The evaluation of L‐arginine solution as a solvent for propolis extraction: The phenolic profile, antioxidant, antibacterial activity, and in vitro bioaccessibility

Abstract

Ethanol has been widely used for the extraction of propolis. Due to its certain disadvantages, there has been an ongoing search to find alternative non‐ethanolic extraction solvents. This study aimed to compare the phenolics, antioxidant, and antibacterial activity of propolis extracts prepared with 70% ethanol (EWE), propylene glycol (PGE), and L‐arginine solution (BE). All extracts were subjected to an in vitro simulated digestion procedure, and the phenolic profile of non‐digested and digested samples was determined by using LC–MS/MS. Additionally, the change in total phenolic (TPC), total flavonoid content (TFC), and antioxidant capacities were determined at each digestion phase. TPC and TFC of non‐digested propolis extracts had similar values, although BE showed higher antioxidant capacity (p < .05). The amount of TPC reached or transformed at the intestinal stage was higher for BE and PG compared to EWE. BE also provided the highest antioxidant capacity assay in digested samples. The most common phenolics were pinocembrin, pinobanskin, galangin, and CAPE in non‐digested extracts. However, their concentration was drastically reduced by digestion, and their recovery (R%) ranged from 0% to 9.38% of the initial amount detected in the non‐digested extracts. Chrysin was the most bioaccessible flavonoid in all extracts. Among phenolic acids, the highest R% was determined for trans‐cinnamic acid (22.14%) from BE. All extracts showed in vitro inhibitory activity against Escherichia coli and Staphylococcus aureus. This study suggests that an L‐arginine solution could be used as an alternative solvent to ethanol and propylene glycol for propolis extraction.

This study aimed to compare the phenolics, antioxidant, and antibacterial activity of propolis extracts prepared with propylene glycol (PGE), 70% ethanol (EWE), and L‐arginine solution (BE). All extracts were subjected to an in vitro simulated digestion procedure, and the phenolic profile of non‐digested and digested samples was determined by using LC–MS/MS. Additionally, the change in total phenolic (TPC), total flavonoid content (TFC), and antioxidant capacities were determined at each digestion phase.

1. INTRODUCTION

Propolis is a bee product that possesses a wide range of biological and pharmacological properties, including antioxidant, antimicrobial, antiparasitic activities, anti‐inflammatory effects, immunomodulatory actions, as well as antitumor, anticancer, antiulcer, hepatoprotective, cardioprotective, and neuroprotective properties. (de L. Paula et al., ²⁰²¹; Dos Santos et al., ²⁰²²; Hafshejani et al., ²⁰²³; Sagdic et al., ²⁰⁰⁷).

Its chemical structure is very complicated, with more than 300 different compounds (Ahangari et al., 2018), including plant resins (~50%), waxes (~30%), aromatic and essential oils (~10%), pollens (~5%), and other substances including amino acids, minerals, sugars, phenols, and terpenes (~5%) (Bogdanov, 2017; de L. Paula et al., ²⁰²²; Ding et al., ²⁰²¹). The biological effects of propolis are mainly attributed to phenolic compounds such as phenolic acids, flavonoids, tannins, stilbenes, coumarins, and quinones (Ahangari et al., 2018). Caffeic acid phenyl ester (CAPE), chrysin, galangin, pinobanksin, and pinocembrin were identified as the major active constituents of propolis (Necip et al., 2023; Yen et al., ²⁰¹⁷).

Due to its resinous structure, propolis has very low solubility in water; therefore, it needs to undergo an extraction process before it can be digested. The choice of extraction solvent is crucial, as it directly influences the quantity and properties of bioactive compounds in the final product, propolis extract (PE). Maceration with 70%–80% aqueous ethanol has been traditionally used for propolis extraction (Bankova et al., 2021; Santos et al., ²⁰²¹). However, the presence of alcohol in the extract limits its application in cosmetic, pharmaceutical, nutraceutical, and pediatric products and leads to the exploration of non‐alcoholic extraction solvents. There are different PEs sold on the market as food supplements produced by alternative natural (e.g., water and olive oil) and alcohol derivative extraction solvents (e.g., propylene glycol and glycerin) (Sagdic et al., 2020).

The selectivity of natural extraction solvents may not be proper for the desired phenolic composition in the PE (Kubiliene et al., 2018; Sagdic et al., ²⁰²⁰). Even though glycerin does not have an ADI value, it has been observed that its potential to dissolve propolis is considerably less than ethanol The adverse effects of propylene glycol consumption have been reported previously (Atayoglu et al., 2023; Bakkaloglu & Arici, ²⁰¹⁹; Sagdic et al., ²⁰²⁰).

Due to the need for an alcohol‐free extraction approach with natural solvents, Balparmak R&D Center applied an alkaline aqueous extraction medium containing L‐arginine, a semi‐essential amino acid, to prepare PE. L‐arginine is used by the human body for important functions like protein synthesis, the urea cycle, tissue repair, and the immune system (Pahlavani et al., 2017). The external intake of L‐arginine was also associated with a healthy vascular system (Gad, 2010).

Since in vitro digestion assays are easy, inexpensive, and repeatable methods of evaluating the stability of various dietary ingredients against digestive fluids, they have been created as an alternative to in vivo studies. The bioaccessibility of phenolics in the digestive tract is considered a critical factor in interpreting their health benefits. Over the years, there have been a few studies investigating the bioaccessibility of PE phenolics mainly focusing on ethanolic extracts (Ozdal et al., 2019; Yen et al., ²⁰¹⁷). The bioaccessibility of phenolics could be influenced by a variety of factors such as the other components in the matrix, the method and the solvent of extraction, and the stage of digestion (Tan et al., 2022). In this study, the phenolic composition, antioxidant capacity, antibacterial activity, and the impact of in vitro gastrointestinal digestion on PE obtained by L‐arginine solution were evaluated and compared to the PE prepared by using ethanol (70%) and mono‐propylene glycol.

