CC BY
26
UDC 582.284:543.061 https://doi.org/10.15407/biotech11.03.069
Ganoderma SPECIES EXTRACTS: ANTIOXIDANT ACTIVITY AND CHROMATOGRAPHY
V. Raks1, 2 1Taras Shevchenko National University of Kyiv, Ukraine
M. Ozturk2 2Mugla Sitki Kocman University, Turkey
O. Vasylchenko3 3National Aviation University, Kyiv, Ukraine
M. Raks4 4National University of Food Technologies, Kyiv, Ukraine
E-mail: raksvictoria@knu.ua
Received 19.02.2018
Research aimed to isolate biologically active compounds from mushrooms fruiting bodies of Ganoderma lucidum, Ganoderma adspersum and Ganoderma applanatum and to estimate their antioxidant activities. Various techniques were used to isolate biologically active compounds. Antioxidant properties were estimated with spectrophotometricaly measuring free radical scavenging activity. High performance liquid chromatography was applied to analyze the isolated extracts. Half maximal inhibitory concentration (IC50) was 8.25±0.88 pg/ml and 1.70±1.13 pg/ml for G. applanatum and G. adspersum, respectively. However, petroleum ether and chloroform extracts of G. lucidum demonstrated highler antioxidant activity with an IC50 about 33.66 ± 3.69 pg/ml. Chromatograms of components of acetone and methanol extracts of G. lucidum were recorded. The main outcome of such chromatograms is the possibility to detect the presence of active components in various mushroom species without the usage of expensive standards.
Key words: Ganoderma species mushrooms, antioxidant activity, high performance liquid chro-matography.
Various mushrooms species are the focus of researchers' interest. Hitherto, lectins, polysaccharides, polysaccharide-peptides, polysaccharide-protein complexes, lanostane-type triterpenoids, phenolics and flavonoids were isolated from some mushroom species [1]. Furthermore, various biological activities such as antioxidant, antibacterial, antifungal [2, 3], antitumor [4], anti-inflammatory [5], cytotoxic [6] and anti-cholinesterase [7] activities of the isolated compounds and/or complexes were investigated. In recent years, more variety of mushrooms were isolated and identified, and the number of mushrooms being cultivated for food or medicinal purposes were increasing rapidly.
Chemicals isolated from mushrooms have significant biological activity that may cause noticeable curative effects on human health and therefore could be used in medicine [8]. Hence, the comparison of mushroom components is highly desirable for the creation of drugs. Chromatography techniques are widely applied to separate
biologically active components in extracts of mushrooms at first [1]. Then, chromatograms of such extracts could be compared to each other and peaks with identical retention times could be identified. Such approach would help to identify the extracts that possess biological activity and ease of their future purification on the way of drug formulation.
The aim of present research is developing of effective methods for preparation of samples containing biologically active compounds. Preliminary sample preparation was performed using solid-liquid, ultrasonic and Soxhlet extractions [9, 10]. Then, antioxidant activity and chromatograms were estimated for all extracts.
Materials and Methods
G. lucidum, G. adspersum, and G. appla-natum fruiting bodies were obtained from Mula, Turkey (Table 1). Petroleum ether, methanol, chloroform and acetone of analytical and gradient grade were supplied by Merck.
G. lucidum (120 g), G. adspersum (385 g), and G. applanatum (1200 g) material were collected from Koycegiz, Mula, dried in the air and crushed into small particle (2-6 mm).
Solid-liquid extraction. Bioactive compounds of G. lucidum (50 g), G. adspersum (175 g) and G. applanatum (400 g) were extracted with a mixture of petroleum ether and chloroform (4:1, v/v, 400 ml, 1 l, 2 l respectively). For the extraction, all biological materials should be covered with these solvents. Then bioactive compounds were sequentially extracted with acetone (1 l), methanol (1 l), and water (1 l) at 25 °C. Each extraction experiment was performed until the solvent became colorless.
Ultrasonic extraction. Bioactive compounds of G. lucidum (10 g), G. adspersum (10 g) and G. applanatum (10 g) were extracted with a mixture of petroleum ether and chloroform (4:1, v/v, 100 ml) at 25 °C for 20 min in triplicates. Then, they were sequentially extracted with acetone (100 ml), methanol (100 ml) and water (100 ml).
