Comparative response of essential oil composition, antioxidant activity and phenolic contents spearmint (mentha spicata L.) under protected soilless vs. open field conditions.
Spearmint (Mentha spicata L.), is one of the most important aromatic herbal plants and cultivated for widely use in food, cosmetic, confectionary, chewing gum, toothpaste, pharmaceutical industries and for essential oil productions. Spearmint is one of most important flavour in the world that coming after vanilla and citrus flavours [31,30,48,45]. Spearmint is used popularly as tea flavouring agent as well as used as fresh and dried for folk medicine such as stimulant and carminative. Spearmint contains a complex mixture of bioactive compounds covering a number of demands for human health and exhibiting different biological properties and activities [15,6].
The essential oil of M. spicata showed strong insecticidal and mutagenic activity . Essential oil is extracted either from freshly harvested mint leaves or from semidried or dried leaves through distillation process for industrial applications.
The essential oil content of dried leaves of spearmint was reported to be approximately (2.5%). Essential oil are mostly menthol (50%) and menthone (10 to 30%) were found to be the major components of essential oil. Spearmint oil contains monoterpenoids like carvone, limonene, menthone, menthol, pulegone, dihydrocarveol and s-carvone. Some of them were found to possess high antioxidant activity . Essential oil compositions of aromatic plants depend on their genetic structure, the climatic factors and the agronomical practices [20, 40,44]. Several factors influence the chemical composition of plant essential oils, including the species, part of the plant, season of harvesting, geographical origin and extraction method [6, 49, 27].
In recent years, there has been growing interest in finding natural antioxidants, phenols as well as flavonoids in extraction oils of spearmint plants because they inhibit oxidative damage and may consequently prevent inflammatory conditions and their important role in the prevention of various degenerative diseases  ageing and neurodegenerative disease . The antioxidants activity of plant extracts are widely used in the food industry as potential inhibitors of lipid peroxidation, as additives in food and cosmetics which become important and interesting object of research because of the increasing usage of natural antioxidants[41,8,23]. These properties provide the basis for many applications in raw and processed food preservation, pharmaceutical products, alternative medicine and natural therapies . Therefore, antioxidants are considered an important nutraceuticals have many health benefits. Phenolic compounds, also known as polyphenol antioxidants, which include flavonoids and phenolic acids, are found naturally in spearmint plants. Phenolic compounds inhibit lipid peroxidation, scavenge free radicals, chelate iron and copper ions. Phenolic compounds exhibit a range of biological activities, including anticancer, antibacterial, antioxidant and antiinflammatory properties .
The leaves of aromatic plants are often dried before extraction to reduce moisture content. During this process, many compounds, which are dragged to the leaf surface by the evaporating water, are lost . Drying may introduce undesirable changes in appearance, texture, flavour and colour that are not in agreement with the increasing demand of consumers for the highest-quality finished product. As many other food process, drying needs to be optimized with respect to process economics and product quality which has led to the development of new drying techniques. The method of drying usually has a significant effect on the quality and quantity of the essential oils from plants . Spearmint belongs to the Lamiaceae family of plants, which is known to store their essential oils on or near the leaf surfaces . One of the preservation methods ensuring microbial safety of biological products is drying. The contact of dried material with hot air causes degradation of important flavor compounds and nutritional substances as well as color alteration .
To our knowledge, few investigations have been made to the chemical constituents of essential oils for spearmint leaves under soilless culture, although this cultivation method has more yield than soil based conditions. Therefore, the first aim of this study, were to evaluate the effects of cultivation method as well as environmental conditions. In addition, few reports were investigated about nutritional, antioxidant, total phenols, flavonoids of spearmint cultivated under soilless culture conditions. The second aims of this study was to evaluated the total flavonoid content, total phenols of spearmint and antioxidant activity under soil-based and soilless culture conditions as well as methanol extracts, and air drying methods.
Materials And Methods
This study was carried out in Jordan University of Science and Technology (JUST) campus during the growing season 2010-2011 in the open field and protected soilless conditions.
