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[email protected] Infectious Disorders - Drug Targets, 2018, 18, 41-45 RESEARCH ARTICLE ISSN: 1871-5265 eISSN: 2212-3989
Infectious Disorders
In vivo Antibacterial and Wound Healing Activities of Roman Chamomile (Chamaemelum nobile)
Drug Targets
BENTHAM SCIENCE
Infectious Disorders - Drug Targets
Hossein Kazemian1,2, Sobhan Ghafourian1, Nourkhoda Sadeghifard1, Reza Houshmandfar3, Behzad Badakhsh4, Asieh Taji5, Aref Shavalipour6, Reza Mohebi1, Hadi Sedigh Ebrahim-Saraie5, Hamidreza Houri6 and Hamid Heidari5,7,* 1
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran; 2Department of Medical Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 3 Faculty of Veterinary Medicine, Ilam University, Ilam, Iran; 4Department of Internal Medicine, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran; 5Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; 6Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran and 7Department of Microbiology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Abstract: Background: Today considerable number of drugs are produced from plants. Several plants with antibacterial and healing applications are used in medicine such as Roman chamomile (Chamaemelum nobile L.). Wound infection is one of the most prevalent infections among infectious diseases around the world. Due to appearance of drug resistance, researchers are now paying attention to medicinal plants. Therefore, this study was designed to investigate the antimicrobial and wound healing properties of C. nobile against Pseudomonas aeruginosa using in vivo conditions. ARTICLE HISTORY Received: October 28, 2016 Revised: December 11, 2016 Accepted: December 26, 2016 DOI: 10.2174/1871526516666161230123133
Methods: Ethanolic extract of C. nobile was provided using standard method. The 5% C. nobile ointment was prepared by dissolving lyophilized extract in eucerin. Forty five male rats were obtained from Ilam university. After anesthetization and wound creation, wounds were infected by P. aeruginosa. The rats were divided into three groups, group I was treated with C. nobile ointment, group II was treated with tetracycline ointment and the third group was treated with base gel as control group. Results: Antibacterial and wound healing activities of C. nobile ointment were more than tetracycline ointment significantly. Our results indicated that extract of C. nobile had effective antibacterial activity and accelerated the progression of wound healing. Conclusion: Our study indicated that antibacterial and wound healing activities of C. nobile ointment were notable. C. nobile therapy in combination with antibiotics can also be useful because medicinal plants contents operate in synergy with antibiotics. These results revealed the value of plant extracts to control antibiotic resistant bacteria in wound infections.
Keywords: In vivo, antibacterial, wound healing, roman chamomile (Chamaemelum nobile), Pseudomonas aeruginosa. *Address correspondence to this author at the Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Zand st., Shiraz, Iran; Tel: +98-9386312941; E-mail:
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Infectious Disorders - Drug Targets, 2018, Vol. 18, No. 1
1. INTRODUCTION Today considerable number of drugs are produced from plants which are effective against several diseases [1]. Medicinal herbs are being progressively investigated by pharmacological studies and various researches demonstrated beneficial therapeutic effect of them [2, 3]. In the developed countries remarkable percentage of the medical drugs are derived from plants or their chemical substances [1]. Several plants with antibacterial and healing applications are used in medicine such as, Roman chamomile (Chamaemelum nobile), which is belonging to the Asteraceae family. Chamomile is one of the oldest, most broadly used and well recognized medicinal plants in the world. Numerous pharmacological activities have been documented for C. nobile including, antibacterial, antiinflammatory and anti-ulcer effects [4, 5]. Wound infection is one of the most common infections among infectious diseases around the world [6]. These infections are difficult to manage, several factors can impact the development of wounds and bacterial agents are the most important. Recent studies indicated that bacteria produce proteolytic enzymes to affect the connective tissue of skin and wound expansion. Bacteria with strong pathogenicity, such as Pseudomonas aeruginosa usually involve in wound Infections [7]. P. aeruginosa as an important cause of wound infections may lead to life threatening infections in these patients [8, 9]. Currently, broad ranges of antibiotics are being used for treating these infections. But, due to increasing misuse of antibiotics and chemotherapeutic agents leading to drug resistance, researchers are now paying attention to extracts of biologically active compounds isolated from plant species in herbal medicine [8, 10]. Medicinal plants can provide safe and effective treatments against many infections. Herbal medicines in wound management creating a suitable environment for natural healing process [3, 11]. In previous study, in vitro antibacterial, antiswarming and anti-biofilm formation activities of C. nobile were described [12]. So, this study was designed to investigate the antimicrobial properties of C. nobile against P. aeruginosa and its wound
Kazemian et al.
