Quality Properties of Acorn Bee Pollen Extracts

Abstract

Acorn bee pollen is a highly nutritious natural food, but information regarding its quality properties is currently lacking. Therefore, in this study, acorn bee pollens were collected from five regions of South Korea, and then acorn bee pollen extracts (ABPe) were prepared, and their quality properties, as well as their components, were analyzed. For the analysis related to the ABPe’s antioxidant activity, the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical, 2,2-diphenyl-1-picrylhydrazyl radical, and nitrite scavenging activities of JNe (sample from Jeonnam) were at 72.7%, 66.8%, and 60.0%, respectively, which were significantly higher than their respective averages of 69.7%, 56.7%, and 55.3% for the other four samples. Moreover, the inhibition activities of JNe on tyrosinase, α-glucosidase, and α-amylase were 72.5%, 92.6%, and 72.4%, respectively, which were significantly higher than their respective averages of 62.7%, 70.9%, and 61.5% for the other four samples. When considering the ABPe components, the total flavonoid content of JNe was 8.4 mg QE/g, which is higher than the average of 4.6 mg QE/g for the other four samples, and exhibited the highest correlation with the quality properties of acorn bee pollen. Furthermore, among the quercetin glycosides in JNe, rutin revealed a higher correlation with the quality properties than the other flavonoids. Therefore, total flavonoid and rutin content are closely associated with the antioxidant activity and enzyme inhibition of the ABPe. In conclusion, these results are expected to be used for evaluating the quality properties of acorn bee pollen as a healthy functional food ingredient or a natural food additive.

INTRODUCTION

Acorn bee pollen, a type of pollen that is widely produced domestically, is used as a protein source for bee larvae and adults and is rich in carbohydrates, proteins, lipids, vitamins, and minerals (Wu et al., 2021). Furthermore, flavonoids are among the major active substances in bee pollen and serve as important markers. Duan et al. (2019) reported the presence of rutin, isoquercitrin, and quercitrin in bee pollen, and Lee et al. (2022) identified rutin, isoquercitrin, hyperoside, and quercetin in acorn bee pollen. The component composition within the pollen particles varies according to the growing environment and type of source plant, and it is known that there are differences in their chemical properties (Choi and Jeong, 2004). In addition, to confirm the functionality of bee pollen, Bang and Ahn (2019) identified its antioxidant activity and anti-angiogenic effects, Papoti et al. (2018) demonstrated its effect on improving prostatitis, and Li et al. (2009) and Fang et al. (2008) confirmed its immune-enhancing activity. Lee et al. (2022) also conducted research on its anti-diabetic and anti-thrombotic effects.

Bee pollen consists of the intine, exine, and pollen coat. The intine is similar to a typical plant cell wall, and the exine is divided into the inner (nexine) and outer (sexine) layers (Wu et al., 2021). The outer layer of the exine is composed of a chemical compound called sporopollenin, which is physically strong and resistant to degradation by animal digestive enzymes, as well as by acids and alkalis. Bees can only digest nutrients on the pollen surface. Whereas in humans, the bioavailability of pollen is very low, approximately 10%－15%, because of the physical and chemical resilience of the pollen’s exine (Pyeon et al., 2018). In addition, there is little information on the quality index component for evaluating acorn bee pollen, even though in South Korea, pollen was previously classified as a health supplement. However, after the enactment of the Health Functional Food Act in 2002, it was included as a health functional food until 2009. Since 2010, it has been recognized under the standards and specifications for food as processed pollen products (including processed pollen and pollen-containing products) (Ministry of Food and Drug Safety, 2023).

This study aimed to compare the total flavonoid contents and the contents of quercetin and its glycosides in acorn bee pollen extracts (ABPe) collected from five regions in South Korea and evaluate their antioxidant activity and enzyme inhibition as quality properties. Thereafter, correlation analyses between the results of antioxidant activity measurements and enzyme inhibition assays, as well as the total flavonoid content, and the amount of each component (quercetin and quercetin glycosides) in the ABPe, were performed. The findings present the ABPe’s quality properties for evaluating their potential value as a healthy functional food ingredient or natural food additive.

