Relationship between total phenolic contents and biological properties of propolis from 20 different regions in South Korea

Background Propolis (or bee glue), collected from botanical sources by honey bee, has been used as a popular natural remedies in folk medicine throughout the world. This study was conducted to assess growth inhibitory effects of ethanol extracts of propolis (EEPs) from 20 different regions in South Korea on human intestinal bacteria as well as their human β-amyloid precursor cleavage enzyme (BACE-1), acetylcholinesterase (AChE) inhibitory, antioxidant, antiproliferative, and anti-human rhinovirus activities. Methods The Bonferroni multiple-comparison method was used to test for significant differences in total polyphenol and flavonoid contents among EEP samples using SAS 9.13 program. Correlation coefficient (r) analysis of the biological activities of EEP samples was determined using their 50 % inhibition concentration or minimal inhibitory concentration values and their polyphenol or flavonoid contents in 20 native Korean EEP samples. Results The amounts of total polyphenol and flavonoids in the Korean EEP samples ranged from 49 to 239 mg gallic acid equivalent (GAE)/g EEP (Brazilian, Chinese, and Australian samples, 127–142 mg GAE/g EEP) and from 21 to 50 mg quercetin equivalent (QE)/g EEP (Brazilian, Chinese, and Australian samples, 33–53 mg QE/g EEP), respectively. Correlation coefficient analysis showed that total polyphenol contents may be negatively correlated with 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity (r = −0.872) and total flavonoid content has no correlation with the activity (r = 0.071). No direct correlation between BACE-1 inhibition, AChE inhibition, or antiproliferative activity and total polyphenol or total flavonoid content in Korean EEP samples was found. Gram-positive and Gram-negative bacteria were observed to have different degrees of antimicrobial susceptibility to the EEP samples examined, although ciprofloxacin susceptibility among the bacterial groups did not differ greatly. Conclusions Further studies will warrant possible applications of propolis as potential therapeutic BACE-1 blocker, antioxidant, antiproliferative agent, and antimicrobial agent.


Background
Propolis (or bee glue), a strongly adhesive, resinous natural substance collected from botanical sources (branches, flowers, pollen, and buds) by honey bee (Apis mellifera L.) [1], has been used as a popular natural remedy in folk medicine throughout the world. Bees use propolis to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance toward intruders [2,3]. Propolis is generally composed of 50 % resin and balsam, 30 % wax, 10 % essential and aromatic oils, 5 % pollen, and 5 % various other substances [3], although the precise composition of raw propolis varies with several factors such as botanical source and geographical zones [1][2][3].
Propolis has been reported to possess a broad spectrum of biological activities, such as anticancer, anticomplement, antihypertensive, antihyperalgesic, hepatoprotective, anti-inflammatory, antioxidant, immunomodulatory, antimicrobial, and antiparasite properties [1,[3][4][5]. The chemical variability, aroma, and color (brown, green, red, and yellow) of propolis significantly depend on its botanical source, age of the honey preparation, geographical zones, and collection season [2,6,7]. In Brazil, propolis from the southeast region and Amazon contains chiefly phenylated phenylpropanoids and polyprenylated benzophenones, respectively, whereas geranyl flavonones have been reported for propolis from the Pacific region, such as Taiwan and Okinawa [7]. Propolis from eastern Mediterranean regions, such as Greece, Crete, and Turkey, may contain predominantly diterpenoids [7]. The variation in the chemical composition of propolis from different origin also causes the diverse biological activities [7]. More than 300 constituents, including aromatic acids and esters, flavonoids (chalcones, flavanones, flavones, flavonols, and dihydroflavonols), waxy acids and terpenoids, were isolated from raw propolis [1,2]. In Brazil, 12 distinct groups of propolis have been classified according to their botanical origin and biological properties [8]. Very little work exists in relation to biological properties of native Korean propolis, although the antioxidant activity of propolis from several regions in South Korea have been described by Ahn et al. [9].
The aim of the current study was to assess total polyphenol and flavonoid contents as well as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, antiproliferative, anti-human rhinovirus (HRV), human β-amyloid precursor cleavage enzyme (BACE-1), and human acetylcholinesterase (AChE) inhibitory activities of ethanol extracts of propolis (EEPs) from 20 different regions in South Korea. In addition, the growth inhibitory effects of the EEP samples on five harmful intestinal bacteria, two nonpathogenic intestinal bacteria, six lactic acid-producing bacteria, and an acidulating bacterium, including Gram-positive bacteria and Gram-negative bacteria, using a microtiter plate-based bioassay and compared with those of ciprofloxacin, a second-generation fluoroquinolone antibiotic with a broad spectrum [10]. The biological activities of the Korean EEP samples were compared with those of EEP samples from Brazil with diverse chemical composition, Australia with various biologically active flavonoids, phenylpropanoids, and stilbenes and prenylated stilbenes, and China with chemical profiles similar to Korean propolis [11]. A correlation between total polyphenol or flavonoid content and biological activities is also discussed.

Propolis samples and extraction
The 20 native Korean propolis samples (P1-20) used in this study are listed in Table 1, along with coordinates. The samples were collected as the crude materials by beekeepers in various regions of South Korea. Propolis samples from Australia, Brazil, and China were purchased from Aussia Pharma (Silverwater, Australia), Uniflora Apicultores Associados (Olimpia, Brazil), and KangSiNong Biotechnology (Wuhan, China), respectively. Because EEP is one of the richest sources of phenolic acids and flavonoids [12], these propolis samples were extracted with ethanol at room temperature for 1 day, and filtered. The combined filtrated was concentrated to dryness by rotary evaporation at 40°C. The ethanol extracts were kept at −20°C until use.

Determination of total polyphenol and flavonoid contents
Total polyphenol contents in EEP samples were determined using Folin-Ciocalteu colorimetric method described by Zongo et al. [13] with slight modifications. In brief, 100 μL of 0.2 N Folin-Ciocalteu reagent was added to 96-well plate (SPL Life Sciences, Pocheon, Gyeonggi, South Korea) containing 20 μL of each EEP in 80 % ethanol for 5 min at room temperature in darkness. The 80 μL of sodium carbonate (75 g/L) was added to each well, and the plate was then incubated for 30 min at room temperature with slightly shaking in darkness. The absorbance was determined at 735 nm using a Ver-saMax microplate reader with SoftMax Pro 5 Software (serial no. SMP500-18672-LWHU) (Molecular Devices, Sunnyvale, CA). EEPs were evaluated at the final concentration of 100 μg/mL. Gallic acid (0-62.5 μg/mL) was applied as the standard, and the calibration equation was Y = 0.0454 X -0.0056 (R 2 = 0.9993), where X is the gallic acid concentration in μg/mL and Y is the absorbance at 735 nm. Total polyphenol contents were expressed as mg of gallic acid equivalents (GAE) per g of EEP samples.
Total flavonoid contents in EEP samples were determined using AlCl 3 colorimetric method [13] adapted to 96-well plate. In brief, 100 μL of 2 % AlCl 3 was added to 100 μL of each EEP in 75 % ethanol, and the plate was then incubated for 15 min at room temperature in darkness. The absorbance was determined at 435 nm using a microplate reader stated previously. EEPs were evaluated at the final concentration of 100 μg/mL. Quercetin (0-50 μg/mL) was used as the standard, and the calibration equation was Y = 0.0343 X + 0.0177 (R 2 = 0.9995), where X is the quercetin concentration in μg/mL, and Y is the absorbance measured at 435 nm. Total flavonoid contents were expressed as mg of quercetin equivalents (QE) per g of EEP samples.

Fluorescence resonance energy transfer enzyme assay
The BACE-1 inhibitory activity of all EEP samples was evaluated according to the methods of Lv et al. [14] and Wang et al. [15]. The assay mixtures consisted of 1 μL of 0.5 μg/μL recombinant human BACE-1, 0.75 μL of 2.5 μg/μL FPS, 47.25 μL of 50 mM sodium acetate (pH 4.5), and EEP (10-2000 μg/mL) in 2 % dimethyl sulfoxide (DMSO). They were incubated for 1 h at 25°C in darkness, followed by adding 16.6 μL of 2.5 M sodium acetate to terminate the reaction. The intensity of fluorescence was determined using a SpectraMax Gemini XS plate reader with Softmax Pro PC/MAC Software (serial no. US 02947) (Molecular Devices, Sunnyvale, CA) at 355 nm excitation and 405 nm emission at room temperature. The inhibition percentage was calculated according to the formula: % inhibition = 100 -[(F S -F S0 )/ (F C -F C0 )] × 100, where F S and F S0 are the fluorescence of samples at 60 min and zero time, and F C and F C0 are the fluorescence of control at 60 min and zero time, respectively [13].

