3.1. Structural Characteristics of the Parent and 3H-Labelled Samples of Fulvic Acid and Lignin Derivative Used in this Study
The samples of peat FA and lignin derivative were characterized for elemental composition, structural group composition, and molecular weight distribution. The obtained values of H/C and O/C atomic ratios for the FA sample used in this study (
Table 1) are indicative of the predominantly aliphatic character of this sample (H/C is 1.27, which is much higher than one). The high O/C ratio (0.68) is, in turn, indicative of the high contribution of oxidized groups into the structure of this sample. These data are in sync with those published for FA in the literature [
47] and with the direct measurements of structural group composition of this sample performed with
13C NMR spectroscopy (
Table 2).
Unlike FA-Vi18, the sample of water-soluble lignin derivative BP-Cx-1 was characterized by a low oxidation degree (O/C ratio of 0.35) and high contribution of aromatic fragments into molecular structures (H/C ratio of 0.75) (
Table 1).
The structural group composition is presented in
Table 2. The FA-Vi18 sample is characterized by the relatively low contribution of aromatic carbon (less than 30% of the total carbon). At the same time, it contained a factor of 1.5 larger amount of aliphatics as compared to aromatic carbon, which explains its low lipophilicity index (0.67; ΣCar/ΣCalk) [
48] and indicates high hydrophilicity of the FA sample. This sample is also characterized by high contribution of carboxyl and carbonyl groups, >25% of the total C, which corroborates well with high oxidation degree of the sample as measured by the elemental analysis. It is typical for fulvic acids.
A distinctive feature of the lignin derivative sample (BP-Cx-1) is the presence of a sharp peak at 55–58 ppm on the
13C NMR spectrum (
Supplementary Figure S1), which is related to methoxy groups—constitutive parts of lignin. High values of spectral density in the range from 108 to 165 ppm of
13C NMR are consistent with the predominantly aromatic nature of this compound. The aromatic carbon content in this sample accounts for 47% of the total C: it represents the main part of BP-Cx-1. This sample contains carboxyl and carbonyl groups formed during the oxidation of the starting lignin material. These two groups constitute 17%, which is significantly less than in the FA-Vi18 sample (25%); these data are in agreement with the lower oxidation of BP-Cx-1. The lipophilicity index of BP-Cx-1 is 1.3, which is two times larger than that of the FA-Vi18 sample. The obtained values indicate the high hydrophobicity of the BP-Cx-1 sample. In general, the structural group composition of the studied compound agrees well with the data for the lignin starting material—a cross-linked polymer composed of phenylpropanoid units [
49].
The molecular compositions of BP-Cx-1 and FA-Vi18 samples were analyzed using FT ICR MS. Van Krevelen diagrams of assigned molecular formulas for BP-Cx-1 and FA-Vi18 are shown in
Figure 1a,b.
The most substantial differences between BP-Cx-1 and FA-Vi18 samples were observed in the high content of low-oxidized species (O/C < 0.5) in the lignin derivative, and in predominance of highly oxidized components (O/C > 0.5) in the sample of fulvic acids. Thus, the lignin-derivative was characterized with the dominance of condensed aromatic compounds and lignin-like species, which occupy the range with the same O/C values (O/C < 0.5) and different H/C values (H/C < 1.0 and 1.0 < H./C < 1.4), respectively. This is consistent with hydrophobic character of the lignin derivative under study.
Despite the substantial differences between the molecular compositions of the samples used in this study, they shared 2881 common formulae out of total 6382 and 6487 formulae assigned to FT ICR MS data on BP-Cx-1 and FA-Vi18, respectively. Van Krevelen diagrams for the unique and common molecular components of BP-Cx-1 and FA-Vi18 are shown in
Figure 1c,d. It can be seen that the both samples shared the common molecular compositions in the range of terpenoic, lignin-like, and flavonoid structures (
Figure 1d). The molecular formulae unique to BP-Cx-1 mainly populate the region of condensed tannins (O/C < 0.5, H/C < 1.0) and phenylisopropanoids (O/C < 0.5, 1.0 < H/C < 1.4). The molecular components that are unique to FA-Vi18 were completely absent in these regions of the van Krevelen diagram. On the contrary, the unique molecular components of FA-Vi18 were mainly represented by hydrolyzed-tannins-like formulae (O/C > 0.5, 1.0 < H/C < 1.4), which were absent in the BP-Cx-1 sample.
