1

1 Institute of Environmental Problems of the North, Ural Branch of the Russian Academy of Sciences

2 Northern Arctic Federal University named after N.N. M.V. Lomonosov

3 Institute of Environmental Problems of the North, Ural Branch of the Russian Academy of Sciences, Northern Arctic Federal University named after V.I. M.V. Lomonosov

Significantly increased interest in biologically active substances of plant origin is explained by a wide range of pharmacological activity of bioactive substances. Among them, a special place is occupied by usnic acid, which has high antimicrobial, antioxidant, antitumor, and immunostimulating properties. In this article, a comparative analysis of methods for extracting usnic acid from a lichen of the genus Cladonia stellaris is carried out. Traditional extraction methods (maceration, percolation), their modifications (use of microwave radiation technique) and modern ones (use of sub- and supercritical solvents) are considered, their advantages and disadvantages are noted. It has been shown that the method of supercritical fluid extraction with carbon dioxide is highly effective, which makes it possible to obtain usnic acid in a high yield (up to 2.39?% by mass of a.s. lichen raw materials), while the extract contains 90–100?% usnic acid.

lichens

extraction methods

usnic acid

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Each species of lichen is characterized by the presence of certain lichen acids (for example, usnic, protolichesteric, lichesteric acids are characteristic of lichens of the genus Cladonia), which serves as their systematic feature. Usnic acid (UA) is a yellow crystalline substance, structurally related to dibenzofurans, has high activity against many pathogenic organisms of a viral, bacterial and fungal nature and has antioxidant, antitumor, immunostimulating and hepatoprotective properties (used as part of dietary supplements for weight loss) , which allows it to be successfully used in the treatment of diseases of various etiologies. Due to these properties, it is used in pharmacology, cosmetics, dentistry and other fields of medicine. However, despite the positive experience of using UA in many areas of clinical medicine, the production of medicines based on it has not been established. Probably, the known methods for isolating bioactive substances from lichen raw materials do not give the desired results. About 70 species of lichens containing usnic acid are known. However, only those of them in which the amount of this acid is at least 0.5% can be of industrial importance. A promising source of usnic acid is the lichen genus Cladonia, in which this compound is the main metabolite.

The classical methods for isolating biologically active compounds from plant materials are extraction methods using organic solvents. These include maceration (infusion), percolation (continuous filtration of the extractant through a layer of raw materials), repercolation. To isolate lichen acids, various organic solvents are used: benzene, acetone, hexane, ethanol, petroleum ether, chloroform, or mixtures thereof to increase the yield of the target product. The advantage of these methods is the simplicity of execution and equipment. The disadvantages include the duration of the extraction process, the high content of impurities in the extracts, the complexity, the use of significant amounts of solvents, often the high toxicity and volatility of the organic solvents used. However, despite these shortcomings, these methods are currently used, but more often in a modified form. Such methods include extraction using the technique of microwave radiation (SHF).

Along with the above traditional extraction methods, modern extraction methods are currently used, such as supercritical fluid extraction (SCFE), extraction with subcritical solvents, accelerated extraction with liquid solvents (ASE), which allow the extraction products to be isolated from plant materials without leading to their destruction and preserving the biological value of all components as much as possible. In this regard, numerous studies carried out in Russia and abroad, devoted to the development of new methods for extracting biologically active substances from natural matrices and the study of their properties, are intensively expanding.

The purpose of this work was a comparative study of the possibility of isolating usnic acid from lichen raw materials by traditional methods and methods using modern technologies.

The objects of this study were lichen thalli of the genus Cladonia stellaris growing in the subarctic territory of the Russian Federation. Lichen samples were collected on Russkiy Kuzov Island, White Sea.

Air-dry lichen raw material, previously purified from mechanical impurities, was crushed in an LN-201 laboratory mill. Elemental analysis of raw materials was carried out on an EvroEA 3000 elemental analyzer (configuration ). The lichen sample contains 42.9 ± 1.7; 6.68 ± 0.27; 1.19 ± 0.05% C, H, and N, respectively, humidity - 6.68%, ash content - 0.73%. To assess the biosafety of raw materials, the content of a number of toxic (including heavy metals), as well as biogenic elements, was determined. The analysis was performed on a sequential wave-dispersive X-ray fluorescence spectrometer XRF-1800. The elemental composition of lichen ash is characterized by a predominant content of biogenic elements: potassium (27.17%), magnesium (5.59%) and phosphorus (7.85%). Other elements (including some heavy metals) such as S, Cl, Ti, Mn, Cr, Sr, Br, Cu, Rb, Ni, Pb are present in amounts less than 1%, which does not significantly affect the vital activity of the lichen and its excretion from him BAV.

Isolation of lichen acids was carried out by various methods:

– extraction with organic solvents by infusion method;

– extraction with organic solvents on a Soxhlet apparatus;

- extraction using microwave technology;

– accelerated extraction with liquid solvents;

– supercritical fluid extraction with carbon dioxide;

– extraction with subcritical carbon dioxide.

Usnic acid was identified by HPLC. Chromatographic separation was performed on an LC-30 Neexera instrument (Shimadzu, Japan). Detection was performed using a spectrophotometric detector, diode array at a wavelength of 280 nm. The samples were dissolved in acetone, filtered and injected into the chromatographic system. Using a standard UA sample from Aldrich, a calibration dependence of the peak area on the concentration in the range from 1 µg/L to 0.1 mg/L was plotted. The dependence is linear with a correlation coefficient of more than 0.99.

Extraction with organic solvents by infusion method

Maceration is an ordinary soaking in a solvent, during which the cell walls of the plant material are loosened and the extracted substances are dissolved. About 5 g of lichen were placed in a flask with ethyl alcohol. The infusion was carried out in an oven at 70°C for 30 minutes. The content of UA in the extract was 24%, and the UA yield from a.s. lichen raw materials - 0.27%. To increase the yield of UA by this method, the duration of the extraction process must be significantly increased.

