Influence of Low Temperature Storage and Ultrasonic Treatment of Placenta on Its Extracts Antioxidant Properties

The experimental data concerning evaluation of low temperature storage and ways of placenta extract obtaining influences on human placenta extract (HPE) antioxidant properties are provided. HPE antioxidant properties were estimated by capacity to reduce the ferric iron and ABTS+ radical, to chelate ferrous ions, by phenolic compound content and the value of catalase and superoxid dismutase activity. Six-month placentae storage at –196°С has been shown not to affect HPE antioxidant activity. Moreover ultrasonic wave's application during obtaining of HPE either decreases the time of extraction or is effective factor of extracts' antioxidant properties preservation.

Встановлено, що екстракти плаценти людини (ЕПЛ) здатні нейтралізувати гідроксильний Human placenta and its preparations are known to possess various therapeutic activities [15,21].This fact is explained by the presence of bioactive substances with antioxidant properties and their ability to neutralize active oxygen species which are a contributing factor in development of various diseases.Placenta contains two kinds of antioxidants: enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and nonenzymatic (chelating proteins, thiols, amino acids, phenolic compounds, uric acid, vitamins and certain hormones (estrogen and melatonin)) [17,27,30].
The main restriction of widening placenta application in clinical practice is a short term of its storage in functionally valid state, i. e. the testing for bacterial and viral contamination is complicated.Extending of placenta storage period and widening perspectives of its clinical usage is possible due to modern cryobiological techniques [9].However, storage of biological objects in a frozen state may also affect the properties and biological activity of HPE compounds, mainly due to macromolecule conformational changes [13,18].
Ultrasound application allows accelerating the extraction, increasing recovery of a substance, to be extracted, lowering placenta preparations cost and enhancing labor productivity [1].Moreover, ultrasound is able to provide the sterilization effect, which is kept after treatment of solution, emulsion, etc. for some time [12].However, ultrasound treatment, depending on power and time, could lead to activity alterations due to high molecular mass substances degradation, influence on protein and nucleic acids structure and functions, protein glycosilation and free radical accumulation [6,14,29].
The aim of the present research was both to study antioxidant properties of extracts obtained from fresh and cryopreserved placenta using ultrasonic waves and to investigate activity of individual HPE fractions.

Materials and methods
Human placentas weighing between 400-600 g (40 weeks of gestation (n = 10) were collected from healthy parturients with their informed consent and tested for viral (HIV, hepatitis B, C) and syphilis contamination.Placentas were stored not longer than 24 hrs at 4°C.Each placenta was cut into two equal parts: the first one ('fresh') from which the extract was obtained immediately; the second one was frozen in plastic bag down to -196°C (in a liquid nitrogen) with the rate of 300 deg/min and stored during 6 months.Thawing was carried out in a water bath at 37°C.
For preparing the extracts either fresh or cryopreserved placenta was thoroughly washed from mucus and blood with NaCl isotonic solution which was substituted several times.The amniotic and chorion membranes were removed from the washed placenta, then it was minced into 3×2 cm pieces.Obtained placenta fragments were plunged into a vessel with physiological solution in 1:5 w/v ratio and stirred for 2-3 min.The upper layer was removed and the fresh portion of physiological solution was added, the procedure was performed 3-4 times.Washed placenta pieces were diluted with physiological solution in 1:1 w/v ratio and homogenized for 3 min using a high-speed homogenizer RT-1 U4.2 (Odessa Experimental Factory of Laboratory Medical Equipment, Ukraine).The obtained homogenate was divided into two parts: the first one was exposed in physiological solution for 12 hrs at 4°C; the second one was treated with ultrasonic waves using ultrasonic disperser USDN-1-U4.2(Ukraine).In order to obtain the extracts using ultrasound the optimal regimen of placenta homogenate treatment was chosen (4 min with 22 kHz).Such a regimen provided the most effective extraction of antioxidant molecules and did not lead to their denaturation.To avoid the homogenate heating the treatment was carried out 2 times during 2 min with the 1 minute interval.Then it was centrifuged at 1500g during 25 min.The supernatant was filtrated through a 0.45 µm membrane filter (Millipore Corp. Carrigtwohill, Ireland).Filtrate, which was an aqueous-saline placenta extract, was poured into tubes for further experiments.
Content of proteins in extracts and individual fractions was measured using direct spectrophotometry by means of recording absorbance at 260 and 280 nm [9].All spectrophotometric measurements were carried out using Pye Unicam SP8000 (UK).
To evaluate total phenolic content the experimental sample (0.1 ml) was mixed with 1.5 mL of Folin reagent (Sigma-Aldrich Chemical) previously diluted with a distilled water in 10 times [28].The obtained mixture was hold for 5 min at 22°C and then sodium carbonate (1.2 mL, 7.5%) (Merk, Germany) was added.After 30 min incubation at 22°C the absorbance at 725 nm was recorded.Gallic acid (Sigma-Aldrich Chemical) was used as a control.
Antiradical activity was calculated using formula: ARA=(A 0 -A)/A 0 ×100%, where A 0 was absorbance at the beginning of the experiment; A -absorbance after certain time period.
Ferrous ion (Fe 2+ ) chelation by HPE was estimated by the Ferrozine assay [3].Experimental sample (0.2 ml) was mixed with a solution of 2 mM FeCl 2 (0.05 ml) (Merck).The reaction was initiated by the addition of 5 mM Ferrozine (0.2 mL) (Sigma-Aldrich Chemical, Germany).Then, the mixture was shaken vigorously and left for 10-15 min at room temperature.Then the sample absorbance was measured spectrophotometrically at 562 nm.The results were expressed as the percentage of inhibition of the Ferrozine-Fe 2+ complex formation.
Superoxide dismutase (SOD) activity in HPE was determined by the enzyme ability to inhibit the reaction of epinephrine autooxidation in alkaline medium [18].Reaction rate was assessed spectrophotometrically by the optical density value of product accumulated during epinephrine autooxidation in the presence of examined sample at 347 nm against the control.
Statistical analysis was carried out using Statgraphics Plus version 2.1 for Windows (Manugistic, USA).All the values of antioxidant activity for each placenta were analyzed in triplicate set.The data were presented as mean for all examined placentas ± standard deviation and analyzed by paired t-test to find out a difference between sample means.Values with P < 0.05 were considered significant.

