Antibacterial activity of Trigonella Foenum- groecum essential oil against skin infection with Staphylococcus aureus: In vitro and in vivo studies

ALKindy Col Med J 2016:Vol. 12 No.1 www.kmjub.com 1 ABSTRACT Background: Multidrug resistant (MDR) Staphylococcus aureus infections have become a major public health concern in both hospital and community settings. Objective: to investigate the antibacterial activity of T. Foenumgroecum essential oil against skin infection with S. aureus and to study probable synergistic activity in combination with Clindamycin. Type of the study: Cross-sectional study. Methods:Antibacterial activity of T. Foenumgroecum essential oil extract (1.2gm/100 μl) was investigated in multidrug resistance (MDR) Staphylococcus aureus specimen isolated from patients with skin infection in Baghdad. T. Foenumgroecum use externally for cellulites and skin inflammation due to the presence of diosgenin .fast liquid chromatography was used to separate these components. Results: Antibiotics combinations revealed that T. Foenumgroecum essential oil with Clindamycin against MDR isolates of S. aureus showed a synergistic effect when used as 1/4 MIC for each antimicrobial. In vivo study was executed to determine antibacterial activity of these compounds by induction of skin infection with Staphylococcus aureus in mice and the treatment begun after 4hrs later and continue to seven days then skin biopsy was taken and sent for histopathological examination. Conclusions: According to the results of this study, we can conclude that T. Foenumgroecum essential oil has remarkable antistaphylococcal activity. Combination of T. Foenum-groecum essential oil with Clindamycin was more effective than Clindamycin alone in treatment of skin infection with Staphylococcus aureus.


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maysaa_ali82a@yahoo.com, Mobile No. 07801881096 ulti-drug resistant (MDR) Staphylococcus aureus infections have become a major public health concern in both hospital and community settings 1,2 . Since the late 1990s, community-associated MDR Staphylococcus aureus emerged as a principal cause of skin and soft tissue epidemics throughout the world 3 . It has been estimated that mortality rates from MDR infections in U.S. hospitals are higher than those from infections caused by HIV/AIDS and tuberculosis combined 4 . Furthermore, there is a considerable medical challenge in treating MDR infections due to the remarkable ability of S. aureus to develop resistance to multiple antibiotics, thus limiting the number of viable therapeutic options 2,5 . The resistant strains of S. aureus are often methicillin-resistant or nafcillin-resistant by virtue of changed penicillin-binding proteins. Therefore, there is an urgent need to develop novel antimicrobials with unique mechanisms of action to combat MRSA.The drug of choice for these staphylococci is vancomycin, to which Gentamicin is occasionally added. Clindamycin or Trimethoprim-sulfamethoxazole can be used to treat non-life -threatening infections caused by these organisms 6 .
T. Foenum-groecum is one of the most widely used ingredient alternative medicine for the treatment of wounds 7 . According to the fact that about 30% of drugs used worldwide are based on natural products and because T. Foenum-groecum has bioactive component with well known antibacterial activity (like diosgenin ) which encourage us to investigate the antibacterial activity of T. Foenum-groecum essential oil against skin infection with S. aureus and to study probable synergistic activity in combination with Clindamycin. Materials and Methods: Isolation and detection of Staphylococcus aureus :All specimens were diagnosed microscopically (Gram stain), morphorgically and biochemically according to standard methods 8,9 , and biochemical tests using commercial kits (GP-VITEK2 Gram positive colorimetric M identification kit) for Staphylococcus aureus bacteria ( BioM erieux, France).
Extraction of T. Foenum-groecum essential oil T. Foenum-groecum essential oil is extracted from seeds of plant (supply from local markets in Baghdad). The oil was extracted by steam distillation from dried plant and yield 1.2gm for T. Foenum-groecum for each 100gm of plant materials 10 .
Assessment of antibacterial activity of T. Foenumgroecum essential oil: The antibacterial activity of essential oil was evaluated using agar well diffusion method with minor modifications 11 . The broth micro dilution method was used to determine minimum inhibitory concentration (MIC). All tests were performed in mueller hinton broth (Salucea (Netherlands) supplemented with Tween 80 (BDH (England) at a final concentration of 0.5% (v/ v) 12 .Fractional inhibitory concentration values (FICs) for antimicrobials combinations was used to determine the effect of antimicrobials combinations on multidrug resistance (MDR) strains of bacteria. FIC values used to assess the synergism between Clindamycin (Saudia Arabia) with T. Foenum-groecum essential oil for S. aureus 13 .
Animals experiment (In vivo method): Thirty five healthy, domestic male mice, weighing 23-25 gram were used in this study; they were obtained from animal house in high institute for infertility diagnosis and assisted reproductive technologies, in the period between August/2014 to October/ 2015. These mice were kept in separated cages; the room temperature was maintained at 20 -25˚C.
Animals grouping: The mice randomly divided into five groups (n=7, each) according to following: Group 1(control): control group infected just by phosphate buffer saline (China).
Group 2(induction group): infected by bacteria without treatment.
Group 4: treated with Clindamycin solution 1% (Saudia Arabia) alone for 7 days. For preparation of inoculate, the bacteria were sub cultured onto brain heart agar (BHA) (Oxoid (England) and incubated at 37°C overnight. Then one colony was inoculated into brain heart broth (BHB) (Oxoid (England) and incubated overnight at 37°C, the overnight culture was diluted 1:100 in fresh BHB and grown until the midexponential phase (approximately 3 hours). The bacteria washed twice and resuspended in sterile phosphate buffered saline (PBS) 15 . Before inoculation the mouse models of bacterial skin infection were sedated with ether. The flanks of the sedated mice were shaved with clippers when necessary and cleansed with an ethanol solution (BDH (England), and then make wound by scalpel cuts. The wounds were subsequently inoculated by 50 μl of the bacterial suspension. Then the mice were returned to their cages and observed. All mice had free access to food and water throughout the duration of the experiments. Animals were observed daily and skin lesion size, swelling, redness, amount of buss were noticed. The treatments with antimicrobial used in this study begin after 4 hrs of bacterial inoculation and continued at the regimens of 7 days 15,16 .  (1).
The effects of T. Foenum-groecum essential oil alone and in combination with Clindamycin against S. aureus in different are shown in table (1) . The treatments with antimicrobial used in this study for in vivo study begin after 4 hrs of bacterial inoculation and continued at the regimens of 7 days. Then after seven day part of lesion area was tested for histopathological examination.
Group 1(control): the mice infected just by phosphate buffer saline, no lesion, no redness, no swelling, no death, and histopathological section showed normal skin without inflammatory cell infiltration, also no vascular congestion, no edema and no necrosis as showed in figure (2).
Group 2 (induction group): in this group the mice infected by S. aureus and left without treatment, skin lesion was occurred (1cm) with redness, swelling and pus, five mice dead in the first day. Under histopathological examination heavy infiltration of dermis and subcutaneous tissue by acute inflammatory cell predominantly neutrofil, market edema with proteinaceous exudates, and vascular congestion was shown as in figure (3).
Group 3 (T. Foenum-groecum): healing begun in third day and four mice died in second day. Histopathological examination showed mild inflammatory cell infiltration (neutrofil) of dermis and subcutaneous tissue, edema with mild vascular congestion as showed in figure (4). Group 4 (Clindamycin): in this group pus, redness and lesion (1 cm) remain until day seven and three mice died in second day. Histopathological examination showed thickness of epidermis, heavy infiltration of dermis and subcutaneous tissue by acute inflammatory cell with necrotic depris, extravagated red blood cell and pinkish oxidate (pus) as shown in figure (5).

