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

Abstract

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.

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)):

$$\mathrm{Relative}\mathrm{concentration}(\%)=\frac{\mathrm{Area}\left(\mathrm{compound}\right)}{{{\displaystyle \sum }}^{}\mathrm{Area}}\times 100$$

(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.

3. Results

3.1. 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. Concentrations of compounds (mean of four replicates), expressed as mg flavonoids/mL, determined from HPLC analyzes.

Nr.	Compound	RT (min)	Conc. (Ba) (mg/mL)	Conc. (Th) (mg/mL)	Conc. (Ya) (mg/mL)	Conc. (Da) (mg/mL)	Conc. (PF) (mg/mL)
1	Rutin	2–3.6	1.843	3.437	2.543	1.049	1.540
2	Quercetin	4.2	0.017	9.379	0.232	0.029	0.061
3	Chrysene	9.8	0.027	0.012	0.004	0.000	0.007
4	Flavone	15.8	0.000	0.000	0.010	0.016	0.002

RT-retention time; Conc.-concentration; Ba-basil; Th-thyme; Ya-yarrow; Da-dandelion; PF-Protofil^®.

.

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.

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 (r² = 0.98).

3.2. 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. Results of GC-MS analysis for Ocimum basilicum (Basil) samples.

No.	A Compound Identified by GC-MS	RT (min)	Kovats Index	Relative Concentration (%)
1	Column	5.198	948	0.00
2	alpha-Pinene	5.686	974	1.29
3	Column	5.944	987	0.00
4	Column	6.173	999	0.00
5	Camphene	6.250	1002	0.15
6	beta-Phellandrene	6.796	1028	0.50
7	beta-Pinene	6.955	1035	1.42
8	Sabinen/beta-pinene	7.037	1039	0.64
9	Bicyclo [3.1.0]hexane, 4-methyl-1-(1-methylethyl)-, didehydro	7.584	1063	0.05
10	Terpinolen	7.830	1074	0.06
11	Limonene	8.101	1086	1.10
12	Dihydrocarveol	8.195	1090	0.08
13	3-Carene/alpha-pinene	8.318	1095	0.27
14	Eucalyptol	8.582	1106	7.31
15	gamma-Terpinen	9.005	1124	0.14
16	Terpinolen	9.740	1155	0.13
17	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-	10.116	1171	0.09
18	Linalool	10.192	1174	0.31
19	Fenchone	10.739	1198	0.08
20	Tetrahydroactinidiolide	11.015	1210	0.05
21	Camphor	12.607	1280	0.08
22	Caprylyl acetate	12.683	1283	0.02
23	Fenchyl acetate	13.136	1303	0.05
24	Chavicol methyl ether	13.870	1337	54.91
25	Bornyl acetate	15.034	1391	0.04
26	alpha-Cubebene	15.239	1400	0.04
27	Copaene	16.015	1437	0.30
28	Di-epi-alpha-cedrene	16.138	1443	0.28
29	beta-Bourbonene	16.362	1454	0.24
30	beta-Elemene	16.479	1459	0.58
31	alpha-Bergamotene	16.984	1484	0.15
32	alpha-Bergamotene	17.290	1498	5.79
33	Caryophyllene	17.401	1504	6.75
34	trans-Caryophyllene/Isocaryophyllene	17.684	1518	0.25
35	beta-Farnesene	17.778	1522	1.74
36	Humulene	18.300	1548	1.88
37	gamma-Muurolene	18.436	1555	0.22
38	(Z)-beta-Farnesene	18.706	1568	0.48
39	Germacrene D	18.906	1578	3.41
40	alpha-Himachalene	19.088	1587	0.54
41	Eremophilene	19.293	1598	0.11
42	Elixene	19.358	1601	0.23
43	beta-Cedrene	19.599	1613	0.10
44	gamma-Cadinene	19.658	1616	0.75
45	beta-Cadinene	19.758	1621	0.19
46	1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z-	19.928	1630	6.33
47	Calamenene	20.275	1648	0.09
48	Caryophyllene oxide	21.790	1728	0.02
49	Palmitic acid	27.519	2068	0.15
50	Ethyl palmitate	27.584	2072	0.41
51	Column	30.163	2262	0.00
52	Arachidic acid	30.598	2299	0.10
53	Ethyl linolenate	30.980	2333	0.08
54	Column	31.755	2407	0.00
55	Column	32.214	2456	0.00
56	Column	33.230	2577	0.00

RT-retention time.

