A Multiparametric Protocol for the Detailed Phytochemical and Antioxidant Characterisation of Plant Extracts

Medicinal and herbal plants are abundant sources of phytochemicals, which are biologically active compounds with potential health benefits. The characterisation of phytochemicals has been the subject of many studies, but there is a lack of comprehensive assays to accurately assess the main phytochemical categories and their antioxidant properties. To address this, the present study has developed a multiparametric protocol comprising eight biochemical assays, which quantify the major categories of phytochemicals, including polyphenols, tannins and flavonoids, as well as their antioxidant and scavenging potential. The presented protocol offers several advantages over other methods, including higher sensitivity and significantly lower cost, making it a simpler and more affordable approach compared to commercial kits. The protocol was tested on two datasets with seventeen distinct herbal and medicinal plants, and the results demonstrated its effectiveness in accurately characterising the phytochemical composition of plant samples. The modular design of the protocol allows its adaptation to any spectrophotometric instrumentation, while all assays are simple to follow and require a minimum number of analytical steps.


Introduction
Since ancient times, plants provide mankind with many remedies and food supplements, and in some countries, medicinal plants may be the primary or sole source of healthcare [1,2]. Some examples of medicinal and herbal plant products include chamomile, ephedra, garlic, ginseng, marijuana and opium. Furthermore, plants have a significant impact on diet in both societal (i.e., veganism) but also in practical contexts in relation to human physiology. The nutritional and medicinal benefits of plants are reflected in their phytochemical components, as plants are able to synthesise a large range of compounds known as phytochemicals [3][4][5]. The metabolites of plants are categorised into two groups: primary and secondary metabolites. The primary metabolites are involved in the major metabolic pathways for plant growth and development, while secondary metabolites serve non-essential purposes in the plants [6,7]. Secondary metabolites are required for plants to survive as these compounds are involved in adaptation and survival mechanisms, but also have been proven to have medicinal properties [4,8]. As a result, medicinal and herbal plants play a critical role in the food and pharmaceutical industries [9].
Several in vivo and in vitro studies have been focused on the beneficial biological actions of plant extracts highlighting their importance in ethnobotany. Most innovative studies emphasise the phytochemical characterisation and identification of biologically active compounds, which can then be synthesised in organic chemistry laboratories or in heterologous hosts [10]. In this effort, several assays for the determination of the major categories of phytochemicals (i.e., polyphenols, tannins, flavonoids, alkaloids, etc.) were combined with assays focusing on the assessment of the antioxidant properties of plant extracts. These methods are useful for the identification of bioactive compounds. To date, there is not an optimised comprehensive protocol to provide a series of analytical options within a general (not detailed) phytochemical characterisation.
The present multiparametric protocol provides targeted assays for the characterisation of the main categories of plant metabolites for eight distinct biochemical protocols with standardised microplate assays. Specifically, flavonoids are quantified by their reaction with aluminium trichloride [11], tannins are assayed by their reaction with vanillin under acidic conditions [12], and polyphenols are assessed by the Folin reagent [13]. In relation to the metal scavenging [14] and the antioxidant potential [15] of plant extracts, the capacity of plant extracts to reduce ions such as iron and copper (Figure 1) was based on assays using the 2,4,6-tri-pyridyl-s-triazine [16] and neocuproine reagents [17], respectively. Both assays require first the metal cation to generate a complex with the reagent, followed by the reduction of the metal cation to generate a chromogenic reduced metal-reagent complex. Finally, assays based on the reduction of the stable DPPH [18], ABTS •+ cation [19] and galvinoxyl radicals are performed using the loss of their colour when scavenged by a phytochemical extract, to assess the % of scavenging capacity ( Figure 1). Overall, the protocols presented underwent extensive troubleshooting and optimisation of the conditions existing in the original methods, previously applied on extracts from the sumac plant [20] aiming to provide a unified gold standard approach.
Methods Protoc. 2023, 6, x FOR PEER REVIEW 2 of 13 categories of phytochemicals (i.e., polyphenols, tannins, flavonoids, alkaloids, etc.) were combined with assays focusing on the assessment of the antioxidant properties of plant extracts. These methods are useful for the identification of bioactive compounds. To date, there is not an optimised comprehensive protocol to provide a series of analytical options within a general (not detailed) phytochemical characterisation. The present multiparametric protocol provides targeted assays for the characterisation of the main categories of plant metabolites for eight distinct biochemical protocols with standardised microplate assays. Specifically, flavonoids are quantified by their reaction with aluminium trichloride [11], tannins are assayed by their reaction with vanillin under acidic conditions [12], and polyphenols are assessed by the Folin reagent [13]. In relation to the metal scavenging [14] and the antioxidant potential [15] of plant extracts, the capacity of plant extracts to reduce ions such as iron and copper (Figure 1) was based on assays using the 2,4,6-tri-pyridyl-s-triazine [16] and neocuproine reagents [17], respectively. Both assays require first the metal cation to generate a complex with the reagent, followed by the reduction of the metal cation to generate a chromogenic reduced metalreagent complex. Finally, assays based on the reduction of the stable DPPH [18], ABTS •+ cation [19] and galvinoxyl radicals are performed using the loss of their colour when scavenged by a phytochemical extract, to assess the % of scavenging capacity ( Figure 1). Overall, the protocols presented underwent extensive troubleshooting and optimisation of the conditions existing in the original methods, previously applied on extracts from the sumac plant [20] aiming to provide a unified gold standard approach.

