Investigation of Chemical Constituents and Antioxidant Activity of Biologically Active Plant-Derived Natural Products.

The aim of this publication is to present rapid screening methods (visual/colorimetric) that will enable quick identification of the presence of biologically active compounds in aqueous solutions. For this reason, 26 plant extracts obtained by ultrasound-assisted extraction were analysed for the content of these compounds. Higher plants, used as a raw material for extraction, are common in Europe and are easily available. The article proposes a comparison of various protocols for the identification of various compounds, e.g., phenolic compounds (phenols, tannins, anthocyanins, coumarins, flavones, flavonoids), vitamin C, quinones, quinines, resins, glycosides, sugars. Initial characterisation of the composition of plant extracts using fast and inexpensive methods allows you to avoid the use of time-consuming analyses with the use of advanced research equipment. In addition, the antioxidant activity of plant extracts using spectrophotometric methods (DPPH, ABTS, FRAP assay) and quantitative analysis of plant hormones such as abscisic acid, benzoic acid, gibberellic acid, indole acetic acid, jasmonic acid, salicylic acid, zeatin, zeatin riboside, and isipentenyl adenine was performed. The obtained results prove that the applied visual methods show different sensitivity in detecting the sought chemical compounds. Therefore, it is necessary to confirm the presence or absence of bioactive substances and their concentration using modern analytical methods.

Rice gluten meal as a substitute for soybean meal in the diets for growing rabbits.

The impacts of different dietary levels of rice gluten meal (RGM) on growth performance, digestibility, carcass characteristics, and blood traits of growing New Zealand White (NZW) rabbits were studied. One hundred and twenty, 6 weeks old weaned male rabbits (body weight; 682 [g] ± 23) were randomly allotted into four groups. The control diet contained 160 [g/kg] soybean meal (SBM), while the other three diets were obtained by replacing 40, 80, and 120 [g/kg] SBM with RGM (RGM40, RGM80, and RGM120, respectively). The results showed that RGM contained higher levels of dry matter (DM), crude protein (CP), ether extract (EE), ash, and gross energy than SBM. RGM contained a high level of arginine followed by leucine and valine as essential amino acids and high levels of glutamic, aspartic acid, and alanine as non-essential amino acids. The obtained results showed that the final body weight of rabbits fed diets containing 40, 80, and 120 [g/kg] RGM was higher than those fed the control diet. The daily weight gain of rabbits fed RGM diets increased (p < 0.05) by 10.50%, 6.50%, and 10.00%, respectively, compared to the control group. Rabbits fed RGM80 showed the highest (p < 0.05) digestibility of DM, organic matter (OM), EE, neutral detergent fibre (NDF), and acid detergent fibre (ADF) compared to the other tested levels. Rabbits fed RGM120 had the highest (p < 0.05) digestible energy (DE) and digestible crude protein (DCP) values. RGM inclusion levels of 40 and 80 [g/kg] increased (p < 0.05) plasma total protein and albumin compared to the control group. Rabbits fed a diet containing RGM40 had the highest (p < 0.05) globulin level. The highest (p < 0.05) plasma urea concentration level was measured in the rabbit group fed the RGM120 diet. Conclusively, RGM could be a valuable ingredient for growing rabbits, as at all the tested levels improved growth performance, digestibility, and nutritional values.

Methods for Rapid Screening of Biologically Active Compounds Present in Plant-Based Extracts.

In recent years, there has been an increased interest in products of natural origin. The extraction procedure of bioactive compounds from plant matrices is a crucial step in the development of useful new bioproducts for everyday life. The utilisation of analyses enabling the rapid identification of the presence of a given group of compounds can be helpful in the early stages of the development of new products in order to save time and reduce costs. Within this article, we have presented a comparison of different, accessible methods for the identification of various bioactive compounds, e.g., saponins, carboxylic acids, oils and fats, proteins and amino acids, steroids, and alkaloids in plant-based extracts. Additionally, the multielemental composition of extracts was also examined. The applied methods allowed for confirmation of the presence of biologically active compounds in bio-products obtained by ultrasound-assisted extraction. At a later stage, these procedures should be supplemented by advanced analytical techniques in order to determine the plant chemicals' content qualitatively and quantitatively.

