Phytochemistry and biological activities of latex from Schubertia grandiflora Mart. (Apocynaceae)

Schubertia grandiflora is a plant species that produces latex from roots to flowers. This study aimed to evaluate the latex of S. grandiflora on qualitative phytochemical analysis and effects of quantitative biological activities. Latex was collected from logs. Phytochemical analysis of precipitation and colorimetry were carried out for several groups of phytogroups. Antifungal activity was performed on Sclerotinia sclerotiorum , Colletotrichum acutatum and C. gloeosporioides , cytotoxic activity on Artemia salina , antioxidant activity on DPPH free radical and determination of inhibition on acetylcholinesterase. The latex proved to be rich in phytochemical groups, inhibition capacity of S. sclerotiorum with 64% and for C. gloeosporioides of 30%, low rate of cytotoxicity with 25% in 1000 µg mL -1 , reduction of DPPH free radical of 67% and AChE inhibition of 86%. Schubertia grandiflora latex proved to be a potential natural phytochemical agent for diverse biological activities through this study.


Introduction
Apocynaceae Juss. family is considered as one of the largest among the Angiosperm botanical families, with more than 5,350 species distributed in 378 genera and five subfamilies found in tropical and subtropical regions of the planet (Endress et al., 2014(Endress et al., , 2018Campos;Farinaccio, 2021). Among this exuberant flora in Apocynaceae, we can mention Schubertia grandiflora Mart. & Zuccarini (1824: 57) found in Cerrado domain environments, Pantanal and Caatinga biomes, and in anthropic areas (side of dirt roads) (Araújo, 2014). This species is characterized by vine-like appearance, white laxex ( Figure 1, A and B), pubescent branches, leaves with oval blade, umbelliform summits, subaxillary, calyx with oval lobes, acuminate, abaxially pubescent, corolla rotaceous, alvescent, abaxially glabrous, adaxially pilose with elongated trichomes, corona with target segments that are longer than the anthers and oval follicles with longitudinally distributed spinous protuberances.
The plant bears flowers between January and March, with fruiting in September (Fontella-Pereira, 2005). It is distributed in more than 16 Brazilian states, including Paraguay, Bolivia and Argentina. In Brazil, there are no scientific records that S. grandiflora has individuals in the states of Acre, Alagoas, Amapá, Espírito Santo, Rio de Janeiro, Rio Grande do Norte, Rio Grande do Sul, Santa Catarina and Sergipe (Flora do Brasil, 2020) .
Schubertia grandiflora produces throughout its length (roots, branches, leaves and flowers) a white latex without aroma that becomes rigid with time. This latex is still unknown at a scientific level regarding its phytochemical composition and possible and attractive biological activities, thus leading, in this study, to determine which are the phytochemical groups and some biological activities of great medical interest.
This study aimed to qualitatively evaluate which groups of phytocompounds and quantitatively some biological activities such as antifungal, antibacterial, cytotoxic, antioxidant and acetylcholinesterase inhibition evaluating the latex extracted from the branches of Schubertia grandiflora.

Identification and deposit of the specimen
The plant species was identified by the second author of this study, and a specimen was deposited in the Herbarium in the Laboratory of Vegetal Systematic belonging, to the Department of Biological Sciences of the Goiano Federal Institute, Rio Verde, Goiás, Brazil, with the following Voucher: HRV 13509.

Latex collection
Latex collection was performed manually in the morning between 7 and 8 h (am) by cutting branches of S. grandiflora with a sterile stainless steel knife. The latex was collected using a sterile disposable syringe and sterile glass vials. The sample was added 1 mL of sterile water to 5 mL of latex (1:5, v/v). The vials containing exudate were kept refrigerated at -2 °C in a thermal box and transported to the laboratory for analysis. 95 g of latex in natura were obtained from 45 samples of S. grandiflora.

