A Comprehensive Biological and Physico-Chemical Characterization of Humic and Fulvic Acids Nanoparticles as a Perspective Drug

This work presents the results of a comprehensive physico-chemical and biological study of hu-mic substances samples – an extract of humic and fulvic acids. The performed loss on drying test showed a 22 times different dry matter content between EHS and FA. The morphology and dis-tribution of particles in the dry residue of the samples assessed using the methods of optical and digital microscopy demonstrated differences in the qualitative features of the microstructures of their surfaces and granulometries. Shimadzu X-ray fluorescence spectrometry revealed Si (8.1 and 1.7%), P (33.5 and 2.7%), S (4.3 and 59.5%), K (1.35 and 2.5%), Ca (10.9 and 3.2%), Mn (0.27 and 0.06%), Fe (11 and 0.05%), Cu (0.16 and 0.45%), Zn (0.06 and 0.02%) in the dry residues of the EHS and FA samples, respectively. A high intensity of the X-ray fluorescence signal for Fe atoms in the EHS sample was demonstrated. The FT-IR spectra for EHS and FA are characterized by simi-lar vibration frequencies that are characteristic of the chromone derivatives (1-benzopyran-4-one). The UV absorption spectrum is characterized by max = 281 nm for FA. The EHS solution showed a fluorescence maximum at em = 560 nm at ex = 280 nm. Using the DLS method, nanoparticles of 1 nm and 200 nm were detected in EHS and FA diluted solutions, which are likely to condition the biochemical and physical properties of humic acids. Using the Spiro-tox-test method, the absence of the toxic effect of humic acids on the cell model of ciliates Sp. am-bigua was established. When the cell model was incubated in a solution of a toxicant of the fluo-roquinolone group, a decrease in toxicity was demonstrated when diluted with the EHS solu-tion. The results of the study of the antiviral activity of EHS and FA showed that the study ob-jects in the culture of Vero-E6 cells, in doses non-toxic to cells, suppress the reproduction of the SARS-CoV-2 virus both in the study of the virucidal effect and in the study of the antiviral activ-ity according to the therapeutic and prophylactic model scheme of injection. The results obtained suggest that standardized drugs based on humic acids may open up new perspectives in their biomedical application as antiviral drugs.