Soxhlet extraction. Bioactive compounds of G. lucidum (50 g), G. adspersum (50 g), and G. applanatum (50 g) were extracted in a Soxhlet apparatus with a mixture of petroleum ether and chloroform (4:1, v/v, 1 l) for 4 h. Then, mushroom materials were sequentially extracted with acetone (1 l), methanol (1 l), and water (1 l).
Each extraction experiment was performed until the solvent became colorless. Sediments were filtered by means of filter paper. Filtrates were concentrated under vacuum (V = 0.5 ml)
using a rotary evaporator and dried in the air. The extracts collected under different techniques started above were subjected to in vitro tests to confirm their antioxidant activities.
1. Extraction of polysaccharides. Solidliquid extraction. After methanol extraction, mushroom materials were collected and extracted with distilled hot water. The polysaccharide extracts were obtained by hot water extraction and precipitation with ethanol.
Mushrooms material was extracted with 500-1000 ml of distilled hot water at +80 °C (until samples became cold). The crude hot water extracts were filtered and finally concentrated under vacuum (V = 50-100 ml) using a rotary evaporator. Then 200-400 ml of ethanol was added to concentrated hot water extracts. Polysaccharides were precipitated overnight at +4 °C. The precipitated polysaccharides were collected after centrifugation (N ve NF800) at 3100 x g for 2 min, and extraction yield was calculated.
Ultrasonic extraction. After methanol extraction, 10 g of material were extracted tree times with 100 ml of distilled water at 80 °C (until samples became cold) for 20 min with ultrasonication. Hot water extracts were filtered and combined (V = 300 ml). Finally, concentrated under vacuum (V = 30 ml) using a rotary evaporator. 120 ml of ethanol was added to concentrated hot water extracts and polysaccharides were precipitated overnight at +4 °C. The precipitated polysaccharides were collected after centrifugation (N ve NF800) at
Table 1. Characteristics of mushroom species collecting through 2014
Number Mushroom species Tree type Region of collection Time of collection
1 G. lucidum Sweetgum Mula, Fethiye September
2 G. adspersum Sweetgum Mula, Fethiye September
3 G. applanatum Mulberry Mula, Koycheiz September
4 G. lucidum Sweetgum Mula, Koycheiz September
5 G. adspersum Walnut Izmir, Balchova October
6 G. adspersum Peach Mula, Ula October
7 G. adspersum Plum Mula, Fethiye September
8 G. lucidum Sweetgum Mula, Marmaris November
9 G. adspersum Sweetgum Mula, Marmaris November
10 G. lucidum Sweetgum Mula, Ula November
11 G. adspersum Mulberry Mula, Koycheiz November
12 G. lucidum Mulberry Mula, Koycheiz November
13 G. adspersum Mulberry Karabalar, Mula November
3100 x g for 2 min, and extraction yield was calculated.
Soxhlet extraction. After methanol extraction, 50 g of material were extracted in a Soxhlet apparatus with 1 l water for 4 h. Finally, concentrated under vacuum to V = 100 ml using a rotary evaporator. 400 ml of ethanol was added to concentrated hot water extracts. Polysaccharides were precipitated overnight at +4 °C. The precipitated polysaccharides were collected after centrifugation (Nuve NF800) at 3100 x g for 2 min, and extraction yield was calculated.
Determination of antioxidant activity. fi-Carotene-linoleic acid assay
The procedure was done according to Ferreira et al. (2006) [7]. A stock solution of fi-carotene and linoleic acid was prepared by dissolving 0.5 mg of fi-carotene in 1 ml of chloroform and adding 25 pl of linoleic acid with 200 mg of Tween-40. The chloroform was evaporated at 40 °C under vacuum using a rotary evaporator. Aerated water (100 ml) was added to the residue.
4 ml of this mixture were transferred into different test tubes containing different concentrations of the sample in ethanol. The zero time-absorbance was measured at 470 nm. The samples were incubated for 2 h at 50 °C together with a blank solution, and four others containing the antioxidants Butylated hydroxyanisole (BHA), a-tocopherol. The absorbance was measured at 470 nm. The bleaching rate (R) of fi-carotene was calculated according to the following equation:
R = (ln a/b)/t,
where ln — natural log, a — absorbance at time zero and b — absorbance at time t (2 h). Antioxidant activity (AA) was calculated in terms of percent inhibition relative to the control, using following equation:
AA = [(Rcontrol — Rsample)/Rcontrol] x 10°.