2.1. Experiment 1:
Seedling of spearmint (Mentha spicata L.) was cultivated in woods beds (120*110*25) in cm (W*L*D), filled with tuff zeolite (xxx0 3-8mm) under protected soilless conditions. The irrigation water and nutrients were delivered to the plants via drip irrigation twice a day for 15 minute (early morning and evening). The nutrient solutions were prepared manually once per two week. The concentration of ions was used in preparation nutrient solution expressed as (mg/L): N: 360, P: 2, K: 283, Ca: 302, Mg: 48, S: 64, Fe: 2.76, Mn: 0.974, B: 0.536, Zn: 0.3, Cu: 0.076, Mo: 0.155.
2.2. Experiment 2:
Seedling of spearmint was transplanted in concrete blocks (95*100*75) in cm (W*L*D) filled with soil mix with peatmoss under open field conditions (soil-based). Peat moss was applied at 33L/[m.sup.2] at the top layer of soil (20cm) to improve for water holding capacity of soil. Inorganic NPK fertilizer has formula [N.sub.2], [P.sub.2][O.sub.5], and [K.sub.2]O with (20:20:20) ratio was applied at the rate 30 g/[m.sup.2] at the planting time. After each harvest of spearmint plants, 25g/[m.sup.2] of NPK fertilizer were added. In addition, 6 g/[m.sup.2] nitrogen fertilizer was added in the form of urea (46%N) after each harvest.
2.3. Preparation method for analysis:
2.3.1. Drying methods of aerial parts:
Drying is the most important step in the preparation of mint leaves for extraction and isolation essential oil by different methods. The samples from spearmint plants under soilless cultivation condition were collected from five harvesting times and the samples from soil-based conditions were collected from two harvesting times. The samples were dried at room temperature for 10days. As many other food process, drying needs to be optimized with respect to process economics and product quality which has led to the development of new drying techniques. Drying may introduce undesirable changes in appearance, texture, flavour and colour that are not in agreement with the increasing demand of consumers for the highestquality finished product.
2.3.2. Isolation of essential oil:
The aerial parts of M. spicata plants were sampled and air-dried at room temperature for 10 days. 100 g dry weight (DW) samples were grossly pulverized and essential oils were isolated by steam distillation for 3 h. Extraction of essential oil dried with anhydrous sodium sulfate and stored in sealed amber flasks at 4[degrees]C until analysis The yield of essential oils is expressed as % (mL 100/ g DW). All experiments were conducted in triplicates and results were expressed based on dry matter weight.
2.3.3 Gas Chromatography Analysis (GC):
The isolated oils were dilution with hexane (C6H12), and sample was injection into the gas Chromatographic analysis. The constituents of essential oils of M. spicata were identified. Four components were analyzed from essential oils of spearmint plants including; menthone, menthol, pulegone and 1, 8-cineole.. Identification of aromatic compounds was based on the calculation of their concentration in ([micro]g /g).
2.3.4. Preparation of the methanolic extracts:
For the preparation of the methanolic extracts, each 0.5grams (three replicates) of dried leaves of M. spicata sample was weighed out, and extracted with 50ml of methanol. Extraction was carried out under shaker at overnight at 30[degrees]C. Each extract was filtered into a 50 ml volumetric flask using whatman filter paper No 42. Volume were completed to mark, and allowed to set in the dark until analysis.
2.4. Determination of total phenolic content:
The total phenolic content of the solvent extracts was determined by the method using Folin-Ciocalteu reagent and Gallic acid as standard to produce the calibration curve . 2000 [micro]l of the plant extract (triplicate) were transferred into a test tube, and then mixed with 2.5ml of 10% Folin-Ciocalteu reagent. After 3 minutes for allowing the reaction to take place, 2000 [micro]l of a 10 sodium carbonate ([Na.sub.2]C[O.sub.3]) was added. The tube were allowed to stand for 1hour at ambient temperature, and the absorption was measured at 760 nm using UV-VIS spectrophotometer (model SpectroScan 50) against a blank, with contained 50ul of menthol in place of sample. Gallic acid was used as calibration standard, and results were calculated as Gallic acid equivalent (GAE mg/g dry weight basis).
