|Year : 2015 | Volume
| Issue : 1 | Page : 10-13
Antibacterial effect and healing potential of topically applied licorice root extract on experimentally induced oral wounds in rabbits
Venus Dilshad Najeeb1, Ali Sultan Al-Refai2
1 Department of Basic Sciences, College of Dentistry, Hawler Medical University, Erbil, Kurdistan Region of Iraq
2 Department of Oral and Maxillofacial Surgery, College of Dentistry, Hawler Medical University, Erbil, Kurdistan Region of Iraq
|Date of Web Publication||2-Feb-2015|
Ali Sultan Al-Refai
Department of Oral and Maxillofacial Surgery, College of Dentistry, Hawler Medical University, Erbil, Kurdistan Region of Iraq
Source of Support: None, Conflict of Interest: None
Background and Objectives: Evidence has accumulated linking the effects of licorice for the treatment of many diseases. The objectives of this study were to evaluate the antibacterial action of licorice root extract against Staphylococcus aureus and to study its healing potential on the experimentally induced oral wounds in the rabbit's cheek mucosa.
Materials and Methods: Different concentrations of licorice root extract were prepared and their antibacterial activities against S. aureus determined by disk diffusion method. The wound had been created using of biopsy punch at the right cheek mucosa of the rabbit. In the study group, these wounds were irrigated with 1 ml of 60 g/100 ml of licorice root extract twice daily. Seven rabbits from the control and the study group were sacrificed in the 3 rd and 6 th day of the experiment, and biopsies were taken for histological observations of the wounds.
Results: There was no sensitivity of S. Aureus to different concentrations of licorice extract. The histological features of oral wounds in the study group showed satisfactory healing potential after twice daily irrigation with 60 g/100 ml of licorice root extract in comparison to control group.
Conclusions: Different concentrations of licorice root extract has no antibacterial action against S. aureus, but licorice can shorten the healing time of the induced oral mucosal wounds in rabbits.
Keywords: Glycyrrhiza , liquorice, mucosal wound
|How to cite this article:|
Najeeb VD, Al-Refai AS. Antibacterial effect and healing potential of topically applied licorice root extract on experimentally induced oral wounds in rabbits. Saudi J Oral Sci 2015;2:10-3
|How to cite this URL:|
Najeeb VD, Al-Refai AS. Antibacterial effect and healing potential of topically applied licorice root extract on experimentally induced oral wounds in rabbits. Saudi J Oral Sci [serial online] 2015 [cited 2020 Aug 9];2:10-3. Available from: http://www.saudijos.org/text.asp?2015/2/1/10/150583
| Introduction|| |
Herbs with medicinal properties are useful and have been used for many years as effective treatment for various diseases. Currently many studies are being conducted to understand these herbs in depth, and there have been numerous reports of the use of these traditional plants for the treatment of oral diseases.
The licorice (liquorice) plant genus name Glycyrrhiza is derived from the ancient Greek word for "sweet root" (Gr. glykos means sweet + rhiza means root), which was later latinized to Liquiritia and eventually to licorice.  Licorice remains one of the oldest and most commonly prescribed herbs and is known to have therapeutic use as a laxative, antitussive, expectorant, antiarthritic, antiallergic, antihepatotoxic, anticholinergic, antiestrogenic, antileukaemogenic, anticarcinogenic and antiatherosclerotic.  The root's main sweet component is glycyrrhizin, the main triterpene saponin of licorice and it's biologically active metabolite of glycyrrhetinic acid, are both known for their antiinflammatory properties. 
Glycyrrhizin, glycyrrhetinic acid and a number of licorice flavonoids were reported to exhibit antiviral, antibacterial activity, and appear to inhibit Helicobacter pylori growth.  In addition to that licorice has soothing effects on acute outbreak of recurrent aphthous stomatitis, as well as preventing its recurrences. 
Wound healing is a complex process and participation of inflammatory cells is extremely crucial. The inflammatory cells promote the migration and proliferation of endothelial cells leading to angiogenesis among connective tissue cells which synthesize the extracellular matrices including collagen; and of keratinocytes leading to re-epithelialization of the wounded tissue.  This study was designed to evaluate the antibacterial effects of different concentrations of aqueous licorice root extract against Staphylococcus aureus swabbed from the surface of the wound and to study the healing potential of aqueous licorice root extract on the experimentally induced oral wound in the rabbit cheek mucosa at different time intervals.
