HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 50 pp. 1–5 (2022) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2022-01 ANTIOXIDANT EFFECT OF HUMIC SUBSTANCES FROM HUNGARIAN LEONARDITE ATTILA CSICSOR*1,2 AND ETELKA TOMBÁCZ1,3 1Doctoral School of Environmental Sciences, University of Szeged, Rerrich Béla tér 1, Szeged, 6720, HUNGARY 2Hymato Products Kft., Kossuth Lajos u. 33, Szentkirályszabadja, 8225, HUNGARY 3Soós Ernő Water Technology Research and Development Center, University of Pannonia, Zrínyi Miklós u. 18, Nagykanizsa, 8800, HUNGARY Humic substances are natural substances that are continuously formed from the decay of plant residues. These materials have a very diverse range of properties, making them versatile. According to many new studies, these humic substances also exhibit antioxidant propensities. The aim of this paper was to shed light on whether humic substances really have antioxidant properties. Keywords: humic substances, humic acid, fulvic acid, himatomelanic acid, antioxidant, leonardite 1. Introduction The majority of the population of Hungary suffers from one of the diseases associated with free radicals, e.g. dis- eases of civilization, obesity, cardiovascular disease, ma- lignant neoplasms, etc. Although it is well-known that our consumption of vegetables and fruit lags far behind those of the other Member States of the European Union, it has been scientifically proven that essential fresh vita- mins and provitamins as well as minerals are fundamental not only for the smooth functioning of the body but also for their antioxidant components. Furthermore, they can play a key role in terms of disease prevention. Berries have become a major player in a number of related re- search papers as they are also essential in the prevention as well as aftercare of cancer and cardiovascular disease. What other options are available and what other sub- stances may still have high antioxidant contents? Based on the aforementioned points, it is also important and justified to study the antioxidant properties of new com- pounds such as humic substances and determine which of their components play a crucial role in the develop- ment of their antioxidant effect, both qualitatively and quantitatively. The aim of this research is to compare the antioxidant properties of humic acids with already well- known natural antioxidants. Firstly, different humic acid Recieved: 26 August 2021; Revised: 16 September 2021; Accepted: 20 September 2021 *Correspondence: csicsor.attila@gmail.com fractions were prepared. The expected results should help to more accurately interpret the complex behavior of hu- mic acids, thereby helping to expand their range of ap- plications. Therefore, if the results are convincing, this could open up new fields with regard to the application of humic acids in the food and cosmetics industries [1]. 2. Humic substances Humic substances are a group of naturally occurring macromolecules found throughout nature, that is, in soil, air, water, carbon deposits and peat. Chemically ill- defined humic substances are natural organic colloids comprised of decomposition products of plant-derived biomass as the result of a process called humification. Their conversion into stabilized humic material is one of the most complex and least understood biogeochemical processes of the carbon cycle. Most natural humic acids (HA) are found in older peat, lignite and juvenile lignite. In Hungary, the HA content of the so-called leonardite, a famous source of HA, is close to 70% in its natural form. (A near-surface deposit of leonardite is located in Dudar, where this geological formation can be mined). In the humification process, dead plant matter that en- ters the soil is broken down by enzymes found in soil bac- teria and fungi, so simple compounds like sugar and am- monia are formed from carbohydrates, fats, proteins and lignin, which on the one hand serve as a source of food for the soil microbes but on the other hand as a source in the formation of humic substances. As a result of biotic and abiotic (condensation and polymerization) processes, https://doi.org/10.33927/hjic-2022-01 mailto:csicsor.attila@gmail.com 2 CSICSOR AND TOMBÁCZ the decomposition products form high-molecular-weight humus compounds, the presence of which is characteris- tic of the soil [2]. On the basis of differences in solubility, humic sub- stances can be divided into several groups such as the main fractions humin, HA, and fulvic acids (FA), as well as the alcohol soluble himatomelanic acids (HY). They are a mixture of similarly behaving, yellow-brown-black, acidic, high-molecular, natural organic substances that are operationally defined. Humic substances themselves contain too many kinds of molecules that can be sepa- rated by changing the solubility conditions. Even though these molecules behave similarly, each fraction has differ- ent properties and their molecular structure is not uniform [2]. The nomenclature of fractions that can be separated according to their solubility is suggested in the guidelines of the International Humic Substances Society (IHSS): • Humin - a black fraction of humic substances that is insoluble in water at any pH. • Humic acids - the fraction of humic substances that is insoluble in water under acidic conditions (pH < 2) but soluble in water at higher pH values. They can be extracted by various alkaline solutions be- fore being precipitated by a strong acid and are dark brown or black in color. • Fulvic acids - the fraction of humic substances that is soluble in water at all pH values. Fulvic acids are light yellow or yellowish brown in color. • Himatomelanic acids - the fraction of humic sub- stances that is soluble in an alcohol. 