A. Kalashnikov1, H. Voitenko1, O. Losiev2, N. Kurdil1, V. Mikhailov1, I. Holovetskyi2, O. Lutsenko3,
1 State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety”, Ministry of Health of Ukraine, Kyiv
2 National University of Life and Environmental Sciences of Ukraine, Chair of Horse Breeding and Bee Keeping, Kyiv, Ukraine
3 National Researcher Center for Radiation Medicine of NAMS of Ukraine, Laboratory of Food Hygiene and Food Safety, Kyiv, Ukraine
ABSTRACT. In the context of increasing anthropogenic load, the ability of the body to maintain high-level resistance to various harmful factors is essential. Thus, the creation of effective tools that can increase the level of adaptive capacity of the human body under conditions of the environmental hazards is a topical issue of modern medicine.
Objective. Scientific substantiation of the technology of creation of enriched natural honey containing bioactive substances — adaptogens, capable to increase adaptive capabilities of the body under conditions of environmental hazards.
Materials and Methods. The material for this study were Ukrainian breed bees. The process of enriching the natural properties of natural honey was carried out in a natural way through the bee family, by feeding colonies of bees with a special composition of natural origin components based on common oat (Avtina satHva) and milk thistle (Silybum marianum).
Results. The analysis of the regulatory framework governing the use of honey with phytoadditives and bee products in Ukraine has proved that currently there is a lack of scientifically grounded technology of natural honey enrichment, which is realized through the bee family by feeding optimally selected qualitative and quantitative composition of high-quality components of natural origin. The authors proposed a model of toxicological and hygienic evaluation of raw materials and finished product based on enriched natural honey, which allows carrying out toxicological support of the process of creating an innovative product — enriched natural honey, and developing a modern technological standard of the finished product according to the evaluation results. Study results have proved that the most promising medicinal raw material for the creation of enriched natural honey is milk thistle (Silybum marianum) extract.
Conclusion. The results of the first stage of the study confirm the prospect of further research on natural adaptogens for the development of innovative products based on natural honey and bioactive substances of plant origin, which will allow obtaining new useful varieties of honey and significantly improving the economic performance of enterprises producing these products.
Key Words: biotechnologies, honey, bioactive substances, adaptogens.
Introduction. This study was performed at the State Enterprise “L. I. Medved’s Scientific Center of Preventive Toxicology, Food and Chemical Safety” of the Ministry of Health of Ukraine within a framework of scientific and research work: “Scientific substantiation of safety of pesticides and agrochemicals, new technologies, substances, materials, products, environmental objects, food products and food raw materials for human health; development of appropriate medical criteria and parameters (sanitary and epidemiological); sanitary-chemical, toxicological and hygienic evaluation, regulation, rationing” (state registration number 0112U001133), fragment: “Toxicological support for creating innovative dietary supplements — adaptogens capable of enhancing the protective capabilities of the body upon harmful impact of the living environment”, time for completion 2018–2020.
We have selected the topic concerning the study of human adaptive capacity from the point of view of toxicology not by chance. The term “adaptation” in the scientific literature is widely used and involved in absolutely all levels of life organization. In modern conditions of rapidly progressing anthropogenic load, the ability of the body to maintain a high level of resistance to the action of a various harmful factors, in particular those of extreme nature, is essential, therefore, the study of adaptive capacity of a human who is adversely affected by environmental factors is a relevant problem.
It is known that, as a final result, adaptation can form increased or decreased resistance to the harmful factor, which is accompanied by the expansion or narrowing of the range of protective and adaptive capabilities of the body. Therefore, it is necessary to create effective tools that can purposefully influence the level of adaptive capacity of the human body.
Products of plant and animal origin, which are part of the so-called group of natural adaptogens are sufficiently comply with these requirements. The advantage of natural products is the content of a complex of bioactive substances (hereinafter — BAS), in general, in the proportions typical for the human body.
However, the significant successes of phytotherapy and honey therapy [1–6], as well as many aspects of the impact of these methods, remain unknown today.
The unique natural and geographical conditions and cultural traditions have formed Ukraine as a state that traditionally produces a significant amount of honey. In recent years, we have been among the top five manufacturers in the world in terms of quantity of these products. Today, honey quality in Ukraine can be properly controlled, and the requirements of the national standard are sufficiently close to the guidelines of the European Directive [7–8].
In Ukraine, a kind of natural honey — honey with phytoadditives is produced, and it is created on the basis of the addition of dried fruits of subtropical crops (previously boiled in sugar syrup) to the composition of natural honey. There is another technology based on the special feeding of bees with BAS-containing syrups (e.g. rosehip syrup).
Thus, previous developments of honey enriched in Ukraine were aimed at obtaining targeted clinical effects, which were enhanced by the special properties of BAS (vitamin C, iodine, unsaturated fatty acids, essential oils) [9–13]. Although, they did not consider the possibility of creating natural honey with directed adaptogenic properties, which confirms the relevance of these studies.
Study object. Honey from the point of view of the content of high-quality components of natural origin — bioactive substances — adaptogens.
Study scope. Adaptogenic properties of enriched natural honey made by feeding bees with the specially selected qualitative and quantitative composition of components of natural origin.
Objective. Scientific substantiation of the technology of creation of enriched natural honey containing bioactive substances — adaptogens, capable to increase adaptive capabilities of the body under conditions of environmental hazards.
Study task
1. Study of the regulatory framework governing the use of honey with phytoadditives and bee products in human nutrition in Ukraine.
2. Scientific substantiation of the technology of natural honey enrichment, which is realized through the bee family by feeding them the optimum selected qualitative and quantitative composition of high-quality components of natural origin.
3. Development of a model of toxicological support (toxicological and hygienic evaluation) of raw materials and finished product created on the basis of enriched natural honey.
Materials and methods. A literary search was performed, relevant literature sources were selected, and information was summarized that made it possible to achieve the objective of the study. The systematic analysis (study and analysis of sources devoted to the investigation of the role of bioactive substances of natural origin in the treatment and prevention of human diseases), as well as the bibliosemantic method (modern principles, world experience and domestic developments were studied for improving the quality of natural honey by enriching its properties using natural components), were used.
During the study, toxicological study methods were also used, namely: chemical and analytical (chromatography, mass spectrometry), medical and biological statistics.
The material for this study were Ukrainian breed bees. Twelve colonies were formed of them, which comprised 3 study groups (n = 3), the first group was the control group, the second group and the third group were the experimental ones. Each group contained 4 colonies. To form study groups, we used the method of balanced group-analogue, so that after their allocation the difference between the groups at the beginning of the experiment was not greater than 5% by parameters: colony power, number of different-age brood, egg-laying of females bees, feedstock and the rate of its consumption.
Procedure for enriching natural honey. The process of enriching treatment and preventive properties of natural honey was carried out in a natural way through the bee family by feeding bee colonies with the optimally selected qualitative and quantitative composition of high-quality components of natural origin, which have a high level of specific activity and provide balanced application and release of the active substances. The common oat (Avéna satíva) and milk thistle (Silybum marianum) were selected as natural components, and pharmaceutical preparations based on them were used. Biologically active substances of the pharmaceutical products Аvеnа Sativa 10:1 (Naturex) and Milk Thistle Dry Extract were transformed into natural honey using worker bees in order to control the increase in its bioactivity.
The experimental part was performed during active apiary period in 2018 at the apiary of the brood bee breeding nursery Private Enterprise “Prybuzki medobory” of the Region of Khmelnytskyi. Using the conditioned reflex of bees, we have trained them to process prepared feed. For feeding bee colonies, we used a candy of monofloral honey from Robinia 50–55% Вх (Вгіх). Candy was prepared using distilled water. The first experimental group was fed exclusively with honey candy (control) according to the developed schedule, see Table 1.