| Materials and Methods|| |
Forty male rabbits (Oryctolagus cuniculus) were used in the experiment, each weighing 0.8-1 kg. The animals were acclimatized for 1 week to the laboratory conditions prior to experimental manipulation. The rabbits had free access to standard laboratory diet and water ad libitum at room temperature of (19-25°C) with controlled lighting (12 h light/12 h dark). The research project was approved by the Scientific Committee at the College of Dentistry/Hawler Medical University. Animals were anesthetized by intramuscular injection of 25 mg/kg ketamine hydrochloride and 5 mg/kg xylazine hydrochloride. Experimental wound was created - by punch biopsy at the right cheek mucosa of the rabbit [Figure 1]a.
|Figure 1: (a) Creation of wound by using of biopsy punch at the right cheek mucosa of the rabbit. (b) Resistant of Staphylococcus aureus to licorice discs at a concentration of 60 g/100 ml in comparison with five different antibiotics used in the study|
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The shade dried licorice roots were purchased from local market in Erbil City, Iraq, powdered by an electric blender, and different weights of licorice powdered root (5, 10, 15, 20, 40, 50, and 60 g) were soaked in 100 ml distilled water for 24 h to produce different concentrations. Every mixture was then passed through filter paper and kept in refrigerator at 4°C. 
Twelve rabbits were used in pilot study; a swab from the induced oral wounds from six animals were taken in the 2 nd day of the experiment and cultured on a conventional media (blood agar) under aerobic condition at 37°C for 24 h. After the incubation period, the most predominant microorganisms colonized the surface of the ulcer was S. aureus, the identification of Staphylococcus isolates was based on colonial morphology and Gram stain characteristics. 
The selected bacteria were inoculated into 10 ml of sterile nutrient broth, and incubated at 37°C for 18 h. Using a sterile cotton swab, the nutrient broth cultures were swabbed on the surface of sterile nutrient agar plates. The antibacterial activity of licorice root extract was determined by the disk diffusion method. A sterile filter paper discs (6 mm in diameter) impregnated with different concentrations of licorice root extract was placed on a surface of nutrient agar plate for sensitivity testing. Standard 6 mm discs containing amoxicillin 25 μg/disc and other types of antibiotics were also used (like clindamycin, vancomycin, amoclave, amikacin) as a positive controls. The plates were incubated at 37°C in three replications in the incubator for 24 h. Zone of inhibition was measured for each extract using digital vernier caliper and the results were recorded. ,
Clinically, the other six rabbits showed natural healing process completion after 7 or 8 days without any treatment. Therefore, the 3 rd and 6 th days of the experiment were selected to examine the histopathological changes of the oral wound healing.
The result of pilot study
The different concentrations of licorice root extract showed no bactericidal effect on S. aureus and no inhibition zones (clear zone of no growth of bacteria around each disc) were recorded with each concentration of licorice even with the highest concentration (60 mg/100 ml) after overnight incubation [Figure 1]b.
Animal grouping, treatment schedule and wound harvesting
The remaining twenty eight animals were randomly divided into two groups: The control group and the study group, 14 rabbits each. Twice a day, the oral wounds of the control groups were irrigated by 1 ml of distal water. Since different concentrations of licorice extract showed no bactericidal effect against selected bacteria, the oral wounds of the study group were irrigated by the highest concentration of licorice root extract (60 mg/100 ml) twice daily. After 3 days and 6 days respectively, seven animals from each group were sacrificed for histological observations of the wounds. The tissues samples were fixed, processed, sectioned, and stained by hematoxylin and eosin stain. 
The total number of inflammatory cells namely neutrophils, lymphocytes, plasma cells and histiocytes were counted at ×40 magnification, and the vascular density were also calculated in five randomly selected microscopical fields at ×20 magnification. The means of all measurements were determined for each animal, and these measurements were also evaluated by two persons blind to the study. Data analysis was made using one-way analysis of variance. Microscopically, the statistical comparisons between the study and control group were done using Student-Newman-Keuls test. P ≤ 0.05 was considered as statistical significant.
| Results|| |
After laboratory processing of the biopsies which were taken from the oral ulcers in the control and study groups, the result showed that 60 mg/100 ml licorice extract when used as a treatment for induced oral wounds in the rabbit's check mucosa could potentiate the healing process as seen in [Table 1].
