In Vitro and In Vivo Antioxidant Activity of Agave sisalana Agro-Industrial Residue

Agave sisalana agro-industrial residue has considerable potential against damage associated with oxidative stress and skin aging. This study aims to demonstrate, in vitro and in vivo, the potential of Agave sisalana agro-industrial residue as a safe and effective alternative for the prevention of damage caused by oxidative stress and aging. The antioxidant activity was evaluated in vitro (total antioxidant capacity, reducing power, DPPH radical scavenging, metal chelating (Fe2+ and Cu2+), and hydroxyl radical scavenging) and in vivo using the Caenorhabditis elegans organism model. The extract showed in vitro antioxidant activity in all tests performed. Tests with C. elegans showed that the extract was able to reduce the intracellular levels of reactive oxygen species (ROS) and increase the survival rate of worms. A downregulation of gst-4::GFP expression suggests a direct action against free radicals. Agave sisalana agro-industrial residue extract (AsRE) can therefore be considered as a source of antioxidant biomolecules, and the use of this agro-industrial residue in a new production process can lead to sustainability and socioeconomic development.


Plant Material
Sisaltec Industry (Natal, RN, Brazil) supplied the sisal residue that was collected from a farm directly from a decortication machine in the city of Pedra Grande, Rio Grande do Norte State, Brazil (5 • 07 56.3" S 35 • 54 31.7" W). The plant material was identified by a botanist (Msc. Alan de Araújo Roque) and the voucher specimen was deposited at the Herbarium of the Federal University of Rio Grande do Norte (UFRN) under number #20544.

Extract Production
The natural fiber (sisal) was mechanically removed from the A. sisalana leaves and the residual material from this process (residue) was pressed, providing a liquid portion. The liquid portion was added to ethanol 95 • GL at a 1:3 (v/v) residue/solvent ratio and stored at 4 • C for 24 h [10]. Subsequently, the liquid extract was filtrated, concentrated, and lyophilized (Christ Alpha 1-2 LD, Osterode am Harz, Germany). The A. sisalana agro-industrial residue extract obtained was named AsRE. The yield was calculated from the initial amount of liquid portion according to Equation (1).

Total Flavonoids
Total flavonoid was determined by the aluminum chloride coloration method [21]. Briefly, 1.0 mL of AsRE (1.0 mg/mL) was added to 1.0 mL aluminum chloride solution (2.5% w/v). The reaction mixture was incubated at room temperature, for 60 min, in the dark. Absorbance was measured at 425 nm using a spectrophotometer (Biospectro SP-220, Curitiba, Brazil). The total flavonoids were calculated with a standard curve made with the quercetin standard as a reference (2.0, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 µg/mL). The assay was realized in triplicate and the results expressed as µg Eq quercetin (EQ)/mg of AsRE.

Total Saponins
The concentration of total saponins was determined by vanillinsulfuric acid method as described by Ribeiro et al. [22]. Thus, 250 µL of sample (10.0 mg/mL diluted in methanol 80% w/v) was added to 250 µL of ethanolic solution of vanillin 8% (w/v) and 2.5 mL of sulfuric acid 72% (v/v). The resulting acid solution was kept at 60 • C for 10 min. Finally, the absorbance at 544 nm was registered using a spectrophotometer (Biospectro SP-220, Curitiba, Brazil). The standard curve was made using a stock solution of diosgenin (0.50 g/L). The assay was realized in triplicate and the results expressed as percentage.

Fourier Transform InfraRed (FTIR) Spectroscopy
The FTIR was recorded in an IR Prestige-21 spectrophotometer (Shimadzu, Kyoto, Japan) with a frequency range of 700 to 4000 cm −1 and a resolution of 4 cm −1 . AsRE (10 mg) was mixed with spectroscopic-grade potassium bromide powder and then pressed into 1.0 mm pellets and submitted to FTIR analyses.