2. MATERIALS AND METHODS

2.1. Chemicals

Mono propylene glycol, 6‐hydroxy‐2,5,7,8‐tetramethyl chroman‐2carboxylic acid (Trolox, 97%), 2,2′‐azinobis (3ethylbenzthiazoline‐6‐sulfonic acid) (ABTS, >98%), 2,4,6‐tris(2‐pyridyl)‐s‐triazine (TPTZ), neocuproine, amylase (A1031), pepsin (P7012), pancreatin (P7545), and bile (B8631) were purchased from Sigma‐Aldrich Ltd (Steinheim, Germany). Methanol Ethyl Acetate NaCl Muller Hinton Agar Muller Hinton Broth (Merck 110293), Nutrient Broth (Merck 105443), Sodium Chloride (Merck 106404), Formic acid (Merck 100264) Copper (II) chloride, ammonium acetate, and L‐Arginine were purchased from Merck KGaA (Darmstadt, Germany).

Phenolic acids and flavonoids used as standards including 3,4‐dimethoxy cinnamic acid, apigenin, chrysin, caffeic acid, caffeic acid phenethyl ester (CAPE), ferulic acid, galangin, gallic acid, genistein, quercetin, kaempferol, isorhamnetin, luteolin, p‐coumaric acid, naringenin, pinobanksin, pinocembrin, and trans‐cinnamic acid were purchased from Sigma‐Aldrich (Steinheim, Germany).

2.2. Preparation of propolis extracts (PE)

5 kg of raw propolis were obtained from a local beekeeper in Yalova, Turkey, in February 2020. Impurities were removed by sorting out the propolis sample, which was then frozen at −18°C for 24 h. Frozen propolis was immediately ground into a powder with a mechanical grinder and stored at −18°C until further processing. The propolis extracts (PE) were prepared by using different solvents: 70% ethanol (EWE), mono propylene glycol (PGE), and L‐arginine solution (BE) (pH = 9.0 ± 0.5). The propolis to solvent ratio was kept constant at 1:5 (w:v) for all solvents, and the extraction continued for 24 h in the dark at room temperature. The wax collected at the top was filtered (Whatman no.1), after storing the suspensions at 4°C for 24 h, and the residue was extracted two more times under the same condition. All supernatants were combined, filtered (0.45 μm), filled up to the same volume with their solvents, and stored at 4°C until analysis.

2.3. Total phenolic content (TPC) and total flavonoid content (TFC) analysis

The total phenolic content (TPC) of samples was evaluated using Folin–Ciocalteu (FC) method (Singleton et al., 1999). 25 μL of diluted PE were mixed with FC reagent, Na₂CO₃ solution (20%), and ultra‐pure water and incubated for 1 h at room temperature, and the absorbance was measured against the blank at 760 nm (Shimadzu 150 UV‐1800 spectrophotometer, Kyoto, Japan). The results were given as mg gallic acid equivalent (GAE)/g raw propolis with a linear range of 0.2–2 mg/mL (R ² = .999). The total flavonoid content (TFC) of samples was evaluated by the method of Andrade et al. (2017). The diluted PE was mixed with 0.1 mL of AlCl₃ and CH₃COONa solution, the mixture was incubated at room temperature for 30 min, and the absorbance was read against blank at 415 nm. The results were given as mg quercetin (QE)/g raw propolis with a linear range of 0.05–0.5 mg/mL (R ² = .999).

2.4. Antioxidant capacity assays

To assess the antioxidant capacity, three different assays were conducted with minor modifications (Karadag et al., 2020). The ABTS radical scavenging activity of PEs was given as μmol TE/g raw propolis with a linear range of 40–799 μM (R ² = .992). The ferric reducing antioxidant power (FRAP) of samples was given as μmol TE /g raw propolis with a linear range of 10–799 μM (R ² = .992). The copper‐reducing antioxidant capacity (CUPRAC) of PE samples was expressed as μmol Trolox Equivalent (TE)/ g raw propolis with a linear range of 100–1998 μM (R ² = .993).

2.5. Simulated in‐vitro gastrointestinal (GI) digestion

In vitro GI digestion was performed according to Minekus et al. (2014) and Lucas‐González et al. (2018) with minor modifications. The change in antioxidant capacity and the amount of phenolics from the EWE, PGE, and BE were determined by LC/MS–MS at each stage of digestion (oral, gastric, and intestinal). 5 mL of PE were mixed with simulated salivary fluid (pH 7.0) and α‐amylase solution (75 U/mL), CaCl₂ (0.75 mM), and vortexed for 2 min at 37°C. The oral bolus was mixed with simulated gastric fluid (SGF) CaCl₂ (0.075 mM), and pepsin solution (2000 U/mL) at a ratio of 1:1 (v:v), pH was adjusted to 3 using HCl. The samples were incubated in a shaking water bath at 37°C for 2 h. Afterward, the gastric chyme was mixed with simulated intestinal fluid, CaCl₂ (0.3 mM), pancreatin solution (100 U/mL), fresh bile (10 mM) at a ratio of 1:1 (v:v), and pH was adjusted to 7 using NaOH. The segments of dialysis bags (MWCO 12,000 Da) filled with sufficient Na₂CO₃ solution (0.1 M) were placed in the SIF medium, and the beakers were kept in a shaking water bath for 2 h at 37°C. The substances that entered the serum (IN) made up the contents of the dialysis bags, while the stuff in the GI tract (OUT) made up the contents outside the bags. The supernatants of the samples taken for the oral, gastric, and intestinal phases were collected after centrifugation at 2480 g for 10 min at 4°C. Analysis was also performed on a blank test tube that included all simulated digestive fluids but no samples. All supernatants were lyophilized and kept at −20°C until further analysis. All procedures were done in triplicate.

The effect of in vitro digestion on the phenolics and antioxidant capacity of PE was evaluated by the Recovery percentage (R%) (Lucas‐González et al., 2018) and Bioaccessibility index (BI%) (Minekus et al., 2014) as shown in Equations (1) and (2)

$$\mathrm{R}\%=\frac{\mathit{\text{IN}}}{\text{Undigested}}\times 100$$ (1)

$$\mathrm{BI}\%=\frac{\text{Intestinal phase}\left(\text{IN}+\text{OUT}\right)}{\text{Undigested}}\times 100.$$ (2)

