Quantification of 6-gingerol, metabolomic analysis by paper spray mass spectrometry and determination of antioxidant activity of ginger rhizomes (Zingiber officinale) Cuantificación de 6-gingerol, análisis metabolómico por espectrometría de masas por paper spray y determinación de la actividad antioxidante de los rizomas de jengibre

Ginger is a plant whose rhizome has a high therapeutic potential in combating various diseases due to the action of several of its constituents. The 6-gingerol, phenolic compounds and carotenoids, act on macrophage modulation, antiplatelet aggregation and immunosuppressive activity. This work aimed to determine the total antioxidant capacity as well as to evaluate the use of paper spray mass spectrometry to obtain fingerprints of ginger samples of conventional and organic cultivation. The results demonstrated that organic farming samples showed higher levels of fiber and total protein, as well as 6-gingerol. One must still give preference to organic Ginger intake since it presented significant levels of 6gingerol, fiber and protein. Several chemical classes such as sugars, fatty acids, phenylpropanoids and flavonoids were identified in organic and conventional ginger through paper spray ionization mass spectrometry. This analysis proved to be a very efficient and fast technique for obtaining fingerprints of ginger, allowing the identification of 19 compounds in the positive mode and 28 in the negative mode.


Ginger sample and material
Rhizomes of ginger utilized in the present work were cultivated in two different forms, conventional and organic, in the Southern region of Brazil. The two samples from conventional cultivation were obtained from São Paulo and Espirito Santo states. The two samples from organic agriculture were collected from two different producers from Minas Gerais state. Samples were transported to the Research Laboratory -Food Chemistry Unit of the Federal University of Minas Gerais. Fresh (in natura) ginger samples were sanitized by immersion in a solution of sodium hypochlorite (5 mg/L of active chlorine) for 15 min at room temperature, washed with distilled water, dried, crushed and, stored at-20°C.

Physical-chemical analyses
Chemical composition (moisture, total lipids, total protein and ash) of the samples was carried out according to the Association of Official Analytical Chemists (AOAC) methods.
Determination of soluble and insoluble dietary fiber was performed using enzyme digestion (alpha-amylase, pepsin and pancreatin perfectly) Sigma®. Carbohydrate content was calculated by percentage difference.

Antioxidant activity and phenolic compounds determination
First, 300 mg of crushed fresh in natura ginger were extracted according to the procedure described by Rufino et al. 2010. The obtained extracts were used to determine the content of phenolic compounds and to evaluate the antioxidant activity. Thus, the phenolic compounds were identified following the procedure of Singleton et al. 1999. Antioxidant Research, Society and Development, v. 9, n. 8, e366984822, 2020(CC BY 4.0) | ISSN 2525 6 activity was assessed according described by Rufino et al. 2010 to FRAP andABTS and, the according AOAC protocol (AOAC, 2018) to DPPH method.

PS-MS fingerprints
The chemical profile analysis of the gingers was done using a mass spectrometer LCQ Fleet (Thermo Scientific, San Jose, CA, USA) equipped with a paper spray ionization source.
All ginger samples were analyzed in positive and negative modes.
The chromatographic paper was cut into an equilateral triangle shape (1.5 cm). The paper was positioned in front of the mass spectrometer entrance. This material was supported by a metal connector and placed 0.5 cm away. The instrument was connected to high-voltage power of the spectrometer through a copper wire. Finally, 2.0 µL of pulp was applied on the border of the triangles, 40.0 µL of methanol was transferred to the chromatographic paper.
The instrument was operated under the following conditions, PS-MS source voltage + 4.0 kV (positive) and -3.0 kV (negative); capillary voltage 40 V; tube lenses voltage 120 V; mass range from 100 to 1000 m/z; transfer tube temperature 275°C. Collision energies used to fragment the compounds ranged from 15 to 35 eV (Campelo et al., 2020; A. L. C. C. Ramos et al., 2020;E. Silva et al., 2020;M. Silva et al., 2019). The fragments obtained in this analysis were identified based on the data described in the literature.

Optimization of the 6-gingerol extraction method
The solvents acetone, methanol and acetonitrile were tested to determine the best extraction method for 6-gingerol (Yu et al., 2007) (Yu et al., 2007). Two types of extractor, ultraturrax and ultrasound were also tested. Subsequently, the samples were centrifuged by varying the speed and time of the centrifuge. From the best absorbance data, the best conditions were validated. The solvent polarity, extraction process and analysis method were chosen according to procedures already described to determine the content of gingerol in ginger rhizomes (Ok & Jeong, 2012;Pawar et al., 2011;Sanwal et al., 2010).
The parameters peak purity, linearity, matrix effects, accuracy and precision, were evaluated. The suitability for the use of the method was assessed as a function of the parameters studied and their acceptability criteria defined (Sanwal et al., 2010). The significance level adopted in hypothesis testing was α = 0.05.