Introduction
Humic substances (HS) are complex, heterogeneous, polydisperse mixtures formed in soils, sediments and natural waters as a result of biochemical reactions during the decomposition and transformation of plant and microbial residues (humification) [1]. The first mention of the important role of humic substances in the functioning of plants and animals is found in the works dated 1761 on agronomic chemistry by the Swedish professor of medicine Johan Gottschalk Wallerius. However, all subsequent decades up to the middle of the 20th century were accompanied by the accumulation of empirical knowledge about the biological role of humic substances in natural soil-forming processes. It was shown that HS are characterized by specificity of composition, structure, properties, depending on the conditions and place of their formation [2][3][4][5][6]. Important components of the humification process are plant lignin, its conversion products, polysaccharides, melanin, cutin, proteins, nucleic acids, and lipids [7]. According to [8], the structure of humic substances as polyfunctional compounds does not have a constant chemical composition, which is explained by the stochastic nature of humification processes [9]. Humic substances are extracted from the soil in the form of alkaline solutions, then subjected to fractionation into humic (HA) and fulvic acids (FA), depending on their different solubility in water [10]. Humic and fulvic acids (fulvus -red-yellow) are the most reactive components of humic substances, hypothetical structural models of which are described in [11][12][13]. Molecules of humic substances of different molecular weights can bind and form a supramolecular humic network structure; the degree of aggregation depends on the pH, ionic strength, and mineral composition of the solution [14]. They are therefore classified as polyelectrolytes ( 3-methyl-10-oxo-1,4-dihydropyrano [4,3-b] chromene-9-carboxylic acid); M.m.= 308.24 g/mol [15].
As can be seen in Figure 1, humic substances belong to anionic polyelectrolytes, since they contain polar functional groups in their structure -carboxyl, hydroxyl (alcohol, phenolic) ones, able to dissociate with the formation of negatively charged particles. The described property determines the ability of HS to form complexes with heavy metal ions like the Lewis acid-base reaction [16]. The ability of HS to occlude metals determines the content of micronutrients in their solutions in bioavailable forms, as well as the plant metabolism. According to [17], HS are able to immobilize heavy metals and increase their migration ability. The authors of [18] demonstrated a high efficiency of extraction of precious metals (gold and platinum group elements, PGE) from alkaline extracts of HS brown coal, in which highly dispersed particles of elemental gold were stabilized by humic and fulvic acids.
In recent decades, there have appeared numerous publications about studies of the pharmacological activity of HS and FA against human immunodeficiency virus HIV-1 [19], influenza virus [20], herpes simplex virus-1 (HSV-1) [21], tick-borne encephalitis vi-rus (TBEV) [22], bacteria Enterococcus faecalis and Klebsiella pneumoniae [23], and phytopathogenic fungi [24]. The authors demonstrated a much higher activity of the humic acids versus fulvic acids: HA > HMA > FA, as well as the dependence on the source of the humic acid release: coal > peloid > peat. The significant role of active components -aliphatic fragments (for HA) and COOH, OH fragments (for FA), which determine a positive correlation with the inhibition rates of HAs and FAs against phytopathogenic fungi, is discussed. The both sequences follow the trend in lipophilicity index (a ratio of aromatic to aliphatic carbon) for these humic materials [25]. Review publications describe the possible mechanisms of HS and FA effect in preventing the replication of the SARS CoV-2 virus by sorption on the virus envelope protein and thereby blocking the sorption of viral particles on the cell surface [26]. Here are currently no drugs that have shown clear and consistent benefits in treating SARS CoV-2, but numerous trials in different countries are underway suggesting that HS may reduce symptoms.
All these data create prerequisites for the creation of new biologically active substances based on natural polyelectrolytes for the pharmaceutical industry. However, the official use of humic preparations in medicine is limited by the lack of registered drugs based on them, due to the complexity of their standardization. For this reason, solving the problem of HS standardization by methods of physico-chemical and biological study is an urgent task of the modern pharmacy.
The purpose of this work is a comprehensive study of solutions and dry matters of humic, fulvic acids by physico-chemical and biological methods for the development of a promising drug for SARS CoV-2.

Determination of dry residue
Since the determination of dry matter in a liquid sample gives a more convenient idea of the chemical composition of the analyzed material, and also allows comparing different samples with similar physico-chemical properties, we carried out a test to determine the content of the residue after drying samples of humic acid (EHS) and fulvic acid ( FA) extracts to a constant mass ( Figure 2). The tests carried out in accordance with [27] resulted in the determination of the dry residue -7.34·10 -2 g/ml and 3.36·10-3 g/ml, as well as weight loss due to water and volatile substances, which was 92.63% and 99.66%, respectively, for EHS and FA. Thus, the EHS and FA samples differ from each other in terms of the content of soluble compounds and dry residue.

Optical microscopy
Since the shape, color and size of crystals of a solid are its individual characteristics [28], we studied the morphology and particle distribution of dry samples, invisible to the naked eye, using optical and portable digital microscopy ( Figure 3). It can be seen that the EHS sample contains several mineral phases with different particle shapes (crystals, fragmental particles, complex agglomerates, extended linear structures), a large proportion of which, 48% and 45%, are particles from 0.5 to 2 mm). The FA dry residue sample is a highly dispersed, homogeneous micro-aggregate composition, a large proportion (61%) of which does not exceed 0.1 mm. When viewed through a binocular lens, FA particles are homogeneous, rounded fragments of minerals.
The analysis and digital visualization [29] of the surface relief of the EHS and FA samples demonstrate differences in the qualitative features of their surface microstructure: elongated bodies, accumulation of discrete mineral fragments in the EHS.

