LC-ESI/QTOF-MS Profiling of Chicory and Lucerne Polyphenols and Their Antioxidant Activities

Chicory and lucerne are used as specialised forages in sheep or dairy production systems in some parts of the world. Recently, these plants are gaining attention as raw materials in the search for natural antioxidants for use in animal feeds, human foods and nutraceutical formulations. The antioxidant potential of these plants is credited to polyphenols, a subgroup of phytochemicals. Therefore, phenolic characterisation is an essential step before their use as ingredients in animal feeds, human food or nutraceutical preparations. In this study, we performed qualitative and quantitative analysis of polyphenols in chicory and lucerne. Profiling of polyphenols from chicory and lucerne was performed by LC-ESI/QTOF-MS with a total of 80 phenolic compounds identified in chicory and lucerne. The quantification of polyphenols was achieved by high performance liquid chromatography, coupled with a photo diode array (HPLC-PDA). Chicoric acid was the major phenolic acid found in chicory, with the highest concentration (1692.33 ± 0.04 µg/g DW) among all the polyphenols quantified in this study. 2-hydroxybenzoic acid was the major phenolic acid found in lucerne, with the highest concentration of 1440.64 ± 0.04 µg/g DW. Total phenolic, flavonoids and total tannin contents were measured, and the antioxidant potential was determined by 2,2-Diphenyl-1-picrylhydrazyl, Ferric Reducing Antioxidant Power, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid, Hydroxyl (OH−) Radical Scavenging Activity, Chelating Ability of Ferrous Ion (Fe2+) and Reducing Power (RPA) assays. Both chicory (8.04 ± 0.33 mg AAE/g DW) and lucerne (11.29 ± 0.25 mg AAE/g DW) showed high values for Hydroxyl (OH−) Radical Scavenging Activity. The current study allowed us to draw a profile of polyphenols from chicory and lucerne. They provided a molecular fingerprint useful for the application of these plant materials in human foods, animal feeds and pharmaceutical formulations.


Introduction
In recent years, attempts to find natural antioxidants for use in animal feeds, human foods and medicines have increased, in order to replace their synthetic counterparts due to their harmful effects to body and consumer choice. Therefore, natural plant materials gain popularity in various industrial sectors like human food, animal feed, cosmetics and pharmaceuticals. Selected plant materials can contribute to nutrition and natural therapeutics for the cure of various ailments, such as inflammation and oxidative stress due to the presence of bioactive compounds like polyphenols. Polyphenols are bioactives formed in plants as defensive compounds against ultraviolet radiation or aggression by pathogens [1]. These phenolic compounds possess various health-promoting properties, for example being antioxidative, anti-inflammatory, antiaging, antibacterial, antidiabetic and anti-mutagenic [2][3][4]. produce greater dry matter yield due to warm weather with adequate rainfall, which is perfect conditions for the assimilation of nutrients and other secondary compounds in the vegetative parts. For both species, samples (vegetative parts) were collected from five locations within a particular paddock located at Hamilton Research Station, Vic, Australia and bulked in a ziplock plastic bag, weighing approximately 1000 g in total. The stage of maturity of both forages during collection time were at pre-bloom. Upon collection, on the same day, samples were brought to the University of Melbourne, Parkville 3030 under refrigerated conditions using an esky with ice. Upon arrival, samples were stored at 4 • C until further processing for grinding and chemical extraction.

Samples Preparation
Within a week of collection, bulk samples (chicory and lucerne) were crushed using a mortar and pestle to make a paste-like consistency, in order to facilitate the extraction of polyphenol compounds and stored at −20 • C for further analysis.

Extraction of Phenolics
Samples pastes were extracted with 80% ethanol, and then the sample-ethanol mixtures were homogenised. Homogenised mixtures were incubated in a ZWYR-240 shaker incubator (Labwit, Ashwood, VIC, Australia). Afterwards, centrifugation was performed on a Hettich Rotina 380R centrifuge machine (Tuttlingen, Germany) for 20 min at 5000 rpm (4 • C). The supernatants were collected and filtered through 0.22 µm syringe filter (PTFE membrane) and stored at −20 • C for the characterisation and quantification of polyphenols.

Antioxidant Assays
Antioxidant activities were determined by previously reported methods [20] using 96-well plates. Absorbance was recorded on Multiskan ® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) and standard curves with R 2 ≥ 0.99 were constructed with standard solutions. Results were reported on dry weight basis.

