Polyphenolic Proﬁling of Green Waste Determined by UPLC-HDMS E

: Valorising green waste will greatly enhance and promote the sustainable management of this large volume resource. One potential way to achieve this is the extraction of high value human health promoting chemicals (e.g., polyphenols) from this material. Our primary aim was to identify the main polyphenols present in four contrasting green waste feedstocks, namely Smyrnium olusatrum , Urtica dioica , Allium ursinum and Ulex europaeus , using UPLC-HDMS E . Polyphenol-rich Camellia sinensis (green tea) was used as a reference material. Samples were extracted and analysed by UPLC-HDMS E , which was followed by data processing using Progenesis QI and EZ Info. A total of 77 high scoring polyphenolic compounds with reported beneﬁts to human health were tentatively identiﬁed in the samples, with abundances varying across the plant types; A. ursinum was seen to be the least abundant in respect to the polyphenols identiﬁed, whereas U. europaeus was the most abundant. Important components with a diverse range of bioactivity, such as procyanidins, ( − )-epigallocatechin, naringenin, eriodictyol and iso -liquiritigenin, were observed, plus a number of phytoestrogens such as daidzein, glycitin and genistein. This research provides a route to valorise green waste through the creation of nutritional supplements which may aid in the prevention of disease.


Introduction
In most countries, green waste typically represents a high volume, low value resource, with most of this material being composted and subsequently spread back to agricultural land to improve soil quality [1]. However, green waste also represents a promising starting material for the direct extraction of valuable compounds and for the chemical and fermentative conversion of this waste into basic chemicals [2]. One of the main issues in valorising this resource, however, is knowledge of what high value products can be obtained in sufficient quantities from different types of green wastes to make it commercially viable. One area that has drawn particular interest has been the extraction of polyphenols [3]. These plant species may also contain many other non-polyphenolic bioactive chemicals which are also worthy of attention. Co-extraction of these would, of course, further enhance the value of waste materials, though this research was focused on polyphenols. A full cost-benefit analysis and life cycle assessment are required to determine the valorisation benefits relative to other synthesis or extraction procedures.
Polyphenols are a naturally occurring group of secondary metabolites which are relatively abundant in plants and which are purported to have many health benefits [4][5][6]. For example, they are thought to play an important role in disease prevention, resulting not only from their antioxidant ability but also their epigenetic influence and their positive impact on the composition of gut microbiota. Due to their complex chemical structures, many of these plant-derived bioactive polyphenols can be difficult to synthesize in large quantities [7]. Green waste offers a potentially cheap feedstock to extract and purify these

Sample Collection
Representative samples of Alexanders (Smyrnium olusatrum), Stinging Nettle (Urtica dioica), Wild garlic (Allium ursinum) and Gorse (Ulex europaeus) were collected from the Lligwy Bay area of Anglesey, Wales, UK (53 • 21 14 N, 4 • 15 47 W) in April 2019. The sample of U. europaeus was separated into separate flower and stem samples in order to observe whether there were any significant differences in the characterisation of these two physically connected structures. These plants were chosen due to their contrasting phylogenies and their frequent presence in municipal and agricultural green waste streams.

Sample Preparation
After collection, each sample was thoroughly washed in LC-MS grade water (Optima) and then freeze-dried (48 h) before being ground to a fine powder. A total of 0.5 g of powder was then placed in a glass beaker containing 10 mL of ethanol and sonicated in an ultrasonic water bath for 30 min before being left to stand for 24 h at 4 • C before being sonicated again for a further 30 min. After the solids had settled out, the supernatant was transferred to a polypropylene tube and centrifuged (10,000 rev min −1 , 30 min). Through heating to 60 • C, the resultant ethanol solution of extracted components was concentrated to 1 mL; that is to say that 1 mL of extract was equivalent to 1 g of initial sample. The sample produced was stored at −20 • C. The plant samples were prepared in quadruplicate.