MATERIALS AND METHODS

Materials

The acorn bee pollen used in this study was collected from apiculture farms in Chungcheongbuk-do (CB), Gyeongsangbuk-do (GB), Gyeongsangnam-do (GN), Jeollabuk-do (JB), and Jeollanam-do (JN) in 2024. In addition, rutin hydrate, quercetin, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺), 1-naphthylamine, sulfanilic acid, tyrosinase from mushroom, α-glucosidase from Saccharomyces cerevisiae, p-nitrophenyl-α-D-glucopyranoside, and α-amylase from porcine pancreas were purchased from Sigma-Aldrich Co. Isoquercitrin, hyperoside, and quercitrin were obtained from Biofron Co.; methyl alcohol from Honeywell Burdick & Jackson; Folin-Ciocalteu reagent, L-tyrosine, and L-ascorbic acid from Junsei Chemical Co., Ltd.; 2,2-diphenyl-1-picrylhydrazyl (DPPH radical) from Thermo Fisher Scientific Inc.; acarbose from Tokyo Chemical Industry Co.; potassium acetate, sodium carbonate, and aluminum nitrate nonahydrate from Samchun Chemical Co.; and arbutin, starch, acetic acid, potassium ferricyanide, and trichloroacetic acid were purchased from the Daejung Chemical Co.

Preparation of ABPe

Acorn bee pollens collected in each region were freeze-dried and extracted with methanol using an ultrasonic bath (Bransonic CPX8800H-E, Emerson Electric Co.) at 20°C and 40 kHz. For this process, 60 mL of 99.9% methanol was added to 3 g of acorn bee pollen, followed by ultrasonic extraction performed three times for 1 h each, and then filtered. The filtered extract was collected and concentrated under reduced pressure to obtain the concentrated ABPe.

Total flavonoid content

The total flavonoid content was measured using a modified method previously described by Moreno et al. (2000). Each ABPe (0.5 mL) was diluted to an appropriate concentration, to which 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 4.3 mL of 80% ethanol were added and incubated for 40 min. The supernatant was collected, and the absorbance was measured at 415 nm using a spectrophotometer. The total flavonoid content was calculated from a standard curve prepared using quercetin dihydrate as an anhydrous form, and results were expressed as mg quercetin equivalent per g (mg QE/g) of sample. Individual flavonoids in the ABPe were analyzed by high performance liquid chromatography (HPLC) according to the previously described method by Park et al. (2020) (Table 1).

Table 1.

Operating conditions of high performance liquid chromatography for flavonoid analysis

Parameter	Condition
Instrument	Agilent 1260 infinity II
Column	CAPCELL PAK C18 UG120
Column temperature	30℃
Flow rate	1.0 mL/min
Detector	DAD (260 nm)
Mobile phase	A: 0.1 % (v/v) Trifluoroacetic acid in water
	B: 100 % Acetonitrile
Gradient profile	Time (min)	Solvent A (%)	Solvent B (%)
	0	85	15
	5	85	15
	30	70	30
	40	70	30
	45	85	15
Injection volume	10 µL

ABTS⁺ radical scavenging activity

The ABTS⁺ radical scavenging activity of each ABPe (100 µg/mL) was measured as previously described by Re et al. (1999). The ABTS⁺ solution was allowed to react in the dark for 12 h to generate ABTS⁺ radicals (final concentration of 2.45 mM). The resulting solution was diluted with phosphate-buffered saline (pH 7.4) to a solution with an absorbance of 0.70±0.02 at 734 nm. Then, 990 µL of the ABTS⁺ solution and 10 µL of the ABPe were combined and reacted approximately at 20°C for 6 min. The absorbance was measured at 734 nm using a spectrophotometer (Optizen Alpha, K LAB Co.).

