Operational Conditions Effects on Extraction Yield of Antioxidants from Iranian Rosemary Plant ZARRIN NASRI Faculty Member of Chemical Technologies Department Iranian Research Organization for Science and Technology No.71, Forsat st. Enghelab Av.,Tehran IRAN zn_nasri@yahoo.com, nasri@irost.org, www.irost.org Abstract: - A member of the Lamiaceae family, rosemary (Rosmarinus Officinalis) plant is wildly used in many food applications due to its antioxidants activity. Antioxidants are added to foods, cosmetics and medicines in low amounts to slow down oxidation process. The antioxidants can be divided into synthetic or natural origin. Studies show that synthetic antioxidants such as BHT and BHA have undesirable effects on human health. So, interest in using the antioxidants from natural sources has been increasing. The main components of rosemary antioxidants are phenolic diterpenes and phenolic acids in which carnosol, carnosic acid, and rosmarinic acid have the most antioxidant activities. Rosemary extracts are available in both water soluble and oil soluble products, also in liquid and powder forms. In this paper the operational conditions influences such as solvent concentration, extraction temperature, extraction time on extraction yield of antioxidants from Iranian rosemary plant is experimentally investigated. The results show that amount of total extract and total phenolic compounds in the extracts increased significantly with increasing temperature. An increase of total extract and also total phenolic contents is observed with extraction time and steady state of process has been obtained after 12 min. Influence of ethanol with different concentrations on total extract and total phenolic contents is investigated. According to the results an ethanol concentration of % showed the highest activity. Key-Words: - Extraction, Rosemary, Rosmarinus Officinalis, Antioxidants 1 Introduction In recent years, an increasing interest in using natural antioxidants such as tocopherols, flavonoids and rosemary (Rosmarinus Officinalis) extracts for preserving foods has been observed, because natural antioxidants avoid the toxicity problems which applying synthetic antioxidants, such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT) causes [1-4]. Natural antioxidants inhibit oxidation of fats by scavenging free radicals. Fig.1 shows the most active compounds of rosemary plant including rosmarinic acid, carnosol, and carnosic acid. Bailey et al. (1999) used a process in which they applied this fact that carnosic acid has poor solubility in water, however, some its salts are significantly more water soluble. In their work rosemary plant extracts are obtained by a water soluble solvent such as ethanol and acetone [5]. Thorsen et al. (23) studied the challenges related to precise identification of carnosic acid, carnosol, and the other phenolic diterpenes in extracts of rosemary plant. Stability of carnosic acid and carnosol dissolved in methanol, dimethyl solfoxide, dimethyl solfoxide: acetonitrile (1:9), acetonitrile:ethylacetate (9:1) is investigated. The results showed that carnosic acid dissolved in dimethyl solfoxide was stable for several days, but carnosol was stable only for a few hours in all solvents [6]. Del Bano et al. (23) studied distribution of six polyphenolic compounds in different parts of rosemary plant [7]. Terpine et al. (29) studied the antioxidants properties of phenolic diterpenes in several rosemary extracts [8]. Hernandez et al. (29) investigated the extracts of rosemary and oregano plants and their effects on oxidation and color of pork batters. The extracts are obtained using ethanol and chloroform as solvents. Their results showed that rosemary extracts have higher antioxidant activity [9]. Weerakkody et al. (21) studied antimicrobial activity of some extracts from ISBN: 978-1-614-137-1
four plants including rosemary using water, ethanol, and hexane as solvents. The results show that solvent type remarkably affects antimicrobial activities of plant extracts [1]. 