Valorization of Cladophora glomerata Biomass and Obtained Bioproducts into Biostimulants of Plant Growth and as Sorbents (Biosorbents) of Metal Ions.

The aim of this study was to propose a complete approach for macroalgae biomass valorization into products useful for sustainable agriculture and environmental protection. In the first stage, the effects of macroalgal extracts and ZnO NPs (zinc oxide nanoparticles) on the germination and growth of radish were examined. Macroalgal extract was produced from freshwater macroalga, i.e., Cladophora glomerata by ultrasound assisted extraction (UAE). The extract was used to biosynthesize zinc oxide nanoparticles. In germination tests, extracts and solutions of ZnO NPs were applied on paper substrate before sowing. In the second stage, sorption properties of macroalga, post-extraction residue, and ZnO NPs to absorb Cr(III) ions were examined. In the germination tests, the highest values of hypocotyl length (the edible part of radish), i.e., 3.3 and 2.6 cm were obtained for 60 and 80% extract (among the tested concentrations 20, 40, 60, 80, and 100%) and 10 and 50 mg/L NPs, respectively. The highest sorption capacity of Cr(III) ions (344.8 mg/g) was obtained by both macroalga and post-extraction residue at a pH of 5 and initial Cr(III) ions concentration of 200 mg/L. This study proves that macroalgae and products based on them can be applied in both sustainable agriculture and wastewater treatment.

Effect of Botanical Extracts on the Growth and Nutritional Quality of Field-Grown White Head Cabbage (Brassica oleracea var. capitata).

Nutraceuticals and functional foods are gaining more attention amongst consumers interested in nutritious food. The consumption of foodstuffs with a high content of phytochemicals has been proven to provide various health benefits. The application of biostimulants is a potential strategy to fortify cultivated plants with beneficial bioactive compounds. Nevertheless, it has not yet been established whether the proposed higher plants (St. John's wort, giant goldenrod, common dandelion, red clover, nettle, and valerian) are appropriate for the production of potential bio-products enhancing the nutritional value of white cabbage. Therefore, this research examines the impact of botanical extracts on the growth and nutritional quality of cabbage grown under field conditions. Two extraction methods were used for the production of water-based bio-products, namely: ultrasound-assisted extraction and mechanical homogenisation. Bio-products were applied as foliar sprays to evaluate their impact on total yield, dry weight, photosynthetic pigments, polyphenols, antioxidant activity, vitamin C, nitrates, micro- and macroelements, volatile compounds, fatty acids, sterols, and sugars. Botanical extracts showed different effects on the examined parameters. The best results in terms of physiological and biochemical properties of cabbage were obtained for extracts from common dandelion, valerian, nettle, and giant goldenrod. When enriched with nutrients, vegetables can constitute a valuable component of functional food.

Cladophora glomerata enriched by biosorption with Mn(II) ions alleviates lipopolysaccharide-induced osteomyelitis-like model in MC3T3-E1, and 4B12 osteoclastogenesis.

Chronic osteomyelitis, a bone infectious disease, is characterized by dysregulation of bone homeostasis, which results in excessive bone resorption. Lipopolysaccharide (LPS) which is a gram-negative endotoxin was shown to inhibit osteoblast differentiation and to induce apoptosis and osteoclasts formation in vitro. While effective therapy against bacteria-induced bone destruction is quite limited, the investigation of potential drugs that restore down-regulated osteoblast function remains a major goal in the prevention of bone destruction in infective bone diseases. This investigation aimed to rescue LPS-induced MC3T3-E1 pre-osteoblastic cell line using the methanolic extract of Cladophora glomerata enriched with Mn(II) ions by biosorption. LPS-induced MC3T3-E1 cultures supplemented with C. glomerata methanolic extract were tested for expression of the main genes and microRNAs involved in the osteogenesis pathway using RT-PCR. Moreover, osteoclastogenesis of 4B12 cells was also investigated by tartrate-resistant acid phosphatase (TRAP) assay. Treatment with algal extract significantly restored LPS-suppressed bone mineralization and the mRNA expression levels of osteoblast-specific genes such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN), osteopontin (OPN), miR-27a and miR-29b. The extract also inhibited osteoblast apoptosis, significantly restored the down-regulated expression of Bcl-2, and decreased the loss of MMP and reactive oxygen spices (ROS) production in MC3T3-E1 cells induced by LPS. Furthermore, pre-treatment with algal extract strongly decreased the activation of osteoclast in MC3T3-E1-4B12 coculture system stimulated by LPS. Our findings suggest that C. glomerata enriched with Mn(II) ions may be a potential raw material for the development of drug for preventing abnormal bone loss induced by LPS in bacteria-induced bone osteomyelitis.

