International Journal of Surgery 17 (2015) 88e98
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Original research
Hydroethanolic Pistacia atlantica hulls extract improved wound healing process; evidence for mast cells infiltration, angiogenesis and RNA stability Mohammad Reza Farahpour a, *, Navideh Mirzakhani b, Jamal Doostmohammadi a, Mahmood Ebrahimzadeh a a b
Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran Department of Pathology, Faculty of Veterinary Medicine, P.O.BOX: 1177, Urmia University, Urmia, Iran
h i g h l i g h t s Topical Topical Topical Topical Topical
administration administration administration administration administration
of of of of of
Pistacia atlantica fascinated the inflammatory phase. P. atlantica up-regulated the mast cells infiltration. P. atlantica accelerate proliferation phase. P. atlantica cause significantly lower RNA damage. P. atlantica cause up-regulated hydroxylproline content.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2014 Received in revised form 2 February 2015 Accepted 16 March 2015 Available online 4 April 2015
In Iranian traditional therapy folk, the Pistacia is used for treatment of wound inflammation. Here in the present study, the In vivo effect of Pistacia atlantica hulls ointment (PAO) on the wound healing process was assessed. Excision and incision wounds were induced in rats. Three different doses of PAO were administrated. Following 3, 7, 14 and 21 days, the tissue samples were obtained and skin irritation ratio, hydroxyproline content, as well as immune cells, fibroblasts, fibrocytes distribution and collagen density were analyzed. Moreover, the cellular RNA damage examined using epi-fluorescent microscope. Hydroethanolic extract of PAO significantly (P < 0.05) increased wound contraction percentage and upregulated hydroxyproline content. The animals in medium and high dose PAO-treated groups exhibited remarkably (P < 0.05) higher fibroblast distribution and significantly (P < 0.05) lower immune cells infiltration. PAO up-regulated mast cells distribution on day 7 and elevated neovascularization in a dose dependent manner. Significantly lower RNA damage was revealed in PAO-treated animals. Our data showed that, PAO shortened the inflammation phase by provoking the fibroblast proliferation. Moreover, PAO enhanced mast cells distribution and infiltration, which in turn promoted the neovascularization. Ultimately, promoted angiogenesis increased RNA stability in different cell types. Thus, Hydroethanolic extract of PAO can be considered as an appropriate compound for wound healing medicine. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Wound healing Hydroethanolic extract Pistacia atlantica Excision wound Incision wound
1. Introduction Since long times ago, almost in all Asian countries-especially in Iran-plants are considered in traditional medication. Although the modern medication advanced during the last decades by using
* Corresponding author. Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia 57159-44867, Iran. E-mail address: [email protected] (M.R. Farahpour).
synthetic drugs, the herbal medicine still plays essential roles in different therapeutic approaches. Indeed, the elevating interest for herbal medication raises based on plants therapeutic properties. Several studies indicated that plants, contain large amounts of natural antioxidant [1], antibacterial [2], antiviral and antiinflammatory contents [3], which are highly important in different aspects of usage. Generally, wound healing is a dynamic process, which different factors such as infection, prolonged inflammation and age are
http://dx.doi.org/10.1016/j.ijsu.2015.03.019 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
M.R. Farahpour et al. / International Journal of Surgery 17 (2015) 88e98
interfered with [4,5]. On the other hand, wound contracture is a complex process that occurs throughout the healing process, which starts with fibroblastic phase. Wound healing consists of 3 phases including; inflammatory, proliferative, and maturation that are largely susceptible to the type and extent of damage, the ability of the tissue to repair as well as different inflammatory factors [4,5]. Further to immune cells-involved pathways during the inflammatory stage of wound healing process, role of mast cells is not ignorable. Mast cells-especially activated mast cells-are highly participated in response to allergens, pathogens and pathogensinduced cytokines [6,7]. Mast cells increase the vascular permeability by secreting different factors such as histamine and vascular endothelial growth factor, which lead to prolonged inflammation with enhancing immune cells infiltration [8]. Thus, administrating agents with antibacterial and anti-inflammatory properties has gained increasing consideration in order to shortening the time necessary for complete healing as well as reducing the risks of undesirable complications. Pistacia from Anacardiaceae family (that comprise about 70 genera and over 600 species) are a perennial evergreen shrubs and trees, which are characterized as xerophytic. The Pistacia atlantica belongs to Anacardiaceae family, which is annually growing from the Mediterranean basin to central Asia, especially in Iran, Turkey, Iraq and Saudi Arabia, which is recorded in tropicus data supplied list and was also accepted in Royal Botanic Garden at 1967 [9]. P. atlantica Desf. Subsp Kurdiac (locally named as pesteye vahshi) grows in West Azerbaijan, Iran. There are several reports indicating that other species of P. atlantica have sedative, restorative effects and possess different pharmacological attributes such as; antiinflammatory, antibacterial, antimicrobial [10,11], antifungal and antihyperlipidemia [12]. In addition, Haghdoost and co-workers reported the PAO exerts beneficial effect on burn wound healing [13]. Therefore, current study was designed in order to evaluate the effect of different concentrations of hydroethanolic extract of the P atlantica hulls on the wound healing process. For this purpose, following antioxidant activity tests for the P. atlantica, the immune cell infiltration, angiogenesis, collagen content, mast cell distribution as well as RNA expression in epithelial cells was analyzed as histological features. Moreover, the acute wound irritation test and hydroxyproline content estimation were performed in order to clearly show the effect of the P. atlantica hulls hydro alcoholic extract on the wound healing process.
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powder was suspended in 600 ml of hydroethanolic solution for 96 h at room temperature. The mixture was filtered using a fine muslin cloth followed by filter paper (Whatman No 1). The filtrate was placed in an oven to dry at 40 C. The obtained clear residue was used for the study. The extracts were kept at 20 C until they were used in the experiment [14,15]. 2.2. Antioxidant activity 2.2.1. Assessment of 2, 2-di (4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) DPPH free radical inhibition was assessed as described by Chen et al. [16] with some modification. Ninety six wells micro titer plates were used and 5 different concentrations of each sample were assessed. A solution of 100 mg/ml of DPPH in methanol was used and all experiments were done in triplicates. After a 45 min incubation at 25 C (heidolph titramax 1000 and incubator 1000, Germany), the absorbances were recorded at 517 nm by using a Powerwave XS Microplate spectrophotometer (Bio-Tek Instruments, Inc. Winooski, VT 05404 United States). The free radical inhibition percentage (In %) was calculated as follow:
In% ¼
Ablank Asample Ablank
100
where Ablank was the absorbance of the control reaction (containing all reagents except the test compound), and Asample was the absorbance of the test compound. After that the concentration which could result in 50% inhibition (IC50) was calculated from the graph plotted of inhibition percentage against samples concentration. 2.2.2. The total phenolic content Total phenolic constituents of samples extracts were determined by modified methods described by Saeed et al., and using FolinCiocalteu reagent and Gallic acid (ranging from 0 to 1000 mg/L) as standard phenolic compound [14]. 2.2.3. Total flavonoids estimation The aluminum chloride method was performed for determination of the total flavonoid content of the extracts [14]. The flavonoid content was expressed as mg of quercetin equivalents per gram of dried extract [14].
2. Materials and methods 2.1. Plant material and extract preparation P. atlantica hulls were collected and picked up by hand from the central district of the region of Urmia, West Azerbaijan province, Iran in at July, latitude: 37 340 , longitude: 44 58'. The plant was authenticated by the Department of Botany Sciences, Agriculture and Natural Resources Research Center, Urmia, Iran. Hulls were separated from the fruit by hand and around 600 g of fresh plant material was dried naturally in shadow on laboratory benches at room temperature (23e24 C) for six days until crisp, and powdered in an electric blender [14,15]. Then 150 g of the plant
2.2.4. Ferric reducing antioxidant power (FRAP) test Assessment of ferric reducing antioxidant power is based on the reduction of ferric tripyridyltriazine (Fe (III) -TPTZ) complex to the ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. Production of Fe (II) -TPTZ results in an intensive blue color, which could be assessed at 593 nm [17]. FRAP reagent was prepared just before each experiment by mixing a, b and c solution in the ratio of 10:1:1 (a: Acetate buffer 300 mM pH 3.6, b: 10 mM TPTZ (2, 4, 6tripyridyl-s- triazine) in 40 mM HCl, c: 20 mM FeCl3. 6H2O). A 20 ml from sample was mixed with 200 ml FRAP reagent, held for 10 min at room temperature and the absorbance was recorded at 593 nm by Powerwave XS Microplate spectrophotometer (Bio-Tek
Table 1 Antioxidant properties, total phenol and total flavonoid contents of Pistacia atlantica hulls. IC50 in DPPH inhibition assay (mg/ml) Total phenols (mg/mg dried extract) Total flavonoid (mg eq Rutin/mg dried extract) Eqþ Fe2 m per mg extract. Pistacia atlantica 7.45 BHT 107.04 Ascorbic acid e
257.8 ± 4.3 e e
112.71 ± 2.34 e e
18294.0 ± 83.6 e 7740.2 ± 64.9
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22.03 21.15 18.56 18.07 16.57
± ± ± ± ±
0.62 0.90 0.71a 0.62b 0.79b
Epithelialization time (days)
Table 3 Effect of hydroethanolic Pistacia atlantica hulls extract on linear incision wound model. All data are presented in Mean ± SD.
9.89 12.81 19.02a 16.76a 35.56b
0.61 0.26 0.00b 0.0b 0.0b ± ± ± ± ± 87.75 88.88 100.00 100.00 100.00 ± ± ± ± ± 83.60 85.50 95.32 99.76 100.00 0.73 1.00 0.15a 0.31a 0.25b 75.00 77.35 84.30 86.15 93.35
± ± ± ± ±
0.73 1.56 1.43a 0.41a 0.16b
79.50 81.40 88.65 94.50 98.80
± ± ± ± ±
2.3.1. Animals and study design Healthy white Wistar male rats weighing approximately 200 g and 9 weeks of age were used in the present study. Twenty four healthy white Wistar rats were selected in order to evaluate the acute toxicity analyses. Two weeks before and during the entire experiments, the animals were housed in individual plastic cages (50 40 20 cm) with an ambient temperature of 23 ± 3 C, stable air humidity, and a natural day/night cycle. The animals were handled on a regular daily basis for 2 weeks prior to the study in order to acclimatize them with testing area and experiments. This could minimize anxiety related to the testing inaccuracies. The rats had free access to standard rodent laboratory food and tap water. The procedures were carried out based on the guidelines of the Ethics Committee of the International Association for the Study of pain [18]. The University Research Council approved all experiments.
