Ultrasound-Assisted Extraction of Fennel Leaves: Process optimization, Thin Layer Chromatography and Cytotoxic Activity of Ethanolic Extract


  • Thukaa zuhair Abdul-lalil Department of pharmacognosy and Medicinal plants, collage of pharmacy, University of Baghdad, Baghdad, Iraq.




Fennel, Ultrasound assisted extraction, Single factor experiment, Caffiec acid, Cytotoxic activity


Foeniculum vulgare commonly known as fennel belonging to the family Umbelliferae, is cultivated throughout the world as a spices as well as medicinal plant possessing antimicrobial, antidiabetic, antioxidant, anti-inflammatory, …etc. A Single Factor Experiment analysis has been employed in this study to optimize caffiec acid extraction from fennel leaves by ultrasound assisted extraction method in which three variables single factor examined which were ethanol concentration, extraction time and ultrasound frequency. The study's objective was to assess the effects of variables chosen on caffiec acid's extraction performance, preliminary evaluated by thin layer chromatography (TLC) and evaluate the cytotoxic effect of optimized ethanolic extract of this plant on human breast cancer (MDA-MB-468) cell line. the experimental results revealed that the most influential extraction variables were: 80% ethanol as extraction solvent, 15 min was the time to achieve the good extraction efficacy and 40 KHz ultrasound frequency and The IC50 value of the cytotoxic effect (P < 0.05) of optimized ethanolic extract of Iraqi Foeniculum vulgare leaves on MDA-MB-468 cell line after 72 h of incubation were significantly high (46.89 µg/ml). The results of this study will be used to create a natural medicine for the symptoms of various diseases and to standardize the processes for extracting caffeic acid from natural items, particularly fennel.


et al.: Plant molecular farming: production of metallic nanoparticles and therapeutic proteins using green factories. Green Chemistry 2019; 21(8): 1845–1865.

Ratananikom K and Premprayoon K: Ultrasonic-assisted extraction of phenolic compounds, flavonoids, and antioxidants from Dill (Anethum graveolens L.) Scientifica 2022; 2022(1):1-6

Barba F. J., Zhu Z., Koubaa M., Sant’Ana A. S., and Orlien V.: Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and by-products: a review. Trends in Food Science & Technology 2016; 49: 96–109.

Belwal T., Ezzat S. M., Rastrelli L. et al.: A critical analysis of extraction techniques used for botanicals: trends, priorities, industrial uses and optimization strategies. Trends in Analytical Chemistry 2018; 100: 82–102.

Picot-Allain C., Mahomoodally M. F, Ak G., and Zengin G.: Conventional versus green extraction techniques—a comparative perspective. Current Opinion in Food Science 2021; 40: 144–156.

Mansinhos I., Goncalves S., Rodriguez-Solana R., Ordonez-Diaz J. L., Moreno-Rojas J. M, and Romano A: Ultrasonic-assisted extraction and natural deep eutectic solvents combination: a green strategy to improve the recovery of phenolic compounds from Lavandula pedunculata subsp. lusitanica (chaytor) franco. Antioxidants 2021;10(4):582-590.

Kumar K., Srivastav S., and Sharanagat V. S.: Ultrasound assisted extraction (UAE) of bioactive compounds from fruit and vegeTable processing by-products: a review. Ultrasonics Sonochemistry 2021; 70:1-8.

Muckensturm B., Foechterlen D., Reduron J. P., Danton P., and Hildenbrand M.: Phytochemical and chemotaxonomic studies of Foeniculum vulgare. Biochemical Systematics and Ecology1997; 25(4) :353–358.

Manonmani R. and Abdul Khadir V. M.: Antibacterial screening on Foeniculum vulgare Mill. International Journal of Pharma and Bio Sciences 2011; 2(4):390–394.

Orhan I. E., ¨ Ozc¸elik B., Kartal M., and Kan Y.: Antimicrobial and antiviral effects of essential oils from selected Umbelliferae and Labiatae plants and individual essential oil components. Turkish Journal of Biology 2012;36(3): 239–246.

Dua A., Garg G., and Mahajan R.: Polyphenols, flavonoids and antimicrobial properties of methanolic extract of fennel (Foeniculum vulgare Miller). European Journal of Experimental Biology 2013; 3(4): 203–208.

Ozbek H., U˘gras¸ S., D¨ulger H. et al.: Hepatoprotective effect of Foeniculum vulgare essential oil,” Fitoterapia 2003;74(3): 317–319.

Oktay M., G¨ulc¸in I., and ¨ K¨ufrevioglu O. I.: Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT-Food Science and Technology 2003; 36 (2) :263–271.

Pradhan M., Sribhuwaneswari S., Karthikeyan D. et al.: In-vitro cytoprotection activity of Foeniculum vulgare and Helicteres isora in cultured human blood lymphocytes and antitumour activity against B16F10 melanoma cell line. Research Journal of Pharmacy and Technology 2008;1(4) :450–452.

El-Soud N. A., El-Laithy N., El-Saeed G. et al.: Antidiabetic activities of Foeniculum vulgare mill. Essential oil in streptozotocin-induced diabetic rats. Macedonian Journal of Medical Sciences 2011; 4(2):139–146.