DPPH free radical scavenging activity
The free radical scavenging activity of extracts was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay described by Blois [11] with slight modifications. In its radical form, DPPH absorbs at 517 nm, but on reduction by an antioxidant or a radical species its absorption decreases. 0.1 mmol-L1 Solution of DPPH in methanol was prepared and 4 ml of this solution was added to 1 ml of sample solution in methanol at different concentrations. Thirty minutes in the dark later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture
indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical of an antioxidant was calculated using the following equation:
DPPH scavenging effect (%) =
= [(Acontrol _ Asample)/Acontroli X 100,
where Acontrol is the absorbance of the DPPH solution and Asample is the absorbance of the sample.
ABTS cation radical decolorization assay
The spectrophotometric analysis of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging activity was determined according to the method of Re et al. [12] with slight modifications. The ABTS was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Oxidation of ABTS commenced immediately, but the absorbance was not maximal and stable until more than 6 h had elapsed. The radical cation was stable in this form for more than 2 days in storage in the dark at room temperature. Before usage, the ABTS solution was diluted to get an absorbance of 0.708 ± 0.025 at 734 nm with ethanol. Then, 160 pl of ABTS solution was added to 40 pl of sample solution in ethanol at different concentrations. After 10 min the absorbance was measured at 734 nm by using a 96-well microplate reader. The percentage inhibitions were calculated for each concentration relative to a blank absorbance (ethanol). The scavenging capability of ABTS was calculated using the following equation:
ABTS scavenging effect =
= [(Acontrol _ Asample)/Acontrol] X 100,
where Acontrol is the initial concentration of the ABTS and Asample is the absorbance of the remaining concentration of ABTS in the presence of sample. The extract concentration providing 50% radical scavenging activity (EC50) was calculated from the graph of ABTS scavenging effect percentage against extract concentration. BHT, a-tocopherol were used as antioxidant standards for comparison of the activity.
Cupric reducing antioxidant capacity (CUPRAC). The cupric reducing antioxidant capacity of the extracts was determined according to the CUPRAC method [13] with slight modifications. To each well, in a 96 well plate, 50 pl of 10 mM Cu (1 l), 50 pl of 7.5 mmol neocuproine, and 60 pl of NH4Ac buffer (1 M, pH 7.0) solutions were added. 40 pl of extract at different concentrations was added to the
initial mixture so as to make the final volume 200 pl. After 1 h, the absorbance at 450 nm was recorded against a reagent blank by using a 96-well microplate reader. Results were given as absorbance and compared with BHA, a-tocopherol used as antioxidant standards.
HPLC analysis. A high performance liquid chromatographic system with multiwavelength spectrophotometer was used for measuring. Analytical RP-column Separon shim-pack VP-ODS (5 pm, 4.6 mm x 150 mm) was used for chromatographic separations. Chromatographic conditions used for methanol extracts were as follows. The mobile phase was a mixture of acetonitrile (A) and 0.1% CH3COOH in water (B). 0 min — 2.0% A; 10 min — 2.0% A, 20 min — 5.0% A, 30 min — 20% A, 60 min — 100% A, 62 min — 100% A, 65 min — 2% A, 68 min — 2.0% A. The mobile phase was degassed in a sonicator, and pumped in gradient mode at a flow rate of 1.5 ml/min at 35 °C. The UV detection was accomplished at 245 nm. Samples of 20 pl were injected into column. The qualitative identification of the compounds present in the samples was based on comparison of retention time and UV spectrum with standards.
Chromatographic conditions used for acetone extracts were as follows.
The mobile phase was a mixture of methanol (A) and water (B). 0 min — 50.0% A; 10 min — 50.0% A, 65 min — 100.0% A, 67 min — 100% A, 70 min — 50% A, 75 min — 50% A. The mobile phase was degassed in a sonicator. The mobile phase was pumped in gradient mode at a flow rate of 1.5 ml/min at 35 °C. The UV detection was accomplished at 245 nm and samples of 20 pl were injected into column. The qualitative identification of the compounds present in the samples was based on comparison of retention time and UV spectrum with standards.