Analytical Instrumentation
HDMS E mode is a data-independent acquisition in which data for all gas phase parent ions, and also fragments (product ions) created, are recorded. This was recorded and saved as continuum data. In addition, a drift cell was used to collect ion mobility data. The Synapt G2-Si (Waters UK, Wilmslow, Cheshire, UK) can be described as a quadrupole time-of-flight mass spectrometer (Q-ToF) which has ion mobility capability added to the ion path. Analytes, which have been separated by the UPLC, were infused into a Z-Spray TM source (Waters UK, Wilmslow, Cheshire, UK). Simultaneously, leucine enkephalin (Tyr-Gly Gly Phe-Leu) was infused via a separate probe, which provided the lock mass data to correct the mass axis drift which occurs during an acquisition. Baffle switching allowed for the selection of which infusion, analyte or lock mass entered the MS.

UPLC Conditions
A Waters I-class UPLC was used for analyte separation with a Waters Cortecs Shield RP18 (2.7 µm × 2.1 mm × 100 mm, Waters UK, Wilmslow, Cheshire, UK) solid core column installed. This column provides high selectivity for phenolic compounds due to the use of imbedded polar carbamate technology. A guard column of the same stationary phase was used for protection of the analytical column. Water with 0.1% acetic acid in reservoir A and MeOH with 0.1% acetic acid in reservoir B were used as the mobile phase. The flow rate, column temperature and injection volume were 0.5 mL min −1 , 40 • C and 1.0 µL, respectively. The starting composition for this eluent was 90% A and 10% B, with a linear change to 1% A with 99% B over the course of 4 min. At the end point, the initial conditions were returned to over a time of 0.2 min.

Synapt G2-Si Conditions
Negative ion data, in a mass range of 50 to 1200 Da, was acquired using resolution mode. The scan time was 0.2 s, with an average of 3 scans and a mass window of ±0.5 Da. mode. The cone voltage was 40 V. The method was set to acquire the lockmass (leucine enkephalin, 554.2615 Da) at regular intervals (30 s). This was not used for immediate mass correction throughout the run but stored for later use in the data processing phase.

Data Processing
Progenesis QI software (NonLinear Dynamics Ltd., Newcastle upon Tyne, UK) was used to process these data. An experimental design was chosen (between subjects) by creating individual groups for the various plant extracts, plus one for the reference (green tea) and one group for blank extracts. As this detail has previously been published [8], only a brief overview is provided here. Post deconvolution, a 5 ppm precursor tolerance was used to compare ions to the ChemSpider Polyphenols database [13] using isotope similarity scores above 90%, an elemental composition of C, O, H only and in an silico fragmentation tolerance of 90%. Similarly, filters were used to reveal only analytes with ANOVA p values ≤0.01 and where blanks were the lowest mean, and, also, scores with a value above 40 were selected for further evaluation. Multivariate analysis (MVA) was conducted through the use of EZInfo (Umetrics, Umeå, Sweden). Matlab (MatWorks Inc., Natlick, MA, USA) was used to create the heat map.

