Achillea Biebersteinii Afan. (Asteraceae) by in Vivo Excision and Incision Models
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Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 474026, 7 pages doi:10.1093/ecam/nep039 Original Article Evaluation of the Wound Healing Potential of Achillea biebersteinii Afan. (Asteraceae) by In Vivo Excision and Incision Models Esra Kupeli¨ Akkol,1 Ufuk Koca,1 Ipek Pesin,1 and Demet Yilmazer2 1 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Turkey 2 Department of Pathology, Dı¸kaps ı Yıldırım Beyazit Education and Research Hospital, Ankara, Turkey Correspondence should be addressed to Esra Kupeli¨ Akkol, [email protected] Received 10 December 2008; Accepted 7 April 2009 Copyright © 2011 Esra Kupeli¨ Akkol et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Achillea species are widely used for diarrhea, abdominal pain, stomachache and healing of wounds in folk medicine. To evaluate the wound healing activity of the plant, extracts were prepared with different solvents; hexane, chloroform, ethyl acetate and methanol, respectively from the roots of Achillea biebersteinii. Linear incision by using tensiometer and circular excision wound models were employed on mice and rats. The wound healing effect was comparatively evaluated with the standard skin ointment Madecassol. The n-hexane extract treated groups of animals showed 84.2% contraction, which was close to contraction value of the reference drug Madecassol (100%). On the other hand the same extract on incision wound model demonstrated a significant increase (40.1%) in wound tensile strength as compared to other groups. The results of histoptological examination supported the outcome of linear incision and circular excision wound models as well. The experimental data demonstrated that A. biebersteinii displayed remarkable wound healing activity. 1. Introduction A. biebersteinii Afan. [Asteraceae, Section: Filipendulinae (D.C.) Boiss] (syn. A. micrantha) is a perennial herb, villose, The genus Achillea (Asteraceae), named after the mytholog- stems erect, simple or branched from the base; 30–60 cm ical Greek warrior Achilles, who used Achillea species for high; leaves up to 10 cm, oblong-lanceolate in outline, pin- healing wounded-soldiers during the Trojan War [1]. The natisect into numerous narrow segments, segments divided genus Achillea comprises of ∼85 species, most of which into minute linear-lanceolate mucronate lobes; the heads are endemic to Europe and the Middle East. Turkish flora are radiate, in large dense compound corymbs; involucre 4- possesses 42 Achillea species and 23 of them are endemic [2]. 5 mm, oblong-ovoid; flowering period, April-May. These species have some interesting properties and are used Several biological activity studies have been performed in cosmetics, fragrances and agriculture, for example, plant on various Achillea species, including antibacterial, antiox- protection [3]. Some Achillea species have been known to idant, anti-inflammatory and antispasmodic activities [10– be ethnopharmacologically used in folk remedies for various 13]. purposes such as hemorrhoid and wound healing [4]. Herbal The aim of the present study was to investigate the in vivo teas prepared from some Achillea species are very often used wound healing activity of A. biebersteinii in order to elucidate in folk medicine as diuretic, for abdominal pain, against traditional use of this plant from the scientific point of diarrhea, flatulence and emmenagog, moreover for wound view. The hexane, chloroform, ethyl acetate and methanolic healing purposses, in Turkey [5–7]. extracts prepared from the aerial parts of the mentioned Achillea biebersteinii is locally named Sarıc¸ic¸ek in Turk- plant were tested in mice and rats for wound healing activity ish, and other species widely used as a folk remedy to treat using in vivo circular excision and linear incision wound abdominal pain, wounds and stomachache as well [8, 9]. models. 2 Evidence-Based Complementary and Alternative Medicine 2. Materials and Methods The vehicle group of animals was treated with the ointment only. The negative control group of animals was 2.1. Plant Material. Achillea biebersteinii Afan. aerial parts not treated with a material. Commercial Madecassol, 0.5 g were collected from Beynam forest, Ankara, Turkey, in (Bayer) was applied as a reference drug. May 2007 and was identified by Prof. Dr M. Vural from the Department of Botany, Faculty of Art and Science, 2.6. Linear Incision Wound Model. All the animals were Gazi University. A voucher specimen was deposited in the anaesthetized with 0.15 cc Ketalar and the back hair of Herbarium of Faculty of Pharmacy, Gazi University (GUE- the rats were shaved by using a shaving machine. Five 2602). centimeters long, two linear-paravertebral incisions were made with a sterile blade through the full thickness of the 2.2. Extraction. Following dissection of the aerial parts of skin at the distance of 1.5 cm from the midline of each side A. biebersteinii Afan., they were