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International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.9, No.10, pp 295-305, 2016
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In Vivo Study on the Hindrance Activity of Cinnamon Extract against Aspergillus niger in Mice
Randa Mohamed Alarousy1,4, Sherein IsmailAbd El-Moez1,
Kawkab Ahmed Ahmed2 and Hany Mohamed Hassan3
1Department of Microbiology and Immunology, National Research Centre, Giza, Egypt
2Department of Pathology, Faculty of Veterinary Medicine, Cairo University,
Giza, Egypt
3Department of Immunology, Animal Reproduction Research Institute
4Department of Medical Laboratories, College of Applied Medical Sciences, Majmaah University, KSA
Abstract : Aim: The antimycotic effect and immune stimulating capacity of natural extract of Cinnamomum zeylanicum plant was evaluated against Aspergillus niger strain.
Methods and results: the herbal extract was prepared from cinnamon park to be examined against Aspergillus niger (ATCC16404)species fungal cell suspension. Mice were injected with both fungal cell suspensions and Cinnamomum zeylanicum extract in a certain regime. Histopathological examination was applied on lung, liver and brain tissues extracted from the experimental animals and histopathological findings revealed the strong fungicidal effect of the herbal extract in the mice's tissues. Phagocytic activity, interleukins 2 & 6 (IL 2& IL6) and tumor necrosis factor(TNF) were measured in mice's blood samples as immunity stimulating efficacy parameters and also results revealed the potent immune stimulating efficacy of the extract and its safetyin vivo.
Conclusion: According to the results of the study, Cinnamomum zeylanicum extract has a strong fungicidal activity against A. niger(ATCC16404)in addition to potent immune stimulating action confirmed experimentally. Results are supporting the efficacy of C. zeylanicum extract as prophylactic agent as well as fungicide.
Significance and impact of the study: our study spotted a light on the advantages of the examined herbal extract as antimycotic substance, the promising results revealed encourages the use of C. zeylanicum extract as pharmaceutical preparation for treating and prevention of mycosis in immunocompromised patients instead of using some chemical antimycotic preparations available for commercial use to avoid the disadvantages of these chemically prepared medications.
Keywords: Cinnamomum zeylanicum– Interleukin 2 – Interleukin 6 – Antimycotic extract – Immunostimulant.
Introduction:
Fungal infection is considered among the main causes of morbidity in immunocompromised patients. The epidemiology of invasive mycotic infections has changed within the last decade (Lehrnbecher et al.,1 2010)
About 20 Aspergillus species out of 185 have been reported as opportunistic infectious agent in both man and animals (Stevenset al., 2 2000)
Although opportunistic fungal agent leads to life – threatening infection in immunosuppressed patients, fungal infection affects both immunosuppressed patients as well immune-competent persons in developing countries (Del Poeta and Chaturvedi, 3 2012) The rising incidence in fungal infections observed in the last decade correlates with increases in invasive medical interventions, long-term hospitalization and with large numbers of immunosuppressed patients due to acquired infection due to human immunodeficiency viral infection or treatment-induced immunodeficiency as prolonged using of cortisone before organ trans-plantation or during anticancer therapy ( Pfaller and Diekema, 4,5 2007 and2010)
It is very hard to distinguish between systemic mycosis from other microbial infection depending on the clinical symptoms. This often leads to delayed appropriate intervention using antifungal therapy(Pfaller,6 2012)
Aspergillus niger ATCC 16404 referencestrain was used in this study.Strain was spread onto Sabouraud's dextrose agar with Chloramphenicol (0.5 g L-1) to inhibit bacterial growth and incubated at 300C for 1 to 3 days.
Black colonies with fluffy down can be transferred to Czapek's solution agar (Difco Lab, Detroit, MI) for a definitive confirmation. A piece of colony was teased apart with a needle, stained, marked and mounted with a cover slip. Species confirmationwas achieved on the basis of morphological criteria upon microscopic examination (Richard and Beneke, 10 1989)
Cell-counting:
Spore concentration of different spore suspensions was adjusted using haemocytometer, according to the classical procedure.