Statistical analysis. All the data on antioxidant activity tests were the average of triplicate analyses. The data were recorded as mean ± standard deviation. Significant differences between means were determined by Student's t-test, P values < 0.05 were regarded as significant.
Results and Discussion
The results for antioxidant activities of extracts collected under different sample preparation techniques are represented in the Tables 2-4. All extracts were tested in the range of their concentrations from 6.25 to 800 mg-l-1. Absorbance for those ones varies in the interval of 10^90 absorbance units with the
standard deviation 0.34^4.
The antioxidant activity of mushroom extracts was compared with those of BHA and a-tocopherol that are used as standards in food and pharmaceutical industry. In ß-carotene-linoleic acid assay, petroleum ether and choroform extracts of G lucidum demonstrated the best antioxidant activity with an IC50: 33.66 ± 3.69 pg/ml, followed by acetone 36.97 ± 2.64 pg/ml, aqueous supernatant (IC50: 76.03 ± 7.96 pg/ml), methanol (IC50: 130.68 ± 28.05 pg/ml), and water extract (IC50: 2966.67 ± 793.85). In DPPH assay, acetone extract of G. lucidum demonstrated the best antioxidant activity with an IC50: 135.24 ± 8.94, followed by methanol 409.94 ± 10.09pg/ ml, aqueous supernatant (IC50 : 5 8 6.51 ± 20.05 pg/ml), petroleum ether and choroform extracts (IC50: 1195.25 ± 88.64). In general, the antioxidant activity of acetone extracts of all mushroom species was found as the highest. The DPPH free radical scavenging activity of the G. lucidum was better for solid-liquid extraction in acetone extracts (IC50: 83.79 ± 1.37). The best activity was found to be in acetone extract of G. lucidum using Soxhlet extraction with ß-carotene-linoleic acid assay (IC50: 18.54 ± 2.38 pg/ml). The best ABTS scavenging activity was found in acetone extract of G. lucidum Soxhlet extraction with an IC50 of 25.07 ± 2.83 pg/ml. The best CUPRAC activity was found in acetone extract of G. lucidum using solid-liquid extraction (IC50: 25.28 ± 0.14 pg/ml).
In ß-carotene-linoleic acid assay, methanol extract of G. adspersum demonstrated the best antioxidant activity with an IC50: 1.70 ± 1.13 pg/ml (Table 4), followed by acetone 9.79 ± 5.73 pg/ml, aqueous supernatant (IC50: 69.19 ± 0.30 pg/ml), petroleum ether and choroform (IC50: 157.17 ± 14.80 pg/ml), and water extracts (IC50: 426.90 ± 24.27). In DPPH assay, acetone extract of G. adspersum demonstrated the best antioxidant activity with an IC50: 10.36 ± 0.69 (Table 4), followed by methanol 36.54 ± 1.15 pg/ml, aqueous supernatant (IC50: 282.85 ± 41.17 pg/ml), petroleum ether and choroform extracts (IC50: 9950.60 ± 100.69). The DPPH free radical scavenging activity of the G. adspersum was better for ultrasonic extraction in acetone extract (IC50: 10.36 ± 0.69). The best ABTS scavenging activity was found in acetone extract of G. adspersum in Soxhlet extraction with an IC50 of 3.18 ± 0.17 pg/ml. The best CUPRAC activity was found in acetone extract of G. adspersum using ultrasonic extraction (IC50: 7.58 ± 1.33 pg/ml).