shade dried. Each 30 g of the vertebral column [14]. The wounds were closed with aerial parts was submitted to successive solvent extractions three surgical interrupted sutures of 1 cm apart. The animals seperatelly with n-hexane, chloroform, ethyl acetate and were divided into four groups. The extracts, the reference methanol at room temperature for a day (500 ml each material (Madecassol) and the vehicle were topically applied solvent). This was repeated in two trials. After filtration, the once in a day through 9 days. The fourth group, negative ◦ extracts were evaporated at 40 C (Buchi, Switzerland) to control group of animals, was not treated with a material. dryness in vacuo. Yields of each extracts were 7.9% for n- All the sutures were removed on the 9th post wound day. hexane, 4.8% for diethyl ether, 1.3% for ethyl acetate and On day 10 all the animals were killed with ether anesthesia. 15.3% for methanol. One linear-paravertebral incised skin was measured using tensiometer (Zwick/Roell Z0.5, Germany) for its tensile 2.3. GC-MS Analysis of the n-Hexane Fraction. Chromato- strength, the other incised skin was sent for histopathological graphic analysis was carried out on Agilent 6890N Network examination [15, 16]. GC system combined with Agilent 5973 Network Mass Selective Detector (GC-MS). The capillary column used was 2.7. Excision Wound Model. This model was used to monitor an HP 5 MS Column (30 m × 0.25 mm × 0.25 μm). Helium wound contraction and wound closure time. Each group wasusedascarriergasataflowrateof1ml/minwith1μl of animals (six-animal each) was anaesthetized by 0.01 cc injection volume. Samples were analyzed with the column Ketalar. The back hairs of the mice were depilated by shaving. ◦ held initially 60 C for 1 min after injection with 1 min hold The circular wound was created on the dorsal interscapular ◦ ◦ time, then increased to 180 Cwith3C/min heating ramp region of each animal by excising the skin with a 5 mm ◦ andkeptat180C for 20 min. The injection was performed biopsy punch; wounds were left open [17]. The extracts, in splitless mode. Detector and injector temperatures were the reference material (Madecassol Bayer) and the vehicle ◦ ◦ 280 C and 250 C, respectively. Run time was 60 min. MS ointment were administered once a day till the wound was scan range was (m/z): 35–500 atomic mass units (AMU) completely healed. The progressive changes in wound area under electron impact (EI) ionization (70 eV). were monitored by a camera (Fuji, S20 Pro, Japan) every other day. Later on, wound area evaluated by using AutoCAD 2.4. Animals. Male, Sprague-Dawley rats (160–180 g body program. Wound contraction was calculated as percentage of weight) and Swiss albino mice (20–25 g body weight) were the reduction in wounded area. A specimen sample of tissue purchased from the animal breeding laboratories of Refik was isolated from the healed skin of each group of mice for Saydam Central Institute of Health (Ankara, Turkey). the histopathological examination [18]. The animals left for 3 days at room conditions for acclimatization. They were maintained on standard pellet 2.8. Histological Study. Sample tissues were fixed in 10% diet and water ad libitum throughout the experiment. A formalin and were embedded in paraffin wax. Serial sections minimum of six animals were used in each group, otherwise (5 μm thickness) of paraffin embedded tissues were cut. The described in procedure. The study was permitted by the tissues were stained by haematoxylin and eosin, which were Institutional Animal Ethics Committee (Gazi University examined by light microscope (Olympus BX51). Ulceration, Ethical Council Project Number: G.U.ET-08.037) and was necrosis and epithelisation were evaluated in the skin tissues. performed according to the international rules considering Alsocongestion,edema,PNL,mononuclearcells,fibroblasts the animal experiments and biodiversity right. and vascularisation were qualitatively evaluated as −,+,++ and +++. 2.5. Wound Healing Activity. Incision and excision wound models were used to evaluate the wound healing activity. 2.9. Statistical Analysis of the Data. The data on percentage For the in vivo wound models, samples were prepared in the wound healing was statistically analyzed using one-way ointment consists of propylene glycol : liquid paraffin(6:1) analysis of variance (ANOVA). The values of P ≤ .001 were and applied topically onto the test animals Extracts were considered statistically significant. prepared as 1% in the ointment, and 0.5 mg of it applied Mann-Whitney U, Kruskal-Wallis and chi-squared tests on the wound sides immediately after wound was created were used for the statistical analysis of the histopathological artificially. data. Evidence-Based Complementary and Alternative Medicine 3 30 30 25 25 20 15 2 20 10 Tensile strength (N) 5 15 0 e Woundareamm 10 Vehicl Madecassol 5 -hexane extract Negative control n Methanol extract Chloroform extract Ethyl acetate extract 0 Material 024681012 ff (day) Figure 2: E ects of the extracts from A.