2.5. Determination of total flavonoids:
The total flavonoid content of the extract was determined by the method described in the literature . The total flavonoid contents of spearmint plants under soilless culture and soil conditions were determined. Each sample (0.5ml) of the plant extract (three replicate) were transferred into 150[micro]l of a NaN[O.sub.2] solution (15%). After 6 minutes to allow the reaction to take place, 150 [micro]l of an Aluminum chloride (Al[Cl.sub.3]) solution (10 %) was added and allowed to stand for 6 minutes, then 2000ul of Sodium hydroxide (NaOH) solution (4%) was added to mixture 0.2ml of distilled water to bring volume to 5 ml and the mixture was thoroughly mixed and allowed to stand for another 15 minutes. The tubes were allowed to stand for 1 hour at ambient temperature, and absorption was measured at 510nm using spectrophotometer against a blank which contain 50 ml of menthol in place sample. Catechin was used as calibration standard with different concentrations 10, 30, 80, 100, 150, 200, 300, 400 and 500mg/l were tested to obtain standard curve; results were expressed as Catechin equivalents (mg Catechin/g dried extract).
2.6. Evaluation of antioxidant (antiradical) activity:
Antioxidant activity was determined according to . Briefly, Free radical 2, 2-diphenyl-lpicrylhydrazyl (DPPH) scavenging effect was determined. The DPPH were soluble in menthol. Fresh DPPH stock solution (25ml) was prepared daily. The solution was prepared by weighing 50mg/100 ml, which represent the amounts we need, then 0.0125g of DPPH was dissolved in 25 ml methanol, which resulted in purple color solution. The mixture was mixed thoroughly and allowed to stand in the dark for 60 minutes. Absorbance at zero time (Ato) at 517nm wavelength was determined. Absorbance then was read at 517 nm, against the blank. The percentage inhibition of DPPH free radical was calculated by the formula:
Percentage inhibition (%) = [(A blank- A sample)/ A blank]* 100
Where, A blank is the absorbance of control reaction (DPPH alone) and A sample is the absorbance of DPPH solution in the presence of the test compound. IC 50 values denote the concentration of the sample, required to scavenge % of DPPH free radicals. Extract concentration providing 50% inhibition (IC50) was calculated from the plot of inhibition percentage against extract concentration. All determinations were carried out in triplicate and the results were averaged
2.7. Statistical analysis:
All Data were statistically analyzed using analysis of variance (ANOVA) according to the statistical package MSTAT-C (Michigan State Univ., East Lansing, MI, USA). Probabilities of significance among treatments and LSD (P [less than or equal to] 0.05) were used to compare means among treatments.
3.1. Composition of essential Oils:
The yields of essential oils for spearmint plant grown under protected soilless and open field conditions were identified relatively to the amount of dried herbs used. The chemical composition of the oils is reported in Table 1 according to their concentration in ([micro]g/g). Results obtained showed that essential oil percentage percentage (w/w) was noticed under protected soilless conditions (3.43%) was higher than that obtained under open field conditions (3.14%).
Essential oils consist of complex chemical mixtures that vary widely in chemical composition. In essential oil of spearmint plant, the main components were 1,8 cineole and pulegone Table 1. It can be conclude from Table 1 that menthone, pulegone and menthole concentrations were not significantly. The chemical composition of spearmint plant is characterized by the presence of oxygenated monoterpenes such as menthol and 1,8cineole.  reported the menthol concentration and other chemical compositions of different mint origin varied in contents. Spearmint oils is rich in 1, 8 cineole and pulegone according to our results in Table 1 shows the 1,8 cineole (32.9[micro]g/g) was significantly high than that under soilless cultivation conditions with value(10.6 [micro]g /g). Pulegone concentration under soilless cultivation was (44.3 [micro]g /g) which higher than that under soil cultivation conditions (20.7 [micro]g /g) and significant. Results in this study differs from those obtained by  who studied the oil composition of peppermint of the same specie sample in Turkey, in which (+)Menthol, menthol, neo-menthol, cineole was revealed to be dominant.
Essential oil yield is a product of biomass yield and essential oil concentration in the plants. The essential oil concentration was not influenced by cultivation methods Table 1. In this investigation, essential oil yield and content for aromatic compounds in both cultivation methods was mainly influenced by biomass yield, therefore, the trends were identical for both content of chemical composition and essential oil yields as in Table 2. The maximum essential oil yield was (64.82 ml/kg) under protected soilless condition which significantly high than that under open field condition (32.65 ml/kg) depending on dry matter of spearmint. Content of chemical composition was listed in Table 2 of their contents in mg/[m.sup.2]. According to Table 2, menthone, pulegone, 1,8 cineole and menthole contents were significantly high under protected soilless condition than that under open field condition.
