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1 Advances in Environmental Biology, 5(6): , 2011 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Interaction Effects of Nitrogen, Phosphorus and Potassium on Growth, Essential Oil and Total Phenolic Content of Sweet Basil 1 Shahram Sharafzadeh, 2 Mahmood Esmaeili, 2 Abdol Hossein Mohammadi 1 Islamic Azad University, Firoozabad Branch, Firoozabad, Iran. 2 Islamic Azad University, Jahrom Branch, Jahrom, Iran. Shahram Sharafzadeh, Mahmood Esmaeili, Abdol Hossein Mohammadi: Interaction Effects of Nitrogen, Phosphorus and Potassium on Growth, Essential Oil and Total Phenolic Content of Sweet Basil ABSTRACT Nutrients as one of the most important environmental factors affect qualitative and quantitative characteristics of medicinal plants. Sweet basil (Ocimum basilicum L.), a member of Lamiaceae family, is an annual plant and is widely used as a vegetable and aromatic plant. A greenhouse experiment was conducted to evaluate the effects of nitrogen, phosphorus and potassium on sweet basil. The plants were grown in pots containing 1/3 soil, 1/3 sand and 1/3 leaf mold (v/v). The treatments were using N (50, 100 and 150 kg/ha), P 2 O 5 (50 and 100 kg/ha) and K 2 O (50 and 100 kg/ha) in a factorial experiment based on complete randomized design (CRD) with three replications. The results indicated that the maximum shoot fresh weight (62.9 g/plant) was achieved on N 150 P 100 K 50. The highest shoot dry weight (7.1 g/plant) was obtained in N 150 P 100 K 100. The yield of the oil was maximum (1.6%) in N 150 P 50 K 100. The maximum oil content (105.3 mg/plant) was shown in N 150 P 100 K 100. The highest total phenolic content (148.4 mg GAE/g dw) was achieved on N 50 P 50 K 50. Key words: medicinal plant, Ocimum basilicum, fertilizers, active substances, greenhouse Introduction Basil is widely used as a vegetable and as aromatic plant and was originated North West India, North East Africa and Middle Asia [4]. It grows in subtropical zone from sea level to altitude of 1800 m. This plant includes at least 60 species and numerous varieties. The Ocimum genus belonging to the Lamiaceae family is characterized by a great variability of both morphology and chemotypes [9,10]. Among all the species, Ocimum basilicum L. (basil or sweet basil) has the most economic importance and is cultivated and utilized through out the world [10]. Basil is an annual herb with cm plant height and pink and white flowers. The useful parts of basil plant are leaves and seeds. The most component of basil is essential oil. Oil yield varies from 0.1 to 0.45% based on ecological and agronomical conditions [1]. Basil has been utilized for its expectorant, carminative and stimulant properties in folk medicine. In addition, it was used as insecticide [2], flea and moth repellent and against to snake, scorpion and insect bite [13]. Fresh and dry leaves of plant are used in food and spice industries. The oil of basil is used in food industries, perfumery, dental and oral products [12,15,19]. The oil of basil has antimicrobial effect [3]. Basil produces a range of polyphenolic compounds including rosmarinic acid, a characteristic it shares with herbs in the genus Lamiaceae. Rosmarinic acid is a cinnamic acid derivative with potent antioxidant activity and known antiviral, antibacterial, and anti-inflammatory properties. An important factor affecting the quantity and Corresponding Author Shahram Sharafzadeh, Department of Agriculture, Islamic Azad University, Firoozabad Branch, Firoozabad, Iran. s.sharafzadeh@iauf.ac.ir ; shahramsharafzadeh@hotmail.com; Tel:

2 quality of the basil yield is the optimum level of fertilization. On a sandy soil of low humus and nutrient content, the best fresh and dry herb yield (40 t/ha and 7.3 t/ha, respectively) from two harvests were achieved with medium doses of NPK elements ( kg/ha). The essential oil yield was highest (40.5 kg/ha) at this fertilization level as well. No beneficial effects of higher doses were found. Increasing N-doses gave a considerable rise both in the fresh and dry yield of sweet basil [20]. Due to the longer growing season and the accelerated growth in countries with a warmer climate about 3 5 sequent harvests of basil can be obtained each year. In this case the higher phytomass requires a higher nutrient supply and an additional N-dressing after the harvests are important in order to enhance the growth [6]. According to the results of Tesi et al. [18], Genovese sweet basil, grown in greenhouse seemed to be sensitive to high concentrations of fertilizers in the irrigation water. At 500 kg/ha N doses the leaf area decreased 42%, compared to a N dose of 100 kg/ha. In this study we applied different levels of nitrogen, phosphorus and potassium in order to determine their effects on growth and active substances of sweet basil. Materials and methods Plant Material and Experimental Conditions: This study was conducted on experimental greenhouse of Islamic Azad University, Jahrom Branch, Iran (53 20' E, 28 38' N; 1100 m above sea level). Basil seeds were sown in pots containing 1/3 soil, 1/3 sand and 1/3 leaf mold (v/v) and kept at 27±2/17±2 