Experimental Design
The protocol procedures (outlined in Figure 2), begin with the initial processing of the plant material (step 1) and the extraction of plant metabolites followed by their subsequent

2.
Extract plant tissues in HPLC-MS methanol:HPLC-MS water (80%:20% v/v) with vigorous vortexing to facilitate the extraction of polar molecules. This approach is sufficient to extract different categories of organic and polar molecules, however, it can be tailored to the applications of the laboratory.

3.
Clear the extract from debris by centrifugation at 18,000× g at 4 • C for 10 min and collect the clear supernatant. An alternative approach for removing debris is filtration using a syringe filter (0.45 µm filter membrane).

4.
The plant extract can be used immediately or concentrated by speed vacuum evaporation/lyophilisation. The extract can be aliquoted prior to use or drying at this point. For reference to the initial amount of plant tissue extracted, a record of the plant material collected, and the volume of extraction solvent used must be kept.

Plant Homogenisation, Extraction and Preparation for Assays (1 h)
1. Collect plants (i.e., leaves, roots, stems, flowers) and either quench by immediate freezing in liquid nitrogen or homogenise tissue, i.e., cutting tissues in slices and oven-drying at 40 °C followed by grinding [20,21]. 2. Extract plant tissues in HPLC-MS methanol:HPLC-MS water (80%:20% v/v) with vigorous vortexing to facilitate the extraction of polar molecules. This approach is sufficient to extract different categories of organic and polar molecules, however, it can be tailored to the applications of the laboratory. 3. Clear the extract from debris by centrifugation at 18,000× g at 4 °C for 10 min and collect the clear supernatant. An alternative approach for removing debris is filtration using a syringe filter (0.45 µm filter membrane). 4. The plant extract can be used immediately or concentrated by speed vacuum evaporation/lyophilisation. The extract can be aliquoted prior to use or drying at this point. For reference to the initial amount of plant tissue extracted, a record of the plant material collected, and the volume of extraction solvent used must be kept.

5.
PAUSE STEP The extracted samples after evaporation are stable at −80 °C. Prior to analysis, the evaporated samples should be reconstituted. 6. Prepare serial dilutions of the methanolic extracts in ddH2O and use for all assays.

7.
CRITICAL STEP Only for the assessment of galvinoxyl radical scavenging the dilutions prepared in methanol citrate. 8. If the extract (from step 4) was evaporated/lyophilised, then it is first reconstituted in ddH2O or other solvents with vigorous vortexing and filtering to remove undissolved particulates, and then appropriately diluted in ddH2O to avoid any interferences.

Quantification of Flavonoids (0.5 h)
• Samples (and catechin standards 10-100 µΜ) are assayed for flavonoids according to Table 1. Incubate mixtures for 10 min at room temperature and measure absorbance at 500 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of catechin nmoles using the corresponding standard curve.

PAUSE STEP
The extracted samples after evaporation are stable at −80 • C. Prior to analysis, the evaporated samples should be reconstituted. 6.
Prepare serial dilutions of the methanolic extracts in ddH 2 O and use for all assays. 7.

Plant Homogenisation, Extraction and Preparation for Assays (1 h)
1. Collect plants (i.e., leaves, roots, stems, flowers) and either quench by immediate freezing in liquid nitrogen or homogenise tissue, i.e., cutting tissues in slices and oven-drying at 40 °C followed by grinding [20,21]. 2. Extract plant tissues in HPLC-MS methanol:HPLC-MS water (80%:20% v/v) with vigorous vortexing to facilitate the extraction of polar molecules. This approach is sufficient to extract different categories of organic and polar molecules, however, it can be tailored to the applications of the laboratory. 3. Clear the extract from debris by centrifugation at 18,000× g at 4 °C for 10 min and collect the clear supernatant. An alternative approach for removing debris is filtration using a syringe filter (0.45 µm filter membrane). 4. The plant extract can be used immediately or concentrated by speed vacuum evaporation/lyophilisation. The extract can be aliquoted prior to use or drying at this point. For reference to the initial amount of plant tissue extracted, a record of the plant material collected, and the volume of extraction solvent used must be kept.

5.
PAUSE STEP The extracted samples after evaporation are stable at −80 °C. Prior to analysis, the evaporated samples should be reconstituted. 6. Prepare serial dilutions of the methanolic extracts in ddH2O and use for all assays.

7.
CRITICAL STEP Only for the assessment of galvinoxyl radical scavenging the dilutions prepared in methanol citrate. 8. If the extract (from step 4) was evaporated/lyophilised, then it is first reconstituted in ddH2O or other solvents with vigorous vortexing and filtering to remove undissolved particulates, and then appropriately diluted in ddH2O to avoid any interferences.

Quantification of Flavonoids (0.5 h)
• Samples (and catechin standards 10-100 µΜ) are assayed for flavonoids according to Table 1. Incubate mixtures for 10 min at room temperature and measure absorbance at 500 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of catechin nmoles using the corresponding standard curve.
CRITICAL STEP Only for the assessment of galvinoxyl radical scavenging the dilutions prepared in methanol citrate. 8.
If the extract (from step 4) was evaporated/lyophilised, then it is first reconstituted in ddH 2 O or other solvents with vigorous vortexing and filtering to remove undissolved particulates, and then appropriately diluted in ddH 2 O to avoid any interferences.

Quantification of Tannins (0.5 h)
• Samples (and catechin standards 10-100 µΜ) are assayed for tannins according to Table 2. Incubate mixtures for 10 min at room temperature and measure absorbance at 500 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of catechin nmoles using the corresponding standard curve.

Quantification of Polyphenols (1 h)
• Samples (and gallic acid standards 10-100 µΜ) are assayed for polyphenols according to Table 3. Incubate mixtures for 40 min at room temperature and measure absorbance at 765 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of gallic acid nmoles using the corresponding standard curve.

