This experimental study was approved by the local Ethics Committee of Shiraz University of Medical Sciences (November15, 2014 - Approval no.IR.SUMS. REC.1393.8050).

Herbal Plant Extract
The O. vulgare leaves were purchased from the Gyahan Darooi Company, Kerman, Iran on October 2015 and authenticated at the Department of Pharmacological Sciences of Kerman University, Kerman, Iran. Traditional Soxhlet extraction was carried out in a standard apparatus (22, 23). For 4 hours, 100 g of O. vulgare leaves were soaked in1000 mL of solvent (water/ethanol 80/20v/v). The extracts were filtered via filter paper and the remaining volume was completely dried in an atmospheric oven at 60°C.

Drug Administration to the Chick Embryos
A total of 28 fertile chicken eggs (Ross 308) with an average egg-weight of 54.8±0.5g were supplied by the Mahan Breeder Company, Kerman, Iran and randomly assigned into four equal treatment groups (n=7). Fertilized chicken eggs were placed at 37.5ºC and 60% relative humidity in a humidified incubator (Belderchin Damavand Co. PLC-DQSH, Tehran, Iran). Experiments were performed on chick embryos until day 18 of the embryonic growth. All treatments were administered as 0.5 mL/egg of a single dose into the yolk sac of each egg. On the 4^th day of the embryonic growth, phosphate buffered saline (group 1) or O. vulgare leaves-extract at doses of 25 (group 2), 50 (group 3) or 100 (group 4) mg per Kg egg weight, were injected into each group of fertilized chicken eggs, as described previously (20, 21, 24-26). Before injection, the extracts were filtered through a syringe filter (0.22 μm) to remove any micro-organism.

Assessment of Pathological Lesions
Embryos were investigated for gross and histopathological injures on the 18^th day of the growing period. At first, embryos were killed by chilling and then examined under the stereo-microscope to study any gross lesions (27, 28). The brain, liver, kidneys, heart, and lungs were fixed in 10% neutral buffered formalin to prepare paraffin-embedded tissues, and then stained with hematoxylin and eosin.

Measurements
The body length and weight of the embryo was calculated via scale (Limestone AT-p735, Iran, range 100 g to ± 0.01 g) and caliper (Mitutoyo-500, Japan, range 0.01 mm). The length was measured from front angle of the head to the apex of the tail (uropygial gland).

Statistical Analysis
Statistical analysis was done via SPSS 20 (SPSS Inc., Chicago, IL., USA). The Statistical tests including Fisher's exact test, One-way analysis of variance, and Tukey's tests were used to evaluate the significant among treatment groups. A P-value<0.05 was considered significant.

 

Macroscopic Findings
The embryos of all experimental and control groups were normal. There was no abnormality on the external body limbs (Figure 1).

Microscopic Findings
In group 2, all the tissues were normal and no microscopic changes were observed (Figure 2). In the embryos of group 3, congestion was seen in the brain, liver, kidneys, heart, and lungs. In the brain and lungs, edema was also seen (Figure 3).
 

Figure 1. The chick embryos treated with phosphate buffered saline (a), 25 mg/Kg egg-weight of O. vulgare leaf-extract (b), 50 mg/Kg egg-weight of O. vulgare leaf-extract (c), and 100 mg/Kg egg-weight of O. vulgare leaf-extract (d) are normal with no gross abnormality on the body surface.

 The lesions in the brain, liver, kidneys, heart, and lungs in the embryos of group 4 were the same as group 3, but the brain edema was much more severe (Figure 4). No microscopic injures were evident in the control embryos. Finally, no significant difference was observed in the embryo weight and the embryo length of the groups that were treated with an O. vulgare leaves-extract compared to the control group (Table 1).

Figure 2. Photomicrograph of the chick embryo treated with 25 mg/Kg egg-weight of O. vulgare leaf-extract. (a) Normal structure of the brain is seen (×200, H&E), (b) Normal structure of the liver is seen (×200, H&E), (c) Normal structure of the kidney is seen (×100, H&E), (d) Normal structure of the heart is seen (×100, H&E), (e) Normal structure of the lung is seen (× 100, H&E).