Results and discussion
A combined approach was chosen in order to evaluate antioxidant activity of experimental samples in order to estimate the contribution of different antioxidant centers.

HPE non-enzymatic antioxidant properties.
Phenolic compounds (vitamin E, ubichinons, tryptophan, tyrosin, phenylalanine) may contribute directly to antioxidative action by scavenging free radicals [10,34].The values of total phenolic content in the extracts obtained either by 12-hr-long exposure or using ultrasound derived both from fresh and cryopreserved placentas did not differ significantly (Table 1).
Data obtained for extracts match with those for their individual fractions.There was no significant difference between phenols content values in individual fractions in all the experimental samples (Fig. 1).
All the experimental samples did not manifest a chelating ability.Previously it has been revealed that extracts derived from placentas at 40 weeks of gestation did not possess such activity in comparison with those of 38 weeks (scheduled Cesarean section).This fact may be associated with free radical accumulation in placenta during pregnancy [25].Thus the change of extract obtaining technique did not affect their chelating ability, we suppose it to be stipulated with the placenta quality.
The ABTS + radical is known to react with most antioxidants including thiols, ascorbic acid, uric acid, phenolics.During this reaction, the blue colored ABTS + radical is converted back to its colorless neutral form [31,32].
Previously we have shown that profile of kinetic of ABTS + radical reduction by the extracts derived from placentae of 40 weeks of gestation obtained by 12-hrlong exposure has an overshooting with a minimum in absorbance at 10 sec followed by a partial recovery of the radical absorbance (Fig. 4) [25].Moreover, in some cases the severe loss of slow scavenging centers activity was indicated (Fig. 4).The observed overshooting may be explained with the occurrence of reversible cross-combination reactions.Ultrasound application did not influence the phenomenon.
Taking into account that in all the cases the ABTS + neutralization kinetic included two phases, the activity of both rapid and slow reducing centers have been assessed.The activity values for slow reducing ABTS + radical centers did not differ significantly for HPEs obtained in different ways.Here, the application of ultrasound for obtaining the extracts have resulted in lowering of both activity of rapid reducing ABTS + radical centers and total antiradical activity (Table 2).
Відомо, що ультразвук виявляє ефект стерилізації, що є дуже важливим при отриманні фармацев-2.HPE enzymatic activity.Enzymatic antioxidants in particular SOD and catalase are known to have a high specificity of action and to be the main link of antioxidant protection in intracellular medium.Analyzing SOD activity (together with the one of catalase, which inactivates the product of SOD reaction -H 2 O 2 ) the antioxidant status of extracts, tissue or the whole organism may be assessed.The SOD activity value characterizes its ability to utilize a superoxide radical [16,33].
The method which has been used in the research allows determining total SOD activity of the sample.SOD activity values for all the examined HPE did not differ significantly (Table 3).Studying SOD activity in different fractions, obtained using gel-chromatography method, have revealed the major contribution of extracellular SOD-3 (m. m. 132 kDa).
The main catalase function is known to be hydrogen peroxide decomposition [16].The analysis of catalase activity have indicated the significantly lower values of such an activity for the extracts obtained from fresh placenta with ultrasound treatment in comparison with HPE f (Table 4).
Thus placenta low temperature storage did not lead to significant changes of HPE antioxidant properties.Also lowering of ferric reducing activity of the extracts might be associated with cryodestruction of low molecular mass antioxidants during storage.
Ultrasound is known to provide a sterilizing effect, which is very important when obtaining pharmaceuticals.Previously it has been shown that in some cases ultrasound can improve the extraction of intracellular substances from plant material and increase antioxidant activity of samples.Moreover, such parameters as vibration frequency and duration of ultrasonic treatment is of great importance.This effect may be
related to the cavitation phenomenon [4,7], causing the generation of hydrogen atoms and hydroxyl radicals in aqueous solutions, which in their turn can either recombine to form hydrogen and hydrogen peroxide or react with substances in the gas phase on the gasliquid interface or in the solution bulk [23,24].Taking into account all above said it can be assumed that a slight decrease both of non-enzymatic antioxidant activity and catalase activity of HPE and their factions after ultrasound treatment may be associated with the generation of free radicals with their following deactivation by placenta antioxidants.However, it should be mentioned that antioxidant activity in this extracts is kept at high level.The results of the studying the fraction of 12 kDa (HPE f and HPE c+us ) concerning preservation of all the studied antioxidant properties and increase of antiradical activity of rapid reducing ABTS + radical centers is of great importance.We have previously shown that this fraction was the most effective in preventing nitrite-induced oxidative stress in erythrocytes [26].

Conclusions
Placenta storage at -196°C during 6 months did not lead to significant changes in HPE antioxidant activity.When comparing two methods of extracts obtaining it has been shown that an ultrasound treatment has been shown to be effective when preserving both non-enzymatic and enzymatic antioxidant properties of the extracts.The slight decrease in extracts antioxidant activity obtained using ultrasound as well as their fractions may be associated either with the free radicals generation or insufficient extraction time during ultrasound treatment.