Quantification of Tannins (0.5 h)
• Samples (and catechin standards 10-100 µΜ) are assayed for tannins according to Table 2. Incubate mixtures for 10 min at room temperature and measure absorbance at 500 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of catechin nmoles using the corresponding standard curve.
CRITICAL STEP Agitate by pipetting to solubilise mixture aggregates. • Incubate mixtures for 10 min at room temperature and measure absorbance at 500 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of catechin nmoles using the corresponding standard curve. • Samples (and gallic acid standards 10-100 µM) are assayed for polyphenols according to Table 3. • Samples (and gallic acid standards 10-100 µΜ) are assayed for cupric-reducing power according to Table 5. • Incubate mixtures for 40 min at room temperature and measure absorbance at 450 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of gallic acid nmoles using the corresponding standard curve.

Quantification of DPPH Radical Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh DPPH reagent at ~1.3 A 515 nm is prepared in methanol:acetic acid pH 5.5 (8:2). Ensure that mixing 200 µL of this DPPH stock is with 100 µL ddH2O as reagent blank gives an absorbance of ~0.8 at 515 nm [22]. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the DPPH stock.

Quantification of ABTS Radical Cation (ABTS •+ ) Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh ABTS •+ reagent at ~1.3 A 734 nm is prepared in ddH2O. Ensure that mixing 200 µL of this ABTS •+ stock with 100 µL ddH2O (will be used as reagent blank) gives an absorbance of ~0.8 at 734 nm. The reason being is that the absorbance of the reagent blank will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case, adjust the dilution/concentration of the ABTS •+ stock.
CRITICAL STEP Agitate by pipetting to ensure homogeneity. • Incubate mixtures for 40 min at room temperature and measure absorbance at 765 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of gallic acid nmoles using the corresponding standard curve. • Samples (and gallic acid standards 10-100 µM) are assayed for ferric-reducing power according to Table 4. • Samples (and gallic acid standards 10-100 µM) are assayed for cupric-reducing power according to Table 5.  • Incubate mixtures for 40 min at room temperature and measure absorbance at 450 nm. The net absorbance derived from the absorbance difference of sample/standard minus reagent blank is converted to equivalents of gallic acid nmoles using the corresponding standard curve.

Quantification of DPPH Radical Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh DPPH reagent at ~1.3 A 515 nm is prepared in methanol:acetic acid pH 5.5 (8:2). Ensure that mixing 200 µL of this DPPH stock is with 100 µL ddH2O as reagent blank gives an absorbance of ~0.8 at 515 nm [22]. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the DPPH stock.

Quantification of ABTS Radical Cation (ABTS •+ ) Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh ABTS •+ reagent at ~1.3 A 734 nm is prepared in ddH2O. Ensure that mixing 200 µL of this ABTS •+ stock with 100 µL ddH2O (will be used as reagent blank) gives an absorbance of ~0.8 at 734 nm. The reason being is that the absorbance of the reagent blank will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case, adjust the dilution/concentration of the ABTS •+ stock.
CRITICAL STEP Before starting the experiment, a fresh DPPH reagent at~1.3 A 515 nm is prepared in methanol:acetic acid pH 5.5 (8:2). Ensure that mixing 200 µL of this DPPH stock is with 100 µL ddH 2 O as reagent blank gives an absorbance of 0.8 at 515 nm [22]. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the DPPH stock.
• Samples (and catechin standards 5-100 µM) are assayed for DPPH radical scavenging capacity according to Table 6. • Incubate mixtures for 10 min at room temperature protected from light and measure absorbance at 515 nm. Calculate the % of DPPH radical scavenging as follows: . The % of DPPH radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.

