Corresponding author: Viktoriia V. Protska ( vvprotskaya@gmail.com ) Academic editor: Maya Georgieva
© 2019 O. A. Kyslychenko, Viktoriia V. Protska, Iryna O. Zhuravel.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kyslychenko OA, Protska VV, Zhuravel IO (2019) HPLC determination of phenolic compounds content in Parmelia sulcata and Parmelia vagans thalli. Pharmacia 66(4): 161-164. https://doi.org/10.3897/pharmacia.66.e35194
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The species of Parmelia genus have long been used in Indian folk medicine for the treatment of bronchitis, ulcers, furunculosis, cardiovascular diseases, urolithiasis, amenorrhea, and also at infectious and inflammatory diseases. In Ukraine, the most common lichens of the Parmelia genus are Parmelia sulcata Tailor and Parmelia vagans Nyl. At the same time, thalli of Parmelia genus lichens belong to the non-officinal and poorly studied types of raw material.
The qualitative composition and the quantitative content of phenolic compounds in Parmelia sulcata and Parmelia vagans thalli was studied by HPLC.
According to the results of the chromatographic analysis, salazinic, fumaroprotocetraric, usnic acids, chloratranorin and atranorin were identified in both types of raw material studied. In addition, protocetraric acid was identified in Parmelia sulcata thalli.
According to the results of the experiment, the total content of identified phenolic compounds in Parmelia sulcata thalli was 2019.71±40.39 g/mol, and in Parmelia vagans thalli it comprised 1754.18±34.77 g/mol.
In the thalli of both studied species of Parmelia genus, fumaroprotocetraric acid dominanted by the quantity. This substance was present in Parmelia sulcata thalli in the amount of 474.00±9.00 g/mol, and in Parmelia vagans thalli – 456.21±8.67 g/mol.
In addition, a significant amount of chloratranorin (408.79±8.99 g/mol) was present in Parmelia sulcata thalli. Quite a high content of atranorin (393.34±8.65 g/mol) and usnic acid (375.31±7.53 g/mol) were defined in Parmelia vagans thalli.
The results obtained can be used in the development of quality control methods for Parmelia sulcata and Parmelia vagans thalli, as well as medicines based on these types of raw materials.
HPLC, lichen, lichen acids, Parmelia sulcata, Parmelia vagans, phenolic compounds
Parmelia Ach. genus includes about 71 species of lichens, which body is built on symbiotic relationship between the hyphae of Ascomycota fungi and cyanobacteria (Sharma 2013;
The species of Parmelia genus have long been used in Indian folk medicine for the treatment of bronchitis, ulcers, furunculosis, cardiovascular diseases, urolithiasis, amenorrhea, and also as an antibacterial, anti-inflammatory and fortifying agent (Cocchietto 2002;
According to the literature data, these lichens contain polysaccharides, anthraquinones, carotenoids, diterpenoids and steroids, aliphatic acids, amino acids, however, compounds of much interest are the secondary metabolites of phenolic nature – lichen acids, which possess a wide range of biological activity (Sharma 2013;
Lichen acids which are represented by salazinic and protocetraric, fumaroprotocetraric usnic acids, atranorin and chloratranorin are oxygen containing heterocyclic compounds by their structure. They synthetized predominantly in mycobiont mycelium from the primary compounds formed in photobiont cell by photosynthesis. According to the literature data biosynthesis of the compounds mentioned above undergoes acetylpolymalonyl pathway and is initiated by PKS gene. This gene is responsible for the lichen acids precursor (b-orsellinic acid) production. Further, from two to three molecules of this acid through the connection through the ether and carbon-carbon bonds form more complex molecules of lichen acids. Boustie J. and Grube 2005;
They show antimicrobial, antifungal, antiviral, antiprotozoal, antioxidant, antitumor, photoprotective, immune modulating, hypolipidemic, hepatoprotective, anti-inflammatory, analgesic properties (Cocchietto 2002;
The results of the research carried out by Indian scientists have shown lichen acids to possess cardiac protective and hypotensive activity by inhibiting hydroxyl-methyl-glutaryl-coenzyme А (HMG-CоА) reductase and angiotensin-converting enzyme (ACE) (
The literature data indicate the Parmelia genus representatives to be prospective in terms of development of new medicinal products with cardiac protective activity. At the same time, Parmelia sulcata and Parmelia vagans thalli are non-officinal in Ukraine, and their chemical composition has not yet been sufficiently studied.
The aim of the current research was the determination of qualitative composition and quantitative content of phenolic compounds by HPLC in Parmelia sulcata and Parmelia vagans thalli.
The Parmelia sulcata and Parmelia vagans thalli, which were used for the analysis, were collected at the territory of the Vyshnivchyk forest district of the State Enterprise “Yarmolyntsi forestry” (Khmelnytskyi region, Ukraine) in 2016–2018.
The study of phenolic compounds in the thalli of the analyzed species was carried out using the HPLC method.
The procedure worked out by A. Gudzenko in 2013 was taken as the basis for qualitative composition and quantitative content determination of phenolic compounds in plant raw material.
