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Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities
G Model BIOPHA 4560 No. of Pages 8
Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
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Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities Zohra Ghlissia,* , Rim Kallelb , Assaad Silac , Bahira Harrabia , Rim Atheymena , Khaled Zeghala , Ali Bougatefc , Zouheir Sahnouna a
Research Unit of Pharmacology and Toxicology of Xenobiotics, Faculty of Medicine, University of Sfax, 3029, Sfax, Tunisie Anatomopathology Laboratory, Habib Bourguiba University Hospital, 3029, Sfax, Tunisie c Unit Enzymes & Bioconversion, National School of Engineers of Sfax, University of Sfax, 3038, Sfax, Tunisie b
A R T I C L E I N F O
Article history: Received 3 October 2016 Received in revised form 10 November 2016 Accepted 11 November 2016 Keywords: Globularia alypum GC–MS Polyphenols Wound healing Anti-inflammatory
A B S T R A C T
Burns are known as one of the most common and destructive forms of injury with a vast spectrum of consequences. Despite the discovery of various antibacterial and antiseptic agents, burn wound healing still has remained a challenge to modern medicine. Plants have been considered as potential agents for prevention and treatment of disorders in recent years. Globularia alypum L. (GA) is widely used in folk medicine against skin diseases and abscesses, however there is no scientific evidence justifying its use. This study aimed to evaluate the wound healing and anti-inflammatory effect, the antibacterial and antioxidant activities, as well as the chemical compositions of GA methanolic extract (GAME). Chemical compounds of GAME were examined by GC–MS. Wound healing effect was assessed by second-degree burn wounds in rats, anti-inflammatory activity was studied by carrageenan-induced rat paw edema, antioxidant activity was estimated by the DPPH, reducing power and b-carotene tests and antimicrobial activity was tested against 6 bacteria. A total of 17 compounds were identified. GAME-treated rats showed an improvement in healing process and carrageenan-induced hind paws edema as assessed by histological and biochemical investigations, compared to the control. A significant antioxidant and antibacterial activities were also observed in GAME-treated rats. GAME revealed a burn wound healing activity probably due to the anti-inflammatory, antimicrobial and antioxidant activities of its phytochemical contents. Thus, this study confirms its traditional use, however further more precise studies are needed for future clinical application. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Traditional plant remedies are at great attention to the search of new drugs for treating various diseases and ailments. Most herbal medicines and their derivative products were often prepared from different plant parts, which contain various phytochemical constituents. The chemical features of these constituents differ considerably among different species, but also to differences in climate conditions (hot temperature, high solar exposure, dryness, short growing season). Globularia alypum L. (GA) is a wild plant
* Corresponding author at: Laboratory of Pharmacology, Faculty of Medicine University of Sfax, 3029, Tunisia. E-mail address: [email protected] (Z. Ghlissi).
belonging to the Globulariaceae family. It is a nanophanerophyte reaching 0.4 to 1 m height. Its main distribution area is the Mediterranean regions, Europe and North Africa. This plant, locally named ‘zriga or Ain Larneb’ is known almost throughout Tunisia, from the sub-humid to the arid, but is rare in the Saharan area. It is used as a concentrated decoction (leaves and branches) to help cure ulcers, as an infusion for colic, and as a poultice to bring abscesses to a head quickly [1]. The poultice is applied to the swelling. In the El Hamma region near Gabès, the leaves are ground to a powder [1], or chewed and then sprinkled or smeared onto a wound to heal it. Some pharmacological activities of GA have been reported such us in vivo hypoglycemic [2], in vitro antioxidant and anti-tuberculosis [3] and in vitro anti-inflammatory and acetylcholinesterase [4]. Conversely, other ethnomedicinal properties of GA have no scientific evidence justifying, such as wound healing
http://dx.doi.org/10.1016/j.biopha.2016.11.051 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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Fig. 1. Base peak chromatogram of Globularia alypum methanolic extract by GC–MS.
effect. Burn wound healing is a complicated process which contains three stages: inflammation, proliferation and remodeling of the extra cellular matrix. The aim of the healing process is to prevent pathogens invasion, confirm the integrity of damaged tissue, and reconstruct the skin physiological function. Taking into account its numerous and different uses in Tunisian traditional practice, we investigated the in vivo wound healing and anti-inflammatory actions, the in vitro antimicrobial and antioxidant activities, as well the GC–MS analysis of GAME.
2. Materiel and methods 2.1. Reagents 1,1-Diphenyl-2-picrylhydrazyl (DPPH), ethylene diamine tetraacetic acid (EDTA), trichloroacetic acid (TCA) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Butylated hydroxyanisole (BHA) and all other chemicals were of analytical grade. For wound healing and anti-inflammatory
Table 1 GC–MS spectral analysis of GAME. No.
