Document Type: Final File


Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran


Achillea eriophora D.C. (Asteraceae), an endemic species in Iran, is used extensively in Iranian folk medicine to treat various ailments, including gastrointestinal disorders, skin inflammations and wound healing. The present study was aimed to investigate the effects of the methanolic extract of  A. eriophora leaves on the stimulation of human fibroblast cell proliferation and migration as two pivotal stages in wound healing process. Methanolic extract was prepared by maceration method. Total phenol and flavonoid contents were measured using spectrophotometry. Cytotoxic and proliferation effects of the extract were evaluated by MTT assay on human fibroblast cells. Moreover, migration of the treated cells was assessed by the closure of a denuded area, made by scratching on the confluent monolayer cultures.  The results of cytotoxicity assay indicated that the methanolic extract did not have any cytotoxic effect on fibroblast cells when used at concentrations up to 2 µg/mL. Human fibroblast proliferation was stimulated by low concentrations of the extract (0.1-0.8 µg/mL), and the highest level of proliferation was observed in the lowest treatment (0.1 µg/mL). Migration of the cells was induced by intermediate concentrations (1-30 µg/mL) of the extract and the most effective dose was estimated at 1 µg/mL. Due to the fact that lower concentration of the extract, which showed the best proliferation and migration stimulatory effects, were not toxic on the human fibroblast cells, the crude methanolic extract from the leaves of Achillea eriophora could be recommended as a potential source for improving the wound healing activity in skin. 


Main Subjects

1.            Hammad H.M., Litescu S.C., Matar S.A., Al-Jaber H.I., and Afifi F.U. Biological activities of the hydro-alcoholic and aqueous extracts of Achillea falcata L. (Asteraceae) grown in Jordan. European. J. Med. Plants, 4: 259-270 (2014).

2.            Saeidnia S., Gohari A.R., Mokhber-Dezfuli N., and Kiuchi F. A review on phytochemistry and medicinal properties of the genus Achillea. DARU, 19: 173-86 (2011).

3.            Nemeth E., and Bernath J. Biological activities of yarrow species (Achillea   spp.). Curr. Pharm. Des., 14: 3151-3167 (2008).

4.            Nirmala S., and Karthiyayini T. Wound healing activity on the leaves of Aachillea  millefolium by excision, incision, and dead space model on adult wistar albino rats. Int. Res. J. Pharm., 2: 240-245 (2011).

5.            Pirbalouti A.G., Koohpayeh A., and Karimi I. The wound healing activity of flower extracts of Punica granatum and Aachillea kellalensis in wistar rats. Acta Poloniae Pharmaceutica  Drug Res., 67: 1070-1110 (2010).

6.            Benedec D., Hanganu D., Oniga I., Filip L., Bischin C. , Silaghi-Dumitrescu R.,  Tiperciuc B., and Vlase L.  Achillea schurii flowers: chemical, antioxidant, and antimicrobial investigations. Molecules, 21: 1050-1061 (2016).

7.            Benedec D., Vlase L., Oniga I., Mot A.C., Damian G., Hanganu D., Duma M., and Silaghi-Dumitrescu R. Polyphenolic composition, antioxidant and antibacterial activities for two Romanian subspecies of Achillea distans  Waldst. et Kit. ex Willd. Molecules, 18: 8725-8739 (2013).

8.            Bainbridge P . Wound healing and the role of fibroblasts. J. Wound Care, 22: 407-412 (2013).

9.            Azwanida N. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med. Aromat. Plants, 4: 777-780 (2015).

10.          Pattanayak S., Mazumder P.M., Sunita P. Total phenolic content, flavonoid content and in vitro antioxidant activities of Dendrophthoe falcata (lf) ettingsh. Res. J. Med. Plant, 6: 136-148 (2012).

11.          Chang C.C., Yang M.H., Wen H.M., and Chern J.C. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food  Drug Anal., 10: 178-182 (2002).

12.          Stockert J.C., Blázquez-Castro A., Canete M., Horobinb R.W., and Villanuevaa A. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochemica, 114: 785-796 (2012).

13.          Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth., 65: 55-63 (1983).

14.          Phan T.T., Hughes M.A., and Cherry G.W. Effects of an aqueous extract from the leaves of Chromolaena odorata (eupolin) on the proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization. Wound Repair Regen., 9: 305-313 (2001).

15.          Bielefeld K.A., Amini-Nik S., and Alman B.A. Cutaneous wound healing: recruiting developmental pathways for regeneration.  Cell. Mol. Life Sci., 70: 2059-2081 (2013).

16.          Akkol E.K., Koca U., Pesin I., and Yilmazer D. Evaluation of the wound healing potential of Aachillea biebersteinii Afan.(Asteraceae) by in vivo excision and incision models. Evid-Based Compl. Alt., 2011: 1-7 (2011).

17.          Roy P., Amdekar S., Singh R., Sharma P., and Singh V. In vivo antioxidative property, antimicrobial and wound healing activity of flower extracts of Pyrostegia venusta (Ker Gawl) Miers. J. Ethnopharmacol., 140: 186-192 (2012).

18.          Sorensen O.E., Cowland J.B., Theilgaard-Mönch K., Liu L., Ganz T., and Borregaard N. Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J. Immunol., 170: 5583-5589 (2003).

19.          Lisse T.S., King B. L., and Rieger S. Comparative transcriptomic profiling of hydrogen peroxide signaling networks in zebrafish and human keratinocytes: Implications toward conservation, migration and wound healing. Sci. Rep., 6: 203-220 (2016).

20.          Loo A. E. K., Ho R., and Halliwell B. Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model. Free Radic. Biol. Med., 51: 884-892 (2011).

