesis-and strengthens their differentiation in different types of neuron cells. These results give support to the hypothesis that a diet made up of foods rich in antioxidant substances could delay the onset of DA or even slow down its evolution (Valente et al., 2009).
Thus, agents that combine antioxidant properties with the inhibition of AChE are expected to find usefulness in the management of AD (Tabet, 2006).

Conclusion
In conclusion, of the 30 species studied 6 stood out taking into account high levels of phenols and better antioxidant and acetylcholinesterase inhibition effects: A. occidentale, C. pentandra, L. racemosa, M. indica, M. urundeuva, T. catappa.
Therefore, these plants can be considered the most promising as sources of phenolic compounds to be used in the treatment of Alzheimer's disease, due to their relevant action of inhibiting free radicals and the enzyme acetylcholinesterase.