DETERMINATION OF Β-ECDYSONE IN INFUSIONS OF DIFFERENT ORGANS OF BRAZILIAN GINSENG (PFAFFIA GLOMERATA) BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY DETERMINAÇÃO DE Β-ECDISONA EM INFUSÕES DE DIFERENTES ÓRGÃOS DO GINSENG BRASILEIRO (PFAFFIA GLOMERATA) POR CROMATOGRAFIA LÍQUIDA DE ALTA EFICIÊNCIA

Pfaffia glomerata (Spreng.) Pedersen is a medicinal plant commonly known as “For everything” and Brazilian ginseng. Currently, only the roots have commercial value, as they are known to contain β-ecdysone. The roots of Pfaffia glomerata are used in Brazilian folk medicine, especially as tonics, aphrodisiacs and antidiabetics. Also, based on ethnobotanical data, such as roots and leaves of the plant previously used as general stimulants, tranquilizers, anti-rheumatic, anti-inflammatory, anti-inflammatory, febrifuges, internal and external healing, hemorrhoidal, in vision and memory, for the treatment of gastric disorders, arthritis, arthrosis, anemia, illness and pain. This study aimed to determine the concentration of β-ecdysone in aqueous infusion of leaves, flowers, roots and stems of 6-month-old and 3-year-old P. glomerata individuals by highperformance liquid chromatography. The leaves had the highest concentrations of β-ecdysone in both ages (1.65 ± 0.14% for the three-year-old individual and 1.06 ± 0.10% for the six-month-old individual). The sample of flowers, root and stem of the three-year-old showed: 1.14 ± 0.10%, 0.68 ± 0.05% and 0.62 ± 0.06% β-ecdysone, respectively . In the six-month-old individual, levels of 0.57 ± 0.02%, 0.51 ± 0.12% and 0.43 ± 0.12% were observed for flowers, root and stem, respectively. The results obtained that the aerial parts of the plant can also be of commercial interest, since the leaves presented the highest concentrations than another parts of the plant at both ages.


INTRODUCTION
Technologies and management practices that add value to local production systems are of fundamental importance for sustainable territorial development (FERNANDES et al., 2016). Sustainable production systems, farming practices and forest use are global challenges addressed by the 2030 Agenda for Sustainable Development of the United Nations (ONU, 2015). A strategy that encompasses these three issues is the use of natural plant products.
Natural products are used to promote health and well-being, whether in drugs, food supplements, cosmetics, teas and aromatherapy (PINTO et al., 2002;2003). This use, which has been used for a long time, currently has science as an ally and reinforces the importance of biodiversity conservation, as we know that the medicinal and therapeutic properties of many plants and animals have not yet been discovered and that they contain potential cures for many diseases that plague the world. Natural products are the only therapeutic resource for part of the Brazilian population, and for more than 2/3 of the planet's population.
According to Pinto e colaboradores (2002) the world herbal medicine market Revista Mundi Engenharia, Tecnologia e Gestão. Paranaguá,PR,v.5,n.7, (GOMES et al., 2006). In the Paraná state, P. glomerata is grown in an area of environmental protection, on islands and floodplains in the Paraná River. glomerata (FREITAS et al., 2004;NAKAMURA et al., 2010). This ecdysteroid is used as a chemical marker of P. glomerata root quality (DE PARIS et al., 2000;FREITAS et al., 2004;NETO et al., 2005;MAZZEO, 2015). Takemoto and coworkers (1967) were the first to identify β-ecdysone in plants, in Achyranthes fauriei roots. A few years later, β-ecdysone was isolated from the roots of P.
glomerata and, since then, the plant has been considered an important source of this bioactive compound (SHIOBARA et al., 1993). Traditionally, only the roots are used for commercial and medicinal purposes, depending on their β-ecdysone content. However, Flores (2006) showed that the extract of both roots and aerial parts of P. glomerata contains βecdysone. Most ecdysteroids occur in low concentrations, less than 0.1% dry weight, particularly in Amaranthaceae (DINAN, 2001;REIXACH et al., 1996;SAVCHENKO et al., 1998). In P. glomerata, β-ecdysone contents range from

Source: Authors, 2020
Samples were dried to 10% moisture in a forced-air oven at 60 °C for about 15 h, crushed and stored separately in a freezer.
Infusions of roots, stems, leaves and flowers were prepared by mixing 1.0 g of sample with 100 mL of boiling water, allowing the mixture to rest for 15 min at room temperature, filtering through a 0.45 µm cellulose acetate membrane and immediately analyzed.

Quantification of β-ecdysone by high-performance liquid chromatography (HPLC)
The whole process for β-ecdysone quantification is summarized in the flowchart displayed in  Source: Authors, 2020 The standard curve was obtained by the analysis of six concentrations of β-ecdysone (30-250 mg L -1 ) in triplicate. Peak areas were plotted as a function of β-ecdysone concentration and the best-fit line was determined by linear regression.
Limits of detection (LOD) and quantification (LOQ) were calculated from the standard curve using Equations (1) and (2) (2) Where SD is the standard deviation of y-intercepts (n = 3) and S is the slope of the standard curve.

RESULTS AND DISCUSSION
Chromatographic analysis showed that β-ecdysone was the major component in aqueous infusions prepared from P. glomerata roots, stems, leaves and flowers of 3-years-old ( Figure 4) and of 6-mounth-old ( Figure 5). The compound had a retention time of 9.6 min. β-ecdysone concentrations were found to be higher in adult individuals than in young or mature plants (Table 1). Although only P. glomerata roots are traditionally used for medicinal and commercial purposes, the highest βecdysone concentrations were detected in leaves.

CONCLUSION
Aqueous extracts of the aerial parts of P. glomerata have significant concentrations of β-ecdysone, the major bioactive component of P. glomerata roots. Stems, leaves and flowers are promising sources of β-ecdysone and could be availed to increase diversity and sustainability to the P. glomerata production chain, using the aerial parts as the roots are used, instead of discarding them.
This research contributes to the objectives of the 2030 Agenda for Sustainable Development by identifying a strategy for innovating and adding value to the P. glomerata production chain in Paraná, Brazil. The study also highlights the importance of bringing together research institutions and producer cooperatives in order to promote territorial development.