Bioactive Phenolic Compounds in the Green Waste Extracts
An Excel file in the supplementary materials (plant extracts XL SM v6) provides a detailed summary of the 77 high-scoring components elucidated through this analysis of green waste. Details such as retention time (min.), normalised abundance, drift time (ms) and accurate mass values are provided for parent ions and their main product ions. Very low ANOVA-p and q values are seen, which indicates a false discovery rate (FDR) close to zero. Mass errors of ≤5 ppm are observed in these data, plus scores of over 80 for isotope similarity.
In cases where it was not possible to distinguish analytes from species of the same accurate mass, the various possibilities are listed. Examples of total ion chromatograms (Figures S1-S6) and example molecular structures of the 29 identified components, with scores of 50.0 and above, are shown in Figure S7. Mass spectra ( Figure S8) are available in the supplementary materials.
Green tea, which was used as a reference material, exhibited the expected polyphenols generally associated with this plant in the published literature. These include (−)epigallocatechin, (−)-epigallocatechin gallate, theaflavin and procyanidin (B1, B2, B3 or B4). Another key component of green tea's polyphenol profile, (−)-epicatechin, was also observed at 1.23 min and m/z 289.0732, with an average abundance of score of 38.1, 6782 average abundance, a mass error of 4.8 ppm and very low ANOVA-p and q values (1.31 × 10 −8 and 3.47 × 10 −9 ). A confirmatory product ion at m/z 245.0808, due to the Processes 2021, 9, 824 4 of 13 loss of 44 Da (CH 3 CHO), was observed [14]. This added further validity to this discovery method. An abundance profile of (−)-epicatechin across the plant extracts can be seen in the supplementary materials showing the greatest abundance in the green tea extract ( Figure S9).
A heat map was created using averaged abundances for each sample type over the 77 identified polyphenol components ( Figure 1). This visual overview of polyphenol identifications shows that abundances are higher in U. europaeus flower and stem extracts than they are in the extracts of S. olusatrum, U. dioica and A. ursinum. Furthermore, it can be seen that A. ursinum leaf has the lowest abundance of the components identified here. It is also noted that individual plant extracts have their own pattern or fingerprint of abundances, with components showing much variation been plant types.
Green tea, which was used as a reference material, exhibited the expected polyphenols generally associated with this plant in the published literature. These include (−)epigallocatechin, (−)-epigallocatechin gallate, theaflavin and procyanidin (B1, B2, B3 or B4). Another key component of green tea's polyphenol profile, (−)-epicatechin, was also observed at 1.23min and m/z 289.0732, with an average abundance of score of 38.1, 6782 average abundance, a mass error of 4.8 ppm and very low ANOVA-p and q values (1.31 × 10 −8 and 3.47 × 10 −9 ). A confirmatory product ion at m/z 245.0808, due to the loss of 44 Da (CH3CHO), was observed [14]. This added further validity to this discovery method. An abundance profile of (−)-epicatechin across the plant extracts can be seen in the supplementary materials showing the greatest abundance in the green tea extract ( Figure S9).
A heat map was created using averaged abundances for each sample type over the 77 identified polyphenol components ( Figure 1). This visual overview of polyphenol identifications shows that abundances are higher in U. europaeus flower and stem extracts than they are in the extracts of S. olusatrum, U. dioica and A. ursinum. Furthermore, it can be seen that A. ursinum leaf has the lowest abundance of the components identified here. It is also noted that individual plant extracts have their own pattern or fingerprint of abundances, with components showing much variation been plant types.

Tentative Identification of Polyphenols
Due to the lack of fragmentation data contained in databases, in silico predication has been used here. As the fragmentation mechanisms of polyphenols are well documented, this can be quite an effective approach. Retro-Diels-Alder (RDA) reactions [15] are the general category here, which also applies to the sugar moiety, which is often seen in polyphenols. Additionally, the mass spectra of conjugated phenolic compounds showed the aglycone ion as result of the loss of sugar moieties such as hexosyl ([M-162] − ) or pentosyl ([M-132] − ). An example of this is seen with the two isomers of isorhamnetin glucoside (3 or 7) that are identified at 1.81 and 2.63 min. Their [M-H] − ions undergo the loss of the hexoside moiety to create product ions of m/z 315.0503 and m/z 315.0508. It is also noted that these isobaric species are not only separated by chromatographic retention time but also by drift time, further confirming that these molecules are different in terms of their collision cross section (CCS). The isomer at 1.81 min is seen in high abundance in U. dioica and the other isomer at 2.63 min is high in S. olusatrum. Both are also fairly abundant in the U. europaeus flower extract. Isorhamnetin-3-glucoside is a major component of Salicornia herbacea, which is traditionally used in Asian medicine and is thought to exhibit multiple nutraceutical and pharmaceutical properties [16]. Isorhamnetin-7-glucoside is suggested to have antitumour activity in a review of topopoisons from weeds [17]. . This is one of the key health components of green tea [18] and was present in this extract in high abundance. Although (−)-epigallocatechin was detected in all extracts, it

