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Wound healing and anti-inflammatory activity of some Ononis taxons
Biomedicine & Pharmacotherapy 91 (2017) 1096–1105
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Original article
Wound healing and anti-inflammatory activity of some Ononis taxons b _ _ Burçin Ergene Öza,* , Gülçin Saltan Işcan , Esra Küpeli Akkolb , Ipek Süntarb , c a Hikmet Keleş , Özlem Bahadır Acıkara a
Ankara University Faculty of Pharmacy, Pharmacognosy Department, 06100, Tandogan, Çankaya, Ankara, Turkey Gazi University Faculty of Pharmacy, Pharmacognosy Department, 06330, Etiler, Yenimahalle, Ankara, Turkey c Afyon Kocatepe University, Faculty of Veterinary Medicine, Pathology Department, 03200, Afyonkarahisar, Turkey b
A R T I C L E I N F O
Article history: Received 23 March 2017 Received in revised form 5 May 2017 Accepted 8 May 2017 Keywords: Fabaceae Inflammation Ononis Wound healing
A B S T R A C T
Ononis species are used for their laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic and antifungal effects as well as against skin diseases for wound healing activity. In the light of this information n-hexane, ethylacetate and methanol extracts prepared from Ononis spinosa L. subsp. leiosperma (Boiss.) Sirj., Ononis variegata L., Ononis viscosa L. subsp. brevifolia (DC) Nym. and Ononis natrix L. subsp. natrix L. were tested for their wound healing, anti-inflammatory and antioxidant activities. Linear incision and circular excision wound models and hydroxypyroline estimation assay were used for the wound healing activity. For the assessment of chronic inflammation FCA-induced arthritis and for acute inflammation carrageenan-induced hind paw edema, TPA-induced ear edema and acetic acidinduced increase in capillary permeability tests were conducted. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging activity assay, reducing power assay and hydroxyl radical (OH ) scavenging assay were used for determining antioxidant activities of the extracts. Results showed that O. spinosa subsp. leiosperma roots ethyl acetate extract exhibited remarkable wound healing activity with the 42.6% tensile strength value on the linear incision wound model and 60.1% reduction of the wound area at the day 12 on the circular excision wound model. Hydroxyproline content of the tissue treated by O. spinosa subsp. leiosperma roots ethyl acetate extract was found to be 41.3 mg/mg. Acetic acid induced increase in capillary permeability test results revealed that O. spinosa subsp. leiosperma roots ethyl acetate extract and O. spinosa subsp. leiosperma roots methanol extract inhibited inflammation by 40.4% and 35.4% values respectively. O. spinosa subsp. leiosperma roots ethyl acetate extract showed 21.2-27.2% inhibition in carrageenaninduced hind paw edema test while did not posses activity on TPA-induced ear edema and FCA-induced arthritis models. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction The genus Ononis L. which is a member of Fabaceae family, grows widespread in Europe, Atlantic Islands, West Asia and North Africa. The genus is represented by more than 75 species all over the world and by 18 species in Turkey. This genus is especially distributed closed to the coasts of Mediterranean Sea [1–3]. It is reported that Ononis species are used for their laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic and antifungal effects as well as against skin diseases for wound healing activity [4,5]. The roots of Ononis spinosa L., Ononis arvensis L., Ononis hircina Jacg. and Ononis antiquorum L. are known to be
* Corresponding author. E-mail address: [email protected] (B. Ergene Öz). http://dx.doi.org/10.1016/j.biopha.2017.05.040 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
used against irritations of the skin, itches, wounds and dermatitis in Central Asia and Russia [6]. The extracts prepared from O. natrix are used as antirheumatismal, antibacterial, diuretic, urolithiatic and to reduce blood pressure [1,7,8]. Ethnobotanical studies revealed that other species of this genus such as O. sicula (Guss.) Hub.-Mor. and O. hirta Desf. were used for the wound healing as well as against cold sore as antiseptic [9]. O. spinosa which is widely known as “kayiskiran” in Turkey, is used as a folk remedy against urinary tract diseases and kidney stones due to its antiinflammatory and diuretic effect as well as against eczema and some other skin disorders for wound healing [10,11].
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Phytochemical studies on Ononis species reveals that these plants contains sugar, tannin, saponin, lectins, flavonoid derivatives (such as trifolirhizin, ononin, formononetin, genistein, genistin, biochanin, daidzin, glycitin, glycitein, sissotrin), phenolic acids (gallic, protocathechic, p-hydroxybenzoic, vanillic, cafeic, syringic, p-coumaric, ferulic, sinapic acid), essential oils (anethol, carvon and menthol as major components), sterols (b-sitosterol, campesterol, stigmasterol, stigmastan-3,5-dien), terpenic substances (b-amyrin, a-onocerin), anthranilic acid and resorcinol derivatives, aromatic lactones and coumarines [1,12–16]. Although there are several ethnopharmacological researches that report the traditional uses of Ononis species, the studies, which scientifically prove the traditional uses are insufficient. In this study, four species of the genus Ononis were selected and it is aimed to assess wound healing and anti-inflammatory activities as well as antioxidant capacities of the extracts. For this purpose; extracts were prepared from the roots and aerial parts of the selected species, namely O. spinosa L. subsp. leiosperma (Boiss.) Sirj., O. variegata L., O. viscosa L. subsp. brevifolia (DC) Nym. and O. natrix L. subsp. natrix L. by using n-hexane, ethylacetate and methanol respectively. Wound healing, anti-inflammatory and antioxidant activities of the extracts were investigated. Linear incision and circular excision wound models and hydroxypyroline estimation assay were conducted for the investigation of wound healing activity. Acute inflammation models i.e. acetic acidinduced increase in capillary permeability test, carrageenaninduced hind paw edema and TPA-induced ear edema models as well as a chronic inflammation model i.e. FCA-induced arthritis model were used for the assessment of anti-inflammatory activity. The antioxidant activities of the extracts were also evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) scavenging activity assay, reducing power assay and hydroxyl radical (OH) scavenging assay
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repeated three times with fresh solvent for each extract. Following the extraction, each extract was filtered and the solvent was removed under reduced pressure at 50 C. 2.3. In vivo biological activity tests 2.3.1. Animals Male Swiss albino mice (20–25 g) and Sprague-Dawley rats (160–180 g) were obtained from Kobay A.Ş. (Ankara, Turkey). The animals were kept in animal room conditions for 3 days and were fed on standard pellet diet and water ad libitum. The groups of six animals were used for the biological activity tests. The animals used for the bioactivity tests were maintained in accordance with the directions of Guide for the Care and Use of Laboratory Animals, and the experiment was approved by the Experimental Animal Ethics Committee of Gazi University (Gazi University Ethical Council Project Number: G.U.ET-11.058). 2.3.2. Preparation of test samples for bioassay The test materials were mixed with an ointment base and topically applied onto the wounded area on dorsal part of the test animals for the assessment of the wound healing activity. The extracts and fractions were mixed with glycol stearate: propylene glycol and liquid paraffin (3:6:1) mixture in a mortar. The sample ointments were applied daily on the wounded area following the formation of the wound. The control group was treated with blank vehicle base, and the negative control was not treated with any product. Madecassol1 (Bayer) (0.5 g) was used as reference drug. The samples were suspended in a mixture of distilled water and 0.5% sodium carboxymethyl cellulose (CMC) and given orally to test animals for the evaluation of the anti-inflammatory activity. The same mixture containing appropriate volumes of the dosing vehicle was given to the control group instead of sample. Indomethacin (10 mg/kg) in 0.5% CMC was used as a reference drug.
2. Materials and methods 2.3.3. Wound healing activity 2.1. Plant material Plant samples were collected from different localities, from South and Central Anatolia. Taxonomic identification of the plants were confirmed by Hayri Duman from Gazi University, Department of Biological Sciences, Faculty of Art and Sciences and Osman Tugay from Selcuk University, Department of Biological Sciences, Faculty of Art and Sciences. The voucher specimens are kept in the herbarium of Ankara University Faculty of Pharmacy (AEF). The localities and dates that the plant materials were collected from and herbarium numbers are shown in Table 1. 2.2. Extraction The aerial parts and roots of the plant materials were seperated and dried at room temperature. Dried and powdered materials (30 g) were extracted with n-hexane (300 mL x 3), ethyl acetate (300 mL x 3) and methanol (300 mL x 3) respectively at room temperature by continuous stirring. 24 h of extraction period was
2.3.3.1. Linear incision wound model. The rats were anaesthetized with 0.15 cc Ketasol1 (Richterpharma). The dorsal area of the rats were shaved and disinfected with 70% alcohol. Two 5 cm-length linear-paravertebral incisions were formed with a sterile blade at the distance of 1.5 cm from the dorsal midline on each side. Three surgical sutures were placed each 1 cm apart. The ointments were topically applied on the wounds once a day for 9 days. On the last day, the sutures were removed and tensile strength of wounded and treated skin was measured by using a tensiometer (Zwick/ Roell Z0.5, Germany) [17–19]. 2.3.3.2. Circular excision wound model. The mice were anaesthetized with 0.15 cc Ketasol1 (Richterpharma). A circular wound was created on the dorsal interscapular region of each mouse by excising the skin with a 5 mm biopsy punch (Nopa instruments, Germany) after the dorsal parts of the mice were shaved. Wounds were left open [20]. Untill the wound was completely healed, test ointments were applied topically once a
Table 1 The localities and dates that the plant materials were collected from and herbarium numbers. Plant Material
Herbarium Number
Locality
Collection Date
Taxonomic Identification
O. spinosa L. subsp. leiosperma (Boiss.) Sirj. O.variegata L. O. viscosa L. subsp. brevifolia (DC) Nym. O. natrix L. subsp. natrix L.
AEF AEF AEF AEF
Konya, Aliçerçi Village-Ahırlı roadside Antalya, Side, Sorgun, seaside Antalya, Akseki, Çukurköy, Karaorman Antalya, Fethiye-Korkuteli road side
June 2011 June 2011 May 2011 May 2012
H. H. H. O.
26000 25998 25997 26328
Duman Duman Duman Tugay
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day and the wound areas were monitored by a camera (Fuji, S20 Pro, Japan) everyday. AutoCAD program was used to evaluate wound area. Wound contraction was calculated as percentage of the reduction in wounded area. On day 12, sample from the tissue was seperated from the healed skin of each group for the histopathological examination [19].