Phytochemical screening
Phytochemical analysis was performed for the main groups of special metabolites present through staining, and qualitative precipitation techniques. With the latex of S. grandiflora, the following tests were performed for: organic acids, reducing and non-reducing sugars, alkaloids, anthraquinones, catechins, depsides and depsidones, coumarins, steroids, phenols and tannins, flavonoids, polysaccharides, proteins, amino acids, purines, foaming and hemolytic saponins, sesquiterpenolactones, organic acids, and azulenes proposed by (David et al., 2019).

Antifungal assay
The agar diffusion method was used to determine the antifungal activity on Sclerotinia sclerotiorum, Colletotrichum acutatum and Colletotrichum gloeosporioides as described by Toigo et al. (2022) adapted. The strains used are: SS12-21, CA15-67 and CG 16-21 respectively, from the second author's mycological bank. The fungal strains were cultured at 20 °C for 10 days for S. sclerotiorum and 28 °C for 3-5 days for the other strains.
A mycelium disc with a diameter of 7 mm was transferred to the center of Petri dishes with a diameter of 10 cm containing sterile potato, dextrose and agar (PDA) medium. Different latex concentrations were used, dissolved in 0.1% Triton X-100 to render doses between 100-500 μL mL -1 , in each plate 500 μL of the concentration was pipetted. The plates were transferred to an incubator at 20 °C (10 days) and 28°C (3-5 days), respectively. The diameter of the zone of inhibition was measured and recorded as an indicator of antifungal activity and expressed in percentage (%) using a digital caliper. The commercial reference fungicide Frowncide 500 SC was used as a positive control (dose of 10 μL mL -1 ).
Triton X-100 emulsifier was also evaluated at the lowest dose under investigation (100 μL mL -1 ). The agar diffusion assays applied against the three fungi were performed in quadruplicate. Mycelial growth was obtained daily until complete fungal growth separately on control plates. The experimental design was completely randomized. The data were submitted to analysis of variance (ANOVA) and the averages of the treatments were evaluated by the Scott-Knott test with 5% of significance using the statistical program ASSISTAT. The percentage of mycelial growth inhibition was calculated by the following equation 1.

Citotoxicity assay
Cytotoxicity assay on Artemia salina was based on the technique described by Araújo et al. (2010). Artemia saline water solution was prepared (500 mL) concentration 35.5 g L -1 for preliminary incubation of 50 mg of A. salina eggs. The solution containing eggs of A. salina was maintained under homogenization on a magnetic table at 50 rpm exposed to white artificial light for 24 h for hatching of the larvae (metanauplii). Soon after, the metanauplii were collected and kept for another 24 h for the nauplii stage.
The exudate solution of S. grandiflora was prepared containing 62.5 mg of exudate in natura, added to 28 mL of saline solution and 2 mL of dimethylsulfoxide (DMSO). Ten nauplii were selected in triplicate, divided into six concentrations (1000; 750; 500; 250; 100 and 50 µg mL -1 ). In each tube, an aliquot of the exudate solution ranging from 3000 to 100 µL was added and the volume was completed to 5 mL with saline solution, where concentrations ranging from 1000 to 1 µg mL -1 were obtained. The data were submitted to analysis of variance (ANOVA) and the averages of the treatments were evaluated by the Scott-Knott test with 5% of significance using the statistical program ASSISTAT.

DPPH reducing activity
The antioxidant assay was performed using the methodology as described by David et al. (2019) adapted, in the reduction of the free radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH). DPPH stock methanolic solution, was prepared with a concentration of 40 µg mL -1 and kept refrigerated at -8 °C in a place free of light. The in natura exudate was diluted in methanol in a 1:5 ratio (v/v). Then, an aliquot of 0.8 mL of the solution was added to 0.2 mL of the DPPH stock solution. The mixture was then kept at rest protected from light for 30 min in a refrigerator at -4 °C. After this time, the sample was read in a UV-Vis spectrophotometer at a wavelength of 555 nm. Distilled water was used as a negative control, and a standard ascorbic acid solution was used as a positive control. The ability to reduce DPPH was expressed in percentage (%) of sequestration according to equation 2. %AA = 100 -{[(Abs sample -Abs blank/Abs control)100]/Abs control} Eq. 2 WHERE: %AA = Abs absorbance of the sample; Abs absorbance of the blank, and Abs absorbance of the control.

