Pharmacognosy Determination of the metabolic profile of Solidago canadensis using UFLC-PDA-ESI-TOF

Species in the Solidago genus are used worldwide due to the therapeutic activities of their flavonoids and terpenoids. Its anti-inflammatory, antirheumatic, analgesic, diuretic, aquaretic, wound-healing properties as well as its ability to relieve burn and insect bites allow use in a variety of situations. This study was carried out to determine the metabolic profile of Solidago canadensis inflorescences (SCAI) to identify the species commercialized in Rio de Janeiro, RJ, Brazil, and evaluate the medicinal potential compared to S. chilensis and SCAI, which is used in North America and Europe. The UFLC-PDA-ESI-TOF revealed the metabolic profile of SCAI, finding the pseudo-molecular ions and characteristic fragments, mainly flavonols (10) such as quercetin, kaempferol, isorhamnetin, and its glycosides, as well as chlorogenic acid (CA). Eleven substances were identified, confirming the compatibility of the metabolic profile presented in varieties canadensis and scabra of european S. canadensis. The both contain the same flavonoid aglycones, and their glycosides are important chemical and biological markers for extracts and products based on species of the genus Solidago. UFLC-PDA-ESI-TOF characterized the extracts, which can help with quality control of raw plant materials and commercialized phytotherapeutics as well as for development of new products and phytomedicines.


Introduction
The ethnopharmacological use of species of the Solidago genus has long been used throughout the world and is related to the therapeutic activities of its flavonoids and terpenoids. Some qualitative and quantitative differences exist in anti-inflammatory, bacteriostatic, and aquaretic properties of different goldenrod species (Foster & Tyler 1999;Avila & Fetrow 2000;Robbers & Tyler 2000).
Solidago species are widely used in Europe to treat urinary tract inflammations, preventing formation or facilitating elimination of kidney stones (Robbers & Tyler 2000;Apáti et al. 2002;Gastaldi et al. 2018). This species naturally occurs throughout the world: Solidago canadensis in North America; Solidago virgaurea in Europe; Solidago gigantea in Asia; and Solidago chilensis in South America (Robbers & Tyler 2000;Sutovská et al. 2013). These species are present in pharmacopoeias, scientific literature, and pharmaceuticals. Solidago is indicated as an anti-inflammatory, antirheumatic, analgesic, diuretic, and aquaretic agent, as well as for healing wounds, burns, and insect bites. The species are often used interchangeably (Robbers & Tyler 2000;Apáti et al. 2002;Apáti 2004;Sutovská et al. 2013;Gastaldi et al. 2018;Valverde et al. 2012).
In Brazil, S. chilensis is the official species of the first edition of FB (Brasil 1926) and on the list of interesting species of the Brazilian public health system (Brasil 2009) with the previous synonymy: S. microglossa. It is also on the therapeutic herbal list of many Brazilian cities, such as RJ and SP (SMSDC 2010), while S. canadensis is commonly used for ornamental purposes.
To analyze complex plant extracts and fractions, characterizing their contents without isolation of the substances, the metabolomic approach has been used with different chromatographic and spectroscopic techniques.
This work was conducted to determine the metabolic profile of S. canadensis inflorescences (SCAI) compared to the officinal Brazilian species S. chilensis (SCMI) in order to the evaluate their medicinal potential compared to the S. canadensis used in North America and Europe and S. chilensis used in Brazil.

Solvents and chemicals
Solidagenone, quercetin, and quercitrin previously isolated from S. chilensis inflorescences and identified through NMR, IR, and MS spectra (Valverde et al. 2009;, and five flavonoids and chlorogenic acid used to investigated the SCAI content and to determine its metabolic profile were the pure standard (Sigma-Merck TM ). All the solvents used were AR (analytical research grade) or spectral grade according with the analysis.