1.96 1.04 1.46a 1.00a 1.35b ± ± ± ± ±
± ± ± ± ±
2.3. Biological activity test
2.23 1.77 2.30a 1.48a 1.82b
70.85 73.85 79.80 82.25 88.95 ± ± ± ± ±
273.6 282.53 385.7 478.81 508.05
Instruments, Inc. Winooski, VT 05404 United States). Different concentrations of FeSO4.7H2O (200, 400, 800, 1200 and 1600 mM) were used as standard solution, which reacted with TPTZ reagent and the absorbance was plotted against various ferrous ion concentrations. The results were expressed as mM Fe2þ equivalents per mg of dried extract. L-ascorbic acid was used as standard antioxidant.
n ¼ 6 animals in each group. Valued are expressed as mean ± S.D. The treated groups are compared by Student t test with the control group. *P < 0.05. Note: a, b are presented significant differences between marked groups.
60.00 68.50 69.80 80.75 85.30 3.53 3.89 1.90a 2.49a 2.11b ± ± ± ± ± 40.15 45.00 56.65 60.85 67.55 2.28 2.14 1.72a 4.85a 2.32b ± ± ± ± ± 27.85 32.50 40.70 42.75 48.30 1.73 2.31 5.28 1.80 2.25a ± ± ± ± ± 17.05 18.90 23.05 23.72 26.25 1.10 1.23 1.59 1.65 1.86 ± ± ± ± ± 10.95 11.15 13.99 14.58 16.95 Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
Statistical mean ± S.D.
Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
n ¼ 6 animals in each group. P < 0.05 and versus Control Note: a, b are presented significant differences between marked groups.
0.49 0.85 0.67a 0.32b 0.0b
Day 20 Day 18 Day 16 Day 14 Day 12 Day 10 Day 8 Day 6 Day 4 Day 2 Groups
Percentage of wound contraction on day
Table 2 Effects of the hydroethanolic Pistacia atlantica hulls extract on circular excision wound contraction area (mm2) and period of epithelialization.
Groups
2.3.2. Estimating acute toxicity In order to find a safe dose for P. atlantica, an acute toxicity study was conducted. Twenty four, weighting 200 g and 7e9 weeks old healthy white Wistar rats were randomly divided into 4 groups (n ¼ 6/group) including: Control, Placebo, 5% PAO-treated and 10% PAO-treated groups. The animals were under surveillance for 30 min 2, 4, 24 and 48 h after the administration in order to estimating the any clinical or toxicological symptoms and any mortality ratio were recorded during two weeks. Hematological, serum biochemical and histological (liver and kidney) parameters were determined based on the standard methods [19]. 2.3.3. Formulation of topical wound application forms The topical applicable ointments were prepared in four different concentrations. All the variants consisted base formulation comprising Eucerin (25%) and Vaseline (75%) in 1: 3 proportions. A surgical wound was created, 150 rats randomly were labeled by none toxic color and divided into five groups (No ¼ 30 rats in each group). Two groups served as Controls: Group 1, which did not receive any chemical/ointment and Group 2 marked as Placebotreated; animals in this group were treated with base formulation. Three different concentrations of PAO were administrated in groups 3, 4 and 5. For this purpose, 1.5, 3 and 5 g from PAO extract were mixed with base formulation [20]. The ointments were topically applied on the wound area once a day, starting from the day of operation, until 21 days post-operation. All rats were monitored
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Table 4 Effect of hydroethanolic Pistacia atlantica hulls extract on wound healing of the dead space wound model. All data are presented in Mean ± SD. Groups
Dead space wound model Hydroxyproline content (mg/mL)
Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
10.99 12.41 14.17 15.44 15.81
± ± ± ± ±
Wet weight of the granulation tissue (mg)
0.3 0.32 0.33 0.32a 0.46a
81.1 85.7 97.9 115.83 124.43
± ± ± ± ±
3.25 4.62 4.57a 4.83a 4.55a
Dry weight of the granulation tissue (mg) 12.89 13.72 15.36 17.01 20.54
± ± ± ± ±
0.57 0.76 0.79 0.52a 0.72a
n ¼ 6 animals in each group. a is presented significant differences (P < 0.05) between marked data in the same column.
Table 5 Mean Distribution of immune cells (IMC), fibroblasts and fibrocytes per one mm2 of the sections in different groups. All data are presented in Mean ± SD. Groups
IMC
Fibroblast a
shaved. Following aseptication the PAO formulations were applied to animals in the experimental groups. After 4 h the skin of each animal was observed for signs of inflammation [21].
Fibrocyte a
Negative control 1.5% (day 3) 3% (day 3) 5% (day 3)
16.52 8.66 12.66 29.00
± ± ± ±
3.31 3.05b 2.88b 6.08c
2.21 2.25 5.00 3.25
± ± ± ±
0.60 0.95a,c 0.71b 0.89a
0.00 0.87 2.26 1.24
± ± ± ±
0.00a 0.25b 0.91c 0.48c,b
Negative control 1.5% (day 7) 3% (day 7) 5% (day 7)
26.10 14.66 12.54 19.32
± ± ± ±
1.21a 3.05b 2.08b 2.01c
4.90 2.28 4.35 7.50
± ± ± ±
1.16a 0.96a 1.70a,b 1.91b
0.41 0.87 1.62 3.74
± ± ± ±
0.02a 0.25b 0.47b 0.46c
Negative control 1.5% (day 14) 3% (day 14) 5% (day 14)
12.61 5.33 4.66 3.65
± ± ± ±
3.00a 1.52b 2.01b 0.57b
5.73 7.00 6.50 9.49
± ± ± ±
0.63a 1.11a 1.28a 1.30b
1.30 2.51 2.75 4.54
± ± ± ±
0.21a 1.00b 0.89b 0.66c
Negative control 1.5% (day 21) 3% (day 21) 5% (day 21)
6.20 4.00 2.63 1.33
± ± ± ±
0.71a 1.00b 0.60b 0.57c
2.11 4.75 2.63 4.85
± ± ± ±
0.22a 1.25b 1.70a,b 0.96b
0.64 1.21 1.63 3.00
± ± ± ±
0.70a 0.62a 0.41a 0.81b
a,b,c are presented significant differences (P < 0.05) between marked data in the same column.
for any wound fluid or any evidence of infection or other abnormalities, until complete epithelialization. 2.3.4. Acute skin irritation test The test was carried out based on Gfller et al. (1985) suggested method. About 314 mm [2] from dorsal fur of each animal was
2.3.5. Wound healing models 2.3.5.1. Circular excision wound model. Each group of animals (n ¼ Sixteen) was anesthetized by intraperitoneal administration of ketamine 5%, 90 mg/kg (Ketaset 5%; Alfasan, Woerden, The Netherlands) and xylazine hydrochloride 2%, 5 mg/kg (Rompun 2%, Bayer, Leverkusen, Germany). The fur was prepared aseptically and the predetermined area was marked on the back of animals. Each rat was fixed on the surgery table in ventral posture. Following surgical preparation a circular surgical full thickness wound was made, 314 mm diameters, on the anterior-dorsal side of each rat [22]. Wound contraction percentage and wound closure time were used to assess the wound-healing property. The wound area was measured by immediate placing of a transparent paper over the wound and tracing it out; the area of this impression was calculated using the graph sheet. The wound healing percentage was calculated by the Walker formula after measuring the wound size [23]. The percentage of wound healing was computed at the beginning of experiments and on days 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 days post-test. Percentage of wound size ¼ Wound area on day X/Wound area on day zero 100 Percentage of wound healing ¼ 100 Percentage of wound size
Fig. 1. Histological feature of experimentally-induced wound on 3 after surgery. A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note the higher magnifications for granulation tissue; Well formed granulation tissue and decreased edema is presented in 5% pistachio-treated group, (Masson-trichrome staining, 400 & 600). FN: fibrinoid necrosis, ED: edema, S: scab.
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Table 6 The scores for tissue edema, collagen production and epidermis migration are presented. Groups
Edema score
Collagen score
Epidermis thickness
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþþ þþ þþþ þþ
þþ
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþ þþ þþ þ
þ þþ þþ þþ
20.62 ± 10.08
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþ þþ þ þ
þ þþ þþþ þþþþ
9.61 ± 3.02a 22.57 ± 9.04b 50.16 ± 12.54c 75.24 ± 12.30d
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþ þ þ
þþ þþþ þþþ þþþþ
20.37 43.89 66.87 83.40
þ
± ± ± ±
4.12a 6.27b 7.23c 6.74d
Note: The Masson trichrome staining was scored into þþþþ: Intensive; þþþ: Mild; þþ: Mild to moderate; þ: Faint; : negative. a, b, c are presented significant differences (P < 0.05) between marked data in the same column.