RT (min)
Compound name
Molecular formula
Relative content (%)
1 2 3 4 5 6 7 8
7.16 10.77 11.78 12.06 14.11 14.63 19.79 21.71
C12H32O3Si3 C12H16O2Si C9H8O2 C9H8O2 C12H16O2Si C14H26O2Si2 C17H34O3Si3 C21H52O6Si5
2.65 1.53 1.32 0.32 23.21 4.70 6.61 12.24
9 10 11 12 13 14 15 16 17
22.05 23.17 25.84 26.52 29.24 29.97 30.12 30.65 38.60
Glycerol, tris (TMS) ether Cinnamic acid, TMS ester Cinnamic acid Cinnamic acid, (E) Cinnamic acid, TMS ester 4-Hydroxyphenylethanol, di-TMS Ethanol, (2-(3,4-dihydroxyphenyl)-, tris(TMS) D-Fructose, 1,3,4,5,6-pentakis-O(TMS) 3,4-Heptadien-2-one, 3,5-dicyclopentyl-6-methylBicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl)Talose, 2,3,4,5,6-pentakis-O-(TMS)Palmitic acid, TMS ester Silane, [(3,7,11,15-tetramethyl-2-hexadecenyl)oxy] TMS Linoleic acid, TMS ester Oleic acid, TMS ester Stearic acid, TMS ester 1-(30 ,50 -dichlorophenyl)-5-oxo-4,4-diphenyl-2-imidazolin-2-yl] guanidine
C18H28O C15H24O2 C22H55NO6Si5 C19H40O2Si C23H48OSi C21H40O2Si C21H42O2Si C21H44O2Si C22H17Cl2N5O
12.46 30.96 0.62 6.07 0.47 0.40 1.48 0.75 0.30
GAME: Globularia alypum methanolic extract.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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Stearic Acid, Palmitic Acid, Squalane, Hydrogenated Vegetable Oil, Persea Gratissima (Avocado) Oil, Algin, Centella Asiatica Leaf Extract, Glycine Soja (Soybean) Oil, Arachis Hypogaea (Peanut) Oil, Triethanolamine, Allantoin, Cetyl Palmitate, Ethylhexylglycerin, Caprylyl Glycol, Tocopheryl Acetate, Disodium EDTA, Retinyl Palmitate, Phenethyl Alcohol, Tocopherol, Parfum (Fragrance). 2.2. Animals Male Wistar rats (250–270 g) were purchased from the Central Pharmacy (SIPHAT, Tunisia) and breeded in the animal house of the faculty of medicine of Sfax-Tunisia. The animals were caged under controlled conditions of light (12 h light/dark cycles), room temperature (23 1 C) and relative humidity (50% 10%) with free access to food and water ad libitum. The general guidelines on the use of living animals in scientific investigations (Council of European Communities, 1986) and the guidelines for care and use of laboratory animals controlled by the Tunisian Research Ministry were followed. 2.3. Plant material The fresh leaves of GA were collected in January 2015 from the region of Beja in the north of Tunisia and were identified at the Department of Botany, in the faculty of sciences of Sfax. The leaves were dried in air shade at room temperature, powdered and extracted by maceration in methanol (10 g/100 ml) at ambient temperature for 24 h with continuous stirring. Thereafter, extract was filtrated with No. 1 Whatmann Millipore filter paper (0.45 mm) and concentrated to dryness with a rotary evaporator at 50 C to give solid residues. The dried GA methanolic extract (GAME) was kept in the dark at 4 C prior analysis. 2.4. Gas chromatography–mass spectrometry (GC–MS) GC–MS analysis of GAME was performed using the equipment Thermo GC-Trace Ultra Version: 5.0, Thermo MS DSQ II. The equipment has a DB 35–MS Capillary Standard non-polar column with dimensions of 30 mm 0.25 mm ID 0.25 mm film. The carrier gas used is Helium with at low of 1.0 ml/min. The injector was operated at 250 C and the oven temperature was programmed as follows: 60 C for 15 min, then gradually increased to 280 C at 3 min. The identification of components was based on Willey and NIST libraries as well as comparison of their retention indices. The constituents were identified after comparison with those available in the computer library (NIST and Willey) attached to the GC–MS instrument. 2.5. in vitro antioxidant activity
Fig. 2. Antioxidant activities (DPPH, reducing power and b-carotene) of GAME at different concentrations. BHA was used as positive control.
The DPPH radical scavenging activity, the reducing power and the ability of GAME to prevent b-carotene bleaching were assessed respectively by the methods of Kirby and Schmidt [5], Yildirim et al. [6] and Koleva et al. [7]. The Butylated hydroxyanisole (BHA) was used as a reference drug [8]. 2.6. in vitro antimicrobial activity
1
evaluations, Acetylsalicylic Acid (AA) and “Cytol Centella ” cream, used as reference drugs, were purchased from local pharmaceutical industry (Pharmacy of Triki Rachid, Road Majida Boulila, Sfax). “Cytol Cyntella” is a dermatological cream produced in Tunisia by Dahlia laboratory (BP 95 Bis 3062 Tunisie. www.labodahlia. com) under laboratory license “CYTOLNAT France”. Composition of “Cytol Centella”: Aqua (Water), Glyceryl Stearate, Hydrogenated Polydecene, Vitis Vinifera (Grape) Seed Oil, Propylene Glycol,
Antibacterial activities of GAME were tested against six strains of bacteria: Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 4698), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Salmonella enterica (ATCC 43972) and Listeria monocytogenes (ATCC 43251). Antibacterial activity was determined by means of agar-well diffusion assay according to the method of Vanden Berghe and Vlietinck [9].
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Table 2 Antibacterial activities of GAME. Diameter of inhibition zone (mm SD) Bacteria Salmonella enterica Escherichia coli Listeria monocytogenes Staphylococcus aureus Klebsiella pneumoniae Micrococcus luteus
GAME (50 mg/ml) Ampicillin (50 mg/ml) 20 0.3 13 0.3 19 0.1 17 0.6 15 0.3 15 0.4 16 0.2 19 0.5 18 0.2 14 0.5 16 0.1 13 0.4
Values represent averages standard deviations for triplicate experiments. GAME: Globularia alypum methanolic extract.
