1. Introduction
Bees are necessary for maintaining ecosystems, contributing to biodiversity through pollination, a vital factor for a wide range of crops and wild plants. Today, it is known that the failure of pollination will lead irremediably to the loss of plant cultures and, as a consequence, food security concerns [
1].
Worldwide, 75% of the crops are pollinated by insects with 57 species (mostly bees) as crucial pollinators for approximately 107 plants [
1,
2].
Honey bees (
Apis mellifera L.) are affected by many diseases, the most important being of fungal and viral origin. The main factors affecting disease are small colony population size, extended winter, reduction of cleaning flights, feed supplements, and the hive’s excessive humidity [
3,
4,
5].
Under these circumstances, nosemosis caused by
Nosema apis Zander and
Nosema ceranae Fries protozoa became the principal threat and the most commonly found in honey bee populations [
6,
7,
8,
9,
10,
11,
12].
During the last decade, allopathic drugs against nosemosis were restricted to a few active substances such as Fumagillin (fumidil), an antibiotic obtained from
Aspergillus fumigatus. Unfortunately, although an efficient product, due to the risk of residues, EMA has excluded this product from use in Europe in February 2016 [
6,
13,
14,
15,
16,
17,
18].
In the given circumstances in the treatment of nosemosis, a reliable backup could be ecologic phytotherapy, the usage of whole herbs or parts, with recognized antiprotozoal activity (like flowers of
Matricaria chamomilla,
Hypericum perforatum or
Achillea millefolium, leaves of
Mentha piperita, or leaves and flowers of
Ocimum basilicum) currently being viewed as a great opportunity [
19,
20,
21,
22].
The Research and Development Institute for Beekeeping has developed an herbal product that presents the blend of essential oils highly efficient against
Nosema spp [
23]. Essential oils used in this product are derived from herbs found in spontaneous flora, which include different cyclic and aliphatic hydrocarbons, triterpenes and sesquiterpenes, phenolic structures, oleanolic acid, flavones, microelements, and the vitamins of B group [
24].
This study aimed to analyze the composition of Protofil® as commercial product suggested was the usage in honey bees’ production, as well as to analyze basil, thyme, yarrow, and dandelion, and to compare them to the aforementioned product, respectively.
2. Materials and Methods
The product
Protofil® plant association is a brownish solution, with a characteristic aromatic odor and taste, designed to combat
Nosema spp., and unique advantage is that it has no contraindications (no intoxication or any side effects) to honey bees [
25,
26].
The sample of the product Protofil® was chemically investigated, directly from the producer, the ICDA (Research and Development Institute for Beekeeping, Bucharest, Romania).
Besides Protofil®, samples of Achillea millefolium, Thymus vulgaris, Ocimum basilicum, and Taraxacum officinale, were chemically investigated as well.
The physicochemical methods used to investigate Protofil® and plants were: Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) of the filtered undiluted or diluted hydro-alcoholic extracts and Mass-Spectrometry (GC-MS) coupled with Gas Chromatography of volatile compounds separated by hydro-distillation-extraction in an organic solvent (SDE).
2.1. RP-HPLC Investigation
RP-HPLC investigation of the flavonoid standards and hydro-alcoholic extract samples was performed on a Jasco apparatus (Abbl&e-Jasco, Bucharest, Romania) equipped with: quaternary pump (PU-2080 Plus); mixing unit (LG-2080-04 Quaternary Gradient); degasser (DG-2080-54 4); spectrophotometric detector (UV-2070 Plus Intelligent UV/VIS Detector); acquire and process computer data (JASCO ChromPass Chromatography Data System, Version 1.7.403.1), through an LC-Net II/ADC interface.
The conditions of analysis were:
Column: Nucleosil 100 C18, 250 × 4.6 mm × mm, 5 μm particle diameter;
UV wavelength: 254 nm; Mobile phase: Acetonitrile: Water = 50:50; Temperature: 25 °C; Flow rate: 1.0 mL/min; Injected volume: 20 μL.
For flavonoids, evaluation of their concentration in hydro-alcoholic extracts were performed using the obtained HPLC calibration curves. The flavonoids’ identification correlated the detection of retention times with the standards matching. Therefore, before analysis, the samples were filtered, and, in most cases, they were diluted (1:100).
The samples’ bioactive compounds concentration was measured using the calibration curves for the available flavonoids, results being expressed as mg of flavonoid compound, separated at the retention times corresponding to the standard/mL of sample.
For the RP-HPLC, the following standards were used:
Rutin (≥94%) (Sigma-Aldrich, Taufkirchen, Germany),
Quercetin (≥95%) (Sigma-Aldrich, Taufkirchen, Germany),
Chrysene (>98%) (Sigma-Aldrich, Taufkirchen, Germany),
Flavone (≥99%) (Sigma-Aldrich, Taufkirchen, Germany).
Standard solutions were obtained by dilution in 96% ethanol (Chimopar, Bucharest, Romania) also HPLC purity solvents being used for the chromatographic analysis: acetonitrile (HPLC grade) (Fluka Chemie, Mūnchen, Germany) and bidistilled water HPLC (Fluka Chemie, Mūnchen, Germany).
2.2. GC-MS Analysis
The GC-MS analysis of SDE-separated volatile compounds implied the use of hexane (GC grade) (Fluka) for the extraction of volatile compounds separated, and anhydrous sodium sulfate (>99%) (Merck) to dry the hexane extract. The Kovats retention indices were calculated based on GC-MS assays performed under the same conditions for a mixture of linear C8–C20 alkanes (Fluka Chemie).