$${\text{ABTS}}^{\text{+}}\text{ radical scavenging activity (\%)=}\left(\text{1-}\frac{{\text{Abs}}_{\text{sample}}-{\text{Abs}}_{\text{blank}}}{{\text{Abs}}_{\text{control}}}\right)\times 100$$

DPPH radical scavenging activity

The DPPH radical scavenging activity of each ABPe (250 µg/mL) was measured using a modified method previously described by Blois (1958). Each 25 µL sample of ABPe was incubated with 975 µL of 0.2 mM DPPH solution in the dark at 37°C for 30 min. The absorbance was then measured at 517 nm using a spectrophotometer.

$$\text{DPPH radical scavenging activity (\%)=}\left(\text{1-}\frac{{\text{Abs}}_{\text{sample}}{\text{-Abs}}_{\text{blank}}}{{\text{Abs}}_{\text{control}}}\right)\text{×100}$$

Nitrite scavenging activity

The nitrite scavenging activity of each ABPe (500 µg/mL) was measured using a modified version of the method previously reported by Nam and Kang (2021). First, 125 µL of 1 mM nitrite solution was combined with 125 µL of ABPe. Then, 0.1 N HCl (pH 1.2) was added to bring the total volume up to 1,250 µL. This solution was allowed to react at 37°C for 1 h. Thereafter, 600 µL of the mixture was mixed thoroughly with 400 µL of 2% acetic acid solution and 200 µL of Griess reagent (a 1:1 mixture of 1% sulfanilic acid and 0.1% 1-naphthylamine prepared in 2% phosphoric acid) and allowed to react at room temperature for 15 min. The absorbance was then measured at 520 nm using a spectrophotometer.

$$\text{Nitrite scavenging activity (\%)=}\left(\text{1-}\frac{{\text{Abs}}_{\text{sample}}{\text{-Abs}}_{\text{blank}}}{{\text{Abs}}_{\text{control}}}\right)\text{×100}$$

α-Amylase and α-glucosidase inhibition activity

The α-amylase inhibition activity of each ABPe (250 µg/mL) was measured using a modified method previously described by Pyo et al. (2020). A mixture of 25 µL of ABPe and 260 µL of porcine pancreatic α-amylase (1.0 U/mL) diluted with 50 mM potassium phosphate buffer (pH 6.8) was pre-incubated at 37°C for 10 min. Then, 260 µL of 0.5% starch solution was added, and the mixture was incubated at 37°C for an additional 10 min. Thereafter, 250 µL of 2 N acetic acid was added to stop the reaction, and 205 µL of 0.75% Lugol solution was added for color development. The absorbance of the developed color was measured at 540 nm using a spectrophotometer.

The α-glucosidase inhibition activity of each ABPe (500 µg/mL) was measured using a method that was adapted from the previously described method by Im and Yoon (2015). Briefly, 20 µL of α-glucosidase from S. cerevisiae (0.2 U/mL) was mixed with 50 µL of ABPe and 330 µL of 50 mM potassium phosphate buffer (pH 6.8) and pre-incubated at 37°C for 15 min. Thereafter, 100 µL of 3 mM p-nitrophenyl-α-D-glucopyranose was added, and the mixture was incubated at 37°C for an additional 20 min. The reaction was stopped by adding 500 µL of 0.1 M NaOH. The absorbance of p-nitrophenol released from the substrate because of the enzyme activity was measured at 405 nm using a microplate reader (BKMPR-1101, Konvision Inc.). Acarbose was used as a positive control.

Tyrosinase inhibition activity

The tyrosinase inhibition activity of each ABPe (500 µg/mL) was measured using a modified method previously described by Horng et al. (2017). In a test tube, 70 µL of 1.5 mM tyrosine and 10 µL of ABPe were sequentially added to 110 µL of a 0.1 M potassium phosphate buffer (pH 6.5). To this solution, 10 µL of mushroom tyrosinase was added, and the mixture was incubated at 37°C for 20 min. The absorbance was then measured at 492 nm using a microplate reader. Arbutin was used as a positive control.

