Chemical evaluation and anti-radical activity of varieties of Morus alba l . ( morera , moraceae ) from Venezuela

Mulberry (Morus alba L.), known as white mulberry, is a plant widely used in medicine and food due to its chemical composition. The qualitative study of the primary and secondary metabolites of the methanolic extracts of the four varieties of Morera was developed by chromatographic profile (TLC) against standards. Quantification was performed through colorimetric assays. All results were analyzed through statistical analysis. The results indicated the existence of similarities between varieties for both primary and secondary metabolites. The radical capacity of the varieties analyzed was also evaluated by finding that the Táchira variety had the highest anti-radical capacity with an IC50 of 553.58 ± 3.23 μg / mL followed by the Maracay IC50 varieties of 1054.01 ± 1.76 μg / mL, Boconó IC50 of 1398.93 ± 2.23 μg / mL and Yu-62 IC50 of 3817.89 ± 18.08 μg / mL. It was found that the use of the thin layer chromatography (CCF) technique was efficient to detect the presence of phenolic compounds, soluble carbohydrates, and amino acids in the four varieties studied. The Yu-62 variety had the highest total phenol contents and proteins; the amino acid content was higher for the Táchira variety, while the soluble carbohydrate content was higher in the Boconó variety. The presence of saponins was not detected in any of the four varieties evaluated.


Introduction
The mulberry is a shrub with shiny-light green leaves, prominent whitish veins on the underside, and an asymmetric base 1 ; purple, white, black, or pink fruits with a length of 5 cm 2 .
It is native to Asian countries, used in sericulture, and introduced to other continents, which has led to the creation and extension of a large number of varieties with excellent biomass production, nutritional quality, and high resistance to attack by pests and diseases in a wide range of climate and soil conditions 3 .
For the genus, Morus a total of 24 species are reported 4 , the best-known being M. alba, M nigra, M indica, M laevigata and M bombycis, which are distributed all over the world, from temperate areas (altitude: 4000 masl ) to tropical (dry and humid) 5 , grouped into five regions 6 : East Asia, Archipelago of Malaysia, Southwest Asia, North, Central, and South America and West Africa.
According to Chan et al. 7 , mulberry foliage is the leading food of silkworms; Its leaves are used as food for livestock and its fruits as food products.
Flavonoids and phenolic compounds, in general, are the main components of the plant in its various organs, also mentioning alkaloids, amino acids, and fatty acids, among others. Various biological activities are reported for the species' leaves, including antioxidants, antimicrobial, skin whitening, cytotoxic, antidiabetic, glycosidase inhibition anti-hyperlipidaemias, anti-atherosclerotic, anti-obesity, cardioprotective, and enhancement cognitive [7][8][9][10][11][12] . Fruits are rich in anthocyanins and alkaloids with antioxidant, antidiabetic, antiatherosclerosis, anti-obesity, and hepatoprotective activities 13,14 . The root's bark contains flavonoids, alkaloids, and stilbenes, with antimicrobial, skin whitening, cytotoxic, anti-inflammatory anti-hyperlipidaemic properties 15 . Other pharmacological properties of M. alba include antiplatelet, anxiolytic, antiasthmatic, anthelmintic, antidepressant, cardioprotective, and immunomodulatory activities. The phytochemistry and pharmacology of the different parts of the mulberry tree confirm its traditional and current uses as forage, food, cosmetics, and medicine, the species being a plant with promising medicinal properties 7 . This work's objective was to study comparatively the chemical composition and root activity of the leaves of four varieties of Morus alba cultivated in Venezuela.