|Table 1: Number of inflammatory cells, fibroblasts cells, and blood vessels in the connective tissue of oral ulcers after three and 6 days postoperatively in both control and study groups per high power field (×40)|
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Histological features of wounds 3 days postoperatively
In the study group the epithelium showed longer epithelial tongue with its full thickness and growing in horizontal direction, while in the control group the wound appear larger because the growth of thin epithelial tongue was in vertical direction [Figure 2]. The inflammatory cells seen in the connective tissue were mainly neutrophils. The total numbers of inflammatory cells and blood vessels in the connective tissue of the study group (109 ± 4.660, 4 ± 0.561) were significantly less (P < 0.05) than that of the control group (161 ± 0.545, 5.5 ± 1.112 respectively), but the number of fibroblasts (34 ± 0.20) in the study group was significantly higher (P < 0.05) than the control group (13 ± 1.291) as seen in [Table 1].
|Figure 2: Three days postoperatively. (a) Control group: Growth of epithelial tongue in vertical direction (arrow) with infiltration of large number of inflammatory cells and blood vessels in the connective tissue (H and E, ×20). (b) Study group: Growth of full thickness epithelial tongue in a horizontal direction (arrow) with less number of inflammatory cells infiltration and blood vessels in the connective tissue (H and E, ×20). Six days postoperatively. (c) Control group: Growth of thin epithelium bellow the necrosis/scab (arrows) with closure of wound region. (d) Study group: The newly formed thick stratified squamous epithelium with rete pegs formation covering the connective tissue (H and E, ×40)|
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Histological features of wounds 6 days postoperatively
In the study and the control group, the granulation tissue cloaked completely by stratified squamous epithelium and complete closure of the wound region was evident. The epithelium in the study group appeared thick and contained rete pegs while the epithelium in the control group appeared thin without rete pegs and growing below the necrosis/scab [Figure 2]. The inflammatory cells seen in the connective tissue were mainly lymphocytes and plasma cells. The number of inflammatory cells and the number of blood vessels in the connective tissue of the study group (55 ± 1.291, 2.75 ± 0.853) was significantly less than the control group (95 ± 1.915, 4.5 ± 0.645 respectively) over the same period (P < 0.05), while the number of the fibroblasts in the study group (82 ± 4.243) was significantly higher (P < 0.05) than that of the control group (64 ± 2.160).
| Discussion|| |
Herbal medicines have been used for many years and this has been historically documented. There have been numerous reports of the use of natural products for the treatment of oral diseases. The results of this study showed that aqueous extracts of licorice root at different concentrations did not exhibit antibacterial activity against the bacterial species tested. Shinwari et al. (2009)  found that 25% aqueous extract of the plant was not effective against S. aureus obtained from Culture Library of Microbiology Laboratory. Geetha and Roy (2012)  prepared ethanolic extracts in the following concentrations (2.5 mg/ml, 5 mg/ml and 10 mg/ml) and found that these concentrations showed antibacterial effect on these selected bacteria. Significant results of the roots methanolic extract as antimicrobial activity especially in 2.5 and 2.0 μg/ml against S. aureus was found by Karomi et al. (2012).  This disagreement may be due to the variation in the type of licorice used, the concentrations and the methods used in the preparation of licorice extract in comparison with this study.
The present study showed that the 60% licorice extract could shorten the healing time for oral wound. No other studies used this concentration with this type of oral ulceration. Galal et al. (2012)  found that licorice extract when mixed with other herbs extract like acacia nilotica revealed improved healing potential of minor aphthae than each substance alone. Haley et al. (2005)  found that oral patch with Glycyrrhiza extract significantly expedited the resolution of minor aphthae.
Glycyrrhizic acid, one of the most important polyphenols, is an antioxidant, and the antioxidant therapy is proposed to improve the healing of wounds. , Topical application of licorice extract on the oral wound has caused significant wound healing due to the strong anti-inflammatory activity with its vital role for reducing nitric oxide and prostaglandin E2 production by macrophage cell. It also inhibited the production of pro inflammatory cytokines and CD14, induced a reduced plasma levels of tumor necrosis factor and interleukin-6 (IL-6), and increased IL-10 production. , In addition to that licorice is reported to stimulate the activation of lymphocytes and this finding suggests that it could be considered as a potential specific immune stimulator.  Licorice contains vitamin E, B complex, pantothenic acid, lecithin, biotin, niacin, manganese, calcium, calcium salts, proteins, and nucleic acids which may aids in wound healing.  In conclusion, different concentrations of licorice extract (5, 10, 15, 20, 40, 50, and 60 g/100 ml distilled water) showed no antibacterial effect on S. aureus swabbed from the surface of the induced oral wound, but the irrigation of these oral wounds by 60% aqueous licorice root extract appeared to improve the healing potential.
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[Figure 1], [Figure 2]