Optimisation of Bee Pollen Extraction to Maximise Extractable Antioxidant Constituents

This paper presents the findings of a comprehensive review on common bee pollen processing methods which can impact extraction efficiency and lead to differences in measured total phenolic content (TPC) and radical scavenging activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) data. This hampers the comparative analysis of bee pollen from different floral sources and geographical locations. Based on the review, an in-depth investigation was carried out to identify the most efficient process to maximise the extraction of components for measurement of TPC, DPPH and FRAP antioxidant activity for two bee pollen samples from western Australia (Jarrah and Marri pollen). Optimisation by Design of Experiment with Multilevel Factorial Analysis (Categorical) modelling was performed. The independent variables included pollen pulverisation, the extraction solvent (70% aqueous ethanol, ethanol, methanol and water) and the extraction process (agitation, maceration, reflux and sonication). The data demonstrate that non-pulverised bee pollen extracted with 70% aqueous ethanol using the agitation extraction method constitute the optimal conditions to maximise the extraction of phenolics and antioxidant principles in these bee pollen samples.


Introduction
Flower pollen are the reproductive cells found in the stamen of plants which are transferred to the stigma of another plant via pollinating agents, such as bees, other insects, wind and water [1]. Bee pollen, on the other hand, is made by worker honeybees combining flower pollen, nectar and bee salivary constituents, and it is transferred to beehives in the form of pollen baskets attached to the bees' hind legs [1,2]. Inside the hives, bee pollen is packed into honeycomb cells and covered with a layer of honey and wax to initiate fermentation to generate bee bread, which is the principal source of nutrients for honeybees [2]. Bee pollen, which is also known as apicultural, bee-collected or corbicular pollen, can be harvested for human consumption with the help of pollen traps. These traps are fixed at the entrance of beehives and collect pollen by stripping the pollen baskets from the hind legs of bees on entry to the hives [3].
Bee pollen provides bees with carbohydrates and other necessary nutrients such as proteins, fats, minerals and vitamins [4]. The secondary metabolite profiles of bee pollens vary significantly, reflecting the botanical and geographical origin as well as the climatic conditions, soil type and beekeeper activities [5,6]. The chemical composition of Daoud  It can be assumed that the adopted extraction process for a bee pollen sample, including the sample pre-treatment, extraction method and solvent, impacts the extraction efficiency of antioxidant constituents and consequently on the level of antioxidant activity measured. The objective of this study was to use the Design of Experiment approach to optimise an extraction process for bee pollen with a view to maximising the extraction of antioxidant constituents as measured by the DPPH, FRAP and TPC assays. The independent variables were sample pulverisation, extraction process (agitation, maceration, reflux or sonication) and extraction solvent (methanol, 70% aqueous ethanol, ethanol or water). Two different bee pollen samples from western Australia, Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla) bee pollen, were used as model bee pollen samples.

Chemicals, Reagents and Pollen Samples
The chemicals and reagents were sourced as follows: Folin and Ciocalteu's phenol reagent 2N, (F9252-1L