X-ray fluorescence spectrometry -elemental analysis
Using energy dispersive X-ray fluorescence (EDXRF), the elemental composition of the test samples in the residue after drying was evaluated ( Figure 4). As can be seen in Figure 4, atoms of the elements Si, P, S, K, Ca, Mn, Fe, Cu, Zn were found in both EHS and FA samples. Noteworthy is the high intensity of the X-ray fluorescence signal for Fe atoms in the EHS sample. It is known that humic substances, participating in the formation of chelates with iron, contribute to plant nutrition [30]. Depending on the solubility and molecular size of HS, humified fractions of organic matter in soil sediments contribute to the creation of a Fe reservoir available to plants [31]. It has been shown [32][33] that the distribution and release of Fe within plants can be controlled if they are supplied with water-soluble Fe-HS complexes in comparison with other natural or synthetic chelates. In case of Fe, highly stable HS complexes mainly include O-containing groups (carboxyl and phenolic) [34]. Fulvic acids are less prone to the formation of insoluble complexes with metals.

Fourier Transform Infrared (FT-IR) Spectroscopy
The vibrational-rotational spectra of the EHS and FA samples obtained by the disk technique with potassium bromide are shown in Figure 5.  The analysis of the IR spectra of the samples under investigation showed the presence of characteristic light transmission bands corresponding to vibrations of bonds between atoms in the structural fragments of alcohol and phenolic hydroxyls, cyclic ethers, carbonyls, and numerous methylene groups [35].
The EHS spectrum is represented by several characteristic bands at 3395, 1595, 1378, and 1081 cm -1 . In a comparative analysis of the transmission spectra of the compounds under investigation, it was found that the EHS sample is characterized by higher values of the extinction coefficients, as a result of which the percentage of light transmission is noticeably reduced, which is probably caused by the nonstoichiometric composition and irregular heterogeneous structure with numerous functional groups. Stretching vibrations of free and bound hydroxyl groups (OH) usually form a broadband region in the frequency range from 3200-3670 cm -1 [36]. However, due to the coordinating influence of iron atoms present in the EHS composition and the formation of complex nanostructures containing Me-O bonds, the vibrations of the O-H bond can be shifted to the lowfrequency region (≈3400 cm -1 ). In addition, the stretching vibrations of primary or associated amino groups and, probably, imines, can also be attributed to the absorption band at ≈3400 cm -1 . The presence of a carbon skeleton manifests itself as a band of stretching vibrations of the C -H bond at 2900 and 2850 cm -1 [37] (Table 1). The obtained vibrational-rotational spectra are determined by the structure of the molecules of humus acids, they are characterized by similar vibration frequencies, the shape of the passbands characteristic of the derivatives of benzo-γ-pyrone (chromone (1benzopyran-4-one). Thus, the infrared spectroscopy method allowed to confirm structural features of investigated compounds [38][39].

Fluorescence (FL) and ultraviolet (UV)-Spectroscopy
It is well known that compounds, which are concentrated benzene nuclei with oxidized pyran (coumarins, chromones), are fluorophores or chromophores due to their high photostability, large Stokes shift, and intense fluorescence with a high quantum yield [40][41][42]. The electronic absorption and fluorescence emission spectra of aqueous dilution of the FA and EHS liquid samples are shown in Figure 6. The fluorescence spectra, which are a tool for investigating the effect of the fulvate chromone structure on its optical properties, were recorded at their own excitation wavelength.
As follows from Figure 6 (a), the maximum absorption of fulvate is 281±2 nm, which is associated with the presence of a chromone heterocyclic nucleus in the structure. As a small organic fluorophore containing several condensed nuclei in a fluorescent open form (see Figure 1 (b), the fulvate molecule produces intense violet (360 nm) and low-intensity green (560 nm) fluorescence at an excitation wavelength of 280 nm. The emission peaks in the short-and long-wavelength regions of the FL spectrum can be caused by excited state intramolecular proton transfer (ESIPT), which is characteristic of fragments with intramolecular hydrogen bonds [43]. Since the EHS solution demonstrated near-ultraviolet transparency and increased far-ultraviolet absorption in the absence of detectable analytical wavelengths ( Figure 6 (c)), it is convenient to characterize the EHS structure by fluorescence spectra (Figure 6 (d)). The photophysical properties of humic acids containing numerous closed cycles, variations of substitutes, and delocalized π-bonds have undergone some changes -the intensity of green fluorescence in the 560 nm region significantly increased.