In Vitro Antioxidant Activity
A single antioxidant assay is not sufficient to comprehensively assess the antioxidant capacity of extracts [23]. Therefore, in order to evaluate the activity of the extract at different stages of the oxidative cascade (initiation, propagation and termination), the antioxidant activity of the extract was evaluated by six different methods: total antioxidant capacity, reducing power, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test, cupric and ferric chelating, and hydroxyl radical scavenging. The IC 50 (concentration required for 50% inhibition) value was determined from the plotted graph of reducing power, scavenging activity (DPPH and Hydroxyl radical) and metal chelating (Cupric chelating and ferric chelating) against the different concentrations of AsRE according to Equation (2).
2.5.1. Total Antioxidant Capacity (TAC) The antioxidant capacity assay was assessed according to the method described by Costa et al. [24]. AsRE, together with a reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate), was incubated at 95 • C for 90 min. The mixture was cooled, and the absorbance measured at 695 nm against a blank. The antioxidant capacity was expressed as ascorbic acid equivalent.

Reducing Power
The reducing power was evaluated as described previously [24]. Briefly, 4.0 mL of reaction mixture, containing different sample concentrations (0.05, 0.1, 0.25, 0.5, and 1.0 mg/mL) in phosphate buffer (0.2 M, pH 6.6), was incubated with potassium ferricyanide (1% w/v) at 50 • C for 20 min. The reaction was stopped by trichloroacetic acid solution (10% w/v). Afterwards, the solution was mixed with distilled water and ferric chloride solution (0.1% w/v). The absorbance was measured at 700 nm against a blank. The mixture was stirred, and the absorbance (700 nm) measured using a microplate reader (BioTek, Winooski, VT, USA). Results were expressed as the percentage of activity observed for 0.1 mg/mL (highest activity) ascorbic acid.
where As = absorbance of the sample; A0 = absorbance of the control.

Ferric Chelating
The ferric chelating effects of AsRE were evaluated as described previously by Decker and Welch [27]. Different concentrations (0.1, 0.3, 0.5, 1.0, 1.5, and 2.0 mg/mL) were added to reagent solution (FeCl 2 2.0 mM, Ferrozine 5.0 mM) and incubated at room temperature for 10 min at a wavelength of 562 nm. EDTA was used as a positive control (0.005-0.05 µg/mL). The results were expressed as a percentage of iron ion chelating according to Equation (5).

Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity was evaluated using Fenton's reaction as described previously [28]. Different concentrations of the AsRE (0.5, 1.0, 1.5 and 2.0 mg/mL) were added to reagent solution (10.0 mM iron sulphate, 10.0 mM EDTA, 2.0 mM sodium salicylate, 30% H 2 O 2 ) and incubated at 37 • C for 1 h. Gallic acid (0.5, 1.0, 1.5 and 2.0 mg/mL) was used as positive control. The hydroxyl radical was investigated by monitoring absorbance at 510 nm. The results were expressed as a percentage of the scavenging of radicals according to Equation (6).
where As = absorbance of the sample; A0 = absorbance of the control; Ab = absorbance of the blank.

Caenorhabditis elegans Maintenance Strains and Extract Treatment
The N2 (wild type) strain of C. elegans was used, which was cultivated in the Nematode Growth Medium (NGM) with Escherichia coli OP50 and maintained at 20 • C [29]. Worms were synchronized by treating pregnant hermaphrodites with lysis solution (hypochlorite 2% and NaOH 10 mM) in order to obtain worms at the L1 stage. AsRE was added to the E. coli OP50 fluid at a final concentration of 1.0, 3.0 and 5.0 mg/mL. The fluid was added to the surface NGM [17].

Bacterial Growth Assay
The bacterial growth of E. coli OP50 was assessed by optical density (OD) readings on the spectrophotometer (Biochrom Libra S22, Holliston, MA, USA). E. coli OP50 was cultivated into sterilized liquid medium with or without the different concentrations of AsRE (1.0, 3.0 and 5.0 mg/mL) in Luria-Bertani medium (LB). The nematodes were inoculated with the E. coli OP50 and separated into groups, respectively. The results expressed the relative levels. The initial values of optical density (OD600) to E. coli OP50 were compared at each point with the OD600 at the zero point of each condition. The samples were transferred to a rocking shaker at 37 • C (Marconi MA 420, Piracicaba, Brazil), and values measured once every 1 h up to 5 h.

Effect of A. sisalana Agro-Industrial Residue Extract on Body Length
The worms synchronized at the L1 stage cultivated in NGM medium plates were separated into groups and submitted to treatment with AsRE at 1.0, 3.0 and 5.0 mg/mL for 48 h at 20 • C. In the control group, nematodes were cultured on the NGM with the E. coli OP50 fluid. In total, 30 worms from each group were photographed and body length measured using the ImageJ software (ImageJ 1.51j8-https://imagej.nih.gov/ij/). The experiment was performed in triplicate and results presented as mean area ± standard error of the mean (SEM).