Acetylcholinesterase inhibition assay
The assay procedure was performed using the recombinant human AChE (R&D system, Minneapolis, MN) according to the manufacture's protocol. In brief, the reaction mixture consisted of 50 μL of AChE (0.044 μg/ mL in 0.1 M sodium phosphate buffer (pH 7.5)) and EEP (10-2000 μg/mL) in 1 % DMSO. The reaction was started by adding 50 μL substrate/DNTB mixture (800 μM ATChI in assay buffer containing 400 μM DTNB). The reaction mixture was incubated at 37°C for 1 h. The absorbance was recorded at 410 nm using a VersaMax microplate reader. The AChE inhibitors donepezil hydrochloride, tacrine, and huperzine A [16] served as positive controls and were likewise formulated.
DPPH radical scavenging assay DPPH free radical scavenging activity of all EEP samples was evaluated according to the method described by Blois [17] with minor modifications. In brief, 100 μL of 0.4 mM DPPH methanl solution was added to 96well plate containing each EEP sample in methanol. Based on the preliminary test results, the radical scavenging activity of each EEP sample was determined with five to seven concentrations ranging from 5 to 2000 μg/mL. After incubation for 30 min at room temperature in darkness, the absorbance was measured at 518 nm by using a VersaMax microplate reader. Ascorbic acid served as a positive control and was likewise formulated. The radical scavenging ability was calculated according to the formula: % DPPH free radical scavenging activity = (1 -A s /A c ) × 100, where A c is the absorbance of the control (without sample) and A s is the absorbance of the sample.
Cancer cell lines and cell proliferation assay The antiproliferative activity of all EEP samples toward the human cancer cell lines examined was evaluated using a MTT assay described by Morgan [18]. In brief, MTT was dissolved in phosphate-buffered saline (PBS) (pH 7.4) at 5 mg/mL and sterile-filtered. Cells were plated at 2 × 10 4 cells per well in 100 μL of complete culture medium containing the test EEP samples (dissolved in DMSO Hybri-Max) in 96-well culture plates. The final concentration of DMSO Hybri-Max in all assays was 0.1 % or less. Based on the preliminary test results, the antiproliferative activity of each EEP sample was determined with five to six concentrations ranging from 15 to 1000 μg/mL. The culture plates were incubated for 2 days in a 37°C in a humidified atmosphere of 5 % CO 2 . The plates were then washed with 100 μL PBS. The 100 μL medium containing 0.05 % MTT was added to each well and then incubated for 4 h at the same condition stated previously. MTT solution was removed and 200 μL DMSO was added to each well. Finally, the plate was shaken for 10 min to dissolve the purple formazan crystals formed. Cisplatin served as positive controls and was similarly formulated. Negative controls only consisted of the DMSO solution only. The optical density values were recorded using a VersaMax microplate reader at a 560 nm and a 670 nm reference.
The anti-HRV activity of all EEP samples was assessed by a SRB assay using CPE reduction [19,20]. In brief, HeLa cells were seeded onto 96-well culture plates at a density of 3 × 10 4 cells per well for 1 day. The culture medium was then removed and the plates were washed with PBS. Subsequently, 90 μL of diluted virus suspension containing CCID 50 of the virus stock was put into the wells, and then 10 μL of MEM supplemented with 30 mM MgCl 2 containing four to five concentrations (0.1-200 μg/mL) of each EEP sample in 0.1 % DMSO was added to produce an appropriate CPE within 2 days after infection. After incubation at 37°C and 5 % CO 2 for 2 days, the plates were washed once with 200 μL PBS. The 100 μL of 0.057 % (w/v) SRB in 1 % acetic acid solution was added to each well and left at room temperature for 30 min. The absorbance was recorded using a VersaMax microplate reader at 562 nm and at 620 nm reference. Ribavirin served as a positive control and negative controls consisted of the DMSO solution. Viral inhibition rate (VIR) (%) was calculated according to the formula [20]: % VIR = (OD tV -OD cV )/(OD cd -OD cV ) × 100, where OD tV is the optical density measured with a given concentration of the EEP sample in HRV infected cells; OD cV is the optical density measured for the control untreated HRV infected cells; OD cd is the optical density measured for the control untreated HRV uninfected cells.