Quantification of Galvinoxyl Radical Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh galvinoxyl radical reagent at ~1.3 A 435 nm is prepared in methanol:citric acid pH 6 (9:1). Ensure that mixing 200 µL of this galvinoxyl radical stock is with 100 µL methanol:citric acid pH 6 (9:1) as reagent blank gives an absorbance of ~0.8 at 435 nm. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the galvinoxyl radical stock.
• Samples (and catechin standards 5-100 µΜ) are assayed for galvinoxyl radical scavenging capacity according to Table 8.  ASolventBlank). The % of galvinoxyl radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.
CRITICAL STEP Before starting the experiment, a fresh ABTS •+ reagent at~1.3 A 734 nm is prepared in ddH 2 O. Ensure that mixing 200 µL of this ABTS •+ stock with 100 µL ddH 2 O (will be used as reagent blank) gives an absorbance of~0.8 at 734 nm. The reason being is that the absorbance of the reagent blank will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case, adjust the dilution/concentration of the ABTS •+ stock. • Samples (and catechin standards 5-100 µM) are assayed for ABTS •+ radical scavenging capacity according to Table 7.
• Incubate mixtures for 40 min at room temperature protected from light and measure absorbance at 734 nm. Calculate the % of ABTS •+ radical scavenging as follows: The % of ABTS radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.   ASolventBlank). The % of ABTS radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.

Quantification of Galvinoxyl Radical Scavenging (1 h)
• CRITICAL STEP Before starting the experiment, a fresh galvinoxyl radical reagent at ~1.3 A 435 nm is prepared in methanol:citric acid pH 6 (9:1). Ensure that mixing 200 µL of this galvinoxyl radical stock is with 100 µL methanol:citric acid pH 6 (9:1) as reagent blank gives an absorbance of ~0.8 at 435 nm. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the galvinoxyl radical stock.
• Samples (and catechin standards 5-100 µΜ) are assayed for galvinoxyl radical scavenging capacity according to Table 8. Table 8. Galvinoxyl radical cation scavenging assay conditions. . The % of galvinoxyl radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.

Troubleshooting
• All protocols presented are straightforward with minimum number of steps similar to kit-based assays. A troubleshooting table (Table 9) is presented for possible caveats.
CRITICAL STEP Before starting the experiment, a fresh galvinoxyl radical reagent at~1.3 A 435 nm is prepared in methanol:citric acid pH 6 (9:1). Ensure that mixing 200 µL of this galvinoxyl radical stock is with 100 µL methanol:citric acid pH 6 (9:1) as reagent blank gives an absorbance of~0.8 at 435 nm. The reason being is that the absorbance of the reagent blank (RB) will be the initial 100% radical which needs to be in the linear absorbance scale of the microplate reader used and also in excess to allow sufficient scavenging. If this is not the case adjust the dilution/concentration of the galvinoxyl radical stock.

Reagents Solvent Blank (µL) Reagent Blank (µL) Sample/Standard (µL)
Methanol • Incubate mixtures for 10 min at room temperature protected from light and measure absorbance at 435 nm. Calculate the % of galvinoxyl radical scavenging as follows: . The % of galvinoxyl radical scavenging is converted to equivalents of catechin nmoles using the corresponding standard curve.

•
All protocols presented are straightforward with minimum number of steps similar to kit-based assays. A troubleshooting table (Table 9) is presented for possible caveats.