2.3.5.2. Incision wound model. Animals were randomly divided into five experimental groups of six animals each: Control, Placebo, 1.5%, 3% and 5% groups. All animals in the experimental groups were anesthetized with the same way mentioned above and 4-cm length incision was made through the skin and cutaneous muscle at a distance about 1.5 cm from the middle on the right side of the depilated back. The wound was closed at 0.5 cm intervals using 3/ 0 nylon (Dafilon, B/Braun, Germany). All the groups were treated the same as mentioned in the excision model. Ointments were applied in manners that fully cover the area, once daily for 9 days. On day 9, sutures were removed and the tensile strength of healed wounds was measured on day 10 by Strongraph mechanical test frame (Toyoseiky Tensile Testing Unit, Model R3, Japan) [24]. Tensile strength was calculated using the following formula: Tensile strength ¼ breaking strength (g)/cross sectional area of skin (mm2)
2.3.6. Dead space wound model and hydroxyproline content estimation Animals were randomly divided into five experimental groups of six animals in each: Group 1 was considered as the Control, did not receive extract; group 2 was considered as the Placebo, received 2 ml of 1% carboxy methyl cellulose (CMC); and groups 3, 4 and 5 were treated orally with 100 mg/kg, 200 mg/kg and 400 mg/kg hydroethanolic extract suspended in 1% w/v CMC, respectively [25]. The dead space wound was created using subcutaneous implanting of polypropylene tubes, 2.5 cm 0.5 cm, in the lumbar region on the dorsal side. The Animals of the experimental groups received the extract from 0 to 9 day post-wounding induction. On day 10 post-wound induction, the created granulation tissue was carefully dissected. Breaking strength and hydroxyproline content were analyzed. 2.3.7. Histopathological study Animals were anesthetized with the same way mentioned above and specimens of skin were taken for further
histopathological analyses on 3, 7, 14 and 21 days after surgery. Sample tissues, excised along with 1e2 mm surrounding normal skin and in a depth of approximately 3 mm, were pinned on a flat cork surface and fixed in neutral-buffered formalin 10%. Then the sample tissues were routinely processed, paraffin wax embedded, sectioned at 5 mm, and stained with Masson's trichrome and examined under light microscopy (Olympus CX31RBSF, Japan) to assess the predominant stage of wound healing. Three parallel sections were obtained from each specimen. Following factors including; Cellular infiltration (immune cells, fibroblasts and fibrocytes), angiogenesis (the number of blood vessels and capillary buds) were quantitatively evaluated per one mm2 of the tissue for each section under 400 magnification. Epithelialization (epithelium thickness), collagen production and density were also evaluated qualitatively and calculated manually. All parameters were analyzed in 5 per high power fields (HPFs) [26,27]. 2.3.7.1. Fluorescent analyses for RNA damage. The RNA damage was assessed using the acridine-orange NO dye (Sigma Aldrich, Germany) according to von Bertalanffy and Bickis method. In brief, the tissues were washed out with ether alcohol and cut by cryostat (8 mm). The prepared sections were fixed by different degrees of alcohol (ethanol) for 15 min. Then the sections briefly were rinsed in acetic acid, 1% aqueous, followed by washing in distilled water. The specimens then were stained in acridine-orange for 3 min and distained in phosphate buffer and ultimately were followed for fluorescent colors differentiation in calcium chloride. The degenerated cells were characterized by loss of RNA and/or with faint red stained RNA. The normal cells were marked with bright red RNA at the apex of the nuclei. In order to reduce the bias problems for staining density, 20sections for each sample were investigated. The RGB plots (color class identification) for red reacted sites were prepared by using image pro-insight image analysis software (version 6.0, media cybernetics) [28]. 2.3.7.2. Mast cells distribution. Mast cells distribution per one mm2 of the tissue was analyzed according to Jagatic and Weiskopf method. Briefly; the slides were deparaffinized and hydrated into water. Then the samples were stained in hematoxylin for 5 min and washed in running water. After that, the slides were stained with acridine-orange for 6 min and after dehydration were mounted in Harleco fluorescent mountant [29]. 2.3.8. Statistical analysis Experimental results were expressed as means ± SEM. Statistical analyses were performed using PASW 18.0 (SPSS Inc., Chicago, IL, USA). Model assumptions were evaluated by examining the residual plot. Results were analyzed using two-way ANOVA. Dunnett's test for pair-wise comparisons was used to examine the effect of time and treatments. Statistical differences were considered significant when P < 0.05. 3. Results 3.1. Antioxidant activity, total phenol and flavonoid contents The extract concentrations providing 50% inhibition (IC50) of DPPH free radicals were determined and compared with that of butylated hydroxytoluene (BHT), as a standard antioxidant. The results for DPPH are presented in Table 1. Assessment of FRAP also revealed that PAO exhibited significantly (P < 0.05) higher antioxidant activity comparing to ascorbic acid as a standard. The phenolic content of the extract was determined based on FolinCiocalteu reagent method. Results showed that the assessed extract yield 257.8 ± 4.3 mg/mg dried extract and 112.71 ± 2.34 mg
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Fig. 2. Histological feature of experimentally-induced wound on 14 after surgery. A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note the newly generated epidermis in control-sham that is completed in 3 and 5% pistachio-treated group. Faint edema remained in deeper dermis of the 1.5% pistachio-treated group, while is significantly limited in 5% pistachio-treated group. Complete layers of the epidermis are generated in 3 and 5% pistachio-treated groups. See the regular basement membrane cells and the spinosume cellular layer. Papilla is completely generated after 14 days in 5% pistachio-treated group. No edema is detectable in 3 and 5% pistachio-treated groups, (Masson-trichrome staining, 400 & 600). EP: epidermis, De: dermis, D.De: deeper dermis, BL: basement layer, Sp: spinosume stratum, Kr: keratin.
eq Rutin/mg dried extract for phenol and flavonoid contents, respectively (Table 1). 3.2. Skin irritation The formulated ointments of PAO, at different doses, did not show any inflammation or noticeable swelling and/or any type of irritation on the skin. 3.3. Wound contraction developed in PAO-treated animals Administration of different doses of PAO in excision wound
model significantly (P < 0.05) increased wound contraction rate compared into non-treated animals. The wound closure achieved by all doses on 6th post wounding day was approximately close to those contractions from reference drug. Comparing the contraction rate between groups showed that, from day 6 until day 16 the 5% PAO-treated animals exhibited significantly (P < 0.05) higher contraction rate (98% on day 16) in comparison to 1.5% (88% on day 16) and 3% (94% on day 16) PAO-treated groups. Meanwhile, the contraction rate of the non-treated group was only 79% 16 days post-injury (Table 2).
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Fig. 3. Histological feature of experimentally-induced wound on 21 after surgery. Fluorescent staining for RNA and Collagen with RGB image plot for red and green reactions in 600 mm for A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note increased collagen synthesis accomplished with enhanced RNA content in 3 and 5% pistachio-treated groups. E: epithelium, Co: collagen, F: hair follicle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Micro vessels distribution per one mm2 of the tissue in different days and different administrated doses, all data are presented in Mean ± SD. Note: a, b, c, d, e, f are presented significant differences between marked groups.
3.4. Linear incision wound model and dead space wound model changed depending on dose In order to evaluate the wound healing activity of the PAO, the linear incision wound model was performed. As reported in Table 3, the PAO-treated animals exhibited significantly (P < 0.05) higher values in comparison to non-treated group. Interestingly, the 5% PAO-treated animals showed the highest values versus 1.5% and 3%
Fig. 5. Mast cells distribution in different groups; PAO enhanced mast cells distribution more prominently on day 7 after wound induction. All data are presented in Mean ± SD and P < 0.05 considered as significant difference. Note: a, b, c presented significant differences between marked data.
PAO-received groups. According to our analyses of the dead space wound model, the hydroxyproline content of the PAO-treated animals increased depending on administrated dose. Accordingly, 3% and 5% PAOreceived animals represented the greatest hydroxyproline content versus to those at 1.5% PAO-treated and non-treated groups. Similar to the results for hydroxyproline, the PAO-treated animals showed
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Fig. 6. Fluorescent photomicrograph for mast cells distribution 7 days after wound induction; A) control, B) 1.5% PAO-treated, C) 3% PAO-treated and D) 5% PAO-treated. Note dose dependently increased mast cell distribution (head arrow) enclosed to vessels (arrows) in treated groups, (Fluorescent staining for mast cells, 400).
Fig. 7. Pistachio atlantica hulls hydroethanolic extract-induced early wound healing process.
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significantly (P < 0.05) higher dry and wet weights of granulation tissue in comparison to non-treated animals (Table 4).
maximum neovascularization on day 7 after wound induction, while the animals in 5% PAO-treated group exhibited massive neovascularization on day 14 (Fig. 4).
3.5. Histopathology result 3.5.1. Immune cells (IMC) infiltration decreased in a dose dependent manner Mean distribution of IMC per one mm2 of the tissue was analyzed in different groups (Table 5). Histological observations showed that on day 3, the mean number of IMC significantly (P < 0.05) decreased in 1.5% PAO-treated animals in comparison to other treated and non-treated groups (Fig. 1). Meanwhile, the situation changed after day 7, when the IMC distribution was detected significantly (P < 0.05) lower in 3% PAO-treated group. Finally, after 14 and 21 days, the animals in 5% PAO-treated group showed the lowest distribution of IMC per one mm2 of the tissue in comparison to those in other test and control groups. 3.5.2. Fibroblast and fibrocyte distribution was dependent to administrated dose Three days after injury, the difference between control and 1.5% PAO-treated group was not statistically noticeable. However, at the same time, the number of fibroblasts and fibrocytes remarkably (P < 0.05) increased in one mm2 of the wound area in 3% PAOtreated group. In contrast, 7, 14 and 21 days after treatment the 5% PAO-treated animals exhibited the highest distribution of fibroblasts and fibrocytes per one mm [2] of the tissue. The data for IMC, fibroblast and a fibrocyte number per one mm2 of the tissue are presented in Table 5. 3.5.3. Collagen deposition, edema and re-epithelialization Assessment of collagen deposition was conducted via scaling the intensity of the blue color in Masson-trichrome stained slides. As represented in Table 6, 7th day after wound induction the collagen deposition started in PAO-treated animals, which was more pronounced in 3 and 5% PAO-treated animals compared to 1.5% PAO-received and non-treated wound-induced groups. In order to reduce the manual examination errors, the fluorescent staining for collagen bundles was conducted and the RGB plot analyses based on classification of fluorescent reactions were performed. The results from the RGB image plot for green collagen content confirmed the results from routine Masson-trichrome histochemistry. Observations revealed that, 7 days after injury the collagen deposition started at 3% and 5% PAO-treated animals and reached to its maximum amount of 14th days after wound induction in 5% PAO-treated group (Fig. 2). Our analyses for edema in tissue showed that PAO at dose levels of 3% and 5%, significantly (P < 0.05) reduced the edema scores on day 7 after wound induction. Surprisingly, 7 days after injury the surface and deeper dermis of the animals in 5% PAO-treated animals exhibited the lowest edema score (þ), while the 1.5% and 3% PAO-treated groups exhibited higher scores (þþ). On days 14th and 21, the reepithelialization started and, albeit with some differences, the animals in all treated groups were revealed with epidermis. The 5% PAO-treated animals showed the highest thickness for epithelium in comparison to other test groups. Regular and complete basement membrane, enhanced stratum spinosume and granulosa layer were identified in 5% PAO-treated animals. In despite of reepithelialization in non-treated animals, the cellular layers of the epidermis were not organized appropriately (Fig. 3). 3.5.4. Neovascularization increased in PAO-treated groups On day 3 after wound induction, the PAO-treated animals showed significant (P < 0.05) increase in neovascularization. Interestingly, animals in 3% PAO-treated group showed the
