The effect of ultrasound method on the extraction of chlorophyll from mulberry leaves

Document Type : Research Article


1 Assistant Professed, Food Additives Department, Food Science and Technology Research Institute, ACECR, Khorasan Razavi, Iran

2 Assistant Professor, Food Additives Department, Food Science and Technology Research Institute, ACECR, Khorasan Razavi, Iran


The aim of this research was optimizing of the chlorophyll extraction by ultrasound method. A Central Composite Design with 6 repeats at the central point was employed to optimize the effect of the ultrasound power (10-100%), solvent to solid ratio (5-10 ml/g), temperature (30-70 °C ) and time (2-10 min) on the total chlorophyll and a* value. Results of optimization showed that a quadratic model with R2=0.922 was significant. The solvent to solid ratio was the most important parameter which positively affected the yield, although increasing the temperature of extraction significantly reduced the yield. Enhancing the ultrasound power considerably increased the yield of chlorophyll. The maximum yield of chlorophyll was obtained under power of 100%, temperature of 30.2 °C,  time of 9.83 min and solvent to solid ratio of  9.99 (ml/g) which was 680.5 mg/100 g d.m. The comparison of conventional and ultrasound methods showed that the yield of chlorophyll extracted by conventional method (460.5 mg/100 g d.m.) was significantly lower than the amount (680.5 mg/100 g d.m.) extracted by ultrasound method. Therefore, the ultrasound method is an efficient method for extraction of chlorophyll.


