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11
Биорадикалы и Антиоксиданты. 2019 Том 6, №1
20
ОРИГИНАЛЬНЫЕ СТАТЬИ
ANTIOXIDANT ACTIVITY OF JUGLANS NIGRA L BARK EXTRACT IN THE EXPERIMENT
Pozdnyakov D.I., Zolotych D.S., Dajronas Zh.V., Vernikovskij V.V., Lyakhova N.S., Pozdnyakova A.E.
Pyatigorsk Medical and Pharmaceutical Institute, a branch Volgograd State Medical University, Pyatigorsk
Abstract
Objective: to evaluate the antioxidant activity of the Juglans nigra L bark extract in the experiment.
Materials and methods: The antioxidant activity of the ethanol extract of the Juglans nigra L bark was evaluated in in vitro tests to determine the scavenging and chelating activity. The antiradical activity of the extract was studied under conditions of generation of DDPH radical, superoxide, hydroxyl and nitrosyl radicals; chelating properties were evaluated for iron binding ability. As referents, ascorbic acid (scavenging activity) and EDTA (chelating properties) were used. The total antioxidant activity of the extract studied, the total content of flavonoids and phenolic compounds was also assessed.
Results: the study found that the studied extract of the bark of Juglans nigra L exhibits high scavenging activity comparable to ascorbic acid and expressed in inhibition of DDPH-radical (75,4%), superoxide radical (79,2%), hydroxyl radical (74,3%), nitrosyl radical (78,1%). Also, the extract studied exhibits iron-chelating properties, which is confirmed by the formation of a stable complex with ferric ions. Such activity of the extract of the Juglans nigra L bark may be associated with a high content of flavonoids (in terms of quercetin - 2,77±0,053 mg/g) and phenolic compounds (in terms of gallic acid - 2,38±0,045 mg/g).
Conclusion: The study showed that the ethanol extract of the Juglans nigra L bark has antioxidant properties, which makes it promising to further study this object in order to create on its basis a product with antioxidant activity.
Key words: antioxidant, herbal extracts, Juglans nigra L, flavonoids
Introduction
The intensification of the free radical oxidation can underlie the aging of the organism, the intensification of the effect of unfavorable environmental factors, as well as the pathogenesis of a number of diseases, for example oncopathology [Valko M., Rhodes C., Moncol J., Izakovic M., Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer // Chem Biol Interact. 2006. Vol. 160, N 1. P. 1-40.], atherosclerosis [Yang X., Li Y., Li Y. et al. Oxidative Stress-Mediated Atherosclerosis: Mechanisms and Therapies // Front in Physiol. 2017. Vol. 8. P. 600.
doi:10.3389/fphys.2017.00600.] , ischemic stroke [Carbone F, Teixeira PC, Braunersreuther V, Mach F, Vuilleumier N, Montecucco F. Pathophysiology and Treatments of Oxidative Injury in Ischemic Stroke: Focus on the Phagocytic NADPH Oxidase // Antioxid & Red Sign. 2015. Vol. 23, N 5. P. 460-489.], arrhythmia [Karagueuzian H.S., Nguyen T.P., Qu Z., Weiss J.N. Oxidative stress, fibrosis, and early afterdepolarization-mediated cardiac arrhythmias // Front in Physiol. 2013. Vol. 4. P. 19. doi:10.3389/fphys.2013.00019.], etc. Correction of oxidative stress is of undoubted scientific and practical interest and can be used as an independent link in pharmacotherapy. In the present, it has been established that the use of agents with antioxidant activity promotes accelerated convalescence in atherosclerosis, ischemic heart disease and diabetes mellitus [Gerber P.A., Rutter G.A. The Role of Oxidative Stress and Hypoxia in Pancreatic Beta-Cell Dysfunction in Diabetes Mellitus // Antioxid & Red Sign. 2012. Vol. 26, N 10. P. 501-518. doi:10.1089/ars.2016.6755.]. Among the antioxidants can be attributed substances of both natural [Xu D.-P., Li Y., Meng X. et al. Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and Resources. Arráez-Román D, Gómez Caravaca AM, eds // Int. J Mol. Scien. 2017. Vol. 18, N 1. P. 96. doi:10.3390/ijms18010096] and synthetic [Poljsak B., Suput D., Milisav I. Achieving the Balance between ROS and Antioxidants: When to Use the Synthetic Antioxidants // Ox. Med. and Cell. Long. 2013. Vol. 2013. P. 956792. doi:10.1155/2013/956792] origin. However, despite the high therapeutic effectiveness of synthetic agents in the scientific community and among practitioners specialists, more attention is paid to compounds of natural genesis [Tao Z., Shi A., Zhao J. Epidemiological perspectives of diabetes // Cell Biochem Biophys. 2015. Vol. 73. P. 181-185.], since along with the equivalent therapeutic efficacy, substances free occurring in nature have a smaller spectrum of undesirable side reactions [Delaviz H., Mohammadi J., Ghalamfarsa G., Mohammadi B., Farhadi N. A Review Study on Phytochemistry and Pharmacology Applications of Juglans Nigra Plant. // Pharmacog. Rev. 2017. Vol. 11, N 22. P. 145-152. doi:10.4103/phrev.phrev_10_17.]. In this connection, the present study is devoted to the evaluation of the antioxidant activity of the extract of the Juglans nigra L bark, which can undoubtedly be attributed to natural products.