time of wounding as 100%. The rate of wound closure of individual animal from control and treated groups was used as an indicator of wound healing. Progressive decrease in the wound size was monitored periodically every 4 days interval using transparent graph sheet and a marker. During the wound healing period, the wound areas were traced manually using transparent paper. Wound areas were then measured using a computer software application for design and drafting (Auto CAD RL 14). Wound contraction, expressed as a percentage of reduction of original wound size, was calculated using the following equation: n Wound closure ð%Þ ¼A0AA 100 where A0 and An are the initial 0
wound area (day 0) and wound area on day n, respectively. 2.7. In vivo anti-inflammatory test 2.7.1. Experimental design The rats were divided into 3 groups (n = 6) pre-treated respectively with 1 ml/kg of sterile saline, 150 mg/kg of AA [10] and 200 mg/kg of GAME [11]. After 1 h of intraperitoneal injection of drug and plant extract, 0.1 ml of carrageenan (1% in normal saline) was subcutaneously injected in the foot pad of the rat right hind paw [10]. Foot volumes were measured up to a fixed mark at the level of the lateral malleolus at 0, 3 and 7 h after the administration of carrageenan with a plethysmometer (TK-101P; Unicom Co., Ltd., Tokyo, Japan) [12]. We estimated carrageenaninduced edema and drug efficacy using the edema rate as an index Edema rate (%) = (foot volume at the measurement time x foot volume at time zero)/foot volume at time zero x 100 [13]. Seven hours after carrageenan injection, the rats were anaesthetized by intramuscularly injection of 50 mg/kg of ketamine and then sacrificed. The blood samples were collected and the serum was separated for the determination of C-reactive protein (CRP). The plantar muscles were dissected to histological analyses. 2.7.2. Determination of C-reactive protein (CRP) The CRP level was carried out according to previous reported method [14]. The serum samples were analyzed by commercially available enzyme-linked immunesorbent assay (ELISA) kit (R&D system, USA) according to the manufacturers’ recommendations. 2.8. Burn wound healing activity 2.8.1. Experiment protocol The back of the twenty rats were shaved under anesthesia. Then a deep circular 15 mm diameter burn wound (177 mm2) was created on their dorsal parts, using an electrical heater (110 C for 10 s). The underlying skin was cleaned with normal saline [15]. The rats bearing second-degree burn wound were divided into 4 groups (n = 5): group 1 (negative control) was just cleaned by a saline solution, group 2 was treated with glycerol (dilution matrix of GAME), group 3 was treated with standard drug “Cytol Centella” cream and group 4 was treated with GAME. After burn induction, the animals were housed individually. The wounds were dressed up and treated every day till completely healed. After sixteen days of treatment, all the rats were anaesthetized (50 mg/kg of ketamine) and then sacrificed. The granulation tissue was excised from the sacrificed animals. A part of wet tissue was preserved for hydroxyproline estimation and another one was fixed in formalin 10% (v/v) and processed for histological examination. 2.8.2. Wound healing area assessment The wound area was traced, measured and the actual value was converted into percent value taking the size of the wound at the
2.8.3. Hydroxyproline level measurement Hydroxyproline content of tissue samples was estimated according to Lee and Tong [16] and values were reported as mg/ g dry weight of tissue. 2.9. Histopathological examination The skin and sub-plantar muscles samples were fixed in 10% neutral buffered formalin solution, embedded in paraffin wax, cut into 5 mm-thick sections, astained with hematoxylin–eosin and finally examined under light microscope. 3. Statistical analysis Data are expressed as mean SD. The statistical significance between groups was assessed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. Statistical significance was set at P < 0.05. 4. Results and discussion 4.1. Chemical compositions of GAME The base peak chromatogram of GAME is depicted in Fig. 1. The identities of GA constituents, their retention time (RT), molecular formula and percentage composition are listed in Table 1. Seventeen compounds were identified: 3 polyphenols (at 30.96%, 6.61% and 0.30%), 6 phenols including 4 phenolic acids (at 23.21%, 1.53%, 1.32% and 12.46%); 2 sugars (at 12.24% and 0.62%); 4 fatty acids (at 6.07%, 1.48%, 0.75% and 0.40%), a glycerol at 2.65% and a phytol at 0.47%. 4.2. Antioxidant activity As illustrated in Fig. 2A, the DPPH activity increased with the increasing concentration of GAME tested. The highest value was 89.30% at a concentration of 50 mg/ml of sample. The reducing power of GAME was also dose dependent and reached a maximum of 1.76 0.07 at 50 mg/ml (Fig. 2B). Similar results were observed
Table 3 Effect of GAME on paw edema and CRP level after a carrageenan injection. Groups Paw edema (mm) CRP(mg/ml) 0h 3h 7h Control 7.28 0.18 Carrageenan 3.25 0.25 6.88 0.51** 6.36 0.55** 10.31 0.45### Carrageenan + AA 3.45 0.21 6.12 0.45** 5.38 0.36* 9.59 0.42¥ Carrageenan + GAME 3.20 0.15 5.67 0.35* 3.77 0.24 8.23 0.13¥¥ Values are expressed as mean SD. *P 0.05 and **P 0.01 compared with zero time. ¥P < 0.05 and ¥¥P < 0.01 compared with carrageenan group; ###P < 0.001 as compared to control. GAME: Globularia alypum methanolic extract; AA: acetylsalicylic acid; CRP: C-reactive protein.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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Fig. 3. (A) Histological sections of edema paws of: (a) saline group showed normal tissue; (b) the carrageenan displayed massive signs of inflammation, (c) carrageenan + AA show moderate signs of inflammation and (d) carrageenan + GAME group exhibited a slight signs of inflammation. Tissues were stained with hematoxylin–eosin and visualized at 400 magnifications. (B) Representative photomicrographs of epidermal and dermal architecture of wounds on the 16th day of rats treated respectively with: saline (a); glycerol (b); “Cytol Centella” cream (c) and GAME (d). Tissues were stained with hematoxylin–eosin and visualized at 400 magnifications.
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for b-carotene activity of GAME that displayed significant increase, the maximum value was 83.66 0.80% at 50 mg/ml (Fig. 2C). Our results are in agreement with some previous studies on antioxidant action of GAME [3,17]. This effect may be attributed to richness of GAME in phenolic constituents. Nine phenolic compounds are identified in this study at a high rate of 76.59%. Additionally, several known antioxidant compound, such as Bicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl) and cinnamic acid, represented the major proportion. 4.3. Anti-bacterial activity The inhibition zones relative to the antimicrobial activity of GAME as represented in Table 2. Three Gram-negative bacteria species (E. coli, S. enterica and K. pneumoniae) and three Gram-
positive bacteria species (L. Monocytogenes, S. aureus and M. luteus) were tested. GAME exhibited an antimicrobial activity against all bacteria cited above. The inhibition zones diameters against the 6 bacteria are too close. The best activities were against S. enterica (20 0.3 mm), E. coli (19 0.1 mm) and K. pneumonia (18 0.2 mm). According to the literature, this is the first antibacterial investigation of GA. These positive results justify therefore the use of this plant in Tunisian folk medicine as a concentrated decoction (leaves and branches) to help cure ulcers and as a poultice (with honey) to bring abscesses to a head quickly [1]. This antimicrobial efficiency of GAME may be due to cinnamic acid compounds, as described previously in relation to other medicinal plant [18,19]. A considerable rate of cinnamic acid compounds (26.38%), with dominance to cinnamic acid-TMS ester (23.21%) is revealed in this study. These composites may be
Fig. 4. (A) Macroscopic appearance of the wound site on days 0, 4, 8, 12 and 16 of rats treated respectively with: saline; glycerol; “Cytol Centella” cream and GAME. (B) Reduction of wound diameter (0–16 days). Each data point represents the mean SD of six rats.