3.5.5. Mast cells distribution differed depending on dose Fluorescent analyses for mast cells showed that, 1.5% and 5% PAO-treated groups represented similar pattern for mast cells infiltration. Accordingly, both groups exhibited massive mast cells distribution on days 3 and 14 post-injury. However, the 5% PAOtreated animals manifested significantly (P < 0.05) higher mast cells infiltration versus 1.5% PAO-received group. In contrast, 3% PAO-treated group exhibited intensive mast cells distribution on day 7 after wound induction (Figs. 5 and 6). 3.5.6. RNA expression increased in PAO-treated groups Special fluorescent analyses for RNA content of normal and necrotic cells was used in order to detect the normal RNA expression in epithelial and dermal cells of all groups. Observations demonstrated that administration of PAO at 3% and 5% concentrations significantly (P < 0.05) increased RNA expression as well as normal RNA content in epithelial and dermal cells. Meanwhile, non-treated groups exhibited significantly (P < 0.05) lower normal RNA versus treated animals. The interactive 3D surface plot and image RGB Plot showed remarkably higher dense red fluorescent reactions (marking normal RNA content) in PAO-treated animals (Fig. 2). 4. Discussion The current study showed that PAO in different doses shortened the time taking to wound healing and up-regulated wound contraction rate after 7 days from wound induction. Moreover, the animals treated with PAO exhibited well-formed granulation tissue, enhanced collagen deposition as well as rapid neovascularization in comparison to non-treated group. Analyzing the mast cells distribution showed that low (1.5%) and high dose (5%) PAO-treated animals manifested massive mast cells infiltration 3 and 14 days after injury induction and those in 3% PAO-received group showed intensive mast cell distribution on day 7 following wound induction. Our results showed that at early stages (on day 3), the 1.5% PAO treated animals showed lower IMC infiltration, while the higher doses (especially 5% PAO) resulted in massive IMC infiltration. The irritant property of PAO in higher doses might be responsible for increased IMC infiltration. Accordingly, the animals in 3% and 5% PAO-treated groups were represented higher edema and delayed angiogenesis in comparison to 1.5% PAO-received group. In the line with this hypothesis, the influence of irritant contents of extracts on recurrent inflammation during the wound healing process was reported previously [30]. In addition, the results from fluorescent analyses of mast cells showed that the animals in 5% PAO-treated group represented massive mast cell distribution on days 3 after wound induction. The role of mast cells in enhancing the cellular infiltration during wound healing has been reported previously [6,7] Mast cells by synthesis of different cytokines and growth factors provoke the proliferative phase and up-regulate the IMC infiltration via enhancing the vascular permeability [31,32]. Therefore, we come close to this fact that the PAO increased IMC infiltration partly via impressing the mast cells, which in turn resulted in intensive IMC infiltration. In continuo, the elevated vascular permeability resulted in edema that was scored higher than those in 1.5% PAO-treated group. Lower mast cells distribution in 1.5% PAO-received animals confirmed this theory. In order to understand the mast cells oppositional role in
M.R. Farahpour et al. / International Journal of Surgery 17 (2015) 88e98
shortening the wound healing process one should note that mast cells are participated in collagen synthesis directly by up-regulating fibroblast's physiologic function via secreting fibroblast growth factor-2 [32,33]. On the other hand, mast cells are known for secreting vascular growth factor (VGF) that stimulates the endothelial cells proliferation, which in turn enhances neovascularization in healing wounds [34]. Increased neovascularization at day 14 following wound induction and it's harmony with mast cells infiltration in 5% PAO-treated animals may be inferred that PAO could provoke the healing process via stimulating mast cells infiltration after 14 days. Increased collagen deposition following 14 days in 5% PAO-treated animals was in good accordance with neovascularization and mast cells distribution. It is important to consider that mast cells secret tryptase and chymase. Tryptase promotes the fibroblast proliferation and stimulates collagen synthesis and chymase can directly cleave the procollagen type I and up-regulate the fibrillation of collagens [35,36]. In good accordance with these findings, our light microscopic analyses showed that the fibroblast and fibrocyte distribution elevated in PAO-treated animals. At the same time, the collagen deposition increased simultaneously with elevated fibroblast distribution as well as mast cells number after 7 and 14 days from wound induction (Fig. 7). Our biochemical analyses revealed that PAO extract contained high amounts of phenol and flavonoid. Flavonoids are known for their antioxidant and anti-inflammatory effects [37,38]. In the line with this fact, there is a negative correlation between pathologically-induced oxidative stress and cellular physiologic function as well as normal DNA and RNA contents. Indeed, IMCs especially the polymorphonuclear cells are main sources of reactive oxygen species (ROS) [39,40]. In order to evaluate the cellular damage we used special fluorescent staining for normal RNA content in epithelial and dermal cells. Observations revealed severe RNA damage in non-treated group. Meanwhile, administrating of PAO significantly decreased RNA damage. Therefore, we can suggest that the flavonoid content of the PAO extract could considerably decrease the inflammation-induced oxidative stress that ultimately inhibited the RNA damage both in epidermal and dermal cells. Furthermore, our analyses showed that administrating of PAO significantly provoked wound contraction ratio versus non-treated animals. Contraction is so essential for reducing the time for healing process because closure of the wound decreases the distance and declines the size of the wound [41]. In this condition it facilitates re-epithelialization process. In this study, the epithelization time was also found to be significantly shorter and the animals in 5% PAC-treated group showed complete layers of epidermis. Increased viability of epithelial cells beside their successful proliferation and migration from wound bed result in to well-formed epithelialization [42]. Rapid re-epithelialization is considered as a hallmark for well wound care. Therefore, the shorter epithelialization time in 5% PAO-treated animals shows its effective impact on epithelial cells proliferation and active migration. Taking together, the surveillance of epithelial cells in PAO-treated groups might be due to its antioxidant property that inhibited oxidative stress-induced cellular damage and facilitated their migration process. 5. Conclusion This study highlighted the wound healing activity of PAO. Our results showed that different doses of the PAO reduced the healing time, facilitated the wound contraction, up-regulated hydroxyproline content and elevated the neovascularization. Moreover, findings of present study represented that PAO increased collagen
97
deposition simultaneously by up-regulating the mast cells and fibroblast distribution. Finally, obtaining better results from high dose administration of PAO suggests that dosing higher concentration contains more constituents that plays major role in shortening healing time. Ethical approval None. Sources of funding None. Author contribution Mohammad Reza Farahpour: Study design, surgical procedures and writing. Navideh Mirzakhani: Pathology procedures, Data collection and data analysis. Jamal Doostmohammadi: Care of animals. Mahmood Ebrahimzadeh: Care of animals. Conflict of interest The authors report no conflict of interest. Guarantor Mohammad Reza Farahpour. Acknowledgment The authors wish to thank laboratory section of the AYANDEH Lab. for laboratory tests and analyses. References [1] Z. Kokanova-Nedialkova, P. Nedialkov, S. Nikolov, The genus chenopodium: Phytochemistry, ethnopharmacology and pharmacology, Pharmacogn. Rev. 3 (6) (2009) 280. ~ ez, M.C. Recio, R.M. Giner, J.M. Prieto, M. Cerda -Nic[2] E.M. Giner-Larza, S. M an ol as, et al., Oleanonic acid, a 3-oxotriterpene from Pistacia, inhibits leukotriene synthesis and has anti-inflammatory activity, Eur. J. Pharmacol. 428 (1) (2001) 137e143. [3] I. Orhan, M. Aslan, B. Sener, M. Kaiser, D. Tasdemir, In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L, Phytomedicine 13 (9) (2006) 735e739. [4] B.P. Nagori, R. Solanki, Role of medicinal plants in wound healing, Res. J. Med. Plant 5 (4) (2011) 392e405. [5] D. MacKay, A.L. Miller, Nutritional support for wound healing. Alternative medicine review, J. Clin. Ther. 8 (4) (2003) 359e377. [6] K.N. Rao, M.A. Brown, Mast cells, Ann. N. Y. Acad. Sci. 1143 (1) (2008) 83e104. [7] T.C. Theoharides, A. Angelidou, K.-D. Alysandratos, B. Zhang, S. Asadi, K. Francis, et al., Mast cell activation and autism, Biochim. Biophys. Acta 1822 (1) (2012) 34e41. [8] A.M. Dvorak, Ultrastructure of Mast Cells and Basophils, Karger Medical and Scientific Publishers, 2005. [9] Royal Botanic Garden E. 1967, 28, 11. [10] M. Ali-Shtayeh, S.I. Abu Ghdeib, Antifungal activity of plant extracts against dermatophytes, Mycoses 42 (11e12) (1999) 665e672. [11] P. Magiatis, E. Melliou, A.-L. Skaltsounis, I.B. Chinou, S. Mitaku, Chemical composition and antimicrobial activity of the essential oils of Pistacia lentiscus var. chia, Planta Med. 65 (08) (1999) 749e752. [12] M. Sanz, M. Terencio, M. Paya, Isolation and hypotensive activity of a polymeric procyanidin fraction from Pistacia lentiscus L, Die Pharm. 47 (6) (1992) 466e467. [13] F. Haghdoost, M.M. Baradaran Mahdavi, A. Zandifar, M.H. Sanei, B. Zolfaghari, S.H. Javanmard, Pistacia atlantica resin has a dose-dependent effect on angiogenesis and skin burn wound healing in rat, Evidence-Based Complementary Altern. Med. (2013), http://dx.doi.org/10.1155/2013/893425. Article ID 893425, 8 pages. [14] N. Saeed, M.R. Khan, M. Shabbir, Antioxidant activity, total phenolic and total
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M.R. Farahpour et al. / International Journal of Surgery 17 (2015) 88e98 flavonoid contents of whole plant extracts Torilis leptophylla L, BMC Complement. Altern. Med. 12 (1) (2012) 221. A.N.A. Abad, M.H.K. Nouri, F. Tavakkoli, Effect of Salvia officinalis hydroalcoholic extract on Vincristine-induced Neuropathy in mice, Chin. J. Nat. Med. 9 (5) (2011) 354e358. F.-A. Chen, A.-B. Wu, P. Shieh, D.-H. Kuo, C.-Y. Hsieh, Evaluation of the antioxidant activity of Ruellia tuberosa, Food Chem. 94 (1) (2006) 14e18. I.F. Benzie, J. Strain, The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay, Anal. Biochem. 239 (1) (1996) 70e76. M. Zimmermann, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (2) (1983) 109e110. A.A. Adeneye, O.P. Ajagbonna, T.I. Adeleke, Preliminary toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats, J. Ethnopharmacol. 105 (2006) 374e379. L. TrivellatoGrassi, A. Malheiros, C. Meyre-Silva, Z. da Silva Buss, € de, et al., From popular use to pharmacological E.D. Monguilhott, T.S. Fro validation: a study of the anti-inflammatory, anti-nociceptive and healing effects of Chenopodium ambrosioides extract, J. Ethnopharmacol. 145 (1) (2013) 127e138. W. Gfeller, W. Kobel, G. Seifert, Overview of animal test methods for skin irritation, Food Chem. Toxicol. 23 (2) (1985) 165e168. A. Mekonnen, T. Sidamo, K. Asres, E. Engidawork, In vivo wound healing activity and phytochemical screening of the crude extract and various fractions of Kalanchoe petitiana A. Rich (Crassulaceae) leaves in mice, J. Ethnopharmacol. 145 (2) (2013) 638e646. H.L. Walker, A.D. Mason Jr., A standard animal burn, J. Trauma 8 (6) (1968) 1049e1051. A. Derakhshanfar, M. Oloumi, M. Mirzaie, Study on the effect of Peganum harmala extract on experimental skin wound healing in rat: pathological and biomechanical findings, Comp. Clin. Pathol. 19 (2) (2010) 169e172. B.S. Nayak, A.L. Udupa, S.L. Udupa, Effect of Ixora coccinea flowers on dead space wound healing in rats, Fitoterapia 70 (3) (1999) 233e236. M. Mashreghi, M.R. Bazaz, N.M. Shahri, A. Asoodeh, M. Mashreghi, M.B. Rassouli, S. Golmohammadzadeh, Topical effects of frog “Rana ridibunda” skin secretions on wound healing and reduction of wound microbial load, J. Ethnopharmacol. 145 (3) (2013) 793e797. I. Süntar, I. Tumen, O. Ustün, H. Keles¸, E.K. Akkol, Appraisal on the wound healing and anti-inflammatory activities of the essential oils obtained from the cones and needles of Pinus species by in vivo and in vitro experimental models, J. Ethnopharmacol. 139 (2) (2012) 533e540. V.L. Bertalanffy, I. Bickis, Identification of cytoplasmic basophilia (ribonucleic acid) by fluorescence microscopy, J. Histochem. Cytochem. 4 (5) (1956)
481e493. [29] J. Jagatic, R. Weiskopf, A fluorescent method for staining mast cells, Arch. Pathol. 82 (5) (1966) 430. [30] R.F. Diegelmann, M.C. Evans, Wound healing: an overview of acute, fibrotic and delayed healing, Front. Biosci. 9 (1) (2004) 283e289. [31] C.L. Gallant-Behm, K.A. Hildebrand, D.A. Hart, The mast cell stabilizer ketotifen prevents development of excessive skin wound contraction and fibrosis in red Duroc pigs, Wound Repair Regen. 16 (2) (2008) 226e233. [32] N. Shiota, Y. Nishikori, E. Kakizoe, K. Shimoura, T. Niibayashi, C. Shimbori, et al., Pathophysiological role of skin mast cells in wound healing after scald injury: study with mast cell-deficient W/Wv mice, Int. Arch. Allergy Immunol. 151 (1) (2009) 80e88. [33] I. Puxeddu, A. Piliponsky, I. Bachelet, F. Levi-Schaffer, Mast cells in allergy and beyond, Int. J. Biochem. Cell. Biol. 35 (12) (2003) 1601e1607. [34] G.J. Younan, Y.I. Heit, P. Dastouri, H. Kekhia, W. Xing, M.F. Gurish, et al., Mast cells are required in the proliferation and remodeling phases of microdeformational wound therapy, Plast. Reconstr. Surg. 128 (6) (2011) 649ee658e. [35] J. Gailit, M.J. Marchese, R.R. Kew, B.L. Gruber, The differentiation and function of myofibroblasts is regulated by mast cell mediators, J. Invest. Dermatol. 117 (5) (2001) 1113e1119. [36] B.L. Gruber, R.R. Kew, A. Jelaska, M.J. Marchese, J. Garlick, S. Ren, et al., Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis, J. Immunol. 158 (5) (1997) 2310e2317. [37] B.S. Nayak, S. Sandiford, A. Maxwell, Evaluation of the wound-healing activity of ethanolic extract of Morinda citrifolia L. leaf, Evidence Based Complementary Altern. Med. 6 (3) (2009) 351e356. [38] T. Okuda, Systematics and health effects of chemically distinct tannins in medicinal plants, Phytochemistry 66 (17) (2005) 2012e2031. [39] M. Getie, T. Gebre-Mariam, R. Rietz, R. Neubert, Evaluation of the release profiles of flavonoids from topical formulations of the crude extract of the leaves of Dodonea viscosa (Sapindaceae), Pharmazie 57 (5) (2002) 320. [40] S. Shetty, S. Udupa, L. Udupa, Evaluation of antioxidant and wound healing effects of alcoholic and aqueous extract of Ocimum sanctum Linn in rats, Evidence Based Complementary Altern. Med. 5 (1) (2008) 95e101. [41] F. Strodtbeck, Physiology of wound healing, Newborn Infant Nurs. Rev. 1 (1) (2001) 43e52. [42] C. Esimone, C. Nworu, C. Jackson, Cutaneous wound healing activity of a herbal ointment containing the leaf extract of Jatropha curcas L. (Euphorbiaceae), Int. J. App. Res. Nat. Prod. 1 (4) (2008) 1e4.