Table 2. Antioxidant activity of the extracts of G. lucidum, G. adspersum, G. applanatum by the fi-carotene-linoleic acid, DPPH, ABTS, and CUPRAC, obtained by solid-liquid extraction
Mushrooms/ standards Extracts ß-carotene-linoleic acid assay !C50a (Pg/ml) DPPH assay IC50 (Pg/ml) ABTS assay IC50 (Pg/ml) CUPRAC IC50 (Pg/ml)
G. lucidum Petroleum ether and choroform 120.70±8.52* ** 1448.76±42.06* ** 890.02±199.93* ** 477.50±14.85* **
Acetone 22.84±1.78* ** 83.79±1.37* ** 27.14±2.24* ** 25.28±0.14* **
Methanol 24.94±0.07 249.09±7.23* ** 35.78±20.56 199.25±48.44* **
Water 260.18±10.00 * ** N.A. 150.70±17.62* ** 26.15±0.68
Aqueous supernatant 124.79±5.82* ** 498.54±119.83 94.61±2.99* ** 86.40±2.26* **
G. adspersum Petroleum ether and choroform 367.35±0.41* ** 6402.63±74.95* ** N.A. 436.33±12.90* **
Methanol 41.20±2.42** 95.09±4.22** 23.23±1.05 31.89±0.77
Water 24.97±3.34* 702.30±32.06* ** 133.93±26.41* ** 40.10±1.14**
G. applanatum Petroleum ether and choroform 265.87±8.47* ** 3653.56±242.66* ** N.A. 422.22±5.17* **
Acetone 20.19±9.50** 3.50±1.68** 4.27±0.97** 1.16±0.17**
Methanol 8.25±0.88 42.17±1.57* ** 3.35±0.94 11.56±0.60*
Water 41.79±1.28* ** 231.62±0.25* ** 79.36±10.54* ** 47.50±2.12* **
Control a-Tocopherol b (standard) Ethanol 0.81±0.01 28.99±0.87 15.37±0.50 64.50±3.94
Control BHA b (standard) Ethanol 0.54±0.04 16.82±0.11
Hereinafter: a — IC50 values represent the means ± standard deviation of three parallel measurements (P < 0.05); b — reference compounds; N.A. — not bioactive; * — level of confident probability between average value, statistical significance of the differences comparing to the first control (a-tocopherol), P < 0.05; ** — level of confident probability between average value, statistical significance of the differences comparing to the second control (BHA), P < 0.05.
In ß-carotene-linoleic acid assay (Table 2), methanol extract of G. applanatum demonstrated the best antioxidant activity with an IC50 : 8.25 ± 0.88 pg/ml (Table 4), followed by acetone 20.19 ± 9.50 pg/ml, water (IC50: 41.79 ± 1.28 pg/ml), petroleum ether and chloroform extracts (IC50: 265.87±8.47 pg/ml). In DPPH assay, acetone extract of G. applanatum demonstrated the best antioxidant activity with an IC50: 3.50 ± 1.68 (Table 2), followed by methanol 42.17 ±
1.57 pg/ml, water (IC50: 231.62 ± 0.25 pg/ml), petroleum ether and choroform extracts (IC50: 3653.56 ± 242.66). The best ABTS scavenging activity was found in methanol extract of G. applanatum in solid-liquid extraction with an IC50 of 3.35 ± 0.94 pg/ml. The best CUPRAC activity was found in acetone extract of G. applanatum using solid-liquid extraction (IC50: 1.16 ± 0.17 pg/ml).