3. Antioxidants The most important physiological role of antioxidants is to neutralize the free radicals that are continuously formed in the Szent-Györgyi-Krebs cycle and counteract the free radicals with different oxidizing forces that en- ter the body. An antioxidant is a substance that inhibits oxidation, more broadly speaking, retards or hinders oxi- dation. They are chemically reducing agents, i.e. electron donors. These materials are usually organic compounds but include metals and organometallic complexes. Many different types of antioxidants exist, which usually work together and do not neutralize free radicals on their own. Although our body itself is able to produce some antiox- idants, sometimes it is necessary for us to ingest these substances from external sources. Together, these antiox- idants already form a very strong line of defence in our body, e.g. vitamin C, Vitamin A, flavonoids, glutathione, resveratrol, unsaturated fatty acids, etc. [3, 4] 4. Methods for measuring the antioxidant capacity The antioxidant capacity is the combined free radical scavenging effect of all antioxidant compounds in the system studied. Since the need for its accurate numer- ical determination is growing, a number of analytical procedures and measurement systems have been de- veloped. Given that methodologies are constantly be- ing modified and refined, nowadays, the number of ap- plied methods exceeds one hundred. In the literature, the majority of studies use several methods to deter- mine the antioxidant capacity [5]. The most common methods of measuring the antioxidant capacity can be divided into two main groups: electron transition-based ones (Ferric iron Reducing Antioxidant Power, Total Polyphenol Content, Copper ion Reducing Antioxidant Capacity, Trolox Equivalent Antioxidant Capacity, and 2,2-Diphenyl-1-picrylhydrazyl (DPPH)) as well as those based on hydrogen atom transfer (Oxygen Radical Ab- sorptance Capacity, total peroxyl radical-trapping po- tential, chemiluminescence-based methods, and photo- chemiluminescence measurements just to mention the most commonly used methods). Although these two types of measurements determine the antioxidant capacity, the results obtained do not nec- essarily have to be correlated with each other, since the reducing power of a sample is not necessarily related to its ability to scavenge for the reaction of test compounds. Electron transfer reactions involve colour changes, from which the antioxidant capacity can be deduced. The essence of these methods is to create a free radical as the result of a reaction. To this free radical, the antioxidant is added at various dilutions leading to a colour change, which is monitored by a spectrophotometer and then the antioxidant capacity of the test substance is calculated from the results obtained. Methods involving hydrogen atom transfer are based on the kinetics of the reaction. Tests measure how effec- tive a sample is against a given free radical, namely its free radical scavenging capacity [5]. 5. Antioxidant effect of humic substances The question may arise as to why humic substances would exhibit an antioxidant effect. Humic acids are chemically very complex mixtures of composite molecules, that is, natural polymers formed during the varying degrees of polymerization of basic build- ing blocks. According to their chemical structure, they are polyhydroxy carboxylic acids with quinone and semiquinone groups. In some respects, they are similar to flavonoids and phenols, in which the so-called flavone skeleton is polysubstituted by hydroxy groups. However, they also have a quinoid structure that is known to be re- sponsible for antioxidant properties. These properties of HA have already been demonstrated in a number of scien- tific publications by both classical analytical methods (re- dox titrations) and instrumental analytical measurements (Electron Spin Resonance) [6]. The general structure of HA and the formulae of some well-known antioxidants are presented in Fig. 1 From Hungarian Journal of Industry and Chemistry ANTIOXIDANT EFFECT OF HUMIC SUBSTANCES 3 Figure 1: Model structure of HA and identical moieties of known antioxidants [2] their structure, it is clear that HA are comprised of a num- ber of groups such as already well-known antioxidants, so will exhibit exceptionally high antioxidant capacities [7]. 6. Results 6.1 Measurement of total phenolic content All measurements of total phenolic content were made according to the method developed by Shetty et al. [8]. During these measurements, only high-quality (a.r.) chemicals were used. The fractions of humic substances were extracted by ourselves from samples of leonardite retrieved from Dudar. Rather than indirectly measuring the antioxidant property of the sample, the method mea- sures its total phenolic content, from which its antioxi- dant capacity can be deduced. The method consisted of diluting 1 ml of the sample in 5 ml of distilled water and 1 ml of 95% ethanol in a test tube before adding 0.5 ml of 50% Folin-Ciocalteu (FC) reagent (half of distilled water in half FC reagents) to each sample. After being stirred for 5 minutes, 1 ml of 5% Na2CO3 was added to the re- action mixture and allowed to stand for 1 hour before the absorbance values of the samples were measured at 725 nm. These absorbance values were then converted into µg gallic acid equivalents in order to compare the mea- sured values with each