Figure 2 visualizes the proportions of occupation densities (OD) in van Krevelen diagrams of general seven chemotypes characteristic for these two samples. These quantitative data confirm the qualitative observations outlined above. It can be seen that the BP-Cx-1 sample is enriched with the most hydrophobic and low oxidized molecular components, such as condensed tannins (OD 0.41 for BP-Cx-1 vs. OD 0.21 for FA-Vi18), phenylisopropanoids (0.26 vs. 0.23), and lipids (0.11 vs. 0.04), as compared to FA-Vi18. The most tangible difference is observed in the comparison of the population densities in the region of hydrolyzed tannins (O/C > 0.5, H/C < 1.4): FA-Vi18 is represented mainly by molecular components of this class, while there are no formulae of this chemotype in BP-Cx-1 (0.03 for BP-Cx-1 vs. 0.37 for FA-Vi18).
Thus, the conducted study on the molecular compositions of the BP-Cx-1 and FA-Vi18 samples allowed us to conclude that BP-Cx-1 is enriched with condensed aromatic hydrophobic components, whereas FA-Vi18 is rich in hydrophilic and oxidized molecular components of predominately hydrolyzed tannins chemotype.
The following molecular weights were obtained for the analyzed samples: Mp = 5.9 kDa for FA-Vi18, and Mp = 4.7 kDa for BP-Cx-1. Due to the high hydrophobicity of BP-Cx-1, it was not possible to achieve its full recovery from the column. The molecular weight of peat fulvic acid correlates well with the data reported for peat HS [
40]. In general, the FA sample can be characterized as predominantly aliphatic, highly oxidized, and with relatively high molecular weight. The lignin derivative, on the contrary, is characterized by high hydrophobicity, a low degree of oxidation, and a lower molecular weight. The radioactivity and UV profiles of both samples used for the subsequent studies are shown in
Figure 3.
As seen in
Figure 3, the radioactivity and UV profiles of both labeled samples are substantially similar. This confirms that the label was incorporated in all components of the molecular ensemble of the FA-Vi18 and BP-Cx-1 samples, which were used in subsequent bioavailability and tissue distribution studies. The suggested structures of the
3H-labeled components are shown in
Supplementary Figure S1 (Supplementary Materials).
3.2. Distribution of the 3H-Labeled Fulvic Acid and BP Cx-1 in the Tissue of Mice
The
3H-labeled samples of peat fulvic acids and of lignin derivative were administered intravenously and by gavage to mice. In the experiments with the
3H-labeled fulvic acids, none of the specific gross changes were observed in the internal organs of the sacrificed animals. Tritium-labeled compounds were detected in all analyzed organs and tissues, including the brain (
Table 3). The tissue distribution profiles after intravenous and gavage administration are shown in
Figure 4a,b. All concentrations of
3H-labeled compounds in the tissues were calculated using eq 1. C
max of [
3H]-FA-Vi18 in all studied tissues was observed at 5 min after intravenous injection and was the highest in kidneys (28.53 mg/kg), blood (14.12 mg/kg), and liver (9.65 mg/kg), while the smallest values were observed in the brain (0.69 mg/kg) of mice.
The highest accumulation of [
3H]-FA-Vi18 was observed in the kidneys (
Figure 4a), reflected in the order of magnitude more significant value of AUC
0-24 for this organ (250.86 h × mg × kg
−1) and tissue-to-blood ratio (2.02), which may be related to the metabolism of FA-Vi18 in this organ. The largest half-lives were obtained for the liver (53.7 h), kidneys (52.2 h), and brain (48.8 h), while the smallest one was for the blood (11.7 h). Thus, when administered intravenously, FA was rapidly distributed from the bloodstream to the organs.