Effect of Agave sisalana Agro-Industrial Residue Extract on Hatchability
The effect of AsRE on the egg hatching of wild type C. elegans was evaluated for hatchability percentage. Embryos resistant to treatment with lysis solution were collected and added to the surface NGM medium in treatment groups at 1.0, 3.0 and 5.0 mg/mL (in E. coli OP50) and the control group only with E. coli OP50. After 24 h at 20 • C, hatchability percentage was calculated to a ratio of progeny number over brood size. Experiments were performed in triplicate and results presented as mean area ± standard error of the mean (SEM). Fluorescence quantification was performed on GloboMax ® -mult detection system (Promega, Madison, WI, USA), with excitation at 490 nm and emission at 510-570 nm. Six readings were taken, with an interval of 30 min between each reading. The experiment was performed in three independent experiments, performed in triplicate and results presented as mean area ± standard error of the mean (SEM).

Oxidative Stress Survival Assay
Wild type worms from the L1 stage were treated with AsRE at concentrations of 1.0, 3.0 and 5.0 mg/mL for 48 h at 20 • C. Subsequently, 50 nematodes per group were transferred to plates with NGM solid medium containing 10 mM tert-butyl hydroperoxide (TBHP). The fraction of surviving nematodes was evaluated for up to 33 h using a stereoscopic microscope by probing the nematodes. This assay was repeated three times with a total number of 150 worms/group and results presented as mean area ± standard error of the mean (SEM).

Expression Levels of the Oxidative Resistance Relative Gene
CL2166 mutant worms carrying an inducible green fluorescence protein (GFP) reporter for gst-4 (gst-4::GFP) were maintained on NGM plates with or without (control) different concentrations of AsRE (1.0, 3.0 and 5.0 mg/mL) for 48 h. The gene expression was determined by fluorescence microscopy (Olympus BX51, BR). The fluorescence intensity was quantified using the ImageJ software (ImageJ 1.51 j8).

Statistical Analysis
All experiments were performed three times. Statistical analysis was performed using GraphPad Prism 8.0.2 (https://www.graphpad.com/scientificsoftware/prism/). Student's t test was used to compare pairs of groups, as well as a one-way ANOVA followed by Tukey's post test. For the hydroxyl radical scavenging test, a two-way way ANOVA followed by Sidak's multiple comparisons was used. Survival curves were analyzed by the log-rank (Mantel-Cox) test.

Chemical Characterization
Saponins and polyphenols are the most widely studied classes of the Agave genus. After the extractive process, the yield for AsRE was 7.08%. The results obtained showed that saponins comprise 18.01% ± 1.81 of AsRE while the concentration of total flavonoids is 0.92 ± 0.26 µg EQ/mg. The infrared spectra of AsRE displayed some characteristic bands compatible with polysaccharides, saponins and phenolics ( Figure 1).

In Vitro Antioxidant Activity
The total antioxidant capacity test (TAC) showed a high activity for AsRE (91.75 mg Eq AA/g). The reducing power assay showed AsRE (1.0 mg/mL) with an activity of 49.13% ± 2.03 in comparison to ascorbic acid (100 µg). When AsRE was assessed for its ability to scavenge DPPH radical scavenging, it showed that it was able to scavenge the radicals in a concentration-dependent manner up to a concentration of 5.0 mg/mL with maximum values of 94.40% ± 1.94 (Figure 2). Starting at the concentration of 4.0 mg/mL and higher concentrations, there is no significant difference between AsRE and the positive control used. In relation to the metal chelating capacity, the extract showed a high cupric chelating power (70% at 0.5 mg/mL). Furthermore, for ferric chelating activity, the extract (1.0 mg/mL) showed 15.10% ± 3.44 chelating activity (Figure 3). AsRE demonstrated hydroxyl radical scavenging in a concentration-dependent manner, reaching 71.38% ± 3.25 at 2.0 mg/mL (Figure 4).
The results presented demonstrate the potential of the extract as an antioxidant raw material with possible intrinsic performances in different phases of the oxidative cascade. The IC 50 values obtained for antioxidant tests can be seen in Table 1.