Correlation between quality properties and analytical components

The correlation analysis between the results of the antioxidant activity measurement and enzyme inhibition assays and the amount of each component (quercetin and quercetin glycosides) in the ABPe was performed using GraphPad Prism 9 (GraphPad Software Inc.).

Statistical analysis

All experimental results were performed in triplicate, and the data are presented as the mean±standard deviation using SPSS 29.0 (IBM Corp). Significant differences between variables were analyzed using one-way analysis of variance, followed by Duncan’s multiple range test. A P-value<0.05 was considered statistically significant. Graphs were created using Sigmaplot 10 (SPSS Inc.).

RESULTS

Total flavonoid content

The results of the total flavonoid content of the ABPe are shown in Table 2. The average total flavonoid content was 5.36±1.77 mg QE/g, and the JNe was found to contain significantly higher flavonoid levels (8.4 mg QE/g) compared to the average flavonoid levels for the other four samples (4.6 mg QE/g). A higher flavonoid content generally increases physiological activities, such as antioxidant effects. This may serve as a fundamental resource for verifying the antioxidant activity of natural products (Yi et al., 2017). Therefore, the JNe was expected to exhibit higher antioxidant activity than the other samples.

Table 2.

Total flavonoid contents of the acorn bee pollen extracts

Sample	ABPe
	CBe	GBe	GNe	JBe	JNe
Total flavonoid contents (mg QE/g)	5.38±0.04b	4.69±0.03d	5.18±0.02c	3.14±0.02e	8.40±0.02a

Different letters (a-e) within each row are significantly different at P<0.05 using Duncan’s multiple range test.

ABPe, acorn bee pollen extracts; CBe, ABPe collected in Chungbuk; GBe, ABPe collected in Gyeongbuk; GNe, ABPe collected in Gyeongnam; JBe, ABPe collected in Jeonbuk; JNe, ABPe collected in Jeonnam.

Flavonoid profile of the ABPe

The retention times of quercetin glycosides in the HPLC chromatogram of the ABPe were identical to those of the standard solution, and the ultraviolet (UV) spectrum of the five flavonoid peaks in the sample matched exactly with the UV spectrum of the flavonoid standards (Fig. 1). The results of the analysis of the quercetin and quercetin glycoside contents in the ABPe using HPLC-DAD in this study are presented in Table 3. The JNe, which exhibited higher antioxidant activities and enzyme inhibition activities compared to the average of the other four samples (Fig. 2 and 3), had the highest content of rutin at 286.1±4.1 µg/mL and hyperoside at 86.2±0.9 µg/mL. Moreover, isoquercitrin exhibited the highest content of 1,557.6±16.0 µg/mL in the GBe. In addition, the highest amounts of quercitrin and quercetin were 116.9±3.7 and 195.6±0.1 µg/mL, respectively, which were found in JBe.

Fig. 1.

High performance liquid chromatography chromatogram and ultraviolet spectrum of quercetin glycosides and quercetin in the acorn bee pollen extract. (A) Authentic quercetin glycosides (a, rutin; b, hyperoside; c, isoquercitrin; and d, quercitrin) and quercetin (e, quercetin) (250 µg/mL). (B) The extract from acorn bee pollen collected in Jeonnam.

Table 3.

Amount of quercetin and quercetin glycosides in the acorn bee pollen extracts

Flavonoids (µg/mL)	Quercetin glycoside	Formula	M.W	ABPe
				CBe	GBe	GNe	JBe	JNe
Rutin	Quercetin-3-rhamnosyl glucoside	C27H30O16	610.52	10.3±0.1c	12.5±0.2c	12.2±0.1c	16.6±0.2b	286.1±4.1a
Hyperoside	Quercetin-3-galactoside	C21H20O12	464.38	56.7±0.6c	23.5±0.3d	21.9±0.2e	59.3±0.9b	86.2±0.9a
Isoquercitrin	Quercetin-3-glucoside	C21H20O12	464.38	1,362.9±13.4b	1,557.6±16.0a	1,174.9±7.2c	965.3±9.8d	837.0±7.4e
Quercitrin	Quercetin-3-rhamnoside	C21H20O11	448.38	35.0±1.0b	17.0±1.1c	20.2±0.2c	116.9±3.7a	5.3±0.3d
Quercetin	Quercetin	C15H10O7	302.24	4.4±0.1d	6.1±0.1b	5.1±0.6c	195.6±0.1a	5.7±0.1b