2.6. LC–MS/MS analysis

The phenolic profiles of undigested and digested samples was determined according to the method of Guzelmeric et al. (2023) by using Waters® Xevo (Waters Corporation, Milford, MA, USA) LC–MS/MS system equipped with a binary pump, a temperature‐controlled column compartment, an autosampler, and a triple‐quadrupole (TQ) MS/MS mass analyzer with ESI ion source. An analytical Cortecs T3 (Waters Corporation Milford, USA) column (2.1 × 150 mm, 1.6 μm) was used for the separation. The mobile phase consisted of two solvents: (A) the acidified water (0.01% acetic acid, v:v), and (B) acetonitrile: methanol (80:20, v:v). The gradient flow rate was 0.25 μL/min. The gradient elution conditions were: 0–1.30 min, 2% B; 1.30–35 min, 2%–55% B; 35–37 min, 55%–95% B; 37–37.01 min, 95%–2% B, and 37.01–40 min, 2% B. The column temperature was 30°C and the injection volume was 5 μL. The eluted samples from LC were analyzed using the ESI‐MS/MS system in multiple reaction monitoring (MRM) mode. The parameters of ESI‐MS/MS were adjusted as follows; the ion source and desolvation temperature were set at 150 and 450°C, respectively. The capillary voltage was 2 kV. Desolvation and cone gas flows were 850 and 50 L/h, respectively. LC–MS/MS data was processed using Waters® Mass‐Lynx software at Target Lynx Program (Waters®). Each phenolic compound was individually injected into the LC–MS/MS system for the determination of retention times, ionization mode, precursor ion energy, capillary voltage, and fragment ions, and optimal MRM transition parameters, such as cone voltage, collision. A stock of standard solutions was prepared in methanol (2 mg/mL), and the calibration curve was prepared at 50, 100, 250, 500, and 1000 μg/L of concentrations. Filtered samples were diluted with acidified methanol: water (1:1, v:v, 0.01% acetic acid) (Table 1).

TABLE 1.

Phenolic profiles of propolis extracts by LC–MS/MS method.

Compounds	Molecular formula	RT (min.)	Ionization mode	Precursor ion (m/z)	Product ion(s) (m/z)	Cone (V)	Collision (eV)	EWE	PGE	BE
								mg/g raw propolis
3,4 dimethoxy cinnamic acid	C11H12O4	19.1	ESI (−)	206.7	102.7	25	20	4.25 ± 0.52b	4.45 ± 0.08ab	4.95 ± 0.06a
Apigenin	C15H10O5	25.0		269	117.3/149/151	40	30/25/25	1.84 ± 0.06b	1.87 ± 0.10ab	2.01 ± 0.05a
Caffeic acid	C9H8O4	10.9		179	135	25	20	3.88 ± 0.35ab	3.88 ± 0.14ab	4.37 ± 0.02a
CAPE	C17H16O4	32.4		283	179/161/135	25	20	6.41 ± 0.04b	6.64 ± 0.11a	6.12 ± 0.03c
Chrysin	C15H10O4	30.9		253	225/209/151	25	20	4.30 ± 0.10b	4.33 ± 0.12ab	4.57 ± 0.04a
Trans‐ferulic acid	C10H10O4	14.7		193	178/149/134	25	20	1.29 ± 0.06b	1.22 ± 0.05b	1.52 ± 0.11a
Galangin	C15H10O5	31.9		269	197/213/227	25	20	7.98 ± 0.13a	7.99 ± 0.17a	4.30 ± 0.42b
Genistein	C15H10O5	24.7		269	133.2/159.2/224.2 240	40	30/20/25/20	0.33 ± 0.06a	0.33 ± 0.02a	0.32 ± 0.04a
Isorhamnetin	C16H12O7	25.9		315	300	25	20	0.98 ± 0.07a	1.05 ± 0.05a	0.05 ± 0.02b
Kaempferol	C15H10O6	25.4		285	93/151/257	25	20	2.01 ± 0.19a	2.13 ± 0.16a	0.04 ± 0.01b
Luteolin	C15H10O6	22.3		285	133/241/267	25	20	0.48 ± 0.10a	0.54 ± 0.16a	0.62 ± 0.16a
Naringenin	C15H12O5	24.2		271	145/151	25	20	1.07 ± 0.03b	1.02 ± 0.02b	1.46 ± 0.07a
p‐coumaric acid	C9H8O3	13.5		163	93/119/147	25	20	1.86 ± 0.07b	1.85 ± 0.05b	2.22 ± 0.02a
Pinobanksin	C15H12O5	24.3		271.2	153/225/253	25	20	6.08 ± 0.14b	6.28 ± 0.21b	10.65 ± 0.32a
Pinocembrin	C15H12O4	31.1		255	151/171/213	25	20	12.54 ± 0.06ab	12.78 ± 0.07a	11.44 ± 0.20c
Quercetin	C15H10O7	22.4		301	150.8/178.9	35	20	1.50 ± 0.03a	1.46 ± 0.07a	0.06 ± 0.04b
Trans‐cinnamic acid	C9H8O2	21.1		147	77/102.8	25	20	2.14 ± 0.06ab	2.09 ± 0.28ab	2.30 ± 0.19a
Total								58.94	59.91	57.00

Note: Data are expressed as mean ± SD of triplicate measurements. Means with different letters in the same row are significantly different (p < .05).

Abbreviations: BE, L‐arginine solution extract of propolis; ESI, electrospray ionization; EWE, 70% ethanolic extract of propolis; PGE, mono propylene glycol extract of propolis; RT, retention time.

2.7. Antibacterial activity with disk diffusion method

Strains of Escherichia coli O157 ATCC 700728 and Staphylococcus aureus ATCC 25923 were obtained from the American Type Culture Collection. The in vitro inhibitory activities of PE were determined by the disc diffusion method (Sagdic et al., 2007). They were inoculated with nutrient agar for 24 h, at 37°C. They were adjusted against 0.5 Mc Farland turbidity standard tubes (1 × 10⁸). 6 mm sterilized discs (Bioanalyse Antimicrobial susceptibility test discs) were impregnated with 30 μL of extracts (without dilution) and placed on Muller Hilton agar plates, and incubated for 24 h, at 37°C. Inhibition zones (mm) on the medium were evaluated. All tests were performed in triplicate. The antibacterial activity of each solvent used for extraction was also assessed for control. The chloramphenicol disc (HIMEDIA Chloramphenicol C300 SD006‐SCT) was used as a positive control.

2.8. Statistical analysis

The data were expressed as the mean ± standard deviation (SD), analyzed by one‐way analysis of variance (ANOVA), and Tukey's post hoc test was used for comparison of the means using SPSS Statistics Software (IBM version 20, USA). All experiments were carried out in triplicate.

