Metabolomic profiling and correlations of supercritical extracts of guarana

Abstract A previous optimization of supercritical extraction from guarana seeds was performed applying orthogonal array design (OA9(34)). The antioxidant and antimicrobial activities of these extracts, as well as metabolomic profiling and correlations from the compounds by statistical analysis were determined. Extracts 1 (40% ethanol; 20 min; 40 °C and 100 bar), 2 (40% methanol; 60 min; 40 °C and 200 bar), and 8 (40% methanol; 40 min; 60 °C and 100 bar) had the highest combined values of antioxidant capacity for the DPPH, FRAP, ABTS and xanthine oxidase system methods, and were identified by chemometric analysis. Similar chemical profiles of the extracts were obtained by LC-DAD-MS, and were identified: methyl-xanthine, (epi)catechin and dimers and trimers of type A and B proanthocyanidins. The heat map analysis showed positive correlation between antioxidant methods DPPH, FRAP and ABTS and with flavan-3-ols and proanthocyanidins. Extract 3 was active against Gram-negative and -positive bacteria and Candida tropicalis. Graphical Abstract


Introduction
Supercritical fluid extraction (SFE) allows the reuse of solvents, greater ease in the steps of recovery, which become faster and cleaner, usually employs low temperatures, and provides products with high added value.Because they are free of unwanted waste and have no risk of thermal degradation, thus featuring numerous advantages over conventional extraction (Ramsey et al. 2009).Several studies have used supercritical extraction for extracting bioactive compounds from natural products, and the addition of co-solvents, such as ethanol and methanol, enables the extraction of more polar compound.
Guarana (Paullinia cupana Kunth, Sapindaceae) is a plant native to the Amazon, which has a variety of pharmacological properties, being used in the food and pharmaceutical industries.Several activities have been attributed to the seeds of guarana and condensed tannins, such as: anticancer, antioxidant, antimicrobial, antidepressant, gastroprotective, and for weight loss, among others.Guarana seeds are chemically rich in caffeine (2.41-5.07%)and tannins (5.0-14.1%),especially those derived from proanthocyanidins (10.7%) and from catechin (5.98%) and epicatechin (3.78%).
The aim was to investigate the antioxidant capacity of supercritical extracts of guarana for different methods by grouping them according to chemometric tools, and analyze their metabolomic profiling and to apply statistical analyses to correlate the chemical constituents and the antioxidant properties.In addition, the antimicrobial activity of these extracts was evaluated for microorganisms used in the food, clinical and industrial fields.

Data analysis of antioxidant activity and total phenolic content (TPC)
The results of the antioxidant activities of the four methods (DPPH, FRAP, ABTS, and xanthine oxidase system) are shown in Table S2.
In this study, the principal components analysis was applied to assess the TPC and antioxidant data of the above cited methods.The two first principal components (PCs) explained 70.08% of the total variance (Figure S1).The PC1 explained up to 46.76% of the total variance.Guarana samples were separated in PC1 according to the differences observed from results of antioxidant activity of the FRAP, ABTS, and xanthine oxidase system methods.PC2 explained 23.32% of total variance, separating the samples based on the results obtained with the DPPH and xanthine oxidase system methods.
In association with the scatter plot (scores and weights) it is possible to know which samples were related to the phenolic compounds and to the antioxidant activity in vitro, so that each sample or samples of the group may be distinguished.The extracts 1, 2, and 8 are characterized by higher levels, while the other extracts are characterized by lower levels of antioxidant activity.
Therefore, from the chemometric analysis it was possible to separate the extracts in two distinct groups.The group corresponding to extracts 1 (ethanol; 20 min; 40 C, 100 bar), 2 (methanol; 60 min; 40 C, 200 bar), and 8 (methanol; 40 min; 60 C, 100 bar) had the highest combined values of antioxidant activity for the DPPH, FRAP, ABTS, and xanthine oxidase system methods.
Several studies have shown that guarana has antioxidant activities (Mattei et al. 1998;Basile et al. 2005;Majheni c et al. 2007;Yamaguti-Sasaki et al. 2007;Dalonso & Petkowicz 2012;Portella et al. 2013;Bittencourt et al. 2013;Bonilla et al. 2017) which have been largely attributed to the polyphenols (particularly tannins).In another study testing crude and semi-purified guarana extracts, it was found higher content of polyphenols, reflecting the analysis for antioxidants, with a low IC 50 and a higher relative antioxidant capacity compared with the other extracts (Yamaguti-Sasaki et al. 2007).
The caffeine standard, evaluated as control in the methods of detection of antioxidant activity, had negative results in all tests.