Spores were harvested by flooding the agar surface with PBS (Sigma) and then filtered and suspended in PBS with 0.01 % Tween 80 (Difco), in serial concentrations. Spore suspensions were stored at 4 0C for up to 5 days till use.
Hundred micro liters of fungal stock solutions from Aspergillus nigerATCC 16404 spores were inoculated into 10 ml of (YPD) broth and cultured for 48 h. at 30oC with agitation. Fungal pellets were re-suspended in sterile saline solution and were adjusted to 105 - 107 spore/ml using haemocytometer to prepare working solution from microorganism spores.
Experimental infection:
Eight to ten week-old male mice were purchased from laboratory animal house of Ramad hospital, Giza, Egypt. The mice were provided with clean water and solid feed. All animal studies were performed in accordance with the guidelines and permission of the animal experiment care, feeding, changing bedding and general good health was observed regularly.
Fifty mice were divided randomly into 5 groups in 5 cages each contains 10 mice and treated as follow:
A1: mice were injected I/Vwith 100µl of 105 - 107 spore/ml of A. niger ATCC 16404 reference strain as control positive.
A2: mice were injected with 100µl of 105 - 107 spore/ml of A. nige rATCC 16404 followed by 100µl of C. zeylanicum extract that was prepared previously three days post infection.
A3: mice were injected with 100µl of 105 - 107 spore/ml of A. niger ATCC 16404 followed by 100µl of plant extract seven days post infection.
A4: mice were injected with 100µl of 105 - 107 spore/ml of A. niger ATCC 16404 followed by 100µl of plant extract ten days post infection.
A5: mice were injected with 100µl of normal saline as negative control group.
Mortalities were determined and recorded daily.
Sampling:
After 14 days, serum and heparinized blood samples were collected via heart puncture from all mice for immunological assays. All mice then were euthanized; lungs, livers and brains of sacrificed mice were removed and fixed in 10% formalin, the organs then were cut and embedded in paraffin then these blocks were cut into 4 µm thick sections and were stained with Hematoxylin and Eosin (H&E) stain for histopathological examination microscopically(Bancroft et al., 121996)
Blood samples were used to measure certain immunological parameters as phagocytic activity (phage); killing activity of macrophages; lymphocyte transformation (Lyt1); interleukin 2 (IL2); interleukin 6 (IL6) and tumor necrosis factor (TNF).
Lymphocyte transformation:
Lymphocyte transformation assay was carried out as described by Denizot and Lang 13 (1986) with some modifications(Maslak and Reynolds, 14 1995)
Determination of phagocytic killing and chemotaxis activities of PMN:
Polymorph nuclear cells were isolated from blood by the method described by Rouse et al15(1998).The mixture of PMN and bacteria (S. aureus) was incubated at 370C for 2 hours with regular stirring and then the mixture was centrifuged at 200 g for 5 minutes at 40C. The supernatant was used to estimate the percentage of bacteria phagocytosed (Woldehiwet and Rowan, 16 1990). The mixture of bacteria and PMN was treated with one cycle of freezing and thawing and the percentage of bacteria killed was estimated according to the formula described by Woldehiwet and Rowan16(1990).The chemotactic index of PMN was calculated using the chemotaxis under agarose technique based upon migration patterns to chemotactic factor (E. coli filtrate)Nelson, 17 1974)
Measurement of IL-2; IL-6 and TNF by enzyme linked immunosorbent assay:
Serum IL-2, IL-6 and TNF-α level was measured by using a polyclonal ELISA kits (Uscn, Life Science Inc., USA) following the manufacturer’s instructions. Briefly, the anti-IL-6 capture polyclonal antibody was absorbed on a polystyrene 96-well plate and the IL-6 present in the sample was bound to the antibody coated
wells. The biotinylated anti-IL-6 detecting pAb was added to bind the IL-6 captured by the first antibody. Avidin-peroxidase (Sigma, USA) was added to the wells to detect the biotinylated detecting antibody and finally 2,2´- azinobis (ABTS; Sigma, USA) substrate was added and a colored product was formed and measured at optical density 405 nm (OD 405) with an ELISA MICROPLATE READER (MODEL 450, Bio-Rad, Chicago, Illinois, USA). A standard curve was generated and calculated. The measurement of TNF- α and IL-2 are similar to that of IL-6. All determinations were performed by full-time technical personnel. Statistical analysis was applied using One Way ANOVA SPSS, POSTHOC C ALPHA (0.05).
Results:
The gross findings of liver, lung and brain those were extracted from mice were depicted in Table (1).
Table (1): Showed the gross findings of extracted organs from experimental animals:
Group |
Liver |
Lung |
Brain |
A1 (+ve) |
Pale/severe pale |
Fleshy/semi normal |
Fragile-granulated-calcified/highly fragile to semi normal. |
A2 (3rd) |
Slight pale |
Normal |
Normal |
A3 (7th) |
Slight normal |
Lobulated lungs |
Slight normal |
A4 (10th) |
Slight pale |
Normal |
Normal |
A5 (-ve) |
Normal |
Normal |
Normal |
Remark: Total mortalities were 30%
Blood samples were normal in all cases.
Histopathological profile:
The histopathological findings of lung tissues from the experimentally infected mice were shown in figures 1, 2, 3, 4, 5 and 6. The profile of liver histopathology is revealed in figures number 7, 8, 9, and 10, while the histopathological findings of mice brain were clear in figures 11, 12, 13 and 14. Comparing the histopathological profiles among the mice groups were applied in order to correlate the fungicidal efficacy of C. zeylanicum extract and the intervention time of its use.
Histopathological examination of lung tissues:
Fig. (1): Lung of mice from group A1 showing marked dilatation and congestion of pulmonary blood vessels (H & E X 100) |
Fig. (2): Lung of mice from group A1 showing focal interstitial inflammatory cells infiltration (H & E X 400). |
Fig. (3): Lung of mice from group A2 showing dilatation and congestion of pulmonary blood vessels (H & E X 400). |
Fig. (4): Lung of mice from group A2 showing focal pulmonary emphysema (H & E X 400).
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Fig. (5): Lung of mice from group A3 showing congestion of perialveolar blood capillaries and focal mononuclear inflammatory cells aggregation (H & E X 400). |
Fig. (6): Lung of mice from group A4 showing congestion of pulmonary blood vessels and interstitial pneumonia (H & E X 400).
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Histopathological findings of liver tissues:
Fig. (7): Liver of mice from group A1 showing congestion of central vein and perivascular inflammatory cells infiltration (H & E X 400). |
Fig. (8): Liver of mice from group A2 showing hydropic degeneration of hepatocytes (H & E X 400). |
Fig. (9): Liver of mice from group A3 showing focal hepatic necrosis associated with inflammatory cells infiltration (H & E X 400).
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Fig. (10): Liver of mice from group A4 showing focal hepatic necrosis associated with inflammatory cells infiltration as well as Kupffer cells activation (H & E X 400). |
Histopathological profile of brain tissues:
Fig. (11): Brain of mice from group A1 showing necrosis of neurons and neuronophagia (H & E X 400). |
Fig. (12): Brain of mice from group A3 showing focal gliosis (H & E X 400). |
Fig. (13): Brain of mice from group A2 showing focal necrosis infiltrated with glia cells (H & E X 400). |
Fig. (14): Brain of mice from group A4 showing focal necrosis of neurons infiltrated with glia cells (H & E X 400). |
Immunological profile:
Phagocytic activity percentages (Phage); lymphocytes transformation capacity (Lyt1); values of interleukin 2 (IL2); interleukin 6 (IL6) and tumor necrosis factor (TNF) were measured to assess the immunological stimulation efficacy of C. zeylanicum extract and to evaluate the significance of intervention time for treating the mycosis using it, results were depicted in tables 2 and 3.Statistical analysis was applied using one way ANOVA/SPSS.
Table (2): Immunological parameters values among all tested groups:
|
|
A1 (n= 3) |
A2 (n= 3) |
A3 (n= 3) |
A4 (n= 3) |
A5 (n= 3) |
Phage |
Mean |
76.4667 |
76.5067 |
80.2667 |
80.8433 |
89.3333 |
SD |
3.86307 |
3.47327 |
4.42982 |
4.76593 |
4.30968 |
|
Kill |
Mean |
70.9333 |
74.1000 |
75.5000 |
76.2667 |
84.9333 |
SD |
3.95517 |
2.75136 |
5.15655 |
4.37531 |
5.21568 |
|
Lyt 1 |
Mean |
1.1833 |
1.2100 |
1.4033 |
1.4600 |
1.6333 |
SD |
.12662 |
.08185 |
.17559 |
.10149 |
.61460 |
|
IL6 |
Mean |
496.0833 |
465.4633 |
374.6167 |
405.5433 |
238.8800 |
SD |
64.98828 |
.48014 |
13.83812 |
14.78130 |
11.62476 |
|
IL2 |
Mean |
193.3133 |
186.5567 |
187.4100 |
179.9467 |
167.4667 |
SD |
9.46088 |
7.75016 |
11.24827 |
9.66580 |
5.09912 |
|
TNF |
Mean |
182.1400 |
168.7167 |
164.4667 |
169.6900 |
138.7267 |
SD |
11.26012 |
10.40734 |
7.79219 |
3.09264 |
10.29692 |
Table (3): Correlation between immunological parameters and intervention time:
Dependent Variable |
(I) group |
(J) group |
Mean Difference (I-J) |
Std. Error |
Sig. |
Phagocytic Activity |
A1 (untreated) |
A2 |
-.00190- |
.14282 |
1.000 |
A3 |
-.18095- |
.16159 |
.908 |
||
A4 |
-.20841- |
.16867 |
.864 |
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A5 (Control) |
-.61270- |
.15912 |
.104 |
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A2 |
A3 |
-.17905- |
.15476 |
.894 |
|
A4 |
-.20651- |
.16213 |
.847 |
||
A5 (Control) |
-.61079- |
.15217 |
.096 |
||
A3 |
A4 |
-.02746- |
.17889 |
1.000 |
|
A5 (Control) |
-.43175- |
.16992 |
.316 |
||
A4 |
A5 (Control) |
-.40429- |
.17666 |
.396 |
|
Killing Activity |
A1
(untreated) |
A2 |
-.13533- |
.11888 |
.899 |
A3 |
-.19516- |
.16034 |
.871 |
||
A4 |
-.22792- |
.14552 |
.711 |
||
A5 (Control) |
-.59829- |
.16150 |
.126 |
||
A2 |
A3 |
-.05983- |
.14421 |
1.000 |
|
A4 |
-.09259- |
.12752 |
.989 |
||
A5 (Control) |
-.46296- |
.14549 |
.223 |
||
A3 |
A4 |
-.03276- |
.16686 |
1.000 |
|
A5 (Control) |
-.40313- |
.18096 |
.417 |
||
A4 |
A5 (Control) |
-.37037- |
.16797 |
.428 |
|
Lymphocyte Transformation (Lyt1) |
A1 (untreated)
|
A2 |
-.02186- |
.07135 |
1.000 |
A3 |
-.18033- |
.10245 |
.620 |
||
A4 |
-.22678- |
.07679 |
.226 |
||
A5 (Control) |
-.36885- |
.29696 |
.848 |
||
A2
|
A3 |
-.15847- |
.09168 |
.645 |
|
A4 |
-.20492- |
.06170 |
.166 |
||
A5 (Control) |
-.34699- |
.29342 |
.869 |
||
A3 |
A4 |
-.04645- |
.09598 |
.999 |
|
A5 (Control) |
-.18852- |
.30249 |
.994 |
||
A4 |
A5 (Control) |
-.14208- |
.29479 |
.999 |
|
Interleukin 6 (IL6) |
A1 (untreated) |
A2 |
.09892 |
.12121 |
.969 |
A3 |
.39240 |
.12393 |
.285 |
||
A4 |
.29249 |
.12431 |
.451 |
||
A5 (Control) |
.83089 |
.12314 |
.070 |
||
A2 |
A3 |
.29348* |
.02583 |
.029 |
|
A4 |
.19357 |
.02758 |
.073 |
||
A5 (Control) |
.73198* |
.02170 |
.003 |
||
A3 |
A4 |
-.09991- |
.03777 |
.288 |
|
A5 (Control) |
.43850* |
.03371 |
.001 |
||
A4 |
A5 (Control) |
.53841* |
.03507 |
.001 |
|
Interleukin 2 (IL2) |
A1 (untreated) |
A2 |
.16917 |
.17679 |
.955 |
A3 |
.14781 |
.21247 |
.993 |
||
A4 |
.33467 |
.19552 |
.640 |
||
A5 (Control) |
.64714 |
.15536 |
.116 |
||
A2 |
A3 |
-.02137- |
.19746 |
1.000 |
|
A4 |
.16550 |
.17909 |
.962 |
||
A5 (Control) |
.47797 |
.13411 |
.151 |
||
A3 |
A4 |
.18686 |
.21439 |
.973 |
|
A5 (Control) |
.49933 |
.17853 |
.308 |
||
A4 |
A5 (Control) |
.31247 |
.15797 |
.538 |
|
Tumor Necrosis Factor (TNF) |
A1
(untreated) |
A2 |
.20977 |
.13834 |
.736 |
A3 |
.27619 |
.12355 |
.427 |
||
A4 |
.19456 |
.10536 |
.609 |
||
A5 (Control) |
.67844* |
.13767 |
.047 |
||
A2 |
A3 |
.06642 |
.11730 |
.998 |
|
A4 |
-.01521- |
.09796 |
1.000 |
||
A5 (Control) |
.46867 |
.13209 |
.132 |
||
A3 |
A4 |
-.08163- |
.07564 |
.910 |
|
A5 (Control) |
.40225 |
.11651 |
.153 |
||
A4 |
A5 (Control) |
.48388 |
.09700 |
.110 |
Discussion:
Cinnamon is famous in traditional Chinese medicine as a carminative, it is used for most gastrointestinal complaints (Nadkarni, 18 1954). In addition, it is often present in traditional Chinese formulas to facilitate the action of the other herbs present, cinnamon is frequently used as a cardio-tonic, which helps to stimulate blood flow Bensky and Gamble19 (1993).Antioxidants properties of cinnamon extract were reported in many previous studies confirming its expressed ferric reducing antioxidant power (FRAP)(Tenoreet al., 20 2011).
The current study describes the effectiveness of C. zeylanicum whole plant extract against Aspergillus nigerATCC 16404 reference strain invivo in experimental laboratory animals.
Preliminary experiments were carried out in vitro using C. zeylanicum essential oil on solid medium through solid medium drop diffusion technique in order to evaluate the effect of some essential oils, including it, on the fungal growth(Alarousy,21 2004). Results showed that C. zeylanicum had the strongest fungicidal effect on fungal cells.
Aspergillus niger, that is known as environmental fungal species, was used as a model in this preliminary study and other species will be used in next studies aspathogenic models.
The antimycoticactivity of phytochemicals found in C. zeylanicum extractmay involve inhibition ofextracellular enzymes synthesis of the cellwall structure of fungal cells, cellular damage that leads to cellular death (Brul and Coote, 22 1999; Burt, 23 2004 and Atandaet al., 24 2006).Atanda, et al.24(2006)and Rasooli, et al.25 (2006) also reported that C. zeylanicum extract is able to interfere with the enzymatic reactions, suchas respiratory electron transport, protein transport and coupledphosphorylation that take place in the mitochondrial membrane.
It was necessary to explore whether the fungicidal effect is restricted on the essential oils only or not, as it would be difficult somehow to assess the fungicidaleffect of C. zeylanicum essential oil in the laboratory animals (in vivo) due to the difficulties of injecting oils in living bodies. In our study, we tried to test this concept and to approve that the fungicidal effect is due to the entire compounds of the plant as well as its essential oil. So, we used the whole plant extract to inject the pre-infected mice with it in order to track the efficacy of the plant extract inside the animal body for its fungicidal effect as well as its capacity for immune system stimulation. Beside, we designed the experiment to use the extract after the infection of mice with Aspergillus nigerATCC 16404 reference strain with different time intervals (three, seven and ten days post infection).
Histopathological profile:
Histopathological examination of lung tissues:
Histopathological lesions are the phenotypic profile representing the direct correlation between defense mechanisms of the host and virulence of infectious agent (Tochigiet al., 26 2013)
Results depicted in table (1) revealed that the lungs from group A1 mice who were injected with the fungal spores only, were the most damaged lungs in comparison to the other four groups used in this study, followed by lungs from group A3 and A4 who received the C. zeylanicum extract seven days and ten days' post infection respectively. Histopathological results confirmed the harmful effect of the mold on lung tissues, examined lung tissues of mice from group A1 revealed marked dilatation and congestion of pulmonary blood vessels (Fig. 1), focal interstitial inflammatory cells infiltration (Fig. 2) and focal pulmonary emphysema. However, examined sections from group A2, who received the C. zeylanicum extract three days post infectionshowed improvement in histopathological picture as the lung showed dilatation and slight congestion of pulmonary blood vessels (Fig. 3) and focal pulmonary emphysema (Fig. 4). Meanwhile, lung of mice from group A3 revealed congestion of perialveolar blood capillaries and focal mononuclear inflammatory cells aggregation (Fig. 5). Examined sections of lung of mice from group A4 showed severecongestion of all pulmonary blood vessels, focal emphysema and interstitial pneumonia (Fig. 6). These findings agreed to those explained by Tochigi et al26 (2013), they identified two distinct patterns of histopathological findings in lung tissues, one of those pattern involves a distinct nodule consisting of demarcated round-shaped coagulation
necrosis, while the other pattern involves fused lobular integration, which corresponds to the usual bronchopneumonia which is characterized histologically by the filling of acute inflammatory exudates with a fungal proliferation in alveoli.
Histopathological findings of liver tissues:
findings of group A1 revealed congestion of central vein, perivascular inflammatory cells infiltration (Fig. 7), Kupffer cells activation and focal hepatic necrosis associated with inflammatory cells infiltration. Meanwhile, liver of mice from group A2 revealed no histopathological changes except hydropic degeneration of hepatocytes (Fig. 8). In spite of the slightly normal gross appearance of livers extracted from groups A3& A4, livers showed more or less similar histopathological changes confined as Kupffer cells activation and focal hepatic necrosis associated with inflammatory cells infiltration (Figs. 9 & 10).
No liver abscesses were detected in the current study, in contrary to the expected results. According to Kumar et al., 27 (2010), the organisms reach the liver either through ascending infection in biliary tract, or vascular seeding, either portal or arterial, or direct invasion of the liver from a nearby source or through penetrating injury.
Histopathological profile of brain tissues:
Histological findings of brain tissues were completely different, the brain tissues from the groups: A1, A3 and A4 showed variable degrees of necrosis as shown in figures (11, 12 and 14). However, 20% only of group A2 showed gliosis as shown in figure 13. These results elucidated the limited fungicidal effect of the examined herbal extract in brain. This might be due to rapid growth of lesions caused by Aspergillus species if reached the brain tissue.
The rapiddevelopment of lesions could be explained by three possible mechanisms. First, the origins of the multiple perforator arteries are in proximity along the parent artery and are presumably vulnerable to an extending vasculo-pathy of the parent artery wall. There is an evidence suggesting that the necessary conditions for the vasculo-pathy are present. First, endothelial cells in culture have been shown to engulf the organism(Paris et al., 281997)and Aspergillus has been seen to infiltrate and destroy the internal elastic lamina of major cerebral arteries (Chouet al., 29 1993). Second, any infarction, thromboembolic or septic, may have associated secondary edema, which increases during the first few days after the arterial occlusion. Third, Aspergillus hyphae were frequently observed within the parenchymal lesions at autopsy; infection of the infarcted tissue may be aggressive, and direct extension into the surrounding brain may progress quickly (DeLoneet al., 30 1999).
Aspergillosis as an invasive fungal infection has an inflammatory properties as revealed through the histopathological profile of the current studyand C. zeylanicum extract showed good fungicidal capacity less or more the expected result, hence cinnamon park extract was reported previously to have strong antimicrobial activity (Shan et al., 31 2007).Comparing the histopathological profiles between groups A1 and A5 revealed the fungicidal activity of C. zeylanicumin vivo and comparing the histopathological profiles of the three treated groups (A2, A3 and A4) using C. zeylanicum extract also showed the significance of early intervention with this herbal extract, as all the results showed the marked improvement of histopathological profile in group A2 which received the treatment three days post infection. Absence of toxicity signs on experimental animals indicated the safety of using C. zeylanicum extract in vivo.
Immunological profile:
To assess the primary capacity of C. zeylanicum extract for stimulating the immune system, values of phagocytic activity (phage); killing activity of macrophage; lymphocyte transformation activity (Lyt1); IL2; IL6 and TNF were measured and compared between the untreated group (A1) and all treated groups (A2; A3; A4 and A5), regardless the intervention time, from one side and withinthe three treated groups (A2; A3 and A4) from the other side.
Results of multiple comparison between all the treated groups (A2; A3 and A4), regardless intervention time, and A1 group were significant except for TNF (p value = 0.047) between A1 and A5 (the accepted mean difference is significant at the 0.05 value). Significance was the least between untreated group (A1) and control
negative group (A5),as for phage (p value = 0.10); for killing capacity (p value = 0.126); for IL6 (p value = 0.07) and for IL2 (p value = 0.116), while for TNF, it didn’t show accepted significance (p value = 0.047) as mentioned before. These results were matching with the reported ones byCarballo et al. 32 (1998).
In the other hand, another factor was correlated to the efficacy of medicinal application of the extract which is the initiation time of using C. zeylanicum extract in vivo. The relation between the immune stimulating capacity of C. zeylanicum and the intervention time of its use was clarified in table (3), as for phage (F= 4.703); killing activity (F= 4.244); Lyt1 (F= 1.178); IL2 (F= 3.689) and TNF (F= 9.296) all values showed significant differences within the three treated groups (A2, A3 and A4). Meanwhile IL6 values were significant only between (A4 & A2) and between (A4 & A3) and there wasn’t significance between the values in groups A2 and A3 (P value = 0.029). Hence, these results indicated the capacity of immune stimulation of the extract in correlation to the intervention beginning time and this is a novel character of C. zeylanicum extract application medicinally for treating latent and/ or chronic fungal infection as well as its usage as prophylaxis by immunocompromised patients. Further studies will be conducted to investigate deeper the time factor effect on the efficacy of the therapeutic application of the extract.
The conclusion from the current study revealed that Cinnamomum zeylanicum extract has strong fungicidal activity and great immune system stimulating efficacy with correlation to intervention time as well. Results proved that the tested natural herbal extract is more advantageous than the synthetic agents due to its biodegradability, potency, absence of toxicity, efficacy as a prophylactic agent for immunocompromised patients and future studies will be done to analyze the herbal components of Cinnamomum zeylanicum and compare them with the currently available antimycotic pharmaceutical preparations.A. nigerATCC 16404 reference strainwas chosen in this study as preliminarytested strain and further investigation will be carried out on other fungal species.
Conflict of interest:
The authors declare that there is no conflict of interest.
References:
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