For the second and third experimental groups, Avena Sativa 10:1 (Naturex) and Milk Thistle Dry Extract were introduced into the main feed, respectively. Honey candy was prepared using distilled water. The following sequence was used for feeding: 150 g of Milk Thistle Dry Extract was dissolved in 1 L of prepared warm water; prepared solution of Milk Thistle Dry Extract + 4 L of Н2О was added to 5 kg of Robinia honey; 50 g of Аvеnа Sativa 10:1 (Naturex) was dissolved in 0.5 L of prepared warm water; prepared solution of Аvеnа Sativa 10:1 + 4.5 L of Н2О was added to 5 kg of Robinia honey. Each group of bee colonies was assessed by a set of biological and economical characters throughout the study.
Table 1
Experimental groups and schedules for feeding bee colonies
Prepared bee colonies were fed with appropriate compositions containing honey during the honey-flowless period. Each bee colony was fed with only one type of solution (feed) according to the above schedule.
The colonies were prepared as follows: all feed frames were temporarily removed from the nest, leaving only the brood (preferably closed) and replaced with empty, clean, light honeycombs. Bees were given as much feed as they could consume per day according to the schedule. Only mature honey was extracted, controlling the entire process with a refractometer. Humidity was maintained below 20%. Medium power bee colonies (8–9 beeways) and those colonies that are kept in vertical type beehouses were used for the production of enriched honey since they are ideal for the production of enriched (multivitaminized) honey by the rapid method. Such a design of beehouse makes it easy to use and access it and, most importantly, there is the ability to control the physiological processes of growth in the number of bees and bringing and storage feed compared to other types. Honey was extracted and then centrifuged. Further, it was refined using special filters. The refined honey was filled in the clean glass containers. Glass or enamelware was used for storage of enriched honey. The enriched honey was stored in clean, dry rooms, protected from direct sunlight, with a humidity of up to 60%, avoiding storage with highly odorous substances. The complete production process took about 5 to 12 days (depending on the quantity and composition of the product).
Results and discussion. According to FAO, Ukraine is one of the world leaders in honey production per capita and export volume, and since 2008 it has been ranked first among European countries and third after China and Argentina. Today, about 400,000 people in Ukraine care for nearly four million beehouses [14–16]. The product line of natural honey, its packaging, marking and storage, sampling and preparation for the study are set out in the State Standards of Ukraine DSTU 4497–2005 “Natural honey. Technical specifications” [9]. The parameters of quality and safety of honey are checked in accordance with the requirements of the national standard DSTU 4497–2005 “Natural honey. Technical specifications”, and in EU countries — according to the Council Directive 2001/110/EC, 96/23/EC, EU Regulations 178/2002, 853/2004, 396/2005 and Codex Alimentarius 12-1981 [7–10].
Honey and bee products are widely used in human nutrition. Honey is known to contain up to 60 different components. Its key active ingredient is easily digested carbohydrates. 100 g of honey contains 335 kcal, that is, it is an important energy product used to quickly restore energy after physical and psychological exertion. The product line of honey-based food is constantly expanding due to the introduction of new technologies, the development of new recipes.
According to DSTU 4497-2005, the quality of natural honey is checked by the following parameters: organoleptic (colour, clarity, consistency, viscosity, crystallization, signs of fermentation, flavour, taste, mechanical impurities); mass fraction of moisture; acidity; method of pollen analysis; mass fraction of reducing sugar and sucrose; diastasis number; the content of hydroxy- or oxymethylfurfural; testing of honey for adulteration (determination of admixtures of sugar syrup, chalk, honeydew in flower honey, presence of starch molasses, determination of naturalness of honey by luminescence analysis, microscopic method of pollen analysis, determination of maturity and dilution with water) and a number of other quality and safety parameters of honey. The national standard of Ukraine for honey with phytoadditives was developed for the first time in 2006 in order to expand the range of new products, this standard is set out in the document “Technical specifications. DSTU 4649:2006” [10].