Plant material and extraction
Solidago canadensis inflorescences were purchased in June 2018 at CADEG (Rio de Janeiro Municipal Market) with the popular description: Tango and the scientific description presumed by the seller as Solidago canadensis. The inflorescences (102 g) were dried and pulverized in a knife mil and extracted according with our previous work, with ether: ethanol (1:1), by dynamic maceration, filtered and concentrated to dryness under reduced pressure at 40 o C furnishing 4.1 g of the inflorescences extract (yield 4.2%) (Valverde et al. 2009).

Metabolic profile analysis
The TLC analysis was performed using 30 μL aliquots of the raw extract and 10 μL of the pure standard which were eluted with BAW (n -butanol:acetic acid:water -upper phase) (4:1:5) for phenolics and flavonoids and with hexane: ethyl acetate (8:2) for other phytochemicals. The TLCs were physically developed under UV lamp and with reagent solutions, such as NP-PEG for phenolics and flavonoids, to characterize their different phytochemicals using sulfuric anisaldehyde (Wagner & Bladt 1996). TLC plates precoated with silica gel F254 (Merck™) were used.
The UFLC-PDA-ESI-TOF analysis was carried out on a KINETEX TM (100×3×2.6 µm) at 30 o C with a solution of TFA (trifluoroacetic acid) (A) and acetonitrile (B) as mobile phase (flow 0.8 mL/min), in a Shimadzu Nexera 30AD equipped with a quaternary pump, detector with photodiode arrangement and automatic sampler. MS was performed using a Bruker Compact Mass Spectrometer (Bruker TM ) spectrometer with electrospray ionization interface (ESI) and quadrupole ion trap analyzer to identify directly and simultaneously the presence of flavonoids in the samples, extract, and pure substances. The ESI-MS spectrum was acquired in the negative mode for the sample and reference compounds (Sigma TM ). Parameters: gas temperature 220 o C, capillary voltage at 4.5 kV, gas mist (N 2 ), flow 10 L/min, and pressure at 5.0 Bar. MS was obtained in the full-scan mode in the 50-800 m/z range.

Results and Discussion
The analysis of SCAI extract compounds by UFLC-PDA-ESI-TOF (Fig. 1)  The structures of these compounds were confirmed by comparing with standard pure compounds such as quercetin, kaempferol, quercitrin, isoquercitrin, and chlorogenic acid, as well as their fragmentation ESI-MS-TOF pattern in experiments and literature data (Saldanha et al. 2013;Brito et al. 2014;Kim et al. 2017;Jang et al. 2016).
Only three aglycone, detected by the pseudomolecular anion [M-H] -, quercetin at m/z 301, kaempferol at m/z 285, and isorhamnetin at m/z 315 were identified in the flavonoid content.
All these are flavonols (Tab. 1), and their occurrence was previously reported in the Solidago genus (Apáti et al. 2002(Apáti et al. , 2004) and its glycosides, but using HPLC. These flavonoids are responsible to the important biological activities of S. chilensis.
The characteristic loss of the sugars in the compounds confirmed the presence of glucose (-162), rhamnose (-146), and rutinose (-308). The identification of all MS detected and identified compounds present in SCAI extract are presented in Figure 2 and Table 1 and explained below.

Identification of compounds
Major compounds detected through the UFLC analysis of the SCAI extract are in Table 1
The results confirmed, with reference substances, fragmentation pattern, UV-vis max, and literature data, that the metabolic profile obtained was similar to the canadensis and scabra varieties of S. canadensis found in Europe, which presents the same flavonoid aglycones and large amounts of their glycosides as described by Apáti (2004).
The UFLC-PDA-ESI-TOF fingerprinting is presently used for rapid quality control purposes to evaluate commercially available medicinal plants widely used by the population, to simultaneously determine their metabolic markers and predict the biological plant performance, their identity, and the confirmation of the metabolic pattern of this plant obtained in a popular market with literature data described for S. canadensis.
This work is basis for the quantification of the identified flavonoids, to determine the