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SUpErCrItICaL FLUID tEChnoLoGIES In ManUFaCtUrInG pLant raW MatErIaL DErIVED proDUCtS rudas a.n. 1, komaristaya V.p. Ecobioton LLC, Kharkov V.N. Karazin Kharkov national university, Kharkov Transition to the technologies built upon principles of natural cycles is one of the approaches of improving the state of the environment. For the technologies, which use organic solvents as extraction or reaction media, the alternative of choice is supercritical fluid technologies using carbon dioxide that are widely implemented over the world in the last decades. The implementation of supercritical fluid technologies on the ground of CO2 is especially relevant for manufacturing ingredients of plant origin for food, pharmaceutical, perfumery and cosmetics industry as exploitation of traditional technologies brings to nothing the naturalness of such products declared by their manufacturers owing to unavoidable presence of organic solvent traces in them.

The task of a series of research conducted on the base of Ecobioton LLC the development of the technologies of manufacturing various products from plant raw materials using supercritical CO2.

Supercritical fluid is the state of substance under pressure and temperature above critical. At supercritical pressures and temperatures the margin between liquid and gaseous phases is absent, and the substance acquires properties intermediate between the properties of gas and liquid. For carbon dioxide the critical are the pressure of 73,8 bar and the temperature of 304,1 K. Viscosity and diffusibility of fluids is close to such in gases, and density and dissolving power to such in liquids. That makes supercritical fluids practically ideal for extraction.

Supercritical CO2 is a non-polar solvent. The spectrum of non-polar substances soluble in supercritical CO2 is wider than for any known organic solvent. It can be widened into polar region by adding small quantities of polar co-solvents (modifiers, entrainers), the preferable of which being ethanol on account of its natural origin.

Total extracts of plant materials obtained using supercritical CO2 by their qualitative and quantitative composition 䳿 Karazin natural science studios of natural substances are more rich than extracts obtained by traditional technologies and do not contain foreign admixtures.

Non-soluble for supercritical fluids are only high molecular weight substances: proteins, oligo- and polysaccharides, nucleic acids, other natural polymers. Nevertheless, they can also be obtained via washing out low molecular weight substances with supercritical solvents. In supercritical media the activity of many enzymes is preserved [1]. Combining supercritical extraction with the technology of immobilized enzymes or with the pretreatment of raw material with hydrolytic enzymes natural polymers can be separated in the individual form.

The unique characteristic of supercritical fluids is also that changing pressure and temperature in the supercritical range one can change the solubility of individual substances in them. It makes possible to selectively extract individual substances from raw material as well as selectively precipitate individual compounds from extracts. In supercritical media there are realized such methods of fine purification of substances as supercritical columnar distillation and supercritical preparative chromatography [2]. Thus, using supercritical fluid technologies practically any compound can be extracted from plant material and isolated in the pure form.

Carbon dioxide is cheap, inert, explosion-proof, non-flammable. Supercritical extraction and separation processes are performed at temperature regimes not causing thermal degradation of natural substances, without light and oxidative effect of oxygen. Supercritical media possess sterilizing effect and prevent microbial contamination of products. Supercritical technologies are characterized by low operational costs. The solvents circulate in closed cycle and are re-used. Waste water and toxic emission to the atmosphere are absent. The equipment for supercritical technologies is hermetic, automated and meets GMP standards. In the market there are suppliers who propose this equipment for acceptable prices [3].

In 2010 we have developed the supercritical technologies of extracting the complex of essential oils (from carrot fruits, nutmeg aril), fatty oils (from seeds of peanut, soybean, rape, flax). The research is carried out on other species of starting plant raw material and products obtained.

1. Kamat S.V., Beckman E.J., Russell A.J. Enzyme activity in supercritical fluids // Critical Reviews in Biotechnology. 1995. V.15, N1. P. 41-71.

2. Clifford T. Fundamentals of supercritical fluids. New York: Oxford University Press, 1999. 210 p.

3. . [. ]. 2010. Mode of access: www.ecobioton.su74.ru thE DEtErMInatIon oF antIoxIDant anD antIraDICaL aCtIVIty oF PORPHYRIDIUM CRUENTUM ExtraCtS By non-SpECIFIC MEthoDS Sadovnic Daniela Institute of Microbiology and Biotechnology of Academy of Sciences of Moldova Microalgae and cyanobacteria contain the antioxidant complexes with high activity, that made possible their survival and evolution. Currently, researches confirm the profitability of using algae to produce raw material for obtaining the natural antioxidants [2, 4, 5, 8, 11].