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Original research
Hydroethanolic Pistacia atlantica hulls extract improved wound healing process; evidence for mast cells infiltration, angiogenesis and RNA stability Mohammad Reza Farahpour a, *, Navideh Mirzakhani b, Jamal Doostmohammadi a, Mahmood Ebrahimzadeh a a b
Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran Department of Pathology, Faculty of Veterinary Medicine, P.O.BOX: 1177, Urmia University, Urmia, Iran
h i g h l i g h t s Topical Topical Topical Topical Topical
administration administration administration administration administration
of of of of of
Pistacia atlantica fascinated the inflammatory phase. P. atlantica up-regulated the mast cells infiltration. P. atlantica accelerate proliferation phase. P. atlantica cause significantly lower RNA damage. P. atlantica cause up-regulated hydroxylproline content.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2014 Received in revised form 2 February 2015 Accepted 16 March 2015 Available online 4 April 2015
In Iranian traditional therapy folk, the Pistacia is used for treatment of wound inflammation. Here in the present study, the In vivo effect of Pistacia atlantica hulls ointment (PAO) on the wound healing process was assessed. Excision and incision wounds were induced in rats. Three different doses of PAO were administrated. Following 3, 7, 14 and 21 days, the tissue samples were obtained and skin irritation ratio, hydroxyproline content, as well as immune cells, fibroblasts, fibrocytes distribution and collagen density were analyzed. Moreover, the cellular RNA damage examined using epi-fluorescent microscope. Hydroethanolic extract of PAO significantly (P < 0.05) increased wound contraction percentage and upregulated hydroxyproline content. The animals in medium and high dose PAO-treated groups exhibited remarkably (P < 0.05) higher fibroblast distribution and significantly (P < 0.05) lower immune cells infiltration. PAO up-regulated mast cells distribution on day 7 and elevated neovascularization in a dose dependent manner. Significantly lower RNA damage was revealed in PAO-treated animals. Our data showed that, PAO shortened the inflammation phase by provoking the fibroblast proliferation. Moreover, PAO enhanced mast cells distribution and infiltration, which in turn promoted the neovascularization. Ultimately, promoted angiogenesis increased RNA stability in different cell types. Thus, Hydroethanolic extract of PAO can be considered as an appropriate compound for wound healing medicine. © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
Keywords: Wound healing Hydroethanolic extract Pistacia atlantica Excision wound Incision wound
1. Introduction Since long times ago, almost in all Asian countries-especially in Iran-plants are considered in traditional medication. Although the modern medication advanced during the last decades by using
* Corresponding author. Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia 57159-44867, Iran. E-mail address: [email protected] (M.R. Farahpour).
synthetic drugs, the herbal medicine still plays essential roles in different therapeutic approaches. Indeed, the elevating interest for herbal medication raises based on plants therapeutic properties. Several studies indicated that plants, contain large amounts of natural antioxidant [1], antibacterial [2], antiviral and antiinflammatory contents [3], which are highly important in different aspects of usage. Generally, wound healing is a dynamic process, which different factors such as infection, prolonged inflammation and age are
http://dx.doi.org/10.1016/j.ijsu.2015.03.019 1743-9191/© 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.
M.R. Farahpour et al. / International Journal of Surgery 17 (2015) 88e98
interfered with [4,5]. On the other hand, wound contracture is a complex process that occurs throughout the healing process, which starts with fibroblastic phase. Wound healing consists of 3 phases including; inflammatory, proliferative, and maturation that are largely susceptible to the type and extent of damage, the ability of the tissue to repair as well as different inflammatory factors [4,5]. Further to immune cells-involved pathways during the inflammatory stage of wound healing process, role of mast cells is not ignorable. Mast cells-especially activated mast cells-are highly participated in response to allergens, pathogens and pathogensinduced cytokines [6,7]. Mast cells increase the vascular permeability by secreting different factors such as histamine and vascular endothelial growth factor, which lead to prolonged inflammation with enhancing immune cells infiltration [8]. Thus, administrating agents with antibacterial and anti-inflammatory properties has gained increasing consideration in order to shortening the time necessary for complete healing as well as reducing the risks of undesirable complications. Pistacia from Anacardiaceae family (that comprise about 70 genera and over 600 species) are a perennial evergreen shrubs and trees, which are characterized as xerophytic. The Pistacia atlantica belongs to Anacardiaceae family, which is annually growing from the Mediterranean basin to central Asia, especially in Iran, Turkey, Iraq and Saudi Arabia, which is recorded in tropicus data supplied list and was also accepted in Royal Botanic Garden at 1967 [9]. P. atlantica Desf. Subsp Kurdiac (locally named as pesteye vahshi) grows in West Azerbaijan, Iran. There are several reports indicating that other species of P. atlantica have sedative, restorative effects and possess different pharmacological attributes such as; antiinflammatory, antibacterial, antimicrobial [10,11], antifungal and antihyperlipidemia [12]. In addition, Haghdoost and co-workers reported the PAO exerts beneficial effect on burn wound healing [13]. Therefore, current study was designed in order to evaluate the effect of different concentrations of hydroethanolic extract of the P atlantica hulls on the wound healing process. For this purpose, following antioxidant activity tests for the P. atlantica, the immune cell infiltration, angiogenesis, collagen content, mast cell distribution as well as RNA expression in epithelial cells was analyzed as histological features. Moreover, the acute wound irritation test and hydroxyproline content estimation were performed in order to clearly show the effect of the P. atlantica hulls hydro alcoholic extract on the wound healing process.
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powder was suspended in 600 ml of hydroethanolic solution for 96 h at room temperature. The mixture was filtered using a fine muslin cloth followed by filter paper (Whatman No 1). The filtrate was placed in an oven to dry at 40 C. The obtained clear residue was used for the study. The extracts were kept at 20 C until they were used in the experiment [14,15]. 2.2. Antioxidant activity 2.2.1. Assessment of 2, 2-di (4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) DPPH free radical inhibition was assessed as described by Chen et al. [16] with some modification. Ninety six wells micro titer plates were used and 5 different concentrations of each sample were assessed. A solution of 100 mg/ml of DPPH in methanol was used and all experiments were done in triplicates. After a 45 min incubation at 25 C (heidolph titramax 1000 and incubator 1000, Germany), the absorbances were recorded at 517 nm by using a Powerwave XS Microplate spectrophotometer (Bio-Tek Instruments, Inc. Winooski, VT 05404 United States). The free radical inhibition percentage (In %) was calculated as follow:
In% ¼
Ablank Asample Ablank
100
where Ablank was the absorbance of the control reaction (containing all reagents except the test compound), and Asample was the absorbance of the test compound. After that the concentration which could result in 50% inhibition (IC50) was calculated from the graph plotted of inhibition percentage against samples concentration. 2.2.2. The total phenolic content Total phenolic constituents of samples extracts were determined by modified methods described by Saeed et al., and using FolinCiocalteu reagent and Gallic acid (ranging from 0 to 1000 mg/L) as standard phenolic compound [14]. 2.2.3. Total flavonoids estimation The aluminum chloride method was performed for determination of the total flavonoid content of the extracts [14]. The flavonoid content was expressed as mg of quercetin equivalents per gram of dried extract [14].