The acetone fraction of G. adspersum showed similar antioxidant activity in the
Table 3. Antioxidant activity of the extracts of G. lucidum, G. adspersum, G. applanatum by the p-carotene-linoleic acid, DPPH, ABTS, and CUPRAC, obtained by Soxhlet extraction
Mushrooms Extracts ß-carotene-linoleic acid assay IC50 (pg/ml) DPPH - assay IC50 (pg/ml) ABTS-+ assay IC50 (pg/ml) CUPRAC IC50 (pg/ml)
G. lucidum Petroleum ether and choroform 88.57±0.67* ** 1566.24±79.64* ** 1045.57±342.46* ** 356.00±5.57v
Acetone 18.54±2.38* 94.72±1.18** 25.07±2.83** 27.68±1.11* **
Methanol 29.08±1.94* 267.45±16.74** 191.22±5.38* 246.17±61.33* **
G. adspersum Petroleum ether and choroform 59.27±4.06* ** 4808.94±296.19* ** N.A. 399.50±0.71* **
Acetone 17.50±2.41* 28.94±5.05* 3.18±0.17** 11.23±0.33*
Methanol 31.35±4.71* 61.76±2.65** 15.53±1.86** 20.78±0.36*
G. applanatum Petroleum ether and choroform 162.59±27.95* ** 5108.02±376.58* ** N.A. 404.50±21.92* **
Acetone 30.72±8.39* 11.94±0.60* 11.94±0.60* 9.40±0.25**
Methanol 70.64±13.73* ** 11.33±0.53** 15.09±0.14** 6.30±0.69*
Table 4. Antioxidant activity of the extracts of G. lucidum, G. adspersum, G. applanatum by the ß-carotene-linoleic acid, DPPH, ABTS, and CUPRAC, obtained by ultrasonic extraction
Mushrooms Extracts ß-carotene-linoleic acid assay IC50 (pg/ml) DPPH - assay IC50 (pg/ml) ABTS-+ assay IC50 (pg/ml) CUPRAC IC50 (pg/ml)
G. lucidum Petroleum ether and choroform 33.66±3.69** 1195.25±88.64* N.A. 316.00±17.35**
Acetone 36.97±2.64* 135.24±8.94** 39.66±1.89* 35.72±0.63*
Methanol 130.68±28.05** 409.94±10.09* ** 40.49±23.20** 370.00±12.53*
Water 2966.67±793.85* ** N.A. 672.02±54.18* ** 566.00±50.09* **
Aqueous supernatant 76.03±7.96* ** 586.51±20.05* ** 119.58±1.34* ** 101.88±11.68*
G. adspersum Petroleum ether and choroform 157.17±14.80* ** 9950.60±100.69* ** N.A. 379.67±3.79**
Acetone 9.79±5.73* 10.36±0.69* ** 17.72±1.60* ** 7.58±1.33* **
Methanol 1.70±1.13** 36.54±1.15* ** 7.67±1.36* ** 13.20±0.33*
Water 426.90±24.27* N.A. 66.38±4.93** 201.50±17.68* **
Aqueous supernatant 69.19±0.30* 282.85±41.17 52.09±2.61* 26.15±0.68* **
G. applanatum Petroleum ether and choroform 724.75±32.58 N.A. N.A. 458.67±7.51**
Acetone N.A. 7.68±0.51* ** 13.08±1.17** 6.08±1.31* **
Methanol 67.84±0.25* ** 5.42±0.83* ** 11.15±2.48* 2.69±0.97* **
Water 338.58±0.92* ** 1566.68±615.75** 116.66±20.34** 52.00±0.45*
Aqueous supernatant 267.85±22.91** 247.89±14.94* 42.67±3.32* 25.07±0.66**
P-carotene-linoleic acid assay in our (IC50 = 9.79 ± 5.73, Table 4) and other scientists research (IC50 = 7.89 ± 0.91 pg/ml, [10]). Authors of work [10] stated that among the extracts, the ethyl acetate fraction of G. adspersum demonstrated the highest activity in the P-carotene-linoleic acid assay (IC50 = 5.63 ± 0.66 pg/ml). In our research the highest antioxidant activity for G. adspersum was observed in methanol fraction (IC50 = 1.7 ± 1.13 pg/ml).
As an example, chromatograms of dry components of acetone extracts of G. lucidum ilustrated in the Fig. 1. Chromatogram in red color shows fingerprint for it. Chromatogram in black color shows the result of treatment of G. lucidum sample dissolved in methanol (40 000 ppm) with ABTS solution in ethanol.
Solutions were mixed in ratio of 1 to 1. Concentration of G. lucidum in solution for both samples were the same (20 000 ppm).
Chromatogram of dry components of methanol extracts of G. lucidum ilustrated in the Fig. 2. Chromatogram shows fingerprint for it.
Thus, three-sample preparation techniques were used for obtaining mushrooms extracts. Antioxidant activity was estimated for all extracts. Antioxidant activities depend on investigated extract and method used for their measuring. Using solid-liquid extraction of G. applanatum and CUPRAC assay, optimal IC50 value is up to 1.16 ± 0.17 pg/ml. Soxhlet extraction of G. adspersum and ABTS assay gives the best IC50 value for this mushroom equal to 3.18 ± 0.17 pg/ml. Using ultrasonic
Fig. 1. Chromatograms of dry components of acetone extracts of G. lucidum
Fig. 2. Chromatogram of dry components of methanol extracts of G. lucidum
extraction of G. adspersum and ß-carotene-linoleic acid assay, methanol extract with the highest activity was found (IC50 1.70 ± 1.13pg/ml).