Statistical analysis
The analysis of variance (single-factor ANOVA) and Tukey test at 5% probability were used to compare the values found in the studies. The software Statistica version 10.0 (StatSoft, Tulsa, OK, USA) was used. Xcalibur version 2.2 SP1 software (Thermo Scientific, San Jose, CA, USA) was used to collect mass spectra results. Table 1 shows the results of the physical-chemical analyses of ginger from each variety (conventional or organic). The moisture content of the samples was 85.0% (conventional cultivation Sao Paulo), 86.2% (conventional cultivation Espírito Santo), 72.3% and 72.4% organic agriculture from Minas Gerais sample 1 and 2, respectively. In the samples of organic ginger a higher protein and fiber content was observed. On the other hand, these samples had lower ash, lipid and carbohydrate content than conventionally cultivated ginger samples.

Physical-chemical assay
Ginger samples showed high percentages of dietary fiber, with levels ranging from 33.83 to 55.80% for conventional and organic ginger, respectively. There is no difference between the same type of cultivation. The samples of organic farming featured more significant levels of soluble fiber when compared with conventional cultivars. In this present work two methods were used to assess the antioxidant activity of ginger as shown in Table 2. As for FRAP methodology, results ranged from 18.9 to 24.5 µM ferrous sulfate g -1 sample and the content of the antioxidant activity in OMG2 was significant than OMG1 ginger.

Phenolic compounds and antioxidant activity
When employing ABTS methods, a significant difference was observed between conventional and organic samples, the latter being significantly higher than the conventional samples (1371.1; 1515.0; 1674.4; 1628.6 mol of Trolox/g of ginger from CSP, CES, OMG1 and OMG2, respectively).
Both the ABTS and the FRAP methods showed better results for the antioxidant activity in samples of organic ginger.

Measurement and determination of 6-gingerol
The chromatographic peak purity was determined after the default and sample scan (a ginger extract) employing a DAD detector. The peak of the spectrum is considered to be homogeneous when the angle of purity is less than the edge of the line.
The figure below presents an example of a calculation peak purity of 6-gingerol on the concentration of 150 µg/mL.
Research, Society and Development, v. 9, n. 8, e366984822, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.4822 So the reading of the standard was homogeny since the value of the angle of purity (0.933) is less than the limit (1.124), indicating that the peak is only an analyte, and there is no overlap of more than one substance in the same peak.
After the examination to confirm the veracity of the data, since the significance of regression (p < 0.001) and the absence of significant deviation from linearity (p > 0.05).
Therefore, the parameters evaluated confirmed the linearity of the usual curve in the range of concentration of analyte from 30 to 180 µg mL -1 .
The comparison of the slope of the straight lines by the t-Test did not indicate a matrix effect (p > 0.05). The t statistic calculated (tb = 0.38) was less than the critical value of t (t = 2.04), confirming the absence of effect of the array. Therefore, the standard curve of gingerol may be used to estimate the amount of gingerol in ginger samples.
Recovery data in two levels of addition of analyte studied (level 1 and level 2) were subjected to the Grubbs test, which indicated the absence of outliers (p >0.05). The average recovery also observed that levels 1 and 2 were 88.15% and 91.5%, respectively. These results are in the range of 80% to 110% accessibility established by the European Commission (2002), indicating appropriate concentration levels of veracity.
Ginger samples of organic farming showed significantly higher levels of 6-gingerol when compared to conventional farming samples; there is no difference between samples within the same culture. Mean values ± standard deviation (n = 6) with equal subscripts * in the same row did not differ significantly (p ≤ 5; Tukey's test). Source: Author.

Chemical constituents identified in ginger by PS-MS
Mass spectra and fragmentation profile of some characteristic ginger ions are shown in    Table 3 presents the proposed identification for signals found in ginger in positive ionization mode and the profile of compounds identified in negative ionization mode is presented in Table 4.  Research, Society and Development, v. 9, n. 8, e366984822, 2020 (CC BY 4. X 493, 359, 313, 295, 269, 197, It can be seen from this table that analysis by (+) PS-MS (Table 3)  As shown in Table 4 It is noteworthy that no studies were found in the literature evaluating the differences between the chemical profile of organic and commercial ginger. Organic ginger samples showed lower moisture content when compared with the conventional samples and increased amounts of protein and fiber with a prevalence of insoluble fiber.

Final Considerations
It is necessary to intake 21 to 43 g of fresh ginger, to achieve the proper amount of gingerols, biologically active substances, responsible for the anti-inflammatory activity of ginger.
Preference must be given to organic ginger intake, since, besides the absence of pesticides, it also presented superior levels of 6-gingerol, dietary fiber and protein when compared with conventionally grown ginger.
PS-MS proved to be an efficient method for determining the chemical constituents of ginger, allowing the identification of 28 compounds belonging to different classes such as sugars, fatty acids, phenylpropanoids and flavonoids.
Moreover, through this method it was possible to verify some differences between the