2.3.3.3. Histopathology. The skin specimens were fixed in 10% buffered formalin, processed and blocked with paraffin The samples were cut into 5 micrometer sections, stained with hematoxylin & eosin (HE) and Van Gieson (VG) stains. A light microscope (Olympus CX41 attached Kameram1 Digital Image Analyze System) was used for the examination of the tissues. The results were given as mild (+), moderate (++) and severe (+++) for epidermal or dermal re-modeling. Re-epithelization or ulcus in epidermis; fibroblast proliferation, mononuclear and/or polymorphonuclear cells, neo-vascularization and collagen depositions in dermis were analyzed to score the epidermal or dermal re-modeling. Van Gieson stained sections were analyzed for collagen deposition. At the end of the examination, the wound healing phases as inflammation, proliferation, and remodeling in all groups were staged according to all of the wound healing processes 2.3.3.4. Hydroxyproline estimation. Isolated tissues were dried in hot air oven at 60–70 C untill a consistent weight was achieved. The tissues were hydrolyzed with 6 N HCl for 3 h at 130 C, the solution was adjusted to pH 7 and subjected to chloramin T oxidation. Absorbance of the colored adduct-product formed with Ehrlich reagent at 60 C was read at 557 nm. The values for the standard hydroxyproline was also reported as mg/mg dry weight of tissue [19,21]. 2.3.4. Anti-inflammatory activity 2.3.4.1. Carrageenan-induced hind paw edema. The method developed by Kasahara et al. [22] was used with some modifications [23]. The suspension of carrageenan (0.5 mg/ 25 mL) (Sigma, St. Louis, Missouri, USA) in physiological saline (154 mM NaCl) was prepared freshly and the suspension was injected into the subplantar tissues of the right hind paw of each mouse, sixty minutes after the oral administration of test sample/ dosing vehicle. 25 mL saline solution was injected into that of the left hind paw as the control. Paw edema was measured in every 90 min during 6 h following the induction of inflammation, a pair of dial thickness gauge callipers (Ozaki Co., Tokyo, Japan) was used to measure the difference in footpad thickness between the right and left foot. Mean values of treated groups and control group were compared and analyzed using statistical methods. Acetic acid-induced increase in capillary 2.3.4.2. permeability. Whittle method was used with some modifications for the determination of the effect of the test samples on the increased vascular permeability induced by acetic acid in mice [24]. Test samples were given orally to the mice in 0.2 mL/20 g body weight. Thirty minutes after the administration 0.1 mL of 4% Evans blue (Sigma, St. Louis, Missouri, USA) in saline solution (iv.) was injected to the tails of each mouse. 0.4 mL of 0.5% (v/v) AcOH was injected ip 10 min after the i.v. injection of the dye solution. The mice were killed by dislocation of the neck after 20 min and the viscera were exposed and irrigated with distilled water, then poured into 10 mL volumetric flasks through glass wool. Each flask was made up to 10 mL with distilled water, 0.1 of 0.1 N NaOH solution was added to the flask and the absorption of the final solution was measured at 590 nm (Beckmann Dual
Spectrometer). A mixture of distilled water and 0.5% CMC was used for the control group. 2.3.4.3. TPA-induced mouse ear edema. 2.5 mg of TPA was dissolved in 20 mL of ethanol (70%) and the solution was applied by an automatic pipette in 20 mL volumes to both anterior and posterior surfaces of the right ear of each mouse. The same volume of ethanol (70%) was applied to the left ear as control at the same time. Indomethacin (0.5 mg/ear) was used as reference drug. Two different measurements were taken as given below [25]. The thickness of each ear was measured using gauge callipers (Ozaki Co., Tokyo, Japan) four hours after the induction of inflammation. The difference between the right and left ears due to TPA application was calculated and inhibition percentage was expressed as a reduction thickness with respect to the control group. At the end of 4 h following the administration, the animals were sacrificed. Discs of 6 mm diameter were cut from each ear and weighed in balance. The swelling was estimated as the difference in weight between the punches from right and left ears and expressed as an increase in the ear thickness. 2.3.4.4. Adjuvant-induced chronic arthritis. The method described by Newbould [26] was used with some modifications. Test samples were administered to rats once a day by a feeding needle for 14 days. 25 mL of Freund’s Complete Adjuvant (FCA) (Sigma, St. Louis, Missouri, USA) was injected into subplantar tissue of the right hind paw of each rat on the third day. A pair of thickness gauge callipers was used to measure swelling in the injected and contralateral hind paws. The measurements were made every day from day 3 to 14, and at the end, on day 21. The differences in Table 2 Effects of the extracts on linear incision wound model. Material
Statistical Mean SEM
Tensile Strength%
Vehicle Negative control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Madecassol1
12.38 1.19 11.86 1.27 12.99 1.26 13.37 1.43 9.94 1.16 11.85 1.91 16.23 1.72 15.42 1.34 11.70 1.28 14.11 1.94 14.78 1.31 11.41 1.57 17.66 0.93 16.77 1.09 11.35 1.34 13.01 1.64 13.20 1.23 10.03 1.72 13.17 1.40 14.04 1.05 11.20 1.38 11.72 1.63 11.84 1.25 10.11 1.86 13.02 1.53 12.84 1.22 19.68 0.82
4.4 – 4.9 7.9 19.7 – 31.1* 24.6 – 13.9 19.4 – 42.6*** 35.5** – 5.1 6.6 – 6.4 13.4 – – – – 5.2 3.7 58.9***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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severity of arthritis between the experimental groups and the control group were statistically analyzed [27].
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2.4. In vitro biological activity tests 2.4.1. Antioxidant activity
2.3.5. Statistical analysis of the data The activities were statistically analyzed using one-way analysis of variance (ANOVA) and Students-Newman-Keuls posthoc test. The values of p 0.05 were considered statistically significant. Histopathologic data were considered to be nonparametric; therefore, no statistical tests were performed.
2.4.1.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The samples and the reference compound (ascorbic acid) were dissolved in methanol and the solutions were mixed with DPPH solution (80 mg/mL). Remaining DPPH amount was measured at 517 nm using spectrophotometer. DPPH inhibition activities were calculated according to the following formula:
Table 3 Effects of the extracts on circular excision wound model. Wound Area (mm2) SEM (Contraction%)
Material Day
0
2
4
6
8
10
12
Vehicle
19.39 1.84
Negative control Hex-NH
20.02 1.61 19.48 1.73
EtOAc-NH
19.36 1.59
MeOH
19.88 1.91
17.91 2.15 (1.6) 18.21 2.17 16.88 2.19 (5.8) 17.21 2.85 (3.9) 17.26 2.26 (3.6) 19.24 2.46 – 16.32 2.48 (8.9) 17.23 2.54 (3.8) 18.61 2.45 – 18.32 2.14 – 18.05 2.15 – 17.96 2.03 – 16.02 1.88 (10.6) 16.29 1.96 (9.0) 18.06 1.99 – 17.88 2.03 (0.2) 17.96 2.14 – 18.82 2.10 – 17.40 1.96 (2.8) 17.26 1.85 (3.6) 17.21 2.08 (3.9) 18.12 2.01 – 17.95 2.13 – 18.03 2.19 – 18.10 1.99 – 16.07 1.91 (10.3)
14.79 2.28 (2.8) 15.20 2.56 14.41 2.10 (2.6) 14.13 2.07 (6.7) 13.85 2.04 (6.4) 16.88 2.70 – 12.17 2.31 (17.7) 12.87 2.46 (12.9) 15.66 1.97 – 13.82 2.24 (6.6) 15.39 2.20 – 16.35 2.16 – 13.28 1.75 (10.2) 14.01 1.85 (5.3) 15.13 2.19 – 14.91 1.69 – 15.16 2.11 – 14.88 2.21 – 15.01 2.16 – 12.91 2.21 (12.7) 13.98 2.03 – 14.95 2.16 – 15.24 2.22 – 15.08 2.20 – 14.92 2.07 – 12.04 1.56 (18.6)
11.22 2.13 (8.9) 12.31 2.08 10.93 2.07 (2.6) 11.10 1.89 (1.07) 10.91 1.26 (2.8) 14.12 2.51 – 8.86 1.42 (21.0) 8.96 1.39 (20.1) 12.41 2.02 – 11.36 1.95 – 10.67 2.07 (4.9) 11.29 1.83 – 10.22 1.79 (8.9) 10.92 1.17 (2.7) 12.26 2.08 – 13.16 1.45 – 12.07 1.88 – 12.06 1.96 – 11.45 2.08 – 9.44 1.60 (15.9) 11.96 1.97 – 11.38 2.16 – 12.06 1.15 – 11.77 1.67 – 11.29 2.10 – 7.01 1.06 (37.5)*
7.25 2.40 (8.3) 7.91 1.74 7.14 1.48 (1.5) 6.99 1.86 (3.6) 7.16 1.13 (1.2) 7.93 2.11 – 5.06 0.91 (30.2)* 5.28 0.95 (27.2) 7.11 2.45 (1.93) 7.08 2.07 (2.3) 6.95 1.93 (4.1) 7.36 2.33 – 5.10 0.91 (29.7) 6.33 1.42 (12.7) 7.12 1.26 (1.8) 8.14 2.43 – 7.05 0.78 (2.8) 7.36 2.26 – 7.48 2.41 – 5.39 0.86 (25.7) 7.04 2.13 – 8.01 2.33 – 7.16 1.21 (1.2) 7.80 1.95 – 7.93 1.97 – 4.08 0.75 (43.7)**