Astaxanthin and other Nutrients from Haematococcus pluvialis-Multifunctional Applications.

Bioactive compounds of natural origin are gaining increasing popularity. High biological activity and bioavailability, beneficial effects on health and safety of use are some of their most desirable features. Low production and processing costs render them even more attractive. Microorganisms have been used in the food, medicinal, cosmetic and energy industries for years. Among them, microalgae have proved to be an invaluable source of beneficial compounds. Haematococcus pluvialis is known as the richest source of natural carotenoid called astaxanthin. In this paper, we focus on the cultivation methods of this green microalga, its chemical composition, extraction of astaxanthin and analysis of its antioxidant, anti-inflammatory, anti-diabetic and anticancer activities. H. pluvialis, as well as astaxanthin can be used not only for the treatment of human and animal diseases, but also as a valuable component of diet and feed.

Field-Scale Evaluation of Botanical Extracts Effect on the Yield, Chemical Composition and Antioxidant Activity of Celeriac (Apium graveolens L. Var. rapaceum).

The use of higher plants for the production of plant growth biostimulants is receiving increased attention among scientists, farmers, investors, consumers and regulators. The aim of the present study was to examine the possibility of converting plants commonly occurring in Europe (St. John's wort, giant goldenrod, common dandelion, red clover, nettle, valerian) into valuable and easy to use bio-products. The biostimulating activity of botanical extracts and their effect on the chemical composition of celeriac were identified. Plant-based extracts, obtained by ultrasound-assisted extraction and mechanical homogenisation, were tested in field trials. It was found that the obtained formulations increased the total yield of leaves rosettes and roots, the dry weight of leaves rosettes and roots, the content of chlorophyll a + b and carotenoids, the greenness index of leaves, the content of vitamin C in leaves and roots. They mostly decreased the content of polyphenols and antioxidant activities in leaves but increased them in roots and conversely affected the nitrates content. Extracts showed a varied impact on the content of micro and macroelements, as well as the composition of volatile compounds and fatty acids in the celeriac biomass. Due to the modulatory properties of the tested products, they may be used successfully in sustainable horticulture.

Current Trends on Seaweeds: Looking at Chemical Composition, Phytopharmacology, and Cosmetic Applications.

Seaweeds have received huge interest in recent years given their promising potentialities. Their antioxidant, anti-inflammatory, antitumor, hypolipemic, and anticoagulant effects are among the most renowned and studied bioactivities so far, and these effects have been increasingly associated with their content and richness in both primary and secondary metabolites. Although primary metabolites have a pivotal importance such as their content in polysaccharides (fucoidans, agars, carragenans, ulvans, alginates, and laminarin), recent data have shown that the content in some secondary metabolites largely determines the effective bioactive potential of seaweeds. Among these secondary metabolites, phenolic compounds feature prominently. The present review provides the most remarkable insights into seaweed research, specifically addressing its chemical composition, phytopharmacology, and cosmetic applications.

Biofortification of hens' eggs with microelements by innovative bio-based dietary supplement.

New bio-based dietary supplement with micronutrients for livestock was elaborated. The new preparation was tested on laying hens to determine the influence of new biological feed additives on the level of trace elements in egg content. The diet of laying hens (Hy-Line Brown, 30 weeks of age) was supplemented with soya bean meal enriched with Cu(II), Zn(II), Fe(II) and Cr(III) by biosorption. A total of 150 laying hens were divided into five groups: one control and four experimental. In the control group, microelements were supplemented in the inorganic form, whereas in experimental groups, Cu, Zn, Fe and Cr were replaced with soya bean meal enriched with a given microelement ion. The feeding experiment was conducted for 12 weeks and was divided into three series. The results showed that adding the new feed additives to the diet of laying hens had an impact on microelement transfer to eggs, in particular with increased dosing. Eggs were biofortified with iron, zinc and copper and to a lesser extent with chromium. The microelements were accumulated primarily in the albumen because soy protein was the carrier of micronutrient ions in hens' diet. Transfer of trace elements to eggs was not linearly dependent on the dosage of biologically bound microelements in the diet.