Static and Dynamic Light Scattering (SLS/DLS)
The measurement of light scattering of the polyelectrolyte samples allows the estimation of molecular and supramolecular parameters that are important for their identification and characterization. The joint use of the DLS and SLS techniques makes it more efficient to determine particle sizes, aggregation phenomena, interparticle interactions, investigation of the structure and relaxation dynamics of complex fluids when the molecular association/dissociation occurs simultaneously [44][45] (Figure 7).  (1:2) sample of FA shows a bimodal distribution with peaks at 1 nm and 180 nm according to DLS data. The volume fraction of micron aggregates in the FA sample is distributed over three size groups: 4 m, 9 m, 12 m. Thus, the EHS and FA nanoparticles found in the investigated samples determine their biochemical and physical properties, which predetermine the transfer through cell membranes by different paths solely because of their size. This quality can be used for the delivery of drugs and nucleic acids into cells [46][47][48].
Further experimental analysis showed the existence of a relationship between the particle size, their biological activity and the efficiency of internalization of nanoparticles by EHS and FA cells.

Biological activity study
The investigation of individual and combined biological activity in accordance with the Arrhenius model of temperature kinetics were carried out using the test culture Spirostomum ambigua [49]. The description of the mechanisms of ligand-receptor interaction in the study of the biological activity of substances makes it possible to quantitatively evaluate the biological response of a cellular biosensor to the effect of a toxicant [50].
The existence of a transition state in the process of ligand-induced death of the test object means that the process of cell death occurs with energy consumption. However, the Spirotox experiment using EHS and FA samples demonstrated the absence of toxicity and a significant lifespan of the cellular biosensor in these media. We assume that the reason for the observed phenomenon lies in the unique ability of humified substances to perceive the effects produced without changing properties for an indefinitely long period, showing the effect of the assimilation potential (AP) [51]. To assess the biological activity of EHS, which showed the AP effect, we selected an object of a drug substance with antibacterial action -moxifloxacin hydrochloride (Mxf HCl) -belonging to the fluoroquinolone group. It was found that ligand-induced cell transitions are linearized in Arrhenius coordinates: where k is the rate constant, Ea is the activation energy, R is the gas constant, A is the preexponential factor, and T is the temperature.
In semilogarithmic coordinates, the tangent of the straight-line slope ln(1/tL) =F(1/T) to the abscissa axis is Ea/R. The values of observed activation energy ( obs Ea) for the test compounds have been found, using Arrhenius coordinates (Table 2). The cytotoxic effect was determined by incubation of the investigated humic acids of various concentrations with Vero-E6 cells for 96 h using the MTS vital dye and visual assessment of the cell monolayer. Based on the data obtained in the investigation of the cytotoxic effect on the culture of Vero-E6 cells using the MTS vital dye, there were constructed analytical curves, from which the CC50 was determined for EHS and FA. The concentration that reduced the optical density value by 50% compared to the control cells was 3.19 μg/ml (EHS series 1), 3.13 μg/ml (EHS series 2) and 3.22 μg/ml (EHS series 3). The FA concentration, which reduced the optical density by 50% compared to the control cells, could not be determined, since it was higher than the 1:2 dilution (Figure 8).  Table 3 shows the results of the investigations carried out to study the virucidal and therapeutic-prophylactic activity of three series of the extract of humic acids in concentrations non-toxic for the culture of Vero E6 cells. Based on the primary data obtained for EHS1, EHS2, EHS3, there were constructed analytical curves, from which 50% inhibitory concentration was determined (IC50). The results for virucidal and therapeutic-prophylactic schemes of injecting the extract of humic substances are shown in Figure 9.  Table 5 shows the results of the cytotoxic and antiviral activities of EHS and FA against the SARS-CoV-2 virus in the Vero E6 cell culture.