3.1. Total Phenols:
The quantitative estimation of the phytochemicals constituents of spearmint show that spearmint plants were rich in total phenols and total flavonoids according to their data shown in Table 3. Total phenols content of methanol extracts of leaf for spearmint plants was evaluated using the Folin-Ciocalteu reagent and expressed as mg Gallic Acid equivalent in milligrams per gram (GAE/g). The results revealed great variation that evaluation of total phenols content of plant essential oils under two cultivation methods, methanol extracts, and air drying method. In the spearmint essential oil, a high content of total phenols (223 mg GAE/g) was obtained under open field conditions which significantly highest than that obtained under protected soilless conditions (114 mg GAE/g). It has been suggested, total phenolic content of plant materials is correlated with their antioxidant activity and total flavonoids content. It is considered that the total phenolic content is due to their high redox potentials, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers .
3.2. Total Flavonoids:
Total flavonoids concentration of methanol extracts of spearmint leaves were evaluated using the AlCl3 reagent and expressed as mg Catechin Equivalent (mg CE/g of dry weight) under two cultivation conditions (soil vs. soilless), methanol extracts, and air drying methods. The presence of total flavonoids in leaf parts of spearmint plant under soil-based cultivations conditions was varying from (149 mg CE/g) and (73 mg CE/g) under soilless cultivation conditions as shown in Table 3.
3.3. Antioxidant Activity:
Antioxidant capacity in spearmint leaves was summarized in Table 3. The antiradical (1/antioxidant) values varied highly significantly in leaf under soil-based cultivation conditions than soilless cultivation conditions. Antiradical of leaf under soil -based cultivation conditions was (8 mg/g) which are highly significant than antiradical of leaf under soilless cultivation condition (3 mg/g).
Essential oil of spearmint plant was used extensively in the food, flavor, and pharmaceutical industrial. The analyses of chemical composition of essential oil revealed the richness and diversity of spearmint essential oils. Pulegone was found as a predominant component in all the essential oils studied, together with 1,8cineole for M. spicata under protected soilless and soil-based conditions in this study. Few of literature about close examination of the chemical composition of essential oil for spearmint plants that reveals composition under protected soilless and open field conditions and the variation for major compounds such pulegone and 1,8cineole quantitative. However, for spearmint plant, a significant quantitative variation of some chemical constituents was observed between oils from both cultivation methods. Oil obtained from cultivated spearmint plant under soilless, was observed a decrease in the content of 1,8cineole than that under open field conditions. Essential oil extraction from spearmint plants under soilless condition was collected from five harvesting times, but under open filed condition was collected from two harvesting times during growing season before blooming stage. Also, the chemical constituents of essential oils was varied under soilless conditions at greenhouse and under soil -based conditions at open field and this explained the variation between concentration of chemical constituents under both cultivation methods due to different environmental conditions. A result comparable with  that was reported the different climatic conditions in the locations affected the quantity of monoterpenes and sesquiterpenes hydrocarbons, contrary to oxygenated monoterpenes. Comparison between our results and the results of other reports showed variations in oil content and oil composition, that could be attributed due to the cultivation methods, environmental conditions including temperature, relative humidity, irradiance and photoperiod, as well as the time of harvesting, stage of harvesting and height of cutting plants. In addition, the components of spearmint oil vary slightly from year to year.
According to , the major compound of M. spicata produced in Greece were betapinene, myrcene 1,8 cineole, dihydrocarvone and carvone. In addition,  reported that, the pulegone, menthofuran and menthyl acetate amounts were lower in the oils of all mints. Essential oils for spearmint under soilless and soil cultivation methods showed richness in pulegone and 1,8cineole, which are the major compounds. Pulegone are usually found in high amounts in flowers  but in our study, they are found in high concentration in leaves. In addition to variation in essential oils concentration from different part of plant, the different geographical origin shows variation in essential oils contents, which the major component was menthol, menthyl acetate and menthone in Serbia by . In other study in Italy, the main components were menthol, menthyl acetate and menthonfuran for spearmint plants. In addition,  reported that menthanol and menthone, which are the major compounds of the essential oils, contents for spearmint from Iran.  Reported that the main component pulegone and other oxygenated monoterpene, 1,8-cineole also showed variation in the different ecologies. Pulegone is also the major component in some wild Mentha species, which is similar to other oxygenated monoterpenes. It is biologically active and can act as an antifeedant .