3. RESULTS AND DISCUSSION

3.1. Total phenolic and total flavonoid content

The total phenolic content (TPC) and the total flavonoid content (TFC) of PEs are given in Table 2. TPC of PEs ranged between 109.87 ± 2.64 and 116.36 ± 3.08 mg GAE/g raw propolis (p > .05). The TFC of PEs was not significantly different and ranged between 45.67 ± 0.06 and 47.54 ± 2.05 mg QE/g raw propolis. Kubiliene et al. (2015) also reported no significant differences in the TPC between PE obtained by PGE and ethanol (70%). Our results were in the range of the study of Ozdal et al. (2019), who compared TPC (27.48–199.70 mg GAE/g) and TFC (30.73–291.75 mg QE/g) of ethanolic extracts of 11 propolis samples collected from different regions. The content of propolis may vary depending on the plant species that honeybees collected the resin. The TPC and TFC values determined in our study were also in the range of Xue et al. (2016), who stated that the TPC and TFC values of the ethanolic extracts of propolis samples from Brazil, Australia, China, and South Korea ranged from 48.5 ± 4.08 to 238.9 ± 4.61 mg GAE/g and 23.5 ± 0.15 to 53.0 ± 0.22 mg QE/g, respectively. Ding et al. (2021) reported that TPC and TFC values of Chinese propolis collected from five regions was ranged from 72.02 to 155.95 mg GAE/g, and 129.68 to 412.83 mg QE/g of ethanolic extracts, respectively. Whereas Iranian propolis samples showed TPC and TFC values ranged from 26.59 to 221.38 mg GAE/g and from 4.8 to 100.03 mg QE per g of ethanolic extract, respectively (Hafshejani et al., 2023).

TABLE 2.

Total phenolic, flavonoid contents, and antioxidant capacities of propolis extracts.

	EWE	PGE	BE
TPC	112.08 ± 2.24a	109.87 ± 2.64a	116.36 ± 3.08a
TFC	47.54 ± 2.05a	46.41 ± 1.46a	45.67 ± 0.067a
CUPRAC	1837.81 ± 46.67c	1803.44 ± 92.07c	2592.29 ± 43.90a
ABTS	2150.92 ± 132.55bc	1940.20 ± 126.72c	4593.52 ± 97.55a
FRAP	1089.65 ± 57.14b	861.88 ± 38.19c	1397.29 ± 53.16a

Note: Data are expressed as mean ± SD of triplicate measurements. Means with different letters in the same row are significantly different (p < .05).

Abbreviations: ABTS, 2,2′‐azino‐bis (3‐ethylbenzothiazoline6‐sulphonic acid); BE, L‐arginine solution extract of propolis; CUPRAC, Copper reducing antioxidant capacity; EWE, 70% ethanolic extract of propolis; PGE, mono propylene glycol extract of propolis; FRAP, ferric reducing antioxidant power expressed as μmol TE/g raw propolis; TFC, total flavonoid contents expressed as mg QE /g raw propolis; TPC, total phenolic contents expressed as mg GAE/g raw propolis.

The aqueous extract of propolis yielded substantially lower TPC and TFC than those of the ethanolic extract in the study of Kubiliene et al. (2018) and Mokhtar (2019). In our study, extraction with L‐arginine solution (BE) yielded similar TPC and TFC values compared to EWE and PGE. This situation could be attributed to the change in ionic strength of the water, which can affect the solubility of compounds (Chong & Chua, 2020).

3.2. Total antioxidant capacity

It was stated that a single assay would not be enough to assess the results for complex matrices that include more than one compound possessing multiple antioxidant mechanisms (Karadag et al., 2009). Therefore, in this study, the results of three different assays (CUPRAC, ABTS, and FRAP) were evaluated (Table 2). Each method serves a different mechanism of the reaction, for example, CUPRAC and FRAP were classified as electron transfer (ET) based methods whereas ABTS assay was classified as a hydrogen atom transfer (HAT) based method (Karadag et al., 2009).

The CUPRAC, ABTS and FRAP values of our samples varied from 1803.44 to 2592.29, 1940.20 to 4593.52, and 861.88 to 1397.29 μmol TE/g raw propolis, respectively. Our ABTS result for PGE and EWE was in the similar range of the 19 PE analyzed by Bonvehí and Gutiérrez (2011) whereas the ABTS, CUPRAC and FRAP values of our ethanolic extract of propolis (EWE) were higher than those determined by Ozdal et al. (2019). Although their TPC and TFC values were similar, BE showed significantly higher antioxidant capacity values than other propolis extracts. The individual phenolic compounds in the extracts depending on the solvent might also display different antioxidant capacities. For example, compared with the other propolis extracts, BE had a significantly higher amount of ferulic acid, p‐coumaric acid, naringenin, and pinobaksin (Table 1). All antioxidant capacity assays were conducted in an aqueous medium, therefore the solubility of the extracts in the reaction medium should be also another important parameter to consider. When the propolis extracts were mixed with the reaction medium of antioxidant assays, the BE sample would have the advantage of enhanced solubility in the reaction medium due to the aqueous nature of its solvent. The pH of BE was around 9, it was reported that the antioxidant capacity of propolis extracts was increased when the extraction was applied in an alkaline condition (Yeo et al., 2015).

3.3. Analysis of individual phenolics in propolis extracts by LC–MS/MS

The individual phenolics and their concentration in the propolis extracts (EWE, PGE, and BE) were given in Table 1. The phenolic profiles of samples were typical for poplar propolis with the presence of flavonoids such as CAPE, pinocembrin, pinobaksin, galangin, apigenin, chrysin, quercetin, and phenolic acids such as ferulic acid, p‐coumaric acid, and cinnamic acid (Guzelmeric et al., 2023; Ozdal et al., ²⁰¹⁹).