healing activity using in vivo conditions to compare with a known antibiotic. 2. MATERIALS AND METHODS 2.1. Plant Material Sample of C. nobile was collected from the natural habitat of Ilam province (west of the Iran) and air-dried at 40°C. The plant was identified and authenticated by clinical research center of Ilam university of medical sciences previously. As described previously, dried flowers were chopped and reduced into well powder, 10 gram (g) of powder was mixed by 120 milliliter (ml) of ethanol 50 %. The extract was provided using a rotating apparatus. The acquired suspension was condensed in vacuum by distillation process [12]. The extract was then freeze-dried and the 5% C. nobile ointment was prepared by dissolving lyophilized extract in eucerin (base gel of ointment). Eucerin contains ceramide and licochalcone that combats the skin dryness and reduces inflammation, irritation, erythema, and itching [13]. 2.2. Experimental animals Forty five male Sprague Dawley rats (weighing 200±30 g) were obtained from the Animal Laboratory of Ilam university. Before conducting the experiment, the rats were housed for a week in standard stainless steel cages at 22-25˚C, 60-70% humidity and 12-hour light/dark cycle. The rats were given free access to a standard laboratory diet and water. The animal experiments were performed according to the Animal Care Committee of Veterinary Organization of Iran. All of experimental procedures were approved by ethical Committee of Ilam university of medical sciences. 2.3. In Vivo Assay The rats were anesthetized with intra-peritoneal injection of ketamine (100 mg/kg) and xylazin (10 mg/kg). The dorsal surfaces of the rats were shaved, and their skin were cleaned with povidone iodine solution and wiped with sterile distilled water. An acute incision of 1.5 cm in length was created by a scalpel blade on the back of the each rat. After wound creation, animals were allowed to recover and housed in sterile cages separately. To infect animals, 100 microliter (μl) of standard 0.5
In vivo Antibacterial Activity of C. nobile
McFarland concentration of P. aeruginosa strain (ATCC 27853) were inoculated to the wound area as described previously [14]. After one day, the wound area was sampled by a sterile swab to confirm P. aeruginosa infection. The rats were then randomly divided into three groups of 15 animals. In group I, the wounds were treated with C. nobile ointment (5%), the wounds of group II were treated with 3% tetracycline ointment (Approval Number: 1228053948, Iran daru Co., Iran) and to treatment of wound in the third group (control group), the base gel (eucerin) was used only. Test drugs were locally used once a day in volume of 0.5 g for 2 weeks. The wound areas were sampled and cultured on conventional media every day to evaluate of antibacterial activity of ointments. In addition, the wound conditions were checked and healing times were recorded every day.
Infectious Disorders - Drug Targets, 2018, Vol. 18, No. 1 43
wounds without infection after 3.1 and 3.8 days in group I and II.
2.4. Statistical Analysis Results were presented as the average time of infection eradication and healing of wounds for each group (means ± S.E.M). The statistical difference between the infection treatment and wound healing times of group I, II and III(control) were calculated by One-way analysis of variance (ANOVA) and Scheffe post test using SPSS software version 21. Results were considered to be significant when P < 0.05.
Fig. (1). Eradicated infection from wounds of animals (culturenegative); A, treated with C. nobile; B, treated with tetracycline.
Table 1. Time required for infection treatment and wound healing.
The sacrificed animals were excluded from analysis.
Animal group
No. of animals
Type of treatment
3. RESULTS Extra wound contamination was not observed. In the other words, only pure colonies of P. aeruginosa were isolated from wound samples in all of the experimental period.