Bacterial Growth Assay
Before the in vivo antioxidant tests, the effect of the extract on the growth of E. coli OP50 bacteria, used as a food source, was evaluated. The toxicity against E. coli OP50 can lead to an increase in the lifespan of C. elegans [17]. The AsRE (1.0, 3.0 and 5.0 mg/mL) did not exhibit antibacterial activity against E. coli OP50.

Intracellular Accumulation of ROS in C. elegans
Nematodes treated with 1.0, 3.0 and 5.0 mg/mL of the extract showed a reduction in intracellular ROS levels of 45.18% (p < 0.0001), 36.84% (p < 0.01) and 44.19% (p < 0.0001), respectively (Figure 6a). However, no statistical differences were observed among the AsRE at different concentrations.

Oxidative Stress Survival Assay
The reduction in intracellular levels of ROS enabled us to evaluate the ability of the extract to increase the survival fraction of worms against the acute stress induced (TBHP). The extract promoted a significant change to the right in the survival curve under acute oxidative stress induced at the concentrations evaluated when compared to the control (p < 0.0001) (Figure 6b). The increase in average life span when compared to the control group (p < 0.0001) can be seen in Table 2. The OS process leads to cumulative damage to biomolecules that promote the aging process and consequently the survival time of C. elegans [13]. Therefore, the extension of the lifespan is strongly associated with enhanced resistance to stress [15]. Thus, we suggest that there is an increase in the survival fraction with a reduction in the ROS levels promoted by the extract. Where: n 1 = number of animals used in experiment; AsRE = A. sisalana agro-industrial residue extract.

Effect of AsRE on the Expression Levels of the Oxidative Stress Relative Gene (gst-4::GFP)
The CL2166 worms were previously treated with 1.0, 3.0 and 5.0 mg/mL of AsRE to assess their ability to modulate the expression of genes related to oxidative stress resistance (gst-4::GFP).
A dose-dependent profile was observed in the negative expression of the gst-4::GFP gene (Figure 7). However, when compared to the control, only the concentration of 5.0 mg/mL showed a significant reduction in the expression of gst-4::GFP, reducing the fluorescence intensity by 27.04% ± 0.04.

