Journal of Experimental Agriculture International

42(2): 79-86, 2020; Article no.JEAI.55254 ISSN: 2457-0591 (Past name: American Journal of Experimental Agriculture, Past ISSN: 2231-0606)

Phytochemical Screening and Betaxanthines of catingicola (Gürke) Byles & Rowley Subsp. salvadorensis (Werderm.) Zappi (Cactaceae)

Rodrigo Garcia Silva Nascimento1, Ana Paula Pereira do Nascimento1, Fabiana dos Anjos Barbosa2, João Henrique Constantino Sales Silva2* and Alex da Silva Barbosa3

1Program of Post-Graduation in Agronomy, Center of Agrarian Sciences, Federal University of Paraiba, Areia-PB, Brazil. 2Program of Post-Graduation in Agrarian Sciences (Agroecology), Center for Humanities, Social and Agrarian Sciences, Federal University of Paraiba, Bananeiras-PB, Brazil. 3Department of Agriculture, Center for Humanities, Social and Agrarian Sciences, Federal University of Paraiba, Bananeiras-PB, Brazil.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors RGSN, APPN, FAB and JHCSS designed the study, wrote the protocol and wrote the first draft of the manuscript. Author ASB managed the analyses of the study and performed the statistical analysis. All authors managed the literature searches, read and approved the final manuscript.

Article Information

DOI: 10.9734/JEAI/2020/v42i230471 Editor(s): (1) Dr. Bishun Deo Prasad, Bihar Agricultural University, India. Reviewers: (1) Camilo Torres-Serna, Universidad Santiago de Cali, Colombia. (2) Agustín Maceda, Mexico. (3) Ochieng O. Anthony, Sumait University, Tanzania. Complete Peer review History: http://www.sdiarticle4.com/review-history/55254

Received 10 January 2020 Accepted 15 March 2020 Original Research Article Published 25 March 2020

ABSTRACT

In the semiarid region of northeastern Brazil, we find several cacti that are very important for the regional fauna and flora, including the “facheiro” (Pilosocereus catingicola (Gürke) Byles & Rowley subsp. salvadorensis (Werderm.) Zappi, a species that belongs to this botanical family and is widespread in the Paraiba semiarid region. The objective of this study was quantify and characterize the chemical constituents of the fruits and cladodes of Pilosocereus catingicola subsp. salvadorensis. The fruits were lyophilized and the samples were subsequently ground. A 2 gram ______

*Corresponding author: E-mail: [email protected];

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aliquot of the lyophilized material was solubilized in 5 mL of 50% MeOH and 50 mmol L-1 of sodium ascorbate and distilled water. The extractive content was monitored by absorbance in a UV-VIS Meter® SP2000 (600 nm) spectrophotometer. Analyzes were performed using the SAS® Program (North Carolina, USA), version 9.2. The constituent groups identified on the stem were the phenolic and tannin groups: steroids, tannins, , and . are the group of alkaloids of greater expression in the fruits of this species, from which the betaxanthines are its main constituents, such as vulgaxanthin III, muscarine and dopaxanthin. Pilosocereus catingicola is a potential source of these metabolites in different parts of the , which, therefore, justifies encouraging the best employment of the species.

Keywords: Cactaceae; chemical constituents; facheiro; flora; metabolites.