Different letters (a-e) within each row are significantly different at P<0.05 using Duncan’s multiple range test.

M.W, molecular weight; ABPe, acorn bee pollen extracts; CBe, ABPe collected in Chungbuk; GBe, ABPe collected in Gyeongbuk; GNe, ABPe collected in Gyeongnam; JBe, ABPe collected in Jeonbuk; JNe, ABPe collected in Jeonnam.

Fig. 2.

ABTS⁺ (A), DPPH (B) radicals, and nitrite (C) scavenging activities of the ABPe. Bars with different letters (a-e) are significantly different at P<0.05 using Duncan’s multiple range test. The IC₅₀ of ascorbic acid in each of the ABTS⁺, DPPH radicals, and nitrite scavenging activity assays was 1.1, 9.1, and 29.2±0.4 µg/mL, respectively. The concentrations of the ABPe for each assay were 100, 250, and 500 µg/mL, respectively. ABTS⁺, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABPe, acorn bee pollen extracts; CBe, ABPe collected in Chungbuk; GBe, ABPe collected in Gyeongbuk; GNe, ABPe collected in Gyeongnam; JBe, ABPe collected in Jeonbuk; JNe, ABPe collected in Jeonnam.

Fig. 3.

Inhibition activities of the acorn bee pollen extracts on α-amylase (A), α-glucosidase (B), and tyrosinase (C). Bars with different letters (a-e) are significantly different at P<0.05 using Duncan’s multiple range test. The IC₅₀ of acarbose was 3,753.3±7.4 and 192.8±2.5 mg/mL in α-amylase and α-glucosidase inhibition activity tests, respectively. The IC₅₀ of arbutin was 40.7±0.5 mg/mL in tyrosinase inhibition activity. The concentrations of the ABPe used for each assay were 250, 500, and 500 mg/mL. ABPe, acorn bee pollen extracts; CBe, ABPe collected in Chungbuk; GBe, ABPe collected in Gyeongbuk; GNe, ABPe collected in Gyeongnam; JBe, ABPe collected in Jeonbuk; JNe, ABPe collected in Jeonnam.

ABTS⁺, DPPH radicals, and nitrite scavenging activity

The ABTS⁺ radical scavenging activity (with 100 µg/mL ABPe), DPPH radicals scavenging activity (with 250 µg/mL ABPe), and nitrite scavenging activity (with 500 µg/mL ABPe) are shown in Fig. 2. In this study, the average ABTS⁺ scavenging activity of ABPe was 69.7±2.6%. The JNe and GBe samples exhibited the highest ABTS⁺ radical scavenging activity (72.7±0.8% and 72.4±0.4%, respectively), and the JBe had the lowest scavenging activity, at 66.5±0.4%. To compare the relative ABTS⁺ radical scavenging activity, the IC₅₀ of ascorbic acid was 1.1 µg/mL, which was used as the positive control. The ABTS⁺ radical scavenging activity of the JBe was comparable to that of ascorbic acid (approximately 2.0 µg/mL).

The average DPPH radical scavenging activity of ABPe was 56.7±6.2%. The highest DPPH radical scavenging activity of 66.8±0.3% was observed in the JNe, and the lowest was 50.5±0.1% in the JBe. To compare the relative DPPH radical scavenging activity, the IC₅₀ of ascorbic acid was 9.1 µg/mL, which was used as the positive control. The DPPH radical scavenging activity of the JBe was comparable to that of ascorbic acid at approximately 9.0 µg/mL.