The sum of the amounts of identified phenolic acids and flavonoids in the three extracts was between 57.00 and 59.91 mg/g of raw propolis, and pinocembrin and pinobanksin were the most abundant phenolics, ranging from 11.44 to 12.78 mg/g and 6.08 to 10.65 mg/g, respectively. Pinobanksin and pinocembrin were reported to be the main flavanones of the European propolis (Hegazi et al., 2000). While pinobanksin content was higher in BE, pinocembrin was determined to be higher in EWE and PGE. The elevated concentrations of galangin and CAPE were also detected in all extracts varying from 4.30 to 7.99 and 6.12 to 6.64 mg/g, respectively. Ozdal et al. (2019) determined the concentration of pinobanksin and CAPE ranged from 0.24 to 3.84 mg/g and 0.4 to 2.86 mg/g, respectively, in propolis ethanol extracts collected from different parts of Turkey. While phenolic acids (p‐coumaric acids, caffeic, and ferulic), which are more soluble in water, were identified at higher concentrations in BE, the level of galangin, isorhamnetin, kaempferol, and quercetin was lower compared with EWE and PGE samples. This could be due to the instability of the flavonol group at high pH compared to other subgroups of flavonoids such as flavanone, flavanol, and flavone. There are three main structural points that determine the reactivity of flavonoids: (i) the C3‐OH group in the C‐ring, (ii) the catechol moiety in the B‐ring, and (iii) the C2 = C3 bond in the C‐ring (Jurasekova et al., 2014). Galangin, isorhamnetin, kaempferol, and quercetin have both C3‐OH and C2 = C3 double bonds in their structure. In addition to these two groups, quercetin also has a catechol group, which makes it very sensitive to higher pH.

3.4. In vitro bioaccessibility of TPC, TFC, and individual phenolics of propolis extracts

During digestion through the simulated oral, gastric fluid, and intestinal fluid phases, the TPC, TFC, and antioxidant capacity of the samples were determined at the end of each digestion stage (Table 3). In all samples, the amount of TPC and TFC released in the oral phase was reduced in the following gastric stage, most probably owing to the low solubility of phenolics in the gastric fluid, at which a low pH value causes the protonation of phenolic compounds and alters them to a more hydrophobic nature. Yen et al. (2017) revealed that the maximum amount of total phenolics and flavonoids in propolis extracts of ethanol, water, and glycerol were noted in the oral stage of digestion. The high enzymatic activity and dialysis filtration in the following digestion phases could lower the phenolic and flavonoid contents (Yen et al., 2017). Consequently, the antioxidant capacity values of all propolis extracts were reduced in the gastric stage, and this reduction was less prominent in BE.

TABLE 3.

Total phenolic, flavonoid contents, and antioxidant capacities of propolis extracts during in vitro gastrointestinal digestion.

Analyses	Oral	Gastric	Intestinal		R%	BI%
			OUT	IN
TPC
EWE	62.22 ± 2.57a	23.87 ± 2.50c	39.14 ± 2.68b	3.46 ± 1.05d	3.08 ± 0.91AB	37.48 ± 1.14B
PGE	76.72 ± 3.57a	22.14 ± 7.11c	49.58 ± 3.27b	2.53 ± 0.65d	2.28 ± 0.44B	46.85 ± 1.11A
BE	55.35 ± 2.26a	35.86 ± 1.37b	47.90 ± 8.51ab	5.18 ± 0.01c	4.52 ± 0.04A	45.01 ± 4.37A
TFC
EWE	32.09 ± 2.96a	14.99 ± 6.60b	16.73 ± 1.46b	2.21 ± 0.48c	5.65 ± 1.14A	73.74 ± 4.58A
PGE	30.34 ± 2.04a	11.18 ± 6.16b	11.83 ± 1.24b	1.85 ± 0.52c	4.63 ± 0.88A	53.88 ± 0.27B
BE	11.62 ± 1.04a	9.66 ± 1.56b	12.51 ± 0.97a	1.98 ± 0.27c	5.20 ± 0.41A	58.15 ± 1.92B
CUPRAC
EWE	672.76 ± 22.16a	251.61 ± 4.61b	231.38 ± 12.90b	21.07 ± 0.37c	1.15 ± 0.03B	14.19 ± 0.82B
PGE	377.21 ± 6.13a	314.75 ± 3.28b	375.75 ± 11.25a	18.09 ± 1.04c	1.01 ± 0.11B	20.85 ± 0.19A
BE	520.11 ± 31.12a	339.00 ± 6.21b	506.26 ± 10.34a	38.07 ± 1.66c	1.47 ± 0.08A	21.56 ± 0.15A
ABTS
EWE	423.70 ± 25.05a	168.26 ± 33.60b	206.18 ± 26.54b	17.83 ± 1.81cC	0.83 ± 0.11C	10.92 ± 1.21C
PGE	544.57 ± 62.84a	93.72 ± 5.41c	200.99 ± 15.10b	53.17 ± 3.06cB	2.75 ± 0.31A	13.63 ± 0.91B
BE	601.74 ± 36.96b	396.80 ± 6.83c	750.75 ± 67.08a	60.94 ± 0.42dA	1.35 ± 0.01B	18.64 ± 0.52A
FRAP
EWE	318.18 ± 13.96a	133.50 ± 3.36b	55.72 ± 2.48c	5.01 ± 0.33d	0.46 ± 0.04B	5.59 ± 0.44C
PGE	198.84 ± 8.02a	64.95 ± 5.35c	183.47 ± 5.07b	7.68 ± 0.58d	0.92 ± 0.08A	21.07 ± 0.33A
BE	216.45 ± 12.81a	162.91 ± 2.98b	216.45 ± 19.49a	10.80 ± 0.80c	0.77 ± 0.08A	17.67 ± 0.37B

Note: Data are expressed as mean ± SD of triplicate measurements. Different lowercase letters in the same row for each propolis extract among digestion phases are significantly different (p < .05). Different capital letters on the same column are significantly different (p < .05) among propolis extracts. R% = (IN/Indigested) * 100, BI% = ((IN + OUT)/Indigested) * 100.

Abbreviations: ABTS, 2,2′‐azino‐bis (3‐ethylbenzothiazoline6‐sulphonic acid); BE, L‐arginine solution extract of propolis; CUPRAC, copper reducing antioxidant capacity; EWE, 70% ethanolic extract of propolis; FRAP, ferric reducing antioxidant power expressed as μmol TE/g raw propolis; PGE, mono propylene glycol extract of propolis; TFC, total flavonoid contents expressed as mg QE/g raw propolis; TPC, total phenolic contents expressed as mg GAE/g raw propolis.