Expected Results
The multiparametric protocol described here can be applied to any type of plant material. Previously, we demonstrated this approach in our article on the sumac root, leaf and stem extracts on an in vitro ethanol toxicity model system [20]. In this study, we provide the biochemical analysis of Greek herbal plant extracts (dataset 1) and other common plants (dataset 2) summarised as indicative exemplars of our outlined approach in Supplementary Excel files with automated calculations to assist the end user. Each sheet contains a respective protocol in addition to a standard curve and sample analysis (for four replicates per samples). The technical reproducibility of the methods presented is reflected on the low coefficient of variance obtained from independent replicates of each sample. This is a result of the simplicity of each assay performed in a minimum number of steps. Simple statistics with unpaired tests can be performed using the Excel files provided. Furthermore, the results can be collectively processed (in their averages per sample) after z-score standardisation to avoid the large differences among data skewing the findings. The results are easily presented in radar charts (Figure 3), which can collectively summarise the parameters measured for a great number of plants. Values were z-score standardised as outlined in the automated Excel file. In the first dataset of the Greek herbal plants, the stinging nettle (Urtica urens, Uu) is the sample in the inner center of the radar charts, thus with the minimum amount of all measurement parameters, while for other samples characteristic increased values such as tannins for Laurus nobilis (Ln) can be easily visualised.
Although there are many biochemical methods available for determining the antioxidant activities of biological materials [27], to our knowledge there has been no attempt so far in the research community to generate a gold standard approach on the outlined topic of a general characterisation of plant extracts. Furthermore, as a general rule for the assays available in the literature, these mostly rely on large reagent and sample volumes, which in turn results in increased consumption of reagents, generating additional lab waste and requiring more samples, which, in turn, would reflect on the sensitivity of the methods [28]. The methods developed here aim to quickly, accurately, and with the highest sensitivity provide results for the general characterisation of plants.
The protocols presented here provide a detailed characterisation of plant extract as their primary focus to identify the main phytochemical categories. However, the specific identification of compounds would require significant and cumbersome analytical instrumentation such as chromatographic separation coupled with mass spectrometry or NMR spectroscopy to achieve the specific elucidation of plant extract chemical composition [29]. Although there have been many advances in hyphenated analytical methods for the quantification and identification of polyphenols, flavonoids and tannins, providing their analytical coverage is limited by matrix effects and the cost of analytical equipment [30]. The protocol presented here could also be part of a more detailed assessment if a user chooses to reconstitute the samples from step 4 following their extraction and proceed towards an HPLC-MS analysis. Although there are many biochemical methods available for determining the antioxidant activities of biological materials [27], to our knowledge there has been no attempt so far in the research community to generate a gold standard approach on the outlined topic of a general characterisation of plant extracts. Furthermore, as a general rule for the assays available in the literature, these mostly rely on large reagent and sample volumes, which in turn results in increased consumption of reagents, generating additional lab waste and requiring more samples, which, in turn, would reflect on the sensitivity of the methods [28]. The methods developed here aim to quickly, accurately, and with the highest sensitivity provide results for the general characterisation of plants.
The protocols presented here provide a detailed characterisation of plant extract as their primary focus to identify the main phytochemical categories. However, the specific identification of compounds would require significant and cumbersome analytical instrumentation such as chromatographic separation coupled with mass spectrometry or NMR spectroscopy to achieve the specific elucidation of plant extract chemical composition [29]. All methods presented here have been modified extensively from their original studies and were further modulated and optimised to promote an analytical approach with the least number of steps and for processing large sample numbers, with results comparable in reproducibility, accuracy and applicability to a number of kits reviewed. The protocols presented result in significant time saving, reducing the total cost for reagents and providing a cost-effective method. As an example, the typical kits available would be limited to usually 200 samples and cost approximately 500 euros for FeRP and polyphenols. The flexibility of the multiparametric protocol also highlights its versatility as different parameters can be handled and the workload can be spread appropriately, or users can focus only on specific methods of their research interest. The feasibility of this protocol is reflected in the Supplementary Excel files, which provide worked-out examples for the fast processing of results generated. All calculations and analyses are easily performed in an automated fashion which allows the user to generate their quantitative results. We foresee the application of this method to the wider research community and a diverse set of research themes such as phytochemistry, toxicology and redox biochemistry.

Conclusions
In summary, the present multiparametric protocol provides a comprehensive and novel approach to assessing the phytochemical and antioxidant properties of plant and food extracts. The methods described here have been explored in the literature extensively with different elaborations and approaches followed as independent assays and yet never as a unified multiparametric approach. The protocol employs eight parameters that cover the three major plant metabolite categories as well as the antioxidant status of plant extracts. While maintaining a high level of accuracy and sensitivity, this approach has the potential to significantly reduce the cost and time related to traditional methods. Furthermore, the protocol is highly adaptable as to allow researchers to focus on specific parameters of interest. The automated calculations and standardised reporting presented in this study improve the reproducibility and reliability of the approach. To our knowledge, this is the first approach of multiple assay protocols which can provide a holistic description of eight parameters covering the three main phytochemical categories of plant metabolites and the antioxidant status of plant extracts.