Juglans nigra L is a deciduous plant of the Juglandaceae family, found everywhere in the countries with a sharp-continental or moderate climate (eastward from the Balkans to South-West China, Eastern Europe, the USA) [Zhao M.-H., Jiang Z.-T., Liu T., Li R. Flavonoids in Juglans nigra L. Leaves and Evaluation of In Vitro Antioxidant Activity via Intracellular and Chemical Methods // The Scientific World J. 2014. Vol. 2014. P. 303878. doi:10.1155/2014/303878.]. Raw Juglans nigra L, in particular leaves, is a source of sesquiterpenes, phenols, phospholipids, sterols, sphignolipids, flavonoids, terpenoids and has a variety of therapeutic effects: antibacterial, anti-inflammatory, antidiabetic, angioprotective, hypoglycemic [Cheniany M., Ebrahimzadeh H., Vahdati K., Preece J.E., Masoudinejad A., Mirmasoumi M. Content of different groups of phenolic compounds in microshoots of Juglans regia cultivars and studies on antioxidant activity // Acta Physiol. Plant. 2013. Vol. 35, N 2. P. 443-450.]. In this connection, it seems promising to study the therapeutic potential
of other parts of the Juglans nigra L plant, which are more economical in the billet, as well as with a greater yield of active substances, for example, bark.
Materials and methods
Preparation of Juglans nigra L bark extract
40% ethanol was used as the extractant. The extraction was carried out in the soxhlet apparatus for 48 hours, the initial volume of the ground raw material was 500 g. The extract was collected and concentrated in a vacuum evaporator. Prior to use, the extract was stored in a desiccator.
Evaluation of the antioxidant properties of the extract of the Juglans nigra L bark. The antioxidant properties of the Juglans nigra L bark extract were evaluated in various in vitro model test systems in the concentration: 1000 ^g / ml; 500 ^g / ml; 250 ^g / ml; 125 ^g / ml; 62.5 ^g / ml.
Determination of the total flavonoids
The total content of flavonoids in the extract was determined by reaction with aluminum chloride [Garg D., Shaikh A., Muley A., Marar T. In vitro antioxidant activity and phytochemical analysis in extracts of Hibiscus rosa-sinensis stem and leaves // Free Radic. Antioxid. 2012. Vol. 2. P. 41-46.]. The reaction mixture contained 1 ml of the solution of the studied extract (1:10) in various concentrations + 0.5 ml of a 1.2% solution of aluminum chloride + 0.5 ml of a saturated solution of potassium acetate. The medium was incubated for 30 minutes. at room temperature, and the absorbance of the samples was measured at 415 nm. The total content of flavonoids was expressed in terms of quercetin (mg / g).