4.31 1.51 (6.5) 4.61 1.44 4.08 0.84 (5.3) 3.80 1.29 (11.8) 3.87 0.81 (10.2) 4.92 1.85 – 2.16 0.65 (49.9)** 3.07 0.89 (28.8) 4.58 1.66 – 4.19 1.92 (2.8) 4.17 1.19 (3.2) 4.48 1.59 – 2.98 0.83 (30.9)* 3.25 0.93 (24.6) 4.27 0.95 (0.9) 5.16 1.40 – 4.86 1.32 – 5.03 1.63 – 5.19 1.74 – 3.20 0.93 (25.8) 4.78 1.89 – 5.95 1.53 – 4.45 0.87 – 4.45 0.87 – 4.69 1.29 – 1.25 0.27 (70.9)**
2.73 0.98 (6.8) 2.93 0.79 2.43 0.54 (10.9) 2.10 0.38 (23.1) 2.30 0.67 (15.8) 2.85 0.71 – 1.79 0.50 (34.4)* 2.03 0.74 (25.6) 2.88 0.99 – 2.61 0.86 (4.4) 2.42 0.92 (11.4) 2.79 0.81 – 1.09 0.21 (60.1)** 1.75 0.42 (35.9)* 2.76 0.99 – 3.01 0.86 – 2.51 0.81 (8.1) 2.82 1.03 – 2.77 0.88 – 1.99 0.96 (27.1) 2.96 0.92 – 2.75 0.90 – 2.91 0.88 – 2.82 0.79 – 2.60 0.91 (4.8) 0.00. 0.00 (100.00)***
NH
Hex-NR
20.11 2.03
EtOAc-NR
19.65 1.57
MeOH
19.42 1.34
NR
Hex-SH
19.47 2.02
EtOAc-SH
19.58 1.99
MeOH-SH
20.01 2.02
Hex-SR
19.93 2.11
EtOAc-SR
19.62 1.46
MeOH-SR
20.14 2.10
Hex-VH
19.95 1.79
EtOAc-VH
19.81 1.59
MeOH-VH
19.40 1.22
Hex-VR
20.18 3.05
EtOAc-VR
20.31 3.13
MeOH-VR
19.59 1.54
Hex-VarH
19.48 2.36
EtOAc-VarH
20.04 2.95
MeOH-VarH
19.46 1.27
Hex-VarR
19.98 2.24
EtOAc-VarR Madecassol
1
19.52 2.30 19.70 1.32
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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Table 4 Effects of the extracts on hydroxyproline content. Material
Hydroxyproline (mg/mg) SEM
Vehicle Negative control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Madecassol1
9.4 1.23 8.5 1.64 11.8 1.22 10.5 1.27 10.8 1.14 9.1 1.17 30.3 0.88* 29.5 1.05 10.2 1.51 12.3 1.32 18.7 1.49 8.8 1.54 41.3 0.51** 32.4 0.83* 9.1 1.95 10.4 1.76 10.1 1.89 8.8 1.46 10.3 1.61 13.6 1.47 9.5 1.38 11.5 1.86 8.3 1.76 10.1 1.12 13.4 1.75 12.1 1.34 45.3 0.49***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
Inhibition(%) = (Acontrol Asample) 100/Acontrol where Acontrol represents the absorbance of the control, and Asample represents the absorbance of the extracts or reference. Experiments were run in duplicate and the results were expressed as inhibitory values [28]. 2.4.1.2. ABTS (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) scavenging activity. The aqueous solution of ABTS was mixed with K2S2O8 (2.45 mM) and the mixture was kept in dark for 12–16 h to generate ABTS+ radical (7 mM). The absorbance of the solution was adjusted to 0.700 (0.030) units at 734 nm. The sample solutions (15 mL) was mixed with the radical solution and reaction kinetic was measured through 15 min in 1 min periods. Concentration against percentage of inhibition was calculated as Trolox equivalent antioxidant concentration (TEAC) [28]. 2.4.1.3. Reducing power assay. 1 mL of the extract solution was mixed with 0.2 M phosphate buffer (pH: 6.6) (2.5 mL) and 1% potassium hexaferricyanide solution (2.5 mL) and the mixture was incubated at 50 C for 30 min. 2.5 mL of 10% trichloroacetic acid (TCA) was added and then the mixture was centrifuged for 10 min. 2.5 mL oft he upper layer, 2.5 mL distilled water and 0.5 mL of 0.1% aqueous ferric chloride were mixed and absorbance of the final solution was recorded at 700 nm. Reducing power of the extracts were expressed as ascorbic acid equivalents (AscAE), mmol ascorbic acid/g sample [29]. All analysis were performed as dublicate and expressed as mean values. 2.4.1.4. Hydroxyl radical (OH) scavenging assay. The mixture of 100 mL FeCl3 and 104 mM EDTA (1:1, v/v) solution was prepared. The reaction mixture contained 100 mL extract solution, 500 mL of 5.6 mM 2-deoxyribose prepared in phosphate buffer solution
Table 5 Inhibitory effect of the extracts on acetic acid-induced increase in capillary permeability test. Material Control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Indomethacin
Dose (mg/kg)
Evans blue concentration (mg/mL) SEM
Inhibition%
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 10
12.01 1.14 11.86 0.96 11.39 0.83 11.91 0.87 12.93 1.14 9.42 0.76 9.16 0.69 13.74 1.08 12.78 1.17 10.02 0.86 13.11 1.19 7.16 0.75 7.76 0.68 12.26 1.21 11.98 1.16 12.30 1.08 13.26 1.50 12.89 1.26 10.36 1.03 14.18 1.85 12.66 1.36 13.10 1.26 14.08 1.92 13.15 1.33 12.05 1.11 5.78 0.24
1.2 5.2 0.8 – 21.6 23.7 – – 16.6 – 40.4** 35.4* – 0.2 – – – 13.8 – – – – – – 51.9***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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Table 6 Effects of the extracts against carrageenan-induced paw edema in mice. Material
Dose (mg/kg)
Control Hex-NH
100
EtOAc-NH
100
MeOH
100
NH
Hex-NK
100
EtOAc-NK
100
MeOH
100
NK
Hex-SH
100
EtOAc-SH
100
MeOH-SH
100
Hex-SR
100
EtOAc-SR
100
MeOH-SR
100
Hex-VH
100
EtOAc-VH
100
MeOH-VH
100
Hex-VR
100
EtOAc-VR
100
MeOH-VR
100
Hex-VarH
100
EtOAc-VarH
100
MeOH-VarH
100
Hex-VarR
100
EtOAc-VarR
100
MeOH-VarR
100
Indomethacin
10
Swelling Thickness (10
2
mm) SEM (Inhibition%)
90 min
180 min
270 min
360 min
47.3 2.7 47.6 3.5 – 48.5 3.1 – 47.0 3.1 (0.6) 45.4 3.6 (4.0) 45.1 2.9 (4.7) 46.8 3.0 (1.1) 49.5 3.1 – 44.3 2.5 (6.3) 44.9 2.7 (5.1) 46.9 3.2 (0.8) 45.1 3.2 (4.7) 45.7 3.0 (3.4) 47.4 2.8 – 48.1 2.9 – 46.2 3.3 (2.3) 50.1 4.6 – 49.5 4.1 – 47.5 3.9 – 49.3 3.8 – 50.1 3.9 – 48.2 3.4 – 48.8 3.6 – 49.0 2.8 – 49.8 3.9 – 30.2 2.4 (36.2)**
50.1 3.3 51.5 2.5 – 51.5 4.2 – 42.7 3.3 (14.8) 52.5 3.5 – 41.6 3.6 (16.9) 44.8 3.0 (10.6) 52.4 3.5 – 49.3 3.3 (1.6) 46.0 3.5 (8.2) 50.3 4.8 – 39.5 2.4 (21.2)* 41.2 3.1 (17.8) 45.9 3.3 (8.3) 51.3 4.4 – 52.5 3.6 – 51.8 3.4 – 50.7 3.2 – 51.2 3.9 – 52.1 2.9 – 51.5 3.8 – 51.7 3.7 – 53.6 3.6 – 49.5 3.8 (1.2) 50.5 2.9 – 30.5 2.1 (39.1)***
53.9 3.1 52.6 3.3 (2.4) 54.2 3.8 – 49.3 3.2 (8.5) 49.7 2.8 (7.8) 38.4 3.0 (28.8) 42.9 2.3 (20.4) 54.8 3.7 – 54.1 3.9 – 53.0 3.4 (1.7) 55.6 3.7 – 40.7 3.0 (24.5)* 47.3 3.3 (12.2) 50.6 3.6 (6.1) 55.7 4.4 – 54.7 3.9 – 55.2 3.8 – 55.7 3.4 – 54.9 3.6 – 57.2 3.1 – 55.0 3.3 – 56.6 3.9 – 56.1 3.8 – 49.5 3.4 (8.2) 54.2 3.7 – 37.6 2.1 (30.2)**
60.2 3.4 59.3 2.7 (1.5) 62.6 4.7 – 56.8 3.8 (5.6) 59.2 3.1 (1.7) 41.9 3.1 (30.4)* 49.7 3.6 (17.4) 61.5 3.9 – 57.8 3.2 (3.9) 62.8 3.9 (4.1) 59.8 4.1 (0.6) 43.8 3.1 (27.2)* 56.7 3.5 (5.8) 59.5 3.8 (1.2) 62.2 4.3 – 62.7 4.5 – 61.8 3.9 – 57.4 3.8 (4.7) 56.6 4.1 (5.9) 60.5 4.4 – 60.3 4.0 – 61.2 3.7 – 61.4 4.3 – 57.6 3.7 (4.3) 64.1 3.9 – 36.8 1.9 (38.9)***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
(50 mM, pH 7.4), 200 mL of FeCl3 and EDTA mixture and 100 mL of 1.0 mM aqueous ascorbic acid solution were mixed by vortexing and incubated at 50 C for 30 min, 1 mL of 2.8% trichloroacetic acid and 1 mL of 1.0% thiobarbituric acid were added to the mixture and incubated at 50 C for another 30 min. The absorbance was measured at 532 nm. The percentage of inhibition was calculated according to the following formula by using the absorbance of the control (Ac) and the absorbance of the sample (As) and expressed as mannitol equivalent (mmolmannitol/gsample)
[28]. Results were given as the mean values of two analysis performed in parallel. [(Ac
As)/Ac] 100
3. Results and discussion Wound healing and anti-inflammatory activity results of nhexane (Hex), ethyl acetate (EtOAc) and methanol (MeOH) extracts
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Table 7 Effects of the extracts against TPA-induced ear edema in mice as measurement of swelling thickness and weight measurement of edema. Material Control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Indomethacin
Dose (mg/ear)
Swelling Thickness (mm) SEM
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
370.4 22.8 345.7 20.6 339.2 25.6 347.8 22.9 386.4 31.2 331.7 26.6 322.3 21.8 375.6 30.4 379.1 31.5 372.4 23.6 384.6 25.9 328.5 23.7 330.4 24.5 371.8 29.6 388.9 26.9 372.7 29.4 377.4 29.6 374.8 27.9 389.3 26.7 362.4 28.0 368.9 29.3 379.1 25.2 366.5 28.9 352.7 27.7 348.4 25.3 137.1 17.2
Inhibition%
Weight Edema (mg) SEM
Inhibition%
6.7 8.4 6.1 – 10.4 12.9 – – – – 11.3 10.8 – – – – – – 2.2 0.4 – 1.1 4.8 5.9 62.9***
28.4 3.1 27.8 3.4 26.5 3.8 26.8 2.6 29.2 3.6 24.7 2.9 23.5 3.3 31.2 3.0 30.7 3.2 29.9 2.6 30.1 3.8 22.4 2.9 23.6 2.2 27.9 3.2 29.6 3.1 30.3 3.4 31.4 3.6 29.0 3.2 33.7 3.8 25.6 3.1 27.1 3.6 28.5 3.5 31.7 3.3 29.8 2.9 28.1 3.6 12.4 2.4
2.1 6.7 5.6 – 13.0 17.3 – – – – 21.1 16.9 1.8 – – – – – 9.9 4.6 – – – 1.1 56.3***
*p < 0.05; **p < 0.01;SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. *** p < 0.001.