The average nitrite scavenging activity was 55.3±3.3%. The highest nitrite scavenging activity, 60.0±0.4%, was observed in the JNe, and the lowest, 50.9±0.6%, was found in the JBe. To compare the relative nitrite scavenging activity, the IC₅₀ of ascorbic acid was 29.2±0.4 µg/mL, which was used as the positive control. The nitrite scavenging activity of the JBe was comparable to that of ascorbic acid at approximately 14.3 µg/mL.

α-Amylase and α-glucosidase inhibition activity

α-Amylase and α-glucosidase are enzymes that convert starches present in food into glucose (Oh et al., 2010). In patients with diabetes, the activity of these enzymes is higher than normal, which results in elevated blood sugar levels. The blood sugar-lowering drug (acarbose) inhibits the breakdown of polysaccharides into monosaccharides in the small intestine, thereby interfering with sugar absorption. The α-amylase and α-glucosidase inhibition activity of ABPe is shown in Fig. 3. The average α-amylase inhibition activity of ABPe (250 µg/mL) was 61.5±9.6%. The GBe and JNe samples exhibited high α-amylase inhibition activities (69.3±1.1% and 72.4±1.5%, respectively), while JBe exhibited the lowest α-amylase inhibition activity (46.6±2.6%). The inhibition activity of acarbose was used as the positive control to compare the relative α-amylase and α-glucosidase inhibition activities. The IC₅₀ of acarbose for α-amylase was 3,753.3±7.4 µg/mL. The α-amylase inhibition activity of the JBe was comparable to that of acarbose (approximately 4,024.5 µg/mL). The average α-glucosidase inhibition activity of ABPe (500 µg/mL) was 70.9±14.5%. The highest α-glucosidase inhibition activity was observed in JNe (92.6±2.1%), while the lowest was found in the JBe sample (48.8±0.5%). The IC₅₀ of acarbose for α-glucosidase was 192.8±2.5 µg/mL. The α-glucosidase inhibition activity of the JBe (500 µg/mL) was 98.8 µg/mL, which was comparable to that of acarbose.

Tyrosinase inhibition activity

Melanin is a protein-based organic pigment that is responsible for human skin and hair color, and its synthesis is regulated by tyrosinases, tyrosinase-related protein (TRP)-1, and TRP-2 (Jung et al., 2009). Tyrosinase is the rate-limiting enzyme for melanin biosynthesis, as it converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into DOPA quinone to synthesize melanin (Jeon et al., 2013). The tyrosinase inhibition activity of ABPe (500 µg/mL) is presented in Fig. 3, with an average inhibition activity of 62.7±5.4%. To compare the relative tyrosinase inhibition activity, the IC₅₀ of arbutin was 40.7±0.5 µg/mL, which was used as a positive control. The JNe sample exhibited the highest tyrosinase inhibition activity (72.5±0.5%), which was similar to the inhibition activity of arbutin (100 µg/mL) that was used as a positive control (75.2±0.4%). The tyrosinase inhibition activity of the JBe (500 µg/mL) was 17.1 µg/mL, which was comparable with that of arbutin.

Correlation between quality properties and analytical components

The antioxidant and enzyme inhibition activities of the ABPe collected from the five regions were measured, and the results of the correlation analysis between the analyzed items, including quercetin and its glycosides, are presented in Table 4. Correlation analysis among the analyzed quality properties revealed that the total flavonoid content was highly correlated with the ABTS⁺ radical scavenging activity (r=0.685), DPPH radical scavenging activity (r=0.893), nitrite scavenging activity (r=0.858), α-amylase inhibition activity (r=0.780), α-glucosidase inhibition activity (r=0.966), and tyrosinase inhibition activity (r=0.903).

Table 4.