Determination of the total phenolic compounds
To 0.5 ml of the studied extract (1: 5), 0.1 ml of a Folin-Chokalteu reagent (0.5n) was added [McDonald S., Prenzler P., Robards K. Phenolic content and antioxidant activity of olive extracts // Food Chem. 2001. Vol. 73. P. 73-84.]. The resulting mixture was incubated at room temperature for 15 minutes. after which a saturated solution of sodium carbonate (2.5 ml) was added. The mixture was exposed for 30 minutes. at room temperature. The absorbance of the samples at 760 nm was measured. The total content of phenolic compounds was expressed in terms of gallic acid (mg / g).
DPPH - scavinging activity.
The effect of the extract studied on the generation of the DPPH radical was evaluated by the Mensor method [Mensor L.L., Menezes F.S., Leitao G.G., Reis A.S., dos Santos T.C., Coube C.S. et al. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method // Phytother Res. 2001. Vol. 15. P. 127-130.]. Analysis: 0.5 ml of methanol 0.4 mM DPPH solution was added 1 ml of the solution of the extract under study at estimated concentrations and incubated at room temperature for 30 minutes. As a reference substance, ascorbic acid was used in similar concentrations. After this time interval, spectrophotometric detection was performed at a wavelength of 518 nm against pure methanol. A solution of DPPH in methanol was taken as a positive control (A0). The percentage inhibition of DPPH radical formation was calculated from formula (1):
% inhibition = Ax / A0 * 100, where
Ax - absorbanse of the extract (referent) sample;
A0 - absorbanse of the positive control sample.
Hydroxyl radical - scavinging activity
The analysis was carried out using the Elizabeth & Rao method, based on the detection of a colored complex of degradation products of 2-deoxyribose with thiobarbituric acid [Elizabeth K., Rao M.N. Oxygen radical scavenging activity of curcumin // Int J. Pharm. 1990. Vol. 58. P. 237-240.]. Generation of the hydroxyl radical was carried out in the Fenton reaction. Analysis: to the incubation medium (0.1 ml 2.8 mM deoxyribose solution + 0.1 ml 0.1 mM EDTA + 0.1 ml 0.1 mM ascorbate solution + 0.1 ml phosphate buffer (pH 7, 4) 1 ml of the extract solution was added at different concentrations, incubated for 60 minutes at 370 C. 1 ml of a 2.8% solution of trichloroacetic acid and 1 ml of a 1% solution of thiobarbituric acid was added to the medium, heated in a water bath (1000C) 20 min After cooling, the optical density of the samples was measured at a wavelength of 532 nm against air.The positive control was the generation medium of the hydroxyl radical without adding the extract being studied. As comparison substance ascorbic acid used in similar concentrations. Percent inhibition was calculated by the formula 1.
Superoxide radical - scavinging activity
The analysis was carried out according to the Winterbourn method [Winterbourn C.C., Hawkins R.E., Brian M., Carrell R.W. The estimation of red cell superoxide dismutase activity // J. Lab. Clin. Med. 1975. Vol. 85. P. 337-341.]. The superoxide radical was generated in the photobroduct reaction of riboflavin. The incubation medium consisted of 0.1 ml of the solution of the extract under study at various concentrations + 0.1 ml of a 1.5 mM solution of nitro-blue tetrazolium + 0.2 ml of 0.1 M EDTA + 0.05 ml of 0.12 mM riboflavin + 2 solution, 55 ml of phosphate buffer (pH 7.4). The resulting mixture was incubated for 5 minutes at 250C. The absorbance of the samples was measured at 560 nm against air. As a reference substance, ascorbic acid was used in similar concentrations. Positive control was the incubation medium without adding the extract (referent). Percentage inhibition was calculated by the formula 1.
Nitrosyl radical scavinging activity
The course of the analysis corresponded to the method described by Marcocci [Marcocci L., Maguire J.J., Droy-Lefaix M.T., Packer L. The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761 // Biochem Biophys Res Commun. 1994. Vol. 201. P. 748-755.]: 2 ml of 10 mM sodium nitroprusside + 0.5 phosphate buffer solution (pH 7.4) + 0.5 ml of the solution of the extract under study in different concentrations were introduced into the cuvette. The resulting mixture was incubated at 250C for 15 minutes. After a period of incubation, 0.5 ml of Griess reagent was added to the medium, incubated for 30 minutes at room temperature. The absorbance of the samples was measured at 546 nm. As a reference substance, ascorbic acid was used in similar concentrations. The percentage of inhibition of the nitrosyl radical was calculated from formula (2):
% inhibition = Ax / A0 * 100, where
Ax - absorbance of the sample before the addition of the Griess reagent;
A0 - absorbance of the sample after the addition of the Griess reagent.
