Pharmacy and Drug Development

Abstract

For the prevention and treatment of associated gastrointestinal and respiratory diseases in young farm animals, a new veterinary drug Trametin Plus is proposed, obtained on the basis of xylographic fungi using biotechnology methods. The properties of biological products obtained on the basis of microorganisms and fungi depend on the biologically active substances that make up their composition. In the literature, there is scattered information about the features of lipogenesis and the composition of fatty acids synthesized by wood-destroying fungi. The paper presents data on the study of the qualitative and quantitative composition of fatty acids, a qualitative analysis of volatile polar and non-polar organic compounds of the drug. It has been established that the total concentration of fatty acids is 70 µg/g of the preparation. Of these, 50.0% are free fatty acids, the esterified acids of which account for 50.0% of the total mass of fatty acids. Among the dominant non-polar volatile components of Trametin Plus, squalene can be noted. Organic aliphatic amino acids such as glycine, arginine, and β-alanine with a small molecular weight are presented as minor non-polar volatile components of Trametin plus. The analysis data confirm the multicomponent nature of the drug, which determines its various biological properties, namely, antibacterial, antiviral, antioxidant and immunostimulant activity, which determines the high therapeutic and prophylactic efficacy of the veterinary drug Trametin Plus.

Introduction

Improving the system of measures to reduce the sickness rate of young farm animals includes the use of new therapeutic and prophylactic drugs. As such a preparation, the new Trametin Plus is proposed, obtained on the basis of the culture liquid during liquid-phase cultivation of the producer Trametes pubescent (Shumach. Fr.) Pilat. strain 0663 from the Collection of BIN RAS L.L. Komarov [1-5].

In the literature, there is information about the features of lipogenesis and the composition of lipids of lower fungi, and algae, while for higher fungi, in particular, for basidiomycetes, similar data are rather scattered. The composition of free fatty acids of the genus Basidiomycetes is poorly studied. Research is limited to single works on the study of lipids of mycothallus [6-16]. However, it is well known that fatty acids have pharmacological activity in relation to various nosologies in both humans and animals. The aim of this work was to study the qualitative and quantitative composition of fatty acids, and qualitative analysis of volatile polar and non-polar organic compounds of the veterinary drug Trametin Plus.

The work was carried out with the financial support of the Federal state budgetary institution "Foundation for the Promotion of the Development of Small Forms of Enterprises in the Scientific and Technical Sphere".

EXPERIMENTAL PART

The determination of fatty acids was carried out according to the method of A.A. Nikonova et al. [17].

Extraction of lipids from Trametin Plus

Lipids were extracted from parallel weighed portions with a Folch mixture (chloroform–methanol, 2: 1, by volume) in plastic Eppendorf tubes with a capacity of 2 ml, shaking and keeping them in an ultrasonic bath (1.2 ml × 3 × 5 min). The extracts were combined in glass tubes for centrifugation, 1.2 ml of water was added, emulsified, and centrifuged at 3000 rpm. Prior to the extraction of lipids from sponges, 100 µL of 8% H2SO4 in water was added to weighed portions (wet weighing 0.02 g at 97% humidity), and after the color changed from green to yellow, lipids were extracted after 2 min. Extraction of lipids from sponges was carried out 1 time, 350 μl of water was added to the extract.

Acid esterification of fatty acids and production of methyl esters of fatty acids (total content) of Trametin Plus

The chloroform layer of the extract (bottom) was transferred into glass penicillin flasks with a capacity of 10 ml, evaporated to dryness in a flow of argon, 4.5 ml of 2% H2SO4 in methanol was immediately added, tightly covered with foil, and placed in a thermostat at 55°С for 1.5 h. After methanolysis fatty acid solutions were cooled to room temperature, 0.8 ml of n-hexane was added. FAMEs were extracted from the resulting solutions with n-hexane (3 mL × 2 × 2 min). Before the second extraction, 1 ml of water was added to the solutions. The extracts were concentrated to 1 ml in an argon flow and dried over anhydrous Na2SO4. The extracts were analyzed by GC-MS.