2. Materials and methods 2.1. Plant material and extract preparation P. atlantica hulls were collected and picked up by hand from the central district of the region of Urmia, West Azerbaijan province, Iran in at July, latitude: 37 340 , longitude: 44 58'. The plant was authenticated by the Department of Botany Sciences, Agriculture and Natural Resources Research Center, Urmia, Iran. Hulls were separated from the fruit by hand and around 600 g of fresh plant material was dried naturally in shadow on laboratory benches at room temperature (23e24 C) for six days until crisp, and powdered in an electric blender [14,15]. Then 150 g of the plant
2.2.4. Ferric reducing antioxidant power (FRAP) test Assessment of ferric reducing antioxidant power is based on the reduction of ferric tripyridyltriazine (Fe (III) -TPTZ) complex to the ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. Production of Fe (II) -TPTZ results in an intensive blue color, which could be assessed at 593 nm [17]. FRAP reagent was prepared just before each experiment by mixing a, b and c solution in the ratio of 10:1:1 (a: Acetate buffer 300 mM pH 3.6, b: 10 mM TPTZ (2, 4, 6tripyridyl-s- triazine) in 40 mM HCl, c: 20 mM FeCl3. 6H2O). A 20 ml from sample was mixed with 200 ml FRAP reagent, held for 10 min at room temperature and the absorbance was recorded at 593 nm by Powerwave XS Microplate spectrophotometer (Bio-Tek
Table 1 Antioxidant properties, total phenol and total flavonoid contents of Pistacia atlantica hulls. IC50 in DPPH inhibition assay (mg/ml) Total phenols (mg/mg dried extract) Total flavonoid (mg eq Rutin/mg dried extract) Eqþ Fe2 m per mg extract. Pistacia atlantica 7.45 BHT 107.04 Ascorbic acid e
257.8 ± 4.3 e e
112.71 ± 2.34 e e
18294.0 ± 83.6 e 7740.2 ± 64.9
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M.R. Farahpour et al. / International Journal of Surgery 17 (2015) 88e98
22.03 21.15 18.56 18.07 16.57
± ± ± ± ±
0.62 0.90 0.71a 0.62b 0.79b
Epithelialization time (days)
Table 3 Effect of hydroethanolic Pistacia atlantica hulls extract on linear incision wound model. All data are presented in Mean ± SD.
9.89 12.81 19.02a 16.76a 35.56b
0.61 0.26 0.00b 0.0b 0.0b ± ± ± ± ± 87.75 88.88 100.00 100.00 100.00 ± ± ± ± ± 83.60 85.50 95.32 99.76 100.00 0.73 1.00 0.15a 0.31a 0.25b 75.00 77.35 84.30 86.15 93.35
± ± ± ± ±
0.73 1.56 1.43a 0.41a 0.16b
79.50 81.40 88.65 94.50 98.80
± ± ± ± ±
2.3.1. Animals and study design Healthy white Wistar male rats weighing approximately 200 g and 9 weeks of age were used in the present study. Twenty four healthy white Wistar rats were selected in order to evaluate the acute toxicity analyses. Two weeks before and during the entire experiments, the animals were housed in individual plastic cages (50 40 20 cm) with an ambient temperature of 23 ± 3 C, stable air humidity, and a natural day/night cycle. The animals were handled on a regular daily basis for 2 weeks prior to the study in order to acclimatize them with testing area and experiments. This could minimize anxiety related to the testing inaccuracies. The rats had free access to standard rodent laboratory food and tap water. The procedures were carried out based on the guidelines of the Ethics Committee of the International Association for the Study of pain [18]. The University Research Council approved all experiments.
1.96 1.04 1.46a 1.00a 1.35b ± ± ± ± ±
± ± ± ± ±
2.3. Biological activity test
2.23 1.77 2.30a 1.48a 1.82b
70.85 73.85 79.80 82.25 88.95 ± ± ± ± ±
273.6 282.53 385.7 478.81 508.05
Instruments, Inc. Winooski, VT 05404 United States). Different concentrations of FeSO4.7H2O (200, 400, 800, 1200 and 1600 mM) were used as standard solution, which reacted with TPTZ reagent and the absorbance was plotted against various ferrous ion concentrations. The results were expressed as mM Fe2þ equivalents per mg of dried extract. L-ascorbic acid was used as standard antioxidant.
n ¼ 6 animals in each group. Valued are expressed as mean ± S.D. The treated groups are compared by Student t test with the control group. *P < 0.05. Note: a, b are presented significant differences between marked groups.
60.00 68.50 69.80 80.75 85.30 3.53 3.89 1.90a 2.49a 2.11b ± ± ± ± ± 40.15 45.00 56.65 60.85 67.55 2.28 2.14 1.72a 4.85a 2.32b ± ± ± ± ± 27.85 32.50 40.70 42.75 48.30 1.73 2.31 5.28 1.80 2.25a ± ± ± ± ± 17.05 18.90 23.05 23.72 26.25 1.10 1.23 1.59 1.65 1.86 ± ± ± ± ± 10.95 11.15 13.99 14.58 16.95 Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
Statistical mean ± S.D.
Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
n ¼ 6 animals in each group. P < 0.05 and versus Control Note: a, b are presented significant differences between marked groups.
0.49 0.85 0.67a 0.32b 0.0b
Day 20 Day 18 Day 16 Day 14 Day 12 Day 10 Day 8 Day 6 Day 4 Day 2 Groups
Percentage of wound contraction on day
Table 2 Effects of the hydroethanolic Pistacia atlantica hulls extract on circular excision wound contraction area (mm2) and period of epithelialization.
Groups
2.3.2. Estimating acute toxicity In order to find a safe dose for P. atlantica, an acute toxicity study was conducted. Twenty four, weighting 200 g and 7e9 weeks old healthy white Wistar rats were randomly divided into 4 groups (n ¼ 6/group) including: Control, Placebo, 5% PAO-treated and 10% PAO-treated groups. The animals were under surveillance for 30 min 2, 4, 24 and 48 h after the administration in order to estimating the any clinical or toxicological symptoms and any mortality ratio were recorded during two weeks. Hematological, serum biochemical and histological (liver and kidney) parameters were determined based on the standard methods [19]. 2.3.3. Formulation of topical wound application forms The topical applicable ointments were prepared in four different concentrations. All the variants consisted base formulation comprising Eucerin (25%) and Vaseline (75%) in 1: 3 proportions. A surgical wound was created, 150 rats randomly were labeled by none toxic color and divided into five groups (No ¼ 30 rats in each group). Two groups served as Controls: Group 1, which did not receive any chemical/ointment and Group 2 marked as Placebotreated; animals in this group were treated with base formulation. Three different concentrations of PAO were administrated in groups 3, 4 and 5. For this purpose, 1.5, 3 and 5 g from PAO extract were mixed with base formulation [20]. The ointments were topically applied on the wound area once a day, starting from the day of operation, until 21 days post-operation. All rats were monitored
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Table 4 Effect of hydroethanolic Pistacia atlantica hulls extract on wound healing of the dead space wound model. All data are presented in Mean ± SD. Groups
Dead space wound model Hydroxyproline content (mg/mL)
Control Placebo Pistacia atlantica 1.5% Pistacia atlantica 3% Pistacia atlantica 5%
10.99 12.41 14.17 15.44 15.81
± ± ± ± ±
Wet weight of the granulation tissue (mg)
0.3 0.32 0.33 0.32a 0.46a
81.1 85.7 97.9 115.83 124.43
± ± ± ± ±
3.25 4.62 4.57a 4.83a 4.55a
Dry weight of the granulation tissue (mg) 12.89 13.72 15.36 17.01 20.54
± ± ± ± ±
0.57 0.76 0.79 0.52a 0.72a
n ¼ 6 animals in each group. a is presented significant differences (P < 0.05) between marked data in the same column.
Table 5 Mean Distribution of immune cells (IMC), fibroblasts and fibrocytes per one mm2 of the sections in different groups. All data are presented in Mean ± SD. Groups
IMC
Fibroblast a
shaved. Following aseptication the PAO formulations were applied to animals in the experimental groups. After 4 h the skin of each animal was observed for signs of inflammation [21].
Fibrocyte a
Negative control 1.5% (day 3) 3% (day 3) 5% (day 3)
16.52 8.66 12.66 29.00
± ± ± ±
3.31 3.05b 2.88b 6.08c
2.21 2.25 5.00 3.25
± ± ± ±
0.60 0.95a,c 0.71b 0.89a
0.00 0.87 2.26 1.24
± ± ± ±
0.00a 0.25b 0.91c 0.48c,b
Negative control 1.5% (day 7) 3% (day 7) 5% (day 7)
26.10 14.66 12.54 19.32
± ± ± ±
1.21a 3.05b 2.08b 2.01c
4.90 2.28 4.35 7.50
± ± ± ±
1.16a 0.96a 1.70a,b 1.91b
0.41 0.87 1.62 3.74
± ± ± ±
0.02a 0.25b 0.47b 0.46c
Negative control 1.5% (day 14) 3% (day 14) 5% (day 14)
12.61 5.33 4.66 3.65
± ± ± ±
3.00a 1.52b 2.01b 0.57b
5.73 7.00 6.50 9.49
± ± ± ±
0.63a 1.11a 1.28a 1.30b
1.30 2.51 2.75 4.54
± ± ± ±
0.21a 1.00b 0.89b 0.66c
Negative control 1.5% (day 21) 3% (day 21) 5% (day 21)
6.20 4.00 2.63 1.33
± ± ± ±
0.71a 1.00b 0.60b 0.57c
2.11 4.75 2.63 4.85
± ± ± ±
0.22a 1.25b 1.70a,b 0.96b
0.64 1.21 1.63 3.00
± ± ± ±
0.70a 0.62a 0.41a 0.81b
a,b,c are presented significant differences (P < 0.05) between marked data in the same column.
for any wound fluid or any evidence of infection or other abnormalities, until complete epithelialization. 2.3.4. Acute skin irritation test The test was carried out based on Gfller et al. (1985) suggested method. About 314 mm [2] from dorsal fur of each animal was
2.3.5. Wound healing models 2.3.5.1. Circular excision wound model. Each group of animals (n ¼ Sixteen) was anesthetized by intraperitoneal administration of ketamine 5%, 90 mg/kg (Ketaset 5%; Alfasan, Woerden, The Netherlands) and xylazine hydrochloride 2%, 5 mg/kg (Rompun 2%, Bayer, Leverkusen, Germany). The fur was prepared aseptically and the predetermined area was marked on the back of animals. Each rat was fixed on the surgery table in ventral posture. Following surgical preparation a circular surgical full thickness wound was made, 314 mm diameters, on the anterior-dorsal side of each rat [22]. Wound contraction percentage and wound closure time were used to assess the wound-healing property. The wound area was measured by immediate placing of a transparent paper over the wound and tracing it out; the area of this impression was calculated using the graph sheet. The wound healing percentage was calculated by the Walker formula after measuring the wound size [23]. The percentage of wound healing was computed at the beginning of experiments and on days 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 days post-test. Percentage of wound size ¼ Wound area on day X/Wound area on day zero 100 Percentage of wound healing ¼ 100 Percentage of wound size
Fig. 1. Histological feature of experimentally-induced wound on 3 after surgery. A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note the higher magnifications for granulation tissue; Well formed granulation tissue and decreased edema is presented in 5% pistachio-treated group, (Masson-trichrome staining, 400 & 600). FN: fibrinoid necrosis, ED: edema, S: scab.