Evaluation of iron-chelating activity
The basis for this analysis was taken by the method proposed by Benzie & Strain [Benzie I.F., Strain J.J. The ferric reducing ability of plasma (FRAP) as a measure of —antioxidant power": The FRAP assay // Anal Biochem. 1996. Vol. 239. P. 70-76.]. The reaction medium contained 1 ml of 0.05% methanol solution of o-phenanthroline + 2 ml of iron (III) chloride (200 ^M) + 2 ml of various concentrations of the extract under study. The resulting mixture was incubated for 10 minutes at room temperature. The absorbance of the samples was measured at 510 nm. EDTA (metal chelator) was used as the reference substance in similar concentrations. Positive control was the incubation medium without adding the extract (referent). The percentage inhibition was calculated by the formula 1.
Determination of total antioxidant activity
The total antioxidant activity was evaluated in the Mo (VI) oxidation reaction in Mo (V) [Prieto P., Pineda M., Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of Vitamin E // Anal Biochem. 1999. Vol. 269. P. 337-341.]. To 4 ml of the solution of the extract under study at different concentrations 4 ml of a phosphorus-molybdenum reagent was added. The resulting mixture was heated in a water bath (950C) for 90 minutes. After cooling to room temperature, the absorbance of the samples at 695 nm against air was measured. As a reference substance, ascorbic acid was used in similar concentrations. The percentage inhibition was calculated by the formula 1.
Statistical methods
The results of the experiment were processed statistically using the software package "STATISTICA 6.0". The data were expressed as M ± SD. The half-inhibition coefficient (IC50) was calculated by the probit analysis method.
Results
The total antioxidant activity, scavinging and chelating properties of the Juglans nigra L bark extract were evaluated in the range of double dilutions of concentrations of 62,5-1000 ^g / ml.
Estimating the total content of phenolic compounds and flavonoids in the studied extract, it was established that the total content of phenolic compounds in the extract of the Juglans nigra bark in terms of gallic acid was 2,38 ± 0,045 mg / g, with the total concentration of flavonoids in terms quercetin was 2,77 ± 0,053 mg / g.
Evaluation of the DPPH radical of the inhibitory properties of the studied extract showed that the extract at a concentration of 1000 ^g / ml inhibited the formation of the DPPH radical by 75,4%, while ascorbic acid at similar concentrations showed inhibitory properties with respect to the DPPH radical at 77,2% (Fig. 1). The IC50 value of the extract was slightly higher than that of ascorbic acid and was 435 ^g / ml vs 377 ^g / ml in ascorbic acid.
Fig. 1. DPPH - radical scavinging activity of the Juglans nigra L bark extract
(Note: E Jr - Juglans nigra L bark extract)
Estimating the ability of the extract under study to suppress the formation of a superoxide radical, it is established that the investigated object at a concentration of 1000 ^g / ml suppresses the generation of the superoxide radical (Fig. 2) by 79.2%, which is insignificantly higher than the ascorbic acid (76,3%), The IC50 for the extract was 293,8 ^g / ml, and for ascorbic acid 356,5 ^g / ml.
10 -I-\-1-1-1-1
0 200 400 600 800 1000
Co ne eut ration, ink g ml
Fig. 2. Superoxide - radical scavinging activity of the Juglans nigra L bark extract
(Note: E Jr - Juglans nigra L bark extract)
By ability to inhibit the formation of hydroxyl radical (Fig. 3), the extract studied also slightly exceeded ascorbic acid, as evidenced by the percent inhibition of hydroxyl radical formation for the studied extract (74,3%) and ascorbic acid (70,2%). The IC50 value in assessing the hydroxyl radical scavinging properties in the studied object was 478,2 ^g / ml, while in ascorbic acid this value was 486,8 ^g / ml.
