The rhododendrons of the Ericaceae family are widespread in the northern hemisphere and constitute a large part of vascular plants. There are eight different subgenera and more than 800 species of this genus. However, only five species of
Honey has been reported to have an inhibitory effect on around sixty species of bacteria including aerobes and anaerobes, gram-positives and gram-negatives (Manyi-Loh, Clarke, & Ndip, 2011). Insufficient knowledge of the antimicrobial agents in honey and theirinfluence on bactericidal efficiency hinder overall applicability of natural honey. Researchers have attempted to resolve the mechanism of action of honey's antimicrobial effect and appraised the additition of honey components to bactericidal activity against pathogenic bacteria.
Besides toxic diterpenes,
The aim of this research is to investigate
Pollen and honey materials of
For each
Honey samples (50 g) and raw pollen samples were chopped into small pieces (50 g) and then extracted with 250 mL of 95% ethanol through continuously stirring with a digital orbital shaker (SHO-2D, DAIHAN Scientific Co., Ltd., S. air-conditioning booth, Grotech brand, GR8 model, Unitroniks) at 180 rpm and 24°C with 18/6 light/dark period (single extraction). The suspension was filtrated, and the supernatant was separated after centrifugation at 10,000 rpm for 15 min. The ethanolic solution was then concentrated in a rotary evaporator under reduced pressure at 40°C to obtain the crude extract in paste form and kept in a dry and dark place at 4°C until use (Chang et al., 2002).
Pollen samples were prepared according to the method described by Louveaux, Maurizio, & Varwohl (1978). 10 g of pollen sample was dissolved in 20 mL of distilled water, divided into two centrifuge tubes of 15 mL, and centrifuged for approximately 10 min at 4000 rpm. The same procedure was repeated after distilled water was added to the sediment. A glycerine - water mixture (1:1) 5 mL was added to the sediment and was left to rest for 30 min prior to centrifugation. The sediment was then removed with the aid of a stylet, embedded in glycerine jelley and deposited on a microscopic slide sealed with paraffin wax. Pollen analysis was performed under light microscope in order to classify the samples as monofloral or not.
Scanning electron microscopy (SEM) images were recorded using Hitachi model SU1510. For SEM evaluation, properly dried pollen of each cultivar for electron microscopy shot stubs were secured with double-sided carbon tape glued on and fixed samples were coated with 15 nm gold-palladium (SEM coating system, sputter). SEM imaging was conducted at 5–15 kV voltage at l000×.
The AOAC method was used for determining such physicochemical features as moisture, acidity and sucrose content (AOAC, 1990). Hydroxymethyl furfural (HMF) was determined after the addition of sodium bisulphate to the clarified honey samples. Absorbance was measured at 284 and 336 nm using a UV/Vis spectrophotometer. Diastase activity was determined using a buffered solution of soluble starch and honey incubated in a thermostatic bath at 40°C. Afterwards, 1 mL aliquot of this mixture was removed at 5 min intervals and the absorption of the sample was followed at 660 nm (Official Method 958.09) (AOAC, 1990). The diastase value was calculated using the time taken for the absorbance to reach 0.235, and the results were expressed in Gothe degrees as the amount (mL) of 1% starch hydrolyzed by an enzyme in 1 g of honey in 1 h. Single measurements were performed on homogenized honey and pollen samples for physicochemical analyzes.
The following nineteen standards of phenolic compounds were analyzed using HPLC (Elite LaChrom Hitachi, Japan): gallic acid, protocatechuic acid,
The antimicrobial activity of the samples were studied against
Antibacterial and antifungal activities were measured using methods of disc diffusion on agar plates (Ertürk, 2006). All bacterial strains were grown in Mueller Hinton Broth medium (Merck) for 24 h at 37°C, and fungal strains were grown in Sabouraud Dextrose Broth (Difco) at 30°C for 48 h. Bacterial suspension with a turbidity of 0.5 McFarland and fungal suspension with a turbidity of 1.0 McFarland standards were prepared. Thus, the concentration was adjusted to 108 cells/mL for bacterial suspensions and to 3×108 cells/mL for fungal suspensions. Sterile paper discs (6 mm in diameter) were then placed on the agar for 30 μL of each sample (40 mg/mL) to be loaded. 100 units of nystatin for fungus and Ampicillin and Cephazolin for bacteria, all obtained from a local pharmacy, were used as positive controls, and alcohol was used as a negative control. Inhibition zones were determined after incubation at 27°C for 48 h. Inhibition zones of different organisms by different samples were measured with the help of a digital caliper to estimate the potency of antibacterial and antifungal substance and then tabulated. All measurements were performed on triplicate samples.
