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

Csicsor, Attila

Csákiné Tombácz, Etelka

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HUNGARIAN JOURNAL OFINDUSTRY AND CHEMISTRYVol. 50 pp. 1–5 (2022)hjic.mk.uni-pannon.huDOI: 10.33927/hjic-2022-01ANTIOXIDANT EFFECT OF HUMIC SUBSTANCES FROM HUNGARIANLEONARDITEATTILA CSICSOR*1,2 AND ETELKA TOMBÁCZ1,31Doctoral School of Environmental Sciences, University of Szeged, Rerrich Béla tér 1, Szeged, 6720,HUNGARY2Hymato Products Kft., Kossuth Lajos u. 33, Szentkirályszabadja, 8225, HUNGARY3Soós Ernő Water Technology Research and Development Center, University of Pannonia, Zrínyi Miklós u.18, Nagykanizsa, 8800, HUNGARYHumic substances are natural substances that are continuously formed from the decay of plant residues. These materialshave a very diverse range of properties, making them versatile. According to many new studies, these humic substancesalso exhibit antioxidant propensities. The aim of this paper was to shed light on whether humic substances really haveantioxidant properties.Keywords: humic substances, humic acid, fulvic acid, himatomelanic acid, antioxidant, leonardite1. IntroductionThe majority of the population of Hungary suffers fromone 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 thatour consumption of vegetables and fruit lags far behindthose 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 fundamentalnot only for the smooth functioning of the body but alsofor their antioxidant components. Furthermore, they canplay a key role in terms of disease prevention. Berrieshave become a major player in a number of related re-search papers as they are also essential in the preventionas well as aftercare of cancer and cardiovascular disease.What other options are available and what other sub-stances may still have high antioxidant contents? Basedon the aforementioned points, it is also important andjustified to study the antioxidant properties of new com-pounds such as humic substances and determine whichof their components play a crucial role in the develop-ment of their antioxidant effect, both qualitatively andquantitatively. The aim of this research is to compare theantioxidant properties of humic acids with already well-known natural antioxidants. Firstly, different humic acidRecieved: 26 August 2021; Revised: 16 September 2021; Accepted: 20September 2021*Correspondence: csicsor.attila@gmail.comfractions were prepared. The expected results should helpto 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, thiscould open up new fields with regard to the application ofhumic acids in the food and cosmetics industries [1].2. Humic substancesHumic substances are a group of naturally occurringmacromolecules found throughout nature, that is, in soil,air, water, carbon deposits and peat. Chemically ill-defined humic substances are natural organic colloidscomprised of decomposition products of plant-derivedbiomass as the result of a process called humification.Their conversion into stabilized humic material is one ofthe most complex and least understood biogeochemicalprocesses 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, afamous 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 andlignin, which on the one hand serve as a source of foodfor the soil microbes but on the other hand as a source inthe formation of humic substances. As a result of bioticand abiotic (condensation and polymerization) processes,2 CSICSOR AND TOMBÁCZthe decomposition products form high-molecular-weighthumus 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 themain fractions humin, HA, and fulvic acids (FA), as wellas the alcohol soluble himatomelanic acids (HY). Theyare a mixture of similarly behaving, yellow-brown-black,acidic, high-molecular, natural organic substances thatare operationally defined. Humic substances themselvescontain too many kinds of molecules that can be sepa-rated by changing the solubility conditions. Even thoughthese molecules behave similarly, each fraction has differ-ent properties and their molecular structure is not uniform[2]. The nomenclature of fractions that can be separatedaccording to their solubility is suggested in the guidelinesof the International Humic Substances Society (IHSS):• Humin - a black fraction of humic substances thatis insoluble in water at any pH.• Humic acids - the fraction of humic substances thatis insoluble in water under acidic conditions (pH< 2) but soluble in water at higher pH values. Theycan be extracted by various alkaline solutions be-fore being precipitated by a strong acid and are darkbrown or black in color.• Fulvic acids - the fraction of humic substances thatis soluble in water at all pH values. Fulvic acids arelight yellow or yellowish brown in color.• Himatomelanic acids - the fraction of humic sub-stances that is soluble in an alcohol.3. AntioxidantsThe most important physiological role of antioxidantsis to neutralize the free radicals that are continuouslyformed in the Szent-Györgyi-Krebs cycle and counteractthe free radicals with different oxidizing forces that en-ter the body. An antioxidant is a substance that inhibitsoxidation, more broadly speaking, retards or hinders oxi-dation. They are chemically reducing agents, i.e. electrondonors. These materials are usually organic compoundsbut include metals and organometallic complexes. Manydifferent types of antioxidants exist, which usually worktogether 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 thesesubstances from external sources. Together, these antiox-idants already form a very strong line of defence in ourbody, e.g. vitamin C, Vitamin A, flavonoids, glutathione,resveratrol, unsaturated fatty acids, etc. [3, 4]4. Methods for measuring the antioxidantcapacityThe antioxidant capacity is the combined free radicalscavenging effect of all antioxidant compounds in thesystem studied. Since the need for its accurate numer-ical determination is growing, a number of analyticalprocedures 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 commonmethods of measuring the antioxidant capacity can bedivided into two main groups: electron transition-basedones (Ferric iron Reducing Antioxidant Power, TotalPolyphenol Content, Copper ion Reducing AntioxidantCapacity, Trolox Equivalent Antioxidant Capacity, and2,2-Diphenyl-1-picrylhydrazyl (DPPH)) as well as thosebased 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 themost commonly used methods).Although these two types of measurements determinethe antioxidant capacity, the results obtained do not nec-essarily have to be correlated with each other, since thereducing power of