Extract Preparation
The bee pollen samples had already been dried, processed, and packed commercially, and no further treatment was undertaken prior to their analysis. To obtain pulverised samples (75- 150 µm), the crude pollen grains were milled for 5 min using a commercial grinder (Breville Coffee Grinder Model BCG200). An amount of 0.5 g of pollen samples (crude, and non-pulverised) were separately extracted using ethanol, methanol, deionised water, and 70% ethanol in water, and the following extraction procedures: Agitation. Agitation extraction was performed based on a protocol reported by Aleksieva et al. [72] with minor modifications. The extraction was carried out over 2 h in 7 mL of solvent using a hotplate magnetic stirrer (LLG Uni stirrer 3, John Morris Group) operating at a speed of 1500 rpm, with the temperature set at 40 • C. After 2 h, the solvent was decanted, and was replaced with fresh solvent and the extraction process was repeated two more times for the sample.
Maceration. Maceration extraction was performed based on a protocol reported by Kaškonienė et al. [95] with minor modifications. The bee pollen sample was macerated in 7 mL of extraction solvent at room temperature (25 • C) at a speed of 160 rpm shaking (Memmert Shaker Bath, Model WNB 22) over three days and solvent changes every 24 h.
Reflux. Reflux extraction was performed using an Electromantle reflux set-up and the protocol reported by Cheng et al. [81] with slight modifications. The method employed 21 mL of solvent and an extraction temperature determined by the boiling point of the respective solvent. Extraction was performed once over 2 h for each sample.
Sonication. Sonication extraction was performed following a procedure developed by Yan et al. [79] with slight modifications. The extraction was performed with 21 mL of solvent using a probe sonicator (Sonics Vibra Cell Model VCX130) operating at 130 Watts and 20 kHz. The amplitude was set at 100% and the extraction process was carried out once for 30 min on an ice bath.
Following extraction, the solvent was filtered (Whatman #4 filter paper) and, if the extraction process was repeated, filtrates were combined, and made up to 25 mL with the respective solvent. The resulting solutions were stored at −85 • C until further analysis. Every extraction process was carried out in triplicate for each pollen sample, and the responses combined to determine the mean. Table 2 summarises the independent variables studied along with their corresponding abbreviations.

Determination of Total Phenolic Content
A sugar solution was used as a blank in order to account for the potential sugar matrix interference in the assay. The sugar solution was prepared by diluting 2 g of a sugar stock solution (21.625 g of fructose, 18.125 g of glucose, 1.000 g of maltose, 0.750 g of sucrose and 8.500 g of water) to 5 mL (40%) with deionised water. The solution was stored under refrigeration and used within a week. A dilute Folin-Ciocalteu reagent was prepared by mixing 1 mL of Folin-Ciocalteu reagent with 30 mL deionised water. A 0.75% anhydrous sodium carbonate solution was prepared by mixing 0.1875 g Na 2 CO 3 in 25 mL water. A 2 mg/mL gallic acid stock solution was prepared by dissolving 200 mg of gallic acid in 100 mL deionised water and standards ranging in concentration from 0.18 mg/mL to 0.06 mg/mL were prepared by diluting the stock with water.
The TPC assay was performed based on the methodology described by Liberato et al. [116] with minor modifications. In brief, for the analysis, 200 µL of pollen extract or 100 µL of gallic acid standard spiked with 100 µL of sugar solution were placed in a test tube followed by the addition of 1 mL of the diluted Folin-Ciocalteu reagent. The mixture was allowed to react for 5 min before 800 µL of Na 2 CO 3 was added. The mixture was kept in the dark for 2 h before the absorbance was measured at 760 nm (Carry 60 Bio UV-Vis spectrophotometer) using 100 µL of water spiked with 100 µL of sugar solution along with other TPC reagents as a blank. The analysis was carried out in triplicate and the mean results for each sample were obtained. The antioxidant activity was expressed as mg of gallic acid equivalent (GAE) per g of pollen.