Extraction with organic solvents on a Soxhlet apparatus

During percolation, the solvent passes (leaks) through a layer of crushed raw materials and “washes out” the target components. A cartridge with a weight of about 5 g of lichen was placed in a Soxhlet apparatus. Acetone, ethanol, or chloroform (chemically pure grade) were used as the extractant; the duration of percolation was 30–60 min (table).

Yield of UA during extraction with various solvents on a Soxhlet apparatus

Despite its simplicity, traditional extraction does not allow one to obtain high yield UA by simple extraction, since the lichen plant cell with this extraction method remains intact and impenetrable, in addition, the use of toxic and flammable organic solvents makes this technology unsafe.

Extraction using microwave technology

To intensify the process of extracting biologically active substances, the impact on raw materials of various force fields is used. One of the effective ways to extract plant materials is microwave treatment in a microwave field. Technological parameters of the BAS extraction process in the microwave field: specific power 350 W/h; liquid module 1/15; extractant - ethyl alcohol. The duration of extraction varied from 5 to 20 min. The nature of the impact of the microwave field is similar to the intensive moisture-thermal treatment carried out by combining treatment with live steam and conductive heating, but the destruction of the structure under the influence of the microwave field occurs to a greater extent, which makes it possible to intensify the impregnation of the pores of plant raw materials with a liquid extractant and, accordingly, significantly speed up the process. extraction. During extraction with ethanol for 10 minutes, the UA yield reaches a maximum value of 1.36% of the a.s. mass. lichen raw materials (Fig. 1), while increasing the purity of the target product (the content of AA in the extract was 30%).

Rice. Fig. 1. The effect of microwave treatment on the yield of UA (% of the mass of raw materials) with varying the duration of extraction

The use of microwave technology for the extraction of UA made it possible to reduce the extraction time to 10 minutes, in comparison with traditional methods for extracting biologically active substances, while the yield and purity of the target product increase significantly.

Accelerated extraction method with liquid solvents

The accelerated solvent extraction method is a relatively new technology that uses elevated temperature and pressure to increase the rate and degree of extraction of target components from samples with different matrices. Extraction was performed on an ASE 350 instrument, Dionex USA. A sample of crushed lichen weighing 1 g, mixed with 1 g of diatomaceous earth, was placed in a 10 ml cell. Extraction was carried out at temperatures of 80, 100, 150°C and a pressure of 100 atm. Extraction parameters: solvents of different nature and polarity (water, acetone, ethanol), heating the cell for 5 min, keeping the sample at a given temperature for 5 min, extractant volume 10 ml.

It was shown that water is a poor solvent of usnic acid; the yield of usnic acid does not exceed 0.08% (Fig. 2).

The use of ethanol and acetone as an extractant (subcritical conditions) shows comparable results, and the UA yield reaches 2.77–2.82%, while the UA content in the extract was 20–30%. With an increase in the extraction temperature, the yield of UA increases. With ASE extraction, the process time is reduced to several minutes, sample preparation is significantly accelerated, and small amounts of solvent are required for its implementation. Thus, ASE is a promising method for isolating lichen acids, in particular, UA, and varying the process parameters can significantly increase the yield of the target component.

Rice. 2. Yield of UA (% wt. wt. raw material) in the extract obtained by the ASE method

Supercritical fluid extraction method

Supercritical fluid extraction was performed using an MV-10ASFE unit (Waters, USA). Supercritical carbon dioxide was used as an extractant. The SCFE process was carried out in a dynamic mode, in a wide range of temperatures (40–80 °C) and pressures (10–35 MPa). Extraction time 20 min. The extract after decompression was dissolved in a stream of dousing solvent (acetone, flow rate 2 ml/min). The use of a make-up solvent prevents the solid components of the extract from being carried away with the flow of carbon dioxide gas. Supercritical carbon dioxide is a stable and inert substance that exhibits chemical indifference to the processed raw materials and extracted substances. Also, its advantages are low cost and the possibility of repeated use. The use of carbon dioxide instead of organic solvents increases the environmental safety of production, as well as the degree of purity of the products obtained.

An increase in temperature from 40 to 80 °C leads to an increase in the efficiency of extraction, while the content of solids in the isolated extract increases from 1 to 2% of the mass of the a.s. raw materials taken for analysis. An increase in pressure from 10 to 35 MPa leads to a 2-fold increase in the yield of the target product (Fig. 3).

The extract obtained using CO2 in the supercritical state contains 90–100% usnic acid and is characterized by its high yield relative to the raw material – 0.52–2.39%. In addition, obtaining extracts using supercritical CO2 is economically beneficial, since this method makes it possible to produce sufficiently concentrated (or in solid form) extracts of high purity usnic acid.

Extraction using subcritical CO2

An extract of lichen acids can also be obtained using subcritical CO2 as an extractant (pressure 7 MPa, temperature 20 °C, CO2 supply rate 0.1 kg/h, CO2 consumption 100 kg/kg raw material). Extract yield 0.52% of a.s. raw materials, the extract contains 85% usnic acid and is characterized by a high yield of UA relative to raw materials - 1.02%. In addition, milder conditions (compared to SCFE) exclude isomerization processes during extraction, which contributes to the preservation of the biological activity of the isolated biologically active substances. Also, the advantage of using subcritical CO2 as an extractant is the reduction in energy costs for increasing the pressure and heating CO2.

Rice. 3. Influence of pressure and temperature of SCFE on the yield of UA (% of raw material a.s.)

