Mentha piperita: Essential Oil and Extracts, Their Biological Activities, and Perspectives on the Development of New Medicinal and Cosmetic Products.

This review aims to analyze Mentha piperita L. as a potential raw material for the development of new health-promoting products (nutraceuticals, cosmetics, and pharmaceutical products). A lot of scientific publications were retrieved from the Scopus, PubMed, and Google Scholar databases which enable the study and generalization of the extraction procedures, key biologically active compounds of essential oil and extracts, biological properties, and therapeutic potential of M. piperita, along with perspectives on the development of its dosage forms, including combinations of synthetic active substances and herbal preparations of M. piperita. The results of this review indicate that M. piperita is a source rich in phytoconstituents of different chemical nature and can be regarded as a source of active substances to enhance health and to develop medicinal products for complementary therapy of various conditions, especially those related with oxidant stress, inflammation, and moderate infections. Essential oil has a broad spectrum of activities. Depending on the test and concentration, this essential oil has both anti- and prooxidant properties. Gram-positive bacteria are more sensitive to the essential oil of M. piperita than Gram-negative ones. This review also considered some facets of the standardization of essential oil and extracts of M. piperita. Among the identified phenolics of extracts were caffeic acid, rosmarinic acid, eriocitrin, luteolin derivates (luteolin-7-O-rutinoside, luteolin-7-O-glucoronide), and hesperidin. The concentration of these phenolics depends on the solvent used. This review also considered the relationships between the chemical component and biological activity. The results showed that the essential oil and extracts reduced inflammation in vitro by inhibiting the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and in vivo by reducing the paw edema induced using carrageenan injection in rats. Therefore, herbal preparations of M. piperita are promising medicinal and cosmetic preparations for their usage in skincare and oral cavity care products with antimicrobial, anti-inflammatory, and wound-healing properties. This plant can also be regarded as a platform for the development of antibacterial preparations and combined anti-inflammatory and cardioprotective medicinal products (synthetic active substances plus herbal preparations). This review could be considered for the justification of the composition of some medicinal products during their pharmaceutical development for writing a registration dossier in the format of Common Technical Document.

Cornelian Cherry (Cornus mas) Powder as a Functional Ingredient for the Formulation of Bread Loaves: Physical Properties, Nutritional Value, Phytochemical Composition, and Sensory Attributes.

In the current study, Cornelian cherry powder (CCP, Cornus mas) was investigated as a functional ingredient for bread production. Experimental bread loaves were prepared using five levels of CCP (0, 1, 2, 5, and 10% w/w) to replace wheat flour in bread formulation. The final products were analyzed regarding their proximate composition, content of selected biologically active substances, antioxidant activity (AA), volume, and sensory attributes. Increasing the incorporation of CCP led to significantly (p < 0.05) higher concentrations of carbohydrate, ash, energetic value, total polyphenols, phenolic acids and AA, and reduced fat and protein contents (p < 0.05). Moreover, up to 5% addition of CCP positively affected the volume (642.63 ± 7.24 mL) and specific volume (2.83 ± 0.02 cm3/g) of bread loaves, which were significantly (p < 0.05) higher compared to the control (no addition of CCP; 576.99 ± 2.97 mL; 2.55 ± 0.002 cm3/g). The sensory attributes chewiness, crumb springiness, bitterness, and sourness had lower scores (p < 0.05) in bread formulated with 10% CCP compared to the control. Overall, results show that the bread loaves produced with up to 5% CCP addition were considered the preferred formulation among the experimental samples tested, taking into consideration their composition, bioactive content, sensory, and physical properties.

Effect of Priestia endophytica on the metabolites accumulation in chicory and lettuce plants cultivated in vitro.

The influence of the culture medium without bacterial cells, obtained after the cultivation of endophytic bacteria Priestia endophytica UCM B-5715, on the growth and synthesis of some metabolites in lettuce and chicory seedlings under in vitro conditions was studied. Bacteria were cultivated in liquid LB medium at 37 ºC for 24 h with periodic stirring. The culture fluid was separated from the cell biomass. For preparing the test solution, the supernatant was sterilized by filtration through a filter with a pore diameter of 0.2 µm (Sartorius, Minisart) and diluted with sterile distilled water. The 20% culture fluid (30 µl/plant) was applied to 3-day-old seedlings. In 28 days root and shoot weights of treated chicory plants were 54.3 ± 6.9 and 260.0 ± 20.2 mg, respectively (8.0 ± 0.7 and 91.4 ± 7.0 mg for the control plants). Total flavonoid content and antioxidant activity increased only in chicory plants after the addition of the test solution. Significant changes in the metabolism of treated plants were detected. In the treated lettuce plants asparagine content increased compared to the control (90 vs 22 µg/g, p < 0.1). The median content of fructose was also higher in treated lettuce and chicory plants (1469 vs 73 µg/g and 2278 vs 1051 µg/g). Therefore, the use of culture fluid obtained after the cultivation of P. endophytica UСM B-5715 stimulated the growth of lettuce and chicory plants, affecting the synthesis of some compounds in single-treated plants. These results indicate the potential of compounds excreted during bacterial growth to create natural growth stimulators.

The consumption of sea buckthorn (Hippophae rhamnoides L.) effectively alleviates type 2 diabetes symptoms in spontaneous diabetic rats.

Sea buckthorn (Hippophae rhamnoides L.) is described by various beneficial effects as it contains several bioactive substances characterized by antioxidant effects. These effects are closely related to the reduction of oxidative stress that is involved in the development of the disease. One such diseases is Diabetes mellitus, the prevalence of which is growing and is associated primarily with diet, lack of exercise and/or genetics. This study intends to examine the effects of sea buckthorn and metformin on body weight, water and feed intake, glycaemia, insulinemia, sorbitol accumulation and cataract development in Zucker diabetic fatty rats, which represent an animal model of type 2 Diabetes mellitus, as well as to characterize the individual content of bioactive substances and the antioxidant activity of sea buckthorn. Particular concentrations were applied (500 and 1000 mg.kg-1 body weight of sea buckthorn, and combinations with 150 mg.kg-1 body weight of metformin) by gastric gavage. The total antioxidant capacity and bioactive compounds were determined by spectrophotometric analysis. The best results of the study showed suppression of hyperglycaemia, water intake, decreased sorbitol levels in the lens of the eyes after sea buckthorn treatment. Determination of bioactive compounds showed significantly higher values in dry berries when compared to fresh berries of sea buckthorn and high total antioxidant capacity. Our results represent an interest in sea buckthorn and its potential use in the treatment of Diabetes mellitus as well as other experimental studies.

Spectroscopic Discrimination of Bee Pollen by Composition, Color, and Botanical Origin.

Bee pollen samples were discriminated using vibrational spectroscopic methods by connecting with botanical sources, composition, and color. SEM and light microscope images of bee pollen loads were obtained and used to assess the botanical origin. Fourier transform (FT) mid- and near-infrared (FT-MIR, FT-NIR), and FT-Raman spectra of bee pollen samples (a set of randomly chosen loads can be defined as an independent sample) were measured and processed by principal component analysis (PCA). The CIE L*a*b* color space parameters were calculated from the image analysis. FT-MIR, FT-NIR, and FT-Raman spectra showed marked sensitivity to bee pollen composition. In addition, FT-Raman spectra indicated plant pigments as chemical markers of botanical origin. Furthermore, the fractionation of bee pollen was also performed, and composition of the fractions was characterized as well. The combination of imaging, spectroscopic, and statistical methods is a potent tool for bee pollen discrimination and thus may evaluate the quality and composition of this bee-keeping product.

Heavy Metals Presence in the Soil and Their Content in Selected Varieties of Chili Peppers in Slovakia.

Capsicum chili peppers are popular vegetables in Slovakia. They provide a supply of health-promoting substances, but contaminated vegetables can pose a serious health risk to the people who consume them. Therefore, the aim of this study was to determine the content of heavy metals (Mn, Zn, Cr, Cu, Ni, Cd, Pb and Hg) in the soil as well as in selected varieties of the genus Capsicum grown in southern Slovakia. The results were compared with the limit values given by the Law no. 220/2004 (valid in the SR) as well as threshold values proposed by the European Commission (EC) (2006). The gained result showed that the total content of Cd (1.64 mg/kg) as well as the available mobile forms of Cd (0.12 mg/kg) and Pb (0.26 mg/kg) was exceeded on the soil on which Capsicum cultivars were grown. The limit values of other monitored heavy metals (Mn, Zn, Cr, Cu, Ni, and Hg) were not exceeded. The studied species of the genus Capsicum did not accumulate monitored heavy metals. It can be stated that consumption of chili peppers does not pose any risk for human health.