Collection and treatment
The Morus alba varieties studied were collected in the greenhouse of the Institute for Advanced Studies (IDEA), located at an altitude of 1179 meters above sea level, 10° 25'59" N, 66°52'59" W, in the Baruta municipality, Miranda state of the Metropolitan District of Caracas, where they are identified as Maracay (Ma 001), Táchira (Ta 002), Boconó (Bo 003) and Yu-62 (Yu 004).
The varieties used were collected from plants with a height of no more than 50 cm in height and little biomass, with leaves of approximately 8 cm long.
Two procedures were carried out: In the first, samples of 10 leaves of each plant per variety (three plants) were taken, and they were dried in the open air for one week, then crushed and macerated in methanol for the preparation of the corresponding extract. In the second procedure, 10 leaves were taken from each plant (total 30); they were cleaned with water and alcohol for transfer to the laboratory in a cellar with liquid nitrogen. The leaves were macerated with liquid nitrogen and stored in a refrigerator at a temperature of -80 ° C for later use.
Chemical evaluation and anti-radical activity of varieties of Morus alba l. (morera, moraceae) from Venezuela Extraction and analysis of varieties Protein extraction: 100 mg of the sample placed in Eppendorf tubes were added 1mL of buffer constituted by Tris-HCl, urea, EDTA disodium salt, tween 20, sodium diacid phosphate and ß mercapto-ethanol. It was stirred to homogenize; it was centrifuged at 10,000 rpm for 10 min separating the solid (soluble proteins).
Extraction of carbohydrates and amino acids (as free proline) 100 mg of the sample placed in Eppendorf tubes were added 1 mL of 80% ethanol; It was stirred until homogenized, it was centrifuged at 4000 rpm for 8 min evaporated in the hood at room temperature until obtaining 1mL aqueous phase: carbohydrates and amino acids. The process was repeated twice.

Extraction of Phenols (soluble and bound to the cell wall)
100 mg of the sample placed in Eppendorf tubes, 500 µL of methanol were added, it was stirred until homogenized, it was centrifuged at 12000 rpm for 15 min. The supernatant contained soluble phenols. 250 µL of 2M NaOH was added to the solid residue, it was stirred until homogenized, incubated at 70 ° C for 16 hours, neutralized with 250 µL of 2M HCl, centrifuged, and the supernatant consisted of phenolic compounds bound to the cell wall.
Qualitative analysis of the varieties TLC performed methanolic extracts, and the solvent systems and standards used were the following: Quantification of primary and secondary metabolites Proteins A 100 µL aliquot was taken from the initial extraction and initially diluted to a volume of 10 mL with water. A 1mL aliquot was taken, and then 1 mL of Bradford's reagent (Coomassie blue in sulfuric acid) was added and stirred until homogenized. It could settle. As a standard, bovine serum albumin solutions of different concentrations were prepared, and the same treatment as the sample was applied. Spectrophotometric analysis was performed for quantification using the Bradford method 17 . The values of the samples were obtained from the corresponding calibration curve. The results were expressed as mg / g of fresh mass equivalent to bovine serum albumin.

Total carbohydrates
A 100 µL aliquot of the sample from the extraction was taken and diluted with distilled water to 1 mL. 0.5 mL of a 5% phenol solution and carefully 2.5 mL of concentrated sulfuric acid were added. It was stirred until homogeneous and allowed to stand for 30 min. Different concentrations of glucose solutions were prepared as a standard, and the same treatment as the sample was applied. The Dubois method 18 carried out quantification. The maximum wavelength of absorption was 492 nm. The values of the samples were obtained from the calibration curve. The results were expressed in mg / g of fresh mass equivalent to glucose.
Soluble phenols bound to the cell wall (total phenols) 100 µL of the plant extract was taken and diluted with 1mL with distilled water. Subsequently, 100 µL of the Folin-Ciocalteau reagent was added, and it was left to stand for 5 min. 600 µL of 1M NaOH saturated with Na2CO3 was im-

Amino acids, such as free proline
A 20 µL aliquot of the plant extract was taken and diluted to a volume of 0.5 mL with distilled water. This was mixed with 0.5 mL of ninhydrin and 0.5 mL of acetic acid. It was stirred to homogenize and subsequently placed in a water bath at 100 ° C for 1h. It could cool. 2 mL of toluene was added, and it was stirred vigorously. L-proline solutions of different concentrations were prepared and the same treatment as the sample was applied as a standard. For quantification, a colorimetric method was used using ninhydrin as a reagent. The maximum absorption length was 520 nm 16 . The values of the samples were obtained from the corresponding calibration curve. The results were expressed in mg / g of fresh mass equivalent to L-proline. mediately added and left to incubate at room temperature for 1h. Chlorogenic acid solutions of different concentrations were prepared as a standard. The same treatment as the sample was applied. Quantification was carried out by the Folin -Cioucalteu spectrophotometric method 19,20 . The values of the samples were obtained from the corresponding calibration curve. The results were expressed in mg / g of fresh mass equivalent to chlorogenic acid.