Thus, the results of the quantitative extraction of usnic acid by various methods showed that traditional methods (maceration, percolation) are ineffective and laborious, and the obtained extracts contain a large amount of by-products. New technologies (extraction with supercritical and subcritical solvents, ASE method) can significantly increase the yield and improve the quality of the target product. Our studies have shown the feasibility of using the technique of supercritical fluid extraction, which makes it possible to extract usnic acid in the form of a solid extract in one technological stage, while the content of usnic acid in the extract is 90–100%.

The study was carried out with the financial support of the FASO of Russia within the framework of the theme (project) No. 0410-2014-0029 "Physical and chemical foundations for studying the main regularities of the fundamental cycle "structure - functional nature - properties" of natural polymer matrices", as well as within the framework of a scientific project of a comprehensive program of the Ural Branch of the Russian Academy of Sciences No. 0410-2015-0021 "New approaches to a comprehensive assessment of the state and evolution of forest and wetland ecosystems in the western segment of the Arctic" using the equipment of the Center for Collective Use of the NO "Arktika" (NarFU) with the financial support of the Ministry of Education and Science of the Russian Federation (Unique identifier of works RFMEFI59414X0004) and equipment TsKP CT RF-Arctic (IEPS, IFPA Ural Branch of the Russian Academy of Sciences).

Reviewers:

Poskotinova L.V., Doctor of Biological Sciences, Associate Professor, Head. Laboratory of Biorhythmology, Institute of Natural Adaptations, Ural Branch of the Russian Academy of Sciences, Arkhangelsk;

Khabarov Yu.G., Doctor of Chemistry, Professor of the Department of Pulp and Paper Technology, FSAEI HPE “Northern (Arctic) Federal University named after I.I. M.V. Lomonosov, Arkhangelsk.

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The biosynthesis of didymic acid, as well as of some other polycyclic lichen substances (in particular, usnic acid and strepsilin considered in this chapter), is probably carried out by condensation of acetyl residues with the formation of the corresponding phenolic compounds521, which then undergo oxidative dimerization and various further transformations.

It should, however, be noted that the drug obtained from the lichen Evernia i - irunastri and containing, along with usnic acid (see p.

Let us now consider the experimental data that led to the conclusion that formula (333) correctly reflects the structure of the usnic acid molecule.

At the same time, proceeding from formulas (333) of hI (349), it is possible to quite satisfactorily explain all the properties and transformations of usnic acid known so far, which makes us recognize these formulas as correct.

However, these studies began to develop most intensively only in the period 1930 - 1940, when a very significant number of reports appeared 1409 ms-i - u, devoted both to usnic acid itself and to a number of its cleavage products.

It is interesting, however, that the antibiotic properties of usnic acid do not depend on the spatial structure of its molecule: () -, (-) - and () - usnic acids have almost the same activity. It is taken up with Et2O and, after separation from other lichen substances, purified by crystallization from petroleum ether.

Usnic acid (333) and its diacetyl derivative (350) are titrated as monobasic acids, although they do not contain a carboxyl group. Usnic acid has three active hydrogen atoms, despite the presence of only two phenolic hydroxyls. It follows that its acyclic group contains an enol hydroxyl, which has strongly acidic properties.

Although usnic acid was first isolated in 1843 from Ramilina fraxinea and L/snea borbata, its chemical structure remained unknown for nearly a century. Numerous works have been devoted to the structure of usnic acid; however, clarity regarding the main structural features of this compound and its transformation products was introduced as a result of later studies by Robertson, Asahina and Schepf and their collaborators.

Using this formula, we can explain the behavior of usnic acid as a 1 3-diketone with respect to reagents that give characteristic reactions with a carbonyl group. Formula XIX makes it possible to explain the behavior of usnic acid during titration and in the determination of active hydrogen by the Chugaev-Tserevitinov method. Based on this structure, it is easy to explain the formation of products of hydrolytic cleavage of usnic acid.

Her thallus has the appearance of erect or prostrate bushes, the terminal branches are always bluish-black. It contains a large amount of usnic acid (up to 8%), reindeer especially willingly eat it.

The structure of most O-heterocyclic antibiotics has now been fully established, and many of them have been obtained synthetically. The chemical study of such antibiotics as patulin, usnic acid, griseofulvin, geodin, citrinin, and sesamin required considerable effort, and the formulas proposed for them had to be repeatedly revised. As a typical example, we can cite usnic acid, the establishment of the structure of which required more than sixty years of research, and its synthesis was carried out another 15 years later. On the other hand, an undeserved amount of attention has recently been paid to the study of rather ordinary transformations, for example, of such a relatively simple compound.

Due to the high antibiotic activity of a number of lichens (their extracts), the substances contained in lichens deserve special attention. Biological activity is explained primarily by the presence of d-usnic acid, found in the species usnea, evernia, letaria and par-melia. About 70 other compounds have also been isolated from lichens, which can be classified as depsides, depsidons, dibenzofurans, and lactonic acids. For identification, it makes sense, along with acids isolated from lichens, to analyze chromatographically the products of their hydrolysis.

Cetraria islandica
Taxon: Parmelia family ( Parmeliaceae)
Other names: Iceland moss
English: iceland moss

Botanical description

Not one plant individual, but a symbiosis of two organisms, of which one belongs to the kingdom of fungi (mainly marsupials), and the other to green or blue-green algae. Both of these organisms are so closely related to each other that they supposedly belong.
Icelandic cetraria or is a perennial foliose lichen, the bushes are erect, less often prostrate, they stand from almost compact vertical lobes. The lobes are irregularly ribbon-shaped, leathery-cartilaginous, narrow, flat, up to 10 cm high and 0.3-5.0 cm wide, with short dark cilia, greenish-brown or with various shades of brown, depending on the lighting, at the base with reddish spots, dull or shiny on the underside, sometimes lighter or the same color on both sides. The underside is abundantly covered with white spots (pseudocyfelames) of various shapes. The edges of the blades are somewhat wrapped up. Cilia at the base are large (sometimes they are completely absent), drying out, they become dark brown.
In cetraria, apothecia, or fruiting bodies, sometimes develop at the ends of strongly expanded lobes. They are plate-shaped, brownish, almost the same color as the thallus, with a flat or somewhat convex disc up to 1.5 cm with a somewhat serrated edge. Apothecia develop sacs filled with spores that can be seen under a microscope. Spores are unicellular, colorless, 8 pcs. in each bag, elliptical shape.
Cetraria Icelandic, like most species of the genus Cetraria, has an extremely slow growth compared to other representatives of lichens. For the development of this species, favorable conditions are needed on the one hand, for the fungus, on the other hand, for the algae. However, sometimes these conditions are unfavorable. Most lichens of the genus Cetraria are characterized by intercalary growth, i.e. any part of the lichen can give rise to new individuals, which in the conditions of the Arctic occurs in a rough-mechanical and vegetative way. With the help of wind, deer and human activity, pieces of Icelandic moss are spread across the tundra to large areas, until they cling to the substrate, fragments of moss begin to grow in the form of new individuals (K. A. Rassadin, 1950).

Distribution of Icelandic cetraria

Cosmopolitan flora element of the globe. This moss is widely distributed in Europe, Asia, Africa, America and Australia. This is a typical representative of pine forests, open barren spaces. Cetraria is distributed throughout the northern hemisphere to the Arctic zone. Icelandic moss grows in the tundra, dry pine forests of the northern part of the forest zone, in all high mountains (alpine moss-lichen tundra), rising to a height of 1500 m above sea level and higher. Icelandic moss is widespread in stony and grassy areas, in peat bogs, high mountain glades, in mountain forests, sometimes on the bark of old stumps. It is found in Northern and Central Europe, in the tundra and forest zone of Siberia, in Ukraine - in the Carpathians. In Europe, in addition to the Carpathians, it grows in the Alps, the Balkans and the Pyrenees. It grows on the soil itself, less often on rotten bark and on old stumps. In the northern part of Russia, cetraria is more widely distributed in the European than in the Asian part. It also grows in the mountains of the Caucasus, Altai, Sayan and the Far East.
grows mainly on sandy, unshaded places, forming pure thickets in podekuly. It is also often found in pine forests and in heather thickets, where it grows in small groups and single specimens among other lichens, mosses and higher plants. Cetraria is a typical plant of swamps, forest tundra and tundra, where it grows together with other lichens.
Cetraria Icelandic is a polymorphic species, in which, depending on lighting, moisture and other factors, both the color and the size of the blades change. Cetraria Icelandic develops only in conditions of frequent air in ecologically clean regions. Due to this factor, cetraria is an indicator of cleanliness in industrial industrial areas. This factor can find direct practical application in our time in solving global environmental problems.

Collection and preparation of medicinal raw materials of cetraria

In medicine, the dried thallus of Icelandic moss is used ( Lichen islandicus), which has a slight peculiar smell and a bitter-mucilaginous taste. Cetraria thallus is harvested during summer and dry autumn. When harvesting, the thallus of the cetraria is torn off from the substrate (soil or tree bark), the freshly harvested thallus is cleaned of impurities, dried in the sun or in the shade, laid out in a thin layer (3-5 cm) on paper or cloth.
Cetraria thallus can be harvested during the entire growing season, but this type of raw material is mainly harvested in the summer.
Store dried raw materials in boxes with paper lining or in tightly closed jars in a dry, cool room (raw materials are very hygroscopic).
The dry thallus of the cetraria soaked in water should become slimy, and the decoction should turn into jelly after cooling.
As some manuals on the procurement of medicinal raw materials indicate, the natural resources of the cetraria in Ukraine, Russia and some other countries significantly exceed the need for this type of raw material.

Biologically active substances

The study of the chemical composition of the Icelandic cetraria began several hundred years ago, and today it is quite well studied.
In Icelandic cetraria, as in most other lichens, a significant amount of biologically active substances is synthesized. The thallus consists mainly of carbohydrates, among which there are chitin, lichenin, isolichenin, sucrose, mannitol galactomannan, umbilicin, hemicellulose, erythritol and other carbohydrates.
The thallus of the Icelandic cetraria can accumulate up to 50-80% of polysaccharides, which dissolve upon extraction with hot water, forming a thick mass. Lichenin- a linear polysaccharide, hydrolysis gives glucose, dissolves in hot water, does not turn blue from iodine.
Isolichenin has a similar chemical structure, dissolves in cold water, turns blue from iodine.
Icelandic cetraria and other lichens contain organic acids of various composition, which are called lichen acids. It is the acids that give the lichen a bitter taste and determine its tonic and antibiotic properties.
In addition to lichen acids, the Icelandic moss thallus contains naphthoquinone (juglone), pentacyclic triterpene fridelin, proteins, vitamins C and B12, fats, wax, gum, pigments, and minerals.
An interesting fact is the presence of antiscorbutic vitamin C, which is contained in Cetraria cucullata in an easily digestible state. Such a discovery was made by the Russian doctor Granatik, who worked for several years in the north of the Far East. On the basis of experiments carried out on guinea pigs and observations on scurvy patients, he found that vitamin C remains unchanged in dried lichen raw materials for 3 years. Because to Cetraria cucullata close Cetraria nivalis and Cetraria islandica, then these species can be considered a potential source of ascorbic acid (Rassadin K. A., 1950).