prepared from O. spinosa subsp. leiosperma (S), O. variegata (Var), O. viscosa subsp. brevifolia (V) ve O. natrix subsp. natrix (N) roots (R) ve aerial parts (H) are given in Tables 2–8. The ethyl acetate extract of O. spinosa subsp. leiosperma roots (EtOAc-SR) possessed remarkable wound healing activity with 42.6% tensile strength value on linear incision wound model (Table 2) and 60.1% reduction of the wound area at the day 12 on circular excision wound model (Table 3). According to hydroxyproline estimation; hydroxyproline content of the tissue treated by EtOAc-SR was found to be 41.3 mg/mg (Table 4). According to the results of acetic acid-induced increase in capillary permeability test, EtOAc-SR and MeOH-SR were found to exhibit remarkable anti-inflammatory activity with the inhibition values of 40.4% and 35.4%, respectively (Table 5). Carrageenaninduced hind paw edema test results revealed that the ethyl acetate extract of O. natrix (EtOAc-NR) possessed 30.4% inhibition at 360. minute while EtOAc-SR showed 21.2% inhibition at 180. minute, 24.5% inhibition at 270. minute and 27.2% inhibition at 360. minute (Table 6). However, the extracts did not posses activity on the TPA-induced ear edema (Table 7) and the chronic inflammation model which was FCA-induced arthritis models (Table 8). According to the histopathological data, it was established that EtOAc-SR majorly provided the dermal integrity (Fig. 1, Table 9). In vitro antioxidant activity tests revealed that none of the extracts possessed remarkable antioxidant activity (Table 10). Nevertheless the antioxidant capacities of tested extracts were found to be consistent with the wound healing activity results. Although ethnobotanical studies reveals that Ononis species are traditionally used for different purposes such as laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic, antifungal and wound healing effects; there are not enough of the studies which scientifically prove these uses. According to the literature data, the aerial parts of O. spinosa was found to possess analgesic, antimicrobial and antioxidant activities [11,30,31]. Furthermore, anti-inflammatory activity of O. spinosa, O. tridentata, O. sessilifolia
and O. basiadnata [32–34]; antimicrobial activity of O. natrix, O. hirta and O. sicula [7,9,35] and wound healing activity of O. sessilifolia, O. basiadnata and O. macrosperma [34] were reported. According to the study of Dannhart et al. [33], O. spinosa root Table 8 Effects of the active extracts on FCA-induced arthritis model. Day
Swelling Thickness SEM (Inhibition%) Control
EtOAc-NR (100 mg/kg)
EtOAc-SR (100 mg/kg)
Indomethacin (10 mg/kg)
3
311.1 21.04
4
361.9 30.4
5
324.6 22.8
6
311.6 21.3
7
281.5 21.7
8
258.4 20.6
9
250.8 19.9
10
224.7 18.9
11
200.1 15.8
12
227.4 17.1
13
237.6 14.9
14
249.7 19.0
21
306.1 21.8
314.5 29.6 – 387.1 20.3 – 324.9 19.8 – 309.8 19.0 (0.6) 280.7 19.8 (0.3) 251.6 18.5 (2.6) 244.3 17.6 (2.6) 231.9 19.1 – 207.3 20.7 – 232.7 18.8 – 248.6 19.2 – 251.0 20.8 – 257.1 19.6 (16.0)
319.2 20.6 – 328.1 18.4 (9.3) 304.8 18.7 (6.1) 290.6 16.4 (6.7) 275.4 20.8 (2.2) 249.3 19.4 (3.5) 237.5 19.9 (5.3) 230.5 19.7 – 225.4 19.3 – 231.4 22.8 – 244.1 25.2 – 247.0 21.6 (1.1) 255.4 20.5 (16.6)
204.3 16.5 (34.3)** 231.4 10.3 (36.1)*** 222.4 14.4 (31.5)** 207.4 9.2 (33.4)*** 191.6 15.6 (31.9)*** 188.7 16.3 (26.9)** 172.4 15.4 (31.3)*** 170.6 16.1 (24.1) 169.2 16.6 (15.4) 181.5 16.4 (20.2) 185.6 14.7 (21.9) 199.1 15.3 (20.3) 202.6 17.3 (33.8)***
EtOAc-NR: ethyl acetate extract of O. natrix subsp. natrix roots; EtOAc-SR: ethyl acetate extract of O. spinosa subsp. leiosperma roots.
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Fig. 1. The view of the wounded tissues treated with sample/vehicle under light microscobe. Skin sections show hematoxylin & eosin (HE) stained epidermis and dermis. The original magnification was x 100 and the scale bars represent 100 mm for figures. Data are representative of 6 animal pergroup. A: Negative Control. B: Vehicle. C: EtOAc-SR. D: Madecassol.
Table 9 Histopathologic data of the wounded tissues treated with test sample/vehicle. Groups
Vehicle Negative Control EtOAc-SR Madecassol1
Wound Healing Process S
U
++/+++ +++ ++ +
++/+++ +++ +
Wound Healing Phases RE
FP
CD
MN
PM
NV
IF
PF
RF
+/++ +++
++/+++ ++/+++ +++ ++
+ + +++ +++
++ ++ ++/+++ ++
+++ +++ + +/++
++ ++ ++ ++
++/+++ +++ ++ +
++ ++ ++/+++ ++
+/++ ++
HE- and VG-stained sections were scored as mild (+), moderate (++), and severe (+++) for epidermal and/or dermal remodeling. S: scab, U: ulcus, RE: re-epithelization, FP: fibroblast proliferation, CD: collagen depositions, MN: mononuclear cells, PM: polymorphonuclear cells, NV: neovascularization, IP: inflammation phase, PP: proliferation phase, and RP: remodeling phase.
Table 10 Antioxidant activity results of the extracts. Material Extracts Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH
DPPH IC50 (mg/mL)
ABTS IC50 (mg/mL)
Increase of reducing power (%)
OH-radical inhibition IC50 (mg/mL)
222.31 81.24 62.76 186.91 55.41 93.47 205.77 138.48 117.29 243.36 42.17 58.78 324.05 121.15 128.46 145.71 101.14 94.90 308.83
256.29 96.23 69.81 221.13 63.44 99.16 221.17 121.14 127.25 233.32 41.65 63.51 308.89 138.91 145.02 157.66 109.47 99.38 348.41
26.33 31.47 39.94 17.38 31.84 19.81 15.32 20.91 19.57 13.29 39.13 30.88 12.60 22.74 21.08 18.35 17.32 28.56 16.81
205.42 88.60 78.16 195.67 61.19 91.75 196.56 131.92 110.31 240.47 45.55 51.19 319.95 167.87 130.51 137.32 95.54 96.27 284.64
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Table 10 (Continued) Material
DPPH IC50 (mg/mL)
ABTS IC50 (mg/mL)
Increase of reducing power (%)
OH-radical inhibition IC50 (mg/mL)
EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR
116.49 103.16 245.51 133.18 95.32
107.18 157.83 249.57 148.88 99.71
19.20 25.37 17.85 20.96 22.05
107.66 100.35 226.40 138.88 103.12
Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma l roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots.
Table 11 Data of the bioactive extract on different test models. Bioactivity Test Model
Tested Parameter
Data of Bioactive Extract (EtOAc-SRa )
Wound Healing Activity Linear incision wound model Circular excision wound model Hydroxyproline estimation
Tensile strength on day 10 Contraction% on day 12 Hydroxyproline amount
42.6% 60.1% 41.3 mg/mg
Anti-Inflammatory Activity Acetic acid-induced increase in capillary permeability test Carrageenan-induced paw edema in mice
Inhibition% Inhibition% in 360 min
40.4% 27.2%
a
EtOAc-SR: ethyl acetate extract of O. spinosa subsp. leiosperma roots.