The MIC values represent the lowest honey and pollen extract concentration that completely inhibits the growth of microorganisms and were determined through the micro-well dilution method (Ertürk, 2006). All the extracts were dissolved in 70% ethanol and water, and then the dilution series were prepared in a 96-well plate (Corning). A Tris buffer (Amresco 0826-500G) mixture (1:4) was mixed at 30°C with an equal amount of broth solution (Sabouraud Dextrose Agar (Oxoid) for fungi and Mueller Hinton broth (Merck) for bacteria. Each honey and pollen sample was tested at concentrations of 6000, 3000, 1500, 750, 375, 187.5, 93.75 and 46.75 μg/mL. Inoculants were obtained from an overnight broth culture of the test organism. The broth culture was incubated at 35°C until it achieved the turbidity of the 0.5 McFarland standards (usually 24–48 h hours). The inoculum of each bacterium was prepared, and the suspensions were adjusted to 108 CFU/mL for bacteria and 107 CFU/mL for fungi. After solubilization, each well was inoculated with 5 μL of freshly prepared bacterial suspension of 1×108 bacteria, 1×107 fungus/mL, and incubated at 37°C for 24 hours. Amoxicillin and Cefazolin was used as positive control for bacteria and nystatin was used for fungi at 1500, 750, 375, 187.5, 93.75 46.75, 23.375 and 11.687 μg/mL concentrations, while 70% ethanol was used as negative control. Then, 30 μL of 3-(4, 5-dimethyl-thiazol-2-yl)-2.5-diphenyl-tetrazolium bromide (MTT) at a final concentration of 0.5 mg/mL freshly prepared in water was added to each well and incubated for 30 min. The change to red colour indicated that the bacteria were biologically active. The MIC was taken to the well, where no change of colour in MTT was observed and the MIC values were given as mean of triplicate analysis.
All antioxidant activity studies were performed on triplicate measurements.
Total phenolic content of the honey and pollen samples were determined by Folin-Ciocalteu assay (Singleton & Rossi, 1965) and expressed as gallic acid equivalent (GAE).
DPPH free radical scavenging activities of the extracts were tested by following the bleaching of the purple-coloured methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) at 517 nm after the addition of the extracts at different concentrations to DPPH solution prepared in methanol. Scavenging activity value obtained for each concentration was calculated using the following equation:
The FRAP assay was performed following the method based on the principle of reducing the Fe (III)—TPTZ complex in the presence of antioxidants to form blue Fe (II)—TPTZ complex and measurement of maximum absorbance at 595 nm (Oyaizu, 1986). FRAP values for honey and pollen samples were expressed as Trolox equivalents (mM Trolox/g honey).
AChE and BuChE inhibitory activities were measured with the Ellman et al. method (Ellman et al., 1961). Electric eel AChE and equine serum BuChE were used as enzymes, while acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates. The percentage of AChE/BuChE inhibition was determined by comparison of the samples’ reaction rates relative to the blank (methanol as extraction solvent in phosphate buffer pH 8) using the following equation:
Inhibition potentials of the extracts on 2,2′-azobis-(2-amidinopropane)-dihydrochloride (ABAP)-induced lipid peroxidation were also investigated. For this purpose, linoleic acid solutions were treated with 0.1 mg/mL of samples in the presence of ABAP and the absorbance change at 234 nm with time was monitored (Palacios et al., 2011). Lipid peroxidation inhibition potential was expressed as percentage taking into account the change between absorbance values at the beginning and at the end of the period.
To explore how pollen extracts benefited hydroxyl radical-mediated DNA damage, plasmid DNA pUC18 (Thermo Scientific) was used. Pollen extracts (concentration range from 6.25 to 50 mg/mL) were dissolved in tetrahydrofuran (THF, final concentration % 0.1), and honey extracts (concentration range from 3.125 to 50 mg/mL) were dissolved in dimethyl sulfoxide (DMSO, final concentration % 0.1). 20 μL of reaction mixture was prepared containing 0.25 μg/μL plasmid DNA pUC18, 1 μL 3% H2O2 and extracts of pollen and honey in Tris-EDTA (TE) buffer. H2O2 and 0.1% tetrahydrofuran treated plasmid DNAs were used as control groups. The prepared mixtures for each pollen and honey extract were incubated at 37°C for 24 h. Then, 2 μL of loading dye (bromophenol blue (0.025%) and sucrose (4%) in H2O) was added to the mixture (10 μL total volume) and the obtained mixtures were loaded on to the 1% agarose gel. Electrophoresis process was performed for 90 min at 80 V in TBE buffer (Trisma base, boric acid, EDTA) running buffer (pH 8). The gel was imaged under UV light (Akbaş et al., 2013).
