Phytochemical screening, Sun Protection Factor (SPF) and sugar analysis of jatobá fruits (Hymenaea martiana Hayne): A native medicinal plant from the San Francisco Valley

Hymenaea is a genus of the Fabaceae family, highly distributed from Central America to South America, mainly in the Amazon basin, with about 25 species described in the Americas. Plants of the this genus are known in Brazil as “jatobá”, “jetaí”, “jataí-uva”, “jetaíba” and are largely distributed in Brazil, especially in the Brazilian biome called “cerrado” (savanna-like vegetation) (Minas Gerais, Bahia, Goias, and Tocantins) and the Amazon forest (Boniface et al., 2017). Dne species of this genus can be found in Brazilian Caatinga, Hymenaea martiana, with a great phytochemical and medicinal potential (Dliveira et al., 2016; Dliveira et al., 2018).


Introduction
Hymenaea is a genus of the Fabaceae family, highly distributed from Central America to South America, mainly in the Amazon basin, with about 25 species described in the Americas. Plants of the this genus are known in Brazil as "jatobá", "jetaí", "jataí-uva", "jetaíba" and are largely distributed in Brazil, especially in the Brazilian biome called "cerrado" (savanna-like vegetation) (Minas Gerais, Bahia, Goias, and Tocantins) and the Amazon forest (Boniface et al., 2017). Dne species of this genus can be found in Brazilian Caatinga, Hymenaea martiana, with a great phytochemical and medicinal potential (Dliveira et al., 2016;Dliveira et al., 2018).
Being characterized as a region of economic and agricultural importance in the Brazilian Caatinga Biome, the San Francisco Valley is a region in the west of the state of Pernambuco and north of Bahia, bordering the San Francisco River, with semiarid vegetation and climate (Sá et al., 2009). The particular characteristics of this biome, like the high solar incidence and long periods of drought, can interfere directly in the vegetation morphology and especially with its secondary metabolites composition, since some phenolic compounds such as flavonoids may play a special role in the chemoprotection against ultraviolet radiation and external aggressions (Markham et al., 1998).
Among the native medicinal plants found in the São Francisco Valley, with importance in the local traditional medicine, Hymenaea martiana Hayne (Fabaceae) can be highlighted, popularly known as "jatobá" in Northeastern Brazil. Shanley & Medina (2005) characterize this plant as a large tree, 15-20 m high, with dense foliage and thick bark, straight trunk, about 2 m in diameter.
The traditional use of jatobá has been reported, and the alcoholic extract from the barks of H. martiana has been used in the treatment of inflammations and rheumatism, and also as antinociceptive and analgesic (Neves et al., 1993;Gazzaneo et al., 2005). Some substances that have been linked to the pharmacological activities are flavonoids (Carneiro et al., 1993;Silva et al., 2012).

Phytochemical screening, Sun Protection Factor (SPF) and sugar analysis of jatobá fruits (Hymenaea martiana Hayne): A native medicinal plant from the San Francisco Valley
potential of the fruits and seeds of Hymenaea, with important sugars and phenolic compounds in the chemical composition.
Phenolic compounds, such as flavonoids, can absorb ultraviolet radiation (UV) and this class of substance can play an important role against UV radiation from sunlight . The UV radiation can be subdivided into three bandsI: UVA, UVB and UVC. The UVA radiation can be classified as an inducer of oxidative processes in the skin and the use of photoprotectors against UVA radiation has been stimulated, aiming at protection against skin diseases (Popim et al., 2008).
Sunscreens derived from natural products and cosmetic products have been developed using plant extracts and oil as raw material due to their photoprotective activity, and the popular acceptance has been positive .
Nevertheless, according to a literature review, neither phytochemical nor biochemical study on the fruit from Hymenaea martiana was found, and the photoprotective activity was not evaluated. On light of this, this study aimed to determine sugars and secondary metabolites, as well as analyze the sunscreen potential of Hymenaea martiana fruits.

Plant material
The fruits of Hymenaea martiana Hayne were collected in Petrolina, Pernambuco, Brazil, in July 2015, and were identified by the Federal University of São Francisco Valley Herbarium (HVASF), with a voucher specimen n° 6444, coordinates 09"11'04.30 ° S, 040"18'05.40 ° W, 357 m high. The fruits were separated into two plant materials (pulp and seeds) and were pulverized using a mill (Quimis  , SP 31, Diadema, Brazil).