extract exhibited anti-inflammatory activity by the inhibition of 5lipoxygenase enzyme. This study showed that O. spinosa subsp. leiosperma roots, which were traditionally used for wound healing, possessed wound healing and anti-inflammatory activities on different models (Table 11). Literature data also support the results of this study. This study proved the wound healing effect of O. spinosa roots as well as its anti-inflammatory activity. 4. Conclusion In this study, it was aimed to evaluate the wound healing and anti-inflammatory activities of 4 taxons of the genus Ononis as well as their antioxidant potential. For this purpose, different activity test models were used. The results of wound healing activity tests and anti-inflammatory activity tests were consistent. The antioxidant capacity of the extracts was not found to be significant. The ethyl acetate extract prepared from roots of O. spinosa subsp. leiosperma possessed remarkable bioactivity at the wound healing models as well as acute inflammation models, namely acetic acidinduced increase in capillary permeability test and carrageenaninduced hind paw edema model. The extracts did not show activity at TPA-induced ear edema model which could mean that the antiinflammatory activity process was not due to the inhibition of leukotriene synthesis but the inhibition of arachidonic acid metabolites. The extracts did not found to possess activity at chronic inflammation model. Conflict of interest None. Acknowledgements Prof. Dr. Hayri Duman and Prof. Dr. Osman Tugay are acknowledged for the identification of the plant material. The experimental part of this study was financially supported by
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Original article
Wound healing and anti-inflammatory activity of some Ononis taxons b _ _ Burçin Ergene Öza,* , Gülçin Saltan Işcan , Esra Küpeli Akkolb , Ipek Süntarb , c a Hikmet Keleş , Özlem Bahadır Acıkara a
Ankara University Faculty of Pharmacy, Pharmacognosy Department, 06100, Tandogan, Çankaya, Ankara, Turkey Gazi University Faculty of Pharmacy, Pharmacognosy Department, 06330, Etiler, Yenimahalle, Ankara, Turkey c Afyon Kocatepe University, Faculty of Veterinary Medicine, Pathology Department, 03200, Afyonkarahisar, Turkey b
A R T I C L E I N F O
Article history: Received 23 March 2017 Received in revised form 5 May 2017 Accepted 8 May 2017 Keywords: Fabaceae Inflammation Ononis Wound healing
A B S T R A C T
Ononis species are used for their laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic and antifungal effects as well as against skin diseases for wound healing activity. In the light of this information n-hexane, ethylacetate and methanol extracts prepared from Ononis spinosa L. subsp. leiosperma (Boiss.) Sirj., Ononis variegata L., Ononis viscosa L. subsp. brevifolia (DC) Nym. and Ononis natrix L. subsp. natrix L. were tested for their wound healing, anti-inflammatory and antioxidant activities. Linear incision and circular excision wound models and hydroxypyroline estimation assay were used for the wound healing activity. For the assessment of chronic inflammation FCA-induced arthritis and for acute inflammation carrageenan-induced hind paw edema, TPA-induced ear edema and acetic acidinduced increase in capillary permeability tests were conducted. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging activity assay, reducing power assay and hydroxyl radical (OH ) scavenging assay were used for determining antioxidant activities of the extracts. Results showed that O. spinosa subsp. leiosperma roots ethyl acetate extract exhibited remarkable wound healing activity with the 42.6% tensile strength value on the linear incision wound model and 60.1% reduction of the wound area at the day 12 on the circular excision wound model. Hydroxyproline content of the tissue treated by O. spinosa subsp. leiosperma roots ethyl acetate extract was found to be 41.3 mg/mg. Acetic acid induced increase in capillary permeability test results revealed that O. spinosa subsp. leiosperma roots ethyl acetate extract and O. spinosa subsp. leiosperma roots methanol extract inhibited inflammation by 40.4% and 35.4% values respectively. O. spinosa subsp. leiosperma roots ethyl acetate extract showed 21.2-27.2% inhibition in carrageenaninduced hind paw edema test while did not posses activity on TPA-induced ear edema and FCA-induced arthritis models. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction The genus Ononis L. which is a member of Fabaceae family, grows widespread in Europe, Atlantic Islands, West Asia and North Africa. The genus is represented by more than 75 species all over the world and by 18 species in Turkey. This genus is especially distributed closed to the coasts of Mediterranean Sea [1–3]. It is reported that Ononis species are used for their laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic and antifungal effects as well as against skin diseases for wound healing activity [4,5]. The roots of Ononis spinosa L., Ononis arvensis L., Ononis hircina Jacg. and Ononis antiquorum L. are known to be
* Corresponding author. E-mail address: [email protected] (B. Ergene Öz). http://dx.doi.org/10.1016/j.biopha.2017.05.040 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
used against irritations of the skin, itches, wounds and dermatitis in Central Asia and Russia [6]. The extracts prepared from O. natrix are used as antirheumatismal, antibacterial, diuretic, urolithiatic and to reduce blood pressure [1,7,8]. Ethnobotanical studies revealed that other species of this genus such as O. sicula (Guss.) Hub.-Mor. and O. hirta Desf. were used for the wound healing as well as against cold sore as antiseptic [9]. O. spinosa which is widely known as “kayiskiran” in Turkey, is used as a folk remedy against urinary tract diseases and kidney stones due to its antiinflammatory and diuretic effect as well as against eczema and some other skin disorders for wound healing [10,11].
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Phytochemical studies on Ononis species reveals that these plants contains sugar, tannin, saponin, lectins, flavonoid derivatives (such as trifolirhizin, ononin, formononetin, genistein, genistin, biochanin, daidzin, glycitin, glycitein, sissotrin), phenolic acids (gallic, protocathechic, p-hydroxybenzoic, vanillic, cafeic, syringic, p-coumaric, ferulic, sinapic acid), essential oils (anethol, carvon and menthol as major components), sterols (b-sitosterol, campesterol, stigmasterol, stigmastan-3,5-dien), terpenic substances (b-amyrin, a-onocerin), anthranilic acid and resorcinol derivatives, aromatic lactones and coumarines [1,12–16]. Although there are several ethnopharmacological researches that report the traditional uses of Ononis species, the studies, which scientifically prove the traditional uses are insufficient. In this study, four species of the genus Ononis were selected and it is aimed to assess wound healing and anti-inflammatory activities as well as antioxidant capacities of the extracts. For this purpose; extracts were prepared from the roots and aerial parts of the selected species, namely O. spinosa L. subsp. leiosperma (Boiss.) Sirj., O. variegata L., O. viscosa L. subsp. brevifolia (DC) Nym. and O. natrix L. subsp. natrix L. by using n-hexane, ethylacetate and methanol respectively. Wound healing, anti-inflammatory and antioxidant activities of the extracts were investigated. Linear incision and circular excision wound models and hydroxypyroline estimation assay were conducted for the investigation of wound healing activity. Acute inflammation models i.e. acetic acidinduced increase in capillary permeability test, carrageenaninduced hind paw edema and TPA-induced ear edema models as well as a chronic inflammation model i.e. FCA-induced arthritis model were used for the assessment of anti-inflammatory activity. The antioxidant activities of the extracts were also evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) scavenging activity assay, reducing power assay and hydroxyl radical (OH) scavenging assay
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repeated three times with fresh solvent for each extract. Following the extraction, each extract was filtered and the solvent was removed under reduced pressure at 50 C. 2.3. In vivo biological activity tests 2.3.1. Animals Male Swiss albino mice (20–25 g) and Sprague-Dawley rats (160–180 g) were obtained from Kobay A.Ş. (Ankara, Turkey). The animals were kept in animal room conditions for 3 days and were fed on standard pellet diet and water ad libitum. The groups of six animals were used for the biological activity tests. The animals used for the bioactivity tests were maintained in accordance with the directions of Guide for the Care and Use of Laboratory Animals, and the experiment was approved by the Experimental Animal Ethics Committee of Gazi University (Gazi University Ethical Council Project Number: G.U.ET-11.058). 2.3.2. Preparation of test samples for bioassay The test materials were mixed with an ointment base and topically applied onto the wounded area on dorsal part of the test animals for the assessment of the wound healing activity. The extracts and fractions were mixed with glycol stearate: propylene glycol and liquid paraffin (3:6:1) mixture in a mortar. The sample ointments were applied daily on the wounded area following the formation of the wound. The control group was treated with blank vehicle base, and the negative control was not treated with any product. Madecassol1 (Bayer) (0.5 g) was used as reference drug. The samples were suspended in a mixture of distilled water and 0.5% sodium carboxymethyl cellulose (CMC) and given orally to test animals for the evaluation of the anti-inflammatory activity. The same mixture containing appropriate volumes of the dosing vehicle was given to the control group instead of sample. Indomethacin (10 mg/kg) in 0.5% CMC was used as a reference drug.
2. Materials and methods 2.3.3. Wound healing activity 2.1. Plant material Plant samples were collected from different localities, from South and Central Anatolia. Taxonomic identification of the plants were confirmed by Hayri Duman from Gazi University, Department of Biological Sciences, Faculty of Art and Sciences and Osman Tugay from Selcuk University, Department of Biological Sciences, Faculty of Art and Sciences. The voucher specimens are kept in the herbarium of Ankara University Faculty of Pharmacy (AEF). The localities and dates that the plant materials were collected from and herbarium numbers are shown in Table 1. 2.2. Extraction The aerial parts and roots of the plant materials were seperated and dried at room temperature. Dried and powdered materials (30 g) were extracted with n-hexane (300 mL x 3), ethyl acetate (300 mL x 3) and methanol (300 mL x 3) respectively at room temperature by continuous stirring. 24 h of extraction period was
2.3.3.1. Linear incision wound model. The rats were anaesthetized with 0.15 cc Ketasol1 (Richterpharma). The dorsal area of the rats were shaved and disinfected with 70% alcohol. Two 5 cm-length linear-paravertebral incisions were formed with a sterile blade at the distance of 1.5 cm from the dorsal midline on each side. Three surgical sutures were placed each 1 cm apart. The ointments were topically applied on the wounds once a day for 9 days. On the last day, the sutures were removed and tensile strength of wounded and treated skin was measured by using a tensiometer (Zwick/ Roell Z0.5, Germany) [17–19]. 2.3.3.2. Circular excision wound model. The mice were anaesthetized with 0.15 cc Ketasol1 (Richterpharma). A circular wound was created on the dorsal interscapular region of each mouse by excising the skin with a 5 mm biopsy punch (Nopa instruments, Germany) after the dorsal parts of the mice were shaved. Wounds were left open [20]. Untill the wound was completely healed, test ointments were applied topically once a
Table 1 The localities and dates that the plant materials were collected from and herbarium numbers. Plant Material
Herbarium Number
Locality
Collection Date
Taxonomic Identification
O. spinosa L. subsp. leiosperma (Boiss.) Sirj. O.variegata L. O. viscosa L. subsp. brevifolia (DC) Nym. O. natrix L. subsp. natrix L.
AEF AEF AEF AEF
Konya, Aliçerçi Village-Ahırlı roadside Antalya, Side, Sorgun, seaside Antalya, Akseki, Çukurköy, Karaorman Antalya, Fethiye-Korkuteli road side
June 2011 June 2011 May 2011 May 2012
H. H. H. O.
26000 25998 25997 26328
Duman Duman Duman Tugay
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day and the wound areas were monitored by a camera (Fuji, S20 Pro, Japan) everyday. AutoCAD program was used to evaluate wound area. Wound contraction was calculated as percentage of the reduction in wounded area. On day 12, sample from the tissue was seperated from the healed skin of each group for the histopathological examination [19].