In the case of the oral administration, the Cmax in most tissues was reached in half an hour (blood, liver, kidneys, heart, spleen, ovary). The maximum concentration in the thymus was observed after 1 h, and in the brain after two hours. The highest concentrations were obtained in the kidneys (5.6 mg/kg), liver (2.02 mg/kg), and blood (1.99 mg/kg). The most considerable accumulation indicated by the AUC
0-48 values of (25.32 h × mg × kg
−1) and the values of tissue-to-blood ratio of 2.8 was observed in the kidneys after the intravenous injection (
Table 4).
Almost identical AUC
0-inf for blood after oral and intravenous administration indicates a high absolute bioavailability of FA-Vi18 after oral administration. The half-life of [
3H]-FA-Vi18 in the blood after oral administration (41 h) significantly exceeds that after intravenous administration (11.7 h). This, along with a high AUC
0-t value for blood, compared to organs, indicates the relatively slow distribution of the orally administered drug from the bloodstream (
Figure 4b).
In the case of [3H]-BP-Cx-1, the brownish coloration of tissues and internal organs, except for the thymus, was observed in the necropsied animals intravenously administered with [3H]-Bp-Cx-1. Sixty minutes after intravenous injection of [3H]-BP-Cx-1, dark-brown staining of the contents of the small intestine and cecum was observed, most pronounced 12 h after the injection. This might suggest intestinal excretion of [3H]-Bp-Cx-1. No other gross changes of internal organs were observed. In mice necropsied 24 h after oral administration of [3H]-BP-Cx-1, it was observed inside the lumen of the ascending colon and in the cecum. No specific macroscopic changes of internal organs were noted.
The hydrophobic [
3H]-BP-Cx-1 shows a slightly different pattern of organ distribution than [
3H]-FA-Vi18 (
Figure 4c,d). It showed approximately equal accumulation of the drug in the liver and kidneys with intravenous and oral administration. When administered intravenously, the value for the liver was 396.7 mg/kg (T
max = 5 min) and 343.3 mg/kg for the kidneys (T
max = 120 min), with accumulation coefficients of 2.56 and 2.21, respectively (
Table 5). When administered orally, the drug displayed maximum concentration in these organs 120 min after injection. It was 16.13 mg/kg with an accumulation factor of 5.98 for the liver and 17.73 mg/kg with 6.57 for the kidneys (
Table 6). These results indicate that [
3H]-BP-Cx-1 is metabolized both by the liver and kidneys.
Compared to the fulvic acid, the lignin derivative has a higher affinity for ovarian tissues (tissue-to-blood ratio was 0.29–0.17 for [3H]-FA-Vi18 and 0.75–0.43 for [3H]-BP-Cx-1 with the intravenous and oral route introduction, respectively) and spleen (tissue-to-blood ratio 0.25–0.32 and 1.5–1.6 for the two drugs, respectively).
As in the case of [
3H]-FA-Vi18, relatively low concentrations of [
3H]-BP-Cx-1 were found in the heart (5.4 mg/kg with intravenous and 0.16 mg/kg for gavage administration) and in the brain (6.3 mg/kg). We also found a delayed dynamics of accumulation and excretion of [
3H]-BP-Cx-1 from organs (
Figure 4c,d): a significant decrease in concentration during the observation period with intravenous administration occurred only in the blood (half-life 6.8 h), liver (half-life 14.6 h), and kidneys (half-life 15.5 h) (
Table 5). The elimination half-life of the gavage administered [
3H]-BP-Cx-1 significantly exceeded that of the intravenous one and was the largest for the kidneys (226.8 h) (
Table 6). AUC
0-inf for blood after gavage and intravenous administration accounted for 11.6%, indicating low bioavailability of gavage-administered [
3H]-BP-Cx-1. The primary data on kinetics of tissue distribution of [
3H]-FA-Vi18 and [
3H]-BP-Cx-1 in mice are given in
Supplementary Tables S1 and S2, respectively.