Evaluation of the antioxidant potential of Capsicum annuum L., C. baccatum L. and C. chinense Jacq. cultivars.

BACKGROUND: Economically important vegetables are a strong source of antioxidants with different characteristics. Capsicum L. (pepper) is an important agricultural plant because of its economical, medicinal, and nutritional values. METHODS: This study aimed to test antioxidant parameters in the fruits of 9 cultivars of Capsicum annuum L. (CA 01-09), 7 cultivars of C. baccatum L. (CB 01-07), and 11 cultivars of C. chinense Jacq. (CC 01-11). The antioxidant activity of the investigated Capsicum cultivars was measured, along with the free radical scavenging activity (FRSA), using the DPPH method, and the molybdenum reducing power (MRP) was expressed as mg TE (Trolox equivalent) per g of DW (dry weight). Total polyphenol content (TPC), expressed as mg GAE (gallic acid equivalent) per g of DW, total flavonoid content (TFC), expressed as mg QE (quercetin equivalent) per g of DW, and total phenolic acid content (TPAC), expressed as mg CAE (caffeic acid equivalent) per g of DW, were the basic antioxidant parameters of antioxidant activity in this study. RESULTS: All investigated Capsicum extracts exhibited FRSA from 1.45 (CC-06) to 8.21 (CC-05) mg TE/g and MRP from 24.84 (CA-06) to 198.21 (CB-07) mg TE/g. The TPC of the tested extracts ranged from 10.13 (CB-03) to 38.68 (CB-07) mg GAE/g. The TFC of the studied samples showed values from 5.73 (CB-03) to 27.32 (CB-07) mg QE/g and TPAC from 2.24 (CB-03) to 13.07 (CC-07) mg CAE/g. A very strong correlation was found in the investigated cultivars between TPC and TPAC (r = 0.932, 0.839 and 0.848, respectively), and between TPC and TFC (r = 0.921, 0.982 and 0.939, respectively). Very strong relations were also found between TPC and FRSA (r = 0.820) in the C. annuum cultivars and between TPC and MRP (r = 0.898) in the C. baccatum cultivars. CONCLUSIONS: This study found useful results concerning the antioxidant potential of the fruits of Capsicum cultivars. The data obtained demonstrate the strong antioxidant activity of cultivars of Capsicum, which can be used in the food industry because of the commercial importance of these fruits.

Assessment of antioxidant and antimicrobial activities of Crambe spp. during vegetation.

BACKGROUND: The search for new plant raw material as a potential source of antioxidants is still ongoing. This study aimed to evaluate the antioxidant and antimicrobial capacity of the plant raw material of Crambe spp. during vegetation. METHODS: The free radical scavenging activity and molybdenum reducing power of the extracts were used to determine antioxidant activity. The quantification of polyphenol compounds was conducted with Folin-Ciocalteu reagent. Flavonoids and phenolic acids were also determined. The disc diffusion method was used to determine antimicrobial activity. RESULTS: It was determined that the free radical scavenging activity, assessed using the DPPH-method, was 4.38-8.20 mg TE/g DW, the molybdenum reducing power of the extracts was 40.07-129.12 mg TE/g DW, total polyphenol content was 20.24-70.88 mg GAE/g DW, total flavonoid content was 5.73-29.92 mg QE/g DW, and phenolic acid content was 3.00-10.63 mg CAE/g DW. Antimicrobial activity depended on the stage of growth and the part of the plant used. CONCLUSIONS: Crambe spp. possess the antioxidant and antimicrobial potential to mean that they could be used in pharmaceutical studies and the food industry.

Comparison of Heavy Metal Intake by Different Species of the Genus Allium L.