Discussion
The humus acids are high molecular weight polyelectrolytes, the structural units of which are represented by aromatic condensed systems with side chains and heterocycles [52]. The physico-chemical properties of the humus acids, including humic, fulvic, and other acids extracted by alkali solutions were investigated with the involvement of a significant fleet of equipment [53]. However, the properties of the humic and fulvic acids can vary depending on the medium of their formation -humification [54]. This is the reason for many publications about the properties of the humus acids. All this leads to an unambiguous decision on the need for standardization and development of quality control methods for the components of the humus acids.
This problem is especially relevant in connection with the data appearing in the literature on the significant biological activity of the components of humus acids -antitumor, antibacterial, antifungal, antiviral [55][56][57]. Due to the massive spread of new strains of the coronavirus infection, the development of drugs that suppress the replication of the SARS-CoV-2 virus is urgent [58].
The results of this study of the antiviral activity showed that the study objects in the culture of Vero-E6 cells, in doses non-toxic to cells, suppress the reproduction of the SARS-CoV-2 virus both in the study of the virucidal effect and in the study of the antiviral activity according to the therapeutic-prophylactic model scheme of injection. The difference in activity of different EHS series can be related to its heterogeneity and sophisticated complex composition (see Figure 1). According to the criteria for evaluating the antiviral   1,20 -1,05 -0,90 -0,75 -0,60 Virus titer (% of control) lg C,mg/ml /ml effect of drugs in vitro, compounds, in which the decrease in the virus titer in concentrations non-toxic for the cell culture is at least 1.5-2.0 lg and SI≥8, exhibit pronounced activity.
The properties and widespread medical application of nanoparticles to enhance contrast in magnetic resonance imaging, in immunoassays and drug delivery are well known from the literature [59]. The internalization of nanoparticles and their absorption by cells, depending on the size, have been shown [60] The results of our research have shown that the composition of the components of humus acids includes occluded metal nanoparticles in addition to the organic structure of nonstoichiometric composition and an irregular heterogeneous structure with many functional groups. Apparently, their presence determines their properties as nanocontainers [61]. Objects that are not characterized by nanosized particles completely lose the described properties [62]. Our studies emphasize that the consideration of the humus acids and their components -humic and fulvic acids from the standpoint of metal-organic nanosized objects can explain the detected virucidal activity, as well as become a potential object for creating a promising standardized drug.