Previous experience of enriching natural honey with additional bioactive components included an attempt to optimize its chemical composition by adding a finished food product, so as not to lose its basic properties, but rather to improve them [17]. For example, the introduction of pre-boiled in sugar syrup and dried fruits of subtropical crops to the recipe allowed to enrich the product with such valuable micronutrients as potassium, which is found in considerable amounts in figs, vitamin C — in jujube, iodine — in the fruits of feijoa. The introduction of walnuts, the kernels of which contain unsaturated fatty acids, has a positive effect on the biochemical processes in the myocardium, it is known that it is unsaturated fatty acids that trigger metabolic processes in the heart muscle. Introduction of the extract of myrtle leaves to the recipe enhances the antiseptic properties of honey, and rose petals boiled in sugar syrup can enrich the product with rose essential oil with antiseptic and reparative properties.
Despite the considerable experience, the efficiency of the combined use of honey, medicinal plants (MPs) and products based on them is still underestimated. Combinations of honey with syrups or extracts of MPs, such as rose hips, where technology and compounding allow preserving all valuable nutrients and obtain a natural food product with a predetermined composition are the most common.
Honey quality is a concept that covers a fairly wide range of characteristics and properties of this unique product. During the storage of honey, from the moment of extraction, many natural biochemical processes take place in it. This results in constant changes in its composition, and the product is called “living”. It is well known that when processing nectar from flowers, carbohydrates in the stomach of the bee change, and the residual elements of nectar, trace elements, vitamins, essential oils and colouring substances that provide the taste and aroma of honey remain unchanged. However, if the bees preserve such valuable substances as vitamins, then medical honey, in addition to the known curative properties, becomes a multivitamin product also. It was proved that bees play the role of unique “pharmacists”, as a result, there are many considerations to create bee foods enriched with vitamins and other BASs in order to obtain enriched honey. Therefore, unlike the existing technologies of artificial addition of BASs to natural honey, we were offered the technology of enrichment of treatment and preventive properties of natural honey in a natural way. It is realized through the bee family by feeding them the optimum selected qualitative and quantitative composition of high-quality components of natural origin. Such an approach provides a significant effect in the treatment of various diseases, as well as a high level of the specific activity of the tool to balance the introduction and release of active substances.
In order to model the adaptogenic properties of natural honey, we have selected medicinal plants with a scientifically proven adaptogenic action and undoubted clinical efficacy: common oat (Avéna satíva), purple echinacea (Lat. Echinacea purpurea), wormwood (Artemisia vulgaris), milk thistle (Lat. Silybum marianum), common chicory ( Cichorium intybusL). For each specified MP, we have defined BASs content of which should be evaluated in the obtained enriched honey.
The study was divided into 12 main stages, that is, it is formed a comprehensive model of toxicological support or toxicological and hygienic assessment of the process of modelling and creation of an innovative product — enriched natural honey.
At the first stage of the study, the selection of remedial products based on plant raw materials was carried out. Considering a long-term clinical experience of success for certain natural BASs — adaptogens, we have selected common oat (Avéna satíva) and milk thistle ( Silybum marianum) for the enrichment of natural honey. Stages of the study are provided in Table 2.
Table 2
Model of toxicological support (toxicological and hygienic assessment) and main stages of the process for modelling chemical composition of bioactive substances — adaptogens in the composition of natural honey
Note: MPRM, medicinal plant raw material; BAS, bioactive substance
The clinical effect of phytopreparations made on the basis of BASs of plant origin is due to adaptogenic properties having a number of fundamental advantages over chemicals : active ingredients are formed in a living cell , therefore, they are better adapted to functioning of the cells in human body and biochemical processes than synthetic; living cell always contains other substances that enhance or smooth remedial action; medicinal plants have an effect not only on one organ or symptoms but on the whole body — they restore functions of endocrine system , activate immune system , improve body defence, have general curative, antitumour, anti-inflammatory and other actions [5, 6]. Furthermore, advantages of plant BASs include their low toxicity that is especially important in the treatment of chronic diseases, gives the possibility of their long term home-use. Remember that most of them are accessible and inexpensive.