The use of cetraria in medicine

The first information about the use of Icelandic cetraria as a medicinal raw material dates back to the distant past. The first indications of the use of lichens in medicine were known in Egypt as early as 2000 BC.
Since the Middle Ages, Icelandic moss has been widely used in folk medicine in the countries of Northern Europe - Iceland, Norway, Sweden - as an enveloping remedy for bronchitis. Means of cetraria in the form of infusions or decoctions were also used by the peoples of the Scandinavian countries as bitterness to stimulate appetite. They treated dysentery, dyspepsia, chronic and other disorders of the gastrointestinal tract. Icelandic moss was also known as an emollient, nourishing and general tonic. Cetraria thallus was also widely used in the treatment of pulmonary tuberculosis, whooping cough, bronchitis, laryngitis, bronchial asthma and other bronchopulmonary diseases. In addition, cetraria preparations were used for malignant tumors, bleeding, and as a means of reducing excessive sexual excitability in nymphomaniac women.
As an external agent, cetraria was used in the form of lotions from a decoction for wounds, burns, ulcers, infected wounds, hydradenitis, abscesses, boils, acne, microbial eczema.

The first written mention of the use of Icelandic moss as a medicinal raw material appeared in the 17th century. Second half of the 18th and first half of the 19th centuries were the period of the most widespread use of Icelandic moss as a remedy. Among all known lichens, some authors of the time especially highly valued the Icelandic cetraria. In particular, in 1809, Luyken wrote that this moss is in first place among the most medicines. Pointing to the possibilities of therapeutic use of cetraria, including in tuberculosis, Luyken noted that for the antiseptic effect, drugs with cetraria stand out among all the drugs known at that time. In the XVIII and XIX centuries. Cetraria was a well-known traditional remedy in the treatment of pulmonary tuberculosis, and its thallus was included in most of the then European pharmacopoeias.

At the end of XIX and beginning of XX century. due to the intensive development of scientific and practical medicine, doctors began to use medicines with cetraria less often, however, the healing properties of this moss were indicated only in some herbalists.

In 1919, A. A. Elenkin and V. E. Tishchenko wrote the first scientific monograph "Iceland moss and other useful lichens of the Russian flora." The book was submitted for printing to the publishing house of the Petrograd branch of the Russian Food Science and Technology Institute. However, this book was not published due to the liquidation of this institute. In the same year, V. N. Lyubimenko, on the basis of the above manuscript, published the article "Icelandic moss as a food product", and later A. A. Yelenkin in the monograph "Lichens as an object of pedagogy and scientific research" touched upon the problems of practical application of Icelandic cetraria in the food industry . During the period of intervention and civil war in the USSR in the 1920s, which caused famine in certain regions of the country, the peoples of the Russian north used the thallus of Icelandic moss as an additional food product. Removing bitter substances from the cetraria with soda or alkali and drying the peeled thallus, they mixed it into flour and baked bread. Among many northerners, the cetraria of the time was known as bread moss.

Pharmacological properties of cetraria

Biologically active substances of cetraria have anti-inflammatory, softening and expectorant effects.
Icelandic moss polysaccharides have the ability to protect the mucous membrane of the respiratory tract from the effects of various chemical factors.
In the 40s of the last century, it was found that Icelandic cetraria and other lichens have antibiotic activity. This period can be considered the beginning of intensive study and application of cetraria in scientific pharmacy and medicine.

For the first time, the antibacterial activity of extracts from various lichens was noticed by Birdholder and Evans and co-workers in 1944-1945. They tested aqueous, water-buffered, ethereal, alcoholic, and chloroform extracts and suspensions of nearly 100 species of US lichens. A large number of them were active against Staphylococcus aureus and Bacillus subtilis. Against gram-negative bacteria, most of the tested lichen products showed a negative effect. The researchers made the assumption that the antibiotic activity of lichens is due to the presence of lichen acids in them. However, this fact has not been experimentally confirmed. In 1947, Stol, Renz and Lacktka studied the antimicrobial activity of glucose-alkaline extracts - suspensions obtained from 58 species of lichens in the Swiss flora - and established a noticeable activity against Staphylococcus aureus in 38 species. In 1952, K. O. Vartia found antimicrobial activity in 75 out of 149 studied species of lichens in the Finnish flora.
The study of the antimicrobial activity of individual individual lichen substances began in 1945, when P. R. Burkholder et al. reported activity of usnic acid against Staphylococcus aureus. In 1946, V. C. Barry found that roccelic acid, isolated from Lecanora sordida, has little activity Mycobacterium Phlei and Mycobacterium tuberculosis bovis. However, its monoesters and monoamides have the ability to completely inhibit the growth of tuberculosis bacteria at a dilution of 1:500,000.

In 1949, Stoll et al reported the antibacterial activity of some lichen acids. It should be noted that most microbiologists paid attention only to individual lichen acids isolated at that time.
Later it was found that certain lichen acids exhibit pronounced antimicrobial activity. Usnic acid is especially valuable, which has strong antibiotic activity. Its sodium salt at a dilution of 1:2,000,000 inhibits the growth of mycobacterium tuberculosis and other gram-positive microorganisms (staphylococci, streptococci). In terms of antibacterial activity, usnic acid is about 3 times inferior to streptomycin. It has been established that lichen extracts act mainly on gram-positive acid-resistant bacteria. And only a few, as an exception, into separate gram-negative species. In particular, Vartia believes that the activity of lichen extracts against gram-negative bacteria in some cases is due to the decomposition products of individual lichen substances. S. Shibata et al. indicate that the antibacterial action of aqueous extracts of lichens differs from that of individual substances. Therefore, in their opinion, it is quite possible that substances that are insoluble in water can be carriers of antibacterial properties.
In the process of studying the technology of medicines that are made from cetraria, it was found that when making a decoction, only cetraric acid passes into the water, while usnic acid does not.