acetylcholinesterase inhibition
The colorimetric method for determining AChE inhibition followed as

Results and Discussion
Phytochemical analysis conducted on the S. grandiflora plant látex, revealed the presence of constituents which are known to exhibit medicinal as well as physiological activities. Analysis of the plant latex revealed the presence of phytochemicals namely phenols, organic acids, alkaloids, no-reducing sugars, steroids, tannins, carboxilic acids, foaming saponins and hemolytic saponins.
The phenolic compounds possess biological properties such as anti-microbial agents (Hussain et al., 2011), anti-apoptosis, bactericides, antiaging, anti-carcinogen, anti-inflammation, anti-atherosclerosis, cardiovascular protection and improvement of endothelial function, inhibition of angiogenesis and cell proliferation activities (Han et al., 2007;Yadav;Agarwala, 2011). Alkaloids have been closely associated with medicinal uses since centuries and one of their common biological properties is their cytotoxicity (Nobori et al., 1994), analgesic, antispasmodic and bactericidal activities (Hussain et al., 2011).
It was also observed that the latex contains saponins which are known to contribute inhibitory effect against inflammation (Just et al., 1998). Saponins has the property of precipitating and coagulating red blood cells. Some of the characteristics of saponins include formation of foams in aqueous solutions, hemolytic activity, cholesterol binding properties (Sodipo et al., 2000;Okwu, 2004;Hussain et al., 2011). Table 1. Phytochemical prospection of the Schubertia grandiflora latex.

Phytochemical groups Reaction
Phenols + The latex demonstrated effective antifungal activity against S. sclerotiorum and C. gloeosporioides with maximum growth inhibition of 64 and 30% at the maximum concentration of 500 µL mL -1 , respectively. For C. acutatum, no inhibition activity was observed for this strain at different latex concentrations, demonstrating that this variety is resistant to S. grandiflora phytocompounds (Table 2). Oliveira-Tavares et al. (2019) obtained promising results for Jatropha multifida latex on Candida grablata and Candida tropicalis with MIC of 100 µg mL -1 . Corroborating our phytochemical findings, the antifungal action observed in Table 2 suggests that phenolic compounds, organic acids, alkaloids and tannins have antifungal action (Duarte et al., 2014;Menezes Filho et al., 2021;De Morais et al., 2021). The lethality bioassay on A. salina is used with reliable evidence about the toxicity of a given cell sample, especially in humans and animals. Our results demonstrates that S. grandiflora latex has a low rate of cytotoxicity even at the highest concentration of 1000 µg mL -1 with only 25% mortality. After 24 h of exposure (Table 3). In the negative control DMSO there was no mortality in any of the evaluated larvae, and in the positive control using potassium dichromate there was 100% mortality. In the study, by Menezes Filho et al.
(2022) evaluating the latex of Manilkara zapota, the researchers obtained mortality between 65 and 100% on A. salina, with lethal concentration (LC50) of 17.9 µg mL -1 . The percentage of DPPH free radical inhibition was 67.36% and AChE with 86.13% for S. grandiflora latex. The most widely used DPPH and AChE activity assay is the 96-well microplate assay. This is due to the economy of reagents which are mostly expensive but generates quick results in a short time.

Conclusions
Schubertia grandiflora latex presents a formidable number of groups of phytocompounds, in addition to important antifungal activity against Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, low cytotoxic action against Artemia salina, an important antioxidant agent in the reduction of free radical DPPH and strong AChE inhibition activity. New studies should be carried out evaluating other biological activities using latex from S. grandiflora, which proved to be attractive at a scientific level with important results in fungal and cytotoxic inhibition, in combating the action of free radicals and in inhibiting acetylcholinesterase.

Author contributions
Márcia Divina Vidal Silva: project writing, latex collection, laboratory analysis, and article writing. Antonio Carlos Pereira de Menezes Filho: identification and collection of the plant specimen, laboratory analysis, corrections, translation and submission.

Conflicts of interest
There are no conflicts of interest.

Ethical approval
Not applicable.