Intestinal bacterial strains and growth inhibitory assay
Five harmful bacteria, two nonpathogenic bacteria, six lactic acid-producing bacteria, and an acidulating bacterium used in this study are listed in Table 2. Stock cultures of the bacterial strains were stored on BHI broth (pH 7.6) containing 25 % glycerol (v/v) at −70°C. The cultures of Escherichia coli ATCC 11775 and Staphylococcus aureus ATCC 12600 were incubated at 37°C for 1 day under aerobic condition, while the cultures of the other 12 bacterial strains were incubated at 37°C for 1 day in an atmosphere of 5 % H 2 , 15 % CO 2 , and 80 % N 2 in a FA-6 anaerorator (serial no. 98072851) (Hirayama, Tokyo, Japan) [21]. For bioassay, bacterial suspensions containing 1 × 10 5 colony-forming unit (CFU)/mL were prepared in EG agar using 24 h subcultures in BHI broth.
A microtiter plate-based bioassay in sterile 96-well plates was used to determine the minimal inhibitory concentrations (MICs) of all EEP samples toward the organisms, as described by Sarker et al. [22]. In brief, initial EEP samples were prepared in DMSO, and a twofold dilution series was then formulated in 50 μL BHI broth. Subsequently, 10 μL bacterial suspension of each strain was added. Ciprofloxacin served as a positive control and was similarly formulated. Negative controls consisted of the DMSO solution only. Treated and control plates were incubated under the same conditions as those used for bacterial cultures for 24 h. Then, 10 μL of resazurin solution (270 mg resazurin in 40 mL sterile distilled water) was added to each well.

Data analysis
MIC (mg/mL) was defined as the lowest concentration of EEP that visually inhibited bacterial growth using resazurin indicator. The BACE-1 inhibitory, AChE inhibitory, and free radical scavenging activity was expressed as 50 % inhibition concentration (IC 50 , μg/ mL) of the EEP that is required to cause 50 % BACE-1, AChE, and DPPH inhibition, respectively. The antiproliferative activity was expressed as 50 % inhibition concentration (IC 50 , μg/mL) of the EEP that is required to reduce the viability of cells to 50 % compared to the controls. The IC 50 values of the EEP samples were calculated using Prism 5 software program (GraphPad Software, La Jolla, CA). The IC 50 values were considered to be significantly different from one another when their 95 % confidence limits did not overlap. Results were expressed as mean ± standard error (SE) of triplicate samples of three independent experiments. The Bonferroni multiple-comparison method was used to test for significant differences in total polyphenol and flavonoid contents among EEP samples using SAS 9.13 program (SAS Institute, Cary, NC). Correlation coefficient (r) analysis of the biological activities of EEP samples was determined using their IC 50 or MIC values and their polyphenol or flavonoid contents in 20 Korean EEP samples.

Total polyphenol and flavonoid contents of propolis samples
The total polyphenol and flavonoid contents in 20 Korean EEP samples were compared with those of Australian, Brazilian, and Chinese EEP ones ( Table 3). The total polyphenol contents (F = 92.79; df = 22, 46; P < 0.0001) and flavonoid contents (F = 68.66; df = 22, 46; P < 0.0001) in 23 EEP samples significantly differed. The total polyphenol contents in 20 Korean EEP samples ranged from 48.5 to 238.9 mg GAE/g EEP. EEP samples from P10, P13, P3, and P1 showed higher total polyphenol contents (238.9-219.5 mg GAE/g EEP) than those from other regions. The total polyphenol content of EEP from P20 was the lowest of any of the propolis examined. The total polyphenol content of EEP samples from Brazil, China, and Australia was between 127 and 142 mg GAE/g EEP. The total flavonoid contents in Korean EEP samples ranged from 20.8 to 49.8 mg QE/g EEP. EEPs from P9, P20, and P2 showed higher total flavonoid contents (49.8-40.5 mg QE/g EEP) than those from other regions. The total flavonoid content of EEP from P17 was the lowest of any of the propolis examined. The total flavonoid content of EEP samples from China, Australia, and Brazil was between 33 and 53 mg QE/g EEP.

In vitro BACE-1 inhibitory activity
Because BACE-1 is a critical enzyme in the amyloid precursor protein (APP) amyloidgenic pathway that generates β-amyloid, the main component of amyloid plaque in the brain of AD [23], the BACE-1 inhibitory activity of all EEP samples was elucidated using a fluorescence resonance energy transfer enzyme assay (

Human acetylcholinesterase inhibitory activity
Because AChE is one of the major targets of AD [16], the inhibitory activity of all EEP samples was compared with that of three anticancer agents (

DPPH free radical scavenging activity
The antioxidant activity of all EEP samples was compared with that of an antioxidant agent ascorbic acid using a DPPH radical scavenging assay (

Antiproliferative effect on cancer cell lines
The antiproliferative activity of all EEP samples was compared with that of a commercial anticancer agent cisplatin using a MTT assay (

Anti-human rhinovirus activity
The antiviral activity of all EEP samples was compared with that of the antiviral agent ribavirin using a SRB assay (Data not shown). EEP sample from Brazil was 5.9 and 5.1 times more toxic toward HRV-2 (IC 50 , 12.6 μg/ mL) and HRV-4 (15.4 μg/mL) than ribavirin, respectively. IC 50 of the other 22 EEP samples was >100 μg/mL toward two HRVs.

Growth-inhibiting effect on intestinal bacteria
The growth inhibitory effects of all EEP samples on five harmful and two nonpathogenic intestinal bacteria as well as six lactic acid-producing bacteria and an acidulating bacterium examined were compared with those of the commercial antibiotic ciprofloxacin (
In the current study, the total polyphenol and flavonoid contents of 20 native Korean EEP samples were between 49 and 232 mg/g and between 21 and 50 mg/g, respectively, although the polyphenol and flavonoid contents of propolis stated previously were 31 and 431 mg/g and between 3 and 183 mg/g, respectively. Certain Korean propolis samples examined possessed considerable total polyphenol and flavonoid contents, as compared with either Australian, Brazilian, or Chinese propolis samples examined. This finding indicates that propolis with high polyphenol and flavonoid contents have to be selected for commercial propolis products because of the biological significance of the polyphenols and flavonoids [1,2,5]. BACE-1 [36] and AChE [16] are two of the major targets of AD. There are two major neuropathological hallmarks of AD including neurofibrillary tangles consisting of hyperphosphorylated tau protein and extracellular amyloid plaques accumulation of β-amyloid peptides. β-Amyloid was generated via sequential proteolytic processing of APP by BACE-1 and γ-secretase in APP amyloidogenic processing pathway [37,38]. BACE-1 is a prime drug target for inhibiting β-amyloid generation because it is responsible for initiating βamyloid production [39]. No information, however, is available concerning the BACE-1 inhibitory activity of propolis and its constituents. Brazilian propolis improved cognitive function in the patients with mild cognitive impairment that was deemed as a prodromal stage of AD [40]. In addition, inhibition of AChE, responsible for the breakdown of acetylcholine in the neural synapse, is a possible strategy for treatment of AD, which is characterized by a decline in cognitive function and mental atrophy. Propolis with a high phenol content may be an alternative for prevention and/or retardation of AD symptoms because phenols and flavonoids inhibit AChE activity [41]. The anti-AChE activity was reported in propolis from India (IC 50 , 43.46 μg/mL) [42] and Morocco (43-743 μg/mL) [32]. In the current study, IC 50 of 20 native Korean EEP samples was between 15.6-327.3 μg/mL and the anti-AChE activity of the EEPs was lower than that of either donepezil hydrochloride, tacrine, or huperzine A. Certain Korean propolis samples exhibited good BACE-1 inhibitory activity (IC 50 , < 100 μg/mL) and were more pronounced in the inhibitory activity than either Australian, Brazilian, or Chinese propolis examined. Many peptidomimetics and heterocyclic compounds have been evaluated as BACE-1 inhibitors [43,44]. However, none of these have been successfully developed as anti-AD drugs. These results verify that the Korean propolis merit further study as a potential anti-AD agents.
The antimicrobial activity of propolis toward various pathogens have been well noted [3][4][5]. In humans, many species of bacteria (~500-1000 species) reside in the intestinal tract as a complex and dynamic ecosystem [60]. Major functions of the microbiota include metabolic activities that result in salvage of energy and absorbable nutrients, trophic effects on intestinal epithelia (cell proliferation and differentiation) and on immune structure and function, and protection of the colonized host against invasion by alien microbes (barrier effect) [61]. The microbiota might also be an essential factor in certain pathological disorders, including multisystem organ failure, colon cancer, and inflammatory bowel diseases [61]. In addition, prolonged treatment with antibiotics has often produced resistance to the drugs by pathogenic microorganisms [62], which is a major global public health problem in both developed and developing countries. Sometimes, serious side effects of antibiotics occur, such as taste disturbances, nausea, diarrhea, dyspepsia, headache, and angioedema, as well as disturbance of human gastrointestinal microflora [62,63]. Alternative antibacterial agents with novel modes of action and low toxicity are urgently needed. It has been reported that propolis samples from Argentina [64], France [65], and Greece and Cyprus [29] is effective toward Gram-positive bacteria, exerting a limited activity toward Gram-negative bacteria. Boyanova et al. [66] reported that Bulgarian EEP inhibited 97 % (29 of 30 strains) of the nonspore-forming Gram-positive bacteria and 91 % (40 of 44 strains) of the Gram-negative bacteria.
In the current study, the Gram-positive and Gramnegative bacteria were observed to have different degrees of antimicrobial susceptibility to the EEP samples examined, although ciprofloxacin susceptibility among the bacterial groups did not differ greatly. EEP samples from P6, P9 and P11 and P12 exhibited pronounced growth inhibitory activity toward pathogenic Grampositive bacteria C. difficile ATCC 9689 and C. paraputiricum ATCC 25780, respectively. EEP samples from P8 and P9 and P14 were active toward nonpathogenic Gram-negative bacteria B. fragilis ATCC 25285 and E. coli ATCC 11775, respectively, whereas EEP samples from P12 and P19 were active toward both Gram-negative bacteria. However, the propolis samples from Austrailia, Brazil, and China were ineffective toward five harmful bacteria and two nonpathogenic bacteria. Boyanova et al. [66] reported that Bulgarian EEP inhibited 35 % (7 of 20 strains) of Clostridium strains, including C. difficile (13 strains) and C. perfringens (2 strains), and 82 % (9 of 11 strains) of B. fragilis group strains. Ugur et al. [67] found growth inhibitory activity of EEP samples from Brazil and Bulgaria toward E. coli MU 8, MU 11, and MU 23, whereas no inhibitory activity of EEP samples from Argentina toward E. coli ATCC 25922 [64]. Interestingly, six lactic acid-producing bacteria were less susceptible than either harmful or nonpathogenic bacteria to Korean EEP samples. Detailed tests are needed to fully understand the different susceptibility of the EEPs to bacteria. This finding indicates that low concentrations of active Korean propolis extracts could be used in fermented or nonfermented products and drink products, aiming to selectively inhibit the growth of pathogenic bacteria.
Correlation between biological activity and phenolic compound contents has been well studied. High correlation between the total phenolic and flavonoids content and the free radical scavenging activity was reported in propolis samples from Argentina [46], Greece and Cyprus [29], Japan [31], and Poland [33]. Negative or no direct correlation between them was reported in propolis from Morocco [32] and in propolis from Brazil [68] and Greece [48], respectively. For the AChE inhibition, negative correlation between the total phenolic and flavonoids content and IC 50 was reported in propolis samples from Morocco [32]. For the antiproliferative activity toward MCF-7 cell line, negative correlation between the total polyphenol content and IC 50 was reported, whereas positive correlation between the total flavonoid content and IC 50 was reported [56]. For the antimicrobial activity, strong correlation between the total phenolic and flavonoids content and MIC was reported in propolis from Spain [69], whereas no direct correlation between them was also reported in propolis from Brazil [68] and Greece and Cyprus [29]. In the current study, correlation coefficient (r) analysis showed that total polyphenol contents may be negatively correlated with DPPH free radical scavenging activity (r = −0.872) and total flavonoid content has no correlation with the activity (r = 0.071) ( Table 9). No direct correlation between BACE-1 inhibition, AChE inhibition, or antiproliferative activity and total polyphenol or total flavonoid content in Korean EEP samples was found.

Conclusions
Korean propolis-derived preparations could be useful in food and beverages to prevent various diseases in which free radicals, neurodegenerative causes, or pathogenic bacteria are implicated as propolis administration to humans does not lead to side effects [12]. For the practical use of the preparations as novel propolis-derived products to proceed, further research is needed to establish safety to humans and whether the biological activities are exerted in vivo after consumption of the products by humans. Propolis, administered orally to mice at levels up to 4000 mg/kg/day for 2 weeks, had no effect, although it has been identified clinically as an allergen [3]. Lastly, detailed tests are needed to understand how to improve biological potency and stability for eventual commercial development.