Identification of the constituents from extracts by LC-DAD-MS
From the guarana extracts, twenty-one metabolites were detected and identified, including methylxanthines (MX), flavan-3-ols, and proanthocyanidins (PA) (Table S3, Figures S2-S4).The chromatographic peaks 3, 4, and 10 revealed an intense band at 270 nm in the UV spectra (Table S3).From the accurate mass spectra, the molecular formula was confirmed as C 7 H 8 N 4 O 2 (3, 4) and C 8 H 12 N 4 O 2 (10).Thus, these substances were confirmed by the literature data and by injection of the authentic standard as theobromine (3), theophylline (4), and caffeine (10).
The peaks 7-8, 11-12 and 14-21 exhibited an intense band at the wavelength 280 nm in the UV spectra.The compounds 7 and 12 showed the deprotonated ion at m/z 289 (relative to C 15 H 14 O 6 ) and fragmentation profile compatible to (epi)catechin (Nocchi et al. 2017) S3), respectively.The peaks 16 and 18 showed the deprotonated ions at m/z 863 relative to C 45 H 36 O 18 confirming A-type trimeric PA.The typical fragmentation pathway was used to structural characterization of PA, and they confirm the presence of two hydroxyl groups in B ring.The other typical fragmentations were used to suggest the structures, such as the losses of flavan-3-ol units by quinone methide reactions.For example, the fragment ions at m/z 289 and 285 from the compounds 11 and 20.Thus B-type dimeric procyanidin (8, 11), B-type dimeric profisetinidin-procyanidin ( 14), A-type dimeric procyanidin (15,17,(19)(20)(21), and A-type trimeric procyanidin (16, 18) were identified in the extracts.However, proanthocyanidins like ( 14) was not identified at this moment.
The extracts revealed high similarity of the chemical composition (Figures S5 and  S6), but the ion intensities of metabolites from the extracts were different, which were lower intensities in the extracts 7 and 4 (Figure S7).From chromatograms of the extracts at 270 nm, it was possible to observe an intense peak relative to caffeine (10) in all the samples (Figure S5) which was the main identified compound in the samples.Pateiro et al. (2018) investigated the chemical profile of hydro-ethanolic extracts from guarana seeds and found 92 compounds present by means of UHPLC-ESI/QTOF mass spectrometric screening of GSE.Tyrosols and other lower-molecular-weight phenolics were the most represented (n ¼ 30), followed by hydroxycinnamic acids (n ¼ 19), flavones and flavan-3-ols (being 15 and 11 annotations, respectively).Among tyrosol equivalents, tyrosol, 3,4-DHPEA-AC, p-HPEA-AC, furan-and hydroxycoumarins together with phenolic terpenes (such as tymol and syringaldehyde) were the most abundant.Regarding hydroxycinnamic acids, caffeic and dihydrocaffeic acids, homovanillic, cinnamic and sinapic acids were the predominant and most frequently annotated, followed by the hydroxyphenylacetic acid.The predominant phenolic compound among alkylphenols was 4-ethylcatechol, while pelargonidin and its glycosylated derivatives were the most found among anthocyanins.Trihydroxyflavone, apigenin and luteolin were the most representative of flavones group.
In the work by Santana et al. (2019), the authors conclude that the recovery of target compounds in SFE was lower than the recovery in PLE (pressurized liquid extraction) because of poor affinity of polar structure of catechins and methylxanthines in non-polar carbon dioxide, even with the presence of a hydroethanolic cosolvent.