Red alga Porphyridium cruentum contain some components with pronounced antioxidant properties, such as enzymes with antioxidant properties, glutathione complex, phycobiliproteins and carotenoids, sulphated polysaccharides, etc. Each of these components has different mechanisms of antioxidant action and different ways to eliminate free radicals [11,14].

Aim of investigations: determination of antioxidant and anti radical activity of the extracts of Porphyridium cruentum by the various mechanisms of reducing and inhibiting of free radicals production..

Object of study: red microalgae Porphyridium cruentum CNM-AR-01, deposited in National Collection of Non-Pathogenic Microorganisms of Institute of Microbiology and Biotechnology of Academy of Sciences of Moldova.

To obtain water-ethanol extracts from porfiridium biomass, ethanol in concentrations of 10%, 20%, 40%, 55%, 65%, 75% and 96% has been selected. Hydro-ethanolic extracts were obtained under conditions of constant temperature (18-20 0C) and continuous agitation. Ratio biomass: solvent was 1:10, and extraction time-24 hours.

To establish the anti radical antioxidant potential of obtained extracts the usual and non-specific methods of antioxidant activity were adapted and applied. Anti radical activity of the extracts was determined spectrophotometrically using DPPH radical (2,2-diphenil-1-picrilhidrazil) [1] and ABTS (2,2 azinobis 3-ethylbenzothiazoline-6-sulfonic acid) [10]. The antioxidant activity was determined colorimetrically, using the reaction of the phosphomolibden reagent reduction [9]. As an equivalent the ascorbic acid was used. Phenol 䳿 Karazin natural science studios content was determined by the Folin-Ciocalteu reaction [12]. As an equivalent gallic acid was used. Biochemical tests were performed using spectrometer UV / VIS T80 +, PG Instruments LTD.

Thus, determination of the antioxidant activity of porfiridium hydro-ethanolic extracts using the phosphomolibdenic reagent, allows to establish the active structural components and to participate in oxidationreduction reactions as a reductant. According to the obtained results, antioxidant activity of the investigated hydroethanolic extracts range from 1.06 mmol to 8.29 mmol of ascorbic acid / mg active substance. For determination of ability of extracted active components to participate in the radicals annihilation reactions by hydrogen donor anti radical mechanism, radical DPPH was used. Test results demonstarted anti radical capacity of ethanolic extracts with ethanol concentration of 10-20% to reduce DPPH radical to 40%. Extracts of 55% and 70% ethanol concentration reduced DPPH radical by approximately 20% and 40% extracts, 65% and 96% ethanol have anti radical activity from 10% to 13% inhibition. The coefficient of correlation between anti radical activity of extracts and the ethanol concentration is rather low R2 = 0.5417. The decrease of anti radical activity has occurred suddenly, with 70% inhibition activity at the level of 40% ethanol extract. Therefore, Porphyridium biomass extracts have the ability to reduce free radicals and anti radical activity of components of the extracts with the lowest concentration of ethanol.

One of the most common methods for determination of the anti radical activity is the reaction of radical cation ABTS + reduction. For extracts from Porphyridium biomass, which present complex of bioactive substances can be important to determine the ability to participate in the annihilation of free radicals reactions by reduction of reactions mechanism (electron donor). The test is specific for both lipid soluble components and for the watersoluble. High anti radical activity of all extracts against ABTS + radical was determined. 10% ethanol extract inhibits 65% of the radical ABTS + and 20% alcohol extract produced 45% inhibition. And again, 40% ethanol extract is less active with the anti radical effect only 33% inhibition. Extracts of 55% and 70% ethanol have similar anti radical activity of 40% inhibition. Thus, extracts of Porphyridium biomass have the ability to reduce free radicals by hydrogen transfer mechanism and electron transfer and antioxidant active ingredients of the extracts with ethanol minimum concentration.