Routine determination by HPTLC method
Once the thin layer chromatography was developed applying the conditions required for the analyzed sample and varying the concentration of the standard, maintaining a seeding volume of 3 µL in each case, a photo was taken of each plate at a wavelength of 254 nm using a UV radiation lamp and maintaining the same focal distance for each of them. Said photo was subjected to an executable program in MATLAB supplied by the laser laboratory of the School of Chemistry of the UCV Faculty of Sciences for the construction of the corresponding calibration curve.

Statistical analysis
Analysis of variance and test of means were performed on all the variables under study, applying the Di Rienzo, Guzmán, and Casanova's test (DGC). For this, the statistical program In-foStat 2013 (Di Rienzo et al.) 21 .

Results
Qualitative analysis. Visualization of the chemical profile of the varieties Figure 1 shows the chromatographic profiles of the different primary and secondary metabolites evaluated: amino acids, carbohydrates, and flavonoids, indicating the solvent systems, developers, and standards used.
Quantification of free L-proline and proteins Table 1 shows the results for the concentration of amino acids expressed as L-proline and proteins for the three replicates analyzed in the four varieties studied and the statistical analysis results.

Quantification of soluble carbohydrates and phenols
The quantification of soluble carbohydrates and total phenols is described in table 2, where the results of the statistical analysis are also expressed.
Routine quantification by HTLC in the Morus alba varieties: Maracay and Boconó and anti-radical activity Table 3 shows the results of the routine quantification in the varieties that chromatographically gave positive results for this flavonoid, in addition to the determination of the anti-radical activity with DPPH, for the evaluation of the antioxidant activity of the extracts of the varieties under study also indicates the results of the statistical analysis.

Discussion Qualitative analysis. Visualization of the chemical profile
The primary and secondary metabolites (amino acids, carbohydrates, proteins, and phenolic compounds) produce benefits to other living organisms that feed on the plants that contain them, facilitating their growth and development. In some studies, it has been shown that feeding different animals with mulberry leaves produces an increase in the meat's quality and other characteristics of the animals [22][23][24] .
Of the 20 essential amino acids present in mulberry leaves, the concentration levels of Proline, Glutamine, Glycine, and Valine 25 are considerable. It is possible to observe on the chromatographic plate (fig 1-I) a series of purple-violet spots associated with these compounds and a yellow-orange spot of Rf 0.36 corresponding to L-proline. Additionally, the arginine pattern Rf 0.17 could be observed. By comparing the chromatographic profile of the four varieties studied with the L-proline standard, this amino acid's presence could be detected, with different intensities, in all the samples studied. Concerning carbohydrates, a very complex composition of monosaccharides and polysaccharides is proposed, highlighting the concentrations of glucose and sucrose 26 ; The qualitative analysis of the varieties Táchira, Maracay, Boconó and Yu-62, (fig 1-II), compared with the sucrose and glucose standards, showed spots coinciding with the standards, which shows their presence.
In the phenolic compounds for the chromatographic analysis, analysis conditions were used that allowed the detection of both flavonoids and glycosides thereof, considering previous reports of the detection of these metabolites in the species [27][28][29] .     The increase in the concentration of amino acids and L-proline is an indicator to evaluate plants' stress state because, in these conditions, they tend to accumulate free amino acids as a defense mechanism, reducing protein synthesis.
The results achieved the% (DM) values of L-proline below those obtained for the Cuban varieties (table 1), with the Maracay variety being the lowest value. Regarding the comparison between them, the analysis of variance and the tests of means obtained allowed to observe significant differences of the Táchira and Boconó varieties for Yu-62 and Maracay for P <0.01.
Proteins are essential factors for studies related to the nutritional contribution of forage species for animal feed. Plants of the genus Morus have a high protein content (14-28% on a dry basis) 25,31-32 , making them an exciting alternative in the agricultural area.
In the analyses carried out for the mulberry varieties, low protein values were reached (Table 1), comparable with those found in the Cuban varieties evaluated by Cáceres et al. 33 , which obtained values close to the Maracay, Boconó, and Táchira varieties. According to the variance analysis and the test of means, significant differences could be observed P <0.01 between them, the Maracay being the one that presented the highest protein concentration and the Yu-62 the lowest.
Different authors have pointed out the influence of some factors on protein variability in the genus Morus. Some consider that the highest crude protein values depend on the regrowth age and the time of year 25 , while others have outlined the influence of climatic conditions and the use of fertilizers 30,32,[34][35][36] . In this case, it is considered that when the plants were in the greenhouse, it could have affected the regrowth age and the non-use of fertilizers.