Total Phenolics Content (TPC)
Total phenolic content was determined by the Folin and Ciocalteu's method [21] with slight modifications using a 96-well plate. Sample (25 µL) was mixed with 25 µL Folin's Reagent (diluted to 1:3 with water) and allowed to incubate at 25 • C for 5 min. Finally, water (200 µL) and 10% (w/w) Na 2 CO 3 solution (25 µL) were added. This mixture was incubated for 60 min at 25 • C. Absorbance was recorded at 765 nm with a microplate reader. Measurements were made in triplicate, and quantification was done by constructing a standard curve (0-200 µg/mL gallic acid).

Total Flavonoids Content (TFC)
Total flavonoid content for all sample extracts was measured by the AlCl 3 colorimetric method [22] with slight modifications using a 96-well plate. 80 µL sample extract was mixed with 80 µL of 2% AlCl 3 solution. 120 µL of aqueous solution of sodium acetate (50 g/L) was added. This mixture was incubated for 150 min at 25 • C. Absorbance was taken at 440 nm. Measurements for all samples were made in triplicate, and quantification was done by constructing a standard curve (0-50 µg/mL quercetin).

Total Tannin Contents (TTC)
The total tannin content of samples was measured through a colorimetric method [23] with minor changes. Sample extracts (25 µL) was mixed with 4% vanillin solution (150 µL). 25 µL of H 2 SO 4 solution (32%) was added. This mixture was incubated at 25 • C for 15 min. Absorbance was taken at 500 nm. Measurements were made in triplicate and quantification were done by constructing a standard curve (0-1000 µg/mL catechin solution).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
The radical scavenging potential of the samples was estimated by using a previously reported method [24] with slight modifications. 260 µL 0.1 M DPPH solution was mixed with 40 µL sample extract, and allowed to incubate at 25 • C for 30 min. Absorbance was taken at 517 nm. Measurements for all samples were made in triplicate and quantification was done by constructing a standard curve (0-50 µg/mL ascorbic acid).

Ferric Reducing Antioxidant Power (FRAP) Assay
Ferric reducing potential was estimated by a previously used method [24] with modifications in a 96-well plate. 300 mM acetate buffer, 20 mM ferric chloride and 10 mM TPTZ were mixed in 10:1:1 (v/v/v) ratio to prepare FRAP reagent. 280 µL of FRAP reagent was added to 20 µL of sample extract and incubated for 10 min at 37 • C. Absorbance was recorded at 593 nm. Measurements were made in triplicate and quantification was done by constructing a standard curve (0-50 µg/mL ascorbic acid).

2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Radical Scavenging Assay
The potential of samples to scavenge ABTS radical was determined by ABTS + radical cation de-colorisation method [24] with slight modifications. 7 mM ABTS solution was mixed with 140 mM K 2 S 2 O 8 solution, and allowed to incubate in the dark for 16 h for the generation of ABTS + ions in the solution. The absorbance of this solution was adjusted to 0.70 ± 0.02 by dilution with ethanol. After this, 10 µL of sample extracts were mixed with ABTS + solution (290 µL), and allowed to incubate at 25 • C for 6 min. Absorbance was taken at 734 nm. Measurements for all samples were made in triplicate, and quantification was done by constructing a standard curve (0-150 µg/mL ascorbic acid).

Hydroxyl (OH − ) Radical Scavenging Activity Assay
The hydroxyl (OH − ) radical scavenging potential of samples was determined by translating the method of Pavithra and Vadivukkarasi [25] to a 96-well plate method. 50 µL of sample was mixed with 50 µL of 6 mM Ferrous sulphate solution and 50 µL of 6 mM hydrogen peroxide (H 2 O 2 ) solution in a 96-well plate. The mixture was incubated for 10 min at 25 • C. After incubation, 50 µL of 6 mM 3-hydroxybenzoic acid solution was added. Absorbance was recorded at 510 nm. Measurements for all samples were made in triplicate and quantification was done by constructing a standard curve (0-300 µg/mL ascorbic acid).

Chelating Ability of Ferrous Ion (Fe 2+ )
The chelating ability of samples was determined by translating the method of Pavithra and Vadivukkarasi [25] to a 96-well plate method. 100 µL of sample was mixed with 80 µL of 2 mM ferrous chloride solution in a 96-well plate. 70 µL of 5 mM Ferrozine solution was added and mixed well. The mixture was allowed to stand for 10 min at 25 • C. Absorbance was taken at 562 nm. Measurements for all samples were made in triplicate, and quantification was done by constructing a standard curve (0-10 µg/mL EDTA solution).