Group 5 (T. Foenum-groecum + Clindamycin):
swelled and pus continued until day six but with less degree than when treated with Clindamycin alone, no death was occurred. Histopathological examination showed moderate edema, few inflammatory cell infiltrations and no vascular congestion as shown in figure (6).
Discussion: In the present study, correct identification rate of S. aureus in this study was representing 100% (58/58) other study found that correct identification rates of S. aureus were 99.5% 17 18 . Zone of inhibition of T. Foenum-groecum essential oil against S. aureus was ranging from 12 to 22 mm of concentrated essential oil (100%), and some essential oil did not have any antibacterial effect against seven isolates. Other study mentioned that antibacterial activity of T. Foenumgroecum essential oil against gram positive and gram negative bacteria have zone of inhibition above 7 mm in diameter was considered as a positive result 19 .
In this study it was found that MIC of T. Foenumgroecum essential oil ranging from 0.6-1.2 gm/100µl. Other investigations revealed that MIC values of T. Foenum-groecum essential oil were ranging from 0.8 -6.4 gm/100µl against both gram positive and gram negative bacteria 19 . Synergistic effect was seen from combination of Clindamycin with T. Foenum-groecum essential oil against most S. aureus isolates as shown in table (1).
The growth of the organism was clearly observed in all inoculated mice. Lesions cultures was confirmed the infections by bacteria. After usage of the plants as topical treatment for one week, the lesions and wounds were healed dramatically. Control groups were used to prove that healing was not spontaneously. In recent years, different reports from different countries were indicated that there were antimicrobial activities of medicinal plants, for many years, the effect of herbal medicine on burn wound has been noted. Herbal products seem to possess moderate efficacy and are less expensive as compared with synthetic drugs. Many plants and plants-derived products have been shown to possess potent wound-healing activity 26 .
In-vivo-sensitivity of the plants studied on the infected mice proved to be very active. All the infected mice were Most of the medicines are mixture of many plants, but none of these traditional ointments were scientifically studied. In the current study, T. Foenum-groecum extract was compared with Clindamycin as the standard treatment for burn wounds in mice. The actual mechanism of improved healing is still unclear, the probable mechanism are providing necessary material for healing, increasing blood flow to burn area, decreased inflammatory response, and decreasing rate of infection. A new skin medication can be introduced by usage of herbal medicines with fewer adverse effects and shorten the period of healing thus decrease the rate of hypertrophic scar. The result findings denotes of T. Foenum-groecum in healing of burn injuries as an inexpensive and available herbal medicine 25 .