Discussion
The search for new molecules with biological activity has been intensified in studies with plant extracts due to their diverse and complex chemical constitution. In a previous study, our group showed that A. sisalana extract contained polyphenols (2.53%) and sugars (65%) [10]. Now, in this present study, to advance the chemical characterization of this extract, the total saponins and total flavonoids were analyzed. The infrared analyses also indicated the presence of sugar (OH, C-H, C-O-C, C-O, C=O) [23,30,34,35], saponins (CO, C-O-C, C=O) [32,33], and flavonoids [36] in AsRE.
It is interesting to highlight that among the phenolic compounds of the genus Agave, flavonoids, homoisoflavonoids and phenolic acids were the most common phenolic compounds found [7]. Therefore, we hypothesize that the low amount of total flavonoids could indicate a richness of phenolic acid in the extract. Furthermore, the presence of saponin characterization data was an expected result regarding the historical importance and occurrence of these metabolites in the genus. Taken together, these features are compatible with the expected chemical profile of A. sisalana hydroethanolic extract and these biomolecules demonstrated a strong correlation with biological activities, such as antioxidant activity [5,8].
In particular, molecules with antioxidant capacity are used in order to combat the damage caused by oxidative stress and to prevent aging [37,38]. Many studies with extracts and their components have demonstrated the correlation between antioxidant activity and primary and secondary metabolites of plants such as polyphenols [39,40], saponins [8] and polysaccharides [41][42][43][44].
Phenolic compounds are constituted of one or more aromatic rings containing variable hydroxyl groups, and are, therefore, potentially capable of stabilizing reactive species by forming resonance-stabilized phenoxy radicals [45]. The activity of saponins, on the other hand, has been related to their glycosylated C-3 hydroxyl group [8], while polysaccharides can donate atoms and stop chain reactions by turning radicals into stable non-harmful molecules. According to Huang et al. [44], the carboxyl and carbonyl groups, present in polysaccharides, decrease the dissociation energy of the O-H bond, which results in increased proton release, potentiating the antioxidant activity of these compounds.
The antioxidant activity of AsRE was evaluated in three stages: initiation (Total Antioxidant Capacity, power reducing and DPPH radical scavenging), propagation (cupric and ferric chelating), and termination (hydroxyl radical scavenging) [46].
The total antioxidant capacity test showed an activity for the AsRE of 91.75 mg EqAA/g, which is considered high antioxidant activity [24]. Moreover, AsRE showed high reducing power when compared with positive control ascorbic acid (49.13% ± 2.03). A previous study using isolated polysaccharides of agro-industrial A. sisalana showed a power reduction of 37% compared to ascorbic acid [5]. Reducing properties are generally associated with the presence of biomolecules, which exhibit antioxidant activity by scavenging effect on the reactive species [17], donating hydrogen atoms to break chain reactions [5].
AsRE showed high DPPH radical scavenging activity when compared to the positive control ( Figure 2). Polysaccharides isolated from this material demonstrated the ability to interact with oxidative processes acting as an antioxidant agent [5,11]. Furthermore, Ribeiro et al. [8] pointed out a more significant activity for the extract when compared to the isolated compounds. These authors highlighted the low ratio of saponins isolated from the leaves of A. sisalana with the elimination of DPPH radicals (IC 50 > 10.0 mg/mL), while the extract containing saponins and polyphenols showed results (IC 50 = 1.4 mg/mL) similar to those observed for AsRE (IC 50 = 1.44 mg/mL). Therefore, it is possible to imply that the diversified chemical composition of this plant, when compared to isolated molecules, may favor a greater antioxidant potential. The significant results of the total antioxidant capacity and reducing power in vitro indicate that AsRE has the ability to interact with the systems and give electrons to minimize the attack of radicals [47], which correlates with the ability to eliminate radicals [5].
AsRE demonstrated chelating activity of cupric and iron ( Figure 3). Several studies showed plant extracts with iron and/or cupric chelating activity. These authors relate this chelating activity to the presence of molecules such as polyphenols [48] and polysaccharides [5,24,42,49]. The evaluation of the metal chelating potential is important due to transition metals, free of redox activity, such as Cu 2 + and Fe 2 +, which can be extremely pro-oxidant, meaning that they can react with hydrogen peroxide and catalyze the formation of hydroxyl radical through the Fenton reaction [37]. Therefore, when AsRE acts as a metal chelator, it is indirectly combating the damage caused by the hydroxyl radical. However, there are two types of antioxidant mechanism against the hydroxyl radical: one suppresses the generation of the hydroxyl radical and the other scavenges the hydroxyl radicals generated [24]. Due to this, the potential of AsRE for radical hydroxyl scavenging was also evaluated ( Figure 4). The scavenging ability of AsRE for hydroxyl radicals increased with the concentration within the range of 0.5 e 2.0 mg/mL. The rate that AsRE and gallic acid scavenged hydroxyl radicals at 2.0 mg/mL was 71.38% and 100%, respectively. Zhang et al. [5] showed the scavenging effect on the hydroxyl radical by polysaccharides of A. sisalana residue. However, the activity found for these metabolics was around 33% at 4.0 mg/mL. Biomolecules and extracts of other sources also demonstrated activity for scavenging hydroxyl radicals. Cheng et al. [50] showed the activity of flavonoids from Carex meyeriana on scavenging the hydroxyl radical. Melocactus zehntneri ethanolic extract containing phenolic compounds, saponins and polysaccharides showed 100% hydroxyl scavenging activity for polar extract (using 100 µg/mL), whereas the non-polar extracts showed an activity of around 20% [46]. Therefore, AsRE exerted their antioxidant activity in three stages: initiation, propagation and termination.