1. INTRODUCTION a source of phytonutrients that are important for our health because they present a high content Since then, different types of spontaneous of total extractable in their ripe and herbs have been used as key ingredients in stage, which results in relevant antioxidant the production of medicines to treat various activity. Other cacti also have an elevated diseases worldwide [1]. that antioxidant activity in their fruits, such as occur naturally in plants are responsible for the Pilosocereus gounellei A. Weber ex K. Schum. color, smell and taste of the various plant Bly. ex Rowl. [8]. structures. They are used in the production of medicines and many have antimicrobial Nonetheless, such bioactive compounds are not characteristics such as organic acids, tannins, limited only to the fruits; roots and stems can flavonoids, alkaloids, saponins, among others also present groups of chemical constituents in [2,3]. several concentrations. Usually, plant secondary metabolites are synthesized in response to Brazil is deemed the third greatest diversity stress conditions and can work as a draw for center of the Cactaceae family [4]. Many of those pollination through its contribution for the species are endemic to the Northeastern vegetable’s pigmentation, which, similarly to the Caatinga and still require further studies. human immunological system, protects the plant According to Barbosa [5], the Paraiba Caatinga from ultraviolet light and pathogens [6]. is characterized as a forest formation with the endemism of several cacti, from which, some Given the above and the lack of information on genera are highlighted and are more the screening of phytochemicals in cacti, the representative, including Pilosocereus. According purpose hereof was to quantify and characterize to this author, the Pilosocereus catingicola the profile of chemical constituents of (Gürke) Byles & Rowley subsp. salvadorensis morphological parts of Pilosocereus catingicola (Werderm.) Zappi is a species that belongs to (Gürke) Byles & Rowley subsp. salvadorensis this genus and is widespread in the Paraiba (Werderm.) Zappi. semiarid region. 2. MATERIALS AND METHODS In the semiarid region, many species have potential for exploration due to their ability of 2.1 Stem and Fruit Collection producing fruits with singular characteristics and that are able to compete in the market, both The morphological parts of P. catingicola subsp. internal and external [6]. According to the author, salvadorensis were collected from three forest researchers oriented to the employment of fruit fragments of the Caatinga, located in the species that are native of the Caatinga enable following different phytophysionomies at the the production of more knowledge and the mesoregion of the Paraiba Agreste: Arara, Areial, development of new social technologies, inciting and Boa Vista, PB. This region’s vegetation is the discovery of said biome’s potentialities. composed by the Caatinga with semi-deciduous and deciduous species, usually from the Paraiba Melo et al. [7] after their evaluation of the Agrestes. It has a tropical rainy climate and dry nutritional and functional quality of Cereus summer, and an average annual rainfall of 666.1 jamacaru DC., reported that this ’ fruits are mm. This Caatinga region is characterized by the

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practice of wood selective cutting and cattle dissolved into 1 mL 100% MeOH and stored at - grazing during the dry season. Paths are found 20°C. The extractive content was monitored all over the vegetation for the transit of animals, through absorbance in a UV-VIS Meter® SP2000 vehicles and people. at 600 nm spectrophotometer (Sanford, North Carolina, USA) at the Laboratory of Physiology The second stage of the work was composed of and Post-harvest of the Federal University of the collection of the material in field (stem and Paraíba, Campus III - Bananeiras, PB, through fruit), prioritizing young plants and their side the method of HPLC and MS. ramifications, whose length was between 4 and 5 cm, which enabled better handling without 2.4 Extractions, Concentration and damaging the source plant. The processing of Screening the cladodes and fruits was performed in a phytosanitary clinic located at the Center of After grounding and storage, 150 g of the Human, Social, and Agricultural Sciences of the material was split into four 37 gram samples, Federal University of Paraíba (UFPB), Campus which, later, were inserted into 200 ml of ethanol III, Bananeiras/PB, where the stem samples of at 95%. After the extractions at every 24 hours Pilosocereus catingicola were submitted to for three days, totalizing 72 hours, the phytosanitary treatments, such as washing the concentration of the ethanolic extract was fruits and cladodes in running water. initiated at the rotary evaporator (rotary evaporator TE-210), at a temperature of 45°C 2.2 Extraction and Storage and stored in glass ampules. After this stage, Modeling they were taken to the Center of Biotechnology (Cbiotec), located at the Federal University of The betalain extraction, storage, and profile Paraíba, Campus I, João Pessoa-PB, where they modeling was performed at the Laboratory of were submitted to phytochemical screening. Vegetal Physiology, UNIMELB, Melbourne, VIC, Australia. To test and optimize the betalain The data were submitted to Analysis of Variance. extraction, two factors were analyzed: time and To differentiate and quantify the betalain mass. The time values of 20, 70, and 120 min., variation, the ANOVA with the “One-way” and the mass values of 0.5, 1 and 1.5 g of criterion was performed based on the ratio of the lyophilized fruits were based on Maran et al. [9] area between the peaks for each studied and Sanchez-Gonzalez et al. [10]. For the population. The analyses were performed in the extraction of the content of batalaína, a linear SAS® Program (North Carolina, USA), version model used by Barbosa [5]. 9.2, licensee by the Federal University of Paraíba, 2013. 2.3 Content Determination and Betalain Profile of the Fruits 3. RESULTS