Effect of the New Plant Growth Biostimulants Based on Amino Acids on Yield and Grain Quality of Winter Wheat.

Field and laboratory experiments were carried out in 2012-2013, aimed at evaluating the influence of new products stimulating plant growth based on amino acids on crop yield, characteristics of grain and content of macro- and micronutrients in winter wheat (Triticum aestivum L.). The tests included two formulations produced in cooperation with INTERMAG Co. (Olkusz, Poland)-AminoPrim and AminoHort, containing 15% and 20% amino acids, respectively, and 0.27% and 2.1% microelements, respectively. Field experiments showed that the application of products based on amino acids influenced the increase of grain yield of winter wheat (5.4% and 11%, respectively, for the application of AminoPrim at a dose 1.0 L/ha and AminoHort at dose 1.25 L/ha) when compared to the control group without biostimulant. Laboratory tests showed an increase of technological characteristics of grain such as ash content, Zeleny sedimentation index and content of protein. The use of the tested preparations at different doses also contributed to the increase of the nutrients content in grains, in particular copper (ranging 31-50%), as well as sodium (35-43%), calcium (4.3-7.9%) and molybdenum (3.9-16%). Biostimulants based on amino acids, tested in the present study, can be recommended for an efficient agricultural production.

Using XRF and ICP-OES in Biosorption Studies.

In this work, a method of recalculation of results of X-ray fluorescence (XRF) technique to Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) method was elaborated for biosorption studies. Equations that calibrate XRF to ICP-OES were determined, as a biosorbent strawberry, blackcurrant and raspberry seeds after supercritical CO2 extraction were used. ICP-OES showed a better precision and lower detection limits than XRF. The latter technique is cheaper, requires minimal sample preparation and gives faster results. Linear regression of the data gave almost 1:1 correlations without additional correction (for Cu r² = 0.9998, Mn r² = 0.807, Zn r² = 0.979). Calibration and quantification of intensities of XRF was obtained using ICP-OES measurements after samples digestion with HNO3 in a microwave system. High positive correlations were estimated for Cu, Mn, Zn. It was demonstrated that XRF technique can be used together with other well established techniques (ICP-OES) to produce quantitative data from biosorption studies. Elaboration of cheap and quick analytical methodology is an important aspect in development of new processes and products based on biosorption process.

The Cladophora glomerata Enriched by Biosorption Process in Cr(III) Improves Viability, and Reduces Oxidative Stress and Apoptosis in Equine Metabolic Syndrome Derived Adipose Mesenchymal Stromal Stem Cells (ASCs) and Their Extracellular Vesicles (MV's).

This study investigated in vitro effects of freshwater alga Cladophora glomerata water extract enriched during a biosorption process in Cr(III) trivalent chromium and chromium picolinate on adipose-derived mesenchymal stromal stem cells (ASCs) and extracellular microvesicles (MVs) in equine metabolic syndrome-affected horses. Chemical characterisation of natural Cladophora glomerata was performed with special emphasis on: vitamin C, vitamin E, total phenols, fatty acids, free and protein-bound amino acids as well as measured Cr in algal biomass. To examine the influence of Cladophora glomerata water extracts, in vitro viability, oxidative stress factor accumulation, apoptosis, inflammatory response, biogenesis of mitochondria, autophagy in ASCs of EMS and secretory activity manifested by MV release were investigated. For this purpose, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, TEM, FIB observations, mRNA and microRNA expression by RT-qPCR) were applied. The extract of Cladophora glomerata enriched with Cr(III) ions reduced apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of endoplastic reticulum stress. Moreover, it was found, that Cladophora glomerata and Cr(III) induce antioxidative protection coming from enhanced SOD activity Therefore, Cladophora glomerata enriched with Cr(III) ions might become an interesting future therapeutic agent in the pharmacological treatment of EMS horses.

The Influence of Spirulina platensis Filtrates on Caco-2 Proliferative Activity and Expression of Apoptosis-Related microRNAs and mRNA.