Quantification of soluble carbohydrates and total phenols
Carbohydrates are the metabolites found in greater quantity in plants, playing an essential role since they provide the necessary energy for the various vital functions in which they participate, mention the formation and growth of tissues, and be associated with recovery plant after pruning. The photosynthetic process mediates the source of soluble carbohydrates in the plant; therefore, environmental factors such as access to water, leaf surface (size of the leaves), and temperature, among other factors, play an essential role [37][38][39] .
The fact that the studied varieties were in a greenhouse where the incident radiation was necessary, together with a cool environment provided by the non-direct interaction with other plants and a constant watering during the analysis period, allows the studied varieties in the nursery phase they carry out their photosynthetic process efficiently, being evidenced by the size of the leaves despite not observing a great leaf mass.
The variety with the highest concentration of carbohydrates was Boconó, while Táchira had the lowest value ( Table 2). The analysis of variance showed significant differences for P <0.01 between the varieties.
Generally, as a product of their normal secondary metabolism, all plants can biosynthesize a high number of phenolic compounds, some of which are essential for their physiological functions and others are useful to defend themselves against stressful situations (water, light, ) 10,40 .
In the results obtained (table 2), the highest concentrations of this metabolite belong to the category of phenols linked to the cell wall in which the environmental conditions to which the species are subjected will have a direct impact. Being under the same conditions, the response obtained is characteristic of each variety.
Soluble phenols had the lowest concentrations, which can be associated with defense against pathogenic organisms that directly affect the internal cell system, as shown in some reports in the literature [41][42][43] .
The variance analysis and the test of means showed highly significant differences (P <0.01) between the varieties for soluble phenols. The highest concentration was obtained in Táchira, while the others behaved statistically similar among them. In the case of phenols bound to the cell wall, there were significant differences between varieties; the Yu-62 and Boconó cultivars presented the highest values and surpassed Táchira and Maracay, a similar behavior to each other.

Routine HPTLC Quantification and Anti-Radical Activity
According to Polumackanycz 10 , rutin, quercetin, and apigenin are the main bioactive phenolic compounds present in mulberry leaves. Flavanols are the ones that contribute the most to the antioxidant activity in the leaves of the Morus alba species (Flaczyk et al. 44 ), the most representative compound being rutin (quercitin-3-rutinoside). In the qualitative tests carried out by TLC, rutin could not be visualized in the Boconó and Yu-62 varieties.
The values obtained for Táchira and Maracay are shown in table 3. Significant differences were observed for P <0.01 between the two varieties, with Maracay presenting the highest value. The concentrations found were low compared to those published in the bibliography 41,[44][45] , although the climatic conditions develop differently from the study object, which influences the results.
Regarding anti-radical activity, the highest IC 50 is the one corresponding to the lowest activity value. It was to be expected that the trend that should be seen in the antioxidant activity would be the same visualized for the content of total phenols; however, the results obtained showed different trends in which the contribution of other metabolites present in some of the varieties could influence, whose synergistic or summative effect contributed to the results found. The analysis of variance showed highly significant differences (P <0.01) between the varieties. Some authors 46,47 have reported distinct IC 50 values for varieties of the genus Morus, but in the comparison between these and the results obtained, the analysis conditions, the type of extract (extraction method and solvent used), the concentration of DPPH used, and reaction time, among many others.
However, the differences between the varieties studied, where the agroecological conditions are the same, are used, maybe due to each variety's intrinsic characteristics.
A joint analysis of the four varieties allowed to establish that the Táchira variety presented the highest content of amino acids (L-proline), soluble phenols, and anti-radical activity; the Macacay variety exhibited the highest protein content and Boconó the highest concentration of carbohydrates, so each of them can be used depending on the objective to be pursued: for animal or therapeutic feeding purposes.

Conclusions
The analysis of the four varieties allowed to establish that the Táchira variety presented the highest content of amino acids (L-proline), soluble phenols, and anti-radical activity; the Macacay variety exhibited the highest protein content and Boconó the highest concentration of carbohydrates, so each of them can be used depending on the objective to be pursued: for animal or therapeutic feeding purposes.