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therefore able to inhibit the growth of microorganisms and fight against bacterial infection. 4.4. Anti-inflammatory activity The injection of carrageenan exhibited redness and swelling on the right paw of different groups, especially after 3 h of experiment. The maximum of paw size was mainly observed in the untreated group. After 7 h of experiment, the paw size deceased with 40% in the GAME-treated group, compared to value at 3 h (Table 3). The macroscopic results of the paw edema size progress were confirmed through the biological and microscopic examination. As showed in Table 3, the CRP level in serum samples was found to increase after 7 h of the study in carrageenan-treated rats. However, a significant reduction of CRP level was found in serum samples of AA-group (P < 0.05) and GAME rats (P < 0.01). Histological, the paw tissue of the control group showed normal histological sections (Fig. 3Aa), while the carrageenan-treated group displayed massive signs of inflammation, with disruption of tissue structure and increased number of inflammatory cells (Fig. 3Ab). The treatment of animals with GAME produced a significant decrease in the inflammatory cells (Fig. 3Ac) however, slight improvements in edema tissue were observed in the reference group (Fig. 3Ad). These results clearly demonstrate the anti-inflammatory effect of GAME and confirm some previous limited investigations [4,20] ant its traditional applies against ulcers, abscesses and swelling [1]. Anti-inflammatory effect of GAME may be equally related to its cinnamic acids content. Earlier works reported that cinnamic acids displayed an interesting antiinflammatory activity through the inhibition of nitric oxide (NO) production [21–23]. In the inflammation process, NO is produced from L-arginine by the inducible NO synthase. Peroxynitrite, formed by the rapid reaction between superoxide and NO, is a toxic substance that contributes to tissue injury in inflammatory diseases [24]. The anti-inflammatory mechanism of cinnamic acids may be through the L-arginine-NO pathway because cinnamic aldehyde can inhibit the NO production. This result is encouraging to prompt us to separate the molecules responsible for this activity. 4.5. Wound healing effect The macroscopic appearance and size variation of burn wounds of GAME-treated rats and other groups were presented in Fig. 4. At day 4, the untreated burn injured animals showed large unhealed wound areas (Fig. 4A). At day 16, wounds were covered with unevenly dried burn eschar and exhibited wound contraction. In comparison, GAME-treated rats frequently showed increased wound coverage by Day 4. At day 16, increased wound coverage was observed in GAME-treated animals. Furthermore, wound area decreased significantly in GAME-group on the day 12 (P < 0.01) and day 16 (P < 0.001), compared to untreated group (Fig. 4B). The visual observations are confirmed with hydroxyproline content variation. As reported in Table 4, hydroxyproline level increased in
Table 4 Hydroxyproline content in different groups of rats.
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the group treated with GAME (26.23 1.90 mg/g of tissue) compared to the untreated group (16.93 1.54 mg/g of tissue). This augmentation of hydroxyproline content in GAME treated group implies faster rate of wound healing process in this group than in the untreated one. Histopathological examinations of wound tissues on 16th day (Fig. 3B) are in accordance with macroscopic findings. Tissue sections of wound areas of untreated rats revealed invasive inflammatory cells with no epithelial layer (Fig. 3Ba). In healed biopsies of CC-treated group there was significant tissular amelioration, the epidermal layers were structured but there was a persistence of considerable signs of inflammation (Fig. 3Bc). However, in tissues sections of GAME, the layers of skin were clearly identified; epidermis and dermis were normal (Fig. 3Bd). The results of GC–MS indicated two dominant fatty acids: Palmitic and Oleic acids and two moderate fatty acids: Linoleic and Stearic acids. Previous research has demonstrated the efficacy of fatty acid agents in accelerating wound healing [25,26]. Membrane fluidity plays an important role in the process of wound healing. The polyunsaturated fatty acids can alter the composition and function of membrane rafts through eicosanoid-independent mechanisms [27]. Lipid peroxidation, prostaglandin production, and regulation of expression of some inflammation related genes are also involved in wound healing [28–30]. The pro-inflammatory effect of oleic and linoleic acids may speed up the wound-healing process [31]. Both fatty acids may also generate other lipoid mediators, such as intermediate hydroperoxides and lipoxins, which can alter the immune response and thereby alter tissue repair [32]. Moreover, some researchers have found that palmitic acid and oleic acid have antibacterial activity [33,34]. Antioxidant effect may also help to control wound oxidative stress and thereby accelerate wound healing [35]. Therefore, the antioxidant effect of GA extracts reported in the literature and showed in this study may play an important role in wound healing. GAME-group showed better contraction percentage than reference group during the first and the second measurement days (day 4 and day 8) which correspond to the cell hemostasis and cell inflammation phases of wound-healing process, while reference group produced the highest healing percentage during the last six days of treatment, that were in accordance with the proliferation and maturation phases [36,37]. These findings are in agreement with those of previous studies showing that Vitis Vinifera and Centella Asiatica extracts exerted considerable wound-healing activity in the late stage of wound-healing [38,39]. GAME showed significant improvements over the reference drug “Cytol Centella” and the untreated group for both morphological and biochemical wound parameters. Burn wounds are an extensive loss of cells and tissues that make the repair process more complicated [40]. For this reason, many factors can affect wound healing by interfering with one or more phases in this process, thus causing improper or impaired tissue repair. Burn wound healing improvement of GAME, clearly explains its different intervention due to its antioxidants, anti-inflammatory and antibacterial properties due to its richness on different composites; phenols, carbohydrate and fatty acids.
5. Conclusion
Group
Hydroxyproline (mg/g)
Original tissue Control Glycerol CC GAME
30.23 2.20** 16.93 1.54** 17.34 1.45** 21.52 2.05* 26.23 1.90
Values are mean SD (n = 6). *P < 0.1 and **P < 0.05, as compared to GAME-treated group.