Main Subjects

[1] Martins, N., Roriz, C. L., Morales, P., Barros, L., Ferreira, I. C. (2016). Food colorants: Challenges, opportunities and current desires of agro-industries to ensure consumer expectations and regulatory practices. Trends food sci. technol., 52, 1-15.
[2] Gostner, J. M., Becker, K., Ueberall, F., Fuchs, D. (2015). The good and bad of antioxidant foods: An immunological perspective. Food Chem. Toxicol., 80, 72–79.
[3] Patakova, P. (2013). Monascus secondary metabolites: Production and biological activity. J. Ind. Microbiol. Biotechnol., 40(2), 169–181.
[4] Schoefs, B. t. (2002). Chlorophyll and carotenoid analysis in food products. Properties of the pigments and methods of analysis. Trends food sci. technol., 13(11), 361-371.
[5] سازمان ملی استاندارد و تحقیقات صنعتی، 1392. افزودنی‌های خوراکی مجاز- رنگ‌های خوراکی- فهرست و ویژگی‌های عمومی. شماره 740. 66.
[6] باقری، س. ل.؛ رادی، م.؛ و امیری، ص. (1392) بررسی ساختار شیمیایی و پایداری کلروفیل به عنوان یک رنگیزه طبیعی، بیست و یکمین کنگره ملی علوم وصنایع غذایی ایران. دانشگاه شیراز.
[7] Delgado-Vargas, F., Paredes-López, O. (2002). Natural colorants for food and nutraceutical uses: CRC Press.
[8] سحری، م. ع. (1381) شیمی ترکیبات رنگی (در مواد غذایی)، چاپ اول،  انتشارات دانشمند، ص 23-44.
[9] مومنی، ت. ک. (1378) کلروفیل و پایداری آن، موسسه تحقیقات جنگلها و مراتع، سارنگ، 1-117.
[10] Humphrey, A. (2004). Chlorophyll as a color and functional ingredient. J. Food Sci.; 69: 422-425.
[11] Hughes, J. H., Latner, A. L. (1936). Chlorophyll and haemoglobin regeneration after haemorrhage. .J Physiol.; 86: 388–395.
[12] Sato, M., Fujimoto, I., Sakai, T., Kimura, R., Murata, T. (1979). An antioxidative component in the commercial preparations of sodium copper chlorophyllin. Yakugaku Zasshi; 99: 1055–1057.
[13] Sato, M., Imai, K., Kimura, R., Murata, T. (1984). Effect of sodium copper chlorophyllin on lipid peroxidation. VI. Effect of its administration on mitochondrial and microsomal lipid peroxidation in rat liver. Chem. Pharm. Bulletin; 32: 16–722.
[14] Humphrey, A. (1980). Chlorophyll. Food Chem.; 5: 57–67.
[15] Wang, L. Weller, C.L. (2006). Resent advances in extraction of nutraceuticals from plants.Trends Food Sci. Technol., 17,300-312.
[16] Annegowda, H., Bhat, R., Min-Tze, L., Karim, A., Mansor, S. (2012). Influence of sonication treatments and extraction solvents on the phenolics and antioxidants in star fruits. J. Food Sci. Tech., 49(4), 510-514
[17] Tiwari, B., Patras, A., Brunton, N., Cullen, P., O’Donnell, C. (2010). Effect of ultrasound processing on anthocyanins and color of red grape juice. Ultrason. Sonochem., 17(3), 598-604.
[18] Mackinney, G. (1941). Absorption of light by chlorophyll solutions. J. biol. Chem, 140(2), 315-322.
[19] Rouhani, S., Alizadeh, N., Salimi, S., Haji-Ghasemi, T. (2009). Ultrasonic Assisted Extraction of Natural Pigments from Rhizomes of Curcuma Longa L. extraction, 6, 7.
[20] Cuoco, G., Mathe, C., Archier, P., Chemat, F., Vieillescazes, C. (2009). A multivariate study of the performance of an ultrasound-assisted madder dyes extraction and characterization by liquid chromatography-photodiode array detection. Ultrason. Sonochem, 16(1), 75-82.
[21] Shirsath, S., Sonawane, S., Gogate, P. (2012). Intensification of extraction of natural products using ultrasonic irradiations—a review of current status. Chem. Eng. Process.: Process Intensif., 53, 10-23.
[22] صابریان، ح.؛ حسینی، ف.؛ بلوریان، ش. (1396). بهینه‌سازی شرایط استخراج رنگ طبیعی کلروفیل از یونجه و بررسی ویژگی‌های کمی و کیفی آن در مقایسه با منابع گیاهی مختلف، مجله علوم و صنایع غذایی (در نوبت چاپ).
 [23] Kong, W., Liu, N., Zhang, J., Yang, Q., Hua, S., Song, H., Xia, C. (2014). Optimization of ultrasound-assisted extraction parameters of chlorophyll from Chlorella vulgaris residue after lipid separation using response surface methodology. J. Food Sci. Tech.; 51(9): 2006-2013.
[24] Özkan, G., Bilek, S. E. (2015). Enzyme-assisted extraction of stabilized chlorophyll from spinach. Food chem.; 176:152-157.
[25] Arnon, D. (1949). Copper enzymes in isolated chloroplasts, Plant physiol.; 24: 1-16.
 [26] Saberian, H., Hamidi-Esfahani, Z., Abbasi, S. (2013). Effect of pasteurization and storage on bioactive components of Aloe vera gel. Nutr. Food Sci., 43(2), 175-183.
[27] Hill, W.J. Hunter, W.G. 1966. A review of response methodology: A literature survey, Technimetrics, 8 (4): 571-510.
[28] Chen, F., Sun, Y., Zhao, G., Liao, X., Hu, X., Wu, J., Wang, Z. (2007). Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography–mass spectrometry. Ultrason.Sonochem., 14(6), 767-778.
[29] یلمه، م.؛ حبیبی نجفی، م. ب.؛ حسینی، ف.؛ فرهوش، ر. (1393). ارزیابی اثر ضدباکتریایی رنگ آناتو بر سالمونلا انتریتیدیس موجود در سس مایونز، مجله علوم غذایی و تغذیه جلد 11، شماره 44، ص 17-22.
[30] Martino, E., Ramaiola, I., Urbano, M., Bracco, F., Collina, S. (2006). Microwave-assisted extraction of coumarin and related compounds from Melilotus officinalis (L.) Pallas as an alternative to Soxhlet and ultrasound-assisted extraction. J. Chromatography A, 1125(2), 147-151.
[32] Von Elbe, J., Schwartz, S. (1996). Colorants (Cilt 2). New York: Marcel Dekker: Food Chem.
[33] پدرام نیا، ا.؛ شریفی، ا.؛ توکلی پور، ح. (1389) بهینه‌سازی فرایند استخراج آنتوسیانین زرشک در حضور امواج فراصوت، مجله نوآوری در علوم و فناوری غذایی، شماره 4، ص 45-52.
[34] Zou, Y., Xie, C., Fan, G., Gu, Z., Han, Y. (2010). Optimization of ultrasound-assisted extraction of melanin from Auricularia auricula fruit bodies. Innov. Food Sci. Emerg. Technol., 11(4), 611-615.
[35] Wang, J., Sun, B., Cao, Y., Tian, Y., Li, X. (2008). Optimisation of ultrasound-assisted extraction of phenolic compounds from wheat bran. Food Chem., 106(2), 804-810.
[36] Agarwal, P. C., Pruthi, J. S. (1968). A study of factors governing the recovery and quality of pectin from fresh mandarin orange waste (peel and pomace). Indian Food Packer; 22(3): 5–9.
[37] Wasmund, N., Topp, I., Schories, D. (2006) Optimising the storage and extraction of chlorophyll samples. Oceanologia; 48:125–144.
 [38] Sivakumar, V., Anna, J. L., Vijayeeswarri, J., Swaminathan, G. (2009). Ultrasound assisted enhancement in natural dye extraction from beetroot for industrial applications and natural dyeing of leather. Ultrason.  Sonochem., 16(6), 782-789.
 [39] He, B., Zhang, L.-L., Yue, X.-Y., Liang, J., Jiang, J., Gao, X.-L., Yue, P.-X. (2016). Optimization of Ultrasound-Assisted Extraction of phenolic compounds and anthocyanins from blueberry (Vaccinium ashei) wine pomace. Food Chem., 204, 70-76.
[40] خواجه نوری، م.؛ حقیقی اصل، ع. (1393) بررسی استخراج ترکیبات طبیعی گیاهان با کمک امواج میکروویو و فراصوت، مجله فناوری‌های نوین غذایی، 1 (3)، ص 81-91.