Determination of Antioxidant Activity using Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP reagent was prepared by mixing in proportions of 1:1:10 (v/v/v) 10 mM TPTZ (0.31 g dissolved in 100 mL of 40 mM HCl), 20 mM FeCl 3 ·6H 2 O (0.5406 g dissolved in 100 mL of deionised water) and 300 mM acetate buffer pH 3.6 (3.1 g C 2 H 3 NaO 2 , 16.00 mL of glacial acetic acid dissolved in 1000 mL of deionised water). The reagent was freshly prepared and incubated at 37 • C prior to each test. For the standard curve, a 2 mM stock solution of FeSO 4 ·7H 2 O was prepared by dissolving 55.6 mg of FeSO 4 ·7H 2 O in 100 mL of deionised water. Standards ranging in concentration from 1200 µM to 200 µM were prepared prior to each experiment, stored on ice, and used within 2 h. The standard at 600 µM was used as a positive control in each experiment.
The FRAP assay, which is based on the reduction of ferric 2,4,6-tris(2-pyridyl)-1,3,5triazine [Fe(III)-TPTZ] to ferrous complex at low pH followed by a spectrophotometric analysis, was performed according to the protocol described by Almeida et al. [60] with minor modifications. In brief, 20 µL of pollen extract or standards were mixed with 180 µL of FRAP reagent in a 96-well microplate, incubated at 37 • C for 30 min and the absorbance of the reaction mixture was determined at 620 nm (BMG Labtech POLARstar Optima Microplate Reader). The mean of triplicate analysis results was calculated and the FRAP activity was determined on the interpolation of the standard curve and expressed as µmol Fe 2+ equivalent (Fe +2 E)/g FW of pollen.

Determination of Antioxidant Activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
This colorimetric assay utilises 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, which are purple in colour; the colour decays in the presence of antioxidant agents, seen in a change in absorbance at 517 nm. The DPPH reaction mixture was made up of 0.130 mM DPPH reagent (5.1262 mg of DPPH in 100 mL of methanol), 100 mM NaC 2 H 3 O 2 buffer pH 5.5 (7.355 g of NaC 2 H 3 O 2 and 0.621 g of HC 2 H 3 O 2 ) and bee pollen extract. Trolox in a concentration range of 600-100 µM (aqueous, pH adjusted to pH 7.0 to completely solubilize in water) was used as the standard, with the 400 µM standard acting as a positive control throughout all tests.
The DPPH assay adopted in this experiment is based on the protocol described by Karabagias et al. [117] with minor modifications. In brief, 10 µL of bee pollen extract or Trolox standards were placed in a 96-well microplate, followed by the addition of 100 µL of NaC 2 H 3 O 2 buffer and 190 µL of 0.130 mM methanolic DPPH solution. The reaction mixture was kept in the dark for 120 min before the absorbance was measured at 520 nm using the microplate reader. The mean radical scavenging activity of triplicate samples was expressed as the Trolox equivalent (TE), calculated by a linear regression analysis, and then expressed as µmol Trolox equivalent per g of pollen.

Statistical Analysis
Statistical analysis of the data was carried out using Design Expert 12 (StatEase Inc., Minneapolis, MN, USA) and two-way t-test or one-way ANOVA was analysed using Graphpad Prism 9 (GraphPad Software, San Diego, CA, USA). Pareto plots of regression coefficients of the independent variables were generated using Microsoft Excel. The signal to noise ratio was set at 5 times the standard deviation of observations for each response (n = 3). The model was developed based on the regression analysis of the statistical significance of variables, and the model coefficient was significant when the F value was larger than the critical F value (p < 0.05). The relative influence of factors was identified by comparing the magnitude of regression coefficients. Correlations between responses were established using Spearman regression analysis. When significant interactions between factors were identified, a two-way t-test or a one-way analysis of variance with Tukey's post hoc comparison was used to identify differences between the groups, and the statistical significance was set at p > 0.05.

Results
A thorough literature review of 101 published articles on the most widely used conditions for extracting antioxidant principles from bee pollen (Table 1) indicated a lack of a consistent approach. Researchers used a variety of antioxidant assays and analysis standards, and also, the manner by which the results were expressed varied greatly, making it difficult to conduct comparative analyses of findings across research groups. Furthermore, there appears to be no agreed protocol to guide the extraction process itself, which is required to establish baseline data for bee pollen of Australian origin in order to compare their antioxidant activity with other bee pollen samples. The gaps in information form the basis for this study, which aimed to optimise the extraction conditions for bee pollen collected in western Australia to enable the maximum extraction of antioxidant constituents as measured by the TPC, DPPH and FRAP assays. The Design of Experiment approach examined three independent variables: sample pulverisation, extraction solvent, and the extraction process. The two pollen types provided sample diversity to enable the development of a generalised extraction protocol that may be adopted for all types of bee pollen collected in western Australian and beyond.