GAME demonstrated richness and especially various chemical compounds which are the source of these different biological activities (antioxidant, anti-inflammatory, anti-microbial and wound healing effect). These results justify its traditional use to treat infections and injuries. Additional studies must be performed for isolation of bioactive compounds present in GAME for future clinical application.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities Zohra Ghlissia,* , Rim Kallelb , Assaad Silac , Bahira Harrabia , Rim Atheymena , Khaled Zeghala , Ali Bougatefc , Zouheir Sahnouna a
Research Unit of Pharmacology and Toxicology of Xenobiotics, Faculty of Medicine, University of Sfax, 3029, Sfax, Tunisie Anatomopathology Laboratory, Habib Bourguiba University Hospital, 3029, Sfax, Tunisie c Unit Enzymes & Bioconversion, National School of Engineers of Sfax, University of Sfax, 3038, Sfax, Tunisie b
A R T I C L E I N F O
Article history: Received 3 October 2016 Received in revised form 10 November 2016 Accepted 11 November 2016 Keywords: Globularia alypum GC–MS Polyphenols Wound healing Anti-inflammatory
A B S T R A C T
Burns are known as one of the most common and destructive forms of injury with a vast spectrum of consequences. Despite the discovery of various antibacterial and antiseptic agents, burn wound healing still has remained a challenge to modern medicine. Plants have been considered as potential agents for prevention and treatment of disorders in recent years. Globularia alypum L. (GA) is widely used in folk medicine against skin diseases and abscesses, however there is no scientific evidence justifying its use. This study aimed to evaluate the wound healing and anti-inflammatory effect, the antibacterial and antioxidant activities, as well as the chemical compositions of GA methanolic extract (GAME). Chemical compounds of GAME were examined by GC–MS. Wound healing effect was assessed by second-degree burn wounds in rats, anti-inflammatory activity was studied by carrageenan-induced rat paw edema, antioxidant activity was estimated by the DPPH, reducing power and b-carotene tests and antimicrobial activity was tested against 6 bacteria. A total of 17 compounds were identified. GAME-treated rats showed an improvement in healing process and carrageenan-induced hind paws edema as assessed by histological and biochemical investigations, compared to the control. A significant antioxidant and antibacterial activities were also observed in GAME-treated rats. GAME revealed a burn wound healing activity probably due to the anti-inflammatory, antimicrobial and antioxidant activities of its phytochemical contents. Thus, this study confirms its traditional use, however further more precise studies are needed for future clinical application. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Traditional plant remedies are at great attention to the search of new drugs for treating various diseases and ailments. Most herbal medicines and their derivative products were often prepared from different plant parts, which contain various phytochemical constituents. The chemical features of these constituents differ considerably among different species, but also to differences in climate conditions (hot temperature, high solar exposure, dryness, short growing season). Globularia alypum L. (GA) is a wild plant
* Corresponding author at: Laboratory of Pharmacology, Faculty of Medicine University of Sfax, 3029, Tunisia. E-mail address: [email protected] (Z. Ghlissi).
belonging to the Globulariaceae family. It is a nanophanerophyte reaching 0.4 to 1 m height. Its main distribution area is the Mediterranean regions, Europe and North Africa. This plant, locally named ‘zriga or Ain Larneb’ is known almost throughout Tunisia, from the sub-humid to the arid, but is rare in the Saharan area. It is used as a concentrated decoction (leaves and branches) to help cure ulcers, as an infusion for colic, and as a poultice to bring abscesses to a head quickly [1]. The poultice is applied to the swelling. In the El Hamma region near Gabès, the leaves are ground to a powder [1], or chewed and then sprinkled or smeared onto a wound to heal it. Some pharmacological activities of GA have been reported such us in vivo hypoglycemic [2], in vitro antioxidant and anti-tuberculosis [3] and in vitro anti-inflammatory and acetylcholinesterase [4]. Conversely, other ethnomedicinal properties of GA have no scientific evidence justifying, such as wound healing
http://dx.doi.org/10.1016/j.biopha.2016.11.051 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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Fig. 1. Base peak chromatogram of Globularia alypum methanolic extract by GC–MS.
effect. Burn wound healing is a complicated process which contains three stages: inflammation, proliferation and remodeling of the extra cellular matrix. The aim of the healing process is to prevent pathogens invasion, confirm the integrity of damaged tissue, and reconstruct the skin physiological function. Taking into account its numerous and different uses in Tunisian traditional practice, we investigated the in vivo wound healing and anti-inflammatory actions, the in vitro antimicrobial and antioxidant activities, as well the GC–MS analysis of GAME.
2. Materiel and methods 2.1. Reagents 1,1-Diphenyl-2-picrylhydrazyl (DPPH), ethylene diamine tetraacetic acid (EDTA), trichloroacetic acid (TCA) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Butylated hydroxyanisole (BHA) and all other chemicals were of analytical grade. For wound healing and anti-inflammatory
Table 1 GC–MS spectral analysis of GAME. No.
RT (min)
Compound name
Molecular formula
Relative content (%)
1 2 3 4 5 6 7 8
7.16 10.77 11.78 12.06 14.11 14.63 19.79 21.71
C12H32O3Si3 C12H16O2Si C9H8O2 C9H8O2 C12H16O2Si C14H26O2Si2 C17H34O3Si3 C21H52O6Si5
2.65 1.53 1.32 0.32 23.21 4.70 6.61 12.24
9 10 11 12 13 14 15 16 17
22.05 23.17 25.84 26.52 29.24 29.97 30.12 30.65 38.60
Glycerol, tris (TMS) ether Cinnamic acid, TMS ester Cinnamic acid Cinnamic acid, (E) Cinnamic acid, TMS ester 4-Hydroxyphenylethanol, di-TMS Ethanol, (2-(3,4-dihydroxyphenyl)-, tris(TMS) D-Fructose, 1,3,4,5,6-pentakis-O(TMS) 3,4-Heptadien-2-one, 3,5-dicyclopentyl-6-methylBicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl)Talose, 2,3,4,5,6-pentakis-O-(TMS)Palmitic acid, TMS ester Silane, [(3,7,11,15-tetramethyl-2-hexadecenyl)oxy] TMS Linoleic acid, TMS ester Oleic acid, TMS ester Stearic acid, TMS ester 1-(30 ,50 -dichlorophenyl)-5-oxo-4,4-diphenyl-2-imidazolin-2-yl] guanidine
C18H28O C15H24O2 C22H55NO6Si5 C19H40O2Si C23H48OSi C21H40O2Si C21H42O2Si C21H44O2Si C22H17Cl2N5O
12.46 30.96 0.62 6.07 0.47 0.40 1.48 0.75 0.30
GAME: Globularia alypum methanolic extract.
