Composition and Efficacy of a Natural Phytotherapeutic Blend against Nosemosis in Honey Bees

Honey bees are essential to sustaining ecosystems, contributing to the stability of biodiversity through pollination. Today, it is known that the failure of pollination leads irremediably to the loss of plant cultures and, as a consequence, inducing food security issues. Bees can be affected by various factors, one of these being Nosema spp. which are protozoans specifically affecting adult honey bees and a threat to bee populations around the world. The composition of the phytotherapeutic product (Protofil®) for treating nosemosis was analyzed from a biochemical point of view. The most concentrated soluble parts in the phytotherapeutic association were the flavonoids, most frequently rutin, but quercetin was also detected. Additionally, the main volatile compounds identified were eucalyptol (1.8-cineol) and chavicol-methyl-ether. To evaluate the samples’ similarity–dissimilarity, the PCA multivariate statistical analysis, of the gas-chromatographic data (centered relative percentages of the volatile compounds), was applied. Statistical analysis revealed a significant similarity of Protofil® with the Achillea millefolium (Yarrow) samples and more limited with Thymus vulgaris (Thyme) and Ocimum basilicum (Basil), and, respectively, a meaningful dissimilarity with Taraxacum officinale (Dandelion). The results have shown a high and beneficial active compounds concentration in the analyzed herbs. High similarity with investigated product recommending the Protofil®, as the treatment compatible with producing organic honey.


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].

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).
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).
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.

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).

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.
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.

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.

HPLC Curve Calibration for Standard Compounds
To evaluate the concentration of the flavonoid compounds in hydro-alcoholic extracts, calibration curves for the available flavonoids, rutin, quercetin, chrysene, and flavone, were determined. In the case of rutin, the HPLC analysis of the standard solutions indicated the chromatographic peak presence in the retention time range of 2-3 min (most probably, a mixture of isomers due to the presence of two chromatographic peaks that were analyzed together). The quercetin chromatographic peak was detected to 4.2 min, the HPLC examination of chrysene and flavone, assigning peaks, after 9.8 and, respectively, 15.8 min. The HPLC results for standards are presented in Figure 1.

HPLC Curve Calibration for Standard Compounds
To evaluate the concentration of the flavonoid compounds in hydro-alcoholic extracts, calibration curves for the available flavonoids, rutin, quercetin, chrysene, and flavone, were determined. In the case of rutin, the HPLC analysis of the standard solutions indicated the chromatographic peak presence in the retention time range of 2-3 min (most probably, a mixture of isomers due to the presence of two chromatographic peaks that were analyzed together). The quercetin chromatographic peak was detected to 4.2 min, the HPLC examination of chrysene and flavone, assigning peaks, after 9.8 and, respectively, 15.8 min. The HPLC results for standards are presented in Figure 1.

Evaluation of the Flavonoids' Concentration
Concentrations of the studied samples (mean of four replicates, expressed as mg flavonoid available/mL sample) are shown in Table 1. HPLC chromatograms of undiluted samples and etalons, for Ocimum basilicum, Thymus vulgaris, Achillea millefolium, and Taraxacum officinale are presented in Figure 2, and the chromatogram for the associated conditioning Protofil ® , in Figure 3.

Evaluation of the Flavonoids' Concentration
Concentrations of the studied samples (mean of four replicates, expressed as mg flavonoid available/mL sample) are shown in Table 1.  Figure 2, and the chromatogram for the associated conditioning Protofil ® , in Figure 3.  The most concentrated were the flavonoids (expressed as rutin) separated at the beginning of the chromatogram due to the higher hydrophilicity of these compounds, containing saccharide residues, followed by polyphenolic flavonoids of the quercetin type.
Chrysene, a bis-phenolic compound, and similar structures separated at high retention times were detected in medium-low concentrations, while flavone a non-phenolic compound were detect in extremely low concentrations. Analyzing the data for the four herb samples leads to results close to the Protofil's obtained data, except in the case of quercetin (probably due to inappropriate rutin separation).
The HPLC separation of the flavonoid compounds studied, on the C18 nonpolar column, correlates well with their hydrophobicity, with retention times increasing with hydrophobicity, expressed as the logarithm of the octanol/water partition coefficient, calculated with the QSAR Properties program in the HyperChem 5.1 package (log Prutin = 1.61, log Pquercetin = 0.28, log Pchrysin = 1.75 and log Pflavone = 2.32). The best correlation is polynomial of order 2 (r 2 = 0.98).