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Table 6 The scores for tissue edema, collagen production and epidermis migration are presented. Groups
Edema score
Collagen score
Epidermis thickness
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþþ þþ þþþ þþ
þþ
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþ þþ þþ þ
þ þþ þþ þþ
20.62 ± 10.08
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþþ þþ þ þ
þ þþ þþþ þþþþ
9.61 ± 3.02a 22.57 ± 9.04b 50.16 ± 12.54c 75.24 ± 12.30d
Negative control 1.5% (day 3) 3% (day 7) 5% (day 7)
þþ þ þ
þþ þþþ þþþ þþþþ
20.37 43.89 66.87 83.40
þ
± ± ± ±
4.12a 6.27b 7.23c 6.74d
Note: The Masson trichrome staining was scored into þþþþ: Intensive; þþþ: Mild; þþ: Mild to moderate; þ: Faint; : negative. a, b, c are presented significant differences (P < 0.05) between marked data in the same column.
2.3.5.2. Incision wound model. Animals were randomly divided into five experimental groups of six animals each: Control, Placebo, 1.5%, 3% and 5% groups. All animals in the experimental groups were anesthetized with the same way mentioned above and 4-cm length incision was made through the skin and cutaneous muscle at a distance about 1.5 cm from the middle on the right side of the depilated back. The wound was closed at 0.5 cm intervals using 3/ 0 nylon (Dafilon, B/Braun, Germany). All the groups were treated the same as mentioned in the excision model. Ointments were applied in manners that fully cover the area, once daily for 9 days. On day 9, sutures were removed and the tensile strength of healed wounds was measured on day 10 by Strongraph mechanical test frame (Toyoseiky Tensile Testing Unit, Model R3, Japan) [24]. Tensile strength was calculated using the following formula: Tensile strength ¼ breaking strength (g)/cross sectional area of skin (mm2)
2.3.6. Dead space wound model and hydroxyproline content estimation Animals were randomly divided into five experimental groups of six animals in each: Group 1 was considered as the Control, did not receive extract; group 2 was considered as the Placebo, received 2 ml of 1% carboxy methyl cellulose (CMC); and groups 3, 4 and 5 were treated orally with 100 mg/kg, 200 mg/kg and 400 mg/kg hydroethanolic extract suspended in 1% w/v CMC, respectively [25]. The dead space wound was created using subcutaneous implanting of polypropylene tubes, 2.5 cm 0.5 cm, in the lumbar region on the dorsal side. The Animals of the experimental groups received the extract from 0 to 9 day post-wounding induction. On day 10 post-wound induction, the created granulation tissue was carefully dissected. Breaking strength and hydroxyproline content were analyzed. 2.3.7. Histopathological study Animals were anesthetized with the same way mentioned above and specimens of skin were taken for further
histopathological analyses on 3, 7, 14 and 21 days after surgery. Sample tissues, excised along with 1e2 mm surrounding normal skin and in a depth of approximately 3 mm, were pinned on a flat cork surface and fixed in neutral-buffered formalin 10%. Then the sample tissues were routinely processed, paraffin wax embedded, sectioned at 5 mm, and stained with Masson's trichrome and examined under light microscopy (Olympus CX31RBSF, Japan) to assess the predominant stage of wound healing. Three parallel sections were obtained from each specimen. Following factors including; Cellular infiltration (immune cells, fibroblasts and fibrocytes), angiogenesis (the number of blood vessels and capillary buds) were quantitatively evaluated per one mm2 of the tissue for each section under 400 magnification. Epithelialization (epithelium thickness), collagen production and density were also evaluated qualitatively and calculated manually. All parameters were analyzed in 5 per high power fields (HPFs) [26,27]. 2.3.7.1. Fluorescent analyses for RNA damage. The RNA damage was assessed using the acridine-orange NO dye (Sigma Aldrich, Germany) according to von Bertalanffy and Bickis method. In brief, the tissues were washed out with ether alcohol and cut by cryostat (8 mm). The prepared sections were fixed by different degrees of alcohol (ethanol) for 15 min. Then the sections briefly were rinsed in acetic acid, 1% aqueous, followed by washing in distilled water. The specimens then were stained in acridine-orange for 3 min and distained in phosphate buffer and ultimately were followed for fluorescent colors differentiation in calcium chloride. The degenerated cells were characterized by loss of RNA and/or with faint red stained RNA. The normal cells were marked with bright red RNA at the apex of the nuclei. In order to reduce the bias problems for staining density, 20sections for each sample were investigated. The RGB plots (color class identification) for red reacted sites were prepared by using image pro-insight image analysis software (version 6.0, media cybernetics) [28]. 2.3.7.2. Mast cells distribution. Mast cells distribution per one mm2 of the tissue was analyzed according to Jagatic and Weiskopf method. Briefly; the slides were deparaffinized and hydrated into water. Then the samples were stained in hematoxylin for 5 min and washed in running water. After that, the slides were stained with acridine-orange for 6 min and after dehydration were mounted in Harleco fluorescent mountant [29]. 2.3.8. Statistical analysis Experimental results were expressed as means ± SEM. Statistical analyses were performed using PASW 18.0 (SPSS Inc., Chicago, IL, USA). Model assumptions were evaluated by examining the residual plot. Results were analyzed using two-way ANOVA. Dunnett's test for pair-wise comparisons was used to examine the effect of time and treatments. Statistical differences were considered significant when P < 0.05. 3. Results 3.1. Antioxidant activity, total phenol and flavonoid contents The extract concentrations providing 50% inhibition (IC50) of DPPH free radicals were determined and compared with that of butylated hydroxytoluene (BHT), as a standard antioxidant. The results for DPPH are presented in Table 1. Assessment of FRAP also revealed that PAO exhibited significantly (P < 0.05) higher antioxidant activity comparing to ascorbic acid as a standard. The phenolic content of the extract was determined based on FolinCiocalteu reagent method. Results showed that the assessed extract yield 257.8 ± 4.3 mg/mg dried extract and 112.71 ± 2.34 mg
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Fig. 2. Histological feature of experimentally-induced wound on 14 after surgery. A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note the newly generated epidermis in control-sham that is completed in 3 and 5% pistachio-treated group. Faint edema remained in deeper dermis of the 1.5% pistachio-treated group, while is significantly limited in 5% pistachio-treated group. Complete layers of the epidermis are generated in 3 and 5% pistachio-treated groups. See the regular basement membrane cells and the spinosume cellular layer. Papilla is completely generated after 14 days in 5% pistachio-treated group. No edema is detectable in 3 and 5% pistachio-treated groups, (Masson-trichrome staining, 400 & 600). EP: epidermis, De: dermis, D.De: deeper dermis, BL: basement layer, Sp: spinosume stratum, Kr: keratin.
eq Rutin/mg dried extract for phenol and flavonoid contents, respectively (Table 1). 3.2. Skin irritation The formulated ointments of PAO, at different doses, did not show any inflammation or noticeable swelling and/or any type of irritation on the skin. 3.3. Wound contraction developed in PAO-treated animals Administration of different doses of PAO in excision wound
model significantly (P < 0.05) increased wound contraction rate compared into non-treated animals. The wound closure achieved by all doses on 6th post wounding day was approximately close to those contractions from reference drug. Comparing the contraction rate between groups showed that, from day 6 until day 16 the 5% PAO-treated animals exhibited significantly (P < 0.05) higher contraction rate (98% on day 16) in comparison to 1.5% (88% on day 16) and 3% (94% on day 16) PAO-treated groups. Meanwhile, the contraction rate of the non-treated group was only 79% 16 days post-injury (Table 2).
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Fig. 3. Histological feature of experimentally-induced wound on 21 after surgery. Fluorescent staining for RNA and Collagen with RGB image plot for red and green reactions in 600 mm for A) control-sham, B) 1.5% pistachio-treated, C) 3% pistachio-treated and D) 5% pistachio-treated. Note increased collagen synthesis accomplished with enhanced RNA content in 3 and 5% pistachio-treated groups. E: epithelium, Co: collagen, F: hair follicle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Micro vessels distribution per one mm2 of the tissue in different days and different administrated doses, all data are presented in Mean ± SD. Note: a, b, c, d, e, f are presented significant differences between marked groups.
3.4. Linear incision wound model and dead space wound model changed depending on dose In order to evaluate the wound healing activity of the PAO, the linear incision wound model was performed. As reported in Table 3, the PAO-treated animals exhibited significantly (P < 0.05) higher values in comparison to non-treated group. Interestingly, the 5% PAO-treated animals showed the highest values versus 1.5% and 3%
Fig. 5. Mast cells distribution in different groups; PAO enhanced mast cells distribution more prominently on day 7 after wound induction. All data are presented in Mean ± SD and P < 0.05 considered as significant difference. Note: a, b, c presented significant differences between marked data.
PAO-received groups. According to our analyses of the dead space wound model, the hydroxyproline content of the PAO-treated animals increased depending on administrated dose. Accordingly, 3% and 5% PAOreceived animals represented the greatest hydroxyproline content versus to those at 1.5% PAO-treated and non-treated groups. Similar to the results for hydroxyproline, the PAO-treated animals showed
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Fig. 6. Fluorescent photomicrograph for mast cells distribution 7 days after wound induction; A) control, B) 1.5% PAO-treated, C) 3% PAO-treated and D) 5% PAO-treated. Note dose dependently increased mast cell distribution (head arrow) enclosed to vessels (arrows) in treated groups, (Fluorescent staining for mast cells, 400).
Fig. 7. Pistachio atlantica hulls hydroethanolic extract-induced early wound healing process.
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significantly (P < 0.05) higher dry and wet weights of granulation tissue in comparison to non-treated animals (Table 4).
maximum neovascularization on day 7 after wound induction, while the animals in 5% PAO-treated group exhibited massive neovascularization on day 14 (Fig. 4).