).

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. Results of GC-MS analysis for Thymus vulgaris (Thyme) samples.

No.	A Compound Identified by GC-MS	RT (min)	Kovats Index	Relative Concentration (%)
1	solvent	5.254	951	0.00
2	alpha-Thujene	5.454	962	2.06
3	alpha-Pinene	5.683	974	2.94
4	Camphene	6.247	1002	1.39
5	beta-Terpinen	6.8	1028	0.05
6	beta-Pinene	6.952	1035	0.58
7	beta-Pinene	7.035	1039	2.51
8	alpha-Thujene	7.581	1063	0.43
9	alpha-Terpinen	7.828	1074	2.14
10	Limonene	8.104	1086	0.99
11	beta-Phellandrene	8.321	1095	0.45
12	Eucalyptol	8.556	1105	57.63
13	gamma-Terpinen	9.015	1124	15.03
14	Terpinolen	9.743	1155	0.19
15	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-	10.113	1171	0.11
16	Dehydro-p-cymene	10.583	1191	0.11
17	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-	11.018	1210	0.06
18	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, cis-	12.616	1280	0.18
19	Methyl chavicol	13.874	1337	0.14
20	Thymol methyl ether	13.991	1342	1.68
21	2-Isopropyl-1-methoxy-4-methylbenzene	14.15	1350	1.61
22	Bornyl acetate	15.031	1391	0.06
23	Ylangene	15.883	1431	0.06
24	Copaene	16.012	1437	0.20
25	beta-Bourbonene	16.359	1453	0.14
26	Ylangene	17.293	1499	0.07
27	Caryophyllene	17.387	1503	5.10
28	Alloaromadendren	17.716	1519	0.15
29	Humulene	18.292	1548	0.20
30	gamma-Muurolene	18.686	1567	0.48
31	alpha-Muurolene	18.803	1573	0.07
32	Germacrene D	18.903	1578	0.07
33	alpha-Muurolene	19.22	1594	0.57
34	gamma-Cadinene	19.661	1617	0.61
35	beta-Cadinene	19.761	1622	0.76
36	alpha-Muurolene	20.107	1639	0.07
37	Calamenene	20.272	1648	0.30
38	Caryophyllene oxide	21.788	1728	0.15
39	5,9,9-Trimethyl-spiro[3.5]non-5-en-1-one	27.029	2035	0.06
40	Palmitic acid, ethyl ester	27.605	2073	0.49
41	Cholesterol, trifluoroacetate	30.677	2306	0.00
42	Linolenic acid, methyl ester	31.006	2335	0.11
43	2,4,4,6,6,8,8-Heptamethyl-1-nonene	32.699	2511	0.00

RT-retention time.

).

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. Results of GC-MS analysis for Achillea millefolium (Yarrow) samples.

No.	A Compound Identified by GC-MS	RT (min)	Kovats Index	Relative Concentration (%)
1	alpha-Pinene	5.681	974	0.22
2	Camphene	6.251	1002	0.78
3	Yomogi alcohol	7.42	1056	0.26
4	3-Thujene	7.59	1063	0.17
5	Terpinolen	7.837	1074	0.46
6	4-Oxo-beta-isodamascol	7.913	1077	0.62
7	beta-Terpinen	8.319	1095	0.17
8	Eucalyptol	8.577	1106	25.14
9	2-Carene	9.012	1124	1.12
10	2-Norpinanol, 3,6,6-trimethyl-	9.652	1151	0.43
11	3-Thujanone	10.91	1205	3.17
12	Isopulegol	11.021	1210	0.69
13	alpha-Thujone	11.133	1215	0.79
14	4-Oxo-beta-isodamascol	11.679	1239	0.85
15	cis-Sabinol	11.779	1243	0.37
16	Verbenyl ethyl ether	11.867	1247	0.79
17	Lavandulol	12.061	1255	0.57
18	Lavandulol	12.208	1262	0.79
19	4-Oxo-beta-isodamascol	12.42	1271	0.58
20	Camphor	12.596	1279	37.52
21	E-3,5-Dimethylhex-2-en-1,2-dicarboxylic acid	13.031	1299	1.25
22	Isobornyl formate	13.142	1304	1.01
23	Chavicol methyl ether	13.877	1337	3.27
24	trans-Chrysanthenyl Acetate	14.288	1356	0.21
25	Isobornyl acetate	15.028	1390	0.35
26	cis-Carvyl Acetate	16.345	1453	0.36
27	Capric acid, ethyl ester	16.82	1476	1.13
28	9-Cedranone	19.811	1624	0.34
29	Spathulenol	21.656	1721	0.45
30	Caryophyllene oxide	21.785	1728	1.95
31	2-Cyclohexene-1-carboxaldehyde,2,6-dimethyl-6-(4-methyl-3-pentenyl)	22.437	1763	0.16
32	4(equatorial)-n-Propyl-trans-3-oxabicyclo[4.4.0]decane	22.496	1766	0.21
33	gamma-Eudesmol	22.614	1773	1.94
34	beta-Guaiene	22.772	1782	0.58
35	alpha-Eudesmol	23.148	1803	2.47
36	Humulane-1,6-dien-3-ol	23.425	1818	0.78
37	Aristolone	23.742	1836	0.24
38	Ethyl myristate	24.347	1871	0.16
39	Palmitic acid, ethyl ester	27.602	2073	3.37
40	Ethyl Oleate	30.504	2291	0.28
41	Linoleic acid ethyl ester	30.663	2305	2.19
42	Ethyl linolenate	30.992	2334	1.79
43	Column	31.962	2429	0.00
44	Column	32.584	2498	0.00

RT-retention time.

).

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. Results of GC-MS analysis for Taraxacum officinalis (Dandelion) samples.

No.	A Compound Identified by GC-MS	RT (min)	Kovats Index	Relative Concentration (%)
1	Bicyclo[2.1.1]hexan-2-ol, 2-ethenyl-	5.146	945	0.59
2	p-Xylene	5.252	951	2.42
3	Octane, 1-chloro-	5.458	962	0.43
4	alpha-Thujene	5.693	975	0.53
5	Isovaleraldehyde, diethyl acetal	5.945	987	1.72
6	Linalyl propionate	6.239	1002	0.64
7	Pentane, 1,1-diethoxy-	7.003	1037	1.41
8	Eucalyptol	8.572	1106	5.72
9	trans-Verbenol	9.03	1125	0.40
10	Chavicol methyl ether	14.012	1343	0.46
11	Bicyclo[2.2.1]heptane, 2-cyclopropylidene-1,7,7-trimethyl-	17.385	1503	0.66
12	2,3-Dehydro-4-oxo-beta-ionone	19.688	1618	0.50
13	Ethyl laurate	20.863	1679	1.15
14	Ethyl myristate	24.371	1872	2.17
15	Oxirane, 2-methyl-2-(1-methylethyl)-	25.117	1916	0.93
16	Methyl 2-methylhexanoate	26.016	1971	0.71
17	Ethyl palmitate	27.614	2074	62.49
18	Eicosane	28.084	2106	1.31
19	2,6-Pyrazinediamine	30.522	2292	0.55
20	Ethyl stearate	30.622	2301	1.02
21	Ethyl linolate	30.687	2307	8.13
22	Methyl linolenate	31.016	2336	6.06

RT-retention time.

) was recorded, respectively.

The volatile compounds GC-MS analysis is presented in Figure 4 and Figure 5.

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. Results of GC-MS analysis for Protofil^® samples.

No.	A Compound Identified by GC-MS	RT (min)	Kovats Index	Relative Concentration (%)
1	p-Xylene	5.253	951	0.05
2	1-Isopropyl-4-methylbicyclo[3.1.0]hex-2-ene/alpha-Phellandrene	5.458	962	0.72
3	alpha-Pinene	5.687	974	1.20
4	Valeraldehyde, diethyl acetal	5.946	988	0.07
5	Camphene	6.252	1003	0.56
6	beta-Terpinen	6.798	1028	0.07
7	beta-Pinene	6.957	1035	0.39
8	beta-Pinene	7.039	1039	0.95
9	3-Carene	7.585	1063	0.15
10	alpha-Terpinen	7.832	1074	0.24
11	Limonene	8.102	1086	0.55
12	beta-Phellandrene	8.32	1095	0.24
13	Eucalyptol	8.531	1104	28.61
14	gamma-Terpinen	9.013	1124	5.86
15	Terpinolene	9.747	1155	0.10
16	4-Isopropyl-1-methyl-2-cyclohexene-1-ol	10.118	1171	0.08
17	Linalool	10.2	1175	0.44
18	3,4-Dimethylstyrene	10.57	1190	0.08
19	Thujone	10.911	1205	0.12
20	4-Isopropyl-1-methyl-2-cyclohexene-1-ol	11.022	1210	0.07
21	4-Oxo-beta-isodamascol	11.68	1239	0.06
22	1,3-Dioxolane, 2,2-dimethyl-4,5-bis(1-methyl phenyl)-	12.209	1262	0.05
23	Tetrahydroactinidiolide	12.415	1271	0.04
24	Camphor	12.597	1279	1.73
25	E-3,5-Dimethylhex-2-en-1,2-dicarboxylic acid	13.026	1298	0.04
26	alpha-Terpineol	13.138	1304	0.07
27	Chavicol methyl ether	13.784	1333	28.09
28	Thymol methyl ether	13.978	1342	1.43
29	2-Isopropyl-1-methoxy-4-methylbenzene	14.136	1349	0.80
30	Bornyl acetate	15.035	1391	0.09
31	Thymol	15.799	1427	0.05
32	Thymol	15.958	1434	7.19
33	Carvacrol	16.252	1448	0.77
34	Carvacrol	16.739	1472	0.06
35	1-Cyclopropene-1-pentanol, à,î,î,2-tetramethyl-3-(1-methyl phenyl)-	16.992	1484	0.07
36	alpha-Bergamotene	17.274	1498	0.89
37	Caryophyllene	17.386	1503	3.29
38	beta-Farnesene	17.785	1523	0.27
39	Humulene	18.296	1548	0.49
40	gamma-Muurolene	18.696	1568	0.34
41	Germacrene D	18.901	1578	0.35
42	Eugenol methyl ether	18.978	1582	0.83
43	gamma-Cadinene	19.659	1616	0.48
44	beta-Cadinene	19.759	1622	0.40
45	cis-alpha-Bisabolene	19.924	1630	1.21
46	Calamenene	20.27	1648	0.22
47	Ethyl laurate	20.764	1674	0.05
48	Cadala-1(10),3,8-triene	20.952	1683	0.04
49	Spathulenol	21.657	1721	0.07
50	Caryophyllene oxide	21.792	1728	0.55
51	delta-Cadinol	22.127	1746	0.06
52	12-Oxabicyclo[9.1.0]dodeca-3,7-diene, 1,5,5,8-tetramethyl-, [1R-(1R	22.444	1764	0.05
53	Cubenol	22.626	1774	0.04
54	tau-Cadinol	22.779	1782	0.16
55	Cadalene	24.113	1858	0.08
56	Ethyl myristate	24.324	1870	0.15
57	Hexahydrofarnesyl acetone	25.082	1914	0.12
58	Ethyl pentadecanoate	25.987	1969	0.08
59	Naphthalene, 1,2,3,4,4a,5,6,7-octahydro-4a-methyl-	27.027	2035	0.08
60	Ethyl palmitate	27.591	2072	4.45
61	Ethyl (9E)-9-hexadecenoate	27.762	2084	0.13
62	Nonadecane	28.061	2104	0.05
63	cyclopentane carboxylic acid, 4-hexadecyl ester	28.672	2147	0.08
64	Ethyl heptadecanoate	29.125	2181	0.04
65	2-Piperidinone, N-[4-bromo-n-butyl]-	29.565	2214	0.06
66	4-Butoxy-2,4-dimethyl-2-pentene	30.411	2283	0.08
67	Ethyl Oleate	30.494	2290	0.20
68	Ethyl stearate	30.594	2298	0.20
69	9,12-Octadecadienoic acid, ethyl ester	30.652	2304	1.06
70	Linolenic acid, ethyl ester	30.981	2333	1.37
71	Linolenic acid, ethyl ester	31.357	2368	0.05
72	2,4,4,6,6,8,8-Heptamethyl-2-nonene	31.998	2433	0.57
73	(Z)-7-Hexadecenal	32.403	2477	0.04
74	2,4,4,6,6,8,8-Heptamethyl-1-nonene	33.226	2577	0.26

RT-retention time.

).

3.3. 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.

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.