GC-MS Analysis of Volatile Compounds' Relative Concentration
For basil extract, (the most significant from the set of analyses), 56 components (expressed as abundance of 10,000) were separated, the most concentrated compound identified being chavicolmethyl-ether (55%) ( Table 2). The most concentrated were the flavonoids (expressed as rutin) separated at the beginning of the chromatogram due to the higher hydrophilicity of these compounds, containing saccharide residues, followed by polyphenolic flavonoids of the quercetin type.
Chrysene, a bis-phenolic compound, and similar structures separated at high retention times were detected in medium-low concentrations, while flavone a non-phenolic compound were detect in extremely low concentrations. Analyzing the data for the four herb samples leads to results close to the Protofil's obtained data, except in the case of quercetin (probably due to inappropriate rutin separation).
The HPLC separation of the flavonoid compounds studied, on the C18 nonpolar column, correlates well with their hydrophobicity, with retention times increasing with hydrophobicity, expressed as the logarithm of the octanol/water partition coefficient, calculated with the QSAR Properties program in the HyperChem 5.1 package (log P rutin = 1.61, log P quercetin = 0.28, log P chrysin = 1.75 and log P flavone = 2.32). The best correlation is polynomial of order 2 (r 2 = 0.98).

GC-MS Analysis of Volatile Compounds' Relative Concentration
For basil extract, (the most significant from the set of analyses), 56 components (expressed as abundance of 10,000) were separated, the most concentrated compound identified being chavicol-methyl-ether (55%) ( Table 2).
In the case of volatile compounds in the GC-MS of Thymus vulgaris (Thyme), 43 chromatographic peaks were identified, the most concentrated being eucalyptol and γ-terpinene (Table 3).  The most concentrated volatile components in the Achillea millefolium (Yarrow) specimens were: camphor (relative concentration of 37.5%) and eucalyptol (25%), the total GC-separated compounds, in this case, being 44, some of which derived from the column (especially at the high separation temperatures cases) ( Table 4). Table 4. Results of GC-MS analysis for Achillea millefolium (Yarrow) samples. The total concentration of active compounds in the hexane extracts of dandelion was identified. Upon identification of total active compounds, relative concentration was determined. The highest concentration of 5.7% eucalyptol, and 62.5% ethyl palmitate (Table 5) was recorded, respectively.     The Protofil ® analysis, described the most relevant absolute concentration, totaling 74 different components, as well as their absolute abundance in the hexane extract. The highest recorded concentration of active ingredients was eucalyptol (28.6%), followed by chavicol-methyl-ether (28.1%), The Protofil ® analysis, described the most relevant absolute concentration, totaling 74 different components, as well as their absolute abundance in the hexane extract. The highest recorded concentration of active ingredients was eucalyptol (28.6%), followed by chavicol-methyl-ether (28.1%), while the lover concentration of thymol (7.19%), and gamma-Terpinen (5.86%), was also present in the investigated sample (Table 6).

Statistical Multivariate Principal Component Analysis (PCA) of GC Data
PCA reveal that Protofil ® had a significant similarity with yarrow, more limited similarity with thyme and basil, and little similarity with dandelion. Data variation described 53% for PC1, and 34% for PC2 and, for this classification, the chavicol-methyl-ether, and α-muurolen were essential, as a first main component. Eucalyptol concentration had significance, as a following main component ( Figure 6). Analyzing the outcome of the chromatographic analyses used in this study, HPLC and GC-MS, an approximately equal proportion for the four distinct studied components in the Protofil ® association it was ascertained.

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. Analyzing the outcome of the chromatographic analyses used in this study, HPLC and GC-MS, an approximately equal proportion for the four distinct studied components in the Protofil ® association it was ascertained.

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].

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.