Allium vegetables belong to the most widespread and commonly grown vegetables in Slovakia. In this study, we dealt with the content of selected heavy metals (Zn, Mn, Cr, Cu, Ni, Pb, and Cd) in cultivated garlic (Záhorský, Lukan, Makoi), onion (Amika, Red Matte, Diamant), and leek (Bavaria, Golem, Tango). We determined the total content of heavy metals (aqua regia), and available-mobile forms of heavy metals (1 M NH4NO3) in soils. Analytical final procedure was flame AAS. The results were compared with the limit values given by the Law no. 220/2004 (valid in the SR) as well as threshold values proposed by the European Commission (EC) (2006). Gained result showed that the total content of Cd (1.26 mg.kg-1) as well as the available mobile forms of Cd (0.16 mg.kg-1) and Pb (0.64 mg.kg-1) was exceeded on the soil on which Allium species were grown. Determined contents of heavy metals in garlic were in the range of 0.03-0.07 mg Cd.kg-1 FM (fresh matter), 0.02-0.11 mg Pb.kg-1 FM (fresh matter), in onion ranged from ND (not detected) to 0.03 mg Cd.kg-1 FM (fresh matter), 0.09-0.2 mg Pb.kg-1 FM (fresh matter), and in leek were in the range of, 0.07-0.13 mg Cd.kg-1 FM (fresh matter), and 0.3-0.5 mg Pb.kg-1 FM (fresh matter). Based on the obtained BAF values, we can conclude that the genus Allium does not belong to the accumulator of evaluated hazardous metals. Safe food production requires constant monitoring of hazardous metals in raw materials of plant origin, as these metals can have serious negative effects on human health.

Biological properties of sea buckthorn (Hippophae rhamnoides L.) derived products.

BACKGROUND: Sea buckthorn is a good and possible source of a wide range of bioactive compounds with     a positive effect on the human body, especially polyphenols and carotenoids. METHODS: The present study aimed to evaluate the antioxidant and antimicrobial activities of Hippophae rhamnoides L. products - 100% oil, 100% juice, dry berries, and tea (dry berries, leaves, and twigs). The antioxidant activity was determined by DPPH radical scavenging and molybdenum reducing antioxidant power methods. The content of phytochemicals (polyphenols, flavonoids, carotenoids) was measured by colorimetric methods. The detection of antimicrobial activity was carried out using the disc diffusion method and an evaluation of minimal inhibition concentration against three species of Gram-negative bacteria: Escherichia coli CCM 3988, Salmonella enterica subsp. enterica CCM 3807, Yersinia enterocolitica CCM 5671, and three Gram-positive bacteria: Bacillus thuringiensis CCM 19, Listeria monocytogenes CCM 4699, Staphylococcus aureus subsp. aureus CCM 2461. RESULTS: All the tested sea buckthorn products had a high antioxidant activity. The highest content of total polyphenols (204.26 mg GAE/g), flavonoids (30.00 mg QE/g), and carotenoids (0.34 mg/g) were identified in 100% sea buckthorn juice. Maximal values of the antioxidant activity were found using the DPPH method for 100% oil (8.75 mg TE/g) and molybdenum reducing power for tea (196.41 mg TE/g). All the tested products showed strong antimicrobial activity against the tested bacteria, confirmed by both methods - disc diffusion (especially for Yersinia enterocolitica CCM 5671) and minimal inhibitory concentration. CONCLUSIONS: The pronounced antioxidant and antibacterial properties of sea buckthorn products indicate the importance of sea buckthorn application in health promotion and agricultural practice.

Evaluation of the antioxidant activity and phenolic content of Chinese quince (Pseudocydonia sinensisSchneid.) fruit.