2.3.3.3. Histopathology. The skin specimens were fixed in 10% buffered formalin, processed and blocked with paraffin The samples were cut into 5 micrometer sections, stained with hematoxylin & eosin (HE) and Van Gieson (VG) stains. A light microscope (Olympus CX41 attached Kameram1 Digital Image Analyze System) was used for the examination of the tissues. The results were given as mild (+), moderate (++) and severe (+++) for epidermal or dermal re-modeling. Re-epithelization or ulcus in epidermis; fibroblast proliferation, mononuclear and/or polymorphonuclear cells, neo-vascularization and collagen depositions in dermis were analyzed to score the epidermal or dermal re-modeling. Van Gieson stained sections were analyzed for collagen deposition. At the end of the examination, the wound healing phases as inflammation, proliferation, and remodeling in all groups were staged according to all of the wound healing processes 2.3.3.4. Hydroxyproline estimation. Isolated tissues were dried in hot air oven at 60–70 C untill a consistent weight was achieved. The tissues were hydrolyzed with 6 N HCl for 3 h at 130 C, the solution was adjusted to pH 7 and subjected to chloramin T oxidation. Absorbance of the colored adduct-product formed with Ehrlich reagent at 60 C was read at 557 nm. The values for the standard hydroxyproline was also reported as mg/mg dry weight of tissue [19,21]. 2.3.4. Anti-inflammatory activity 2.3.4.1. Carrageenan-induced hind paw edema. The method developed by Kasahara et al. [22] was used with some modifications [23]. The suspension of carrageenan (0.5 mg/ 25 mL) (Sigma, St. Louis, Missouri, USA) in physiological saline (154 mM NaCl) was prepared freshly and the suspension was injected into the subplantar tissues of the right hind paw of each mouse, sixty minutes after the oral administration of test sample/ dosing vehicle. 25 mL saline solution was injected into that of the left hind paw as the control. Paw edema was measured in every 90 min during 6 h following the induction of inflammation, a pair of dial thickness gauge callipers (Ozaki Co., Tokyo, Japan) was used to measure the difference in footpad thickness between the right and left foot. Mean values of treated groups and control group were compared and analyzed using statistical methods. Acetic acid-induced increase in capillary 2.3.4.2. permeability. Whittle method was used with some modifications for the determination of the effect of the test samples on the increased vascular permeability induced by acetic acid in mice [24]. Test samples were given orally to the mice in 0.2 mL/20 g body weight. Thirty minutes after the administration 0.1 mL of 4% Evans blue (Sigma, St. Louis, Missouri, USA) in saline solution (iv.) was injected to the tails of each mouse. 0.4 mL of 0.5% (v/v) AcOH was injected ip 10 min after the i.v. injection of the dye solution. The mice were killed by dislocation of the neck after 20 min and the viscera were exposed and irrigated with distilled water, then poured into 10 mL volumetric flasks through glass wool. Each flask was made up to 10 mL with distilled water, 0.1 of 0.1 N NaOH solution was added to the flask and the absorption of the final solution was measured at 590 nm (Beckmann Dual
Spectrometer). A mixture of distilled water and 0.5% CMC was used for the control group. 2.3.4.3. TPA-induced mouse ear edema. 2.5 mg of TPA was dissolved in 20 mL of ethanol (70%) and the solution was applied by an automatic pipette in 20 mL volumes to both anterior and posterior surfaces of the right ear of each mouse. The same volume of ethanol (70%) was applied to the left ear as control at the same time. Indomethacin (0.5 mg/ear) was used as reference drug. Two different measurements were taken as given below [25]. The thickness of each ear was measured using gauge callipers (Ozaki Co., Tokyo, Japan) four hours after the induction of inflammation. The difference between the right and left ears due to TPA application was calculated and inhibition percentage was expressed as a reduction thickness with respect to the control group. At the end of 4 h following the administration, the animals were sacrificed. Discs of 6 mm diameter were cut from each ear and weighed in balance. The swelling was estimated as the difference in weight between the punches from right and left ears and expressed as an increase in the ear thickness. 2.3.4.4. Adjuvant-induced chronic arthritis. The method described by Newbould [26] was used with some modifications. Test samples were administered to rats once a day by a feeding needle for 14 days. 25 mL of Freund’s Complete Adjuvant (FCA) (Sigma, St. Louis, Missouri, USA) was injected into subplantar tissue of the right hind paw of each rat on the third day. A pair of thickness gauge callipers was used to measure swelling in the injected and contralateral hind paws. The measurements were made every day from day 3 to 14, and at the end, on day 21. The differences in Table 2 Effects of the extracts on linear incision wound model. Material
Statistical Mean SEM
Tensile Strength%
Vehicle Negative control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Madecassol1
12.38 1.19 11.86 1.27 12.99 1.26 13.37 1.43 9.94 1.16 11.85 1.91 16.23 1.72 15.42 1.34 11.70 1.28 14.11 1.94 14.78 1.31 11.41 1.57 17.66 0.93 16.77 1.09 11.35 1.34 13.01 1.64 13.20 1.23 10.03 1.72 13.17 1.40 14.04 1.05 11.20 1.38 11.72 1.63 11.84 1.25 10.11 1.86 13.02 1.53 12.84 1.22 19.68 0.82
4.4 – 4.9 7.9 19.7 – 31.1* 24.6 – 13.9 19.4 – 42.6*** 35.5** – 5.1 6.6 – 6.4 13.4 – – – – 5.2 3.7 58.9***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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severity of arthritis between the experimental groups and the control group were statistically analyzed [27].
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2.4. In vitro biological activity tests 2.4.1. Antioxidant activity
2.3.5. Statistical analysis of the data The activities were statistically analyzed using one-way analysis of variance (ANOVA) and Students-Newman-Keuls posthoc test. The values of p 0.05 were considered statistically significant. Histopathologic data were considered to be nonparametric; therefore, no statistical tests were performed.
2.4.1.1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The samples and the reference compound (ascorbic acid) were dissolved in methanol and the solutions were mixed with DPPH solution (80 mg/mL). Remaining DPPH amount was measured at 517 nm using spectrophotometer. DPPH inhibition activities were calculated according to the following formula:
Table 3 Effects of the extracts on circular excision wound model. Wound Area (mm2) SEM (Contraction%)
Material Day
0
2
4
6
8
10
12
Vehicle
19.39 1.84
Negative control Hex-NH
20.02 1.61 19.48 1.73
EtOAc-NH
19.36 1.59
MeOH
19.88 1.91
17.91 2.15 (1.6) 18.21 2.17 16.88 2.19 (5.8) 17.21 2.85 (3.9) 17.26 2.26 (3.6) 19.24 2.46 – 16.32 2.48 (8.9) 17.23 2.54 (3.8) 18.61 2.45 – 18.32 2.14 – 18.05 2.15 – 17.96 2.03 – 16.02 1.88 (10.6) 16.29 1.96 (9.0) 18.06 1.99 – 17.88 2.03 (0.2) 17.96 2.14 – 18.82 2.10 – 17.40 1.96 (2.8) 17.26 1.85 (3.6) 17.21 2.08 (3.9) 18.12 2.01 – 17.95 2.13 – 18.03 2.19 – 18.10 1.99 – 16.07 1.91 (10.3)
14.79 2.28 (2.8) 15.20 2.56 14.41 2.10 (2.6) 14.13 2.07 (6.7) 13.85 2.04 (6.4) 16.88 2.70 – 12.17 2.31 (17.7) 12.87 2.46 (12.9) 15.66 1.97 – 13.82 2.24 (6.6) 15.39 2.20 – 16.35 2.16 – 13.28 1.75 (10.2) 14.01 1.85 (5.3) 15.13 2.19 – 14.91 1.69 – 15.16 2.11 – 14.88 2.21 – 15.01 2.16 – 12.91 2.21 (12.7) 13.98 2.03 – 14.95 2.16 – 15.24 2.22 – 15.08 2.20 – 14.92 2.07 – 12.04 1.56 (18.6)
11.22 2.13 (8.9) 12.31 2.08 10.93 2.07 (2.6) 11.10 1.89 (1.07) 10.91 1.26 (2.8) 14.12 2.51 – 8.86 1.42 (21.0) 8.96 1.39 (20.1) 12.41 2.02 – 11.36 1.95 – 10.67 2.07 (4.9) 11.29 1.83 – 10.22 1.79 (8.9) 10.92 1.17 (2.7) 12.26 2.08 – 13.16 1.45 – 12.07 1.88 – 12.06 1.96 – 11.45 2.08 – 9.44 1.60 (15.9) 11.96 1.97 – 11.38 2.16 – 12.06 1.15 – 11.77 1.67 – 11.29 2.10 – 7.01 1.06 (37.5)*
7.25 2.40 (8.3) 7.91 1.74 7.14 1.48 (1.5) 6.99 1.86 (3.6) 7.16 1.13 (1.2) 7.93 2.11 – 5.06 0.91 (30.2)* 5.28 0.95 (27.2) 7.11 2.45 (1.93) 7.08 2.07 (2.3) 6.95 1.93 (4.1) 7.36 2.33 – 5.10 0.91 (29.7) 6.33 1.42 (12.7) 7.12 1.26 (1.8) 8.14 2.43 – 7.05 0.78 (2.8) 7.36 2.26 – 7.48 2.41 – 5.39 0.86 (25.7) 7.04 2.13 – 8.01 2.33 – 7.16 1.21 (1.2) 7.80 1.95 – 7.93 1.97 – 4.08 0.75 (43.7)**