3.5. Histopathology result 3.5.1. Immune cells (IMC) infiltration decreased in a dose dependent manner Mean distribution of IMC per one mm2 of the tissue was analyzed in different groups (Table 5). Histological observations showed that on day 3, the mean number of IMC significantly (P < 0.05) decreased in 1.5% PAO-treated animals in comparison to other treated and non-treated groups (Fig. 1). Meanwhile, the situation changed after day 7, when the IMC distribution was detected significantly (P < 0.05) lower in 3% PAO-treated group. Finally, after 14 and 21 days, the animals in 5% PAO-treated group showed the lowest distribution of IMC per one mm2 of the tissue in comparison to those in other test and control groups. 3.5.2. Fibroblast and fibrocyte distribution was dependent to administrated dose Three days after injury, the difference between control and 1.5% PAO-treated group was not statistically noticeable. However, at the same time, the number of fibroblasts and fibrocytes remarkably (P < 0.05) increased in one mm2 of the wound area in 3% PAOtreated group. In contrast, 7, 14 and 21 days after treatment the 5% PAO-treated animals exhibited the highest distribution of fibroblasts and fibrocytes per one mm [2] of the tissue. The data for IMC, fibroblast and a fibrocyte number per one mm2 of the tissue are presented in Table 5. 3.5.3. Collagen deposition, edema and re-epithelialization Assessment of collagen deposition was conducted via scaling the intensity of the blue color in Masson-trichrome stained slides. As represented in Table 6, 7th day after wound induction the collagen deposition started in PAO-treated animals, which was more pronounced in 3 and 5% PAO-treated animals compared to 1.5% PAO-received and non-treated wound-induced groups. In order to reduce the manual examination errors, the fluorescent staining for collagen bundles was conducted and the RGB plot analyses based on classification of fluorescent reactions were performed. The results from the RGB image plot for green collagen content confirmed the results from routine Masson-trichrome histochemistry. Observations revealed that, 7 days after injury the collagen deposition started at 3% and 5% PAO-treated animals and reached to its maximum amount of 14th days after wound induction in 5% PAO-treated group (Fig. 2). Our analyses for edema in tissue showed that PAO at dose levels of 3% and 5%, significantly (P < 0.05) reduced the edema scores on day 7 after wound induction. Surprisingly, 7 days after injury the surface and deeper dermis of the animals in 5% PAO-treated animals exhibited the lowest edema score (þ), while the 1.5% and 3% PAO-treated groups exhibited higher scores (þþ). On days 14th and 21, the reepithelialization started and, albeit with some differences, the animals in all treated groups were revealed with epidermis. The 5% PAO-treated animals showed the highest thickness for epithelium in comparison to other test groups. Regular and complete basement membrane, enhanced stratum spinosume and granulosa layer were identified in 5% PAO-treated animals. In despite of reepithelialization in non-treated animals, the cellular layers of the epidermis were not organized appropriately (Fig. 3). 3.5.4. Neovascularization increased in PAO-treated groups On day 3 after wound induction, the PAO-treated animals showed significant (P < 0.05) increase in neovascularization. Interestingly, animals in 3% PAO-treated group showed the
3.5.5. Mast cells distribution differed depending on dose Fluorescent analyses for mast cells showed that, 1.5% and 5% PAO-treated groups represented similar pattern for mast cells infiltration. Accordingly, both groups exhibited massive mast cells distribution on days 3 and 14 post-injury. However, the 5% PAOtreated animals manifested significantly (P < 0.05) higher mast cells infiltration versus 1.5% PAO-received group. In contrast, 3% PAO-treated group exhibited intensive mast cells distribution on day 7 after wound induction (Figs. 5 and 6). 3.5.6. RNA expression increased in PAO-treated groups Special fluorescent analyses for RNA content of normal and necrotic cells was used in order to detect the normal RNA expression in epithelial and dermal cells of all groups. Observations demonstrated that administration of PAO at 3% and 5% concentrations significantly (P < 0.05) increased RNA expression as well as normal RNA content in epithelial and dermal cells. Meanwhile, non-treated groups exhibited significantly (P < 0.05) lower normal RNA versus treated animals. The interactive 3D surface plot and image RGB Plot showed remarkably higher dense red fluorescent reactions (marking normal RNA content) in PAO-treated animals (Fig. 2). 4. Discussion The current study showed that PAO in different doses shortened the time taking to wound healing and up-regulated wound contraction rate after 7 days from wound induction. Moreover, the animals treated with PAO exhibited well-formed granulation tissue, enhanced collagen deposition as well as rapid neovascularization in comparison to non-treated group. Analyzing the mast cells distribution showed that low (1.5%) and high dose (5%) PAO-treated animals manifested massive mast cells infiltration 3 and 14 days after injury induction and those in 3% PAO-received group showed intensive mast cell distribution on day 7 following wound induction. Our results showed that at early stages (on day 3), the 1.5% PAO treated animals showed lower IMC infiltration, while the higher doses (especially 5% PAO) resulted in massive IMC infiltration. The irritant property of PAO in higher doses might be responsible for increased IMC infiltration. Accordingly, the animals in 3% and 5% PAO-treated groups were represented higher edema and delayed angiogenesis in comparison to 1.5% PAO-received group. In the line with this hypothesis, the influence of irritant contents of extracts on recurrent inflammation during the wound healing process was reported previously [30]. In addition, the results from fluorescent analyses of mast cells showed that the animals in 5% PAO-treated group represented massive mast cell distribution on days 3 after wound induction. The role of mast cells in enhancing the cellular infiltration during wound healing has been reported previously [6,7] Mast cells by synthesis of different cytokines and growth factors provoke the proliferative phase and up-regulate the IMC infiltration via enhancing the vascular permeability [31,32]. Therefore, we come close to this fact that the PAO increased IMC infiltration partly via impressing the mast cells, which in turn resulted in intensive IMC infiltration. In continuo, the elevated vascular permeability resulted in edema that was scored higher than those in 1.5% PAO-treated group. Lower mast cells distribution in 1.5% PAO-received animals confirmed this theory. In order to understand the mast cells oppositional role in
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shortening the wound healing process one should note that mast cells are participated in collagen synthesis directly by up-regulating fibroblast's physiologic function via secreting fibroblast growth factor-2 [32,33]. On the other hand, mast cells are known for secreting vascular growth factor (VGF) that stimulates the endothelial cells proliferation, which in turn enhances neovascularization in healing wounds [34]. Increased neovascularization at day 14 following wound induction and it's harmony with mast cells infiltration in 5% PAO-treated animals may be inferred that PAO could provoke the healing process via stimulating mast cells infiltration after 14 days. Increased collagen deposition following 14 days in 5% PAO-treated animals was in good accordance with neovascularization and mast cells distribution. It is important to consider that mast cells secret tryptase and chymase. Tryptase promotes the fibroblast proliferation and stimulates collagen synthesis and chymase can directly cleave the procollagen type I and up-regulate the fibrillation of collagens [35,36]. In good accordance with these findings, our light microscopic analyses showed that the fibroblast and fibrocyte distribution elevated in PAO-treated animals. At the same time, the collagen deposition increased simultaneously with elevated fibroblast distribution as well as mast cells number after 7 and 14 days from wound induction (Fig. 7). Our biochemical analyses revealed that PAO extract contained high amounts of phenol and flavonoid. Flavonoids are known for their antioxidant and anti-inflammatory effects [37,38]. In the line with this fact, there is a negative correlation between pathologically-induced oxidative stress and cellular physiologic function as well as normal DNA and RNA contents. Indeed, IMCs especially the polymorphonuclear cells are main sources of reactive oxygen species (ROS) [39,40]. In order to evaluate the cellular damage we used special fluorescent staining for normal RNA content in epithelial and dermal cells. Observations revealed severe RNA damage in non-treated group. Meanwhile, administrating of PAO significantly decreased RNA damage. Therefore, we can suggest that the flavonoid content of the PAO extract could considerably decrease the inflammation-induced oxidative stress that ultimately inhibited the RNA damage both in epidermal and dermal cells. Furthermore, our analyses showed that administrating of PAO significantly provoked wound contraction ratio versus non-treated animals. Contraction is so essential for reducing the time for healing process because closure of the wound decreases the distance and declines the size of the wound [41]. In this condition it facilitates re-epithelialization process. In this study, the epithelization time was also found to be significantly shorter and the animals in 5% PAC-treated group showed complete layers of epidermis. Increased viability of epithelial cells beside their successful proliferation and migration from wound bed result in to well-formed epithelialization [42]. Rapid re-epithelialization is considered as a hallmark for well wound care. Therefore, the shorter epithelialization time in 5% PAO-treated animals shows its effective impact on epithelial cells proliferation and active migration. Taking together, the surveillance of epithelial cells in PAO-treated groups might be due to its antioxidant property that inhibited oxidative stress-induced cellular damage and facilitated their migration process. 5. Conclusion This study highlighted the wound healing activity of PAO. Our results showed that different doses of the PAO reduced the healing time, facilitated the wound contraction, up-regulated hydroxyproline content and elevated the neovascularization. Moreover, findings of present study represented that PAO increased collagen
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deposition simultaneously by up-regulating the mast cells and fibroblast distribution. Finally, obtaining better results from high dose administration of PAO suggests that dosing higher concentration contains more constituents that plays major role in shortening healing time. Ethical approval None. Sources of funding None. Author contribution Mohammad Reza Farahpour: Study design, surgical procedures and writing. Navideh Mirzakhani: Pathology procedures, Data collection and data analysis. Jamal Doostmohammadi: Care of animals. Mahmood Ebrahimzadeh: Care of animals. Conflict of interest The authors report no conflict of interest. Guarantor Mohammad Reza Farahpour. Acknowledgment The authors wish to thank laboratory section of the AYANDEH Lab. for laboratory tests and analyses. References [1] Z. Kokanova-Nedialkova, P. Nedialkov, S. Nikolov, The genus chenopodium: Phytochemistry, ethnopharmacology and pharmacology, Pharmacogn. Rev. 3 (6) (2009) 280. ~ ez, M.C. Recio, R.M. Giner, J.M. Prieto, M. Cerda -Nic[2] E.M. Giner-Larza, S. M an ol as, et al., Oleanonic acid, a 3-oxotriterpene from Pistacia, inhibits leukotriene synthesis and has anti-inflammatory activity, Eur. J. Pharmacol. 428 (1) (2001) 137e143. [3] I. Orhan, M. Aslan, B. Sener, M. Kaiser, D. Tasdemir, In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L, Phytomedicine 13 (9) (2006) 735e739. [4] B.P. Nagori, R. Solanki, Role of medicinal plants in wound healing, Res. J. Med. Plant 5 (4) (2011) 392e405. [5] D. MacKay, A.L. Miller, Nutritional support for wound healing. Alternative medicine review, J. Clin. Ther. 8 (4) (2003) 359e377. [6] K.N. Rao, M.A. Brown, Mast cells, Ann. N. Y. Acad. Sci. 1143 (1) (2008) 83e104. [7] T.C. Theoharides, A. Angelidou, K.-D. Alysandratos, B. Zhang, S. Asadi, K. Francis, et al., Mast cell activation and autism, Biochim. Biophys. Acta 1822 (1) (2012) 34e41. [8] A.M. Dvorak, Ultrastructure of Mast Cells and Basophils, Karger Medical and Scientific Publishers, 2005. [9] Royal Botanic Garden E. 1967, 28, 11. [10] M. Ali-Shtayeh, S.I. Abu Ghdeib, Antifungal activity of plant extracts against dermatophytes, Mycoses 42 (11e12) (1999) 665e672. [11] P. Magiatis, E. Melliou, A.-L. Skaltsounis, I.B. Chinou, S. Mitaku, Chemical composition and antimicrobial activity of the essential oils of Pistacia lentiscus var. chia, Planta Med. 65 (08) (1999) 749e752. [12] M. Sanz, M. Terencio, M. Paya, Isolation and hypotensive activity of a polymeric procyanidin fraction from Pistacia lentiscus L, Die Pharm. 47 (6) (1992) 466e467. [13] F. Haghdoost, M.M. Baradaran Mahdavi, A. Zandifar, M.H. Sanei, B. Zolfaghari, S.H. Javanmard, Pistacia atlantica resin has a dose-dependent effect on angiogenesis and skin burn wound healing in rat, Evidence-Based Complementary Altern. Med. (2013), http://dx.doi.org/10.1155/2013/893425. Article ID 893425, 8 pages. [14] N. Saeed, M.R. Khan, M. Shabbir, Antioxidant activity, total phenolic and total
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