Separate production of methyl esters of esterified and free fatty acids under conditions of changing the pH of Trametin Plus

To weigh portions of isolated lipids (0.2 g wet weight) were added 2 ml of 0.4 M NaOH solution in methanol, and placed in an ultrasonic bath for 5 min. The resulting MEs of esterified FAs were extracted with n-hexane (3 ml × 2), washed with water, dried over anhydrous Na2SO4, and concentrated in an argon flow to a volume of 1 ml. Extracts of ME FEA were analyzed by GC. To the remaining alkaline solution 3 ml of water and 0.15 g of copper (II) sulfate ground in a porcelain mortar was added, and then shaken for 5–10 min until the blue color of Cu2SO4 turned green, Cu (OH)2 (up to pH 6.0), without overkeeping the solution to avoid the transition of Cu (OH)2 to black CuO, which can oxidize FA. Free FAs were extracted with n-hexane (3 ml × 2). Hexane was evaporated and the acid esterification of FFA was carried out. The extracts were analyzed by GC-MS.

Analysis of fatty acid methyl esters (qualitative) by gas chromatography with mass spectrometric detection of extracts

The extracts were analyzed on a 6890B GC System, 7000C GC/MS Triple Quad chromato-mass spectrometer (Agilent, United States) with an Optima-17 column (30 m × 0.25 mm, 0.25 µm) from Macherey-Nagel (Germany). Injector temperature 280 °С; quadrupole temperature 150 °С; ion source temperature 230 °С; type of ionization - electron impact; ionization energy 70 eV; sample volume 2 µl in splitless mode; temperature of the GC-MSD interface unit 310 °С; column flow 2.54 ml/min. Chromatography was carried out under conditions of heating the column from 80°C (hold 0.5 min) to 290°C at a rate of 2°C/min and then heating to 310°C (hold 5 min) at a rate of 5°C/min. Total analysis time 115 min. Peaks were detected in the m/z range of 40–500. Mass spectra were identified using the NIST Mass Spectral Search Program for the NIST Mass Spectral Library (V. 2.2) software.

Quantitative determination of FAME fatty acids by GC-MS

Quantitative determination of FAMEs was carried out by the internal standard method, for which 50 μL of a standard solution of dodecyl ether (C20H42O) in n-hexane (1 mg/mL) was added to the extracts before analysis. The detector was calibrated in the ΣFAME range from 40 to 540 μg per sample using standards (Supelco, USA) “35 F.A.M.E. Mix, C4–C24, 100 mg neat” and “Methyl cis-4,7,10,13,16,19-Docosahexaenoic ester, 10 mg/mL in heptane” (70–1000 µg per sample). Calibration factors have been determined for individual acids and for groups of acids (saturated, monounsaturated, polyunsaturated).

Extraction of non-polar organic components of Trametin Plus

Extraction of non-polar components of the sample was carried out from 1.0 g weighed portions of Trametin. The portion of the preparation was placed in a glass vial and extracted with 3 ml of n-hexane three times (1 min). The extracts were combined, washed with distilled water, dried over anhydrous sodium sulfate, concentrated in an argon flow to a volume of 0.5 ml, and analyzed by GC-MS in the full scan mode of the mass spectrum using the NIST Library of Mass Spectra software and the Mass Spectral Search Program for the NIST Mass Spectral Library (V. 2.2).

Extraction of polar organic components of Trametin Plus

Method 1. Extraction of the polar components of the 

sample was carried out from weighed portions of Trametin Plus weighing 1.0 g. ml), methanol (5 ml) and distilled water (10 ml) under vacuum. The elution of the sample concentrated on the cartridge was carried out with ethanol (5 ml) under vacuum. The extract was concentrated in a flow of argon to a volume of 0.5 ml and analyzed by GC-MS in full mass spectrum scanning mode using the NIST Mass Spectral Library software and Mass Spectral Search Program for the NIST Mass Spectral Library (V. 2.2).

Method 2. Extraction of the polar components of the sample was carried out from weighed portions of Trametin Plus weighing 1.0 g. Hydrochloric acid HCl was added to the weighed portion of the preparation to pH 2.0. Extraction was carried out with 3 ml of methylene chloride three times for 1 min. The extracts were combined, washed with distilled water, dried over anhydrous sodium sulfate, concentrated in an argon flow to a volume of 0.5 ml, and analyzed by GC-MS in the full scan mode of the mass spectrum using the NIST Library of Mass Spectra software and the Mass Spectral Search Program for the NIST Mass Spectral Library (V. 2.2).