2 Materials and Methods Rosemary plant used in this paper has been provided from Agricultural Jahad Organization, Hamedan, Iran. The plant was stored at a dark and cold place until use. The plant was ground using a mill to be able to pass through a sieve with mesh size of.5 mm. All chemicals used in this study were grade analytical. rosmarinic acid carnosol 2.1 Extraction Method In this research the conventional solid liquid extraction at atmospheric pressure has been carried out. Ethanol is a very effective and harmless solvent for extraction of effective components from medicinal plants. Due to these facts, rosemary plant was extracted with ethanol as a solvent in this study. The solvent including different concentration of aqueous ethanol is used. The solvent is added to samples of rosemary plant in a glass container with tight lid. The extraction is performed in a shakerincubator at 2 rpm. After extraction process the extract was filtered. Two samples from the extracts have been obtained to determine total extract yield. The remainder extract was concentrated by vacuum rotary evaporator and was dried in oven until reaching constant weight. The dried extracts were maintained at -4 C until analyses. The extraction procedure was conducted two times and all of the extracts were analyzed twice. carnosic acid Fig.1 Most active compounds of rosemary plant Zhang et al. (21) studied antioxidative stability of sunflower oil supplemented with carnosic acid in comparison with synthetic antioxidants. Their results showed that carnosic acid exhibited more antioxidant activity in comparison with BHA and BHT [11]. Hossain et al. (211) optimized the operational conditions of three plants including rosemary extraction using accelerated solvent extraction method to maximize the antioxidant activity of the extracts [12]. In this paper the effects of process conditions such as solvent concentration, extraction temperature, extraction time on extraction yield of antioxidants from Iranian rosemary plant for the first time is experimentally investigated. 2.2 Determination of Total Phenolics Folin-Ciocalteau (FC) colorimetry has been used to determine total phenolic contents of extracts. It is based on required sample to inhibit oxidation of the reagent. FC method has been adopted as a standard procedure for total phenolic contents in all references. The products of chemical reduction of the reagent exhibit a blue colour which has a wide absorbance in 765 nm. The intensity of absorbance is proportional to phenolic concentration. In this research the FC method described by Waterhouse (22) with some modification is used [13]. The reaction mixture contained 1 ml of diluted rosemary extract and 5 ml Folin-Ciocalteu reagent has been prepared. The mixture after mixing was kept at ambient temperature for 1 to 8 min. Then 15 ml 2 % sodium carbonate solution was added. The final mixture was diluted to ml deionized water. The mixture was kept in dark at ambient temperature for 2 hr to complete reaction. Then the absorbance at 765 nm was measured on a UV-VIS ISBN: 978-1-614-137-1 81
spectrophotometer (Perkin Elmer precisely, Lambda). 3 Results and Discussion Antioxidant activity of plants is proportional to phenolic contents, so the plants that have more phenolic components have higher antioxidants activity. In this paper the criteria is determination of phenolic contents. The study shows that main antioxidant effect of rosemary extract is related to phenolic diterpenes such as carnosol, carnosic acid and methyl carnosate and phenolic acids such as rosmarinic and caffeic acids. This is important that antioxidant activity of plant extracts containing more than one antioxidant component compares with antioxidant activity of standard antioxidants to determine interaction between the antioxidants. The standard antioxidant used in this research is Gallic acid. 3.1 Calculation of Yields The total extract yield was calculated by the following formula. mextract TE = m extract,max (1) where TE Yield is total extract yield, m extract is the mass of dry extract of the sample, and m extract,max is the maximum mass of dry extract obtainable from the plant. Also, the total phenolic yield of the extracts was calculated from the following formula. TPextract TP = TP extract,max (2) where TP Yield is total phenolic content yield, TP extract is total phenolic contents of the sample and TP extract,max is the maximum phenolic components obtainable from the plant. 3.2 Determination of Antioxidants Content of Rosemary Plant In order to determine the antioxidants content of the plant, mg of rosemary plant were mixed into ml aliquots of ethanol:methanol:2-propanol (9:5:5 (v/v)) and extracted in an ultrasonic bath for 1 h. The result showed that total antioxidant of used rosemary plant is 166.7 mggae /gr,dry plant. 