Reagents Setup
Reagents are listed in order of appearance for each protocol. 1M ammonium acetate: Prepare fresh by dissolving 3.86 g ammonium acetate (MW: 77.08 g/mol) in 25 mL ddH 2 O and after complete dissolution, adjust volume to 50 mL. !CAUTION The solid ammonium acetate will occupy some volume of the solution.
Amount of 100 mM acetic acid pH 5.5: Prepare fresh by dissolving 0.42 g sodium acetate anhydrous (MW: 82.04 g/mol) in ddH 2 O. Adjust the pH to 5.5 and the final volume to 50 mL.
DPPH radical: Prepare fresh by dissolving 25 mg DPPH (MW: 394.32 g/mol) in 10 mL methanol. Dilute this stock with methanol:acetic acid pH 5.5 (8:2) and filter (0.22 µM). Dilute further this stock with methanol:acetic acid pH 5.5 (8:2) to a value of~1.3A at 515 nm. This value is set to be in the linear range of the spectrophotometer/microplate when diluted 1.5× in the assay (mixing 200 µL from the DPPH with 100 µL ddH 2 O). DPPH is a stable radical which absorbs at 515 nm and when scavenged decolourises (Figure 1 ABTS radical cation (ABTS •+ ): Mix the 14 mM ABTS and the 5 mM potassium persulphate reagents 1:1 and incubate in the dark at RT for 12 h before use. ABTS •+ is a stable radical formed by potassium persulphate radical initiation. Dilute further this stock with ddH 2 O to a value of~1.3 A at 734 nm. This value is set to be in the linear range of the spectrophotometer/microplate when diluted 1.5× in the assay (mixing 200 µL from the ABTS radical cation with 100 µL ddH 2 O). ABTS •+ is a stable radical formed by potassium persulphate radical initiation which absorbs at 734 nm and when scavenged decolourises ( Figure 1).
Amount of 100 mM citric acid pH 6: Prepare fresh by dissolving 0.1921 g sodium acetate anhydrous (MW: 192.12 g/mol) in ddH 2 O. Adjust the pH to 6 and the final volume to 100 mL.
Galvinoxyl radical: Prepare fresh by dissolving 50 mg DPPH (MW: 394.32 g/mol) in 10 mL methanol. Dilute this stock with methanol:citric acid pH 6 (9:1) and filter (0.22 µM). Dilute further this stock with methanol:citric acid pH 6 (9:1) to a value of~1.3 A at 435 nm. This value is set to be in the linear range of the spectrophotometer/microplate when diluted 1.5x in the assay [mixing 200 µL from the galvinoxyl radical with 100 µL methanol:citric acid pH 6 (9:1)]. Galvinoxyl radical is a stable radical which absorbs at 435 nm and when scavenged decolourises (Figure 1).

Standard Curves for Assays
Catechin: Prepare a 30 mM stock solution in methanol by dissolving 8.79 mg catechin (MW: 290.27 g/mol) in 1 mL methanol. Dilute the 30 mM stock 300x to 100 µM and follow to 10 µM in ddH 2 O. Prepare a series of dilutions of standards from each stock for 1-10 µM and 10-100 µM, respectively, for the linear standard curves.
Gallic acid: Prepare a 10 mM stock solution by dissolving 17.1 mg gallic acid (MW: 170.12 g/mol) in 10 mL methanol. Dilute the 10 mM stock solution to 100 µM and follow to 10 µM in ddH 2 O. Prepare a series of dilutions of standards from each stock for 1-10 µM and 10-100 µM, respectively, for the linear standard curves.