EHS and FA Samples
A natural complex of humic-fulvic acids isolated from low-lying peat, sapropel and some varieties of brown coal (leonardite) using the technology of the VimaVita Company (Sistema-BioTechnologii LLC, RF). The HS concentrate containing humic acids (HA), himatomelanic acids (HMC), fulvic acids (FA), humus acids (HFA) and structural analogs of humic substances was obtained by the method of oxidative-hydrolytic destruction of lignin-containing raw materials (solid-phase fermentation) with subsequent purification [63]. As a result of high-intensity acoustic cleaning, a concentrated viscous colloidal disperse system of humic substances of dark brown color was obtained for research. (рН=7.98±0.1).
Fulvic acid extract (Terra Aquatica, France) in the form of a liquid, transparent solution of brown color, obtained by extraction from a special type of brown coal ̶ leonardite (рН=5,77±0,1).
To study the physico-chemical properties of HS and FA, aqueous solutions of the initial concentrate were prepared. All solutions of humic substances fractions were stored at +4°C. 4.1.2. Cell Culture. The study was carried out on a finite kidney cell line of the African green monkey (Chlorocebus aethiops) Vero-E6. MEM medium with glutamine (Capricorn Scientific GmbH, Germany) containing 10% and 2% fetal calf serum (Biosera, USA) respectively and gentamicin (50 μg / ml) (Capricorn Scientific GmbH, Germany) was used as a growth medium (GM) for growing cells and as a supporting medium (SM) for setting up a reaction. Vero-E6 cells were cultured in 96-well plates in a volume of 100 μl of GM for 24 h at 37°C in an atmosphere with 5% CO2. The inoculation dose was 18,000 cells/well. 4.1.3. Virus. The study used the SARS-CoV-2 human coronavirus, passage 3, with infectivity of 10 7.5 TCID 50/ml. Strain description: hCoV-19/Russia/Moscow-PMVL-12/2020 (EPI_ISL_572398) GISAD: PMVL-12. Booking reference EPL_ISL_572398. 4.1.4. Study Design. The study of the antiviral activity of the study objects was carried out in accordance with the requirements of the Pharmacological State Committee of the Russian Federation for the experimental (preclinical) study of new pharmacological substances. The study included: determination and assessment of the cytotoxic effect of the study objects on the morphology and viability of Vero-E6 cells, as well as the study of the antiviral activity against SARS-CoV-2.

Determination of the cytotoxic effect of the study objects on the cell culture Vero -E6.
Multiple dilutions of the study objects were prepared in plates by titration in 7-8 wells with a step of × 2 on GM medium. The solutions of the study objects were transferred in 200.0 μl into the wells of the test plates with cells. Each point was tested in 4 parallel rows of plate wells. 200 μl of SM without study objects was used as a control of the state of the cell culture. The plates were incubated for 96 hours at 37±0.5°С in atmosphere with 5% СО2. The state of the cell monolayer was visually assessed to identify destructive changes and changes in the morphology of the cell monolayer. After 96 hours, the culture medium was removed and 100 μl of GM and 20 μl of vital dye (MTS) (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, G3582) were added to each well. After incubation for 3 hours at 37±0.5°C, the results were recorded on a BIO-RAD reader at a wavelength of 490 nm, the reference wave was 630 nm. The concentration of the test substance, which reduces the optical density value by 50% compared to the control cells, was taken as the 50% cytotoxic dose (CC50).

In Vitro Study of the Antiviral Activity of EHS and FA.
At the second stage of the study, the virucidal activity against SARS-CoV-2 of three series of the extract of humic substances was studied, as well as their antiviral activity according to a therapeutic-prophylactic model scheme. Each series of the drug was tested at three concentrations. The antiviral activity of the Fulvic drug was studied using a therapeutic-prophylactic model scheme at 4 concentrations. The selection of concentrations for the studies of antiviral activity was carried out on the basis of the results of the study of cytotoxic action within the range of concentrations that are not toxic to cells (i.e., lower than the CC50 value).
When studying the virucidal effect, the selected dilutions of the drugs (according to CC50) were mixed with dilutions of the virus (from 10 -1 to 10 -7 ) in equal volumes of 100 μl and incubated for 1 hour at 37±0.5°C in atmosphere with 5% CO2 and transferred to a plate with a monolayer of washed cells. To study the antiviral activity according to the therapeutic-prophylactic model scheme, working dilutions of the study objects were added to the cells 1 hour before infection with the virus and incubated at 37±0.5°C in an atmosphere of 5% CO2.
As a control, we used SARS-CoV-2 virus dilutions from 10 -1 to 10 -7 without adding study objects. Each drug concentration was tested in four parallel rows of plate wells. The plates were incubated for 96 hours at 37±0.5 о С in atmosphere with 5% СО2. The antiviral activity was assessed visually under a microscope 96 hours after infection by inhibition of the cytopathic effect (CPE) of the virus on the cells. The result was assessed by Δlgmaxthe maximum decrease in the value of the infectious viral dose in the experiment in comparison with the control expressed in decimal logarithms.