4.31 1.51 (6.5) 4.61 1.44 4.08 0.84 (5.3) 3.80 1.29 (11.8) 3.87 0.81 (10.2) 4.92 1.85 – 2.16 0.65 (49.9)** 3.07 0.89 (28.8) 4.58 1.66 – 4.19 1.92 (2.8) 4.17 1.19 (3.2) 4.48 1.59 – 2.98 0.83 (30.9)* 3.25 0.93 (24.6) 4.27 0.95 (0.9) 5.16 1.40 – 4.86 1.32 – 5.03 1.63 – 5.19 1.74 – 3.20 0.93 (25.8) 4.78 1.89 – 5.95 1.53 – 4.45 0.87 – 4.45 0.87 – 4.69 1.29 – 1.25 0.27 (70.9)**
2.73 0.98 (6.8) 2.93 0.79 2.43 0.54 (10.9) 2.10 0.38 (23.1) 2.30 0.67 (15.8) 2.85 0.71 – 1.79 0.50 (34.4)* 2.03 0.74 (25.6) 2.88 0.99 – 2.61 0.86 (4.4) 2.42 0.92 (11.4) 2.79 0.81 – 1.09 0.21 (60.1)** 1.75 0.42 (35.9)* 2.76 0.99 – 3.01 0.86 – 2.51 0.81 (8.1) 2.82 1.03 – 2.77 0.88 – 1.99 0.96 (27.1) 2.96 0.92 – 2.75 0.90 – 2.91 0.88 – 2.82 0.79 – 2.60 0.91 (4.8) 0.00. 0.00 (100.00)***
NH
Hex-NR
20.11 2.03
EtOAc-NR
19.65 1.57
MeOH
19.42 1.34
NR
Hex-SH
19.47 2.02
EtOAc-SH
19.58 1.99
MeOH-SH
20.01 2.02
Hex-SR
19.93 2.11
EtOAc-SR
19.62 1.46
MeOH-SR
20.14 2.10
Hex-VH
19.95 1.79
EtOAc-VH
19.81 1.59
MeOH-VH
19.40 1.22
Hex-VR
20.18 3.05
EtOAc-VR
20.31 3.13
MeOH-VR
19.59 1.54
Hex-VarH
19.48 2.36
EtOAc-VarH
20.04 2.95
MeOH-VarH
19.46 1.27
Hex-VarR
19.98 2.24
EtOAc-VarR Madecassol
1
19.52 2.30 19.70 1.32
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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Table 4 Effects of the extracts on hydroxyproline content. Material
Hydroxyproline (mg/mg) SEM
Vehicle Negative control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Madecassol1
9.4 1.23 8.5 1.64 11.8 1.22 10.5 1.27 10.8 1.14 9.1 1.17 30.3 0.88* 29.5 1.05 10.2 1.51 12.3 1.32 18.7 1.49 8.8 1.54 41.3 0.51** 32.4 0.83* 9.1 1.95 10.4 1.76 10.1 1.89 8.8 1.46 10.3 1.61 13.6 1.47 9.5 1.38 11.5 1.86 8.3 1.76 10.1 1.12 13.4 1.75 12.1 1.34 45.3 0.49***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
Inhibition(%) = (Acontrol Asample) 100/Acontrol where Acontrol represents the absorbance of the control, and Asample represents the absorbance of the extracts or reference. Experiments were run in duplicate and the results were expressed as inhibitory values [28]. 2.4.1.2. ABTS (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) scavenging activity. The aqueous solution of ABTS was mixed with K2S2O8 (2.45 mM) and the mixture was kept in dark for 12–16 h to generate ABTS+ radical (7 mM). The absorbance of the solution was adjusted to 0.700 (0.030) units at 734 nm. The sample solutions (15 mL) was mixed with the radical solution and reaction kinetic was measured through 15 min in 1 min periods. Concentration against percentage of inhibition was calculated as Trolox equivalent antioxidant concentration (TEAC) [28]. 2.4.1.3. Reducing power assay. 1 mL of the extract solution was mixed with 0.2 M phosphate buffer (pH: 6.6) (2.5 mL) and 1% potassium hexaferricyanide solution (2.5 mL) and the mixture was incubated at 50 C for 30 min. 2.5 mL of 10% trichloroacetic acid (TCA) was added and then the mixture was centrifuged for 10 min. 2.5 mL oft he upper layer, 2.5 mL distilled water and 0.5 mL of 0.1% aqueous ferric chloride were mixed and absorbance of the final solution was recorded at 700 nm. Reducing power of the extracts were expressed as ascorbic acid equivalents (AscAE), mmol ascorbic acid/g sample [29]. All analysis were performed as dublicate and expressed as mean values. 2.4.1.4. Hydroxyl radical (OH) scavenging assay. The mixture of 100 mL FeCl3 and 104 mM EDTA (1:1, v/v) solution was prepared. The reaction mixture contained 100 mL extract solution, 500 mL of 5.6 mM 2-deoxyribose prepared in phosphate buffer solution
Table 5 Inhibitory effect of the extracts on acetic acid-induced increase in capillary permeability test. Material Control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Indomethacin
Dose (mg/kg)
Evans blue concentration (mg/mL) SEM
Inhibition%
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 10
12.01 1.14 11.86 0.96 11.39 0.83 11.91 0.87 12.93 1.14 9.42 0.76 9.16 0.69 13.74 1.08 12.78 1.17 10.02 0.86 13.11 1.19 7.16 0.75 7.76 0.68 12.26 1.21 11.98 1.16 12.30 1.08 13.26 1.50 12.89 1.26 10.36 1.03 14.18 1.85 12.66 1.36 13.10 1.26 14.08 1.92 13.15 1.33 12.05 1.11 5.78 0.24
1.2 5.2 0.8 – 21.6 23.7 – – 16.6 – 40.4** 35.4* – 0.2 – – – 13.8 – – – – – – 51.9***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
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Table 6 Effects of the extracts against carrageenan-induced paw edema in mice. Material
Dose (mg/kg)
Control Hex-NH
100
EtOAc-NH
100
MeOH
100
NH
Hex-NK
100
EtOAc-NK
100
MeOH
100
NK
Hex-SH
100
EtOAc-SH
100
MeOH-SH
100
Hex-SR
100
EtOAc-SR
100
MeOH-SR
100
Hex-VH
100
EtOAc-VH
100
MeOH-VH
100
Hex-VR
100
EtOAc-VR
100
MeOH-VR
100
Hex-VarH
100
EtOAc-VarH
100
MeOH-VarH
100
Hex-VarR
100
EtOAc-VarR
100
MeOH-VarR
100
Indomethacin
10
Swelling Thickness (10
2
mm) SEM (Inhibition%)
90 min
180 min
270 min
360 min
47.3 2.7 47.6 3.5 – 48.5 3.1 – 47.0 3.1 (0.6) 45.4 3.6 (4.0) 45.1 2.9 (4.7) 46.8 3.0 (1.1) 49.5 3.1 – 44.3 2.5 (6.3) 44.9 2.7 (5.1) 46.9 3.2 (0.8) 45.1 3.2 (4.7) 45.7 3.0 (3.4) 47.4 2.8 – 48.1 2.9 – 46.2 3.3 (2.3) 50.1 4.6 – 49.5 4.1 – 47.5 3.9 – 49.3 3.8 – 50.1 3.9 – 48.2 3.4 – 48.8 3.6 – 49.0 2.8 – 49.8 3.9 – 30.2 2.4 (36.2)**
50.1 3.3 51.5 2.5 – 51.5 4.2 – 42.7 3.3 (14.8) 52.5 3.5 – 41.6 3.6 (16.9) 44.8 3.0 (10.6) 52.4 3.5 – 49.3 3.3 (1.6) 46.0 3.5 (8.2) 50.3 4.8 – 39.5 2.4 (21.2)* 41.2 3.1 (17.8) 45.9 3.3 (8.3) 51.3 4.4 – 52.5 3.6 – 51.8 3.4 – 50.7 3.2 – 51.2 3.9 – 52.1 2.9 – 51.5 3.8 – 51.7 3.7 – 53.6 3.6 – 49.5 3.8 (1.2) 50.5 2.9 – 30.5 2.1 (39.1)***
53.9 3.1 52.6 3.3 (2.4) 54.2 3.8 – 49.3 3.2 (8.5) 49.7 2.8 (7.8) 38.4 3.0 (28.8) 42.9 2.3 (20.4) 54.8 3.7 – 54.1 3.9 – 53.0 3.4 (1.7) 55.6 3.7 – 40.7 3.0 (24.5)* 47.3 3.3 (12.2) 50.6 3.6 (6.1) 55.7 4.4 – 54.7 3.9 – 55.2 3.8 – 55.7 3.4 – 54.9 3.6 – 57.2 3.1 – 55.0 3.3 – 56.6 3.9 – 56.1 3.8 – 49.5 3.4 (8.2) 54.2 3.7 – 37.6 2.1 (30.2)**
60.2 3.4 59.3 2.7 (1.5) 62.6 4.7 – 56.8 3.8 (5.6) 59.2 3.1 (1.7) 41.9 3.1 (30.4)* 49.7 3.6 (17.4) 61.5 3.9 – 57.8 3.2 (3.9) 62.8 3.9 (4.1) 59.8 4.1 (0.6) 43.8 3.1 (27.2)* 56.7 3.5 (5.8) 59.5 3.8 (1.2) 62.2 4.3 – 62.7 4.5 – 61.8 3.9 – 57.4 3.8 (4.7) 56.6 4.1 (5.9) 60.5 4.4 – 60.3 4.0 – 61.2 3.7 – 61.4 4.3 – 57.6 3.7 (4.3) 64.1 3.9 – 36.8 1.9 (38.9)***
SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. * p < 0.05. ** p < 0.01. *** p < 0.001.
(50 mM, pH 7.4), 200 mL of FeCl3 and EDTA mixture and 100 mL of 1.0 mM aqueous ascorbic acid solution were mixed by vortexing and incubated at 50 C for 30 min, 1 mL of 2.8% trichloroacetic acid and 1 mL of 1.0% thiobarbituric acid were added to the mixture and incubated at 50 C for another 30 min. The absorbance was measured at 532 nm. The percentage of inhibition was calculated according to the following formula by using the absorbance of the control (Ac) and the absorbance of the sample (As) and expressed as mannitol equivalent (mmolmannitol/gsample)
[28]. Results were given as the mean values of two analysis performed in parallel. [(Ac
As)/Ac] 100
3. Results and discussion Wound healing and anti-inflammatory activity results of nhexane (Hex), ethyl acetate (EtOAc) and methanol (MeOH) extracts
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Table 7 Effects of the extracts against TPA-induced ear edema in mice as measurement of swelling thickness and weight measurement of edema. Material Control Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR Indomethacin
Dose (mg/ear)
Swelling Thickness (mm) SEM
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
370.4 22.8 345.7 20.6 339.2 25.6 347.8 22.9 386.4 31.2 331.7 26.6 322.3 21.8 375.6 30.4 379.1 31.5 372.4 23.6 384.6 25.9 328.5 23.7 330.4 24.5 371.8 29.6 388.9 26.9 372.7 29.4 377.4 29.6 374.8 27.9 389.3 26.7 362.4 28.0 368.9 29.3 379.1 25.2 366.5 28.9 352.7 27.7 348.4 25.3 137.1 17.2
Inhibition%
Weight Edema (mg) SEM
Inhibition%
6.7 8.4 6.1 – 10.4 12.9 – – – – 11.3 10.8 – – – – – – 2.2 0.4 – 1.1 4.8 5.9 62.9***
28.4 3.1 27.8 3.4 26.5 3.8 26.8 2.6 29.2 3.6 24.7 2.9 23.5 3.3 31.2 3.0 30.7 3.2 29.9 2.6 30.1 3.8 22.4 2.9 23.6 2.2 27.9 3.2 29.6 3.1 30.3 3.4 31.4 3.6 29.0 3.2 33.7 3.8 25.6 3.1 27.1 3.6 28.5 3.5 31.7 3.3 29.8 2.9 28.1 3.6 12.4 2.4
2.1 6.7 5.6 – 13.0 17.3 – – – – 21.1 16.9 1.8 – – – – – 9.9 4.6 – – – 1.1 56.3***
*p < 0.05; **p < 0.01;SEM: Standard error of the mean. Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots. *** p < 0.001.