Figure 3. Photomicrograph of the chick embryo treated with 50 mg/Kg egg-weight of O. vulgare leaf-extract. (a) Dilation of the Virchow- Robin space (arrow) is seen in the brain (×200, H&E), (b) Congestion is seen in the brain (×200, H&E), (c) Congestion is seen in the liver (×200, H&E), (d) Renal congestion is seen (×200, H&E), (e) Congestion is seen in the heart (×100, H&E), (f) Pulmonary edema (arrows)is seen (×100, H&E).

Table 1. Effect of in-ovo injection of O. vulgare leaf-extract on embryo-weight/egg-weight and the body length of the chick embryo

Discussion

Nowadays, herbal remedies are considered in primary healthcare programs. In spite of various properties, their side effects still need to be investigated (1-3). The present study shows that systemic alterations can be induced by in-ovo inoculation of chick embryo, using the leaf-extraction of O. vulgare. The most important changes induced by the leaf-extract of O. vulgare was microscopic lesions. Various histopathological injuries were seen in the brain, liver, kidneys, heart, and lungs’ tissues, and the severest lesion was noticed in embryos which received the high dose of the extract (equal/higher than 50 mg/Kg).
The histopathologic alterations indicated that the chick embryo is susceptible to the application of O. vulgare leaf-extract during the embryonic development. Amongst the histopathological lesions, congestion and edema were noticed in the brain which has to be given attention, due to O. vulgare leaf-extract side effect on the central nervous system. Interestingly, histopathological injures were observed in embryos without any gross lesions, even the ones treated with a high dose of O. vulgare (50 and 100 mg extract/Kg egg-weight). Thymol and carvacrol are the major biochemical in Origanum with proven antioxidant and antimicrobial properties (29). Carvacrol has shown to have anti-proliferative property in HeLa cells (29), but thymol has beneficial effects on the antioxidant features of the brain in rat (30). Thymol and carvacrol have also anti-inflammatory activity on human macrophages (THP-1) (31). The use of oregano’s essential oils provides a promising perspective on the prevention of cell disorders (32), but its high concentration (higher than 30 μg/mL) can cause a reduction in cell viability (31). Genotoxic effect of Origanum and carvacrol have been described elsewhere (33). Furthermore, Origanum toxicity on some animals and insects has been reported (34-37). For example, Nasr et al. (38) showed that O. vulgare caused significant changes in physiological parameters in diamondback moth. It was also shown that Origanum possessed anti-angiogenic activity (39). Thus, it can be inferred that the congestion in the embryo is through its activity on the cardiovascular system. Different alterations have been reported after an application of Origanum compounds in animal models. Administration of aqueous extract of O. vulgare in a mouse model caused a retardation in the embryo development (40).
In this investigation, alterations were observed after in-ovo administration of O. vulgare leaf-extract, which could be the result of major or active chemical compounds present in this plant. Origanum phytochemical analyses exhibited various volatile and aroma organic compounds including linalool, ocimene, caryophyllene, germacrene, bisabolene, and spatulenol (41, 42). Another hypothesis for the alterations induced by O. vulgare leaves-extract could be due to the apoptotic effect of this plant. For instance, Savini et al. (43) showed that O. vulgare could induce apoptosis in the colon cancer cells (caco2) of human. In addition of the O. vulgare active chemicals, other metabolites might also be correlated with the adverse histopathological effects. Furthermore, specific inherent activities of leaf-extract have been postulated to be associated with its effects. Therefore, new studies are warranted to discover O. vulgare biochemical composition and mechanisms in the embryo.

 

Conclusion

Histopathological lesions that had occurred in a dose-dependent manner during O. vulgare administration suggest the susceptibility of chick embryo to the O. vulgare leaf-extract. Injures might be due to different pathways including the effects of chemical compounds, apoptotic effect, biochemicals, and mechanisms that contribute to the inherent properties. Therefore, more attention should be given to the toxic effects of O. vulgare during fetal development period.

 

Acknowledgements

The authors would like to thank the Vice-chancellor of Research at Shiraz University of Medical Sciences (grant No. 93-01-26-8050) for their financial support. The authors also wish to thank Mr. S. Hasanzadeh for his kind cooperation in tissue slide preparation and Mr. H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.

 

Conflicts of Interest

Authors declared no conflict of interests.