Analysis of the Optimisation Process
A multilevel factorial design was implemented using Design Expert 12 (StatEase Inc., Minneapolis, MN, USA), with sample pulverisation, extraction solvent, and the extraction process as independent variables. The conditions selected for each variable were based on the popularity of use, as reflected in the literature review ( Table 1). The responses (dependent variables) measured were the TPC, DPPH, and FRAP antioxidant activities. As summarised in Table 3 (the full data set is available as Supplementary Material, Table S1), the extraction variables for the multilevel factorial design (categorical) were selected at two levels for pulverisation (A, crude = −, pulverised = +), four levels for extraction solvent (B, 70% ethanol: 30% = E70:30, ethanol = EtOH, water = H 2 O, and methanol = MtOH), and four levels for the extraction process (C, agitation = A, maceration = M, reflux = R, and sonication = S). The total runs consisted of 32 experimental points, and each point was triplicated. The sequence of the experiments was randomised, where the random numbers were generated by the Design Expert 12 software.
Pulverisation (Variable A) did not show any significant effect on the TPC, DPPH and FRAP data for the bee pollen (p value > 0.05) and was therefore removed from further analysis. Both solvent type (Variable B) and the extraction process (Variable C) were found to have significant impacts on the TPC, DPPH and FRAP antioxidant activity of bee pollen (p value < 0.05). A significant interaction between the solvent type and extraction process (Variables B and C) was observed for the TPC and FRAP responses (p value < 0.05), whereas the interaction of these variables did not influence the DPPH antioxidant activity.
However, the larger coefficients obtained for extraction solvent relative to those for the extraction process indicate that the selection of solvent has a dominant effect on all three responses based on the tabulated regression coefficients (see Table 4), also seen in Pareto charts (Figures 1-3). The relationships between the independent variables and TPC, DPPH and FRAP assays are further illustrated in three-dimensional graphs (Figures 4-6). Figure 4 shows the results for the TPC assay of the bee pollen samples. The highest responses were observed when a solvent of 70% ethanol (E70:30) was coupled with reflux (R) or agitation (A) as the extraction process (p value < 0.05). These conditions may therefore represent optimal parameters for the extraction of the bee pollen samples for the TPC assay. For the DPPH antioxidant activity (Figure 5), the extraction solvent of 70% ethanol (E70:30) coupled with agitation (A) were the best extracting conditions for the bee pollen samples (p value < 0.001). Figure 6 shows that the extraction solvent of 70% ethanol (E70:30) coupled with maceration (M) produced the highest FRAP activity (p value < 0.05), and may therefore be considered as the combination of solvent type and the extraction process of choice for the FRAP assay of the bee pollen samples.      The relationships between the independent variables and TPC, DPPH and FRAP assays are further illustrated in three-dimensional graphs (Figures 4-6). Figure 4 shows the results for the TPC assay of the bee pollen samples. The highest responses were observed when a solvent of 70% ethanol (E70:30) was coupled with reflux (R) or agitation (A) as the extraction process (p value < 0.05). These conditions may therefore represent optimal parameters for the extraction of the bee pollen samples for the TPC assay. For the DPPH antioxidant activity (Figure 5), the extraction solvent of 70% ethanol (E70:30) coupled with agitation (A) were the best extracting conditions for the bee pollen samples (p value < 0.001). Figure 6 shows that the extraction solvent of 70% ethanol (E70:30) coupled with maceration (M) produced the highest FRAP activity (p value < 0.05), and may therefore be considered as the combination of solvent type and the extraction process of choice for the FRAP assay of the bee pollen samples.