BACKGROUND: Neglected and underutilized plant species could serve as a valuable source of natural bioactive compounds. The objective of this study was to evaluate the biological activity of Chinese quince (Pseudocydonia sinensis Schneid) genotypes of Ukrainian and Slovak origin. METHODS: The content of the total antioxidant activity (DPPH method and molybdenum reducing antioxidant power), total polyphenol, flavonoid and phenolic acid compounds in the pulp and peel of Chinese quince were compared across five genotypes from Slovakia and three from Ukraine. RESULTS: All tested samples exhibited DPPH• radical scavenging activities with values from 6.17 to 9.56 mg TEAC (Trolox equivalent antioxidant capacity) per gram of dry matter (DM). Antioxidant activity, measured using the molybdenum reducing antioxidant power method, ranged from 69.82 to 225.04 mg TEAC per gram of DM. Total polyphenol content was from 34.73 to 82.02 mg GAE (gallic acid equivalent), while total flavonoid content was from 0.50 to 26.72 mg QE (quercetin equivalent) per gram DM. Phenolic acid content varied from 1.12 to 8.39 mg CAE (caffeic acid equivalent) per gram DM. The peel extracts contained the highest content of bioactive compounds when compared with the pulp extract (from 15.30 to 32.60%). All observed parameters differed significantly between the genotypes. Strong positive correlations (p ≤ 0.05) were observed between the content of phenolic acids and flavonoids in the peel in plants from Slovakia (r = 0.951, r = 0.928, respectively); between the phenolic acid and antioxidant capacities detected using the MRP method – r = 0.950 and r = 0.955 for peel and pulp, respectively; between the determination of antioxidant activity by the DPPH and MRP methods in the peel and pulp in plants from Ukraine (r = 0.986, r = 0.998, respectively). Significantly positive correlations were found between all the parameters in the samples of Ukrainian origin. CONCLUSIONS: The results showed that all fruit extracts exhibited strong antioxidant activities, which generally correlated positively with the total phenolic content. This study demonstrates that Chinese quince fruit grown in Ukraine and Slovakia is a perspective source of valuable polyphenol content with high antioxidant activity and is a valuable fruit for use in the agriculture and food industries.

Cornelian cherry fruit improves glycaemia and manifestations of diabetes in obese Zucker diabetic fatty rats.

Cornelian cherry (Cornus mas L.) was in the past frequently used in Slovak Republic; meanwhile fell into oblivion despite the fact that it is known as antidiabetic supplement. However, there is no research investigated its effect on animal model of Diabetes mellitus (DM) 2 type as it is Zucker diabetic fatty (ZDF) rats. Thus, the aim of this study was to determine the effect of C. mas fruit given orally on the development of DM symptoms in ZDF rats. In the experiment male ZDF rats (fa/fa) and their age-matched non-diabetic lean controls (fa/+) were used aged 12 weeks. Male ZDF rats were administered C. mas in two doses (500 and 1000 mg/kg body weight) using a gastric gavage for 10 weeks. One group of diabetic animals served as positive control and received only distilled water. We found significant decrease of glucose level after oral administration of C. mas in dose of 1000 mg/kg bw in pre-diabetic state of animals (until 7th week of the experiment) and significant restriction of water intake in both C. mas groups against the diabetic control. We presume that the higher dose of Cornelian cherry could be beneficial and helpful in prevention of diabetic symptoms when consumed regularly in young animals.

Flavonoid content and antioxidant activity of Artemisia vulgaris L. "hairy" roots.

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris "hairy" roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe3+ as compared to the non-transformed plants. Some "hairy" roots accumulated flavonoids up to 73.1 ± 10.6 mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4 ± 5.0 mg RE/g DW). DPPH-scavenging activity of some "hairy" root lines was 3-3.8 times higher than such one of the roots of the control plants. The Fe3+-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most "hairy" root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe3+ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p ≥ 0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris "hairy" roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.

Antioxidant activities and phenolic compounds in fruits of various genotypes of American persimmon (Diospyros virginiana L.).