21.          Kurahashi T., and Fujii J. Roles of antioxidative enzymes in wound healing. J. Dev. Biol., 3: 57-70 (2015).

22.          Sökmen A., Sökmen M., Daferera D., Polissiou M., Candan F., Ünlü M., and Akpulat H.A. The in vitro antioxidant and antimicrobial activities of the essential oil and methanol extracts of Aachillea biebersteini Afan.(Asteraceae). Phytother. Res., 18: 451-456 (2004).

23.          Alizadeh O.F., Albadi S., Javidnia K., and Miri M. Study of antioxidant activity and total phenolic content of 22 plants from Compositae family. Res. Pharm. sci., 7: S766 (2012).

24.          Varasteh-kojourian M., Abrishamchi P., Matin M. M., Asili J., Ejtehadi H., and Khosravi F. Antioxidant, cytotoxic and DNA protective properties of Achillea eriophora D.C. and Achillea biebersteinii Afan. extracts: A comparative study. Avicenna J. Phytomed., 7: 157-168 (2017).

25.          Benedek B., Kopp B., and Melzig M.F. Aachillea millefolium. Sl–is the anti-inflammatory activity mediated by protease inhibition? J. ethnopharmacol., 113: 312-317 (2007).

26.          Karlova K. Accumulation of flavonoid compounds in flowering shoots of Aachillea collina becker ex. Rchb. Alba during flower development. J. Hortic. Sci. UZPI, 33: 158-162 (2006).

27.          Tuberoso C.I.G., Montoro P., Piacente S., Corona G., Deiana M., Dessì M.A., Pizza C., and Cabras P. Flavonoid characterization and antioxidant activity of hydroalcoholic extracts from Aachillea ligustica All. J. Pharm. Biomed. Anal., 50: 440-448 (2009).

28.          Csupor‐Löffler B., Hajdú Z., Zupkó I., Réthy B., Falkay G., Forgo P., and Hohmann J. Antiproliferative effect of flavonoids and sesquiterpenoids from Aachillea millefolium SL on cultured human tumour cell lines. Phytother. Res., 23: 672-676 (2009).

29.          Sghaier M.B., Skandrani I., Nasr N., Franca M.G.D., Chekir-Ghedira L., and Ghedira K. Flavonoids and sesquiterpenes from Tecurium ramosissimum promote antiproliferation of human cancer cells and enhance antioxidant activity: A structure–activity relationship study. Environ. Toxicol. Pharmacol., 32: 336-348 (2011).

30.          Siekmann T.R.L., Burgazli K. M., Bobrich M.A., Nöll G., and Erdogan A. The antiproliferative effect of pinostrobin on human umbilical vein endothelial cells. Eur.  Rev. Med. Pharmacol. Sci., 17: 668-672 (2013).

31.          Kim S.J., Lim M.H., Chun I.K., and Won Y.H. Effects of flavonoids of Ginkgo biloba on proliferation of human skin fibroblast. Skin Pharmacol. Physiol., 10: 200-205 (1997).

32.          Dinda M., Mazumdar S., Das S., Ganguly D., Dasgupta U.B., Dutta A., Jana K., and Karmakar P. The water fraction of Calendula officinalis hydroethanol extract stimulates in vitro and in vivo proliferation of dermal fibroblasts in wound healing. Phytother. Res., 30: 878-891 (2016).

33.          Tsuruya M., Niwano Y., Nakamura K., Kanno T., Nakashima T., Egusa H., and Sasaki K. Acceleration of proliferative response of mouse fibroblasts by short-time pretreatment with polyphenols. Appl. Biochem. Biotechnol., 4: 777-780 (2014).

34.          Chen P.N., Chu S.C., Chiou H.L., Kuo W.H., Chiang C.L., and Hsieh Y.S. Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer lett., 235: 248-259 (2006).

35.          Shen K.H., Hung S.H., Yin L.T., Huang C.S., Chao C.H., Liu C.L., and Shih Y.W. Acacetin, a flavonoid, inhibits the invasion and migration of human prostate cancer du145 cells via inactivation of the p38 mapk signaling pathway. Mol. Cell Biochem., 333: 279-291 (2010).

36.          Seo G.Y., Lim Y., Koh D., Huh J.S., Hyun C., Kim Y.M., and Cho M. TMF and glycitin act synergistically on keratinocytes and fibroblasts to promote wound healing and antiscarring activity. Exp. Mol. Med., 49: 167-180 (2017).

37.          Cho M., Yoon H., Park M., Kim Y.H., and Lim Y. Flavonoids promoting hacat migration: I. Hologram quantitative structure–activity relationships. Phytomedicine., 2: 560-569 (2014).

38.          Kim Y.M., Huh J.S., Lim Y., and Cho M. Soy isoflavone glycitin  promotes human dermal fibroblast cell proliferation and migration via TGF-β signaling. Phytother. Res., 29: 757-769 (2015).

39.          San Miguel S.M., Opperman L.A., Allen E.P., Zielinski J., and Svoboda K.K. Bioactive antioxidant mixtures promote proliferation and migration on human oral fibroblasts. Arch. Oral. Biol., 56: 812-822 (2011).

40.          Harishkumar M., Masatoshi Y., Hiroshi S., Tsuyomu I., and Masugi M. Revealing the mechanism of in vitro wound healing properties of Citrus tamurana extract.  Bio. Med. Res. Int., 2013: 1-8 (2013).

41.          Agar O.T., Dikmen M., Ozturk N., Yilmaz M. A., Temel H., and Turkmenoglu F.P. Comparative studies on phenolic composition, antioxidant, wound healing and cytotoxic activities of selected Achillea L. species growing in Turkey. Molecules., 20:17976-18000 (2015).