Correlation of TPC, DPPH and FRAP Antioxidant Activity
Bee pollen has been reported to contain many types of polyphenols [99] which are strongly correlated to the antioxidant activity of bee pollen [9]. In this study, a strong positive correlation was observed between the TPC and DPPH data, TPC and FRAP data as well as the DPPH and FRAP data, with correlation values of ρ = 0.6925 (p < 0.001), ρ = 0.7295 (p < 0.001) and ρ = 0.6520 (p < 0.001), respectively. Thus, the presence of polyphenols, captured in the pollen's TPC, appears to positively influence its antioxidant capacity expressed in the DPPH and FRAP assays. However, it needs to be acknowledged that there are other pollen constituents, such as carotenoids, that could also influence the antioxidant capacity [82,106].

Choosing Optimum Conditions
The optimisation of the extraction process to maximise the three dependent variables was determined by employing a new variable desirability, which represents all responses simultaneously. Desirability is an objective function that is determined by ranked responses and its value ranges from zero to one, with one being most desirable. When there are several responses, the individual goals are combined to generate one desirability function, which was automated by Design Expert software 12 (StatEase, Inc. Minneapolis, MN, USA). The numerical optimisation based on the goal of the study finds a point that maximises the desirability function. The criteria adopted to determine the desirability function for this study are to maximise all responses. Figures 7-9 provide predicted TPC, DPPH and FRAP values obtained for the three optimum conditions identified for the extraction of bee pollen samples.

Correlation of TPC, DPPH and FRAP Antioxidant Activity
Bee pollen has been reported to contain many types of polyphenols [99] which are strongly correlated to the antioxidant activity of bee pollen [9]. In this study, a strong positive correlation was observed between the TPC and DPPH data, TPC and FRAP data as well as the DPPH and FRAP data, with correlation values of ρ = 0.6925 (p < 0.001), ρ = 0.7295 (p < 0.001) and ρ = 0.6520 (p < 0.001), respectively. Thus, the presence of polyphenols, captured in the pollen's TPC, appears to positively influence its antioxidant capacity expressed in the DPPH and FRAP assays. However, it needs to be acknowledged that there are other pollen constituents, such as carotenoids, that could also influence the antioxidant capacity [82,106].

Choosing Optimum Conditions
The optimisation of the extraction process to maximise the three dependent variables was determined by employing a new variable desirability, which represents all responses simultaneously. Desirability is an objective function that is determined by ranked responses and its value ranges from zero to one, with one being most desirable. When there are several responses, the individual goals are combined to generate one desirability function, which was automated by Design Expert software 12 (StatEase, Inc. Minneapolis, MN, USA). The numerical optimisation based on the goal of the study finds a point that maximises the desirability function. The criteria adopted to determine the desirability function for this study are to maximise all responses.  Figure 4 for TPC data, Figure 5 for DPPH data, and Figure 6 for FRAP data (desirability = 0.925).
Based on the model, the crude/non-pulverised pollen sample, extracted with 70% ethanol:30% water (E70:30) by agitation (A) as the extraction process produced the highest desirability (0.92) (Figure 7), followed by the crude/non-pulverised pollen sample, extracted with 70% ethanol:30% water (E70:30) by maceration (M) (desirability = 0.893) (Figure 8), and finally, the non-pulverised pollen sample, extracted with 70% ethanol:30% water (E70:30) by reflux (R) (desirability = 0.883) (Figure 9). These three conditions can therefore be considered to represent the optimal combination of sample pre-processing treatment, solvent type and extraction process to yield the maximum extraction of constituents from the bee pollen samples for the TPC, DPPH and FRAP assays. The bee pollen extracts prepared using the crude/non-pulverised pollen sample, extracted with 70% ethanol:30% water (E70:30) by agitation (A) as the extraction process were found to have an average total phenolic content of 20.86 mg GAE/g, DPPH antioxidant activity of 320.11 µmol TE/g and 342.28 µmol Fe +2 E/g FRAP activities, respectively. These values are very close to the predicted values of 20.83 mg GAE/g, 324.52 µmol TE/g and 351.78 µmol Fe +2 E/g, respectively, demonstrating the good fit of the chosen model.

Discussion
Commonly, the initial stage in studying the chemical composition and/or bioactivity of natural products, including bee pollen, includes a pre-extraction step, in which the material undergoes drying in order to preserve the biomolecules present in the sample [21]. This is often followed by grinding the dried material using a mortar and pestle, electric blender or various mills to decrease the particle size to enhance surface contact with the extraction solvent [21]. Particles that are too fine will, however, adsorb onto filters and impede filtration [118]. In this study, the effect of the pulverisation of bee pollen samples on its total phenolic content and associated antioxidant activity as captured by DPPH and FRAP assays was analysed. On the basis of these findings, the pulverisation process can be omitted from the extraction protocol.
The selection of solvent is crucial for solvent extraction, with selectivity for the target compounds, the target compound's solubility as well as cost and safety to be considered. Based on the law of similarity and intermiscibility, solvents with a polarity value near the polarity of the solute are likely to perform better [118]. Water, along with a range of alcoholic and organic solvents such as methanol, ethanol, acetonitrile, acetone, hexane and diethyl ether are commonly utilised in the extraction of bioactive compounds [20]. The current study aimed to analyse the impact of different solvents, including water, methanol (M), ethanol (E) and the combination of ethanol and water at a ratio of 70:30 (v/v) (E70:30) on the total phenolic content as well as DPPH and FRAP antioxidant activity of pollen samples collected from western Australia. The findings of the study demonstrate that the extraction solvent had the strongest influence on the responses of the dependent variables (p <0.05). Among the solvents tested, extracts prepared with 70% ethanol:30% water (v/v) demonstrated the highest activity across all performed assays.
Based on the interactions observed in this study, it appears that TPC values are dependent on solvent type and the extraction process, and it can be concluded that TPC can be maximised when the pollen extraction is carried out either using 70% ethanol:30% water coupled with agitation or reflux. DPPH antioxidant activity is maximised following agitation as an extraction process in any of the investigated solvents, whereas FRAP antioxidant activity is highest when pollen is extracted with maceration coupled with 70% ethanol:30% water. Bee pollen contains 13-55% of carbohydrates [6][7][8] and it can be assumed that some can be carried over into the extract by polar solvents. Consequently, the prolonged heating of bee pollen at a high temperature might lead to the formation of Maillard products from these carbohydrates during processing [3]. Therefore, an extraction method that does not expose the pollen sample to prolonged high temperatures can be considered favourable. Maceration is an easy process in extracting antioxidant principles; however, it takes 72 h to complete, as compared to agitation, which only requires 6 h. Reflux is a very promising method of extraction, as it only requires 2 h to perform; however, depending on the chosen solvent, it might require high temperature in order to operate. Thus, in this light, agitation can be recommended as the optimal extraction process to maximise antioxidant principles obtained from bee pollen samples.
The term 'phenolic' or 'polyphenol' is chemically defined as a substance that possesses an aromatic ring bearing one or more hydroxyl substituents, including functional derivatives such as esters, methyl esters and glycosides. These bioactive compounds are extensively found across the plant kingdom and are closely linked with the sensory and nutritional quality of fresh and processed plant foods, including bee products such as honey and bee pollen [9]. Keskin and Özkök reported various multifloral bee pollen samples from the Czech Republic to have a total phenolic content ranging from 15.2 mg to 22.73 mg GAE/g pollen [38], whereas Mayda, Özkök et al. reported TPC values of 26.69 ± 0.595 and 43.42 ± 0.779 4 mg GAE/g pollen [58]. TPC for multifloral bee pollen from Morocco was reported as 45.96 ± 0.51 mg GAE/g pollen [63] and samples obtained from Hungary had TPC values ranging from 9.15 ± 0.12 to 13.63 ± 0.11 mg GAE/g pollen [72]. TPC values (mg GAE/g pollen) for the two western Australian monofloral pollen samples investigated as part of this study following the optimised extraction protocol by using non-pulverised samples extracted with 70% ethanol:30% via agitation was 20.86 mg GAE/g. With this, the TPC value of the western Australian pollens are broadly within typical ranges found for a range of bee pollen samples from a wide geographical spread. It needs to be highlighted, though, that comparisons between data generated in different studies need to be treated with caution, as the chosen extraction condition (solvent and extraction method) will impact on the obtained TPC data. Furthermore, the method used in the analysis of TPC in this study utilised a Folin-Ciocalteu method in slightly basic medium (0.75% sodium carbonate) as compared to most researchers that used 7.5% sodium carbonate. This amendment to the common assay protocol was found to be necessary, since reducing sugars present in alcoholic and aqueous pollen extracts can also be reduced by the reagent, and thus, lead to an overestimation of TPC. [119] The optimised extraction protocol was also used to assess the antioxidant activity of the two western Australian pollen samples investigated in this study. Despite relatively high correlations between FRAP, DPPH and TPC values found in this study, it can be argued that in vitro antioxidant capacity should not be determined by means of a single antioxidant test model because of the diverse types of antioxidant that might be present in the sample as well as the intricacy of the natural product matrix and the variety of free radical reaction mechanisms involved in oxidation. Complementary antioxidant assays might, therefore, produce richer data [78]. Thus, in this study, the antioxidant potential of bee pollen extracts was determined by means of two different radical scavenging assays, namely DPPH and FRAP.
Using the DPPH assay, Rocchetti and Castiglioni reported Magnolia and Lamium bee pollen from Italy to have antioxidant activities of 11.9 and 134.7 µmol TE/g pollen, respectively [78]. Mărghitaş et al. reported DPPH antioxidant activities ranging from 135 to 2814 µmol TE/g for various monofloral pollens from Romania [28] and Saral et al. found DPPH scavenging activities between 13.87 and 15.04 mg TE/g for multifloral pollen from Turkey [74]. DPPH data generated in this study for the western Australian pollen samples were 320.11 µmol (equivalent to 80.12 mg) TE/g following the optimised extraction protocol using non-pulverised pollen extracted with E70:30 by agitation. These findings are within the range of values reported by others.
Using the FRAP assay, Zuluaga-Domínguez et al. reported 87.2 ± 15.6 µmol Trolox/g for multifloral pollen from Colombia [37], whereas Saral et al. found a FRAP activity ranging from 8.69 to 84.89 µmol Fe 2+ E/g for multifloral bee pollen from Turkey [74]. In this study, following the optimised extraction protocol by using non-pulverised pollen extracted with E70:30 by agitation, FRAP antioxidant activity was found to be 342.28 µmol Fe +2 E/g, which is higher than the values reported by Saral et al. However, the comparison of FRAP values appears even more difficult, not only because the results are dependent on the chosen extraction conditions but also because the studies use different reference standards (Trolox or Fe 2+ ) to express their results.

Conclusions
Based on a thorough review of the extant literature, a number of common bee pollen processing steps, solvents and extraction methods were identified, which can all impact on extraction efficiency and thus result in different TPC, DPPH and FRAP values. The study reports on an in-depth investigation into the optimisation of the most popular extraction conditions for maximum TPC, DPPH and FRAP antioxidant activity using two bee pollen samples from western Australia. The effects of pulverisation, the chosen solvent (70% aqueous ethanol, ethanol, methanol and water) as well as the adopted extraction process (agitation, maceration, reflux and sonication) were determined in order to optimise the extraction parameters. The study's findings demonstrate that non-pulverised pollen extracted with 70% aqueous ethanol coupled with agitation as the extraction method constitutes the best conditions in order to maximise the extraction of phenolics and antioxidant principles in these bee pollen samples.