BACKGROUND: American persimmons (Diospyros virginiana L.) are known as a widespread cultivar which were traditionally used by Native Americans as a food source, and since ancient times have been used in folk medicine. The objective of this study was to evaluate the antioxidant activity and phenolic content of Diospyros virginiana genotypes. METHODS: The content of the total antioxidant activity and phenolic compounds from the fruits of the American persimmon (Diospyros virginiana L.) of six genotypes were compared. Antioxidant activity (AOA) was measured using three different photometric methods – DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) and FRAP (ferric-reducing antioxidant power). Total phenolic content (TPC) was evaluated using Folin-Ciocalteu reagent assay. RESULTS: The results for AOA (μMol Trolox/g) determined by the DPPH method varied from 51.68 (DV-05) to 100.87 (DV-03), those obtained by the ABTS method varied from 65.40 (DV-05) to 142.26 (DV-03), and those obtained by the FRAP method varied from 45.06 (DV-05) to 109.30 (DV-03). The results for TPC varied from 590.75 ±27.98 mg/100 g (DV-05) to 1325.12 ±77.30 mg/100 g (DV-03). The highest results for AOA and TPC were achieved for the fruits of genotypes DV-03 and DV-05. A positive linear correlation was found between antioxidant activity and total phenolic content in the examined plant material. CONCLUSIONS: The results showed that all fruit extracts exhibited strong antioxidant activities, which generally correlated positively with the total phenolic content. This study demonstrates the potential of the fruits of Diospyros virginiana grown in Ukraine as a possible source of valuable polyphenol content, with high anti- oxidant activities and health-promoting properties. The high contents of phenolic compounds and significant linear correlation between the values of the concentration of phenolic compounds and antioxidant activity indicated that these compounds contributed to the strong antioxidant activity.

Optimization of ultrasound-assisted extraction of bioactive compounds from wild garlic (Allium ursinum L.).

Ultrasound-assisted extraction was used for extraction of bioactive compounds and for production of Allium ursinum liquid extract. The experiments were carried out according to tree level, four variables, face-centered cubic experimental design (FDC) combined with response surface methodology (RSM). Temperature (from 40 to 80 °C), ethanol concentration (from 30% to 70%), extraction time (from 40 to 80 min) and ultrasonic power (from 19.2 to 38.4 W/L) were investigated as independent variables in order to obtain the optimal conditions for extraction and to maximize the yield of total phenols (TP), flavonoids (TF) and antioxidant activity of obtained extracts. Experimental results were fitted to the second order polynomial model where multiple regression and analysis of variance were used to determine the fitness of the model and optimal condition for investigated responses. The predicted values of the TP (1.60 g GAE/100 g DW), TF (0.35 g CE/100 g DW), antioxidant activity, IC50 (0.71 mg/ml) and EY (38.1%) were determined at the optimal conditions for ultrasound assisted extraction: 80 °C temperature, 70% ethanol, 79.8 min and 20.06 W/L ultrasonic power. The predicted results matched well with the experimental results obtained using optimal extraction conditions which validated the RSM model with a good correlation.

Variability of Corylus avellana, L. CorA and profilin pollen allergens expression.

Corylus avellana is the source of inhalant allergies induced by hazel pollen as well as food allergies induced after ingestion of hazelnuts. In this study, real-time PCR approach was used to analyse expression of hazel pollen allergens on the molecular level. Relative quantity of hazelnut allergens Corylus avellana, L. CorA and Corylus avellana, L. pollen profiling in samples from different Ukraine areas were determining and comparing. Differences among the levels of both analysed allergen transcripts were found for hazel CorA and profillin. In both cases, the expression within the urbanized growth conditions was higher when compared to the sample from village area. The average expression for CorA was 0.84 times higher than for profilin and the results are very variable depending on the place of growth. Expression levels here were within the range of 2.957 up to the 52.936. Profilin expression was the highest in the sample from the polluted place of growth-cement plant area with the value of 52 times higher when compared to the sample from the village area. In this study, comparison of expression levels of hazel CorA and profiling pollen allergens was performed for the first time. Real-time PCR assay developed in this study proved the sensitivity for detection of the changes of the hazel pollen allergens expression levels and could benefit labs by fast and reproducible detection method of these allergens.

Changes in expression of BetV1 allergen of silver birch pollen in urbanized area of Ukraine.

The aim of the study was to determinate the level of expression of silver birch allergen Betv1 in pollen samples from different Ukraine areas by RT-qPCR SYBR Green assay. Protocol for quantifying the expression of Betv1 allergen was developed when testing of three housekeeping genes-cyclophylin, alpha-tubulin and transcription factor CBF1. Samples from urbanized area was analysed by real-time PCR when a sample from forest growth conditions was used as a calibrator. Real-time PCR based quantifying of Betv1 provides a useful method for rapid and sensitive analyses of this silver birch allergen. Our results show higher expression levels in samples from central parts of urbanized area as housing estates when compared to the samples from borders of the urbanized area.