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Stearic Acid, Palmitic Acid, Squalane, Hydrogenated Vegetable Oil, Persea Gratissima (Avocado) Oil, Algin, Centella Asiatica Leaf Extract, Glycine Soja (Soybean) Oil, Arachis Hypogaea (Peanut) Oil, Triethanolamine, Allantoin, Cetyl Palmitate, Ethylhexylglycerin, Caprylyl Glycol, Tocopheryl Acetate, Disodium EDTA, Retinyl Palmitate, Phenethyl Alcohol, Tocopherol, Parfum (Fragrance). 2.2. Animals Male Wistar rats (250–270 g) were purchased from the Central Pharmacy (SIPHAT, Tunisia) and breeded in the animal house of the faculty of medicine of Sfax-Tunisia. The animals were caged under controlled conditions of light (12 h light/dark cycles), room temperature (23 1 C) and relative humidity (50% 10%) with free access to food and water ad libitum. The general guidelines on the use of living animals in scientific investigations (Council of European Communities, 1986) and the guidelines for care and use of laboratory animals controlled by the Tunisian Research Ministry were followed. 2.3. Plant material The fresh leaves of GA were collected in January 2015 from the region of Beja in the north of Tunisia and were identified at the Department of Botany, in the faculty of sciences of Sfax. The leaves were dried in air shade at room temperature, powdered and extracted by maceration in methanol (10 g/100 ml) at ambient temperature for 24 h with continuous stirring. Thereafter, extract was filtrated with No. 1 Whatmann Millipore filter paper (0.45 mm) and concentrated to dryness with a rotary evaporator at 50 C to give solid residues. The dried GA methanolic extract (GAME) was kept in the dark at 4 C prior analysis. 2.4. Gas chromatography–mass spectrometry (GC–MS) GC–MS analysis of GAME was performed using the equipment Thermo GC-Trace Ultra Version: 5.0, Thermo MS DSQ II. The equipment has a DB 35–MS Capillary Standard non-polar column with dimensions of 30 mm 0.25 mm ID 0.25 mm film. The carrier gas used is Helium with at low of 1.0 ml/min. The injector was operated at 250 C and the oven temperature was programmed as follows: 60 C for 15 min, then gradually increased to 280 C at 3 min. The identification of components was based on Willey and NIST libraries as well as comparison of their retention indices. The constituents were identified after comparison with those available in the computer library (NIST and Willey) attached to the GC–MS instrument. 2.5. in vitro antioxidant activity
Fig. 2. Antioxidant activities (DPPH, reducing power and b-carotene) of GAME at different concentrations. BHA was used as positive control.
The DPPH radical scavenging activity, the reducing power and the ability of GAME to prevent b-carotene bleaching were assessed respectively by the methods of Kirby and Schmidt [5], Yildirim et al. [6] and Koleva et al. [7]. The Butylated hydroxyanisole (BHA) was used as a reference drug [8]. 2.6. in vitro antimicrobial activity
1
evaluations, Acetylsalicylic Acid (AA) and “Cytol Centella ” cream, used as reference drugs, were purchased from local pharmaceutical industry (Pharmacy of Triki Rachid, Road Majida Boulila, Sfax). “Cytol Cyntella” is a dermatological cream produced in Tunisia by Dahlia laboratory (BP 95 Bis 3062 Tunisie. www.labodahlia. com) under laboratory license “CYTOLNAT France”. Composition of “Cytol Centella”: Aqua (Water), Glyceryl Stearate, Hydrogenated Polydecene, Vitis Vinifera (Grape) Seed Oil, Propylene Glycol,
Antibacterial activities of GAME were tested against six strains of bacteria: Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 4698), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Salmonella enterica (ATCC 43972) and Listeria monocytogenes (ATCC 43251). Antibacterial activity was determined by means of agar-well diffusion assay according to the method of Vanden Berghe and Vlietinck [9].
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Table 2 Antibacterial activities of GAME. Diameter of inhibition zone (mm SD) Bacteria Salmonella enterica Escherichia coli Listeria monocytogenes Staphylococcus aureus Klebsiella pneumoniae Micrococcus luteus
GAME (50 mg/ml) Ampicillin (50 mg/ml) 20 0.3 13 0.3 19 0.1 17 0.6 15 0.3 15 0.4 16 0.2 19 0.5 18 0.2 14 0.5 16 0.1 13 0.4
Values represent averages standard deviations for triplicate experiments. GAME: Globularia alypum methanolic extract.