There are some reports about the chemical composition for spearmint plants that reported, essential oil compositions of aromatic plants depend on their the climatic factors, genetic structure and the agronomical practices [40,44,20].  Also, reported about factors that influence on essential oils quantity and quality like the most leaf area, size of epidermal cells and number of oil glands per unit area which was the most important factors that responsible for higher essential oil contents of plant and biomass yield. All reported studies related to essential oils composition of genus Mentha in general was under soil conditions and few studies were conducted about the chemical composition of essential oils under protected soilless conditions. M. spicata has numerous chemical races throughout the world [31,46]. Pulegone, an oxygenated monoterpene, is the main component of mint oil produced from wild plants. Pulegone-rich mint oil has been used as emmenagogue and abortifacient in folk medicine .  Stated pulegone-rich chemotypes in wild spearmint originated from Turkey. In this study, Jordanian spearmint was rich in pulegone and 1,8 cineole and it is attributable to a new chemotypes for Jordanian mint. Dried product quality entirely depends on different unit operations involved in drying process. Drying process should be undertaken in closed equipment to improve the quality of the product . Drying methods, the other reasons for variation in chemical compositions of essential oil of these compounds, due to the influence by drying methods. Air -dry method was suitable for samples that collected from spearmint plants under open field conditions due to low moisture content, which is lower in comparison to that samples which collected under soilless condition which that have higher moisture content. During air-dry method at room temperature 30[degrees]C, dark brown yellow Splatter was appeared on leaves were collected under soilless. Moreover, under soil condition no Splatter was appeared on and still green color. Leaves drying process for agricultural products correlates moisture content of the material at exposure to a constant relative humidity and temperature condition and drying parameters [34,47].
Phyto-chemicals are known to exhibit several health beneficial activities such as antioxidant, antiinflammatory, antitumor, and antimicrobial [25, 39, 33]. Spearmint plant under protected soilless conditions have a high moisture content with ranged (74.60 to 80.03 moisture %) than that spearmint under soil-based cultivation and the moisture content (56.25 to 68.68 of moisture %). Drying might have accelerated more bound phenolic compounds releasing from the breakdown of cellular constituents [11, 12]. However, the air-drying temperature method might be the reason for the lowest phenolic, flavonods contents, and antioxidant activity of spearmint under soilless culture conditions. Spearmint under soilless culture conditions has more moisture content. The effects of drying process on the phenolic substances of foods were studied before. Some of them have reported that phenolics decreases [3, 50]. Total phenolic content for spearmint was significantly higher under open field condition than that under protected soilless conditions. In addition, Total flavonoids of spearmint plants under soil cultivation were higher than under soilless cultivation but not significant. Spearmint have a great attention is given to research of the quantity and activity of antioxidants in leaves part; Antioxidants are biologically active substances, which remove excessive free radicals, decreasing the lipid oxidation . Antioxidant activity for spearmint plants was highly significant under open field conditions than that under protected soilless conditions.  Reported that the M. spicata oil exhibited radical scavenging activity with the IC50 as 87.89 [micro]g/ml. The air-drying methods was effaced on biological activities of spearmint that harvested under soilless conditions, for explain this results, during dry process under room temperature at 30[degrees]C the dark brown color appears on the samples of plants and that may be effected on the content of antioxidant, total phenols as well as flavoniods. Moreover, the dark brown color was not appearing on the samples during the air dry process for spearmint under soil based conditions.  Reported during drying process, many compounds, which are dragged to the leaf surface by the evaporating water, which are lost. In this study, spearmint under soilless culture has high moisture content and the air-drying process at room temperature 30[degrees]C may be lost these compounds during the dried stage before analyzed. The air-dry method was not suitable for spearmint plants production under soilless conditions and can be dry by freezing methods for preservation quality and quantity of the essential oils. Mint belongs to the Lamiaceae family of plants, which are known to store their essential oils on or near the leaf surfaces . The method of drying usually has a significant effect on the quality and quantity of the essential oils from plants . Differences in the harvest time, environmental and cultivation conditions may generate differences in the antioxidant capacity between spearmint under soilless and open field . The dry matter of spearmint under soilless conditions was collected from five harvesting times during the growing season. While, under open field, the dry sample was collected from two harvesting times during the growing season.
