Phytochemical, antioxidant and anticholinesterase profiles of Musanga cecropioïdes R. Br. (Urticaceae) from Côte d'Ivoire

This study is devoted to Musanga cecropioides (Urticaceae), a tropical plant whose leaves and root bark are used in traditional medicine in the treatment of various pathologies. The objective of this work was to study qualitative and quantitative composition of phenolic compounds in extracts of the leaves (McF) and root bark (McR) Musanga cecropioides and to evaluate their antioxidant and anticholinesterase properties. Phytochemical screening using TLC showed that the extracts contain, along with phenolic compounds such as coumarins, flavonoids and tannins, other bioactive phytocompounds namely sterols, terpenes and alkaloids. Quantitative analysis of phenolic phytoconstituents by spectrophotometry showed that contents of total flavonoids and polyphenols in leaves (7.753% and 119.389 mg EAG / g, respectively) are higher than those in the root bark (1.41% and 105.944 mg EAG / g, respectively). The antioxidant activity of total and selective extracts evaluated by FRAP and DPPH methods was found to be significant compared to vitamin C. All the extracts of Musanga cecropioides showed good anticholinesterase activity with percentages of inhibition of the acetylcholinesterase (AChE) ranging from 51.952 to 63.589%.


Introduction
The Urticaceae family, with more than 2000 species of morphological diversity, is distributed in tropical and temperate regions, with the majority of general and species in tropical Asia. They are herbaceous plants, usually deciduous and sometimes creepers shrubs. The Musanga cecropioïdes (Urticaceae) species, widespread in the tropical forests of Africa from Guinea to the Congo Basin [1], is a plant used in traditional medicine in West and Central Africa for the treatment of pathologies such as constipation, rheumatism, cough, schizophrenia, lung infections, leprosy, high blood pressure and malaria [2,3]. Numerous scientific studies have shown that extracts from the various organs of M. cecropioides exhibit hypotensive, hypoglycemic, antidiabetic, antidiarrhoeal and antibacterial properties [4][5][6][7]. In Côte d'Ivoire, according to information gathered from ethnobotanical surveys, the root bark of M. cecropioides are used to treat mental illnesses. In view of this virtue of M. cecropioides, we took an interest in it, and this, by considering studying chemical composition of its organs and their inhibitory activity of acetylcholinesterase (AChE). Phytocompounds such as alkaloids, terpenoids, cardiac glycosides, saponins and coumarins have a beneficial effect on the central nervous system by increasing the activation of cholinergic neurons and stimulating cognitive functions such as acetylcholinesterase inhibitors (AChE) [7,8]. This makes it possible to consider them promising in the prevention of neurodegenerative diseases of the Alzheimer type. Indeed, cholinergic deficiency is a hallmark of Alzheimer's disease (AD), which is associated with the selective death of cholinergic neurons in the initial stages of the disease [9]. We know that the brain is more sensitive to damage caused by free radicals than other tissues. The effectiveness of antioxidant system in the brain gradually decreases with aging, and in the brains of patients with AD, this decrease occurs even more strongly. All of this determines the advisability of using natural antioxidants in the prevention and treatment of AD [10].

Plant material
The plant material consists of the leaves and root bark of M. cecropioides (Cecropiaceae) collected in Zokolilié (Lakota, south-western Côte d'Ivoire), and identified at the National Floristic Center (CNF) of Abidjan under the No. 17543. After cleaning with water and drying in a ventilated room for 15 days, the organs were pulverized using an electric grinder and kept in tightly closed glass jars.

Determination of total phenols (PT)
PT was quantified in the crude extracts (McF and McR) by the spectrophotometric method [14]. 1 ml of extract diluted 1 / 50 added 1.5 ml of a solution of Na2CO3 (17%, m / v) and 0.5 ml of Folin-Ciocalteu reagent (0.5 N). The whole is incubated at 37°C for 30 min and the absorbance read at 720 nm against a blank without extract taken as a reference. A calibration with different concentrations of gallic acid (from 1.95 μg / ml to 62.5) is carried out under the same conditions. The PT contents are calculated according to formula (1) and expressed in μg equivalent of gallic acid per g of dry matter (μg EAG / g DM).
V: volume of the crude extract (ml); C: mean concentration (µg / ml); d: dilution factor; m: mass of the pulverized dry matter (g).

Determination of total flavonoids (FT)
The quantification of FT is performed by spectrophotometry [15,16]. To 2 ml of each crude extract diluted to 1 / 20th were added 100 μl of NEU reagent, the absorbance is measured at 404 nm and compared to that of quercetin (0.05 mg / ml) taken as standard. The percentage of FT is calculated according to expression (2) and converted into µg equivalent of gallic acid per g of dry matter (μg EAG / g DM) % FT = [(0,05 × Aext× d) / Aq× Cext] × 100 (2) Aext: Absorption of the extract; Aq: Absorption of quercetol; Cext: Concentration of the extract (2.5 mg / ml); d: dilution

Hydrolyzable tannins (TH)
This method is based on the formation of the violet-red coloring complex between tannins and iron trichloride (FeCl3), the absorbance of which is read with a UV-visible spectrophotometer (Analytic AL800) [17]. 0.4 g of powder are macerated in 20 ml of MeOH (80%) for 18 h. To 1 ml of the macerate are added 3.5 ml of a solution of FeCl3 (0.01 M in 0.001 M HCl) and the absorbance is read at 660 nm. The TH content is calculated to formula (3): Abs: absorbance, E mole: 2169 of gallic acid (constant expressed in mole), M: mass = 300, V: volume of the extract used, P: mass of the sample.

Condensed tannins (TC)
To 400 μl of sample (0.5 mg / ml) are added 1.5 ml of vanillin solution (4% in MeOH) and 0.8 ml of concentrated HCl. After 15 min of incubation, the absorbance is read at 500 nm [18,19]. The TC contents are deduced from the calibration curve produced with the catechin (0-150 µg / ml) and expressed in µg of catechin equivalent per mg of dry extract (µg ETC / mg).

DPPH test
The antioxidant activity is evaluated by the reduction of the DPPH radical according to the methodologies described in the literature [20,21]. The reference antioxidant used is ascorbic acid (vitamin C) prepared under the same conditions as the samples.

FRAP test
The method is based on the reduction by the antioxidant of the ferrous tripyridyltriazine complex TPTZ-Fe 3 + to TPTZ-Fe 2 + of blue color, the absorbance of which is read at 593 nm with a UV-visible spectrophotometer (Analytic AL800). The standard curve was constructed from the Trolox calibration curve. The results are expressed in mM Trolox equivalent [22,23].

Evaluation of anticholinesterase activity
The inhibition of acetylcholinesterase (AChE) evaluated to methods described literature [24,25]. 10 μl of AChE (1 U / ml) and 150 μl of methanolic extract (2 mg / ml) and 1.80 ml of Tris-HCl buffer. The whole was incubated at 37 ° C for 15 min. Subsequently, 20 μl DTNB (0.01M) and 10 μl ATCI (75 mM) was added, and incubated at room temperature for 5 min. The control mixture (150 μl of galanthamine (Reminyl®) and a blank (150 μl of MeOH) was prepared under same conditions as the extracts. The absorbances was read with a spectrophotometer at 412 nm every 30 s for 6 min. AChE inhibition (I) calculated as a percentage (%) to formula:

Phytochemical profile of selective extracts
The phytochemical screening by TLC of the selective extracts of. M cecropioides has shown the presence of several phytocompounds using appropriate specific developers ( Table 1). Liebermann-Bürchard and Godin reagents and sulfuric vanillin demonstrated sterols, terpenes, oleanan-type triterpenes and ursane in hexane and CHCl3 extracts [11]. The yellow molecular fingerprints perceptible to the naked eye (Table 1) revealed by 5% (w / v) of methanolic KOH solution indicate the presence of coumarins. This coloration may intensify or turn blue or green under UV light / 365 nm [11,13]. KOH solution (5%) allow to identify 1, 2-dioxyanthracene (Rf = 0.45) under UV at 365 nm as a purple spot in the CHCl3 extract of the root bark. [11]. The alkaloids, flavonoids and condensed tannins were revealed in the AcOEt and n-BuOH extracts. Indeed, Dragenddor'ff's reagent revealed alkaloids in orange spots, while AlCl3 revealed flavonoids in yellow to the naked eye and blue under UV / 366nm. Condensed tannins were detected by FeCl3 as gray spots [13]. Comparing our results with those of studies of extracts from the bark of the trunk of M. cecropioides from Nigeria, we observed some similarity in the presence of tannins, flavonoids and alkaloids [26]. In addition, Togolese researchers have also shown the presence of phenols, coumarins, tannins, flavonoids and alkaloids in the leaves and in the stem bark of the species from Gabon [27].

Total phenols and flavonoids content
The PT content is obtained from the linear regression equation for gallic acid (y = 0.0232 x + 0.0002; R2 = 0.9983) and expressed in g EAG / g DM. The values obtained show that the leaf extract (McF) contains more PT (119.389 mg EAG / g) compared to the McR root bark (105.944 mg EAG / g) (Figure 1). The FT contents are also higher in the leaves (7.753%) than in the root bark (1.41%).
A study carried out on the leaves and stem bark of the species from Gabon showed the contents of total phenols (40.69 ± 6.43 and 40.26 ± 3.12 mg / g, respectively) lower compared to to those found in our case [27]. In general, the PT and FT are higher in the leaves than in the stems and roots, which is explained by the fact that the synthesis of flavonoids occurs under UV irradiation, therefore in the part of the plant. The most exposed to solar rays [28,29].

Hydrolyzable (TH) and condensed (TC) tannin content
In plants, TH and TC occur simultaneously, with the predominance of one class. In our case, the quantification of the tannins showed that TH and TC are present in two parts of M. cecropioides with the predominance of TC (Figure 2). Previously, depending on the result of the reaction with FeCl3 solution, TC was found to predominate in the study extracts (Table 1). However, the highest tannin content is found in the aerial part of M. cecropioides, the lowest in its underground part. Tannins in significant quantities accumulate, mainly in underground organs, in the bark and wood of trees and shrubs, less often in the leaves (green and black tea, strawberry, bramble, raspberry) [30]. Knowing that these leaves are great classics of herbalists thanks to their richness in tannins, we can also recommend using the leaves of M. cecropioides in the form of herbal teas.

Antioxidant DPPH and FRAP profiles
There are different methods for determining the antioxidant activity (AOA) of plant extracts. The use of a single method does not give a reliable result and the data obtained by different methods are not always comparable [31]. In this regard, in this work, we have used two methods for the determination of the antioxidant activity of crude and selective extracts of the leaves and root bark of M. cecropioides: the method based on the interaction of antioxidants with the 2,2diphenyl-1-picrylhydrazyl radical (DPPH) and the FRAP method based on the determination of the reducing capacity of extracts when they interact with the Fe complex with tripyridyltriazine. The results of the measurements are shown in Figures 3 and 4.  (Figure 3). However, the values obtained are lower than that of vitamin C (49.49-88.49%), taken as standard. For a better appreciation of this potential, the median concentration of sample which reduces 50% of the DPPH • (CR50) was determined and the values are recorded in Table 2. This value is all the more important (effective), if the graphically determined CR50 is small.

Figure 3
Percentages of reduction of DPPH • by crude and selective extracts The extracts with AcOEt and n-BuOH of two studied organs of M. cecropioides have CR50 values close to the value of vitamin C. This anti-free radical potential could be explained by the presence of phenolic compounds such as flavonoids, coumarins and tannins in these extracts ( Table 1; Figures 1 and 2). The results obtained agree with those obtained with the extract of the root bark and leaves of the species from Gabon which respectively presented CR50 equal to 0.29 ± 0.02 µg / ml and 6.36 ± 0.89 µg / ml [27].
The FRAP test made it possible to evaluate antioxidant activity in vitro by measuring their reduction potential from Fe 3+ to Fe 2+ [32] as a function of the calibration line of Trolox (standard antioxidant) y = 0.9864 x + 0, 0353 (R 2 = 0.9884). The antioxidant activity of extracts of M. cecropioides, determined by their reducing power and that of quercetin (standard) are illustrated by histograms in Figure 4.  (Figures 1 and 2). Indeed, FRAP antioxidant activity could be linked to the presence of these compounds because they are antioxidants recognized by their reducing power [32]. However, the antioxidant activity of the study extracts is lower than that of quercetin (359.690 µM TE / g MS). Also, the FRAP antioxidant activity of M. cecropioides is lower than that of the leaves of different varieties of green tea (272-1144 μM TE / g) [33].
As a result of this study, it was found that regardless of the method used (DPPH or FRAP), the extract of the leaves of M. cecropioides showed higher antioxidant activity than that of its root bark. The leaves of M. cecropioides can be considered a source of phenolic antioxidants.

Anticholinesterase activity
The inhibitory activity from 0 to 360s of the extracts of M. cecropioides tested on AChE gave more or less variable percentages of inhibition (PI) compared to Reminyl® (Galanthamine, commonly used reference alkaloid) (Figure 5). . By comparing the inhibitory activity of the AChE enzyme by extracts tested with that shown by galanthamine (69.380 -73.098%), we can say that the extracts with AcOEt and n-BuOH signed a good anticholinesterase activity, which could be due to the synergistic action of the phytocompounds present in the extracts tested ( Table 1; Figures 1 and 2). Indeed, the extracts contain secondary metabolites such as alkaloids, terpenoids, coumarins, flavonoids and tannins which are the main families of compounds recognized as AChE inhibitors [8,33].

Conclusion
The results of our study showed that extracts from the leaves and root bark of M. cecropioides contain several bioactive phytocompounds which are responsible for their DPPH and FRAP antioxidant activities with overt AChE enzyme inhibitory activity. These results could explain the therapeutic virtues widely attributed to this species in unconventional medicine.