0 100 200 300 400 500 600 700 800 900 1000
Concentration, ink g. ml
Fig. 3. Hydroxyl - radical scavinging activity of the Juglans nigra L bark extract
(Note: E Jr - Juglans nigra L bark extract)
The percent inhibition of nitrosyl radical formation (Fig. 4) for the extract studied at a concentration of 1000 ^g / ml was 78.1%, which was slightly less than the referent, ascorbic acid (81.6%). The IC50 values for this test object and ascorbic acid were 152 ^g / ml and 183 ^g / ml, respectively.
EJr A Ascorbic acid
Concentration, inkginl
Fig. 4. Nitrosyl - radical scavinging activity of the Juglans nigra L bark extract
(Note: E Jr - Juglans nigra L bark extract)
The chelating properties (Fig. 5) of the investigated object were comparable with those of EDTA, which was expressed in approximate IC50 values (for the extract and EDTA studied, this index was 131.4 ^g / ml and 137.6 ^g / ml, respectively), as well as close the percent inhibitory activity at the maximum test concentration (1000 ^g / ml, the percentage of inhibitory activity for the extract studied was 83.8%, for EDTA 81.5%).
The total antioxidant activity of the extract was comparable to that of ascorbic acid (Fig. 6). The IC50 value for the studied object was 485.4 ^g / ml, and ascorbic acid 422.5 ^g / ml.
Fig. 5. Iron-chelating activity of the Juglans nigra L bark extract
(Note: E Jr - Juglans nigra L bark extract)
Fig. 5. Total antioxidant activity of the Juglans nigra L bark extract in comparsion ascorbic acid (Note: E Jr - Juglans nigra L bark extract)
Discussion
Oxidative stress is a typical pathophysiological process that plays a significant role in the pathogenesis of a huge number of diseases. A direct relationship between the initiation of oxidative stress and the development of atherosclerosis of coronary heart disease, type II diabetes, arterial hypertension, muscular fatigue, ischemic stroke, etc. is established. Oxidative stress is defined as an imbalance in the pro / antioxidant system, with an increase in the formation of free radicals (FR) and a violation of their inactivation [Halliwell B. Free radicals and antioxidants: updating a personal view // Nutr. Rev. 2012. Vol. 70, N 5. P. 257-265. doi: 10.1111/j.1753-4887.2012.00476.x.]. Prooxidants are represented by active forms of oxygen (ROS): superoxide radical, hydroxyl radical, hybrid free radicals of nitric oxide and oxygen, etc. [Li S., Tan H.-Y., Wang N. et al. The Role of Oxidative Stress and Antioxidants in Liver Diseases // Int. J Mol. Scien. 2015. Vol. 16, N 11. P. 26087-26124. doi:10.3390/ijms161125942.]. Free radicals can have both endogenous and exogenous origin. The main source of endogenous CP is the mitochondrial respiratory chain [Radi R., Peluffo G., Alvarez
M.N., Naviliat M., Cayota A. Unraveling peroxynitrite formation in biological systems // Free Radical Biology and Medicine. 2001. Vol. 30, N 5. P. 463-488.]. Violation of the functional activity of enzymatic systems of NADPH oxidase (NOX), xanthine oxidase (XO) and inducible nitric oxide synthase (iNOS) leads to hyperproduction of FR with initiation of cell damage. It is known that a superoxide radical is formed as a result of one-electron oxygen oxidation reactions in the mitochondrial respiratory chain and from 2% to 5% of the total electron flux goes to its production [Casteilla L., Rigoulet M., Penicaud L. Mitochondrial ROS metabolism: modulation by uncoupling proteins // IUBMB Life. 2001. Vol. 52, N 3-5. P. 181-188.] (the latter claim is controversial, since according to Staniek and Nohl [Staniek K., Nohl H. H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system // Bioch. et Biophys. Acta. 1999. Vol. 1413, N 2. P. 7080.] superoxide consumes 0.5% -1% of the total amount of mitochondrial oxygen). Hyperproduction of the superoxide radical instigate damage to mitochondrial DNA and proteins, which leads to their death and, accordingly, to cell destruction. In addition, reacting with NO, the superoxide radical forms a cytotoxic peroxonitrite, which has a direct alterative effect on the cells, and also initiates a secondary damage wave by destroying DNA, inactivating enzymes, etc. [Santos J.M., Mohammad G., Zhong Q., Kowluru R.A. Diabetic Retinopathy, Superoxide Damage and Antioxidants // Curr. pharm.biotech. 2011. Vol. 12, N 3. P. 352-361.]. Also important for the development of oxidative stress are divalent metal ions, in particular iron and copper, which catalyze the formation of SR in the Fenton reaction [Imlay J.A. Pathways of oxidative damage // Ann. Rev. of Microb. 2003. Vol. 57. P. 395-418.]. Exogenous antioxidants enter the body from the environment, with food or through radioactive contamination.