The protocol for extraction was adapted from The fruit Pilosocereus catingicola can be Stintizing et al. [10], with modifications, characterized by several colors, due to the consisting in 2 g of the lyophilized pericarp (fruit) combination of two betalain pigments, in the with the addition of 5 mL of 50% MeOH and 50 colors red/purple, and the yellow/orange mmol L-1 of sodium ascorbate. Initially, the pigment, which correspond to and lyophilized fruit was ground and then inserted betaxanthines, respectively. into 50 milliliter tubes. Each extraction was repeated five times to obtain the maximum of The linear effect was seen regarding time and betalains of the solid stage. After the mass (Fig. 1), in which the extraction time solubilization, the material was stirred in vortex increased stability (absorbance) and betalain for 5 min. at 25°C, and then centrifuged at 25°C concentration, and the mass had a significant and 2000 RCF. effect on the extracted content (P < 0.01). The increase of lyophilized dry mass did not increase The supernatant was collected with the the concentration of betalains extracted in the assistance of a 1000 microliter semiautomatic solid stage (P < 0.01), that is, the behavior was pipette. The collected material was concentrated inversely proportional between mass and a total vacuum centrifuge (SpeedVac) and then extracted contents.

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It was not possible to detect a significant effect in Liebermann-Burchard, at 0.12, 0.25, and 0.50%, the interaction between time and mass, and both gelatin (0.5%) and FeCl3 (2%), magnesium presented the same tendency regarding the ribbon, fluorescence, and foam. The presence of betalain content. Based on the betalain content, several classes of metabolites, steroids, tannins, the extraction was explained by the linear model: flavonoids, and saponins reveal that the roots, B= 11.55850213 − 0.03922998 t − 0.00004203 t2 like the stem and fruits, have important groups of − 0.66033773 m − 2.92546663 m2 + 0.04457684 bioactive compounds. mt. According to this model, to achieve maximum efficiency in the extraction, we can employ 1.2 g The results of sample II were similar to sample of lyophilized dry fruit for a period of 173 I’s, in which were identified steroids, tannins, minutes. flavonoids, and saponins. In sample III, it was identified the presence of steroids and saponins, Table 1 presents the phytochemical compounds and, in sample IV, steroids, flavonoids, and that were identified and quantified, varying saponins. Additionally, the absence of tannins is between medium and low intensities and seen both in samples III and IV, and flavonoids negative reaction to the following tests: only in sample III.

Fig. 1. Betalain content of the fruit of Pilosocereus catingicola subsp. salvadorensis in response to the extraction time and dry fruit mass

Table 1. Identification of the compounds in the phytochemical screening of the samples I, II, III, and IV 1 Samples Steroids Tannin E (%) 0.12; Gelatin (0.5%) FeCl3 (2%) Magnesium ribbon Fluorescence Foam 0.25; 0.50 0.5; 1.0; 2.0 0.5; 1.0; 2.0 (Shinoda) (Taubouk) I + + + - + + - + + + ++ ++ II + + + - - + - - + - ++ ++ III - - + ------+ IV - + + ------+ + + Legend: (E) Liebermann-Burchard; “++” (medium intensity), “+” (low intensity), “-” (negative reaction)

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Through the analysis of the fruits’ extracts After the submission of the samples to the employing reverse-phase high-performance phytochemical screening, it can be noticed that liquid chromatography (RP-HPLC), it was those with thorns presented a greater amount of possible to divide and identify components of the compounds in the cladodes in comparison with betaxanthin group at a wave length of 470 nm, the samples in which the thorns were removed below the maximum absorption for this pigment, (Table 2). Nonetheless, the amount of flavonoids which, according to Castellanos-Santiago and remained the same for the samples with and Yahia [11], would be a wave length of 480 nm for without thorns. betaxanthins. Consequently, based on the absorbance reading in the wave length 4. DISCUSSION employed, the betaxanthins present absorbance affinity, enabling the detection and identification According to Castellanos-Santiago [11], the of the four following substances of the betacyanin concentrations for the juice of the betaxanthin group: vulgaxanthin III, histidine, pulp reconstituted with water, of several ecotypes muscarine and dopaxanthine (Fig. 2). of the fruit, are between 0.05 and 5.29 mg/g, and the betaxanthin concentrations are between 0.12 The analysis of the phytochemical screening and 2.86 mg/g. These authors also state that the provided a clear visualization for the yellow/orange ecotypes have a greater determination of the compounds present in the betaxanthin concentration in comparison with the facheiro’s stems, both with and without thorns. purple ecotypes, therefore stressing the idea that According to the clinical analysis, chemical a greater amount of these pigments will result compounds are seen in the samples of facheiro in yellow and orange colors, as the beta- cladodes with and without thorns, including: cyanins represent fruits with reddish and saponins, flavonoids, steroids, and tannins, from purple colors. The betacyanin amount was very which the former is the most representative one. different of the amount of betaxanthins

Table 2. Chemical compounds found in cladodes of Pilosocereus catingicola subsp. salvadorensis with and without thorns