In order to characterize some of the key features of the honey samples, the parameters moisture, pH, proline content and electrical conductivity were evaluated (Tab. 1) and compared with the limits set by the Turkish Food Codex where applicable (Turkish Food Codex, 2005).
Biochemical content and physicochemical parameters of honey samples
Samples | ||||||
---|---|---|---|---|---|---|
No | Analysis | Result | Result | Result | Limits | Method |
1 | Fructose (g/100g) | 35.40 | 38.80 | 38.80 | - | IHC, 2009 |
2 | Glucose (g/100g) | 30.20 | 27.30 | 28.30 | - | IHC, 2009 |
3 | Fructose + Glucose (g/100g) | 65.60 | 66.10 | 67.10 | Minimum 60 | |
4 | Fructose / Glucose | 1.17 | 1.42 | 1.37 | 0.9–1.45 | |
5 | Sucrose (g/100g) | 1.70 | 3.40 | 4.30 | Maximum 5 | IHC, 2009 |
6 | Maltose (g/100g) | 1.70 | 1.80 | 0.80 | - | IHC, 2009 |
7 | Proline (mg/kg) | 550.9 | 248.8 | 923.0 | Minimum 300 | TS 13357 |
8 | Number of diastases | 10 | 8.0 | 15.5 | Minimum 8 | IHC, 2009 |
9 | Moisture (g/100g) | 21.61 | 22.35 | 22.31 | Maximum 20 | |
10 | Brix (g/100g) | 76.81 | 76.09 | 76.13 | - | |
11 | pH | 4.0 | 3.8 | 4.3 | - | |
12 | Electrical Conductivity (mS/cm) | 0.712 | 0.304 | 1.20 | Maximum 0.8 | |
13 | Free Acidity (meq/kg) | 24 | 15 | 24.0 | Maximum 50 | |
14 | HMF (mg/kg) | 0.60 | 0.50 | 0.90 | Maximum 40 |
The results of the pollen analysis confirmed that all the
The phenolic compounds contained in both honey and pollen obtained from the
The amount of phenolic compounds (mg/g sample) in honey and pollen samples
Standards | ||||||
---|---|---|---|---|---|---|
Gallic acid | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
Protocateuic acid | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
Catechin | 3.76 | N.D. | N.D. | N.D. | N.D. | N.D. |
Caffeic acid | N.D. | 6.33 | N.D. | N.D. | N.D. | N.D. |
Syringic acid | N.D. | N.D. | N.D. | N.D. | N.D. | 21.88 |
Epicatechin | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
2.30 | 3.93 | 3.31 | N.D. | N.D. | N.D. | |
Ferulic acid | N.D. | 6.07 | 8.26 | N.D. | 38.19 | N.D. |
Rutin | N.D. | N.D. | 40.47 | N.D. | N.D. | N.D. |
Myerecetin | N.D. | N.D. | N.D. | 506.96 | 497.08 | 624.27 |
Resveratrol | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
Daidzein | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
Luteolin | N.D. | N.D. | N.D. | 16.91 | 207.61 | N.D. |
t-Cinnamic acid | 0.57 | 0.86 | 1.08 | 14.59 | 63.93 | 34.89 |
Hesperetin | N.D. | N.D. | N.D. | N.D. | N.D. | 39.83 |
Chyrisin | 16.81 | 27.44 | 20.16 | N.D. | N.D. | 112.89 |
Pinocembrin | 2.31 | 4.11 | 2.71 | N.D. | N.D. | 43.90 |
Phenylethyl caffeate | 3.70 | 4.84 | 2.34 | N.D. | N.D. | 63.17 |
N.D.: Not determined
In this study, the crude samples of
Zones of inhibition (mm) showing the antimicrobial activity of honey and pollen samples
Samples | S.a. | B.c. | K.p. | E.c. | C.f. | L.m. | E.f. | C.a. | S.c. | P.a. |