HPLC conditions were developed for getting the chromatograms of extracts. Such chromatograms might be used to detect the presence of presence active components in various mushrooms species without usage of expensive standards.
Acknowledgements
The authors would like to acknowledge the technical help provided by Taras Shevchenko National University of Kyiv. One of the authors (V. Raks) would also like to thank to The Scientific and Technological Research Council of Turkey (TUBiTAK) for supporting with the grant; namely, Fellowships for Visiting Scientists and Scientists on Sabbatical Leave (TUBITAK-BIDEB-2221).
REFERENCES
1. Yahia E. M., Gutiérrez-Orozco F., Moreno-Pérez M. A. Identification of phenolic compounds by liquid chromatography-mass spectrometry in seventeen species of wild mushrooms in Central Mexico and determination of their antioxidant activity and bioactive compounds. Food Chem. 2017, 226, 14-22.
2. Ma G., Yang W., Zhao L., Pei F., Fang D., Hu Q. A Critical Review on the Health Promoting Effects of Mushrooms Nutraceuticals. Food Sci. 2018. Human Wellness. In press, accepted manuscript.
3. Anita Klaus, Maja Kozarski, Jovana Vunduk, Predrag Petrovic, Miomir Niksic. Antibacterial and antifungal potential of wild basidiomycete mushroom Ganoderma applanatum. Lek. Sirovine. 2017, 36, 37-46.
4. Elkhateeb WA.,Zaghlol G.M., El-GarawaniI. M., Ahmed E. F., Rateb M. E., Abdel Moneim A. E. Ganoderma applanatum secondary metabolites induced apoptosis through different pathways: In vivo and in vitro anticancer studies. Biomed. Pharm. 2018, 101, 264-277.
5. Komura D. L., Carbonero E. R., GracherA. H. P., Baggio C. H., Freitas C. S., Marcon R., Santos A. R. S., Gorin P. A. J., Iacomini M. Structure of Agaricus spp. fucogalactans and their anti-inflammatory and antinociceptive properties. Bioresource Technol. 2010, 101, 6192-6199.
6. Cheng C.-R., Yue Q.-X., Wu Z.-Y., Song X.-Y., Tao S.-J., Wu X.-H., Xu P.-P., Liu X., Guan S.-H., Guo D.-A. Cytotoxic triterpenoids from Ganoderma lucidum. Molecules. 2018, 23, 649. doi: 10.3390/molecules23030649 101, 264-277.
7. Ferreira A., Proenca C., Serralheiro M. L. M., Araujo M. E. M. The in vitro screening for
acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J. Ethno Pharm. 2006, 108, 31-37.
8. Asakawa Y., Nagashima F., Hashimoto T., Toyota M., Ludwiczuk A., Komala I., Ito T., Yagi Y. Pungent and bitter, cytotoxic and antiviral terpenoids from some bryophytes and inedible fungi. Nat. Prod. Commun. 2014, 9, 409-417.
9. Sakamoto S., Kikkawa N., Kohno T, Shimizu K., Tanaka H, Morimoto S. Immunochromato-graphic strip assay for detection of bioactive Ganoderma triterpenoid, ganoderic acid A in Ganoderma lingzhi, Fitoterapia. 2016. doi: 10.1016/j.fitote.2016.08.01.
10. Tel-Qayan G. l, Öztürk M, Duru M. E, Rehman M. U, Adhikari A., Türkoglu A., Choudhary M. I. Phytochemical investigation, antioxidant and anticholinesterase activities of Ganoderma adspersum. Industr. Crops Prod. 2015, 76, 749-754.
11. Blois M. S. Antioxidant determinations by the use of a stable free radical. Nature. 1958, 181, 1199-1200.
12. Re R., Pellegrini N., Proteggente A., Pannala A., Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Rad. Bio. Med. 1999,26, 1231-1237.
13. Apak R., Güglü K., Özyürek M., KarademirS.E. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, Using their cupric ion reducing capability in the presence of neocuproine CUPRAC Method. J. Agr. Food Chem. 2004, 52, 79707981.
