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19PHARMACEUTICS
PHYTOCHEMICAL RESEARCH OF VAGRANT PARMELIA THALLI AS A PROSPECTIVE
SOURCE OF CERTAIN NUTRIENTS
Kyslychenko O.
PhD, associate professor, doctoral student of the Department of Chemistry of Natural Compounds of the National University of Pharmacy, Ukraine
Protska V.
PhD, assistant of the Department of Chemistry of Natural Compounds of
National University of Pharmacy, Ukraine
Zhuravel I.
Doctor of Pharmaceutical Science, professor, professor of Department of Chemistry of Natural Compounds of National University of Pharmacy, Ukraine
Abstract.
12 fatty acids were found by gas chromatography in vagrant Parmelia thalli, including 7 unsaturated fatty acids. The quantitative content of unsaturated fatty acid was proved to be 6.3 times higher than that of saturated ones. The quantitative content of 19 mineral elements was determined by atomic absorption spectrophotometry as being in total 1066.77±26.67 ^g/100 g. The most numerous in the herb were potassium, calcium and silicon. Heavy metals were present within normal limits.
Ion exchange liquid column chromatography identified in vagrant Parmelia thalli 6 nonessential, 4 conditionally essential and 8 essential amino acids. The total content of these compounds in the herb was 49.43±1.23 mg/g, of them about one third belonged to essential amino acids.
The obtained data will be used in the development of novel drugs based on vagrant Parmelia thalli.
Keywords: Parmelia vagans, Parmeliaceae, fatty acids, macro- and microelements, amino acids.
Introduction
Nutrients are essential bioactive compounds. They include sugars, amino acids, fats (among them fatty acids), vitamins, mineral elements and water. They ensure human body normal functioning. In particular, they are structural elements of cell membranes, substrates for biochemical synthesis of enzymes, hormones and neu-rotransmitters within organism, may accelerate or slower metabolism [1, 2]. Mineral elements, unsatu-rated linoleic and linolenoic acids, certain amino acids and vitamins are incapable of synthesizing within human body and thus belong to essential nutrients [1-7].
At the same time such nutrients as fatty acids, mineral elements and amino acids, possess certain therapeutic effect. Polyunsaturated fatty acids show anti-inflammatory, antibacterial and anti-tumor effect [3, 4]. Certain mineral elements as potassium and sodium participate in neurotransmission, ferric ions in cell respiration processes, whereas zinc ions favor healing of wounds, possess anti-oxidant and gastroprotective properties [5]. Certain amino acids show hepatoprotec-tive, immunomodulatory, anesthetic activity, favor insulin synthesis [6, 7].
Besides, the above essential nutrients substantially affect cardiovascular system. Polyunsaturated fatty acids are known to possess cardioprotective and anti-arrhythmic activity, increase high-density lipoprotein concentration in blood, thus, diminishing the risk of atherosclerosis [3, 4]. Potassium, sodium and magnesium ions control arterial pressure and affect heart rate [5]. Amino acids asparagine, tyrosine and tryptophane are precursors of catechol amines, which in turn diminish cardiac rhythm variability and normalize arterial pressure [6, 7].
The lichens belong to symbiotic organisms which consist of a mycobiont connected to one or several pho-tobionts (algae and cyan bacteria) [8, 9]. They usually
grow on rock formations or as tree epiphytes [8]. Parmelia vagans Nyl. is one of 70 representatives of Parmelia Ach. species, Parmeliaceae Zenker family [9]. According to literature data, parmelias show a wide range of bioactivity, in particular, anti-oxidant, anti-tumor, anti-diabetic, hypocholesterinemic, hypoli-pidemic, cardioprotective, diuretic, anti-viral, anti-fertile, spasmolytic, anti-inflammatory and antipyretic activity [9-14]. Folk medicine uses them to treat coughing, heart diseases, tooth pain and for wound healing [9, 10, 14].
The recent study of American authors showed that ethyl acetate extracts from vagrant parmelia thalli suppressed anti-bacterial activity in relation to microorganisms Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae, but did not hamper growth of Escherichia coli and Klebsiella pneumonia [15]. In the course of rat gastric ulcer ethanol model experiments Russian pharmacologists found that area of gastric mucosa ulcer diminished by 77% under the effect of vagrant parmelia thalli aqueous extract [17].