Qualitative analysis of polar and non-polar components of samples by gas chromatography with mass spectrometric detection

The extracts were analyzed on a 6890B GC System, 7000C GC/MS Triple Quad chromato-mass spectrometer (Agilent, United States) with an Optima-17 column (30 m × 0.25 mm, 0.25 µm) from Macherey-Nagel (Germany). Injector temperature 280 °С; quadrupole temperature 150°C; ion source temperature 230 °С; type of ionization - electron impact; ionization energy 70 eV; sample volume 2 µl in 5:1 split mode. Division 12.691 cm3/min. The temperature of the GC-MSD interface unit 310 °С; column flow 3.02 ml/min. Chromatography was carried out under column heating conditions from 45°C (hold for 3 min) to 310°C (hold for 15 min) at a rate of 7°C/min. Total analysis time 56 min. Peaks were detected in the m/z range of 40–500. Mass spectra were identified using the NIST Mass Spectral Search Program for the NIST Mass Spectral Library (V. 2.2) software.

Table 1 shows the masses of samples of the drug Trametin Plus. The extractants, sample preparation method, analyzed sample components are indicated.

The Discussion of The Results

When analyzing free fatty acids (FFA), esterified fatty acids (EFA) and total fatty acids (TFA) of the veterinary drug Trametin Plus in the form of their methyl esters (ME). Among them, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were analyzed.

On the chromatograms of ME FFA extracts, 15 fatty acid peaks were identified, of which all 15 peaks were identified. On the chromatograms of extracts of ME FFA, 18 peaks of fatty acids were identified, of which all 18 peaks were identified. The chromatograms of extracts of ME OFA showed 18 fatty acid peaks, of which all 18 peaks were identified. In total, 18 fatty acids were identified in the samples of the veterinary drug Trametin Plus. Of these, 12 saturated fatty acids, 5 monounsaturated fatty acids and one polyunsaturated essential ω-6 polyunsaturated linoleic acid, presented in minimal quantities. Chromatographic characteristics of identified acids are presented in Table 2, qualitative composition and quantitative composition - in Table 3.

The share of esterified acids, that is, acids that are in 

the preparation in a bound form as acid residues of triglycerides and phospholipids accounts for 45.0% of all fatty acids of the preparation. The share of free unbound fatty acids accounts for 55.0% of all fatty acids of Trametin Plus (Table 3).

Table 2: Retention time tR and calibration coefficients k of identified FAs

The main share of all fatty acids of the drug (82.0%) falls on saturated fatty acids. The share of monounsaturated fatty acids accounts for 16.0% of all acids, the share of polyunsaturated fatty acids is 2.0% (Table 3). There is a redistribution in the composition of fatty acids depending on the degree of their saturation between free and bound acids. So, for free acids, the share of saturated fatty acids is 67.0%, and the share of unsaturated acids is 33.0%; for bound acids, saturated fatty acids account for 82.0%, while unsaturated acids account for 18.0%. The predominance of unsaturated acids in the free non-esterified form was noted (Table 3).

Table 3 shows that the average concentration of fatty acids in the drug Trametin plus is about 70.0 mcg/g of the veterinary drug. The concentration of saturated fatty acids is about 57.0 mcg/g. The concentration of unsaturated fatty acids is about 12.0 mcg/g. The concentration of free fatty acids is about 41.0 mcg/g, the concentration of esterified fatty acids is about 34.0 mcg/g of the preparation.

Table 3: Qualitative and quantitative (mcg/g) composition of fatty acids of Trametin Plus

Note: TFA1 is the total content of fatty acids (free and esterified) obtained by a direct method (see point 2); TFA2 is the total content of fatty acids (free and esterified), obtained by summing the values of the contents of FFA and EFA in the samples obtained according to point 3.

The percentage of fatty acids is shown in Table 4.