3.3 Effect of Solvent Concentration In order to determine the effect of solvent concentration on the yields of extraction, experiments have been performed in various solvent concentrations. The other process conditions such as solvent to plant ratio, 2 cc/gr, extraction time, 12 min, shaking speed, 2 rpm, and temperature, 7 C were constant in all experiments. Fig.2 shows the effect of solvent concentration on two yields. The results show that optimal total phenolic contents and total extract has been obtained by extraction with ethanol %. The results show the same trend for two yields. The curves might be fitted with the following equations. TE = 1E-7 (SC) 5-3E-5 (SC) 4 +.27 (SC) 3 -.68 (SC) 2 +.784 (SC) + 45.229 TP = -3E-6 (SC) 4 +.4 (SC) 3 -.63 (SC) 2 -.862 (SC) + 17.4 In these equations SC is solvent concentration. 9 7 5 3 2 1 R 2 =.954 R 2 =.8328 2 Ethanol Concentration (%) Fig.2 Effect of solvent concentration on total phenolic contents and total extract yields TE Yield (%) TP Yield (%) Poly. (TP ) Poly. (TE ) 3.4 Effect of Temperature In order to determine influence of temperature on the yields of extraction, experiments have been performed in various temperatures. The other process conditions of experiments including solvent to plant ratio, 2 gr/cc, solvent concentration, % (optimal conditions), extraction time, 12 min, shaking speed, 2 rpm have been constant in all experiments. Fig.3 depicts the effect of temperature on two yields. It can be observed that increasing temperature increases both yields significantly. The results suggest the same trend for two yields. Because the mass transfer rate of active components depends on their solubility in the solvent, heating can increase the mass transfer rate. Also, increasing temperature causes decrease in solvent viscosity and ISBN: 978-1-614-137-1 82
therefore increase different rate. The curves might be fitted with the following equations. TE =.44 (T) 2-3.637 (T) + 66.874 (R 2 =1) extract and total phenolic compounds in the extract. A steady state of process has been obtained after 12 min and an ethanol concentration of % is the best concentration for extraction antioxidants from Iranian rosemary. TE =.29 (T) 2-1.8653 (T) + 37.582 (R 2 =1) In these equations T is temperature (C). 9 7 5 3 2 1 R 2 = 1 R 2 = 1 3 5 7 Temperature (C) TE TP Poly. (TE ) Poly. (TP ) Fig.3 Effect of temperature on total phenolic contents and total extract yields 3.5. Effect of Extraction Time In order to determine the effect of extraction time on the yields of extraction, experiments have been carried out in various extraction time. The other process conditions such as solvent to plant ratio, 2 cc/gr, solvent concentration, % (optimal conditions), temperature, 7 C (optimal conditions), and shaking speed, 2 rpm were constant in all experiments. Fig.4 shows the effect of extraction time on two yields. The results show that two periods with different mechanisms can be identified. Firstly, the extraction rate is high, leads to fast increase in antioxidants extraction. Then mass transfer rate decreases, so the curve is parallel to the abscissa. At the first period, due to fast mass transfer, the large part of phenolic contents has been extracted. The results show the same trend for two yields. The steady state condition has been obtained after 12 min. 4. Conclusion In this paper the effect of process conditions including solvent concentration, extraction temperature, extraction time on extraction yield of antioxidants from Iranian rosemary plant is investigated. The results showed that increasing temperature leads to increase in extraction of total 2 2 3 Time (min) TE TP Fig.4 Effect of extraction time on total phenolic contents and total extract yields References: [1] R. Bruni, M. Muzzoli, M. Ballero, M. C. Loi, G. Fantin, and F. Poli, Tocopherols, Fatty Acids and Sterols in Seeds of Four Sardinian wild Euphorbia Species, Fitoterapia, vol. 75, 24, pp. 5 61. [2] M. J. Frutos and J. A. Hernandez-Herrero, Effects of Rosemary Extract (Rosmarinus Officinalis) on the Stability of Bread with an Oil, Garlic and Parsley Dressing, Lebensmittel- Wissenschaftund Technologic, vol. 38, 25, pp. 651 655. [3] A. R. Hras, M. Hadolin, Z. Knez, and D. Bauman, Comparison of Antioxidative and Synergistic Effects of Rosemary Extract with A-tocopherol, Ascorbyl Palmitate and Citric Acid in Sunflower Oil, Food Chemistry, vol. 71, 2, pp. 229 233. [4] R. J. Williams, J. P. E. Spencer, and C. Rice- Evans, Antioxidants or Signalling Molecules? Free Radical Biology and Medicine, Flavonoids, vol. 36, 24, pp. 838-849. [5] D. T. Bailey, S. L. Richheimer, V. R. Bank, and B. T. King, High Purity Carnosic Acid from Rosemary and Sage Extracts by PH- Controlled Prediction, US5859293A, Jan. 12, 1999, pp. 1-8. [6] M. A. Thorsen and K. S. Hildebrandt, Quantitative Determination of Phenolic Diterpenes in Rosemary Extracts Aspects of Accurate Quantification, Journal of ISBN: 978-1-614-137-1 83
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