The system of endogenous antioxidant protection, which includes both enzymatic (SOD, catalase, glutathione peroxidase, peroxiredoxins) and a non-enzymatic component (glutathione, thioredoxin, a-tocopherol), can prevent the destruction of cells under the action of FR [Poljsak B. Strategies for Reducing or Preventing the Generation of Oxidative Stress // Ox. Med. and Cell. Long. 2011. Vol. 2011. P. 194586. doi:10.1155/2011/194586.]. Proceeding from this, there are two main strategies for correcting oxidative stress: reducing the formation of free radicals or increasing the activity of enzymes of endogenous antioxidant protection.
In this study, it has been found that the extract obtained from the Juglans nigra L bark has a pronounced scavinging and chelating activity comparable to that of the referents (for scavinging properties- ascorbic acid and for chelating properties -EDTA). In various in vitro tests, the studied extract suppressed generation of the superoxide radical, hydroxyl and nitrosyl radicals, and also exhibited iron- chelating properties. In addition, it was found that the studied extract exhibits high overall antioxidant activity comparable to ascorbic acid. Also in the extract of the Juglans nigra L bark, a significant content of flavonoids and phenolic compounds was established, which can underlie the scavinging and chelating activity of the extract, which makes this object promising for further study.
Conclusion
The study showed that the ethanolic extract of the Juglans nigra L bark possesses high antioxidant activity (in vitro) comparable to ascorbic acid (scavinging
properties) and EDTA (chelating activity), which makes this object promising for further research, with the aim of developing an antioxidant agent.
Conflicts of interest
The authors statement no conflict of interest with the submitted manuscript.
References
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3. Casteilla L., Rigoulet M., Penicaud L. Mitochondrial ROS metabolism: modulation by uncoupling proteins // IUBMB Life. 2001. Vol. 52, N 3-5. P. 181-188.
4. Cheniany M., Ebrahimzadeh H., Vahdati K., Preece J.E., Masoudinejad A., Mirmasoumi M. Content of different groups of phenolic compounds in microshoots of Juglans regia cultivars and studies on antioxidant activity // Acta Physiol. Plant. 2013. Vol. 35, N 2. P. 443-450.
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19. Radi R., Peluffo G., Alvarez M.N., Naviliat M., Cayota A. Unraveling peroxynitrite formation in biological systems // Free Radical Biology and Medicine. 2001. Vol. 30, N 5. P. 463-488.
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21. Staniek K., Nohl H. H2O2 detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system // Bioch. et Biophys. Acta. 1999. Vol. 1413, N 2. P. 70-80.
22. Tao Z., Shi A., Zhao J. Epidemiological perspectives of diabetes // Cell Biochem Biophys. 2015. Vol. 73. P. 181-185.
23. Valko M., Rhodes C., Moncol J., Izakovic M., Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer // Chem Biol Interact. 2006. Vol. 160, N 1. P. 1-40.
24. Winterbourn C.C., Hawkins R.E., Brian M., Carrell R.W. The estimation of red cell superoxide dismutase activity // J. Lab. Clin. Med. 1975. Vol. 85. P. 337-341.
25. Xu D.-P., Li Y., Meng X. et al. Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and Resources. Arráez-Román D, Gómez Caravaca AM, eds // Int. J Mol. Scien. 2017. Vol. 18, N 1. P. 96. doi:10.3390/ijms18010096
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27. Zhao M.-H., Jiang Z.-T., Liu T., Li R. Flavonoids in Juglans nigra L. Leaves and Evaluation of In Vitro Antioxidant Activity via Intracellular and Chemical Methods // The Scientific World J. 2014. Vol. 2014. P. 303878. doi:10.1155/2014/303878.