Evaluation 0.12 - 0.25 - 0.50 2.0% 2.0% Foam Cladodes Levels Steroids Tannins Flavonoids Saponins A1 + +++ ++ +++ Presence of A2 + +++ ++ ++ thorns A3 ++ ++ ++ + A4 +++ +++ + ++ A1 + + ++ ++ Absence of A2 + ++ ++ + thorns A3 ++ +++ + + A4 + ++ ++ + Legend: “+++” (high intensity); “++” (medium intensity); “+” (low intensity)

Fig. 2. Chromatographic profile of betaxanthins in fruits of Pilosocerus catingicola subsp. Salvadorensis

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quantified, which explain the reddish/purplish catingicola, [16] identified the same components, color of the fruit’s pulp of Pilosocereus which implies that is common to find these catingicola in the stage of advanced ripening. compounds in the species and that it is a potential source of these metabolites. Maran et al. [9], evaluating the temperature, time, mass, and pH in the extraction process of Recent studies in registered the use of betalains of Opuntia ficus-indica fruits, noticed Cereus jamacaru DC. (Cactaceae) root tea to that this factors affected this variable and treat urinary infections. According to Santana determined the temperature of 42°C for the [15], the vegetables synthesize and build up a extraction, during an extraction period of 115 great diversity of phenolic compounds acting on minutes, employing 1.2 g of dry mass and a the plant’s protection. Its antioxidant potential solution pH of 6.9. Under these conditions, it was can also prevent DNA damages caused by -1 possible to extract 41.54 mg . carcinogens or free radicals. Still according to the author, in the popular medicine, the root has These results support the possible utilization of been used to treat respiratory and renal this fruit in the food industry as a source of diseases, and as diuretics. These studies show natural hydrosoluble pigments, as suggested [11] the importance of the Cactaceae family, with the fruits of different varieties of Opuntia especially the Pilosocereus catingicola subsp. spp., which can be found in different salvadorensis species, which, like the root of concentrations due to the several varieties Cereus jamacaru, also has secondary metabolite employed, ripening stage, edaphoclimatic groups in its roots, as seen in the results of the conditions and/or cultural practices [12]. The tables, with potential for pharmacological use, greatest commercially explored source of considering its antioxidant, anti-inflammatory, betalains is beets (Beta vulgaris L.). and similar effects. Nonetheless, sensory studies already shown that the geosmin compound exhales a essence The Opuntia fruits have several colors due to the characteristic smell of earth, which can limit its presence of pigments designed by betalains, use as pigment [10]. The data presented herein which are divided into two great groups: suggest the potential of the facheiro fruit as a betaxanthins of yellow-orange color and betalain source with commercial, technological, betacyanins of red-violet color, in which the most and functional value. Further studies about well known in both groups are I and toxicity and pigment cost are necessary. , respectively [17]. The fruits of Pilosocerus catingicola subsp. salvadorensis The difference in the presence of compounds present betalains, both betaxanthins and found in the samples in Table 1, can be , which are natural nitrogen explained, according to Santana [13], by the fact compound pigments located in the plants’ that the quantity of these compounds was vacuoles [16]. possibly too low to be identified in the tests. In addition, the secondary metabolites of plants According to Volp et al. [18], fruits of cactus pear may change, depending on environmental stimuli (Opuntia fícus-indica), which also has betalains, and the time and time of collection. make LDL-cholesterol particles more resistant to the oxidative process when incorporated to them. According to Lima Neto et al. [14], the According to these authors, important biological phytochemical screening has the purpose of activities are related to the antioxidant proprieties performing the preliminary detection and contsaained in natural pigments that protect prospection of the different chemical constituents against oxidative damages, has anti- of plants employing proper solvents, enabling the inflammatory effects, and prevent chronic identification of classes of secondary metabolites diseases. Naturally colored food has important of pharmacological interest. Before the resulting bioactive compounds that promote health and phytochemical profile of the gross ethanolic welfare by preventing and frequently assisting in extracts of roots of Pilosocereus catingicola the cure of diseases. subsp. salvadorensis, employing the methodology of Matos [15] and adapted by Silva et al. [19] in their evaluation of the Barbosa [16], it is possible to identify the antibacterial, antioxidant, and cytotoxic activity of presence of the main classes of secondary the species Opuntia cochenillifera (L.) Mill, metabolites. Performing the same study on the reported, through the detection of chemical gross ethanolic extracts of roots of Pilosocereus substances present in the gross ethanolic

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