• Ulfah Anwar Indonesian Red Cross Kampar Regency Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
  • Siti Pauliena Mohd Bohari Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia




Wound healing, Manihot esculenta, therapeutic ultrasound, scratch assay, MTT assay


The aim of this research is to investigate the wound healing process in in vitro by combining the Manihot esculenta aqueous extract and therapeutic ultrasound. Firstly, the optimization seeding densities of HSF cell 1184 in six-well plate, and then followed by the scratch assay experiment. The scratched that made was treated with the remedial treatments (Manihot esculenta aqueous extract only; ascorbic acid+ therapeutic ultrasound; Manihot esculenta aqueous extract+ ascorbic acid; Manihot esculenta aqueous extract+ therapeutic ultrasound and also the combination of these three materials). The rate of wound closure was observed and analysed at a time interval of 0, 2, 4, 6, 8, 10 and 24 h by using image J software. Then, the cells viability were analysed using the MTT assay. The result showed that Manihot esculenta aqueous extract coupled with specific dose therapeutic ultrasound represents a significantly high rate of wound closure at 96.10 % with the cell numbers at 5.44×105 cells/mL when compared to the other combination therapy. The finding of this study revealed that Manihot esculenta aqueous extract 200 µg/mL and the therapeutic ultrasound specific dose (3 MHz, 300 mWatt/cm2, 50% in 5 min) have the potential in accelerating wound healing process of cells in in vitro.


Diegelmann, R. F, Evans, M. C. 2004. Wound Healing: An Overview of Acute, Fibrotic and Delayed Healing. Front Biosci. 1(4): 283-289.

Krishnasamy, M. 2006. Technology Review : Utrasound Assisted Wound Care. 1-11.

Nicks, B. A., Ayello, E. A., Woo, K., Nitzki-George, D., Sibbald, R. G. 2010. Acute Wound Management: Revisiting the Approach to Assessment, Irrigation, and Closure Considerations. Int. J. Emerg. Med. 3(4): 399-407.

Mustoe, T. A,, O’Shaughnessy, K., Kloeters, O. 2006. Chronic Wound Pathogenesis and Current Treatment Strategies: A Unifying Hypothesis. Plast. Reconstr. Surg. 117(7 Suppl): 35S-41S.

Moffatt, C., & Rabe, E. 2003. Understanding Compression Therapy. 1-17.

Tan, J., Abisi, S., Smith, A., & Burnand, K. G. 2007. A Painless Method of Ultrasonically Assisted Debridement of Chronic Leg Ulcers: A Pilot Study. European Journal of Vascular and Endovascular Surgery. 33: 234-238.

Ennis, W. J. et al. 2005. Ultrasound Therapy for Recalcitrant Diabetic Foot Ulcers: Results of a Randomized, Double-blind, Controlled, Multicenter Study. Ostomy Wound Manage. 51(8): 24-39.

Kwiatkowska, B., Bennett, J., Akunna, J., Walker, G. M., Bremner, D. H. 2011. Stimulation of Bioprocesses by ultrasound. Biotechnol Adv. 29: 768-80.

Muhammad, A. A., Aimi, N., Pauzi, S., Arulselvan, P., Abas, F., Fakurazi, S. 2013. In Vitro Wound Healing Potential and Identification of Bioactive Compounds from Moringa oleifera Lam. 1-10.

Wadankar, G. D., Malode, S. N., Sarambekar, S. L. 2011. Traditionally Used Medicinal Plants for Wound Healing in the Washim District, Maharashtra (India). International Journal of PharmTech Research. 3(4): 2080-2084.

Nisa, V. M., Meilawaty, Z., Astuti, P. 2013. The Effect of Cassava Leaves Extract (Manihot esculenta) on Gingival Wound Healing Rats. Artik. Ilm. Has. 1-7.

Nurdiana, A. R. 2013. Uji Ekstrak Daun Singkong (Manihot esculenta) Terhadap Jumlah Neutrofil pada Proses Penyembuhan Luka Tikus ( Rattus norvegiccus ). 1-58.

Restrepo, S., Duque, M. C., Verdier, V. 2000. Characterization of Pathotypes among Isolates of Xanthomonas axonopodis pv. manihotis in Colombia. Plant Pathol. 49:680-687.

Choi, H.-I., Park, J.-I., Kim, H.-J., Kim, D.-W., & Kim, S.-S. 2009. A Novel L-ascorbic Acid and Peptide Conjugate with Increased Stability and Collagen Biosynthesis. BMB Reports. 42(11): 743-746.

Schmidt, R. J., Chung, L. Y., Andrews, A. M., Turner, T. D. 1993. Toxicity of L-ascorbic Acid to L929 Fibroblast Cultures: Relevance to biocompatibility Testing of Materials for Use in Wound Management. J. Biomed. Mater. Res. 27: 521-530.

Meilawaty, Z. 2013. Potensi Ekstrak Daun Singkong (Manihot Utilissima) dalam Memodulasi Ekspresi COX-2 Pada Monosit Yang Dipapar LPS. 1-16.

Anibijuwon, I. I., Udeze, A. O. 2009. Antimicrobial Activity of Carica Papaya (Pawpaw Leaf) on Some Pathogenic Organisms of Clinical Origin from South-Western Nigeria. Ethnobotanical Leaflets. 13: 850-64.

Al-Rofaai, A., Rahman, W. A., Sulaiman, S. F., & Yahaya, Z. S. 2012. Veterinary Parasitology in vitro Ovicidal and Larvicidal Activity of Methanolic Leaf Extract of Manihot esculenta (cassava) on Susceptible and Resistant Strains of trichostrongylus colu-briformis. Veterinary Parasitology. 190(1-2): 127-135.

Al-Ansari A–Mehdi, S., Abdulkareem, M. a. 2014. Some Plant Extracts Retarde Nitrification in Soil. Acta Agric. Slov. 103(1): 5-13.

Chew, K., Aida, W. 2011. Effect of Ethanol Concentration, Extraction Time and Extraction Temperature on the Recovery of Phenolic Compounds and Antioxidant Capacity of Centella Asiatica Ex-tracts. Int. Food Res. J. 18: 571-578.

Parekh, J., Jadeja, D., Chanda, S. 2005. Efficacy of Aqueous and Methanol Extracts of Some Medicinal Plants for Potential Antibacterial Activity. Turk J Biol. 29: 203-210.

James, O., Friday, E. T. 2010. Phytochemical Composition, Bioactivity and Wound Healing Potential of Euphorbia heterophylla (Euphorbiaceae) Leaf Extract. Int. J. Pharm. Biomed. Res. 1(1): 54-63.

Marie-magdeleine, C., Udino, L., Philibert, L., Bocage, B., Ar-chimede, H. 2010. Veterinary Parasitology In vitro Effects of Cassava (Manihot esculenta) Leaf Extracts on Four Development Stages of Haemonchus contortus. Vet. Parasitol. 173(1-2): 85-92.

Yusuf, U. F., Okechukwu, P. N. 2013. Anti-inflammatory, Analgesic and Anti-pyretic Activity of Cassava Leaves Extract. Asian J Pharm Clin Res. 6(4): 89-92.

Zhou, H., Weir, M. D., Ph, D., Xu, H. H. K. 2011. Effect of Cell Seeding Density on Proliferation and Osteodifferentiation of Umbilical Cord Stem Cells on Calcium Phosphate Cement-Fiber Scaffold. Tissue Eng. 17(21-22): 2603-2613.

Rahmat, A., Kumar, V., Fong, L. M., Endrini, S. 2003. Determination of Total Antioxidant Activity in Three Types of Local Vegetables Shoots and the Cytotoxic Effect of Their Ethanolic Extracts against Different Cancer Cell Lines. Asia Pac J Clin Nutr. 12(3): 308-311.

Liang, C-C., Park, A. Y., Guan, J-L. 2007. In Vitro Scratch Assay: A Convenient and Inexpensive Method for Analysis of Cell Migration In Vitro. Nat. Protoc. 2(2): 329-333.

Hulkower, K. I., Herber, R. L. 2011. Cell Migration and Invasion Assays as Tools for Drug Discovery. Pharmaceutics. 3(1): 107-124.

Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. 2004. A High-Throughput Cell Migration Assay using Scratch Wound Healing, A Comparison of Image-based Readout Methods. BMC Biotechnol. 4(21): 1-9.

Freshney, R. I. 2005. Culture of Animal Cells: A Manual of Basic Technique. Fifth Edition. New Jersey: John Wiley & Sons, Inc.

Mahdzir, N. S. B. 2015. Therapeutic Ultrasound as an Alternative Treatment for Wound Healing in In Vitro. 1-91.

Speed, C. 2001. Therapeutic Ultrasound in Soft Tissue Lesions. Rheumatology (Oxford). 40(12): 1331-6.