Spirulina platensis (SP) is a blue-green microalga that has recently raised attention not only as a nutritional component, but also as a source of bioactivities that have therapeutic effects and may find application in medicine, including cancer treatment. In the present study we determined the cytotoxic effect of S. platensis filtrates (SPF) on human colon cancer cell line Caco-2. Three concentrations of SPF were tested-1.25%, 2.5%, and 5% (v/v). We have found that the highest concentration of SPF exerts the strongest anti-proliferative and pro-apoptotic effect on Caco-2 cultures. The SPF negatively affected the morphology of Caco-2 causing colony shrinking and significant inhibition of metabolic and proliferative activity of cells. The wound-healing assay showed that the SPF impaired migratory capabilities of Caco-2. This observation was consistent with lowered mRNA levels for metalloproteinases. Furthermore, SPF decreased the transcript level of pro-survival genes (cyclin D1, surviving, and c-Myc) and reduced the autocrine secretion of Wnt-10b. The cytotoxic effect of SPF involved the modulation of the Bax and Bcl-2 ratio and a decrease of mitochondrial activity, and was related with increased levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Moreover, the SPF also caused an increased number of cells in the apoptotic sub-G0 phase and up-regulated expression of mir-145, simultaneously decreasing expression of mir-17 and 146. Obtained results indicate that SPF can be considered as an agent with anti-cancer properties that may be used for colon cancer prevention and treatment.

Chemical Characterization of Enteromorpha prolifera Extract Obtained by Enzyme-Assisted Extraction and Its Influence on the Metabolic Activity of Caco-2.

The green seaweed Enteromorpha prolifera was used as a feedstock for the production of enzymatic hydrolysate using cellulase. The selection of the conditions for enzymatic hydrolysis of the biomass was carried out for different enzyme doses and incubation periods. The obtained extract was examined in terms of its multielemental composition, content of polyphenols and antibacterial properties (tested against Escherichia coli and Staphylococcus aureus). Additionally, its influence on the metabolic activity of human colon epithelial cells (Caco-2) was analyzed. The tested concentrations of extract using an in vitro model were 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The hydrolysis yield in the most suitable experimental conditions (8-h process and 50 and 100 µL of cellulase) was 36%. Micro- and macroelements were poorly extracted from the algal biomass. Total phenolic content was 55 mg of gallic acid equivalent per 100 g of dry mass of extract. The cytotoxic effect of extracts, related to the inhibition of the metabolic activity of Caco-2, was noted only after 24 h. In turn, cultures of Caco-2 propagated with extracts for 72 h were characterized by significantly elevated metabolism (the concentration of extracts ranged from 62.5 to 1000 µg/mL, p < 0.05). Obtained results indicated the high biological activity of the prepared extracts; however, the observed effects did not occur in a dose-dependent manner.

Plant Growth Biostimulants, Dietary Feed Supplements and Cosmetics Formulated with Supercritical CO2 Algal Extracts.

The review paper presents the use of algal extracts as safe and solvent-free components of plant growth biostimulants, dietary feed additives and cosmetics. Innovative technology that uses extracts obtained by supercritical CO2 extraction, as a method of isolation of biologically active compounds from algal biomass, is presented. An important part of the complete technology is the final formulation of the product. This enabled realization of the further step which was assessment of the utilitarian properties of the extract-based products. The extracts were analysed for the presence of biologically active molecules (e.g., plant hormones, polyphenols) which provide useful properties such as antioxidant, antiviral, anti-inflammatory and antibacterial. The bio-products were tested in germination tests and underwent field trials to search for plant growth biostimulatory properties. Tests on animals (laying hens experiments) were conducted to assess pro-health properties of new dietary feed supplement. Another application were cosmetic formulations (dermatological tests). The results of the application tests were very promising, however further studies are required for the registration of the products and successful implementation to the market.

Biofortification of milk and cheese with microelements by dietary feed bio-preparations.