Usnic acid in small doses, it has the ability to kill pathogens of tuberculosis and some other gram-positive bacteria.
Of great importance is fumaro-protocetraric acid, which is considered one of the most active antimicrobial factors of cetraria. In addition to the above, German scientists consider protocetraric acid isolated from an aqueous extract of cetraria to be a strong immunomodulator that promotes the activation of the immune system (Huovinen, 1989).
In the free state and in the form of salts, D-protolichesteric acid is active on Helicobacter pylori(at a concentration of 16 - 64 mcg / ml). Obviously, the therapeutic efficacy of Icelandic moss in gastric and duodenal ulcers is at least partially associated with this effect. Protolichesteric acid suppresses the proliferative response of lymphocytes to stimulation with mitogens, and therefore it may be a potential tool for the treatment of autoimmune diseases.

High antibacterial and antifungal activity is also shown by naphthoquinones contained in a small amount in Icelandic cetraria.
The mucus and acids contained in the moss exhibit.

The pentacyclic triterpene fridelin and protolichesteric acid exhibit anti-inflammatory properties. The latter is an inhibitor of arachidonic acid 5-lipoxygenase (ED50 = 8.4 µg/ml), due to which it inhibits the synthesis of leukotrienes, which are important inflammatory mediators.

Clinical Application

The first pharmaceutical preparation called Evozin based on lichen acids was created in Germany in the 50s. It had a pronounced antimicrobial activity due to the presence of evernic and usnic acids in the composition. The specified drug was used in clinical conditions for the treatment of other diseases caused by pathogenic microorganisms.
For the treatment of pulmonary tuberculosis, German scientists proposed the drug eosin-2, which, in addition to evernic and usnic acids, included such lichen acids as atronarinic, physodic, caperic acids.
A mixture of usnic acid with streptomycin was used to treat tuberculosis and skin diseases.
In Japan, an antibiotic preparation from lichens was obtained, which is used in the treatment of actinomycosis.
Due to its emollient and expectorant properties, due to the significant content of mucous substances in therapeutic practice, Icelandic moss is a good remedy for bronchitis with a debilitating cough, pulmonary tuberculosis, and bronchial asthma.
In Finland, a method for obtaining a remedy for asthma, cough and runny nose based on Icelandic moss using extracts from yarrow herb, dandelion root, juniper fruit, cinquefoil rhizomes, horsetail herb, coltsfoot herb, bearberry leaves and willow bark has been patented.

In 1956, a preparation based on usnic acid was obtained in the USSR sodium usninate, which in the form of alcohol and oil solutions is used as an effective antimicrobial agent in the treatment of suppuration of wounds, burns, cracks. Despite the positive effect of usnic acid on the healing process of infected wounds, the bacterial flora on the wound surface decreases and disappears slowly, continuing to exist until the end of complete epithelization. The drug Binan is active on different strains of Staphylococcus aureus (titer from 1:45 to 1:35,000), hemolytic staphylococcus (titer from 1:100,000 to 1:350,000). The drug showed high bacterial activity, is stable during storage, but is quite toxic. Recommended only as an external agent in the treatment of infected wounds, if the surface of the wound is very large. This method was also effective in the treatment of acute inflammation of the soft tissues.

Sodium usninate, dissolved in fir balsam ( Balm Binan), is an excellent tool that can be effectively used in surgical practice for tissue transplantation. In particular, it has been established that the indicated form of sodium usninate has the ability to fix and prevent infections of free skin grafts without affecting its regenerative properties, also eliminates an unpleasant odor during wound suppuration and promotes rapid healing of significant donor sites during skin transplantation.

Binan balm was proposed to be used in the treatment of cervical erosion and indicated that it has the ability to stimulate the process of epithelialization on the cervix after diathermo-surgical intervention. Clinical observations in the treatment of cracks in the nipples of women in labor indicate the complete disappearance of purulent mastitis. Positive results have also been obtained in clinical studies using Binan to prevent differences in surgical sutures. Binan was also recommended for the treatment of athlete's foot and other skin diseases. However, despite the above effectiveness, the drug Binan was never introduced into clinical practice and is not used in medicine.

Crushed thallus of cetraria in the form of tablets is considered promising for topical use in stomatitis. Moss tablets were applied to patients on days 1-5 after surgery on the nasal cavity (drying and inflammation of the oral mucosa occurred due to breathing only through the mouth). With the use of 10 tablets per day (0.48 g per day), the drying of the mucosa, the amount of plaque on it, signs of inflammation of the oral mucosa, tongue and lymph nodes, pain and hoarseness of the voice decreased. No side effects were observed with this treatment.

Lichen decoction also acts as an enveloping, soothing and wound healing agent. It exhibits a pronounced therapeutic effect in gastrointestinal diseases, including diarrhea, and digestive disorders. In clinical studies, it was found that in patients with gastric ulcer, taking an alcohol extract of cetraria before meals eliminates the pain associated with eating. Obviously, this is due to the enveloping effect of the drug. Before preparing the decoction, the thallus is soaked in cold water to remove bitterness, then 2 teaspoons of chopped thallus are poured into 2 cups of cold water, brought to a boil and boiled for 5 minutes, filtered and drunk during the day.

The bitterness contained in the decoction of Icelandic moss excites the appetite,. Therefore, a decoction of cetraria is used as a general tonic during the rehabilitation period after serious illnesses. But the bitterness of Icelandic moss, unlike similar compounds of other plants, has not been widely used in gastroenterology, primarily due to the problems of the raw material base (slow growth of moss, its destruction, difficulties in growing).