Reducing Power Assay (RPA)
The reducing power of the samples was determined by translating the method of Pavithra and Vadivukkarasi [25] to a 96-well plate method. 10 µL of sample, 25 µL of 0.2 M phosphate buffer (pH 6.6) and 25 µL of Potassium ferricyanide solution (1% w/v) were added sequentially in a 96-well plate, and incubated for 20 min at 25 • C. Following incubation, 25 µL of TCA solution (10% w/v) was added. After this, 85 µL of water and 8.5 µL of Iron (III) chloride solution (0.1% w/v) and incubated for 15 min at 25 • C. Absorbance was recorded at 750 nm. Measurements for all samples were made in triplicate, and quantification was done by constructing a standard curve (0-150 µg/mL ascorbic acid).

Statistics Analysis
Results were reported as mean ± standard deviation of the values of three independent analyses. The students' t-test was performed using Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA) to test statistical significance by comparing the means (significant difference at p ≤ 0.05).

Polyphenols Estimation from Chicory and Lucerne Extracts (TPC, TFC and TTC)
Chicory and lucerne are rich in polyphenols. The polyphenol content of chicory and lucerne was determined as TPC, TFC and TTC. The results showed that polyphenolic contents varied considerably in chicory and lucerne (Table 1). Lucerne showed a significantly higher TPC (0.71 ± 0.01 mg GAE/g) and TTC (1.32 ± 0.08 mg CE/g) as compared to chicory, with TPC and TTC values of 0.44 ± 0.04 mg GAE/g and 0.84 ± 0.03 mg CE/g respectively. The TPC of lucerne was determined previously by Zagórska-Dziok et al., [27] in the range of 3.52 mg GAE/g to 73.5 mg GAE/g using different concentrations of water-glycerine extracts of lucerne. Our values for TPC of lucerne were lower than the already reported values. The difference could be explained by the use of different solvents for extraction and the methods applied for determination. No significant difference was observed in total flavonoid content (0.07 ± 0.01 mg QE/g in chicory and 0.07 ± 0.01 mg QE/g in lucerne) in both samples (p ≤ 0.05). Table 1. Total phenolics content (TPC), total flavonoids content (TFC) and total tannins content (TTC) of chicory and lucerne.

Phenolic Content
Chicory Lucerne Results are reported on a dry weight basis; n = three replicates per sample. The terms mg GAE/g, mg QE/g and mg CE/g for milligrams of gallic acid equivalents, milligrams of quercetin equivalents and milligrams of catechin equivalents, respectively. Within a row, significant difference (p ≤ 0.05) is indicated by superscript letters ( a,b ).
Kaur and coworkers [28] also determined TPC of chicory extracts, and were found in the range of 23.4 to 62.5 mg GAE/100 g dry weight (0.234 to 0.625 mg GAE/g dry weight) using different solvents for extraction. Our results for TPC of chicory (0.44 ± 0.04 mg GAE/g dry weight) is within the range already reported by Kaur and coworkers.

Antioxidant Activity
Chicory Lucerne Results are reported on a dry weight basis; n = three replicates per sample. The terms mg AAE/g and mg EDTAE/g stand for milligrams of ascorbic acid equivalents and mg of Ethylenediaminetetraacetic acid. a,b Denotes p ≤ 0.05.
No significant difference was observed for DPPH and FRAP values for chicory and lucerne. DPPH and FRAP values of chicory are 0.12 ± 0.01 mg AAE/g and 0.01 ± 0.01 mg AAE/g respectively. Meanwhile, lucerne showed DPPH and FRAP values as 0.13 ± 0.01 mg AAE/g and 0.02 ± 0.01 mg AAE/g, respectively.

Polyphenols Profile of Chicory and Lucerne
Profiling of polyphenols from chicory and lucerne were performed by verifying m/z value from mass spectra in positive ([M + H] + ) and negatiM+He ([M − H] − ) ionisation modes and compounds with mass error less than 10 ppm were selected for the verification of m/z for characterisation using the personal compound database library. 80 polyphenols were identified in chicory and lucerne extracts, with 14 phenolic acids, 52 flavonoids, three lignans, one stilbene and 10 other polyphenols (Table 3). Higher diversity of polyphenols was found in lucerne extract with a total of 56 compounds (Table S2-Supplementary materials), as compared to the chicory extract in which 29 polyphenols (Table S1-Supplementary materials) were identified. Flavonoids and phe-nolic acids were the main polyphenol subgroups in both plant extracts. Stilbenes was only identified in the chicory extract (Supplementary materials- Figure S1).