Qualitative analysis of volatile polar and non-polar organic compounds of the veterinary drug Trametin Plus was carried out using the NIST Database mass spectrum library. A very complex composition of the components is noted. In particular, this applies to hexane and ethanol extracts of the drug. A significant portion of the peaks in the chromatograms are inhomogeneous peaks that cannot be identified by simple comparison with the NIST mass spectra libraries. A significant part of the peaks is recorded as homogeneous peaks, but also cannot be identified by comparing the mass spectra of these peaks with the library ones since the mass spectra of the peaks of Trametin Plus and the library spectra have significant differences.

Table 4: Qualitative and quantitative (% of the total) composition of the fatty acids of Trametin Plus

Most likely this is due to the complexity of the object and the uniqueness of the biological compounds that make up its composition, some of which may yet be simply unknown to science. Detailed identification of the components of the drug requires serious long-term scientific research, thorough and detailed sample preparation for each class of compounds, study of the structure of compounds using various methods of chromatography, mass spectrometry, nuclear magnetic resonance, etc. A number of peaks of volatile components in the chromatograms of these extracts can be identified with a probability of ≥70.0%. These compounds are presented below (Tables 5, 6).

Non-polar volatile components of the sample, soluble in n-hexane, are represented by compounds with small molecular weights Mr ≤350 (Table 5). Among them are pyrans, furans, alcohols, aldehydes, ketones, heterocyclic compounds, saturated aliphatic hydrocarbons (saturated n-alkanes), unsaturated diene hydrocarbons, including squalene, aromatic compounds, indole (Table 5)

The polar volatile components of the sample, soluble in ethyl alcohol, are represented by compounds with small molecular weights Mr ≤310 (Table 6). Among them, acetic acid, acetoin - one of the products of butanediol fermentation, acetic anhydride, furfural - a product of xylose dehydrogenation (Table 6), heterocyclic compounds furanone and pyranone, aldehydes, including phenylacetaldehyde, peptides, ketones, simple sugars and amino acids, aromatic compounds.

Table 5: Non-polar organic components of the drug Trametin Plus according to the NIST library

Table 6: Polar organic components of the drug Trametin plus according to the NIST library

Volatile amino acids of Trametin Plus are presented as minor components of the sample. Among them are glycine and sarcosine in the form of the dipeptide glycylsarcosine, where sarcosine is a methyl derivative of the amino acid glycine, arginine, and β-alanine, presented in the form of an amide of the amino acid β-alanine and pantoic acid (group B vitamin).

Among these compounds, there are compounds that have undeniable and sufficiently strong biological activity. Activity is manifested as anti-inflammatory, antibacterial, antiviral, antioxidant, enveloping, immunomodulatory, and antitumor effects. At the same time, a number of compounds have undoubted toxicity in animal experiments, according to the literature data, including hepatotoxicity, carcinogenicity, general toxic effect, and neurotoxicity.

Conclusion

A quantitative analysis of fatty acids of the veterinary drug Trametin Plus was carried out. It is shown that the total concentration of fatty acids is 70 µg/g of the preparation. Of these, 50.0% are free fatty acids, most of which have a powerful antibacterial, antiviral, and antitumor effect, while esterified (bound) acids, which account for 50.0% of the total mass of fatty acids, to a greater extent, have an enveloping effect. and anti-inflammatory action. At the same time, essential polyunsaturated fatty acids are practically absent in the preparation. According to the literature, a high content of free fatty acids can be characteristic of a number of algae [6], micellar fungi [7], and their analysis plays an important role in diagnostic medicine [8].

Among the dominant non-polar volatile components of the drug Trametin Plus there is a triterpene compound - squalene C30H50 (reliability of determination by GC-MS method ≥95.0%). This substance has a powerful antioxidant, immunostimulating, enveloping surface-active effect, protecting the mucous membranes of the body.

The simplest organic aliphatic amino acids such as glycine (M = 75 g/mol), arginine (M = 174 g/mol), β-alanine (M = 89 g/mol) with a small molecular weight are presented as minor non-polar volatile components of Trametin plus.

The remaining organic components of the drug are able to have a complex effect on the body. The toxicity of some components to animals may be considered to establish the doses and timing of the use of the drug Trametin Plus.