2.3. Separation of Volatile Compounds by Hydrodistillation-Extraction (SDE)
The GC-MS analysis of the separated volatile compounds from hydro-alcoholic extracts by hydrodynamic extraction in hexane (SDE) allowed the relative percentage concentrations of the components to be evaluated using the area method (Equation (1)):
For the analysis of the separated volatile compounds, an HP 6890 Series GC (Hewlett Packard), coupled with an HP 5973 Mass Selective Detector mass spectrometer was used.
The GC assay conditions were: Column: HP-5MS, L = 30 m, inner diameter 0.25 mm, film thickness 0.25 μm; Temperature program: 50 to 250 °C at a speed of 6 °C/min; Injector temperature: 280 °C; Detector temperature: 280 °C; Injection volume: 2 μL; Carrier gas: He.
For the MS detector, an EE energy of 70 eV was used, at a source temperature of 150 °C, scanning range of 50–300 amu, with the speed of 1 s−1 for mass spectrometry, and the obtained spectra, compared with a NIST/EPA/NIH Mass Spectral Library 2.0 database (2002). For data acquisition, version B.01.00/98, of HP Enhanced Chem Station G1701BA software was used, the data processing, being completed utilizing the HP Enhanced Data Analysis program. Hydro-alcoholic samples (~800 mL) were prepared and the condensed volatile compounds, extracted in an SDE system, in 20 mL hexane. The method lasted four hours, and the separated hexane extract was dried. Dry hexane extracts were then GC-MS analyzed, determining the relative percentage concentration of the volatile compounds.
2.4. Statistical Multivariate Principal Component Analysis (PCA) of GC Data
Multivariate analysis of gas chromatography data for hexane extracts of volatile compounds, allowed a classification of samples based on volatile compounds and their relative concentrations, identifying the similarity of these samples. To assess the investigated samples similarity–dissimilarity, the multivariate statistical data analysis—Principal component analysis (PCA), of gas-chromatographic data, was used, the GC data being used for analysis, and validated by cross-validation method.
4. Discussion
Considering treatment with antibiotics is now forbidden in European countries, control of nosemosis has to be completed mainly by employing defensive and alternative measures. Additionally, if a beehive is critically impaired by nosemosis, the strategy, from an economic point of view and in many countries, is to destroy those colonies, although losses could become sizeable. Nevertheless, in the literature, there are presented efforts to combat nosemosis, original phytotherapeutic conditionings being proposed, a present study trying to be part of this cause by proposing this phytotherapeutic approach.
Research shows that food supplements are common in beekeeping [
24]. Research was conducted to evaluate the brood development from colonies, which were fed with different naturals supplement added in supplementary food compared to product
Protofil®. According to result of this research, after the winter period and during the period of preparation for principal honey harvest, the best results were obtained for
Protofil® and
Echinacea [
24].
For instance, thymol was among the first natural substances studied in the beehive infections [
27,
28,
29] as well as various thymol links [
30]. In our results, the volatile compounds analyzed in
T. vulgaris were
eucalyptol and
γ-terpinene.
For example, Maistrello et al. [
19], had evaluated the effectiveness of different phyto compounds, like resveratrol, thymol, vetiver essential oil, and lysozyme, to control nosema in honeybees. The results revealed that bees, fed especially with thymol, which is also identified in our study, and resveratrol considerably reduced infection rates and extending longevity. Thymol and resveratrol have therefore been shown to be effects for control of nosemosis [
19].
Mărghitaş et al. [
23] investigated the influence of nettle, thyme and
Echinacea, fresh juice of onion and garlic, and
Protofil® as supplementary feed in artificially weakened bee colonies. The most effective results in this field experiment were recorded in bees supplemented with nettle [
23].
In another study,
N. ceranae infection was stopped with the use of oxalic acid syrup, in laboratory and field studies, being proposed by authors, as an alternative control strategy [
16].
Yucel and Dogaroglu [
31] studied comparatively, for three years, the activity of Fumagillin, and thymol in
N. apis infection, in 208 honey bee colonies. The results confirm the present investigation with the aim of phytotherapy efficiency and underlining the importance of alternative treatments in honey bees [
31].
The observed low mortality, as well as the honey production, which also brings the organic honey’s benefits, does validate the
Protofil® use judiciousness, as a reliable phytotherapeutic choice. This observation is significant from the organic product consumers and the beekeepers’ economic point of view because research has shown consumers’ higher willingness to pay for organic honey [
32]. The efficacy of
Protofil® for treating nosemosis was demonstrated on 15 colonies. The mortality values compared to the honey production/categories/total quantity, confirmed the judiciousness of treatments with
Protofil® [
33].
Cola [
34] tested to caraway,
Protofil®, fresh juice of onions, garlic, stinging nettle, thyme,
Echinacea, and selenium on the bee families artificially weakened by removing the existing population of 3/4 from initial. It was found that the most significant influence in this research had a stinging nettle, which was in agreement with earlier findings [
24].
5. Conclusions
The chromatographic analyzes completed on plant extracts from different botanical families revealed that the most concentrated soluble components in the alcohol–water mixture were flavonoids, most often rutin, identified in high concentrations in most of the studied samples (except the thyme), but also its corresponding aglycons. The most significant volatile compounds identified were eucalyptol (1,8-cineol) and chavicol-methyl ether, for Lamiaceae (basil and thyme) samples and camphor for Asteraceae (yarrow) family. Representatives of the Compositae family were less concentrated in the volatile compounds (except thyme, significant from this point of view).
The results of our study revealed a considerable similarity of Protofil® with with A. millefolium, less so with T. vulgaris and O. basilicum, while they were significantly different from T. officinale. The results revealed a high concentration of beneficial active components of herbs in Protofil®, and the promised benefits of organic honey, with no residues, plus the lack of undesirable effects, but the further research are still necessary.