Statistical Data Processing
The calculation of the values of 50% cytotoxic concentration (CC50) and 50% effective concentration (IC50) was performed by methods generally accepted for biological research using the Microsoft Excel 5.0 and GraphPad Prism 6.01 software package. The 4-parameter equation of the logistic curve (menu items "Nonlinear regression" -"Sigmoidal doseresponse (variable slope)") was adopted as a working model for the CC50 analysis. The 4parameter equation of the logistic curve (menu items "Nonlinear regression" -"log (inhibitor) vs. response (variable slope)") was adopted for the analysis of IC50. Based on the data obtained, SI was calculated using the equation: SI = СС50/IC50.

Loss on drying (LOD)
The determination of the mass loss of extract of humic substances and liquid fulvic acid on drying was carried out according to the requirements [27]. For this purpose, the accurately measured weight of the tested liquid substance, pre-dried and brought to constant weight, was placed in an evaporating dish (porcelain). The drying was carried out in at 105±5 0 C for 6 hours in a drying oven BINDER FD (Germany) that provides uniform heat treatment of the entire usable chamber volume [65]. The weight of the sample dish was determined and recorded every hour by removing the dish from the oven and allowing it to cool at room temperature in a desiccator for 30 minutes. The loss on drying (%) was calculated according to the Table 6 and following equation: were m1 is the weight of the measuring cup brought to a constant weight (g); m2 is the weight of the measuring cup containing the tested sample before drying (g); m3 is the weight of the measuring cup containing the tested sample after drying (g).

Optical Microscopy (OM)
Determination of the size, shape, and granulometric composition of the dry residue in the EHS and FA samples was carried out using a microscope with a special binocular attachment (Altami BIO 2, Russia) with magnification 10X (linear field of view 20 mm). To do this, a sample of dry matter was distributed on a glass slide without adhesion of particles. The preliminary calibration was carried out using a micrometer object with a scale of 1DIV = 0.01 mm. The particles were observed in separate fields of view. The length was measured on microscopic images and the shape of the particles was determined using the Altami Studio 3.3 software [66].

Digital Microscopy (DM)
The surface structure of powdery substances obtained after drying the EHS and FA samples was investigated using a portable USB digital microscope LX200 (Levenhuk DTX 50, USA) to determine the size of objects from 1 to 50 μm [67]. The microscope is equipped with a built-in digital 1.3 megapixel camera connected to a computer. The advantage of the digital microscope is the express diagnostics of large sample areas without sample preparation. The analysis of the structure, relief and defects of the layers adjacent to the surface allowed us to identify the objects under study.

X-ray Fluorescence (XRF)
An energy dispersive X-ray fluorescence spectrometer (EDX-7000P, Shimadzu Europa GmbH) based on a silicon drift detector with thermoelectric cooling equipped with the PCEDX-Navi software package was used to carry out the nondestructive elemental composition of powder and liquid EHS and FA samples. The range of elements measured by the X-ray fluorescence method is from 11Na to 92U; the X-ray generator is a tube with Rh-anode, current 1-1,000 µA; the irradiated area controlled by the collimator was 10 mm [68]. Pelleted powder or liquid sample of EHS and FA was placed in a closed cuvette covered with a mylar (lavsan) film in air atmosphere and placed exactly in the center of the instrument window. The intensity of the secondary fluorescent radiation was measured to determine the elemental composition of the sample. The study time was 50 seconds for each element (group).

Fourier-Transform IR Spectroscopy
To obtain and analyze the vibrational spectra of the EHS and FA samples in the spectral range from 4,000 to 750 cm -1 , an IR Fourier spectrophotometer (Agilent Cary 630, USA) with a transmission attachment was used [69]. Sample preparation -solid residue after drying -for spectrum recording was carried out in accordance with the requirements [70]. For this, about 1 mg of dry residue was triturated with 400 mg of carefully ground and dried potassium bromide until uniform state and compressed for 3-5 minutes to obtain a disk diameter of about 13 mm to have a spectrum of a suitable intensity.