At the intestinal phase (IN+OUT), compared to the previous stage of digestion, the increase in TPC and TFC could be related to the presence of digestive enzyme and bile salt that could also affect the wax residues in the extracts, therefore increasing the release of some phenolics partitioned in the wax residue. Additionally, the higher level of pH at this stage could provide higher solubility for propolis compounds. Except for the FRAP value of EWE, all samples had higher antioxidant capacity values in the intestinal stage (Table 3). The measured value in the IN fraction was lower than the OUT fraction, which can be due to the dialysis filtration process (Yen et al., 2017). In our study, the TPC losses in post‐gastric digestion (PG) were 78.70%, 79.84%, and 69.18% for EWE, PGE, and BE, respectively, whereas in the study of Turkut et al. (2019), TPC losses in post‐gastric digestion were 85.72% and 92.11% for ethanol and mono‐propylene glycol propolis extracts, respectively. Compared with EWE, BE and PGE provided a higher amount of TPC at the intestinal phase, and 37.48%–46.85% of the initial TPC of the propolis extracts were accounted for in the intestinal phase (BI%). Only 2.28%–4.52% of the TPC determined in the non‐digested extracts could pass through the dialysis membrane and be considered to enter serum (R%) after simulated GI digestion (Table 3). The BI% of TPC for propolis extracts prepared by water, ethanol, and propylene glycol was between 17.42% and 29.59% (Turkut et al., 2019). In another study, the BI% of TPC ranged from 3.6% to 33.05% for ethanol extracts of 11 different Turkish propolis samples (Ozdal et al., 2019). EWE provided the highest TFC at the intestinal phase. The amount of TFC recovered in the intestinal phase (BI%) ranged from 53.88% to 73.74% for all extracts and compared with the initial amount in the non‐digested extracts 4.63% to 5.20% of total flavonoids were recovered (R%) in the IN phase of digestion, that was not significantly different among extracts. The BI% of TFC in ethanolic extracts of 11 different propolis was reported to change from 1.42% to 136% by Ozdal et al. (2019).

While considering the antioxidant capacities of the compounds reached or transformed in the intestinal stage (IN+OUT) and those entered in the serum (IN), the BE sample provided the highest values by each assay. When calculating their proportion to the initial value in the non‐digested sample extracts (BI%), the results differed depending on the assay. In all extracts, the compounds at the intestinal stage provided only 5.59%–21.6% (BI%) of the initial antioxidant capacities of the related extracts. While with the CUPRAC and ABTS assays, the highest BI% values were determined in BE, it was highest in PGE with the FRAP assay. While considering the antioxidant capacity of the compounds that could pass through the dialysis membrane, the lowest recovery (R%) was detected in EWE with FRAP assay, and the highest recovery was calculated in BE with CUPRAC assay. The change of individual phenolics at each digestion stage determined by LC–MS/MS was given in Table 4. In the non‐digested extracts, pinocembrin, pinobanskin, galangin, and CAPE (Table 1) were detected at higher levels in EWE and PGE, but not in BE. However, in all extracts, their concentration was drastically reduced at the end of the intestinal digestion stage, the recovery (R%) of those flavonoids ranged from 0% to 9.38% of the initial amount determined in the non‐digested samples, and the highest bioaccessibility (BI%) of those compounds was recorded for the EWE. Regarding to the other flavonoids, at the end of digestion, chrysin became the highest bioaccessible flavonoid in all extracts, while quercetin, isorhamnetin, and kaempferol were detected in neither of the samples. Apigenin and genistein were both detected at similar concentrations in all extracts. Luteolin was not detected in PGE, and naringenin was not detected in BE at the end of intestinal digestion. Among all phenolics, the highest recovery (R%) was determined for some phenolic acids of the BE sample, namely 3,4 dimethoxy cinnamic acid (21.16%), ferulic acid (10.34%), p‐coumaric acid (13.30%), and trans‐cinnamic acid (22.14%). The difference in the bioaccessibility of phenolics of propolis extracts could be affiliated with their structures. For example, Li et al. (2023) stated that the average bioaccessibility of phenolic acids was higher than that of flavonoids and the bioaccessibility rate decreased with the increase in the number of hydroxyl substitutions among hydroxybenzoic phenolic acids. In our sample, the bioaccessibility of caffeic acid was also lower than that of p‐coumaric acid. The increase in the concentrations of phenolic acids induced by in vitro digestion conditions might be associated with the cleavage of higher molecular structures to free phenolic acids, for example, the main hydroxycinnamic acids found in the diet, caffeic, p‐coumaric, trans‐cinnamic, and ferulic acids were naturally found as glycosides or esters of quinic acid (Stalmach, 2014). Besides the simple phenolic acids, the presence of some esterified and/or methylated derivatives, such as p‐coumaric acid methyl ester, p‐coumaric acid isoprenyl ester, caffeic acid esters such as caffeic acid phenylethyl ester (CAPE), caffeic acid isoprenyl and benzyl esters, 3,4‐dimethyl‐caffeic acid (DMCA) were reported in propolis extracts (Falcão et al., 2013; Ozdal et al., ²⁰¹⁹). According to Cianciosi et al. (2020), phenolic acids are thought to be more stable in the digestive process than flavonoids. Compared with the higher in vitro bioaccessibility values for honey phenolic acids (p‐coumaric, ferulic, and syringic acids, 15.2%, 20.0%, and 25.3%, respectively), very low bioaccessibility values for pinocembrin (0.5%), naringenin (0%), and quercetin (0%) were reported (Seraglio et al., 2021).

TABLE 4.

Change in the individual phenolics of propolis extracts during in vitro gastrointestinal digestion.