The wound infections of group I and II were eradicated very earlier than control group (P < 0.001). However, three animals in the third group were died because their wound infections were not eradicated until the end of experiment. C. nobile activity against P. aeruginosa was more than tetracycline significantly (P = 0.035). The average time for wound infection eradication (culture-negative) in group I and II was 3.1 and 3.8 days respectively (Table 1). The Fig. 1 indicates
(Mean ± S.E.M)
(Mean ± S.E.M)
15
C.nobile ointment
3.1 ± 0.15 a, b
Group II
15
Tetracycline ointment
3.8 ± 0.20 b
12
Base gel (eucerin)
9.3 ± 0.25
(Control) a
Healing time (days)
Group I
Group III
3.1. Antibacterial Activity
Infection eradication time (days)
5.3 ± 0.18 a, b
6.3 ± 0.21 b
12.1 ± 0.37
Showing significant difference in comparison to group II (P < 0.05) Showing significant difference in comparison to control group (P < 0.05)
b
3.2. Wound Healing Activity A significant increase in wound healing was observed in first group in comparison to second group (P = 0.014). As shown in table 1, the average healing time of the first group (treated with C. nobile ointment) was 5.3 days, whereas in the second group (treated with tetracycline ointment) it took 6.3 days.
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The average healing time of control group was 12.1 days. Nevertheless, three animals of this group were sacrificed after 9, 11 and 12 days. Fig. 2 indicates wound of sacrificed (died) animal.
Fig. (2). Wound of sacrificed animal (treated with base gel only).
4. DISCUSSION In the present study, C. nobile ointment on wounded animals created significant infection treatment and wound healing in compare to tetracycline ointment. In the other words our results indicated that extract of C. nobile had effective antibacterial activity and accelerates the progression of wound healing. These findings were similar to various studies which have demonstrated antibacterial and wound healing properties of medicinal plants [8, 11, 14, 15]. Bacterial biofilm formation has a significant role in infection progression [16, 17]. In previous study, anti-biofilm formation activity of C. nobile against P. aeruginosa was described [12]. This anti-biofilm property may be one of the potential mechanisms causing increase in wound healing. Antibacterial activity of C. nobile, can be helpful to the treatment of antibiotic resistant wound infections. C. nobile therapy in combination with antibiotics can also be useful because medicinal plants contents operate in synergy with antibiotics or possess compounds that able to sensitize the bacteria to ineffective antibiotic [18]. Wound healing is a complicated process including three stages; inflammation, cell proliferation and contraction. Inflammation is a basic process that common among all wound types and appears immediately after the injury [19]. In Graf J.'s study, the anti-inflammatory effects of various medicinal plants such as C. nobile has been reported [20]. It seems that, anti-inflammatory activity of C. nobile led to acceleration of wound healing.
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Wound treatment is difficult to manage because wound is in prone to bacterial infections [7]. Disinfectants and topical antibacterial agents were offered to be effective against infections, but the occurrence of allergic reactions to these agents can impact the rate of skin repair and regeneration time [9]. Therefore, considering the antibacterial activity of C. nobile, this is suitable choice as a disinfectant. Recently, new approaches to infectious disease treatment (specially local infections such as wound infection) have been introduced. Anti-adhesion therapy is one of them. In this method bacteria are not killed but are prevented from causing damage to host by inhibiting adherence to host tissues, a requirement for infectious diseases. Medicinal plants and fruits constituents can be extracted and used as anti-adhesive agents in this strategy [21]. Therefore, for future studies this ability of C. nobile should be evaluated to infections prevention and treatment. CONCLUSION In conclusion, extract of Roman Chamomile had more antibacterial activity against P. aeruginosa and wound healing effect compared to tetracycline significantly. The anti-biofilm formation and anti-inflammatory properties of chamomile may play an important role in antibacterial and wound healing activities. These results revealed the value of plant extracts to control antibiotic resistant bacteria in wound infections. ETHICS APPROVAL AND CONSENT TO PARTICIPATE The study was approved by the ethical Committee of Ilam University of Medical Sciences. HUMAN AND ANIMAL RIGHTS The animal experiments were performed according to The US National Research Council's. CONSENT FOR PUBLICATION Not Applicable. CONFLICT OF INTEREST The authors declare no conflict of interest.
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Infectious Disorders - Drug Targets, 2018, Vol. 18, No. 1 45
ACKNOWLEDGEMENTS
[10]
We would like to thank the Clinical Microbiology Research Center of Ilam University of Medical Sciences members for their friendly cooperation.
[11]
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