prepared from O. spinosa subsp. leiosperma (S), O. variegata (Var), O. viscosa subsp. brevifolia (V) ve O. natrix subsp. natrix (N) roots (R) ve aerial parts (H) are given in Tables 2–8. The ethyl acetate extract of O. spinosa subsp. leiosperma roots (EtOAc-SR) possessed remarkable wound healing activity with 42.6% tensile strength value on linear incision wound model (Table 2) and 60.1% reduction of the wound area at the day 12 on circular excision wound model (Table 3). According to hydroxyproline estimation; hydroxyproline content of the tissue treated by EtOAc-SR was found to be 41.3 mg/mg (Table 4). According to the results of acetic acid-induced increase in capillary permeability test, EtOAc-SR and MeOH-SR were found to exhibit remarkable anti-inflammatory activity with the inhibition values of 40.4% and 35.4%, respectively (Table 5). Carrageenaninduced hind paw edema test results revealed that the ethyl acetate extract of O. natrix (EtOAc-NR) possessed 30.4% inhibition at 360. minute while EtOAc-SR showed 21.2% inhibition at 180. minute, 24.5% inhibition at 270. minute and 27.2% inhibition at 360. minute (Table 6). However, the extracts did not posses activity on the TPA-induced ear edema (Table 7) and the chronic inflammation model which was FCA-induced arthritis models (Table 8). According to the histopathological data, it was established that EtOAc-SR majorly provided the dermal integrity (Fig. 1, Table 9). In vitro antioxidant activity tests revealed that none of the extracts possessed remarkable antioxidant activity (Table 10). Nevertheless the antioxidant capacities of tested extracts were found to be consistent with the wound healing activity results. Although ethnobotanical studies reveals that Ononis species are traditionally used for different purposes such as laxative, diuretic, analgesic, anti-inflammatory, antiviral, cytotoxic, antifungal and wound healing effects; there are not enough of the studies which scientifically prove these uses. According to the literature data, the aerial parts of O. spinosa was found to possess analgesic, antimicrobial and antioxidant activities [11,30,31]. Furthermore, anti-inflammatory activity of O. spinosa, O. tridentata, O. sessilifolia
and O. basiadnata [32–34]; antimicrobial activity of O. natrix, O. hirta and O. sicula [7,9,35] and wound healing activity of O. sessilifolia, O. basiadnata and O. macrosperma [34] were reported. According to the study of Dannhart et al. [33], O. spinosa root Table 8 Effects of the active extracts on FCA-induced arthritis model. Day
Swelling Thickness SEM (Inhibition%) Control
EtOAc-NR (100 mg/kg)
EtOAc-SR (100 mg/kg)
Indomethacin (10 mg/kg)
3
311.1 21.04
4
361.9 30.4
5
324.6 22.8
6
311.6 21.3
7
281.5 21.7
8
258.4 20.6
9
250.8 19.9
10
224.7 18.9
11
200.1 15.8
12
227.4 17.1
13
237.6 14.9
14
249.7 19.0
21
306.1 21.8
314.5 29.6 – 387.1 20.3 – 324.9 19.8 – 309.8 19.0 (0.6) 280.7 19.8 (0.3) 251.6 18.5 (2.6) 244.3 17.6 (2.6) 231.9 19.1 – 207.3 20.7 – 232.7 18.8 – 248.6 19.2 – 251.0 20.8 – 257.1 19.6 (16.0)
319.2 20.6 – 328.1 18.4 (9.3) 304.8 18.7 (6.1) 290.6 16.4 (6.7) 275.4 20.8 (2.2) 249.3 19.4 (3.5) 237.5 19.9 (5.3) 230.5 19.7 – 225.4 19.3 – 231.4 22.8 – 244.1 25.2 – 247.0 21.6 (1.1) 255.4 20.5 (16.6)
204.3 16.5 (34.3)** 231.4 10.3 (36.1)*** 222.4 14.4 (31.5)** 207.4 9.2 (33.4)*** 191.6 15.6 (31.9)*** 188.7 16.3 (26.9)** 172.4 15.4 (31.3)*** 170.6 16.1 (24.1) 169.2 16.6 (15.4) 181.5 16.4 (20.2) 185.6 14.7 (21.9) 199.1 15.3 (20.3) 202.6 17.3 (33.8)***
EtOAc-NR: ethyl acetate extract of O. natrix subsp. natrix roots; EtOAc-SR: ethyl acetate extract of O. spinosa subsp. leiosperma roots.
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Fig. 1. The view of the wounded tissues treated with sample/vehicle under light microscobe. Skin sections show hematoxylin & eosin (HE) stained epidermis and dermis. The original magnification was x 100 and the scale bars represent 100 mm for figures. Data are representative of 6 animal pergroup. A: Negative Control. B: Vehicle. C: EtOAc-SR. D: Madecassol.
Table 9 Histopathologic data of the wounded tissues treated with test sample/vehicle. Groups
Vehicle Negative Control EtOAc-SR Madecassol1
Wound Healing Process S
U
++/+++ +++ ++ +
++/+++ +++ +
Wound Healing Phases RE
FP
CD
MN
PM
NV
IF
PF
RF
+/++ +++
++/+++ ++/+++ +++ ++
+ + +++ +++
++ ++ ++/+++ ++
+++ +++ + +/++
++ ++ ++ ++
++/+++ +++ ++ +
++ ++ ++/+++ ++
+/++ ++
HE- and VG-stained sections were scored as mild (+), moderate (++), and severe (+++) for epidermal and/or dermal remodeling. S: scab, U: ulcus, RE: re-epithelization, FP: fibroblast proliferation, CD: collagen depositions, MN: mononuclear cells, PM: polymorphonuclear cells, NV: neovascularization, IP: inflammation phase, PP: proliferation phase, and RP: remodeling phase.
Table 10 Antioxidant activity results of the extracts. Material Extracts Hex-NH EtOAc-NH MeOH NH Hex-NR EtOAc-NR MeOH NR Hex-SH EtOAc-SH MeOH-SH Hex-SR EtOAc-SR MeOH-SR Hex-VH EtOAc-VH MeOH-VH Hex-VR EtOAc-VR MeOH-VR Hex-VarH
DPPH IC50 (mg/mL)
ABTS IC50 (mg/mL)
Increase of reducing power (%)
OH-radical inhibition IC50 (mg/mL)
222.31 81.24 62.76 186.91 55.41 93.47 205.77 138.48 117.29 243.36 42.17 58.78 324.05 121.15 128.46 145.71 101.14 94.90 308.83
256.29 96.23 69.81 221.13 63.44 99.16 221.17 121.14 127.25 233.32 41.65 63.51 308.89 138.91 145.02 157.66 109.47 99.38 348.41
26.33 31.47 39.94 17.38 31.84 19.81 15.32 20.91 19.57 13.29 39.13 30.88 12.60 22.74 21.08 18.35 17.32 28.56 16.81
205.42 88.60 78.16 195.67 61.19 91.75 196.56 131.92 110.31 240.47 45.55 51.19 319.95 167.87 130.51 137.32 95.54 96.27 284.64
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Table 10 (Continued) Material
DPPH IC50 (mg/mL)
ABTS IC50 (mg/mL)
Increase of reducing power (%)
OH-radical inhibition IC50 (mg/mL)
EtOAc-VarH MeOH-VarH Hex-VarR EtOAc-VarR MeOH-VarR
116.49 103.16 245.51 133.18 95.32
107.18 157.83 249.57 148.88 99.71
19.20 25.37 17.85 20.96 22.05
107.66 100.35 226.40 138.88 103.12
Hex- hexane extracts; EtOAc- ethyl acetate extracts; MeOH- methanol extracts, NH: O. natrix subsp. natrix aerial parts; NR: O. natrix subsp. natrix roots; SH: O. spinosa subsp. leiosperma aerial parts; SR: O. spinosa subsp. leiosperma l roots; VH: O. viscosa subsp. brevifolia aerial parts; VR: O. viscosa subsp. brevifolia roots; VarH: O. variegata aerial parts; VarR: O. variegata roots.
Table 11 Data of the bioactive extract on different test models. Bioactivity Test Model
Tested Parameter
Data of Bioactive Extract (EtOAc-SRa )
Wound Healing Activity Linear incision wound model Circular excision wound model Hydroxyproline estimation
Tensile strength on day 10 Contraction% on day 12 Hydroxyproline amount
42.6% 60.1% 41.3 mg/mg
Anti-Inflammatory Activity Acetic acid-induced increase in capillary permeability test Carrageenan-induced paw edema in mice
Inhibition% Inhibition% in 360 min
40.4% 27.2%
a
EtOAc-SR: ethyl acetate extract of O. spinosa subsp. leiosperma roots.
extract exhibited anti-inflammatory activity by the inhibition of 5lipoxygenase enzyme. This study showed that O. spinosa subsp. leiosperma roots, which were traditionally used for wound healing, possessed wound healing and anti-inflammatory activities on different models (Table 11). Literature data also support the results of this study. This study proved the wound healing effect of O. spinosa roots as well as its anti-inflammatory activity. 4. Conclusion In this study, it was aimed to evaluate the wound healing and anti-inflammatory activities of 4 taxons of the genus Ononis as well as their antioxidant potential. For this purpose, different activity test models were used. The results of wound healing activity tests and anti-inflammatory activity tests were consistent. The antioxidant capacity of the extracts was not found to be significant. The ethyl acetate extract prepared from roots of O. spinosa subsp. leiosperma possessed remarkable bioactivity at the wound healing models as well as acute inflammation models, namely acetic acidinduced increase in capillary permeability test and carrageenaninduced hind paw edema model. The extracts did not show activity at TPA-induced ear edema model which could mean that the antiinflammatory activity process was not due to the inhibition of leukotriene synthesis but the inhibition of arachidonic acid metabolites. The extracts did not found to possess activity at chronic inflammation model. Conflict of interest None. Acknowledgements Prof. Dr. Hayri Duman and Prof. Dr. Osman Tugay are acknowledged for the identification of the plant material. The experimental part of this study was financially supported by
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