		Oral	Gastric	Intestinal		R%	BI%
				IN	OUT
Phenolics (mg/g raw propolis)
3,4 dimethoxy cinnamic acid	EWE	9.54 ± 0.77a	6.77 ± 1.27a	0.53 ± 0.09b	7.71 ± 0.54a	12.41 ± 1.02B	165.29 ± 2.82B
	PGE	8.47 ± 0.03a	3.89 ± 0.93b	0.29 ± 0.10c	6.51 ± 0.38a	8.18 ± 0.20C	153.80 ± 3.03B
	BE	9.24 ± 0.27a	5.99 ± 0.42b	1.04 ± 0.04c	8.93 ± 0.08a	21.16 ± 0.56A	200.29 ± 3.96A
Apigenin	EWE	1.89 ± 0.09a	0.30 ± 0.03c	0.05 ± 0.01d	0.95 ± 0.05b	3.59 ± 0.06A	54.82 ± 3.20A
	PGE	1.62 ± 0.10c	0.35 ± 0.12a	0.04 ± 0.01a	0.95 ± 0.08b	2.06 ± 0.43A	56.27 ± 3.97A
	BE	1.12 ± 0.01c	0.11 ± 0.01ab	0.05 ± 0.02a	0.42 ± 0.15b	2.71 ± 0.76A	28.67 ± 1.24B
Caffeic acid	EWE	3.64 ± 0.29a	4.42 ± 1.68a	nd	0.04 ± 0.01b	nd	nd
	PGE	3.18 ± 0.07a	1.90 ± 0.57b	0.01 ± 0.00c	0.25 ± 0.03c	0.34 ± 0.03A	6.93 ± 0.80A
	BE	nd	3.88 ± 0.50a	nd	3.60 ± 0.81a	nd	nd
CAPE	EWE	3.80 ± 0.28a	0.83 ± 0.14b	0.02 ± 0.01c	0.85 ± 0.36b	0.51 ± 0.00B	17.41 ± 0.16B
	PGE	4.75 ± 0.17a	1.10 ± 0.34b	0.05 ± 0.04c	1.24 ± 0.09b	1.12 ± 0.03A	19.19 ± 0.51A
	BE	0.27 ± 0.01ab	0.05 ± 0.01b	nd	nd	nd	nd
Chrysin	EWE	4.23 ± 0.26a	0.40 ± 0.12c	0.34 ± 0.11c	3.00 ± 0.04b	10.77 ± 0.17A	78.75 ± 1.52A
	PGE	3.67 ± 0.14a	1.01 ± 0.30b	0.27 ± 0.04c	3.26 ± 0.15a	6.25 ± 0.74B	82.85 ± 1.79A
	BE	3.76 ± 0.10a	0.53 ± 0.05c	0.31 ± 0.07c	1.89 ± 0.51b	6.77 ± 1.42B	56.10 ± 0.84B
Trans‐ferulic acid	EWE	1.41 ± 0.18a	1.42 ± 0.47a	0.04 ± 0.01B	0.88 ± 0.09a	2.85 ± 0.87B	69.62 ± 4.85B
	PGE	1.25 ± 0.02a	0.69 ± 0.20b	0.04 ± 0.01c	0.89 ± 0.12ab	2.80 ± 0.93B	84.34 ± 1.78B
	BE	nd	1.62 ± 0.18a	0.16 ± 0.01b	1.77 ± 0.01a	10.31 ± 0.97A	124.34 ± 10.14A
Galangin	EWE	7.01 ± 0.51a	0.72 ± 0.09b	0.02 ± 0.00b	0.04 ± 0.01b	0.30 ± 0.01C	0.78 ± 0.02C
	PGE	7.97 ± 0.33a	1.65 ± 0.59b	0.04 ± 0.02c	0.22 ± 0.01c	0.67 ± 0.01A	3.33 ± 0.15A
	BE	1.07 ± 0.01a	0.07 ± 0.01bc	0.02 ± 0.00c	0.10 ± 0.03b	0.49 ± 0.06B	2.65 ± 0.82AB
Genistein	EWE	0.40 ± 0.02a	0.06 ± 0.01c	0.01 ± 0.00d	0.20 ± 0.02b	3.77 ± 0.46A	70.00 ± 4.56A
	PGE	0.32 ± 0.02a	0.08 ± 0.03c	0.01 ± 0.00d	0.20 ± 0.01b	2.76 ± 0.67A	66.88 ± 4.04A
	BE	0.23 ± 0.01a	0.02 ± 0.00b	0.01 ± 0.00b	0.09 ± 0.04b	4.22 ± 0.96A	44.53 ± 1.44B
Isorhamnetin	EWE	0.21 ± 0.01a	0.15 ± 0.03a	nd	nd
	PGE	0.93 ± 0.05a	0.18 ± 0.07b	nd	nd	nd	nd
	BE	0.01 ± 0.00a	nd	nd	nd
Kaempferol	EWE	0.57 ± 0.17a	0.34 ± 0.02a	nd	nd	nd	nd
	PGE	1.39 ± 0.14a	0.32 ± 0.15b	nd	nd	nd	nd
	BE	nd	nd	nd	nd		nd
Luteolin	EWE	0.38 ± 0.05a	0.16 ± 0.07bc	0.01 ± 0.00c	0.18 ± 0.04b	2.29 ± 0.50A	46.44 ± 0.93A
	PGE	0.34 ± 0.06a	0.11 ± 0.02c	nd	0.17 ± 0.02b	nd	nd
	BE	0.19 ± 0.03a	0.04 ± 0.02bc	0.01 ± 0.00c	0.08 ± 0.01b	1.63 ± 0.04A	13.06 ± 0.98B
Naringenin	EWE	1.63 ± 0.03a	0.79 ± 0.13b	0.07 ± 0.01c	0.77 ± 0.07b	6.63 ± 1.68A	76.95 ± 2.71A
	PGE	1.04 ± 0.04a	0.51 ± 0.12b	0.07 ± 0.01c	0.16 ± 0.01c	6.83 ± 1.21A	23.13 ± 0.02B
	BE	nd	nd	nd	nd	nd	nd
p‐coumaric acid	EWE	2.23 ± 0.11a	2.21 ± 0.66a	0.10 ± 0.03b	1.89 ± 0.16a	5.43 ± 1.69B	98.69 ± 1.90B
	PGE	1.77 ± 0.07a	1.34 ± 0.28a	0.05 ± 0.02b	1.52 ± 0.20a	2.73 ± 0.83B	93.40 ± 2.41B
	BE	2.46 ± 0.25ab	2.33 ± 0.32b	0.29 ± 0.02c	3.22 ± 0.24a	13.30 ± 1.06A	157.71 ± 7.79A
Pinobanksin	EWE	9.69 ± 0.04a	4.66 ± 0.76b	0.49 ± 0.09c	4.38 ± 0.57b	9.38 ± 0.26A	77.94 ± 7.06A
	PGE	5.85 ± 0.26a	2.82 ± 0.69b	0.52 ± 0.10c	1.00 ± 0.04c	8.35 ± 1.31A	24.47 ± 1.98B
	BE	nd	nd	nd	nd	nd	nd
Pinocembrin	EWE	13.15 ± 0.46a	2.35 ± 0.09c	0.72 ± 0.12d	7.05 ± 0.29b	5.72 ± 1.36A	61.94 ± 3.67A
	PGE	12.31 ± 0.32a	3.52 ± 1.09b	0.54 ± 0.13c	2.92 ± 0.07bc	4.24 ± 1.00A	27.19 ± 0.26B
	BE	0.12 ± 0.01a	0.07 ± 0.01a	nd	0.07 ± 0.02a	nd	nd
Quercetin	EWE	nd	0.40 ± 0.14a	nd	nd
	PGE	0.93 ± 0.03a	0.34 ± 0.12b	nd	nd	nd	nd
	BE	nd	nd	nd	nd
Trans‐cinnamic acid	EWE	5.06 ± 0.21a	3.06 ± 0.89bc	0.26 ± 0.03c	4.01 ± 0.05b	12.30 ± 1.54B	193.79 ± 1.97B
	PGE	4.01 ± 0.24a	2.18 ± 0.54b	0.17 ± 0.04c	3.21 ± 0.07a	7.75 ± 0.46B	138.04 ± 1.56C
	BE	4.37 ± 0.35a	2.95 ± 0.15b	0.52 ± 0.01c	4.41 ± 0.03a	22.14 ± 2.77A	225.54 ± 0.40A