Correlations between the metabolomic profile of the extracts and antioxidant activities
An orthogonal PLS-DA was applied to evaluate the chemical differences of the extracts; it combines the covariance and correlation loading profiles.The orthogonal PLS-DA graphic (Figure S8-(A)) is possible to observe that is not clear the separations of all samples from the group 1 (higher antioxidant activity) and 2 (lower antioxidant activity), mainly for the extract 1.In addition, the main compounds that explain the differences of group 1 and 2 are the metabolites 18 and 16 (procyanidin trimers -A type), respectively (Figure S8-(B)).These results suggest that the bond (position) between the flavan-3-ol units should be important for the antioxidant activities.
The high chemical composition similarities between the supercritical extracts of guarana can also be observed on the chromatograms (Figures S2 and S4).Besides the dendogram resulting from hierarchal cluster analyses (Figure S9) revealed higher chemical proximities of the extracts 7-4, 5-3-6-1, and 8-2-9, and the extracts the most antioxidant active (extracts 1, 2, and 8) were positioned closer but the extract 1 showed to have chemical differences in relation to the extracts 2 and 8, like as observed in the orthogonal PLS-DA analyses.The correlation coefficients between antioxidant activities, chemical profiles, and TPC are illustrated in Figure S10.
It was possible to find positive correlations between some compounds and the antioxidant metabolites, but the most part of the correlations were not strong (above 0.5).For the antioxidant activity by DPPH assay, all the correlations were not strong, but the proanthocyanidins 18, 8, and 11 were the highest correlation coefficient, while all the methylxanthines did not reveal correlations (negative).
The dimeric procyanidins 8, 11, 19, 15, and the trimeric procyanidin 18 showed strong correlations with the antioxidant activity by xanthine oxidase assay.The ABTS activity revealed only strong correlation with FRAP activity, while the FRAP antioxidant activity demonstrated with dimeric 8, 11, 21, and trimeric 18 proanthocyanidins.
Interestingly, our results suggested the importance of the linkage between of flavan-3-ol units for the antioxidant properties.The similar compounds (16 and 18) showed different correlation with the antioxidant properties, since the proanthocyanidin 18 showed strong correlation coefficient while 16 revealed negative or weak.The metabolites correlated differently according to the method applied for the antioxidant evaluation (Figure S10).

Antimicrobial analyses
Extract 3 (ethanol:methanol; 40 min; 40 C, 300 bar) was chosen for the antimicrobial tests based on its performance in a previous work (Marques et al. 2016) and due to the similar profile of the extracts obtained in the chemical analysis carried out by LC-DAD-MS.The extract 3 was previously partitioned with n-hexane due to difficult solubility for the antimicrobial assay.The aqueous phase showed activity against a Gramnegative bacterial strain (Acinetobacter baumannii).Additionally, the hexane phase showed activity against Gram-positive bacteria and Candida tropicalis (Table S4).
The antimicrobial activity of guarana extracts has been investigated in several studies (Barbosa & Mello 2004;Basile et al. 2005;Majheni c et al. 2007;Yamaguti-Sasaki et al. 2007;Bonilla et al. 2017).Bonilla et al. (2017) investigated the antimicrobial properties of ethanolic extracts of guarana by the disk diffusion method.According to the results of these authors, guarana showed a slight antimicrobial activity against Staphylococcus aureus and no antimicrobial activity against Escherichia coli.According to the results of Majheni c et al. (2007), the alcoholic extracts possessed greater antimicrobial activity than aqueous extracts of guarana seeds.Several authors suggest that the polyphenols present in guarana are responsible for its pharmacological actions (Otobone et al. 2007;Yamaguti-Sasaki et al. 2007;Portella et al. 2013).No published study associates these actions with moderately or non-polar compounds present in guarana.

Conclusion
Despite the high caffeine content present in all extracts, their antioxidant effect cannot be attributed to caffeine.
The extraction of A-and B-type trimeric and dimeric proanthocyanidins were confirmed, and the B-type dimeric profisetinidin had not been identified so far as metabolites of guarana seeds.
For the first time there is a report on antimicrobial activity from low polarity guarana extracts against Gram-positive microorganisms and Candida tropicalis.
, which were confirmed by injection of authentic standard.The compounds 8/11/15 and 15/17/19-21 showed deprotonated ions compatible to molecular formula C 30 H 26 O 12 and C 30 H 24 O 12 suggesting dimeric PA B-and A-type (Figure