ХАРАКТЕРИСТИКА ЕКСТРАКТ1В ГРИБ1В Р1ЗНИХ ВИД1В РОДУ Ganoderma: АНТИОКСИДАНТНА АКТИВН1СТЬ ТА ХРОМАТОГРАМИ
В. Ракс1, 2 M. Озтурк2 O. А Васильченко3 M. Ракс4
^швський нащональний ушверситет iMeHi Тараса Шевченка, Укра1на 2Мугла Сiтки Кокман унiверситет, Туреччина 3Нащональний авiацiйний унiверситет, Kиïв, Укра1на 4Нащональний унiверситет харчових технологiй, Kиïв, Укра1на
E-mail: raksvictoria@knu.ua
Метою роботи було вид^ення б^лопч-но активних сполук i3 плодових тiл грибiв Ganoderma lucidum, Ganoderma adspersum i Ganoderma applanatum та ощнювання ïхньоï антиоксидантноï активностi. Для видiлення бiологiчно активних сполук використовува-ли рiзнi методи. Антиоксидантш властивост визначали спектрофотометрично, вимiрю-ючи активнiсть захоплення вiльних ради-калiв. Для аналiзу хроматограм вид^ених екстрактiв застосовували високоефективну рiдинну хроматограф^. В результатi ана-лiзу з використанням ß-каротин-лiнолевоï кислоти було визначено високу антиокси-дантну активнiсть метанольних екстрак-тiв. Напiвмаксимальне iнгiбування IC50 для G. applanatum i G. adspersum становило 8,25 ± 0,88 мкг/мл та 1,70 ± 1,13 мкг/мл вщпо-вiдно. Водночас, екстракти петролейного ефiру i хлороформу G. lucidum мали вищу антиоксидантну актившсть: IC50 — близько 33,66 ± 3,69 мкг/мл. Отримано хроматогра-ми компоненив ацетонових i метанольних екстракив G. lucidum. Основною перевагою таких хроматограм е можливiсть виявлення активних компоненив рiзних видiв грибiв без використання високоварисних стандар-тiв.
Ключовi слова: гриби видiв Ganoderma, анти-оксидантна активнiсть, високоефективна рiдинна хроматографiя.
ХАРАКТЕРИСТИКА ЭКСТРАКТОВ ГРИБОВ РАЗНЫХ ВИДОВ РОДА Ganoderma: АНТИОКСИДАНТНАЯ АКТИВНОСТЬ И ХРОМАТОГРАММЫ
В. Ракс1' 2 M. Озтурк2 O. А. Васильченко3 M. Ракс4
1Киевский национальный университет имени Тараса Шевченко, Украина 2Мугла Ситки Кокман университет, Турция 3Национальный авиационный университет, Киев, Украина
4Национальный университет пищевых технологий, Киев, Украина
E-mail: raksvictoria@knu.ua
Целью работы было выделение биологически активных соединений из плодовых тел грибов Ganoderma lucidum, Ganoderma adspersum и Ganoderma applanatum и оценка их антиок-сидантной активности. Для выделения биологически активных соединений использовали различные методы. Антиоксидантные свойства определяли спектрофотометрически, измеряя активность захвата свободных радикалов. Для анализа хроматограмм выделенных экстрактов применяли высокоэффективную жидкостную хроматографию. В результате анализа с использованием Р-каротин-линолевой кислоты была определена высокая антиоксидант-ная активность метанольных экстрактов. Полумаксимальное ингибирование IC50 для G. applanatum и G. adspersum составило 8,25 ± 0,88 и 1,70 ± 1,13 мкг/мл соответственно. В то же время экстракты петролейного эфира и хлороформа G. lucidum имели большую анти-оксидантную активность: IC50 — около 33,66 ± 3,69 мкг/мл. Были получены хроматограммы компонентов ацетоновых и метанольных экстрактов G. lucidum. Основным преимуществом таких хроматограмм является возможность выявления активных компонентов различных видов грибов без использования дорогостоящих стандартов.
Ключевые слова: грибы видов Ganoderma, антиоксидантная активность, высокоэффективная жидкостная хроматография.