The literature data on parmelia chemical composition are fragmentary and were never systematized. The sources state that these lichens accumulate specific pol-ysaccharides (lichenin) and phenol compounds, in particular, lichen acids, depsides, emodin derivatives. Besides, parmelia thalli contain terpenic, including steroid, compounds [9-14].
The aim of this work was a study of qualitative composition and determination of quantitative content of certain nutrients, in particular, fatty acids, mineral elements and amino acids, in vagrant parmelia thalli.
Materials and Methods
The research was conducted on air dried, ground vagrant parmelia thalli collected at Vyshnivtsy Forestry Section, Yarmolyntsi Forest Enterprise, Khmelnitsky Region, Ukraine.
The quantitative composition study and quantitative fatty acids content determination in vagrant par-melia thalli was conducted on Selmichrom-1 gas chromatography unit with a flame ionization detector after preliminary methylation. Unit chromatographic column is made of stainless steel, 2.5 m long, internal diameter 4 mm. Stationary phase was Inerton after preliminary treatment with 10% diethyleneglycol succinate (DEGS), carrier gas was nitrogen [17].
Chromatography unit operation parameters: temperature of column thermostat 180°C, of evaporator -230°C, of detector - 220°C. Nitrogen flow rate was 30 cm3/min. Sample volume of herb under study fatty acids methyl esters solution in hexane was 2 mm3 [17].
Fatty acids methyl esters were obtained by modified Peisker method. Methylation was conducted with a mixture of chloroform, methanol and sulfuric acid in relation 100:100:1. To 30-50 ^l of lipophilic fraction in glass ampoules 2.5 ml methylating mixture were added, ampoules were sealed and placed for 3 hours to thermostat at stable temperature of 105°C. Then the contents of ampoules was transferred to a test tube, a small amount of zinc sulfate powder and 2 ml each of purified water and hexane was added to extract methyl esters. The extract was stirred, settled and hexane phase was filtered off for chromatographic analysis [17].
In chromatograms fatty acids were identified by retention time as compared with standard samples of fatty acids. Reference samples were Sigma Ltd standards of saturated and unsaturated fatty acid methyl esters. Fatty acids content in vagrant parmelia thalli was calculated by normalization [17].
The element composition of the herb under study was determined by atomic absorption spectrophotometry.
The herb was pretreated with sulfuric acid and carbonized in the muffle furnace at a temperature below 500°C. The herb samples were evaporated from graphite electrode craters in AC arc discharge. Spectra excitation source was IBC-28- type unit which generated 16 A current at 60 sec exposure. The obtained spectra were registered on photographic plates of DFS-8 spectrograph with diffraction grating 600 grooves/mm and three-lens slit highlighting system. The spectral line intensity of studied samples and of graduated samples (GS) was measured by MF-1 microphotometer [18].
In photographing spectra AC arc current was 16 A, arcing phase took place at 60°C, arcing pulse frequency was about 100 discharges per second, analytical interval was 2 mm, spectrograph slit width was 0.015 mm, exposure was 60 sec. The spectra were photographed in area 230-330 nm. After that photographic plates were dried and subjected to photometry lines (nm) and adjacent background in experimental sample spectra and graduated standard sample spectra [18].
Upon the results of photometry line and background blackening differences were calculated (
S = — S^) for experimental samples spectra (
SiH) and graduated standard spectra (Sr3) for each element [18].
Then a graduated plot was constructed in the coordinates: line and background blackening difference
mean value (Sr3) - element content in graduated
standard logarithm (lg C), where C is expressed in percent to base [18].
From this plot we found the element content in ash (a, %). The element content in live herb (x, %) was found by formula:
a ■ m
x =-,
M
where m - ash mass (g); M - live herb mass (g); a - element content in ash (%) [18].
In calculation the lower limits of impurities content were considered, as follows: for copper1-10—'4; for cobalt, chromium, molybdenum and manganese 2 ■ 10—4; for nickel and lead 5 ■ 10—4 ; for strontium
and zinc 1 ■ 10 2 % [18].
The qualitative composition and quantitative content of free amino acids were studied by ion exchange liquid column chromatography based on amphoteric properties of amino acids.