Determination of ABTS + radical reduction enables the establishing of TEAC (Trolox equivalent antioxidant capacity) coefficient that is used in comparing of the antioxidant activity of different types of vegetal biomass extracts with the omission of the solvent nature. As a blank antioxidant water-soluble Trolox-tocopherol was tested. Thus, calculations demonstrated that TEAC coefficient determined for Porphyridium cruentum is closer for TEAC set for some plants [3], indicating higher values for extracts with low concentrations of alcohol.

The content of extracted phenols was determined The method of reducing of the Folin-Ciocalteu reagent was used. It was established that phenol concentration decreases with increasing ethanol concentration. In principle, Folin-Ciocalteu reagent is not specific only to phenolic components; it may be reduced by some components such as protein, ascorbic acid, etc. With higher concentrations of ethanol some phenolic components which have high antioxidant activity can be extracted.

If we compare the method of determination of the content of phenols as a method to determine the ability of reducing Folin-Cioaclteu reagent capacity test method for reducing ABTS radical assay (TEAC), we get a real dependence with correlation coefficient R2 = 0, 9563. High correlation coefficient allows to introduce this method in the list of usual methods, simple to use and adapted to the determination of antioxidant activity of obtained extracts [6]. Thus, ethanolic extracts in concentration 10-10% have the greatest capacity to reduce the FolinCiocalteu reagent, which is of 5-6 mmol gallic acid active substance. Antioxidant activity decreases in ethanolic extracts of 40% - 96%, a minimum value is 2.89 mmol gallic acid / mg active substance in 96% ethanolic extract.

Therefore, the capacity of the components of ethanolic extracts from Porphyridium cruentum biomass to participate in oxidation-reduction reaction mechanism of electron donor was established by the used methods, except the method of determination of antioxidant activity by phosphomolobdenic reagent reducing.

In conclusion, Porphyridium cruentum can be used as a source of substances with antioxidative and anti radical activity and ethanol in different concentrations is the optimal solvent for extracting of antioxidant components from algae biomass. Non-specific methods for determination of anti radical activity are informational and representative for Porphyridium biomass ethanol extracts. The method of determination of phenols can be used as a method for determination of the antioxidative activity in the reaction of Folin-Ciocalteu reagent reduction.

Brand-WilliamsW., Cuvelier M.E., Berset C. Use of a Free Radical Method to Evaluate Antioxidant Activity. In: Lebensmittel-Wissenschaft und Technologie. Food Science and Technology. 1995, vol. 28, p. 25-30.

Cepoi L., Rudi L., Miscu V., Cojocari A., Chiriac T., Sadovnic D. Antioxidative activity of ethanol extracts from Spirulina platensis and Nostoc linckia measured bz various method. In: Analele Universitii din Oradea.

2009, vol. XVI, no.2, p. 43-48.

䳿 Karazin natural science studios Craciunescu O., Buzgariu W., Buiculescu R., Coroiu V., Moldovan L. Evaluation of radioprotective capacity of green tea (Camellia sinensis) polyphenols. In: Romanian Biological Sciences. 2005, vol. 3-4, p. 20-27.

Herrero M., Martin-Alvarez P.J., Senrans F.J., Cifuentes A., Ibaez E. Optimization of accelerated solvent extraction of antioxidants from Spirulina platensis microalga. In: Food Chemistry. 2005, vol. 93, p. 417423.

Hua-Bin Li, Ka-Wing Cheng, Chi-Chun Wong, King-Wai Fan, Feng Chen, Yue Jiang. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. In: Food Chemistry.

2007, vol. 102, p. 771776.

Magalhes L.M., Segundo M.A., Reis S., Lima,Jos L.F.C. Methodological aspects about in vitro evaluation of antioxidant properties. In: Analytica Chimica Acta. 2008, vol. 613, p. 1-19.

Mao Lin-Chun, Xin Pan, Fei Que, Xue-Hua Fang. Antioxidant properties of water ant ethanol extracts from hot air-dried and freeze-dried daylily flowers. In: European Food Research and Technology. 2006, vol. 222, p. 236-241.

Plaza M., Cifuentes A., Ibez E. In the search of new functional food ingredients from algae. In: Trends in Food Science & Technology. 2008, vol. 19, p. 31-39.

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 E1. In:

Analytical Biochemistry. 1999, vol. 269, p. 337341.

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