Moreover, spearmint in this study was cultivated under open field, which exposed to more light hours than under greenhouse conditions and the abiotic factors may be affected on biological activities content.  reported that the environmental conditions such as light could play an important role in increasing or reducing the synthesis and in the accumulation of the essential oil in peltate trichome glands of Lamiaceae species such as spearmint.
The results obtained in this study support the notion that essential oils and their chemical compositions of spearmint strongly affected by environmental conditions and agronomic management practices. M. spicata grown under open filed and protected soilless condition was rich in essential oil, pulegone and 1,8 cineole content. Cultivation of spearmint especially under soilless conditions proved to be superior in both oil content and quality, in terms of substantial contents of pulegone. Pulegone and 1,8 cineole was the dominant compound under both cultivation methods. Results of this study suggest that soilless cultivation methods should use for producing highest economic amount of extracted essential oil and pulegone and 1,8 cineole for pharmaceutical, therapeutic and food purposes.
The authors acknowledge financial support from the Deanship of Scientific Research, JUST, Irbid, Jordan, and Mr. Mohammad Al-Omoush from the Faculty of agriculture for his technical assistance.
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(1) Abdelrazzaq Al-Tawaha, (1) Ghazi Al-Karaki, (2) Adnan Massadeh
(1) Jordan University of Science and Technology, Faculty of Agriculture, Department of plant production, 3030 Irbid, Jordan
(2) Jordan University of Science and Technology, Faculty of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, 22110 Irbid, Jordan
Abdelrazzaq Al-Tawaha, Jordan University of Science and Technology, Faculty of Agriculture, Department of plant production, 3030 Irbid, Jordan Tel.: +962795016606; E-mail: email@example.com
Table 1: Essential oils content and concentration of chemical composition ([micro]g /g) as identified in spearmint plants grown in open field and protected soilless conditions. Cultivation methods Essential Menthone 1,8 Cineole oil(% w/w) [micro]g /g [micro]g /g Open field 3.14 0.132 32.9 Protected Soilless 3.43 0.106 10.6 Significance ns ns ** Cultivation methods Pulegone Menthol [micro]g /g [micro]g /g Open field 20.7 0.132 Protected Soilless 44.3 0.106 Significance * ns *: Significant at P [less than or equal to] 0.05, **: Significant at P [less than or equal to] 0.01. Table 2: Essential oils yield (ml/kg) and content of chemical composition (mg /[m.sup.2]) as identified in spearmint plants grown in open field and protected Soilless conditions. Cultivation Fresh herbal Dry herbal Essential oil methods yield yield yield Kg/[m.sup.2] Kg/[m.sup.2] ml/Kg Open field 2.9 1.04 32.65 Protected Soilless 8.3 1.89 64.82 Significance ** ** ** Cultivation Menthone 1,8 Cineole Pulegone methods mg /[m.sup.2] mg /[m.sup.2] mg /[m.sup.2] Open field 0.103 32 20.7 Protected Soilless 0.200 33.4 83.72 Significance * ** * Cultivation Menthol methods mg /[m.sup.2] Open field 0.103 Protected Soilless 0.200 Significance * *: Significant at P [less than or equal to] 0.05, **: Significant at P [less than or equal to] 0.01. Table 3: Total phenols, flavonoids, and Antioxidant activity of leaves for spearmint plants under soil based and soilless culture conditions. Cultivation methods Total phenols Total flavonoids mg GAE/g mg CE/g Open field 223 149 Protected Soilless 114 73 Significance ** ** Cultivation methods Antiradical (1/antioxidant) mg/g Open field 8.7 Protected Soilless 4.5 Significance ** *: Significant at P [less than or equal to] 0.05, **: Significant at P[less than or equal to] 0.01.
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|Title Annotation:||Original Article|
|Author:||Tawaha, Abdelrazzaq Al-; Karaki, Ghazi Al-; Massadeh, Adnan|
|Publication:||Advances in Environmental Biology|
|Date:||May 1, 2013|
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