Considering that AsRE showed excellent in vitro antioxidant activity, the nematode C. elegans was used to evaluate the in vivo antioxidant potential of AsRE. In addition, after the results obtained in the in vitro tests and knowing that high concentrations are used in tests with these worms due to the presence of a thick cuticle [18], the concentrations 1.0, 3.0 and 5.0 mg/mL were used in the tests in vivo.
In first step, bacterial Growth of E. coli OP50 was evaluated. The worms feed on E. coli OP50 and the inhibition of this bacteria can lead to an increase in the lifespan of C. elegans [17]. Consequently, if the extract caused the death of the bacteria or growth inhibition, the worms should be fed with the dead bacteria so that the increase in the lifespan does not interfere with the results of the antioxidant tests performed with this model. However, AsRE did not interfere with the bacterial growth of E. coli OP50, so it was not necessary to feed them with dead bacteria. The non-toxicity of AsRE corroborates the results obtained by Santos et al. [51], which showed that hydroethanolic extracts from A. sisalana leaves and residue have no relevant activity against the bacteria tested. Similar results were observed by Ribeiro et al. [8] when evaluating saponins from A. sisalana to E. coli.
An anthelmintic effect, in vitro, against goat gastrointestinal nematodes of A. sisalana agro-industrial residue has been demonstrated by Domingues et al. [52]. Therefore, the ovicidal and larvicidal in vitro effect of A. sisalana was shown by Botura et al. [53] using gastrointestinal nematodes of goats. However, AsRE did not affect the development of C. elegans nematodes.
The results for the antioxidant capacity ( Figure 6) obtained with the C. elegans model reinforce the results observed in vitro. AsRE was able to act as an antioxidant by reducing the intracellular levels of ROS in wild-type worms and by increasing survival rates after they had been induced to strong oxidative stress. ROS are highly reactive, usually produced by the cellular aerobic metabolism that can increase dramatically when organisms are exposed to different stress conditions [54].
The C. elegans results involving the reduction in intracellular levels of ROS are commonly associated with an increase in the rate of survival of these animals, when induced to stress. Different studies have shown plant extracts with antioxidant activity capable of reducing ROS levels and increasing the C. elegans survival rate. Azevedo et al. [16] demonstrated that the aqueous extract of Uncaria tomentosa leaves was also able to reduce ROS levels and increase the survival rate of C. elegans. Panax notoginseng ethanol extracts also increased lifespan and protected C. elegans against oxidative stress. Similar results were observed by Ribeiro et al. [18] when using ethanol extract from Myrciaria tenella leaves. Moreover, a correlation between stress resistance and lifespan extension has been attributed to polyphenols [13,55] and saponins [56].
However, antioxidants can act directly to reduce ROS levels [16] or to increase resistance to oxidative stress, inducing the transcription of cytoprotective proteins [57]. To understand the mechanisms by which AsRE can act against oxidative stress, glutathione S-transferase (gst-4) gene expression levels were evaluated. gst-4 is a phase II detoxification enzyme that catalyzes the conjugation of several exogenous and endogenous electrophiles with the reduced form of glutathione, resulting in the detoxification of ROS [56].
AsRE promoted a reduction in expression of gst-4 levels in a dose-dependent manner (Figure 7). The capacity to suppress expression of gst-4 is widely interpreted as proof of their ability to reduce oxidative stress in C. elegans [16,56]. Accordingly, it could be suggested that the reduction in intracellular levels of ROS associated with increased resistance to stress and downregulation of gst-4::GFP expression is related to the direct activity of the AsRE against ROS.
AsRE was able to reduce the production of intracellular ROS and increase the mean lifespan of wild-type C. elegans under stress conditions. Since oxidative stress plays a role in many diseases and contributes to aging [12], AsRE may lead to the production of a new raw material against oxidative stress and aging in the near future. This agro-industrial residue can be considered a potential renewable, sustainable, and environmentally friendly source. Hence, it is possible to reincorporate it into the industry in new production processes, which can also generate extra income and contribute to socioeconomic development in semi-arid regions.

Conclusions
A. sisalana agro-industrial residue extract has a diverse chemical composition and proven antioxidant activity. In this study, the antioxidant evaluation, in vitro and in vivo, using the C. elegans organism model showed evidence that the extract can efficiently eliminate reactive species in vitro and in vivo, reducing endogenous levels of reactive species and promoting an increased survival rate of C. elegans under stress, acting directly on the elimination of reactive species. However, new tests should be carried out to explain the relationship between in vivo and in vitro assays. A. sisalana agro-industrial residue extract can be considered a potential antioxidant raw material for different applications in the food, cosmetic and pharmaceutical industries. We can also point out as advantages of this studied extract the ease and availability of obtaining biomass and the fact that the production process of the extract is industrially scalable, fundamental conditions for it to become commercial. This enables the prospect of introducing to the agroindustry a renewable process that promotes sustainability and socioeconomic development and brings benefits to current and future generations.