Evidence of non-covalent complexes in some natural extracts: Ceylon tea and Mate extracts.

Considering the high complexity of natural extracts, due to the presence of organic molecules of different chemical nature, the possibility of formation of non-covalent complexes should be taken into account. In a previous investigation the formation of bimolecular complexes between caffeine and catechins in green tea extracts (GTE) has been experimentally proven by means of mass spectrometric and 1 H nuclear magnetic resonance experiments. The same approaches have been employed in the present study to evaluate the presence of bimolecular complexes in Ceylon tea and Mate extracts. The obtained results show that in the case of Ceylon tea extracts protonated theaflavin is detectable, together with theaflavin/caffein complexes, while caffeine/catechin complexes, already detected in green tea, are still present, but at lower concentration. This aspect is evidenced by the comparison of precursor ion scans performed on protonated caffeine for the two extracts. The spectra obtained in these conditions for GTE and Ceylon tea show that the complexes of caffeine with epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), highy abundant in the case of GTE (signal-to-chemical noise ratio in the range 50-100), are negligible (signal-to-chemical noise ratio in the range 2-3) in the case of Ceylon tea. Mate extracts show the formation of bimolecular complexes involving caffeine but not catechins, and chlorogenic acid becomes responsible for other complex formation. Under positive ion and negative ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid, rutin and kaempferol-3-O-rutinoside. These data indicate that the formation of complexes in natural extracts is a common behaviour and their presence must be considered in the description of natural extracts and, consequently, in their biological activity.


| INTRODUCTION
Can molecules be considered as isolated objects in space? Surely in a very rarefied space, where the density of matter is infinitesimal, the probability of noncovalent interaction between two molecules is very low but not anything. In fact, it has been proven that the formation of molecular complexes is a phenomenon observed in most of the universe, 1 and it represents an essential stage for the formation of complex molecular systems. At atmospheric pressure, because of the much higher frequency of interactions, the formation of numerous complexes takes place, above all, in the condensed phase (solution).
These phenomena have been and are still considered the basis of "chemical evolution." 2 These aspects have been studied by a branch of modern physics, the theory of complexity, 3 emerging in the last decades in scientific research. It studies the so-called complex systems, renouncing linearity assumptions in dynamic systems and investigating their behavior more thoroughly.
The number and frequency of molecular interactions depend on the complexity of the system under study. In the case of natural substrates, consisting of hundreds (or thousands) of different molecular species in the condensed phase, the frequency of interactions will be very high. Thus, a natural substrate should not be considered as a set of isolated molecules but as an entity consisting of species generated from continuous processes of molecular interaction, in a situation of equilibrium dependent on thermodynamic conditions. We have, therefore, to observe a complex system, 3 consisting of the molecules present in the natural extract, which interact in a nonlinear way 4 with each other through the formation of noncovalent bonds.
In this frame, mass spectrometry can be a valid tool to achieve a quite accurate general view of natural extracts, 5  A hypothesis on the peculiar pharmacological behavior of biologically active natural compounds is based on the occurrence of molecular interactions following the rules of supramolecular chemistry. 6 In particular, in the case of caffeine, an interesting review has been recently published 7 on the molecular recognition of caffeine in solution and solid state: A wide variety of receptors has been designed for this aim and found to exhibit a high affinity toward caffeine. The modern bioorganic chemistry concerns the design of synthetic molecules that mimic various aspects of enzyme chemistry and allows to understand their essential roles in biological systems. In this case, molecular recognition of caffeine with polyphenols and carboxylic acids has been described. [7][8][9] Caffeine posseses a number of features, which optimize its effectiveness for complexation with polyphenolic substrates: The phenolic group is a good proton donor. It can be assumed that in complexation with caffeine, hydrogen bonding between the polyphenol (proton donor) and caffeine (proton acceptor) takes place, giving a specific contribution to the stability of the complex. 7 In line with this view, some investigations were performed to establish the presence of caffeine/catechin complexes in green tea extracts (GTEs). 10

| Sample preparation for MS and MS/MS analysis
The solutions analyzed by MS and MS/MS were prepared as follows: Green tea, Ceylon tea, and mate powder extracts were dissolved in Milli-Q H 2 O at a concentration of 1 mg/mL. The solutions were then filtered through a pore size filter of 0.22 μm and further diluted to obtain a 100-μg/mL working solutions in MeOH/H 2 O (50:50 v/v).
Mass spectrometry measurements were performed by using an API 4000 triple quadrupole mass spectrometer (AB SCIEX, Massachusetts). The sample solutions were infused by the use of a programmable syringe pump (KD Scientific, Massachusetts), at a flow rate of 600 μL/h. ESI source parameters were as follows: source temperature, 300°C; curtain gas (nitrogen), 40 psi; and nebulizer gas (air) GS1 and GS2, 10 and 15 psi, respectively.

| MS analysis
Full scan spectra in the positive ion mode were recorded with ion spray voltage set at 4500 V, entrance potential at 10 V, and declustering potential at 20 V; for negative ion measurements, ion spray voltage was set at −4500 V, entrance potential at −10 V, and declustering potential at −20 V.

| MS/MS analysis (precursor ion and neutral loss scans)
For collisional experiments, CAD was set at 4 (arbitrary units); for positive measurements collision, cell exit potential (CXP) and collision energy (CE) were, respectively, 15 V and 30 eV, while for negative measurements, CXP and CE were −15 V and −30 eV respectively.

| Sample preparation for UHPLC ESI-QToF analysis
The ground sample of Ceylon tea (0.25 g) and mate were extracted  Table 1.

| Ceylon tea
Tea has been considered since ancient times to exhibit medicinal properties. [12][13][14][15] Tea beverage contains xanthine derivatives such as caffeine, theophylline, theobromine, and the glutamide derivative theanine. These substances have well-known stimulant properties and have also been reported to have beneficial effects on memory and on the immune system. Tea also contains many nutritional components, such as vitamin E, vitamin C, fluoride, and potassium. The astringency of the beverage is due to phenolic constituents known as catechins, a group of compounds that are closely related to tannins. Tannins are acidic because of the phenolic hydroxyl groups present on the gallic acid moiety. They also act as antioxidants and form complexes or chelates with metals. Most of phenols in tea are catechins (see Figure 1), hydroxylated flavanols, and their gallic acid esters. During fermentation process, if employed, these catechins undergo phenolic oxidative coupling reactions that yield red-colored catechin dimers such as thearubigins.
Catechins are believed to have a range of beneficial health effects 13,15 such as neuroprotective activity and anti-inflammatory, antiulcer, antiviral, antibacterial, and antiparasitic effects. The most studied catechin in relation to health contributing potential is epigallocatechin 3-gallate (EGCG), which constitutes 50% to 75% of the total flavonoid content in green tea.
Ceylon tea is a popular type of black tea. 16 It is known for its bold flavor, but it can vary greatly in taste, depending on where it is grown.
The process of manufacture, after leaf plucking, is based on a series of treatments, ie, withering, rolling (a mechanized process in which the leaf cells are ruptured to release enzymes and bring them in contact with air so that aeration can take place), aeration (sometimes known as "fermentation" or "oxidation"), and drying (the leaf is dried in a dessicator or "firing chamber" at 99°C to 104°C to prevent further chemical changes. This shrinks and darkens the leaf, resulting in the product known as black tea). 16 All these treatments are absent in the case of green tea (GT) production. In this case, after the tea leaves are plucked, they are dried to prevent fermentation, so inhibiting any activity that causes oxidation. Tea leaves are stirred constantly for even drying. Withering is also used, which spreads the tea leaves on racks of bamboo or woven straw to dry in the sun or using warm air. Again, the leaves must be moved around to ensure uniform drying.
At this point, a question arises: Do all treatments required for the production of Ceylon tea impact on the composition, making the extracts different to those of green tea? In more detail, are catechin amounts decreased, and are the bimolecular caffeine/catechin complexes, detected in GTEs, still present?
The 1 H NMR spectra of GTE and Ceylon tea extracts are reported in Figure 2. This comparison shows that the content of catechins (mainly EGCG) originally present in GTEs 10 is drastically reduced in Ceylon tea, whereas caffeine content remains practically unaltered.
Therefore, also bimolecular caffeine/catechin complexes are supposed to be reduced in Ceylon tea extracts. The aromatic proton signal of free gallic acid is significantly increased in Ceylon tea, further indicating degradation of catechins. The aromatic signal of gallic acid was assigned by comparison with the signal arising from a pure sample of gallic acid. In the present work, the drastic decrease of catechin contents in Ceylon tea extracts compared with green tea was verified.
Because of the low amount of catechins in Ceylon tea, the chemical shift variations of the aromatic protons of EGCG or ECG, as we did in a previous work, 10 was not discussed.
In the previous investigation on GTE, it was found that ESI operating in negative ion mode is an effective tool to describe the acid components of the extract introduced by direct infusion, 10 leading in particular to a valid mapping of the polyphenolic species. Consequently, the same approach was employed in the study of Ceylon tea extracts. The ESI-(−) spectra of GTE and Ceylon tea extracts are shown in Figure 3. It is evident that the same species are present in the two extracts, but with major differences in their relative abundances, as emphasized by the data reported in Table 2 Table 2. Lower levels of catechins in the Ceylon tea are observed, confirming the 1 H NMR data evidence on the validity of the direct infusion approach and the related precursor ion scan data. Then it can be assumed that the same behavior is present in the case of Ceylon tea and that the data reported in Figure 4 can be considered valid.
These results can be justified by supposing that the treatments above described for the Ceylon tea production are partially lesive either for catechins and/or for the catechin/caffeine complexes. It must be stressed that catechins and their derivatives are thought to contribute to the beneficial effects ascribed to tea. 13

| Mate
The Ilex paraguariensis plant, called yerba mate plant 17,18 is grown and processed in South America, specifically in northern Argentina, Paraguay, Uruguay, and Southern Brazil. Seeds used to germinate new  The main bioactive compounds present in yerba mate extracts are reported in Table 3,  The 1 H NMR spectrum of a mate extract shows the presence of caffeine, chlorogenic acids, and monomeric sugars (sucrose and glucose). In the aromatic region, a significant amount of chlorogenic acids is detected, while the contribution of gallocatechins is negligible.
The same mass spectrometric methods used in the case of GTE, 10 ie, either LC-MS in high resolution conditions or precursor ion scans performed on protonated caffeine and deprotonated chlorogenic acid, have been employed for the detection of possible noncovalent complexes in mate extracts.
The LC-ESI-(+)MS chromatogram of a mate extract, obtained in high resolution conditions, is reported in Figure 6, while the chromatogram obtained in ESI-(−) conditions is reported in Figure 7. The related results are summarized in Tables 4 and 5. Under positive ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid (species h and k in Table 4, reasonably originating from 3,5-DCQ and 4,5-DCQ that, as shown in Table 3 Table 4. In positive ion conditions, accurate mass measurements allow the identification of malealdehyde, chologenic acid, caffeine, two isomers of dicaffeoylquinic acid (species h and k in Table 4  These suppositions, although unproven, were sufficient to fuel new research interest on CQAs properties. 18,19 Total polyphenols and antioxidant activity of mate infusions have been compared with those of commonly consumed beverages like green and black teas, red, rosé and white wines, and orange juice. 20 Mate has a polyphenol content comparable with tea and orange juice, and when normalized for the polyphenol content of beverages, mate showed an antioxidant activity slightly higher than wines, orange juice, and black tea but lower than green tea. 20 Because of the presence of caffeoylquinic and dicaffeoylquinic acid isomers, mixed mono-, di-, and tri-esters of quinic acid, other hydroxycinnamates, and several quercetin and kaempferol glycosides, the consumption of mate infusions   would significantly contribute to the overall antioxidant intake, with biological effects potentially beneficial for human health (eg, for their hypocholesterolemic, antimutagenic, anti-inflammatory, and antiviral properties).

| CONCLUSIONS
The results obtained in the investigation on the presence of bimolecular, noncovalent complexes in Ceylon tea and mate extracts confirm the data previously achieved for green tea. Mass spectrometry is a