The study of amino acids was performed after pre hydrolysis with hydrochloric acid. To this end, 6 N hydrochloric acid solution was added to the test tube with exactly measured portion of herb and after that the tube was cooled in a liquid nitrogen flow. When the test tube contents were frozen, the air was removed from it under vacuum to avoid amino acid oxidation. Then the test tube was sealed and preserved in thermostat at a constant temperature of 106°C within 24 hours. Then the test tube contents were cooled, totally transferred to a glass weighing box, and hydrochloric acid was evaporated on a water bath. 3-4 ml of deionized water was added to a dried sample, and the evaporation went on. The obtained sample was dissolved in 0.3 N lithium citrate buffer at pH 2.2 and applied on amino acid analyzer ion exchange column, whose cation exchanger was previously equalized with sodium citrate or lithium citrate buffer solution. Amino acids were separated by small-grain spherical shape cation exchangers (resins) of sty-rene-divinyl benzene copolymer with a functional sulfite group [19].
T 339 automatic amino acid analyzer performs all analysis operations in a continuous eluent flow. At the column exit a micropump constantly mixed eluate with ninhydrine reagent (mixture of ninhydrine, buffer solution and stannic chloride in argon atmosphere). The obtained mixture was fed along a capillary pipe to reactor heated to 95°C - 98°C. Then it was fed to flow cell for photocolorimetric determination of obtained color intensity at 440 nm or 560 nm. The photocell signals were registered by potentiometric recorder in the form of chromatograms. The peak area in chromatograms was calculated and compared to amino acid peak areas from known chromatograms, and on this basis the absolute values of content of each amino acid in analyzed sample were calculated [19].
The content of each amino acid in sample in ^M (X1) in analyzed sample was calculated by formula: X1 = S1 / So,
where S1 - amino acid peak area in analyzed sample, So - peak area of this amino acid in solution of standard amino acid wherein the content of each amino acid corresponds to 1 ^M. To express the content in mg the obtained amino acid amount in ^M was multiplied
by corresponding molecular mass [19]. Experimentally we identified 12 fatty acids in va-
grant parmelia thalli, 7 of them belonging to unsatu-Results and Discussion of the results rated fatty acids. Gas chromatogram of vagrant par-
melia thalli fatty acids is shown in Fig.1.
0 200 400 600 800 1000 1200 140Û 1600 1 800 2000 220t 240C 250C 2900 300C
Fig. 1 Gas chromatogram of vagrant parmelia thalli fatty acids
The content of unsaturated fatty acids in vagrant this herb made 13.61±0.34% of the sum of all fatty ac-parmelia thalli was 6.3 times higher than that of satu- ids, whereas contents of unsaturated fatty acids was rated fatty acids. The content of saturated fatty acids in 85.99±2.15%. The quantitative content of saturated and
unsaturated fatty acids is shown in Table 1.
Table 1
Fatty acids content in vagrant parmelia thalli
# Fatty acid description Quantitative content of fatty acids, %
Saturated fatty acids
1 Myristic (tetradecanoic) 0.35±0.01
2 Palmitic (hexadecanoic) 8.08±0.20
3 Stearic (octadecanoic) 3.15±0.08
4 Behenic (docosanoic) 0.80±0.02
5 Lignoceric (tetracosanoic) 1.23±0.03
Unsaturated fatty acids
6 Myristoleic 0.50±0.01
7 Palmitoleic (hexadecenoic) 2.78±0.07
8 Oleic (octadecenoic) 28.50±0.71
9 Linoleic (octadecadienoic) 44.67±1.12
10 Linolenoic (octadecatrienoic) 3.15±0.08
11 Gondoinic (eicosenoic) 0.15±0.01
12 Erucic (docosenoic) 6.24±0.16
Total content of saturated fatty acids, % 13.61±0.34
Total content of unsaturated fatty acids, % 85.99±2.15
Total content of unidentified fatty acids, % 0.40±0.01
Among unsaturated fatty acids linoleic acid prevailed with the content of 44.67±1.12%. Oleic acid content in thalli was 1.5 times less - 28.50±0.71%. Erucic acid content (6.24±0.16%) made 1/14 of the total amount of unsaturated fatty acids in the herb under study. The amount of linolenoic and palmitoleic acids were accumulated in vagrant parmelia thalli almost identically - 3.15±0.08% and 2.78±0.07% respectively.