Previously, it was very common to believe that a decoction of cetraria is a good nutrient, since its carbohydrates (lichenan, isolichenan) are easily absorbed by the body. However, in further studies, this property of polysaccharides isolated from cetraria was not confirmed. Therefore, the use of cetraria as a valuable nutritional product is inappropriate, and at present there are no prospects for its use in the food industry.

Medicines

Bronchial plus for children. Syrup with Iceland moss, chamomile and vitamin C (Dr. Muller Pharma, Germany).
Syrup in bottles of 100 ml.
5 ml (6.5 g) syrup contains liquid extract of Icelandic cetraria (1:10) 0.390 g, liquid extract of chamomile flowers (1:10) 0.260 g, ascorbic acid 0.019 g.
Take 1 tablespoon 3 times a day before meals for inflammation of the upper respiratory tract, accompanied by a cough, for acute and chronic bronchitis, and for influenza.
Bronchialtee 400(TAD, Germany).
Tea granulate, 100 g of which contains 5.4 g of thick aqueous extracts (7.8:1) with 10 g of fennel fruit, 5 g of Icelandic moss, 10 g of thyme herb, 5 g of marshmallow, 7 g of sage leaves and 5 g of linden flowers . It is used for colds, acute and chronic bronchitis. Take 1 cup of tea 2-3 times a day.
Isla Mint Pastillen(Engelhard, Germany).
Pastilles containing 100 mg or 160 mg of water extract (2-4:5) of Icelandic moss.
They are used for irritating coughs, hoarseness, dry mucous membranes, bronchial catarrh, for maintenance therapy of bronchial asthma. Take 1-2 lozenges several times a day, slowly dissolving.
Salus Bronchial Tee #8(Salushaus, Germany).
Tea, 100 g of which contains: fennel fruits - 15 g, Icelandic moss - 11 g, mullein flowers - 4 g, linden flowers - 12 g, primrose flowers - 6 g, deaf nettle flowers - 4 g, thyme herb - 13 g, knotweed grass - 12 g, marigold flowers - 4 g, raspberry leaves - 19 g.
It is used to thin mucus and relieve cough in catarrhs ​​and inflammations of the respiratory system. Take 1 glass of hot tea 4-5 times a day.

Toxicology

Usnic acid, as well as its salts, have a rather toxic effect on the organism of animals. For mice weighing 25 g, subcutaneous administration of 2.0 mg of usnic acid in sesame oil is lethal. When the dose was reduced to 1.5 g, no symptoms of poisoning were observed in these animals.
In humans, the daily administration of 0.1-1.0 sodium usninate had no harmful effect, however, at a daily dose of 3 g, pain in the liver occurred, which stopped when the dose was reduced.

Application in the economy

In the past thallus with Cetraria islandica, as well as other lichens, with the addition of metal salts, were also used as a coloring raw material. The simple production of synthetic aniline dyes quickly supplanted the artisanal production of dyes from creeping lichens.
In 1944, Pepper Lano reported on the production of a high-quality adhesive substance, similar to gelatin, from creeping cetraria, which could replace expensive gum arabic in industrial pharmacy (K. A. Rassadin, 1950).

Based on the materials of the works of B. M. Zuzuk, R. V. Kutsik (Ivano-Frankivsk State Medical University), M. R. Shtokalo (OOO, Lviv).

Photos and illustrations

Usnic acid

Usnic acid
General
Systematic name	2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzofurandione
Chemical formula	C 18 H 16 O 7
Physical properties
Condition (st. conv.)	solid
Rel. molek. weight	348 a. eat.
Molar mass	344.315 g/mol
Density	1.54 g/cm³
Thermal properties
Melting temperature	204°C

Usnic acid is one of the specific lichen substances that are formed during metabolism and are not found in other groups of organisms. The name comes from a genus of lichens Usnea.

Among the properties of usnic acid, as biologically active, for humans, of greatest interest is its antibacterial activity, which has already found application in medicine: the drug binan (sodium salt of usnic acid) is used in the treatment of many diseases, including against tuberculosis, and also as an anti-burn remedy that can be bought in pharmacies. This determined the interest in usnic acid.

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Usnic acid is one of the specific lichen substances that are formed during metabolism and are not found in other groups of organisms. The name comes from a genus of lichens Usnea.

Lichens are well known for their variety of secondary metabolites, the so-called lichen substances. Perhaps the most well-known secondary metabolite of lichens is usnic acid, which is produced during childbirth. Cladonia, Usnea, Lecanora, Ramalina, Evernia, Parmelia, Alectoria and in other genera of lichens. Usnic acid has antiviral, antibiotic, analgesic, anti-tuberculosis and insecticidal activities.

Usnic acid is produced by the lichen mycobiont - this was first shown in the work and subsequently usnic acid was extracted from isolated mycobionts of lichens of the genus Ramalina. Usnic acid was first isolated in 1843 from lichens of the genera Ramalina and Usnea, a year later it was characterized as an individual substance and got its name. Nine decades later, its chemical structure was established.

Usnic acid is produced in lichens in large quantities, accounting for up to 8% of the dry weight of thalli. There are large seasonal fluctuations in the content of usnic acid in lichen thalli: peak levels in late spring and early summer, and generally low levels during autumn and winter. The content of usnic acid correlates with the time of the onset of the summer solstice, levels of solar radiation and temperature conditions, and depends on the place where the lichen grows.

Usnic acid is a yellow crystalline substance, in its structure it belongs to dibenzofuran derivatives and exists in the form of two enantiomeric forms that differ in the configuration of the methyl group at the C 9b atom. The dextrorotatory enantiomer has R-configuration of the angular methyl group and its specific rotation is +478 (with 0.2 CHCl 3 , (deg ml) (g dm) -1). A typical producer of (+)-usnic acid is Usnea longissima, the source of the levorotatory enantiomer of usnic acid can be called Cladonia stellaris(-458, s 0.2 CHCl 3 , (deg ml) (g dm) -1).