time of wounding as 100%. The rate of wound closure of individual animal from control and treated groups was used as an indicator of wound healing. Progressive decrease in the wound size was monitored periodically every 4 days interval using transparent graph sheet and a marker. During the wound healing period, the wound areas were traced manually using transparent paper. Wound areas were then measured using a computer software application for design and drafting (Auto CAD RL 14). Wound contraction, expressed as a percentage of reduction of original wound size, was calculated using the following equation: n Wound closure ð%Þ ¼A0AA 100 where A0 and An are the initial 0
wound area (day 0) and wound area on day n, respectively. 2.7. In vivo anti-inflammatory test 2.7.1. Experimental design The rats were divided into 3 groups (n = 6) pre-treated respectively with 1 ml/kg of sterile saline, 150 mg/kg of AA [10] and 200 mg/kg of GAME [11]. After 1 h of intraperitoneal injection of drug and plant extract, 0.1 ml of carrageenan (1% in normal saline) was subcutaneously injected in the foot pad of the rat right hind paw [10]. Foot volumes were measured up to a fixed mark at the level of the lateral malleolus at 0, 3 and 7 h after the administration of carrageenan with a plethysmometer (TK-101P; Unicom Co., Ltd., Tokyo, Japan) [12]. We estimated carrageenaninduced edema and drug efficacy using the edema rate as an index Edema rate (%) = (foot volume at the measurement time x foot volume at time zero)/foot volume at time zero x 100 [13]. Seven hours after carrageenan injection, the rats were anaesthetized by intramuscularly injection of 50 mg/kg of ketamine and then sacrificed. The blood samples were collected and the serum was separated for the determination of C-reactive protein (CRP). The plantar muscles were dissected to histological analyses. 2.7.2. Determination of C-reactive protein (CRP) The CRP level was carried out according to previous reported method [14]. The serum samples were analyzed by commercially available enzyme-linked immunesorbent assay (ELISA) kit (R&D system, USA) according to the manufacturers’ recommendations. 2.8. Burn wound healing activity 2.8.1. Experiment protocol The back of the twenty rats were shaved under anesthesia. Then a deep circular 15 mm diameter burn wound (177 mm2) was created on their dorsal parts, using an electrical heater (110 C for 10 s). The underlying skin was cleaned with normal saline [15]. The rats bearing second-degree burn wound were divided into 4 groups (n = 5): group 1 (negative control) was just cleaned by a saline solution, group 2 was treated with glycerol (dilution matrix of GAME), group 3 was treated with standard drug “Cytol Centella” cream and group 4 was treated with GAME. After burn induction, the animals were housed individually. The wounds were dressed up and treated every day till completely healed. After sixteen days of treatment, all the rats were anaesthetized (50 mg/kg of ketamine) and then sacrificed. The granulation tissue was excised from the sacrificed animals. A part of wet tissue was preserved for hydroxyproline estimation and another one was fixed in formalin 10% (v/v) and processed for histological examination. 2.8.2. Wound healing area assessment The wound area was traced, measured and the actual value was converted into percent value taking the size of the wound at the
2.8.3. Hydroxyproline level measurement Hydroxyproline content of tissue samples was estimated according to Lee and Tong [16] and values were reported as mg/ g dry weight of tissue. 2.9. Histopathological examination The skin and sub-plantar muscles samples were fixed in 10% neutral buffered formalin solution, embedded in paraffin wax, cut into 5 mm-thick sections, astained with hematoxylin–eosin and finally examined under light microscope. 3. Statistical analysis Data are expressed as mean SD. The statistical significance between groups was assessed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. Statistical significance was set at P < 0.05. 4. Results and discussion 4.1. Chemical compositions of GAME The base peak chromatogram of GAME is depicted in Fig. 1. The identities of GA constituents, their retention time (RT), molecular formula and percentage composition are listed in Table 1. Seventeen compounds were identified: 3 polyphenols (at 30.96%, 6.61% and 0.30%), 6 phenols including 4 phenolic acids (at 23.21%, 1.53%, 1.32% and 12.46%); 2 sugars (at 12.24% and 0.62%); 4 fatty acids (at 6.07%, 1.48%, 0.75% and 0.40%), a glycerol at 2.65% and a phytol at 0.47%. 4.2. Antioxidant activity As illustrated in Fig. 2A, the DPPH activity increased with the increasing concentration of GAME tested. The highest value was 89.30% at a concentration of 50 mg/ml of sample. The reducing power of GAME was also dose dependent and reached a maximum of 1.76 0.07 at 50 mg/ml (Fig. 2B). Similar results were observed
Table 3 Effect of GAME on paw edema and CRP level after a carrageenan injection. Groups Paw edema (mm) CRP(mg/ml) 0h 3h 7h Control 7.28 0.18 Carrageenan 3.25 0.25 6.88 0.51** 6.36 0.55** 10.31 0.45### Carrageenan + AA 3.45 0.21 6.12 0.45** 5.38 0.36* 9.59 0.42¥ Carrageenan + GAME 3.20 0.15 5.67 0.35* 3.77 0.24 8.23 0.13¥¥ Values are expressed as mean SD. *P 0.05 and **P 0.01 compared with zero time. ¥P < 0.05 and ¥¥P < 0.01 compared with carrageenan group; ###P < 0.001 as compared to control. GAME: Globularia alypum methanolic extract; AA: acetylsalicylic acid; CRP: C-reactive protein.
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Fig. 3. (A) Histological sections of edema paws of: (a) saline group showed normal tissue; (b) the carrageenan displayed massive signs of inflammation, (c) carrageenan + AA show moderate signs of inflammation and (d) carrageenan + GAME group exhibited a slight signs of inflammation. Tissues were stained with hematoxylin–eosin and visualized at 400 magnifications. (B) Representative photomicrographs of epidermal and dermal architecture of wounds on the 16th day of rats treated respectively with: saline (a); glycerol (b); “Cytol Centella” cream (c) and GAME (d). Tissues were stained with hematoxylin–eosin and visualized at 400 magnifications.
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for b-carotene activity of GAME that displayed significant increase, the maximum value was 83.66 0.80% at 50 mg/ml (Fig. 2C). Our results are in agreement with some previous studies on antioxidant action of GAME [3,17]. This effect may be attributed to richness of GAME in phenolic constituents. Nine phenolic compounds are identified in this study at a high rate of 76.59%. Additionally, several known antioxidant compound, such as Bicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl) and cinnamic acid, represented the major proportion. 4.3. Anti-bacterial activity The inhibition zones relative to the antimicrobial activity of GAME as represented in Table 2. Three Gram-negative bacteria species (E. coli, S. enterica and K. pneumoniae) and three Gram-
positive bacteria species (L. Monocytogenes, S. aureus and M. luteus) were tested. GAME exhibited an antimicrobial activity against all bacteria cited above. The inhibition zones diameters against the 6 bacteria are too close. The best activities were against S. enterica (20 0.3 mm), E. coli (19 0.1 mm) and K. pneumonia (18 0.2 mm). According to the literature, this is the first antibacterial investigation of GA. These positive results justify therefore the use of this plant in Tunisian folk medicine as a concentrated decoction (leaves and branches) to help cure ulcers and as a poultice (with honey) to bring abscesses to a head quickly [1]. This antimicrobial efficiency of GAME may be due to cinnamic acid compounds, as described previously in relation to other medicinal plant [18,19]. A considerable rate of cinnamic acid compounds (26.38%), with dominance to cinnamic acid-TMS ester (23.21%) is revealed in this study. These composites may be
Fig. 4. (A) Macroscopic appearance of the wound site on days 0, 4, 8, 12 and 16 of rats treated respectively with: saline; glycerol; “Cytol Centella” cream and GAME. (B) Reduction of wound diameter (0–16 days). Each data point represents the mean SD of six rats.