Tentative Identification of Polyphenols
Due to the lack of fragmentation data contained in databases, in silico predication has been used here. As the fragmentation mechanisms of polyphenols are well documented, this can be quite an effective approach. Retro-Diels-Alder (RDA) reactions [15] are the general category here, which also applies to the sugar moiety, which is often seen in polyphenols. Additionally, the mass spectra of conjugated phenolic compounds showed the aglycone ion as result of the loss of sugar moieties such as hexosyl ([M-162] − ) or pentosyl ([M-132] − ). An example of this is seen with the two isomers of isorhamnetin glucoside (3 or 7) that are identified at 1.81 and 2.63 min. Their [M-H] − ions undergo the loss of the hexoside moiety to create product ions of m/z 315.0503 and m/z 315.0508. It is also noted that these isobaric species are not only separated by chromatographic retention time but also by drift time, further confirming that these molecules are different in terms of their collision cross section (CCS). The isomer at 1.81 min is seen in high abundance in U. dioica and the other isomer at 2.63 min is high in S. olusatrum. Both are also fairly abundant in the U. europaeus flower extract. Isorhamnetin-3-glucoside is a major component of Salicornia herbacea, which is traditionally used in Asian medicine and is thought to exhibit multiple nutraceutical and pharmaceutical properties [16]. Isorhamnetin-7-glucoside is suggested to have antitumour activity in a review of topopoisons from weeds [17]. documented in the literature as a potent antioxidant which can aid in the fight against cellular injury due to reactive oxygen species [19], and was present in this study, with the highest abundance being in U. dioica and, to a lesser degree, in S. olusatrum.
To link together some key phytoestrogens, dihydrodaidzein (CSID154076) was elucidated via its +M+e] − . These components, which were shown to be highly abundant in U. europaeus flower, U. europaeus stem and U. dioica, have been associated with protection against major adverse cardiac events in women [20]. The findings support the use of a plant-based diet and the need for future randomized prospective studies examining the influence of glycitin and genistein, as well as daidzein and dihydrodaidzein diets on cardiovascular outcomes. A literature review on the benefits of phytoestrogen supplementation on human cognition was conflicting, with less than half of the included studies showing beneficial effects, though supplementation with soy isoflavones for less than 6 months, irrespective of dose and mode, can improve cognitive performance, with greater impact on women than men. Equol is seen as an important product of daidzein metabolism in this process, though only 30%-50% of the general population are equol producers. This is thought to affect individual responses to isoflavone intervention [21]. Two isomeric glucosides of daidzein, which are known as daidzin (m/z 397.0933, 3.15 min) and puerarin (m/z 397.0938, 2.97 min), are also a soy isoflavones known to convert to equol. On top of this, identified at a lower score, is cis-4-hydroxyequol (m/z 257.0807, 2.32 min) which has activity similar to that of equol itself. Detail is available on glucuronidation via microbial metabolic processes, providing a possible route for how polyphenol glucuronides are formed outside of mammalian metabolism [22,23]. Though the 2 isomers of equol O-glucuronide , the former being prominent in U. europaeus flower and stem and the latter being of greatest abundance in green tea, but also present in smaller amounts in the other extracts. These had similar retention times but were clearly separated by drift time, and are therefore different molecules. Molecules of this type are often associated with red wine as a contribution from the barrel wood [27] and have been shown to have potential health benefits [28]. In the context of this, naringin, the rhamnoglucoside of naringenin, was likewise identified in U. Tetrahydroxyflavanones have been shown to reduce the heme group in cytochrome c, which is a necessary component of the electron transport system and is also involved in apoptotic pathways. The oxidation state of the iron in the heme group is crucial to its specific functions [30]. These compounds can be seen as the addition of a phenol group to naringenin, with two being observed in these data: 3 ,4 ,5,7-tetrahydroxyisoflavanone ([2M-H] − , m/z 575.1184, 1.20 min) and eriodictyol ([M-H] − , m/z 287.0554, 2.19 min). 3 ,4 ,5,7tetrahydroxyisoflavanone was seen significantly only in the reference green tea extract and will therefore receive no further discussion. On the other hand, eriodictyol was present in all of the extracts analysed, with a high abundance in U. europaeus flower and stem, but reasonable amounts were also present in U. dioica and A. ursinum leaf. The potential health benefits of eriodictyol are plentiful [8], with further research showing increasing evidence of its function and benefits. For example, eriodictyol may have therapeutic potential for the treatment of rheumatoid arthritis [31], and a study also shows that eriodictyol may provide a new therapeutic strategy for the management of diabetic retinopathy through the inhibition of high glucose-induced oxidative stress and inflammation in retinal ganglial cells [32]. , which was also confirmed by library spectra [33].