The present work reports studies on biofortification of milk and cheese with microelements. The diet of goats was supplemented with soya-based preparations with Cu(II), Fe(II), Zn(II) and Mn(II), produced by biosorption, instead of mineral salts. In innovative preparations, soya was the biological carrier of microelements. The utilitarian properties of the new preparations were tested in two groups (8 goats in each): experimental and control. The concentration of supplemented microelements was monitored in milk during the experiment. The collected milk was then used to produce cheese by enzymatic and acidic coagulation method. The effect of milk and cheese biofortification in microelements was confirmed. In milk, the level of the following microelements was higher than in the control: Cu(II) - 8.2 %, Mn(II) - 29.2 %, Zn(II) - 14.6 %. In cheese the content of Zn(II) obtained in enzymatic (19.8 %) and in acidic (120 %) coagulation was higher when compared to the control group. By using bio-preparations with microelements it was possible to produce new generation of functional food biofortified with microelements, by agronomic, and thus sustainable and ethically acceptable way. Biofortified milk and cheese can be used as designer milk to prevent from micronutrient deficiencies. Graphical Abstractᅟ.

Mapping chemical elements on the surface of orthodontic appliance by SEM-EDX.

BACKGROUND: During orthodontic treatment, the various elements that constitute the fixed appliance undergo different processes. As a result of a change of the surface, elution/coverage of metals on the surface can be observed in the process of corrosion/passivation. MATERIAL AND METHODS: Scanning electron microscopy with an energy-dispersive X-ray analytical system (SEM-EDX) was used to analyze the composition of stainless steel elements of orthodontic fixed appliances (before and after orthodontic treatment), to obtain the composition of the surface of the elements. The analyzed elements were: brackets (Victory Series APC PLUS 022, 3M Unitek, Monrovia, CA, USA); wires (0.017×0.025, 3M Unitek, Monrovia, CA, USA); and bands (37+, 3M Unitek, Monrovia, CA, USA). RESULTS: The results showed a decrease of chromium and iron contribution to the surface, with increase of oxygen content in used vs. new elements of the appliance. CONCLUSIONS: Our results confirm the formation of oxides (passivation layer) on the surface of stainless steel as a result of the presence of the orthodontic appliance in patients' oral cavities.

Using SEM-EDX and ICP-OES to investigate the elemental composition of green macroalga Vaucheria sessilis.

The biomass of Vaucheria sessilis forms algal mats in many freshwaters. There is a need to find the method of algal biomass utilization. Vaucheria sessilis is a rich source of micro- and macronutrients and can be used as a soil amendment. In the paper, the elemental composition of enriched, via bioaccumulation process, macroalga was investigated. For this purpose, two independent techniques were used: scanning electron microscopy with an energy dispersive X-ray analytical system (SEMEDX) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The biomass was exposed to two microelemental solutions, with Cu(II) and Zn(II) ions. After two weeks of the experiment, macroalga accumulated 98.5 mg of Zn(II) ions in 1 g of dry biomass and 68.9 mg g(-1) of Cu(II) ions. Micrographs performed by SEM proved that bioaccumulation occurred. Metal ions were bound on the surface and in the interior of cells. Mappings of all cations showed that in the case of the surface of biomass (biosorption), the elements constituted aggregations and in the case of the cross section (bioaccumulation) they were evenly distributed. The algal biomass with permanently bound microelements can find an application in many branches of the industry (feed, natural fertilizers, etc.).

Determination of exposure to lead of subjects from southwestern Poland by human hair analysis.

The aim of the present work was to investigate the exposure to lead from various sources by investigation of mineral composition of human scalp hair. The research was carried out on hair sampled from 267 young adults living in Wroclaw (southwest Poland). The effect of the place of residence, diet, and lifestyle on lead content in hair was examined by inductively coupled plasma optical emission spectrometry (ICP-OES). Lead was determined at the wavelength 220.353 nm. These outcomes were reached by linking the results of lead level in hair with the results of questionnaire survey. The mean lead level in hair of the whole examined population was 2.01 ± 2.10 mg kg(-1). Lead can enter the human body mainly by inhalation and gastrointestinal absorption. It was found that consuming cheese, fish, and lettuce caused increased level of lead in hair. On the other hand, drinking of milk, tea, coffee, or lemon resulted in decreased content of lead in hair. Additional source of exposure to lead could be cigarette smoking, distance to the traffic road, painting the walls, amalgam filling. Based on the results, it can be concluded that exposure to lead can occur mainly from eating habits and environmental exposure.