Palmitic acid prevailed among saturated fatty acids with the content of 8.08±0.20%. Stearic acid in the herb under study was accumulated 2.5 times less -3.15±0.08%. The content of lignoceric acid was
1.23±0.03%. As regards myristic (0.35±0.01%), myristoleic (0.50±0.01%), gondoinic (0.15±0.01%) and behenic (0.80±0.02%) acids, their content in vagrant parmelia thalli never exceeded 1% of the total sum of fatty acids in this herb.
The results of element composition proved that 19 macro- and microelements were found in vagrant par-melia thalli, including heavy metals lead, cadmium, cobalt, arsenic and mercury. The total content of mineral elements in this herb was 1066.77±26.67 ^g/100 g. The results of the experiment are shown in Table 2.
Table 2
Content of macro- and microelements in vagrant parmelia thalli
# Element Content of mineral elements, ^g/100 g.
1 Iron 7.00±0.18
2 Silicon 175.00±4.38
3 Phosphorus 45.00±1.13
4 Aluminum 17.00±0.43
5 Manganese 14.00±0.30
6 Magnesium 70.00±1.75
7 Nickel 0.12±0.01
8 Molybdenum 0.04±0.01
9 Calcium 245.00±6.13
10 Copper 0.31±0.01
11 Zinc 14.00±0.35
12 Sodium 28.00±0.70
13 Potassium 450.00±11.25
14 Strontium 1.30±0.03
Total content of mineral elements 1066.77±26.67
Content of heavy metals
15 Lead <0.03
16 Cobalt <0.03
17 Cadmium <0.01
18 Arsenic <0.01
19 Mercury <0.01
The obtained results show that potassium, calcium and silicon mostly accumulated in vagrant parmelia thalli. Potassium content in the herb was 450.00±11.25 ^g/100 g which made 42.18% of the total mineral content in this herb. Calcium content was 1.8 times less and that of silicon 2.5 times less as compared to potassium - 245.00±6.13 ^g/100 g and 175.00±4.38 ^g/100 g respectively. Magnesium and phosphorus content was substantially less - 6.56% and 4.22% respectively of the sum of identified elements, numerically 70.00±1.75 ^g/100 g and 45.00±1.13 ^g/100 g respectively. Sodium content (28.00±0.70 ^g/100 g) in the herb was 1.6 times less than that of phosphorus. The smallest accumulation in vagrant parmelia thalli was observed for iron 7.00±0.18 ^g/100 g.
As for the microelements the largest content in vagrant parmelia thalli referred to aluminum (17.00±0.43 ^g/100 g), which made 1.59% from the total content of
all mineral elements. Manganese and zinc were present almost identically - 14.00±0.30 ^g/100 g and 14.00±0.35 ^g/100 g respectively. Strontium accumulation in vagrant parmelia thalli was 1.30±0.03 ^g/100 g ^g/100 g, and that of copper 0,31±0.01 ^g/100 g almost 4 times less than that of strontium. Only traces of nickel and molybdenum were found in the herb -0.12±0.01 ^g/100 g and 0.04±0.01 ^g/100 g respectively.
The content of heavy metals in the herb under study was within the statutory limits in force in Ukraine [20, 21].
In the course of amino acid analysis in vagrant parmelia herb 18 amino acids were identified, 8 of them belonging to essential amino acids. Vagrant parmelia amino acids chromatogram was obtained by ion exchange liquid column chromatography and is shown in Fig.2.
Fig.2 Vagrant parmelia amino acids chromatogram obtained by ion exchange liquid column chromatography
The total content of all amino acids in the herb was 49.43±1.23 mg/g. The part of essential amino acids was 32.33%, or in numerical expression 15.98±0.40 mg/g. The qualitative composition and quantitative content of amino acids in vagrant parmelia thalli are specified in Table 3.