Sugar analysis using High-performance Liquid Chromatography (HPLC)
The determination and quantification of sugars (glucose, xylose and arabinose) were carried out through HPLC, using a liquid chromatograph equipped with a ProStar 210 pump model (Varian  , Palo Alto, USA), model 7725 manual injector (Rheodyne  , Sigma-Aldrich, St. Louis, USA), with a 20-µL loop. The chromatographic conditions are presented in Table 1.
The sample preparation was carried out with a dilution of 1 g of each sample (pulp and seed) with distilled water, in the rate of 1I:20 (pulp/seedI: solution) (dilution factor = 20). The solution was stirred, allowed to stand for a few minutes and filtered; then, 20 μL was injected into the equipment.

Phytochemical screening of the fruits of H. martiana
Onitially, 100 g of pulp and seeds was added to 500 mL of ethanol 95% in amber flasks, which were then kept in the dark for three consecutive days at room temperature, shaken daily. After the maceration time, the residue was filtered. The procedure was repeated three times. After the process, the extraction solution was concentrated under vacuum (Fisatom  , model 801, São Paulo, Brazil) at 50 °C (Silva et al., 2012).
An aliquot of the extracts from each plant material was solubilized in chloroform and subjected to analysis through thin-layer chromatography (TLC) with silica gel 60 F 254 , with aluminum support, applied with a micropipette and eluted with different solvent systems, according to Wagner & Bladt (1996), seeking to highlight the main groups of secondary metabolites (Table 2).

Assessment of sunscreen activity
The sunscreen activity was performed using the spectrophotometric analysis of the diluted solutions, according to Mansur et al. (1986). The extracts were previously dried in an oven at 40 °C for 60 minutes. Dilutions were prepared with concentrations of 5, 25, 50 and 100 mg.L -1 . Readings from 290 to 320 nm with 5-nm intervals were carried out in a spectrophotometer (Quimis  , model Q898UVDB, Diadema, Brazil), with quartz cells of 1-cm optical path.
Calculations of the Sun Protection Factor (SPF) were made considering the intervals λ determined using the following the equation described by Mansur et al. (1986)I: SPF = Correction Factor x Amount of absorbance 290-320 nm x Erythemogenic  Effect of radiation (λ) x Spectrophotometric reading of sample absorbance (λ).

The EE (λ) (Eritemogenic Effect) and O(λ) (Solar Ontensity) used for the calculation of the SPF (Sun Protection Factor)
were the same found in the literature, and the abs (λ) was the spectrophotometric reading of the absorbance of the sunscreen solution (Mansur et al., 1986). The dilution factor was applied for equivalence correction and CF (Correction Factor) was 10 and the determinations were conducted in triplicates.

Sugar analysis using High-performance Liquid Chromatography (HPLC)
The concentrations of sugars found in the pulp and the seeds of H. martiana are displayed in Table 3.
A polysaccharide has been studied in Hymenaea, xyloglucan, which has been extracted from cotyledons (Buckeridge et al., 1997), seeds (Lima et al., 1993) and leaves (Busato et al., 2001). As one of the main hemicellulosic polysaccharides, xyloglucan is present in primary cell walls of dicotyledonous plants (Mcneil et al., 1984;Fry, 1989;Carpita & Gibeaut, 1993), but it was also found in the gymnosperm (Kakegawa et al., 1998). This carbohydrate is related to important functions such as the control of cell expansion, effects on growth and as a reserve of carbohydrate in seeds (Fry, 1989;Hayashi, 1989;Kai & Petkowicz, 2010). Galactose, glucose, xylose and arabinose were identified in this species, derived from the degradation of this polysaccharide present in seeds of this species (Dmaira et al., 2007).
Regarding the measurements, the pulp has a higher amount of glucose, xylose and xylitol, as found in other fruits (Roesler et al., 2007). The seeds showed other types of sugars, such as arabinose, which have been previously described in H. courbaril (Dmaira et al., 2007), and cellobiose, not yet reported in Hymenaea. According to the literature, this is also the first report of the presence of xylitol in species of this genus.
According to Mussatto & Roberto (2002), xylitol is a non-toxic polyol, classified by the FDA as a safe additive that may be used as a substitute for sugar (sucrose) and that has nutritional properties and benefits to the human health. Ot may also act in the cure or prevention of diseases, with various applications in the dental and medical fields and reports on the efficacy in the prevention and control of dental caries. Being a natural sweetener well tolerated by diabetics, it can be used as an adjuvant in the treatment of lipid metabolism disorder and renal injuries. On addition, xylitol can prevent lung infections, otitis and osteoporosis. On addition to these actions, it has the advantages of high chemical and microbiological stability, being a potential additive for food (Mussatto & Roberto, 2002). Thus, the biotechnological potential of the fruits of H. martiana is evident because of its important carbohydrates in the chemical composition.