Phenolic Acids
A total of fourteen phenolic acids belonging to three different subclasses (hydroxybenzoic acids, hydroxycinnamic acids and hydroxyphenylpropanoic acids) were tentatively identified in chicory and lucerne extracts. Hydroxycinnamic acids and hydroxybenzoic acids were the dominant subgroups of phenolic acids, with seven and five compounds, respectively. Only two compounds belonging to hydroxyphenylpropanoic acids were tentatively identified.

Hydroxycinnamic Acid Derivatives
Hydroxycinnamic acid were the predominant phenolic acids in chicory and lucerne [31,32]. Compounds (6, 7, 8, 9, 10, 11 & 12) were identified as hydroxycinnamic acid derivatives. Compound (12) was identified in negative ionisation mode in both chicory and lucerne extracts at m/z 473.0764 and m/z 473.0696, respectively, and was designated as chicoric acid (C 22 H 18 O 12 ). Chicoric acid has already been reported in methanolic extracts of chicory [17,33]. Compounds (6, 9 & 11) were identified only in the lucerne extract in positive ionisation mode at m/z 165.0548, 399.1288 and 517.1319, and were designated as m-Coumaric acid (C 9 H 8 O 3 ), 3-Sinapoylquinic acid (C 18 H 22 O 10 ) and 1,5-Dicaffeoylquinic acid (C 25 H 24 O 12 ), respectively. m-Coumaric acid was also previously identified in lucerne [34]. Compound (8) was identified in the lucerne extract in negative ionisation mode at m/z 616.1062 and designated as 2-S-Glutathionyl caftaric acid (C 23 H 27 N 3 O 15 S). However, compounds (7 & 10) were identified in chicory extract in positive ionisation mode at m/z 149.0585 and 355.0999 and were designated as Cinnamic acid (C 9 H 8 O 2 ) and 3-Caffeoylquinic acid, respectively (C 16 H 18 O 9 ). 3-Caffeoylquinic acid has also previously been identified in chicory [17]. Out of the seven hydroxycinnamic acids identified in this study, three are in the form of quinic acid derivatives. This agrees with a previous finding that hydroxycinnamic acids mainly exist in conjugated form, such as quinic acid [35].

Hydroxyphenylpropanoic Acids
Two hydroxyphenylpropanoic acids (compound 13 & 14) were identified in negative ionisation mode ([M − H] − ). Compound (13) was identified in both chicory and lucerne extracts in negative ionisation mode at m/z 357.0847 and 357.0819 respectively and designated as Dihydrocaffeic acid 3-O-glucuronide (C 15 H 18 O 10 ). However, compound (14) was only identified in lucerne extract in negative ionisation mode at m/z 275.0218, and designated as Dihydroferulic acid 4-sulfate (C 10 H 12 O 7 S).

Flavonoids and Their Derivatives
Higher diversity of flavonoids derivatives was found among the phenolic compounds identified in chicory and lucerne extracts. A total of 52 flavonoids belonging to seven subgroups were identified in this study.

Anthocyanins Derivatives
Anthocyanins provide protection to arteries and endothelial tissues, inhibit platelet aggregation and reduce the risk of heart diseases [17,[36][37][38]. Chicory and lucerne have been reported to contain different anthocyanin derivatives. The anthocyanin derivatives found in chicory are of special interest due to their beneficial effects on visual capacity, brain cognitive function, obesity and cancer prevention [39,40]. Five anthocyanins (compounds 15, 16,  17, 18 & 19) were detected in this study. Out of the five anthocyanins, one compound (18) was putatively identified in negative ionisation mode at m/z 640.145 and 640.1413 in both chicory and lucerne extracts, and was designated as Delphinidin 3-O-feruloyl-glucoside (C 31 H 29 O 15 ). Compounds (15 & 19) were putatively identified in negative ionisation mode in lucerne extract at m/z 300.0654 and 740.2187 and designated as Peonidin (C 16