C day/night temperatures. The treatments were using N (50, 100 and 150 kg/ha), P 2 O 5 (50 and 100 kg/ha) and K 2 O (50 and 100 kg/ha) in a factorial experiment based on complete randomized design (CRD) with three replications. The fertilizers used in this experiment were ammonium nitrate (33% N), triple super phosphate (46% P 2 O 5 ) and potassium sulphate (50% K 2 O). Fifty percent of nitrogen was used as basic fertilizer and 50% as top-dressing. The soil of pots were tested before applying treatments and soil texture was sandy-loam with PH=7.63, organic C=2.13%, total N=0.2%, available P=16 mg/kg, available K=337 mg/kg and EC=4 mmoh. Each replicate contained 4 pots, each pot with 5 plants. Plants were harvested at full bloom in order to determine shoot fresh weight and were dried at room temperature for shoot dry weight measurements. Essential oil extraction: Isolation of essential oils was performed using 1286 hydrodistillation of 50 g sample of dried shoots (stem, leaf and flower) using a Clevenger-type apparatus over 4 hours. The oils were dried over sodium sulphate and the percentage of the essential oils (w/w) were calculated. Total Phenolic Content Determination: In order to determine the total phenolic content, 250 mg of dried shoots of each replication were ground and dissolved in 10 ml of 80% acetone. Sample extracts were rotated for 1 hour in the dark and centrifuged at 3500 rpm for 10 minutes. The amounts of total phenolics in extracts were determined with the Folin-Ciocalteu reagent using the method of Javanmardi et al. [8]. To 50 µl of each sample, 2.5 ml 1/10 dilution of Folin-Ciocalteu reagent and 2 ml of Na 2 Co 3 (7.5% w/v) were added and incubated at 45 C for 15 minutes. The absorbance of samples was measured at 765 nm using a UV-vis spectrophotometer. Results were expressed as milligrames of gallic acid equivalent per gram of dry weight (mg GAE/g dw). Statistical analysis: The data were subjected to analysis of variance (ANOVA) using MSTAT-C computer software at P < 0.05 and means compared with DNMRT. Results and discussion The fertilizer treatments illustrated significant differences (Fig. 1-5). The results indicated that the maximum shoot fresh weight (62.9 g/plant) was achieved on N 150 P 100 K 50 which was not significantly different when compared to N 150 P 100 K 100 (Fig. 1). The highest shoot dry weight (7.1 g/plant) was obtained in N 150 P 100 K 100 and the lowest value (3.1 g/plant) was in N 50 P 50 K 100 (Fig. 2). The yield of the oil was maximum (1.6%) in N 150 P 50 K 100 which was significantly different when compared to other treatments (Fig. 3). The maximum oil content (105.3 mg/plant) was shown in N 150 P 100 K 100 which was significantly different when compared to other treatments (Fig. 4). The highest total phenolic content (148.4 mg GAE/g dw) was achieved on N 50 P 50 K 50 and other treatments had lower values (Fig. 5). Halva and Puukka [5] reported application of NPK kg/ha as optimum basic fertilization. In addition basil received a benefit from the N-top dressing 80 kg/ha. Due to the cold weather, the fresh yields were quite low. According to Hornok [7] the Hungarian fertilization advice includes three applications of fertilizers: basic fertilization in the autumn N=40 60 kg/ha, P=60 80 kg/ha and K= kg/ha are recommended. Start of the fertilization in the spring at the time of soil

3 preparation with N=40 60 kg/ha and P=18 20 kg/ha. Foliar N-fertilization is applied after the foliage cuttings, in doses of kg/ha. In the United States the suggested ratio of N- P 2 O 5 -K 2 O elements is 1:1:1, with a N-dose of kg/ha, as broadcasts and plowdown. Nitrogen side-dressing at rates of kg/ha are recommended after each harvest [16]. In India, in the practical cultivation, NPK= kg/ha basic fertilization is recommended before the soil preparation. 40 kg/ha nitrogen fertilization is applied as top dressing in two equal doses [17] Nguyen [11] illustrated how nutrient availability and specifically nitrogen fertilization, affects the production of polyphenolic compounds in three cultivars (Dark Opal, Genovese, and Sweet Thai) of basil. Nitrogen fertilization was found to have a significant effect on total phenolic levels in Dark Opal and Genovese basil with statistically higher phenolic contents observed when nutrient availability was limited at the lowest (0.1 mm) applied nitrogen treatment. Similarly, basil treated at the lowest nitrogen fertilization level generally contained significantly higher rosmarinic and caffeic acid concentrations than basil treated at other nitrogen levels. Fig. 1: Effect of different level of N, P and K on shoot fresh weight of basil. Columns with the same letters are not significantly different at 5% level of Duncan's new multiple range test (DNMRT). Fig. 2: Effect of different level of N, P and K on shoot dry weight of basil. Columns with the same letters are not significantly different at 5% level of Duncan's new multiple range test (DNMRT). Fig. 3: Effect of different level of N, P and K on oil yield of basil. Columns with the same letters are not significantly different at 5% level of Duncan's new multiple range test (DNMRT).