---|---|---|---|---|---|---|---|---|---|---|
21.0±0.46 c | 19.6±0.17 d | 19.9±0.50 b | 23.5±0.41 b | 15.3±0.04 b | 23.2±0.76 c | 27.3±0.53 b | 25.4±0.28 b | 26.5±0.84 a | 14.4±0.54 c | |
22.5±0.53 bc | 23.5±0.71 c | 14.0±0.52 e | 17.8±0.66 d | 22.6±1.42 a | 23.3±1.20 c | 21.4±0.79 c | 25.9±0.36 ab | 26.9±0.61 a | 14.4±0.054 c | |
25.8±0.64 a | 25.9±1,00 abc | 17.4±0.29 c | 21.5±0.52 bc | 17.5±1.05 b | 22.5±0.31 c | 25.5±0.38 b | 27.2±0.31 a | 26.5±0.71 a | 15.7±0.34 bc | |
24.1±0.90 ab | 25.6±0.64 abc | 22.6±0.34 a | 23.2±0.57 b | 15.2±1.33 b | 15.2±1.33 d | 26.1±0.80 b | 26.1±0.35 ab | 26.3±0.79 a | 16.8±0.21 bc | |
25.0±0.32 a | 25.0±0.44 bc | 16.1±0.76 cd | 22.9±1.03 b | 17.3±1.14 b | 24.3±0.35 bc | 18.8±0.60 c | 27.0±0.31 a | 27.0±0.35 a | 16.4±0.61 bc | |
23.8±0.37 ab | 26.8±0.69 ab | 15.8±0.29 cde | 27.8±0.04 a | 14.4±0.43 b | 25.7±0.17 bc | 25.5±0.67 b | 26.2±0.30 ab | 26.6±0.60 a | 17.7±0.90 b | |
Ampicillin | 10.0±0.00 d | 26.5±0.29 ab | 15.3±0.09 de | 19.8±0.03 cd | 16.3±0.55 b | 27.5±0.37 b | 34.0±0.58 a | N.D. | N.D. | 28.6±0.23 a |
Cephazolin | 7.0±0.00 e | 27.9±0.55 a | 16.2±0.30 cd | 18.8±0.17 d | 16.2±0.70 b | 32.6±0.38 a | 28.2±0.44 b | N.D. | N.D. | 27.9±0.57 a |
Nystatin | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | 17.3±0.32 c | 17.7±0.55 b | N.D. |
Solvent | 6.0±0.00 e | 6.0±0.00 e | 6.0±0.00 f | 6.0±0.00 e | 6.0±0.00 c | 6.0±0.00 e | 6.0±0.00 d | 6.0±0.00 d | 6.0±0.00 c | N.D. |
Means in column, with different letters differ significantly at P<0.01.
N.D.: Not determined.
Microorganisms: S.a.,
Minimum inhibition concentrations (MIC) expressed as μg /mL of honey and pollen samples to inhibit 100% of the microbial growth in vitro
Samples | S.a. | B.c. | K.p. | E.c. | C.f. | L.m. | E.f. | C.a. | S.c. | P.a. |
---|---|---|---|---|---|---|---|---|---|---|
750≤ | 375≤ | 750≤ | 375≤ | 750≤ | 375≤ | 750≤ | 375≤ | 750≤ | 1500≤ | |
375≤ | 375≤ | 375≤ | 375≤ | 750≤ | 375≤ | 750≤ | 187.5≤ | 187.5≤ | 1500≤ | |
375≤ | 375≤ | 750≤ | 375≤ | 375≤ | 375≤ | 750≤ | 187.5≤ | 187.5≤ | 1500≤ | |
375≤ | 375≤ | 750≤ | 375≤ | 375≤ | 375≤ | 750≤ | 187.5≤ | 375≤ | 750≤ | |
375≤ | 375≤ | 375≤ | 375≤ | 750≤ | 375≤ | 375≤ | 187.5≤ | 375≤ | 750≤ | |
375≤ | 750≤ | 375≤ | 750≤ | 750≤ | 750≤ | 375≤ | 187.5≤ | 187.5≤ | 750≤ | |
Ampicillin | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | N.D. | N.D. | 23.375 ≤ |
Cephazolin | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | 11.687 ≤ | N.D. | N.D. | 23.375 ≤ |
Nystatin | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | 23.375 ≤ | 23.375 ≤ | N.D. |
Solvent | - | - | - | - | - | - | - | - |
N.D.: Not determined. Microorganisms: S.a.,
Total phenolic contents, antioxidative activities based on DPPH and FRAP tests and inhibition potentials on linoleic acid peroxidation (LAP) of the samples are presented in Tab. 5.