References

1. Chkhenkeli V.A. Kalinovich A.E. Shkil N.A. Ecological and biological substantiation of the use of trametin in veterinary practice. Moscow: Pero. 2015.126 p.
   View at Publisher | View at Google Scholar
2. Chkhenkeli V.A., Romanova E.D., Vlasov B.Ya. Prevention of the negative impact of environmental factors on the body of young farm animals and poultry using the veterinary drug Trametin. LAP Lambert Academic Publishing RU: Mauritius 2020. 121 p.
   View at Publisher | View at Google Scholar
3. Chkhenkeli V.A. Mechanisms of action of the drug Trametin on the body of animals with associated infections. St. Petersburg: Prospekt Nauki, 2020.176 p
   View at Publisher | View at Google Scholar
4. Chkhenkeli V. A. Last generation drugs based on xylotrophic fungi of the genus Trametes: discovered effects, mechanisms of action and application. Moscow: Pero. 2014.126 p.
   View at Publisher | View at Google Scholar
5. Chkhenkeli V.A., Ulanskaya A.V. Salmonellosis as a biological factor in the eco-technical system and its prevention using the veterinary drug
   View at Publisher | View at Google Scholar
6. Falk-Petersen, S., Sargent, J. R., Henderson, J., Hegseth, E. N., Hop, H., & Okolodkov, Y. B. (1998). Lipids and fatty acids in ice algae and phytoplankton from the Marginal Ice Zone in the Barents Sea. Polar Biology, 20, 41-47.
   View at Publisher | View at Google Scholar
7. Feofilova, E. P., Usov, A. I., Mysyakina, I. S., & Kochkina, G. A. (2014). Trehalose: Chemical structure, biological functions, and practical application. Microbiology, 83, 184-194.
   View at Publisher | View at Google Scholar
8. Feofilova E.P., Gornova I.B., Memorskaya A.S. et al. Lipid composition of fruiting bodies and deep mycelium of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler) // Microbiology. 1998. Vol.67. No. 5. p. 655–659.
   View at Publisher | View at Google Scholar
9. Belova N.V. Prospects for the use of biologically active compounds of basidiomycetes in Russia // Mycology and Phytopathology. Vol.38. B. 2. p.1-7.
   View at Publisher | View at Google Scholar
10. Babitskaya V.G., Chernook T.V., Shcherba V.V. et al. Characterization of lipids in the deep mycelium of fungi // Journal of the Belarusian State University Ser. 2. Chemistry. Biology. Geography. 2009. Vol. 4. Part 1. p. 101.
    View at Publisher | View at Google Scholar
11. Loshchinina E.A., Tsivileva O.M., Makarov O.E. et al. Changes in the carbohydrate and fatty acid composition of the mycelium of Lentinus edodes during the co-cultivation of Azoprifilum brasilense // Journal
    View at Publisher | View at Google Scholar
12. Tsurkan Ya.S. Selection of microorganisms with a high content of practically significant polyunsaturated fatty acids: dissertation for the degree of Doctor of Philosophy. Almaty. 2015.195 p.
    View at Publisher | View at Google Scholar
13. Duel N.I. Biotechnological bases for the intensification of the cultivation of edible and medicinal macromycetes using low-intensity light: dissertation for the degree of Doctor of Biological Sciences. Kyiv. 2015. 387 p.
    View at Publisher | View at Google Scholar
14. Tsivileva O.M., Nguyen T.F., Vu L.N. et al. Lipid components of the pigmented and deep mycelium of Ganoderma in different climatic zones // Journal
    View at Publisher | View at Google Scholar
15. Ermine T.G. Chemical composition, production method and pharmacognostic characteristics of the mycelium of Inonotus rheades (Hymenochaetaceae): dissertation for the degree of candidate of agricultural sciences. Irkutsk. 2019.208 p.
    View at Publisher | View at Google Scholar
16. Oorzhak U.S. Scientific basis for the rational use of Tinder fungus ordinary. Kyzyl: Publishing House of TuvGU.2020.142 p
    View at Publisher | View at Google Scholar
17. Nikonova, A. A., Shishlyannikov, S. M., Shishlyannikova, T. A., Avezova, T. N., Babenko, T. A., Belykh, O. I., ... & Khutoryansky, V. A. (2020). Determination of free and esterified fatty acids in hydrocoles of different content of polyunsaturated fatty acids by gas–liquid chromatography. Journal of Analytical Chemistry, 75, 1310-1321.
    View at Publisher | View at Google Scholar