4 1288 Fig. 4: Effect of different level of N, P and K on oil content of basil. Columns with the same letters are not significantly different at 5% level of Duncan's new multiple range test (DNMRT). Fig. 5: Effect of different level of N, P and K on total phenolic content of basil. Columns with the same letters are not significantly different at 5% level of Duncan's new multiple range test (DNMRT). According to Sharafzadeh [14], nutrients are able to change oil yield and total phenolic content of garden thyme. The growth, oil yield and phenolic content could be markedly affected by the geographical environment, places that plants is grown, physical and chemical characteristics of soil, seed source, plant age, parts of plant which is used for oil isolation and oil isolation method. References 1. Arabaci, O. and E. Bayram, The effect of nitrogen fertilization and different plant densities on some agronomic and technologic characteristics of Ocimum basilicum L. (Basil). J. Agron., 3: Bowers, W.S. and R.J. Nishida, Potent juvenile hormones mimics from sweet basil. Science, 209: Bozin, B., N. Mimica-Dukic, N. Simin and G. Anackov, Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem., 54: Gill, B.S. and G.S. Randhawa, Effect of transplanting dates and stage of harvesting on the herb and oil yields of French basil (O. basilicum L.). Indian Perfumer, 36: Halva, S. and L. Puukka, Studies on fertilization of dill (Anethum graveolens L.) and basil (Ocimum basilicum L.) I. Herb yield of dill and basil affected by fertilization. J. Agric. Sci. in Finland, 56: Hiltunen, R. and Y. Holm, Basil: The Genus Ocimum. Hardwood Academic Publishers. 7. Hornok, L., Cultivation and Processing of Medidnal Plants. Akademia Kiado, Budapest, Hungary. 8. Javanmardi, J., C. Stushnoff, E. Locke and J.M. Vivanco, Antioxidant activity and total phenolic content of Iranian Ocimum accessions. Food Chem., 83: Lawrence, B.M., A Further Examination of the Variation of Ocimum basilicum L. In Flavors and Fragrances: A World Perspective; Proceeding of the 10th International Congress of Essential oil, Fragrances and Flavours, Washington, DC; Elsevier Science: Amsterdam. 10. Marotti, M., R. Piccaglia and E. Giovanelli, Differences in essential oil composition of basil (Ocimum basilicum L.), Italian cultivars related to morphological characteristics. J. Agric. Food Chem., 44: Nguyen, P.M. and E.D. Niemeyer, Effect of nitrogen fertilization on the phenolic composition and antioxidant properties of basil (Ocimum basilicum L.). Retrieved from 8/niemeyer-vol8.pdf.

5 12. Prasad, G., A. Kuman, A.K. Singh, A.K. Bhattacharya, K. Singh and V.D. Sharma, Antimicrobial activity of essential oils of some Ocimum species and clove oil. Fitoterapia, 57: Serin, E.D. and M. Ozguven, Cukurova koullarinda iki farkli kokenli fesleen (Ocimum basilicum L.) in verim ve ucucu yalari uzerinde aratirmalar. Cukurova Universitesi Ziraat Fakultesi Dergisi, 12: Sharafzadeh, Sh., Effect of nitrogen, phosphorus and potassium on growth, essential oil and total phenolic content of garden thyme (Thymus vulgaris L.). Adv. Environ. biol., 5: Simon, J.E., A.F. Chadwick and L.E. Craker, Herbs: An indexed bibliography The Scientific Literature on Selected Herbs and Aromatic and Medicinal Plants of the Temperature Zone. pp: 7-9, Elsevier:Amsterdam Simon, J.E., Basil. New Crop Factsheet. Retrieved from crop. 17. Srivastava, A.K., French basil and its cultivation in India. Farm Bulletin No. 16. Central Institute Medicinal and Aromatic Plants, Lucknow, p: Tesi, R., G. Chisci, A. Nencini and R.Tallarico, Growth response to fertilization on sweet basil (Ocimum basilicum L.). Acta Hort., 390: Vieira, R.F. and J.E. Simon, Chemical characterization of basil (Ocimum spp.) found in the markets and used in traditional medicine in Brazil. Econ. Bot., 54: Wahab, A.S.A. and L. Hornok, Effect of NPK fertilization on Ocimum basilicum yield and essential oil content. Kerteszeti Egyetem Kozlemenyei, 45: