CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.10 No.9, pp 881-886, 2017
Abstract : The Thymus vulgaris extracts are effective as antibacterial and immunomodulating agents. Thymol is the main monoterpene phenol, isomeric with carvacrol, found in the thyme extract. On the other hand, the intracellular infection of salmonella stimulates macrophage to produce interleukin IL12 which plays an essential role to stimulate naïve CD4 T cells to differentiate Th1 cells for the enhancement of IFN-γ secretion. lFN-γ plays an important role in the activaton of macrophage, as it destroys the phagocytized bacteria. The aim of this study is to determine the effect of Thymus vulgaris extracts on serum IL-12 level and to analyze the ability of Thymus vulgaris extracts to decrease bacterial colonies in the liver of mice infected with S. typhimurium. This study used 20 male mice strain Balb/c. The mice were randomly divided into 5 groups which consisted of 4 mice each; the positive control (mice infected with S.Typhimurium), negative control (mice without infection), and treatment groups of D1, D2, D3 (mice administrated with extract of Thymus vulgaris 250, 500, 750 mg/kg BW and infected with S. typhimurium). Thymus vulgaris extract causes the increase of serum IL12 and decreases S. typhimurium colonies in the liver of mouse samples infected by S.typhimurium bacteria. Keywords : Thymus vulgaris, ,IL-12,S.Typhimurium.
Salmonella Typhi is a Gram-negative bacilli which causes typhoid fever in human. This bacterium may survive in phagosome to escape the immune system. Some complications of typhoid fever are perforation of ileum, bacteremia, and endovascular infection1, 2. An infection of mice with Salmonella typhimurium (S. typhimurium) has consequences to systemic infection and a typhoid fever disease similar to that seen in humans after infected with S. typhi3,4 . Salmonella is a very successful enteric pathogen because it has developed strategies to cope with most of the immune defenses employed by the host during the different phases of the disease5. S. typhimurium results in systemic infection and a disease which shows similar mechanism as S. typhi6.
The genus Thymus, member of the Lamiaceae family, contains about 400 species of perennial aromatic, evergreen or semi-evergreen herbaceous plants with many subspecies, varieties, subvarieties and forms7.In folk medicine, some Thymus spp. are used for their antihelminthic, expectorant, antiseptic, antispasmodic, antimicrobial, antifungal, antioxidative, antivirotic, carminative, sedative, and diaphoretic effects. They are usually administered by infusion or are used externally in baths to cure rheumatic and skin diseases8,9 .Thymus vulgaris essential oil (TEO) is a mixture of monoterpenes. The main compounds of this oil are the natural
10,11
terpenoid thymol and its phenol isomer carvacrol (CVL) . Several studies have been performed with plant
10, 12, 13
extracts , which have antioxidative, antimicrobial, antitussive, expectorant, antispasmodic, and
14.15
antibacterial effects . Terpenoids, flavonoid aglycones, flavonoids glycosides, and phenolic acids were also found in Thymus spp. 16.
The extract of Thymusvulgaris which may contains high levels of flavonoids which have antioxidants and antibacterial that can be useful to improve immune response through increasing IL12 and decreasing bacterial colonies in the liver is analyzed in this study. This is to determine the effect of Thymus vulgaris extract to increase cytokines which play a key role for macrophage activation in phagocytosis of intracellular microbes such as Salmonella infection.
The study required 15 days to accomplish. The 20 male mice were randomly divided into 5 groups each consisting of 4 mice, including the positive control (infected with 108cfuS. typhimurium ), negative control (without infection) and treatment groups ofD1,D2,D3 (mice administrated with extract of Thymus vulgaris 250, 500, 750 mg/kg BW and infected withS. typhimurium) and analyzed by measuring the bacterial colonies in liver, IL-12 blood levels by ELISA after obtaining Ethical clearance approval by the studies Faculty of Medicine Brawijaya University.
Thymus vulgaris was dried, ground and sieved to produce a fine powder. The powder was then weighed each 100 grams to be extracted. The extraction process used the macerated method. Each extraction process required 100 grams of Thymus vulgaris powder which was extracted using ethanol 96%. The extract was left to settle overnight at Pharmacology Laboratory, Brawijaya University. Every mouse received 0.5cc of concentration.
After administered with Thymus vulgaris extract for seven days, the mice were orally infected with 300 μLS. Typhimurium bacteria (concentration 2x108 cells / mL) two times with 2-day interval with the duration of seven days. On the day 15, all mice were sacrificed and the whole blood was taken.
The liver will be taken using a septic technique then performed weighing, after that the network liver is obtained and crushed with a mortar and with 4.5 ml of sterile physiological saline, then the test tube is filled with NaCl 4.5 ml, after that the 0.5 ml of mortar is inserted on the first tube and homogenization using a vortex, after that 0.5 ml of the first tube is then inserted into the second tube and so do until the tube dilution fourth that has been done 1;10 at each dilution, In the last tube solution taken, than samples of each tube 0.1 ml was inoculated on MaCOnKey agar (BSA)order then, calculate the number of colonies on each plate dilution containing 30-300 CFU.calculate CFU\gram of tissue by the formula:
The number of CFU X Dilution X 10 Tissue weight (grams)
Each data was subjected to statistical analysis. The data were analyzed using normality test of the Kolmogorov -Smirnov test. The IL-12 data have p-value of 0.200 ( p> 0.05 ), so it can be concluded that the data IL-12 has a normal distribution. Thus it can be tested by ANOVA, because the assumption of normality of data distribution have been appropriate. Because of the results of ANOVA test there was no significant difference between IL-12 in each treatment group, then no need to proceed with the test of multiple comparisons.
Based on the analysis, the results show that the different doses of Thymus vulgaris extract affect differently on IL-12 level. The existence of the effect of Thymus vulgaris extract started to appear where the IL12 in mice induced Salmonella typhimurium bacteria becomes higher, after the treatment was given in the form of Thymus vulgaris extract started at a dose of 250 mg/kg BW (D1), compared to IL-12 in the positive control group. Then the IL-12 increased more when the mice were given higher doses of 500 mg/kg BW (D2), until the highest doses of 750 mg/kg BW (D3). Thus, based on the descriptive assessment seen from the IL-12 mean, it can be said that the administration of treatment in the form of Thymus vulgaris extract at a dose of 250 mg/kgETV (D1), 500 mg/kgETV (D2), and 750 mg/kgETV (D3) showed different influences, where the higher dose of Thymus vulgaris extract will further increase the IL-12 level. The overall differences in IL-12 in each treatment above can also be depicted in the graphic form as follows.
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40.0
30.0
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Groups
According to the analysis, the findings show that the different doses of Thymus vulgaris extract affect differently on the colony of Salmonella typhimurium. The existence of the effect of Thymus vulgaris extract started to appear where the colony of Salmonella typhimurium in mice induced with Salmonella typhimurium bacteria in the group D1 becomes lowest than positive control group, after the treatment was given in the form of Thymus vulgaris extract started at a dose of 250 mg/kg BW (D1). Then the colony of Salmonella typhimurium becomes zero when the mice were given higher doses of 500 mg/kg BW (D2), and doses of 750 mg/kg BW (D3).
The overall differences in the colony of Salmonella typhimurium in each treatment above can also be depicted in the graphic form as follows.
The average of IL-12 The average of Colony Salmonella typhimurium
250 200 150 100 50 0 -50
Figure2. Graph of the colony of Salmonella typhimurium at each dose of Thymus vulgaris extract
This study showed that the extract of Thymus vulgaris affected the increased levels of IL-12 in groups D1, D2 and D3.Thymusvulgaris extract at a dose of 750 mg/kgBW could increase the IL-12 levels higher than the provision of Thymus vulgaris extract at a lower dose of250 mg/kgBW (D1). Moreover, the dose of500 mg/kg BW (D2) was more effective to increase IL-12 levels than the dose of 250 mg/kgBW. The lower dose250 mg/kg BW (D1) could increase the IL-12 levels compared to IL-12 in the positive control group. Then the IL-12 increased more when the mice were given higher doses of 500 mg/kg BW (D2). Extract at a dose of500 mg/kgBW could increase the average IL-12 levels better than a 750 mg/kgBW and250 mg/kg BW doses. Thymus vulgaris extract at a dose of 250 mg/kgETV (D1), 500 mg/kgETV (D2), and 750 mg/kgETV (D3) showed different influences, where the higher dose of Thymus vulgaris extract provided will further increase the IL-12. The dominant active compound contained in the extract is Thyme contains many active ingradients include thymol, carvacol and flavonoids. Major chemicals of thyme are essential oil (borneol,carvacol,linalool and thymol) bitter principle,tannin ,saponins and triterpenic acids have the potential as super antioxidants, supposed to act as an immunomodulator in modulating the increase of IFN-γ and IL-12 secretion. IFN-γ secretion by macrophages also stimulates the formation of free radicals to destroy components of M. tuberculosis bacteria which are the DNA and the bacteria’s cell walls that play a role in combating the M. tuberculosis bacteria14 in other studies showed that extract of Thymus vulgarison can induce an increasing value of TLR5 and CD14 expression by mice PBMCs, with optimal dose:250mg/kgB.wt, Since the level of cytokines plays an important role during the protective response in a biological system, it is interesting to investigate the effect of Thymus vulgaris on cytokines17. IL-12 has multiple biological activities, and it is a key factor that drives Th1 responses and IFN-γ production. Early application or production of IL-12 during infection may activate Macrophages and augment a host’s cell-mediated immunity while shaping the ultimate antigen-specific immune responses. As a result, IL-12 may play a key role in protection against bacterial and viral infections, and IL-12 immunotherapy could be important in the treatment of diseases where a Th1 response is desirable. Meanwhile, cytokines including IL-12 is short in vivo half-life, and in the development of
18, 19
advanced drug delivery systems
Other studies showed that effect of Thymus vulgaris on DCs co-cultured with T cells in order to determine their impact on DCs function and T-cell responses. Both compounds at 10mg/ml significantly reduced T cell proliferation in MLR. To determine whether thymol and carvacrol modulated the production of cytokines by T cells20 .Alsothe effect of Thymus vulgaris extracts on MLR, they both increased CD40 expression on DCs. CD40 expression is important for DC maturation and function. It is possible that activation of DCs by these extracts has led to release of inhibitory cytokines such as IL-10. This cytokine can inhibit the proliferative responses as well as the production of IFN-γ inImmunomodulatory21. And shown that Thymus vulgaris extracts increase the number of polymorphonuclears, total lymphocytes, T CD4+, CD8+ and NK cells. In this present study showed that Thymus vulgaris improve immune response through the increases serum IL-12 production in mice infected with S. Typhimurium.
Salmonella typhimurium is a facultative intracellular pathogen which causes typhoid fever in humans (the only known natural hosts and reservoir of infection). Typhoid is a systemic disease that its bacteremic phase of disease is characterized by the dissemination of organisms. The most commonsites of secondary infection are the liver and spleen by penetrating the bacteria from intestine to organs such as spleen and liver and make colonies23.
This result showed the difference in dose of Thymus vulgaris extract influence or different effects on the colony of Salmonella typhimurium. The different doses of Thymus vulgaris extract have different effects on the colony of Salmonella typhimurium. The existence of the effect of Thymus vulgaris extract starts to appear where the colony of Salmonella typhimurium in mice induced with Salmonella typhimurium bacteria in the group D1 becomes lowest than positive control group, after the treatment was given in the form of Thymus vulgaris extract starting at a dose of 250 mg/kg BW. Then the colony of Salmonella typhimurium becomes zero when it was given higher doses of 500 mg/kg BW(D2) and 750 mg/kg BW (D3).
Base on the result above, it was concluded that Thymus vulgaris have an ability to improve the immune system by increasing the level IL-12 and can decrease the bacterial colinesin the liver .
The Thymus vulgaris extract at 500 mg/kg dose has a significant effect to increase the levels IL12 and the activity of ETV significantly decreases the S. typhimurium colonies in liver of mice model.
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