Total phenolic content and antioxidative activity values of honey and pollen samples
Samples | Total phenolic content (mg GAE/g sample) | DPPH (SC50; mg/mL) | FRAP (mM TX/g sample) | Inhibition ratio (%) on LAP of the samples at 0.1 mg/mL |
---|---|---|---|---|
0.04±0.01 | 64.002±2.49 | 0.409±0.01 | 14.695±1.25 | |
0.22±0.02 | 35.634±1.95 | 2.025±0.05 | 8.550±0.05 | |
0.92±0.15 | 16.652±0.80 | 3.588±0.06 | 12.563±0.70 | |
25.81±1.85 | 1.054±0.85 | 6.177±0.03 | 9.368±1.03 | |
13.63±0.77 | 1.018±0.83 | 2.676±0.25 | 17.174±1.55 | |
23.10±1.09 | 0.496±0.01 | 4.162±0.30 | 37.158±2.25 |
Although there have been several studies on the potential of acetylcholinesterase inhibition of such
Acetylcholinesterase and butrylcholinesterase inhibition potentials (%) of the samples at 0.5 mg/mL concentration
Samples | AChE | BuChE |
---|---|---|
11.781±1.50 | 0.099±0.02 | |
50.330±2.65 | N.D. | |
5.750±0.96 | 0.790±0.35 | |
3.487±0.03 | 18.855±2.55 | |
8.671±0.85 | 7.601±1.00 | |
10.933±1.04 | 4.343±0.55 | |
Galantamine | 85.203±3.00 | 24.778±3.25 |
N.D.: Not determined
The protective effects of pollen and honey extracts on hydroxyl radical-mediated DNA damage have been investigated and given in Fig. 2 and Fig. 3, respectively.
The evaluation of physicochemical parameters of the honey and pollen samples points out that the samples are within the accepted limits of Turkish Food Codex except for the moisture for all three samples and the proline content of the honey obtained from
According to the results obtained from the HPLC analysis, the cinnamic acid content is remarkable and is present in all honey and pollen samples. Myricetin was detected in all pollen samples, whereas p-coumaric acid, chyrisin, pinocembrin and phenylethyl caffeate were present in all honey samples. Other phenolic compounds syringic acid, ferulic acid, rutin, luteolin and hesperetin were also detected in one or more of the honey and pollen samples.
Medicinal plants and their crops in Turkey are often known by local people, but in the case of honey and pollen obtained from
When we examine the total phenolic contents and antioxidative activities of the honey and pollen samples, it can be interpreted from Tab. 5 that
As mentioned before, there have not been many studies in literature comparing honey and pollen samples from different
Evaluation of lipid oxidation inhibition is another important parameter to evaluate biological antioxidant capacity (Watanabe, Nakajima, & Konishi, 2008). Protocatechuic acid and protocatechuic acid methyl ester isolated from the leaves of
We investigated cholinesterase inhibition potentials of honey and pollen samples and compared the results with galantamine. As can be seen from Tab. 6, the distribution of the results is quite interesting. Namely, AChE inhibition ratios of the honey samples were higher with respect to the pollen samples. On the other hand, BuChE inhibition degrees of the pollen samples were significantly higher; in particular, compared to galantamine, the pollen sample obtained from
The interaction of pollen and honey extracts with DNA may depend on the binding and/or cleavage properties, resulting in changes in three-dimensional DNA conformation. These changes have an impact on the DNA band density and in the rate of migration of DNA in an electric field (Asmafiliz et al., 2013). Plasmid DNA is found in supercoiled circular form I, singly nicked relaxed circular form II and linear form III. Untreated plasmid DNA migrates on the gel with two DNA bands (Akbaş et al., 2013). Interactions with the extract may cause conformational changes on the plasmid DNA and in DNA mobility through agarose gel. In the current study, pUC18 plasmid DNA was treated with 6.25, 12.5, 25, and 50 mg/mL polen extract respectively (Fig. 2). Lanes 1, 2, 3 and 4 were run with pUC18 plasmid DNA untreated with pollen extract as a control, while lanes 5, 6, 7 and 8 pointed out plasmid DNA interacted with increasing concentrations of the extracts in the presence H2O2 condition. Pollen obtained from
In Fig. 3, lanes 1–3 were run with pUC18 plasmid DNA untreated with honey extract as a control, while lanes 4–18 pointed out plasmid DNA interacted with increasing concentrations of the extracts in the presence of H2O2 condition. Honey extracts obtained from
As a result, antibacterial activity studies of monofloral honey samples from three different