a sample is not necessarily related toits ability to scavenge for the reaction of test compounds.Electron transfer reactions involve colour changes,from which the antioxidant capacity can be deduced. Theessence of these methods is to create a free radical as theresult of a reaction. To this free radical, the antioxidantis added at various dilutions leading to a colour change,which is monitored by a spectrophotometer and then theantioxidant capacity of the test substance is calculatedfrom the results obtained.Methods involving hydrogen atom transfer are basedon the kinetics of the reaction. Tests measure how effec-tive a sample is against a given free radical, namely itsfree radical scavenging capacity [5].5. Antioxidant effect of humic substancesThe question may arise as to why humic substanceswould exhibit an antioxidant effect. Humic acidsare chemically very complex mixtures of compositemolecules, that is, natural polymers formed duringthe varying degrees of polymerization of basic build-ing blocks. According to their chemical structure, theyare polyhydroxy carboxylic acids with quinone andsemiquinone groups. In some respects, they are similarto flavonoids and phenols, in which the so-called flavoneskeleton is polysubstituted by hydroxy groups. However,they also have a quinoid structure that is known to be re-sponsible for antioxidant properties. These properties ofHA 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 somewell-known antioxidants are presented in Fig. 1 FromHungarian Journal of Industry and ChemistryANTIOXIDANT EFFECT OF HUMIC SUBSTANCES 3Figure 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, sowill exhibit exceptionally high antioxidant capacities [7].6. Results6.1 Measurement of total phenolic contentAll measurements of total phenolic content were madeaccording to the method developed by Shetty et al.[8]. During these measurements, only high-quality (a.r.)chemicals were used. The fractions of humic substanceswere extracted by ourselves from samples of leonarditeretrieved from Dudar. Rather than indirectly measuringthe 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 ofdiluting 1 ml of the sample in 5 ml of distilled water and1 ml of 95% ethanol in a test tube before adding 0.5 ml of50% Folin-Ciocalteu (FC) reagent (half of distilled waterin half FC reagents) to each sample. After being stirredfor 5 minutes, 1 ml of 5% Na2CO3 was added to the re-action mixture and allowed to stand for 1 hour before theabsorbance values of the samples were measured at 725nm. These absorbance values were then converted intoµg gallic acid equivalents in order to compare the mea-sured values with each other and with other data from theliterature.Whilst measuring the total phenolic content of thesamples, a series of dilutions was made from our sam-ples of known mass, for which the total phenolic contentwas measured. A series of dilutions had to be produced inorder to measure an absorbance value of approximatelyone for each sample. This was necessary to be able tocompare the samples because the three different fractionsyielded one absorbance value at different concentrationsonce the reaction was complete. Furthermore, since thesamples are coloured (brownish - dark brown), the mea-sured absorbance values had to be calibrated in light ofthe background of the samples. Therefore, the sampleswere diluted to the concentration present in the reactionvolume. After measuring the series of samples, their to-tal phenolic content was calculated from the calibrationcurve, which can be seen in Fig. 2, and the results ob-tained are shown in Table 1.The results show that different initial concentrationsof the various samples are required to achieve a similarabsorbance. 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 ofapproximately one. Himatomelanic acid has an averagenumber of phenol groups while FA is comprised of theFigure 2: Calibration curve of gallic acid50 pp. 1–5 (2022)4 CSICSOR AND TOMBÁCZTable 1: The measured and corrected absorbance of the samples and the gallic acid equivalent (GAE) valuesSampleCorrectedconcentration[mg/ml]AbsmeasuredAbscorrectedGAE [mg/ml]from thecalibration curveGAE [mg/g]HY 0.59 1.027 0.953 0.103 174.5FA 1.18 0.999 0.948 0.102 86.8HA 0.35 1.024 1.024 0.11 310.3IHSS FA 0.684 - 1 0.107 156.8IHSS HA 0.212 - 1 0.107 506.1Table 2: The IC50 readings of the different samplesName of thesampleOriginal con-centrationIC50 [µg/ml]HY 1.5 mg/ml 200FA 2 mg/ml 300HA 3.25 mg/ml 460fewest. The results suggest that the studied humic frac-tions are likely to exhibit an antioxidant effect.6.2 DPPH methodMeasurement 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 radicalformed in the reaction mixture loses its colour when itreacts 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 stableradical that is not found in the living organism is used in-stead of a radical formed during the normal metabolismin the cell. Using this method, it is not possible to esti-mate how effective the sample is as an antioxidant withregard to biological radicals [8].During the measurements, 6 ml of a DPPH workingsolution (0.1 mg/ml) was added to one ml of the sample,vortexed and stored in the dark for 30 minutes until thecolour reaction began. After 30 minutes, the absorbanceswere measured at 517 nm. From the measured values thathad previously been corrected, the percentage of inhibi-tion (Inhibition%, Inhib.%) and the value correspondingto 50% inhibition (IC50 value) were calculated. Whilethe measurements were taken, in the same way as whenthe 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 werecalculated and plotted as a function of the concentrationin 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 functionsof the concentration to determine the IC50 valuesTheir IC50 values (Table 2) are promising and compara-ble with other data in the literature [9].7. ConclusionsAlthough the results revealed that the samples made fromdomestic raw materials exhibit antioxidant properties,further studies are needed to gain a complete pictureof 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 ChemistryANTIOXIDANT EFFECT OF HUMIC SUBSTANCES 5REFERENCES[1] Balogh, E.; Hegedus, A.; Stefanovits-Banyai, E.:Application of and correlation among antioxidantand antiradical assays for characterizing antioxidantcapacity of berries Sci. 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Antioxidant Effect of Humic Substances from Hungarian Leonardite

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Antioxidant Effect of Humic Substances from Hungarian Leonardite

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