Phytochemical screening of the fruits of H. martiana
A phytochemical screening was carried out with the extracts and the TLC plates were analyzed. The maceration of the fruits indicated the presence of anthracene derivatives, flavonoids, monoterpenes, diterpenes and naphthoquinones; the maceration of the seeds indicated the presence of anthracene derivatives and flavonoids (Table 4).
The analysis of the pulp extract indicated the presence of anthracenic derivatives, flavonoids, monoterpenes, diterpenes, naphthoquinones, triterpenes and steroids. Although there are no studies with the fruits of H. martiana, previous studies on fruits of other species of the genus identified substances with important pharmacological activities, such as the flavonoid rutin (Peres et al., 2013), the carotenoids β and α-tocopherol (Dias et al., 2013) and diterpenes such as spathulenol, stigmasterol and β-sitosterol, among other terpenes (Aguiar et al., 2010;Dias et al., 2013). The analysis of the extract from the seeds indicated the presence of anthracenic derivatives and flavonoids. However, studies were found in other species of the genus that only indicated the presence of coumarin (ipomopsin and himenain) (Simões et al., 2009) and xyloglucans (Lima et al., 1993;Buckeridge et al., 1997).

Assessment of sunscreen activity
The Sun Protection Factor (SPF) values were calculated according to Mansur et al. (1986) (Table 5).  With the extract obtained from the pulp, SPF values obtained were not significant, with 0.66 ± 0.41. But the extract obtained from the seeds was 4.54 ± 0.11. According to the National Health Surveillance Agency (Brasil, 2012), the minimum value for SPF for sunscreen products is 6.0. Although the values found are lower than the recommended by the national health surveillance agency, the extracts obtained from the fruits of H. martiana could still be used for the future development of sunscreen products, as for example together with chemical filters, which would bring protection within the minimum values recommended, while also providing several benefits to the formulation with the supply of different secondary metabolites containing important medicinal properties. Currently, the inclusion of natural products in the photoprotective formulations is a tendency  and studies have focused on the analysis of chemical constituents containing chromophores and compounds with antioxidant activity (Polonini et al., 2011) such as the phenolic compounds, mainly the flavonoids , substances found in phytochemical screening of the fruits and seeds of the species under study.
Despite the seed extract showed higher value for SPF then the pulp extract, it presented qualitatively less presence of flavonoids and tannins, as shown in Table 4. This fact may be due to the chemical composition of the seeds, with may include wax, oil and fatty acids. Previous study presented the cosmetic potential of the lipid fraction of seeds, as skin moisturizers and suscreens, with significant SPF values related to fatty acids as linoleic and oleic acid in the seeds of coffee beans (Wagemaker et al., 2011). So, the SPF values for the seed extract of Hymenaea martiana may be related not only to the phenolic compounds, but also to the lipid fraction of the seeds. Therefore, more in-depth studies are necessary to quantify and identify the major constituents of these materials, to improve the phytochemical knowledge of the species.

Conclusion
This study shows the great biochemical and medicinal potential of the fruit from Hymenaea martiana. The sugar analysis showed that several important carbohydrates are found in the pulp and seeds. The phytochemical screening identified important bioactive secondary metabolites and the sunscreen activity showed a great potential for the future development of cosmetic formulations.
Thus, bringing new data on the fruits of Hymenaea martiana, this study emphasizes the biotechnological potential of the species, aggregating chemical and medicinal value to a species native to the Caatinga of the São Francisco Valley, an important step for the conservation of its biodiversity.