-Hydroxy-O-desmethylangolensin was identified by its [M-H]
Violanone was observed via its [M-H 2 O-H] − adduct, with a score of 55.8 at m/z 297.0759 and 1.87 min. The main product ion m/z 121.0282 was due to [C 7 H 6 O 2 +e] − . Violanone was predominant in U. europaeus flower and stem extracts, but is also an important component of the fragrant Chinese rosewood (Dalbergia odorifera), which is used in traditional medicine, with claims broadly relating to its antioxidant qualities [34]. Furthermore, it is shown to selectively inhibit phytopathogenic fungi [35] and to be key component in the roots of Pongamia pinnata, which exhibits antioxidant, anticancer, antimicrobial and anti-inflammatory properties through its use in folk medicine [36]. It is noted, too, that the references to violanone in traditional medicinal plants, found in South Asia and China [34][35][36], also encompass the benefits of many other components identified in these plant extracts from Britain, including daidzein, eriodictyol, naringenin, genistein, isoliquir- ing both the prevention and treatment of tumours [37]. The transformation of tectoridin ([M-H] − , m/z 461.1107, 1.55 min), also found in high abundance in U. europaeus flower and stem, to the bioactive compound tectorigenin is efficiently achieved by gut microflora, resulting in anaphylaxis inhibitory action [38]. Additionally, recent research suggests that tectorigenin inhibits airway inflammation and pulmonary fibrosis in allergic asthma [39]. These four molecules are known as chlorogenic acids, which are a polyphenolic family of quinic acid esters of hydroxyl-cinnamic acids such as p-coumaric acid, caffeic acid and ferulic acid. A placebo-controlled double-blind pilot study, conducted in Japan, found that chlorogenic acids decreased arterial stiffness, which is a characteristic of the progression of arteriosclerosis [40]. 4-O-Feruloyl-D-quinic acid was found in high amounts in U. europaeus flower and A. ursinum leaf extracts, whereas 3-feruloylquinic acid was found to be abundant in S. olusatrum and U. dioica. Chlorogenic acid was found to be most abundant in U. europaeus flower and U. europaeus stem, with its stereoisomer, neochlorogenic acid, also being observed ([M-H] − , m/z 353.0876, 1.51 min), which was seen in high abundance in S. olusatrum and U. dioica as was p-coumaroyl quinic acid.
Theaflavin ([M-H] − , m/z 563.1182, 2.56 min) is created when epicatechin and epigallocatechin come in contact with polyphenol oxidase. This analyte is used as an indicator of production quality in the tea industry, and can be present in low quality green tea but is more common in oolong and black teas. It is shown to be one of many components that are responsible for the antioxidant properties of tea [41]. Here, it is seen in the green tea reference but also in significant abundance in the alexander extract.
The two isomers of procyanidin were identified by their [M-H] − adduct, one at 1.18 min (m/z 577.1376) and the other at 1.30 min (m/z 577.1370), with drift times of 4.5570 ms and 4.6113 ms, respectively. These were either B1, B2, B3 or B4 isomers, but which two could not be determined by the technique as described, although, with external standards of the 4 isomers, retention time and drift time comparison could reveal which is which. Both isomers detected were described by the same three product ions, namely [C 22 [6,27,28], and both isomers were shown to be present in U. europaeus flower and stem extracts. These important phytochemicals, which give rise to the red positively charged cyanidin pigment associated with grapes and berries, exhibit beneficial health effects, including anti-inflammatory, anti-proliferative and antitumor activities, with many reports suggesting procyanidin as a promising lead compound for cancer prevention and treatment [42].
Many glucuronides of potent polyphenols have been identified and, as previously mentioned, most research relates to the creation of these via mammalian metabolism rather than their benefits as a result of ingestion. Of the remaining components, the highlights will be discussed, though further details can be found in the supplementary materials. U. europaeus flower extracts were found to have the highest abundance of hesperetin 3 -O-glucuronide and genistein 4 -O-glucuronide, to name but two examples of many. U. europaeus stem had a high abundance of the glucuronide of cis-resveratrol [43], which was the component that initially sparked great interest in polyphenols. Additionally, dihydrodaidzein 7-glucuronide was seen with the highest abundance in U. europaeus stem. 4 -Methyl epigallocatechin-3 -glucuronide was observed primarily in U. dioica extract, although the amount was fairly low. Irisolidone 7-O-glucuronide and feruloyl  [44,45]. These analytes were seen in their highest abundance in U. europaeus stem and, to a lesser extent, U. europaeus flower. Additionally, U. dioica extract contained significant amounts of salicylic acid. Benzoic acid is the functional group in salicylic acid and its derivatives which are responsible for inducing stress tolerance in plants [46]. Although its score is high, its abundance is seen to be low across all of the extracts, with small amounts in U. europaeus flower and stem. On the other hand, the methoxyisoflavone, irisolidone (m/z 314.0786, 2.69 min), is well documented for health benefits, including anti-helicobacter pylori activity, inhibition of prostaglandin E2 production, hepatoprotective effects, anticancer, estrogenic activity, inhibition of JC-1 virus gene expression and anti-inflammatory activity. Irisolidone is created by the intestinal bacterial transformation of kakkalide (ChemSpider ID 4590337), which is a component of traditional medicine [48]. Irisolidone is evident in good abundance in U. europaeus stem and, to a lesser extent, U. europaeus flower. The product ion m/z 281.0451 ([C 16 H 9 O 5 +e] − ) helped to confirm the identity of irisolidone. Two methoxyisoflavanones (without the double bond on the C-ring) were seen lower on the list of identifications ordered by score. 7,2 -Dihydroxy-4 -methoxyisoflavanone (m/z 267.0663, 2.41 min) and 7-Hydroxy-4 -methoxyisoflavanone (m/z 269.0829, 2.07 min) were seen mainly in U. europaeus flower, U. europaeus stem and U. dioica extracts. 7,2 -Dihydroxy-4 -methoxyisoflavanone has been shown to exist in many plants and is an important component of Tibetan medicine [49]. Isoflavanones are rare compared to isoflavones, with 7-Hydroxy-4 -methoxyisoflavanone being the focus of little research in recent years [50], but it could conceivably have similar activity to its more studied analogues.
The isomers, neohesperidin (m/z 591.1716, 2.77 min) and hesperidin (m/z 591.1724, 2.42 min) were identified with a high score by their [M-H 2 O-H] − adducts. These are both derivatives of hesperetin, differing only in the configuration of their rhamnoglucoside moiety and, therefore, cannot, in truth, be specifically identified without external standards, which is made possible due to differences in chromatographic retention times, as well as drift time separation in ion mobility. Hesperidin and derivatives, which are commonly associated with citrus fruits, play an important role in plant defense systems to combat pathogens. It is thought that they may be useful for humans, as they possess antibacterial, antiviral and antifungal activities [51].
Furthermore, hesperidin and derivatives have been shown to have strong activity against the formation of advanced glycation end products which result in the accumulation of random damage in extracellular proteins. This process is known to have deleterious effects on biological functions which are associated with aging and diabetes, such as cataracts, nephropathy, vasculopathy, proliferative retinopathy and atherosclerosis [52]. Large amounts of hesperidin were observed in the U. europaeus stem extracts.