Fluorescence and UV-Spectroscopy
To obtain the fluorescence spectra of a series of dilutions of the EHS and FA samples, we used an AGILENT Cary Eclipse spectrofluorimeter (USA) with two ultrafast scanning monochromators. The excitation wavelength was 280 nm. The fluorescence spectra in the range from 300 to 800 nm with the maxima of violet and green fluorescence was studied. The absorption spectrum of an aqueous dilution blank (1:100) of FA was obtained in the range from 200 nm to 350 nm using AGILENT Cary 60 equipment (USA).

Determination of Particle Sizes of Dispersed Systems
To determine the particle size of dispersed systems, the methods of static and dynamic light scattering of aqueous dilutions of EHS and FA were used.

Static and Dynamic Light Scattering (SLS/DLS)
The particle size analysis (volumetric distribution on an ensemble of particles by size/size spectra) was recorded by static light scattering (Low-angle laser light scattering, LALLS) using a MasterSizer 3600 Ec small-angle laser dispersion meter (Malvern, UK) [71][72]. The optical module of the equipment used makes it possible to determine the size of the dispersed phase particles in the range from 1 μm to 180 μm based on the measurement of the angular dependence of the intensity of the scattered laser light passing through the dispersed sample [73]. For the granulometric analysis of the samples under investigation, there were used dilutions of EHS and FA in water 1:1000 and 1:2, respectively.
A Zetasizer Nano ZSP (Malvern, UK) based on dynamic light scattering (DLS) was used to measure the size of nanoparticles in the EHS and FA samples from 0.1 nm to 10,000 nm. DLS technology measures particle diffusion due to Brownian motion with its subsequent transformation into size according to the Stokes-Einstein equation [74]: were D is the diffusion coefficient, kB is the Boltzmann constant, T is an absolute temperature, η is the liquid viscosity, r is the particle radius. The dispersion analysis was performed using a Malvern ZetaSizer Nano ZS series analyzer.

Spirotox Method
The study of the biological activity of aqueous dilutions of EHS in the presence of fluoroquinolone supplement was carried out using the cell culture Spirostomum ambigua [75][76]. The mechanism of ligand-receptor interaction includes the stage of interaction of the xenobiotic with the cell, the disintegration of the intermediate complex, accompanied by a change in the concentration of the cellular biosensor due to conformational changes in the receptor, degradation, synthesis of new receptors and the formation of the intermediate state C· Ln ( Figure 12).

Conclusions
A comprehensive study of the properties of humus acids drugs -an extract of humic substances and fulvic acid -was carried out using gravimetric, optical and biological methods. The dry matter content of the samples differs by a factor of 22, the weight loss due to volatiles on drying is 92.63%, 63 and 99.66% for EHS and FA, respectively. The Xray fluorescence analysis showed a high content of Fe and Cu in the samples. The fluorescence intensity and the number of determined elements increase significantly when the samples are dried. The elemental composition indicates the possible formation of watersoluble Fe/Cu-HS complexes, which are necessary in nature for plant nutrition. This can explain the absence of toxicity of the humus acids when the cell model is exposed to the Spirotox method. Nanoparticles detected by the DLS technique in the range from 1 nm to 200 nm in the HS samples can have a structure of lipid-like objects with occluded iron penetrating through cell membranes into an organic matrix, which was demonstrated by the suppression of the SARS-CoV-2 reproduction in the Vero-E6 cell culture. The IR spectroscopy has demonstrated the appearance of bands characteristic of Fe nanoparticles. The structure of chromone (benzo-γ-pyrone) in samples of humus acids has also been proven, which is a powerful fluorophore and chromophore according to FL and UV spectroscopy methods. Thus, samples of humate chromone can be of important interest and be promising candidates for practical application in the fields of biomedicine after quality and biological activity tests. ership Program

Conflicts of Interest:
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Sample Availability: Samples of the compounds are available from the authors.