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therefore able to inhibit the growth of microorganisms and fight against bacterial infection. 4.4. Anti-inflammatory activity The injection of carrageenan exhibited redness and swelling on the right paw of different groups, especially after 3 h of experiment. The maximum of paw size was mainly observed in the untreated group. After 7 h of experiment, the paw size deceased with 40% in the GAME-treated group, compared to value at 3 h (Table 3). The macroscopic results of the paw edema size progress were confirmed through the biological and microscopic examination. As showed in Table 3, the CRP level in serum samples was found to increase after 7 h of the study in carrageenan-treated rats. However, a significant reduction of CRP level was found in serum samples of AA-group (P < 0.05) and GAME rats (P < 0.01). Histological, the paw tissue of the control group showed normal histological sections (Fig. 3Aa), while the carrageenan-treated group displayed massive signs of inflammation, with disruption of tissue structure and increased number of inflammatory cells (Fig. 3Ab). The treatment of animals with GAME produced a significant decrease in the inflammatory cells (Fig. 3Ac) however, slight improvements in edema tissue were observed in the reference group (Fig. 3Ad). These results clearly demonstrate the anti-inflammatory effect of GAME and confirm some previous limited investigations [4,20] ant its traditional applies against ulcers, abscesses and swelling [1]. Anti-inflammatory effect of GAME may be equally related to its cinnamic acids content. Earlier works reported that cinnamic acids displayed an interesting antiinflammatory activity through the inhibition of nitric oxide (NO) production [21–23]. In the inflammation process, NO is produced from L-arginine by the inducible NO synthase. Peroxynitrite, formed by the rapid reaction between superoxide and NO, is a toxic substance that contributes to tissue injury in inflammatory diseases [24]. The anti-inflammatory mechanism of cinnamic acids may be through the L-arginine-NO pathway because cinnamic aldehyde can inhibit the NO production. This result is encouraging to prompt us to separate the molecules responsible for this activity. 4.5. Wound healing effect The macroscopic appearance and size variation of burn wounds of GAME-treated rats and other groups were presented in Fig. 4. At day 4, the untreated burn injured animals showed large unhealed wound areas (Fig. 4A). At day 16, wounds were covered with unevenly dried burn eschar and exhibited wound contraction. In comparison, GAME-treated rats frequently showed increased wound coverage by Day 4. At day 16, increased wound coverage was observed in GAME-treated animals. Furthermore, wound area decreased significantly in GAME-group on the day 12 (P < 0.01) and day 16 (P < 0.001), compared to untreated group (Fig. 4B). The visual observations are confirmed with hydroxyproline content variation. As reported in Table 4, hydroxyproline level increased in
Table 4 Hydroxyproline content in different groups of rats.
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the group treated with GAME (26.23 1.90 mg/g of tissue) compared to the untreated group (16.93 1.54 mg/g of tissue). This augmentation of hydroxyproline content in GAME treated group implies faster rate of wound healing process in this group than in the untreated one. Histopathological examinations of wound tissues on 16th day (Fig. 3B) are in accordance with macroscopic findings. Tissue sections of wound areas of untreated rats revealed invasive inflammatory cells with no epithelial layer (Fig. 3Ba). In healed biopsies of CC-treated group there was significant tissular amelioration, the epidermal layers were structured but there was a persistence of considerable signs of inflammation (Fig. 3Bc). However, in tissues sections of GAME, the layers of skin were clearly identified; epidermis and dermis were normal (Fig. 3Bd). The results of GC–MS indicated two dominant fatty acids: Palmitic and Oleic acids and two moderate fatty acids: Linoleic and Stearic acids. Previous research has demonstrated the efficacy of fatty acid agents in accelerating wound healing [25,26]. Membrane fluidity plays an important role in the process of wound healing. The polyunsaturated fatty acids can alter the composition and function of membrane rafts through eicosanoid-independent mechanisms [27]. Lipid peroxidation, prostaglandin production, and regulation of expression of some inflammation related genes are also involved in wound healing [28–30]. The pro-inflammatory effect of oleic and linoleic acids may speed up the wound-healing process [31]. Both fatty acids may also generate other lipoid mediators, such as intermediate hydroperoxides and lipoxins, which can alter the immune response and thereby alter tissue repair [32]. Moreover, some researchers have found that palmitic acid and oleic acid have antibacterial activity [33,34]. Antioxidant effect may also help to control wound oxidative stress and thereby accelerate wound healing [35]. Therefore, the antioxidant effect of GA extracts reported in the literature and showed in this study may play an important role in wound healing. GAME-group showed better contraction percentage than reference group during the first and the second measurement days (day 4 and day 8) which correspond to the cell hemostasis and cell inflammation phases of wound-healing process, while reference group produced the highest healing percentage during the last six days of treatment, that were in accordance with the proliferation and maturation phases [36,37]. These findings are in agreement with those of previous studies showing that Vitis Vinifera and Centella Asiatica extracts exerted considerable wound-healing activity in the late stage of wound-healing [38,39]. GAME showed significant improvements over the reference drug “Cytol Centella” and the untreated group for both morphological and biochemical wound parameters. Burn wounds are an extensive loss of cells and tissues that make the repair process more complicated [40]. For this reason, many factors can affect wound healing by interfering with one or more phases in this process, thus causing improper or impaired tissue repair. Burn wound healing improvement of GAME, clearly explains its different intervention due to its antioxidants, anti-inflammatory and antibacterial properties due to its richness on different composites; phenols, carbohydrate and fatty acids.
5. Conclusion
Group
Hydroxyproline (mg/g)
Original tissue Control Glycerol CC GAME
30.23 2.20** 16.93 1.54** 17.34 1.45** 21.52 2.05* 26.23 1.90
Values are mean SD (n = 6). *P < 0.1 and **P < 0.05, as compared to GAME-treated group.
GAME demonstrated richness and especially various chemical compounds which are the source of these different biological activities (antioxidant, anti-inflammatory, anti-microbial and wound healing effect). These results justify its traditional use to treat infections and injuries. Additional studies must be performed for isolation of bioactive compounds present in GAME for future clinical application.
Please cite this article in press as: Z. Ghlissi, et al., Globularia alypum methanolic extract improves burn wound healing process and inflammation in rats and possesses antibacterial and antioxidant activities, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.11.051
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