Note: Data are expressed as mean ± SD of triplicate measurements. Different lowercase letters in the same row for each propolis extract among digestion phases are significantly different (p < .05). Different capital letters on the same column are significantly different (p < .05) among propolis extract. R% = (IN/Indigested) * 100, BI% = ((IN + OUT)/Indigested) * 100.

Abbreviations: BE, L‐arginine solution extract of propolis; EWE, 70% ethanolic extract of propolis; nd: not determined; PGE, mono propylene glycol extract of propolis.

3.5. Antibacterial activity

The antibacterial activity of propolis has been associated to some of its constituents such as the presence of some flavonoids such as galangin, pinocembrin, and pinobanksin at high concentrations (Ding et al., 2021). All propolis extracts showed antibacterial effects against Gram‐negative and Gram‐positive bacterial pathogens (Table 5). Compared with the positive control, chloramphenicol, all propolis extracts showed weaker antimicrobial activity against both E. coli and S. aureus.

TABLE 5.

Antibacterial activity of propolis extracts.

Samples	Average inhibition zones (mm)
	Escherichia coli	Staphylococcus aureus
BE	9.0 ± 0.09	13.9 ± 0.63
PGE	8.0 ± 0.24	11.1 ± 0.16
EWE	11.4 ± 0.71	19.4 ± 3.13
Chloramphenicol	32.3 ± 0.05	30.6 ± 1.50
BE blank	0.0 ± 0.00	0.0 ± 0.00
PGE blank	0.0 ± 0.00	0.0 ± 0.00
EWE blank	8.8 ± 0.24	11.2 ± 0.38

Note: The results are mean ± standard deviation of replicates (n = 3). Chloramphenicol was used as the positive control.

Abbreviations: EWE, 70% ethanolic extract of propolis; PGE, mono propylene glycol extract of propolis; BE, L‐arginine solution extract of propolis.

The inhibition zones provided by our samples ranged from 11.1 to 19.4 mm against S. aureus and 8.0 to 11.4 against E. coli. Compared with E. coli, S. aureus was reported to be more sensitive to propolis extracts (Bayram et al., 2017). The inhibition zone of ethanolic propolis extracts obtained in Serbia was in the range of 9–13 mm against S. aureus, while only two ethanolic propolis extracts out of 13 samples showed an antibacterial effect against E. coli (Stepanović et al., 2003). Muli and Maingi (2007) reported that the propolis extracts from three regions of Kenya showed the inhibition zone ranged from 8 to 9.5 mm to 9 to 10 mm against S. aureus and E. coli, respectively.

Although the antimicrobial effect of the EWE sample seems to be higher, it should be kept in mind that the solvent of this extract (70% of ethanol), provided the antimicrobial effect by itself (Table 5), whereas the other solvents used in PGE and BE do not have antibacterial effects on their own, and the entire effect was due to propolis extract.

4. CONCLUSION

Although the broad spectrum of pharmacological and biological properties of propolis has been studied and well‐documented in many different studies, during its oral digestion, the choice of extraction solvent becomes a limiting and important factor due to the solubility of the desired bioactive compounds and its safe and extensive use in different consumer products. This study showed that as an alternative to ethanol and propylene glycol, which are the most commonly used solvents on the market, the aqueous L‐arginine solution can be used as an extraction solvent in terms of phenolic composition, antioxidant, and antimicrobial activity. However, when subjected to in vitro simulated digestion conditions, there was a very drastic reduction of all individual phenolics regardless of the extraction solvent used. Therefore, an efficient delivery system should be proposed for propolis extracts that would also protect the phenolics through digestion.

AUTHOR CONTRIBUTIONS

Gizem Mergen Duymaz: Data curation (equal); formal analysis (equal); methodology (equal); validation (equal); writing – original draft (equal); writing – review and editing (equal). Gamze Duz: Data curation (equal); formal analysis (equal); validation (equal); writing – original draft (equal); writing – review and editing (equal). Kubra Ozkan: Data curation (equal); formal analysis (equal); writing – original draft (equal); writing – review and editing (equal). Ayse Karadag: Conceptualization (equal); methodology (equal); validation (equal); writing – original draft (equal); writing – review and editing (equal). Ozlem Yilmaz: Data curation (equal); resources (equal). Ayca Karakus: Data curation (equal); resources (equal). Ozlem Cengiz: Data curation (equal); resources (equal). Ismail Emir Akyildiz: Data curation (equal); resources (equal). Gunay Basdogan: Funding acquisition (equal); writing – review and editing (equal). Emel Damarlı: Funding acquisition (equal). Osman Sagdic: Conceptualization (equal); methodology (equal).

FUNDING INFORMATION

This study did not receive any grant from funding agencies in the public, commercial, etc.

CONFLICT OF INTEREST STATEMENT

There are no conflicts of interest.

Mergen Duymaz, G. , Duz, G. , Ozkan, K. , Karadag, A. , Yilmaz, O. , Karakus, A. , Cengiz, O. , Akyildiz, I. E. , Basdogan, G. , Damarlı, E. , & Sagdic, O. (2024). The evaluation of L‐arginine solution as a solvent for propolis extraction: The phenolic profile, antioxidant, antibacterial activity, and in vitro bioaccessibility. Food Science & Nutrition, 12, 2724–2735. 10.1002/fsn3.3953

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.