The hydroxyl groups of usnic acid are involved in the formation of strong intermolecular hydrogen bonds. The dissociation constants of the hydroxyl groups of usnic acid, determined by spectrophotometric titration, are: pKa 1 4.4 (C 3 -OH), pKa 2 8.8 (C 7 -OH), pKa 3 10.7 (C 9 -OH). The acidity of the environment and the ratio of neutral and anionic forms of usnic acid, according to the researchers, play an important role in the life of the lichen.

The hydroxyl groups of this molecule form strong intramolecular hydrogen bonds, and are also capable of forming intermolecular hydrogen bonds, which can contribute to the rapid transfer of excess energy received by lichens from the Sun to the environment in the form of heat.

The presence of a resorcinol cycle and a system of conjugated carbonyl groups contribute to the fact that the usnic acid molecule absorbs widely in the near UV (320-400 nm), mid-UV (280-320 nm) and far UV (below 280 nm) ranges. It should be noted that this metabolite acts as an effective sunscreen for lichens. This allows lichens, for example, under the condition of long exposure to the sun in hot deserts, to reduce the harmful effects of solar radiation.

The main method for obtaining usnic acid, from the first studies in the 19th century to the present day, is the extraction of lichens with organic solvents and subsequent precipitation from the extract or its recrystallization. Usnic acid is highly soluble in benzene, chloroform, amyl alcohol, glacial acetic acid, sparingly soluble in ethanol, petroleum ether, diethyl ether, and insoluble in water.

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Notes

An excerpt characterizing Usnic acid

Balashev could not answer this and silently bowed his head.
“Yes, in this room, four days ago, Winzingerode and Stein conferred,” Napoleon continued with the same mocking, confident smile. “What I cannot understand,” he said, “is that Emperor Alexander brought all my personal enemies closer to him. I do not understand this. Did he think that I could do the same? - he asked Balashev with a question, and, obviously, this memory pushed him back into that trail of morning anger, which was still fresh in him.
“And let him know that I will do it,” said Napoleon, standing up and pushing his cup away with his hand. - I will drive out of Germany all his relatives, Wirtemberg, Baden, Weimar ... yes, I will drive them out. Let him prepare a refuge for them in Russia!
Balashev bowed his head, showing with his appearance that he would like to take his leave and is listening only because he cannot but listen to what he is told. Napoleon did not notice this expression; he addressed Balashev not as an ambassador of his enemy, but as a man who was now completely devoted to him and should rejoice at the humiliation of his former master.
- And why did Emperor Alexander take command of the troops? What is it for? War is my trade, and his business is to reign, not to command troops. Why did he take on such responsibility?
Napoleon again took the snuffbox, silently walked several times around the room and suddenly unexpectedly approached Balashev and with a slight smile so confidently, quickly, simply, as if he was doing some not only important, but also pleasant for Balashev, he raised his hand to the face of the forty-year-old Russian general and, taking him by the ear, tugged slightly, smiling only with his lips.
- Avoir l "oreille tiree par l" Empereur [To be torn by the ear by the emperor] was considered the greatest honor and mercy at the French court.
- Eh bien, vous ne dites rien, admirateur et courtisan de l "Empereur Alexandre? [Well, why don't you say anything, adorer and courtier of Emperor Alexander?] - he said, as if it was funny to be in his presence someone else courtisan and admirateur [court and admirer], except for him, Napoleon.
Are the horses ready for the general? he added, bowing his head slightly in response to Balashev's bow.
- Give him mine, he has a long way to go ...
The letter brought by Balashev was Napoleon's last letter to Alexander. All the details of the conversation were transferred to the Russian emperor, and the war began.

After his meeting in Moscow with Pierre, Prince Andrei went to Petersburg on business, as he told his relatives, but, in essence, in order to meet there Prince Anatole Kuragin, whom he considered it necessary to meet. Kuragin, whom he inquired about when he arrived in Petersburg, was no longer there. Pierre let his brother-in-law know that Prince Andrei was coming for him. Anatole Kuragin immediately received an appointment from the Minister of War and left for the Moldavian army. At the same time, in St. Petersburg, Prince Andrei met Kutuzov, his former general, always disposed towards him, and Kutuzov invited him to go with him to the Moldavian army, where the old general was appointed commander in chief. Prince Andrei, having received an appointment to be at the headquarters of the main apartment, left for Turkey.
Prince Andrei considered it inconvenient to write to Kuragin and summon him. Without giving a new reason for a duel, Prince Andrei considered the challenge on his part compromising Countess Rostov, and therefore he sought a personal meeting with Kuragin, in which he intended to find a new reason for a duel. But in the Turkish army, he also failed to meet Kuragin, who returned to Russia shortly after the arrival of Prince Andrei in the Turkish army. In the new country and in the new conditions of life, Prince Andrei began to live easier. After the betrayal of his bride, who struck him the more, the more diligently he concealed from everyone the effect made on him, those living conditions in which he was happy were difficult for him, and even more difficult were the freedom and independence that he so cherished before. He not only did not think about those former thoughts that first came to him, looking at the sky on the field of Austerlitz, which he liked to develop with Pierre and which filled his solitude in Bogucharov, and then in Switzerland and Rome; but he was even afraid to recall these thoughts, which opened up endless and bright horizons. He was now interested only in the most immediate, not connected with the former, practical interests, which he seized on with the greater greed, than the former ones were hidden from him. It was as if that endless receding vault of the sky that had previously stood above him suddenly turned into a low, definite vault that crushed him, in which everything was clear, but nothing was eternal and mysterious.