Koppula S. and Kumar H.: Foeniculum vulgare Mill (Umbelliferae) attenuates stress and improves memory in wister rats. Tropical Journal of Pharmaceutical Research 2013; 12(4):553–558.

Shamkant B., Vainav V., and Atmaram H.: Foeniculum vulgare Mill: A review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology. BioMed Research International 2014;2014(1):1-32.

Abdul-Jalil Th. Z.: Screening of rutin from seeds and leaves extract of dill, coriander and fennel cultivated in Iraq. Pharmacy Global IJCP 2013; 3(2): 1-16.

Torres M., Talavera T.A., Andrews H.E., Contreras A.S. and Pacheco N.: Ultrasound assisted extraction for the recovery of phenolic compounds from vegeTable sources. Agronomy 2017; 7(47): 1-19.

Abubakar A.R. and Haque M.: Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes. Journal of Pharmacy and Bioallied Sciences 2020;12(1):1-10.

Bargah R.K.: Preliminary test of phytochemical screening of crude ethanolic and aqueous extract of Moringa pterygosperma Gaertu. Journal of Pharmacognosy and phytochemistry 2015; 4(1): 7-9.

Hind M. Jewely H.M. and Abdul-Jalil Th.Z.: Extraction, isolation and identification of caffeic Acid and p-coumaric acid from n-butanol fraction of Iraqi Osteospermum ecklonis (F. Asteraceae). IJDDT 2022; 12 (2),648-653.

Hamad M.N.: Detection and isolation of Flavonoids from Calendula officinalis (F. Asteraseae) cultivated in Iraq. Iraq J Pharm Sci 2016; 25(2): 1-6.

Moulazadeh A., Ranjbar R., Ardestani A.M., Ranjbar K., Akbar Farjadfar A. et al: Cytotoxic effects of Trachyspermum ammi and Ferula assafoetida on MCF‑7 and MDA‑MB‑468 breast cancer cell lines. Beni-Suef Univ J Basic Appl Sci 2022; 11(147):1-10.

Ibrahem N. M., Mutlag Sh. H.,and Kadhim E.J.: The cytotoxic effect of Iraqi Rhus Coriaria against breast and esophagus cancer cells. Drugs and Cell Therapies in Haematology 2021;10(3):115-129.

Ali Z, Jabir M, Al-Shammari A.: Gold nanoparticles inhibiting proliferation of human breast cancer cell line. Research journal of biotechnology 2019; 14:79-82.

Ogbole OO, Segun PA, Adeniji AJ.: In vitro cytotoxic activity of medicinal plants from Nigeria ethno-medicine on Rhabdomyosarcoma cancer cell line and HPLC analysis of active extracts. BMC complementary and alternative medicine 2017;17(1):1-10.

Singh Sh. P.: A Comprehensive Review on Pharmacological Activity of Foeniculum vulgare. Global Journal of Pharmacy & Pharmaceutical Sciences 2019;7(1):1-5.

Sharopov F., Valiev A., Satyal P., Gulmurodov I., Yusufi S., et al: Cytotoxicity of the essential oil of fennel (Foeniculum vulgare) from Tajikistan. Foods 2017; 6(73):1-11.

AlTemimi A., Lashassassi N., Baharlouei A., Watson D.J. and Lightfoot D.A.: Phytochemicals: extraction, isolation and identification of bioactive compounds from plant extracts. Plants 2017; 6(42): 1-23.

Xu Y. and Pan S.: Effects of various factors of ultrasonic treatment on the extraction yield of all trans-lycopene from red grape fruit (Citrus paradise Macf.). Ultrason. Sonochem 2013; 20: 1026-1032.

Kumara K, Srivastava S, Sharanagatb V.S.: Ultrasound assisted extraction (UAE) of bioactive compounds from fruit and vegeTable processing by-products: A review. Ultrason. Sonochem 2021; 70:1-11.

Xu D., Zhou Y., Zhang J., Li S., Li A. and Li H.: Optimization of ultrasound assisted extraction of natural antioxidant from the flower of Jatropha integerrima by response service methodology. Molecules 2016; 21: 1-18.

Yue T., Shao D., Yuan Y., Wang Z. and Qiang C.: Ultrasound assisted extraction, HPLC analysis and antioxidant activity of polyphenols from unripe apple. J Sep Sci 2012; 35: 2138-2145.

LinaresG and RojasM.L.: Ultrasound-assisted extraction of natural pigments from food processing by-products: A review. Frontiers in Nutrition 2022; 9:1-17.

Çevik D., Kan Y., and Kırmızıbekmez H.: Mechanisms of action of cytotoxic phenolic compounds from Glycyrrhiza iconica roots. Phytomedicine 2019; 58:1-12.

Rezaei-Seresht H., Cheshomi H., Falanji F., MovahediMotlagh F., Maryam Hashemian M. and Mireskandari E.: Cytotoxic activity of caffeic acid and gallic acid against MCF-7 human breast cancer cells: An in silico and in vitro study. AJP 2019; 9(6):574-586.

Zaremba-Czogalla M., Jaromin A., Sidoryk K., Zagórska A., Cybulski M. and Gubernator J.: Evaluation of the in Vitro cytotoxic activity of caffeic acid derivatives and liposomal formulation against pancreatic cancer cell lines. Materials 2020;13(5813): 1-19.