Medicinal products based on milk thistle (Silibinin, Hepabene) have a powerful hepatoprotective effect, normalize metabolic processes in the liver, increase the resistance of hepatocytes to the effects of negative environmental factors, contribute to faster restoration of hepatic parenchyma and its physiological function. Flavonoids of milk thistle, including silibinin, regulate the functional state of the capillary walls, reducing their fragility, improve microcirculation, exhibit anti-allergic, diuretic, antispasmodic, cholesterol-lowering, hypotensive effects, dilate coronary vessels, improve myocardial contractility [18, 20–22]. Hypoazotemic and antitumour activity of milk thistle flavonoids have recently been revealed [19].
The process of natural honey enrichment was carried out in a natural way through the bee colony by feeding the colonies of bees with components of natural origin; Avena Sativa 10:1 (Naturex) and Milk Thistle Dry Extract were introduced to the main feed, respectively. According to the results of the first stage of the study, it was established that bees processed (consume) feed with the same intensity, but bioactive supplements always remained in the feeders in the form of insoluble dry residues, which was not observed in the feeders with the addition of milk thistle extract — Thistle Milk Thistle Extract.
At the second stage of the study, the content of the fed standards in batches of honey was determined. Ferulic acid was chosen as the standard for common oat and silibinin — for milk thistle. The mass concentration of ferulic acid was measured according to EN 15662:2008 [23]. Foods of plant origin – Determination of pesticide residues using GC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE – QuEChERS-method. Limit of quantification of ferulic acid is 0.02 mg/kg. The studies were performed on a Sumadzu LC-30A chromatograph with Sumadzu LCMS-8050 mass detector No. 1083547010US, chromatographic column — Kinetex 2.6 µm Biphlenil, 100*2.1 mm.
The study has established the following: the content of the standard in the obtained honey, which was enriched with Avena Sativa 10:1 (Naturex), was in pitiful concentrations. This did not allow further studies with this substance. On the contrary, the content of the standard in the resulting honey, which was enriched with Milk Thistle Dry Extract, appeared in large quantities, which allowed us to consider milk thistle as a promising material for natural enrichment of honey.
In the future, we are planning a toxicological and hygienic assessment of the finished product. Since honey, like any food product, is characterized by organoleptic and physicochemical quality parameters, it is obligatory to determine honey safety parameters (toxicity, the content of pesticides, antibiotics, radionuclides). At the final stage of the study, it is planned to determine the optimal quantitative composition of bioactive substances — adaptogens in the composition of enriched honey, capable of increasing body adaptation to hazardous environmental factors. Upon completion of the study of the finished product, it is planned to design technical specifications and other regulatory documents for the production and sale of innovative product and dietary supplements made on its basis.
Conclusion
1. Analysis of the regulatory framework governing the use of honey with phytoadditives and bee products in human nutrition has proved that today Ukraine has no scientifically justified technology of natural honey enrichment, which is realized through the bee colony by feeding the optimum selected composition of the components of natural origin.
2. The proposed model of toxicological and hygienic assessment of raw materials and finished product based on enriched natural honey will allow to carry out complex toxicological support of the process of creating an innovative product and to develop a technological standard based on the results of its evaluation
3. To create enriched natural honey, an extract of milk thistle ( Silybum marianum) is promising, as the bioactive components of this plant have characteristics that allow obtaining the final product with a high content of active substances.
4. The use of new forms of enriched honey in Ukraine will allow obtaining a modern tool of individual correction of the adaptive capacity of the human body, namely to simulate the impact on the immunity, metabolism and adaptive capacity of the body, which is adversely affected by environmental factors.
5. The results of the first stage of the study confirm the prospect of developing innovative products based on natural honey and bioactive substances of plant origin, which will allow obtaining new useful varieties of honey and significantly improve the economic performance of enterprises involved in the production of these products.
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Надійшла до редакції 03.09.2019 р.