Correlation coefficients between the quality properties and components of the acorn bee pollen extract

	Total flavonoid	ABTS+	DPPH	Nitrite savenging	α-Amylase inhibition	α-Glucosidase inhibition	Tyrosinase inhibition
Total flavonoid	1.000	0.685**	0.893***	0.858***	0.780***	0.966***	0.903***
Rutin	0.880***	0.589*	0.846***	0.736**	0.581*	0.762**	0.943***
Hyperoside	0.542*	0.093	0.361	0.321	0.137	0.368	0.832***
Isoquercitrin	—0.364	0.149	—0.511	0.001	0.196	-0.157	-0.550*
Quercitrin	—0.749**	—0.776***	—0.673**	—0.809***	—0.881***	—0.862***	—0.432
Quercetin	—0.647**	—0.646**	—0.515*	—0.703**	—0.799***	—0.787***	—0.320
Quercetin glycosides	—0.091	0.429	—0.341	0.322	0.477	0.096	—0.217
Quercetin+quercetin glycosides	—0.434	0.169	—0.657**	0.015	0.146	—0.289	—0.415

*P<0.05, **P<0.01, and ***P<0.001.

ABTS⁺, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH, 2,2-diphenyl-1-picrylhydrazyl.

DISCUSSION

The measurements of the antioxidant activity revealed that JNe exhibited significantly higher scavenging activity across all categories compared to the average of the other four samples. Moreover, the JNe sample exhibited inhibition activities against α-amylase and α-glucosidase as well as tyrosinase inhibition activity, which was confirmed to be significantly higher than the average values of the other four samples.

Furthermore, the total flavonoid contents in the ABPe were evaluated, and JNe was found to contain significantly higher flavonoid levels compared to the average flavonoid levels of the other four samples. Correlation analysis among the analyzed quality properties showed that the total flavonoid content was highly correlated with the ABTS⁺, DPPH radicals, and nitrite scavenging activity, as well as with α-amylase, α-glucosidase, and tyrosinase inhibition activity.

Flavonoids were selected as the target compounds in acorn bee pollen because flavonoid content has a considerable impact on antioxidant activity. Among the flavonoid components, quercetin and quercetin glycosides were predominant in the ABPe. In particular, rutin showed a strong correlation with the ABPe antioxidant activity and enzyme inhibition. Rutin also revealed a high correlation with ABTS⁺, DPPH, and nitrite scavenging activity, as well as α-amylase, α-glucosidase, and tyrosinase inhibition activity. Quercetin and its glycosides exhibit strong antioxidant activity that helps inhibit oxidative damage to cells and aging caused by the oxidation of DNA, proteins, lipids, and other cellular components (Kim et al., 2022). Their bioavailability varies according to the glycoside type (Russo et al., 2012). Previous studies reported that only quercetin, due to its hydrophobic characteristics, is absorbed in the gastrointestinal tract via passive diffusion. However, subsequent studies confirmed that quercetin is rapidly broken down, has low solubility in water, and is easily oxidized, which leads to low stability (Boots et al., 2008). However, quercetin glycosides, which possess hydrophilic characteristics, exhibit higher antioxidant and antimicrobial effects than the aglycone form of quercetin (Lee and Park, 2022). In addition, they have been reported to exhibit higher absorption rates through de-glycosylation processes and carrier-mediated transport in the intestine (Hollman et al., 1995; Lee, 2018). Therefore, rutin content can be used to evaluate the quality of acorn bee pollen. Rutin from bee pollen collected in Spain showed a high correlation with total free amino acids (r=0.98), thereby suggesting that rutin could indicate bee pollen quality from biological and nutritional perspectives (Serra Bonvehí et al., 2001). This is consistent with this study’s results, which found that measuring the rutin content among flavonoids is an appropriate method for evaluating the quality of acorn bee pollen. In conclusion, the total flavonoid and rutin content are closely associated with the antioxidant and enzyme inhibition activities of the ABPe. Therefore, they are anticipated to be used for evaluating the potential value as well as the quality properties of acorn bee pollen as a healthy functional food ingredient or natural food additives.