Table 3
Amino acid composition of vagrant parmelia thalli
# Description of amino acid Amino acid content, mg/g
Nonessential amino acids
1 Y-aminobutyric acid 0.10±0.01
2 Aspartic acid 6.24±0.16
3 Serine 3.60±0.09
4 Glutamic acid 9.67±0.24
5 Proline 2.05±0.05
6 Glycine 3.93±0.09
Conditionally essential amino acids
7 Arginine 1.90±0.05
8 Tyrosine 1.47±0.04
9 Cysteine 0.57±0.01
10 Alanine 3.92±0.10
Essential amino acids
11 Valine 1.01±0.03
12 Histidine 0.83±0.02
13 Threonine 2.82±0.07
14 Methionine 2.34±0.06
15 Phenylalanine 1.87±0.05
16 Isoleucine 0.93±0.02
17 Leucine 2.86±0.07
18 Lysine 3.32±0.08
Total content of amino acids 49.43±1.23
Total content of essential amino acids 15.98±0.40
In vagrant parmelia thalli the highest accumulation was observed for glutamic acid whose content made 19.56% of total content of identified amino acids. The part of aspartic acid was 1.5 times less - 6.24±0.16 mg/g. The difference in content of serine and glycine was tiny - 3.60±0.09 mg/g and 3.93±0.09 mg/g respectively. Proline with 2.05±0.05 mg/g accounted for 4% of the total content of amino acids in the herb under study.
Among 4 conditionally essential amino acids in vagrant parmelia thalli alanine prevailed with 3.92±0.10 mg/g which took 8.0% of total amino acids content. Arginine accumulation in the herb was twice less and tyrosine 2.5 times less - 1.90±0.05 mg/g and 1.47±0.04 mg/g respectively.
Lysine was dominating among unsaturated fatty acids as for the quantitative presence of 3.32±0.08 mg/g in vagrant parmelia thalli. Less accumulation of leucine and threonine was observed - 2.86±0.07 mg/g and 2.82±0.07 mg/g respectively. Vagrant parmelia thalli contained 2.34±0.06 mg/g methionine and 1.87±0.05 mg/g phenylalanine which corresponded to 14% and 12% of the sum of essential amino acids in the herb.
The least present in vagrant parmelia thalli among nonessential amino acids were threonine (0.83±0.02 mg/g) and isoleucine (0.93±0.02 mg/g), as well as tyrosine (1.47±0.04 mg/g) among conditionally essential amino acids. Nonessential y-aminobutyric acid with minimum presence of 0.10±0.01 mg/g accounted for only 1% of amino acids content in the herb.
Conclusion
The qualitative composition and quantitative content of fatty acids, mineral elements and amino acids in vagrant parmelia thalli was studied.
The analysis of herb under study identified 12
fatty acids, 7 of them are unsaturated fatty acids. The quantitative content of unsaturated fatty acids (85.99±2.15%) was 6.3 times higher than that of saturated fatty acids. Among unsaturated fatty acids linoleic and oleic acids prevailed with content of 44.67±1.12% and 28.50±0.71% respectively.
The total content of 19 identified mineral elements was 1066.77±26.67 ^g/100 g. Mostly potassium, calcium and silicon were present. Dominating microelements were aluminum (17.00±0.43 ^g/100 g), zinc (14.00±0.30 ^g/100 g) and manganese (14.00±0.30 ^g/100 g). The content of heavy metals in the herb under study was within the statutory limits in force in Ukraine.
18 amino acids with the total content of 49.43±1.23 mg/g were identified in the herb under study. About one third of this content belonged to essential amino acids. Among essential amino acids in quantitative ratio dominating were lysine (3.32±0.08 mg/g), leucine (2.86±0.07 mg/g) and threonine (2.82±0.07 mg/g).
The obtained results give grounds for statement that vagrant parmelia thalli contain substantial quantity of nutrients, in particular, unsaturated fatty acids, amino acids and mineral elements. The results of the experiment will be subsequently used in the development of novel drugs on the basis of vagrant parmelia herb.
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BLACK CURRUNT FRUITS, CRANBERRY FRUITS AND ONION PEELS ANTHOCYANIDINS
STUDYING USING HPLC
Kuznietsova V.
Candidate of Pharmaceutical Sciences, Associate Professor Dean of Pharmaceutical Department National University of Pharmacy Kharkiv, Ukraine Kyslychenko V. Doctor of Pharmacy, Professor Head of the Department of Chemistry of Natural Compounds
National University of Pharmacy Kharkiv, Ukraine