Sample preparation. 125.0 ml of 70% ethanol were added to 5.0 g of the dried crushed raw material and then heated on a water bath with a reflux condenser for 1 hour. The obtained extract was cooled, filtered through a paper filter with the pore size of 3-5 µm. 5.0 ml of the filtrate were then made up with 70% ethanol to the volume of 10.0 ml (
The chromatographic analysis of the received samples was carried out using the Agilent Technologies 1200 LC/MSD chromatograph with a diode-matrix mass-elective detector. The stainless steel column С18 SunFire was used in the experiment. Its size was 150 mm × 4,6 mm, and the grain size equaled 3.5 µm. The column temperature was 38°С, the detecting wavelengths of the diode-matrix detector were 254 nm, 330 nm, 350 nm, 360 nm, 370 nm (
The samples were ionized by electrospray, scanned in the range of 150-800 mass/charge (positive and negative ionization), the injection speed – 1.0 ml/min, the injection volume – 5 µl, eluation – gradient, mobile phase А – 0.1% formic acid solution in water, mobile phase В – 0.1% formic acid solution in acetonitrile (
Time, min | Mobile phase А, %, (V/V) | Mobile phase В, %, (V/V) |
0→1 | 92 | 8 |
1→15 | 92→70 | 8→30 |
15→25 | 70→0 | 30→100 |
25→33 | 0 | 100 |
33→33,5 | 0→92 | 100→8 |
As a result of the experiment, salazinic, fumaroprotocetraric and usnic acids, atranorin and chloratranorin were identified in Parmelia sulcata and Parmelia vagans thalli. In addition, protocetraric acid was identified in Parmelia sulcata thalli. The HPLC chromatograms of Parmelia sulcata thalli are given in Figure
The results of the HPLC determination of the quantitative content of phenolic compounds in Parmelia sulcata and Parmelia vagans thalli are given in Table
The content of phenolic compounds in Parmelia sulcata and Parmelia vagans thalli.
Component | Parmelia sulcata | Parmelia vagans | ||
---|---|---|---|---|
Retention time, min | Quantitative content, g/mol | Retention time, min | Quantitative content, g/mol | |
Salazinic acid | 27.26 | 388.28±7.77 | 27.26 | 349.78±7.01 |
Protocetraric acid | 28.87 | 374.30±7.11 | – | – |
Fumaroprotocetraric acid | 29.91 | 474.00±9.00 | 29.11 | 456.21±8.67 |
Usnic acid | 30.19 | 344.32±7.23 | 29.99 | 375.31±7.53 |
Chloratranorin | 31.37 | 408.79±8.99 | 31.39 | 270.26±5.68 |
Atranorin | 31.91 | 374.34±7.49 | 32.25 | 393.34±8.65 |
Total content | 2019.71±40.39 | 1754.18±34.77 |
Fumaroprotocetraric acid dominated by the quantitative content in Parmelia sulcata (474.00±09.00 g/mol) and Parmelia vagans (456.21±8.67 g/mol) thalli. In addition, chloratranorin accumulated in significant content in Parmelia sulcata thalli, which comprised 408.79±8.99 g/mol. The content of this compound in Parmelia vagans thalli was 1.5 times lower than in Parmelia sulcata thalli and equaled 270.26±5.68 g/mol. It should be pointed out that the content of chloratranorin in Parmelia sulcata thalli was 1.7 times lower than the content of fumaroprotocetraric acid. The content of usnic acid and atranorin was somewhat higher in this type of the raw material – 375.31±7.53 g/mol and 393.34±8.65 g/mol respectively.
The content of usnic acid (344.32±7.23 g/mol) in Parmelia sulcata thalli was almost at the same level as the one in Parmelia vagans thalli (375.31±7.53 g/mol), but at the same time it was 1.4 times lower compared to the content of fumaroprotocetraric acid, which accumulated in the analyzed raw material in the maximum quantity.
The content of salazinic and protocetraric acids, as well as atranorin in Parmelia sulcata thalli was almost equal and comprised 388.28±7.77 g/mol, 374.30±7.11 g/mol and 374.34±7.49 g/mol respectively. The content of salazinic acid in Parmelia vagans thalli was 349.78 ±7.01 g/mol, which was 1.3 times lower compared to the content of fumaroprotocetraric acid (456.21±8.67 g/mol) in this type of the raw material.
The maximum content of chloratranorin, salazinic and fumaroprotocetraric acids was observed in Parmelia sulcata thalli, while the content of usnic acid and artanorin – in Parmelia vagans thalli.
Salazinic, fumaroprotocetraric, usnic acids, chloratranorin and atranorin were identified in both types of the raw materials studied. In addition, protocetraric acid was identified in Parmelia sulcata thalli. In the thalli of both studied species of Parmelia genus, fumaroprotocetraric acid dominanted by quantity. The content of this substance in Parmelia sulcata thalli was 474.00±9.00 g/mol, and in Parmelia vagans thalli – 456.21±8.67 g/mol.
As the result of the research it was determined that Parmelia sulcata and Parmelia vagans thalli had identical qualitative composition of phenolic compounds. The studied objects insignificantly differ by the quantitative content of these compounds.
The obtained results will be used in the development of quality control methods for Parmelia sulcata and Parmelia vagans thalli, as well as medicines based on these types of the raw materials.