Sorrell, J. M., Caplan, A. I. 2004. Fibroblast Heterogeneity : More than Skin Deep. J. Cell Sci. 17(5): 667-675.

Varkey, M., Ding, J., Tredget, E. E. 2015. Advances in Skin Substi-tutes-Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing. J. Funct. Biomater. 6(3): 547-563.

Otero, M., Favero, M., Dragomir, C. et al. 2014. Human Chondrocyte Cultures as Models of Cartilage-Spesific Gene Regulation. Methods Mol Med. 107(4): 69-95.

Matsubayashi, Y., Razzell, W., Martin, P. 2011. “White Wave†Analysis of Epithelial Scratch Wound Healing Reveals How Cells Mobilise Back from the Leading Edge in a Myosin-II-Dependent Fashion. J. Cell Sci. 124(7): 1017-1021.

Sylvester, V. W. 2011. Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability. Methods Mol Biol. 716: 157-68.

Suzuki, O., Koura, M., Noguchi, Y., Uchio-yamada, K., & Matsuda, J. 2011. Use of Sample Mixtures for Standard Curve Creation in Quantitative Western Blots. Experimental Animals. 60(2): 193-196.

Brescia, P., Sc, M., Banks, P., Ph, D., & Instruments, B. 2009. Quantifying Cytotoxicity of Thiostrepton on Mesothelioma Cells using MTT Assay and the Epoch TM Microplate Spectrophotometer.

Pallant, J. 2010. SPSS Survival Manual: A Step by Step Guide to Data Analysis using SPSS. 3rd ed) Maidenhead: Open University Press/McGraw-Hill.

Malek, S. N. A., Phang, C. W., Ibrahim, H., Norhanom, A. W., & Sim, K. S. 2011. Phytochemical and Cytotoxic Investigations of Alpinia Mutica Rhizomes. Molecules. 16(1): 583–589.

Sajjadi, S. E., Ghanadian, M., Haghighi, M., & Mouhebat, L. 2015. Cytotoxic Effect of Cousinia Verbascifolia Bunge against OVCAR-3 and HT-29 Cancer Cells. Journal of HerbMed Pharmacology. 4(1): 15-19.

Al-Qubaisi, M., Rozita, R., Yeap, S.-K., Omar, A.-R., Ali, A.-M., & Alitheen, N. B. 2011. Selective Cytotoxicity of Goniothalamin against Hepatoblastoma HepG2 Cells. Molecules. 16(12): 2944-2959.

Franco de Oliveira, R., Pires Oliveira, D., Soares, C. P. 2011. Effect of Low-intensity Pulsed Ultrasound on l929 Fibroblasts. Arch. Med. Sci. 7(2): 224-229.

K.-M. Choi et al., 2008. Effect of Ascorbic Acid on Bone Marrow-derived Mesenchymal Stem Cell Proliferation and Differentiation. J. Biosci. Bioeng. 105(6): 586-594.

Unger, P. G. 2008. Low-frequency, Noncontact, Nonthermal Ultra-sound Therapy: A Review of the Literature. Ostomy Wound Manag. 54(1): 57-60.

Aggarwal, B. B., Prasad, S., Reuter, S., Kannappan, R., Vivek, R. 2011. Identification of Novel Anti-inflammatory Agents from Ayurvedic Medicine for Prevention of Chronic Diseas-es: Reverse Pharmacology and Bedside to Bench Approach. Curr Drug Targets. 12(11): 713-794.

Alajmi, M. F., Alam, P. 2014. Anti-inflammatory Activity and Qualitative Analysis of Different Extracts of Maytenus obscura (A. Rich.) Cuf. by High Performance Thin Layer Chromatography Method. Asian Pac. J. Trop. Biomed. 4(2): 152-7.

Ganceviciene, R., Liakou, AI., Theodoridis, A., Makrantonaki, E., Zouboulis, C. C. 2012. Skin Anti-aging Strategies. Dermato-Endocrinology V. 4(3): 308-319.

Schagen, S. K., Zampeli, V., Makrantonaki, E., Zouboulis, C. C. 2012. Discovering the Link Between Nutrition and Skin Aging. Dermatoendocrinol. 4(3): 298-307.

Olczyk, P., Mencner, Å., Komosinska-Vassev, K. 2014. The Role of the Extracellular Matrix Components in Cutaneous Wound Healing. Biomed Res. Int. 1-8.




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