Urolithins have been shown to be effective in cancer chemoprevention [53] though, due to differences in gut microbiota, urolithin production capacity from ellagic acid varies amongst individuals [54]. , identified in this characterisation of plant extracts, play a key role in the biosynthetic pathway that produces vanillin [55]. 3,4-dihydroxybenzadehyde was found here in good abundance in U. europaeus flower, whereas vanillic acid was at its highest abundance in U. europaeus stem.
Lignans have been shown to have anticarcinogenic properties. 7-Hydroxymatairesinol has been shown to be effective in a prostate cancer model in vivo [56]. A study in the Netherlands showed that some plant lignans, can be converted by intestinal microflora into enterolignans, e.g., enterolactone and enterodiol, and may reduce the risk of certain types of cancer, as well as cardiovascular diseases, through anti-oxidant and anti-estrogenic actions [57]. Once again, their highest abundance was to be found in U. europaeus flower and stem.
The flavonoid, pseudobaptigenin, can be seen at [M-H] − , m/z 281.0460, 3.10 min and was present in high abundance in U. europaeus stem and in lower quantities in U. europaeus flower. It is believed that this phytochemical could be used as a prototype for synthesizing new molecules against diabetic cataracts [58]. Although only observed at low abundance in U. europaeus stem, 3 ,4 ,7-trihydroxyisoflavan ([3M-H] − , m/z 773.2626, 2.50 min) was present, but little is published about this trihydroxyisoflavan, which can be produced microbially but is included here for completeness. Found to be high in S. olusatrum, 4 ,4 -dimethylepigallocatechin gallate was observed via its [M-H] − adduct at m/z 485.1076 and 1.46 min. Research shows that methylation of epigallocatechin gallate alters its potency, with a study showing reduced inhibitory effects in macrophages [59], with a further study on cell surface binding abilities showing that dimethylation prevented surface binding completely, suggesting that the hydroxyl groups on the 4 -position in the B ring and the 4 -position in the gallate are crucial for the cell surface binding activity of epigallocatechin gallate [60].
Finally, we observed three related phytochemicals which all contain the chromone structure and that are very prominent in the polyphenol literature.  [29]. Quercetin is present in all the plant samples analysed here, with very high amounts in U. europaeus flower and high amounts in S. olusatrum. Quercetin is a powerful antioxidant that has a well-documented role in reducing different human cancers, and is one of the most abundant antioxidants in the human diet [61]. In fact, isoquercetin, which was found to be most abundant in S. olusatrum, and rutin, which was found in very high amounts in U. europaeus flowers, have also been shown to have powerful anti-mutagenic activity [6].
It is noted from a review relating to the drug discovery potential of these components that the chromones of the previous paragraph have large differences in their chemistry and bioactivity compared to the chroman-4-ones, all due to the C 2 -C 3 double bond [62]. Naturally occurring chroman-4-ones, such as naringenin, naringin and eriodictyol, were evident in abundance across these plant extracts, providing, together with the other identified polyphenols, a diverse range of bioactivity, with much potential to benefit human and animal health [63].

Conclusions
The effective use of UPLC-HDMS E for the detailed analysis of four common, but low value, green waste materials is demonstrated here. This discovery mode characterisation, which led to the identification of 77 polyphenols with well-documented health potential, opens the door for these plants to provide naturally occurring treatments for disease.
Author Contributions: This research was conceptualized by C.M.P., who was also responsible for methodology and formal analysis. The samples were collected, prepared and analysed by C.M.P. The first draft of this manuscript was written by C.M.P. and reviewed and edited by D.L.J. and C.M.P. All authors have read and agreed to the published version of the manuscript.