Quantification of Polyphenols through HPLC-PDA
HPLC is a commonly applied technique for the quantification of polyphenols from various types of samples. The phenolic acids and flavonoids have medicinal importance, and are well known for their high antioxidant capabilities. These are the main compounds responsible for the high antioxidant potential of plant extracts. Therefore, we quantified four phenolic acids (Cinnamic acid, Chicoric acid, 2-hydroxybenzoic acid and m-Coumaric acid) and one flavonoid (Isorhamnetin) in chicory and lucerne, as these compounds are commonly found polyphenols in chicory and lucerne. Table 4 shows the data of targeted polyphenolic compounds quantified in chicory and lucerne. Of the nine targeted polyphenols, six compounds belong to the phenolic acids and three are flavonoids. Two compounds (Cinnamic acid and Isorhamnetin) were detected and quantified only in chicory and three compounds (2-hydroxybenzoic acid, m-Coumaric acid and p-hydroxybenzoic acid) quantified in lucerne only. Two phenolic acids (gallic acid and chicoric acid) were detected and quantified in both chicory and lucerne. The concentration of gallic acid was 38.17 ± 0.03 µg/g DW in chicory and 55.74 ± 0.04 µg/g DW in lucerne. Chicoric acid is the major phenolic acid in chicory, with the highest concentration (1692.33 ± 0.04 µg/g DW) among all phenolic compounds quantified in this study. The concentration of chicoric acid in lucerne was lower (1434.36 ± 0.02 µg/g DW) as compared to chicory. p-hydroxybenzoic acid was quantified only in lucerne (11.55 ± 0.02). Cinnamic acid concentration was 115.00 ± 0.01 µg/g DW in chicory. 2-hydroxybenzoic acid and m-coumaric acid concentrations in lucerne were 1440.64 ± 0.04 µg/g DW and 2.64 ± 0.01 µg/g DW, respectively. Out of the three detected flavonoids, isorhamnetin was quantified in only the chicory extract at a concentration of 641.80 ± 0.03 µg/g DW. Meanwhile, quercetin 3-rhamnoside and epicatechin gallate were detected and quantified in both chicory and lucerne. Quercetin 3-rhamnoside concentration in lucerne was much higher (187.74 ± 0.05 µg/g DW) as compared to chicory (5.50 ± 0.04 µg/g DW). The concentration of epicatechin gallate was 29.28 ± 0.02 µg/g DW and 62.77 ± 0.03 µg/g DW in chicory and lucerne, respectively. Table 4. Quantification of targeted polyphenols in chicory and lucerne by HPLC-PDA analysis.

No.
Compound Name RT Chicory (µg/g DW) Lucerne (µg/g DW) Polyphenol Class The terms RT and DW stands for retention time and dry weight.

Relationship of Phenolic Contents and Antioxidant Activities
Polyphenols found in chicory and lucerne contribute significantly to their bioactive potential, owing to their strong antioxidant activities. Considering the role of phenolic contents in antioxidant potential, we investigated the TPC, TFC and TTC. Lucerne showed higher values of TPC, TFC and TTC as compared to chicory ( Table 1). The DPPH, ABTS, FRAP, OH − Radical Scavenging Ability, Chelating Ability of Fe 2+ and RPA were measured to determine the antioxidant potential of chicory and lucerne. Lucerne showed higher values for the all the antioxidant activities corresponding to its high phenolic contents (TPC, TFC and TTC). Therefore, it could be established that the phenolic contents of the plants highly contributed to their antioxidant activities. Results of the study showed that phenolic contents of chicory and lucerne are significant contributors of their antioxidant potential and bioactive properties. However, the antioxidant effects of different phenolic constituents could vary owing to their concentration, synergistic action and antagonistic actions with other chemical moieties present in chicory and lucerne.

Conclusions
A major finding of this study was that the lucerne has higher level of phenolic compounds (TPC, TFC and TTC) and greater antioxidant potential (DPPH, FRAP, ABTS, Hydroxyl (OH − ) Radical Scavenging Activity, Chelating Ability of Ferrous Ion (Fe 2+ ) and Reducing Power) than chicory. This was supported by the LC-ESI-QTOF/MS analysis, since a higher diversity of polyphenols was observed in the vegetative parts of lucerne (56 compounds) when compared with chicory (29 compounds). Among the polyphenols identified, phenolic acids and flavonoids were the most common polyphenols present in both lucerne and chicory forages. Hence, chicory and lucerne could serve as good sources of antioxidant polyphenols. Moreover, the obtained results could support these plants' utilisation as ingredients of natural feed additives in animal feeds, functional foods and pharmaceutical formulations. However, further experimental work conducted in animals in vivo is needed to understand the mode of actions of these polyphenols in the body, their inclusion levels in the diet and feeding length that can improve the performance or wellbeing of farm animals and humans.