other and with other data from the literature. Whilst measuring the total phenolic content of the samples, a series of dilutions was made from our sam- ples of known mass, for which the total phenolic content was measured. A series of dilutions had to be produced in order to measure an absorbance value of approximately one for each sample. This was necessary to be able to compare the samples because the three different fractions yielded one absorbance value at different concentrations once the reaction was complete. Furthermore, since the samples are coloured (brownish - dark brown), the mea- sured absorbance values had to be calibrated in light of the background of the samples. Therefore, the samples were diluted to the concentration present in the reaction volume. After measuring the series of samples, their to- tal phenolic content was calculated from the calibration curve, which can be seen in Fig. 2, and the results ob- tained are shown in Table 1. The results show that different initial concentrations of the various samples are required to achieve a similar absorbance. In the humic acid samples, since the major- ity of the phenol groups are in this fraction, the lowest ini- tial concentration is required to achieve an absorbance of approximately one. Himatomelanic acid has an average number of phenol groups while FA is comprised of the Figure 2: Calibration curve of gallic acid 50 pp. 1–5 (2022) 4 CSICSOR AND TOMBÁCZ Table 1: The measured and corrected absorbance of the samples and the gallic acid equivalent (GAE) values Sample Corrected concentration [mg/ml] Abs measured Abs corrected GAE [mg/ml] from the calibration curve GAE [mg/g] HY 0.59 1.027 0.953 0.103 174.5 FA 1.18 0.999 0.948 0.102 86.8 HA 0.35 1.024 1.024 0.11 310.3 IHSS FA 0.684 - 1 0.107 156.8 IHSS HA 0.212 - 1 0.107 506.1 Table 2: The IC50 readings of the different samples Name of the sample Original con- centration IC50 [µg/ml] HY 1.5 mg/ml 200 FA 2 mg/ml 300 HA 3.25 mg/ml 460 fewest. The results suggest that the studied humic frac- tions are likely to exhibit an antioxidant effect. 6.2 DPPH method Measurement of the antioxidant capacity based on sta- ble DPPH radical scavenging is one of the first methods. The reaction proceeds as follows: the dark purple radical formed in the reaction mixture loses its colour when it reacts with antioxidants. This method is widely used because the radical- forming molecule DPPH is commercially available, sta- ble as well as not particularly reactive nor aggressive, which is beneficial in the reactions that take place as tak- ing measurements is simple. On the other hand, a stable radical that is not found in the living organism is used in- stead of a radical formed during the normal metabolism in the cell. Using this method, it is not possible to esti- mate how effective the sample is as an antioxidant with regard to biological radicals [8]. During the measurements, 6 ml of a DPPH working solution (0.1 mg/ml) was added to one ml of the sample, vortexed and stored in the dark for 30 minutes until the colour reaction began. After 30 minutes, the absorbances were measured at 517 nm. From the measured values that had previously been corrected, the percentage of inhibi- tion (Inhibition%, Inhib.%) and the value corresponding to 50% inhibition (IC50 value) were calculated. While the measurements were taken, in the same way as when the total phenolic content was measured, a series of dilu- tions was made from a sample of known concentration. From the measured absorbances, after background cor- rection the percentages of inhibition of the samples were calculated and plotted as a function of the concentration in order to determine the IC50 values of the samples (Fig. 3). From the data, it can be concluded that the materi- als prepared and tested exhibit antioxidant properties be- cause they inhibit the decomposition of the DPPH radical. Figure 3: Percentage of inhibition of himatomelanic acid (top), humic acid (middle), and FA (bottom) as functions of the concentration to determine the IC50 values Their IC50 values (Table 2) are promising and compara- ble with other data in the literature [9]. 7. Conclusions Although the results revealed that the samples made from domestic raw materials exhibit antioxidant properties, further studies are needed to gain a complete picture of the antioxidant capacity of these substances. Further- more, in the future, it is our intention to confirm the re- sults collected so far with in-vitro and in-vivo experi- ments. Hungarian Journal of Industry and Chemistry ANTIOXIDANT EFFECT OF HUMIC SUBSTANCES 5 REFERENCES [1] Balogh, E.; Hegedus, A.; Stefanovits-Banyai, E.: Application of and correlation among antioxidant and antiradical assays for characterizing antioxidant capacity of berries Sci. 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CyTA - Journal of Food, 2018, 16(1), 1131-1138 DOI: 10.1080/19476337.2018.1517828 50 pp. 1–5 (2022) https://doi.org/10.1016/j.scienta.2010.04.015 https://www.nccih.nih.gov/health/antioxidants-in-depth https://doi.org/https://doi.org/10.1515/hjic-2018-0008 https://doi.org/https://doi.org/10.1515/hjic-2018-0008 https://doi.org/10.1021/jf030723c https://doi.org/10.1021/jf030723c https://doi.org/10.4141/s00-075 https://doi.org/10.1016/S0032-9592(03)00089-X https://doi.org/10.1016/S0032-9592(03)00089-X https://doi.org/10.1080/19476337.2018.1517828 Introduction Humic substances Antioxidants Methods for measuring the antioxidant capacity Antioxidant effect of humic substances Results Measurement of total phenolic content DPPH method Conclusions