Volume 29, Issue 3 (5-2022)                   RJMS 2022, 29(3): 0-0 | Back to browse issues page

Research code: 961256-133-18-84-2
Ethics code: طبق دستورالعمل اخلاقی موسسه واکسن و سر سازی رازی
Clinical trials code: 961256-133-18-84-2

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Hosseini R, namavari M, Ghalegolab N, Shirazinejad A, Alipour S. The effects of zataria multiflora on angiogenesis of chorioallantoic membrane of chick embryo. RJMS. 2022; 29 (3)
URL: http://rjms.iums.ac.ir/article-1-6646-en.html
, namavari@yahoo.com
Abstract:   (544 Views)
Background: Angiogenesis is a multi-stage phenomenon that involves the initial germination, separation and regeneration of new blood vessels from previous ones and is essential for many physiological functions of the body. If angiogenesis is performed insufficiently or excessively, it can lead to pathological lesions and injuries. In the treatment of various tumors and cancers.  inhibition of angiogenesis proposed as a more effective solution than killing cancer cells directly. Despite significant advances in chemotherapy, the survival rate is unsatisfactory due to drug resistance. Therefore, new strategies should be considered. The use of herbal remedies in the treatment of various diseases has been common since ancient times all around the world. Herbs contain various compounds including flavonoids, tannins, steroids, saponins, alkaloids, terpenoids, quinones, cardiac glycosides and coumarins that these compounds can have potential in the treatment of various diseases caused by pathological lesions of angiogenesis. Recently, many researchers have focused on the discovery of natural and synthetic drugs as anti-angiogenesis compounds so that they can be used as a treatment for various cancers or at least part of tumor treatment protocols. Zataria multiflora is a plant from the mint genus and native to southwest Asia. In various studies biological activities of thyme essential oil including its anti-cancer effects and antioxidant properties have been mentioned. But so far no study has been done on its anti-angiogenic effects as a target in cancer control in the embryonic chicken egg model. The aim of this study was to investigate the effects of aqueous extract of Zataria multiflora and its hydroalcoholic extract on angiogenesis and morphological changes on embryonic chicken eggs as a laboratory model.
Methods: After complete washing of the prepared plant, 5 g of thyme dried powder was boiled in 100 ml of sterile distilled water at 100 ° C for 5 minutes and then the liquid obtained is filtered and it was kept at 4 ° C until its use. The hydroalcoholic extract was also prepared from Barij Essential Oil Company, which is commercially available as gastrolite drops. After counting the vero cells, 7000 cells were considered for each 96-well plate. To determine the suitable dose, different dilutions of extracts were prepared as follows: 1000 µg/ml, 500 µg/ml, 250 µg/ml and 125 µg/ml and from hydroalcoholic extract, dilutions of 1/10, 1/100, 1/1000 and 1/10000 were used in DMEM culture medium. After the desired time, the supernatant was replaced with 50 μl of 12 mM MTT solution and incubated for 4 hours. After this time, 200 μl of dimethyl sulfoxide was used to dissolve the formazan crystals in each well. The plate's optical absorption was read at 570 nm with an ELISA reader.
In ovo inoculation:
30 Specific Pathogen Free (SPF) embryonated chicken eggs were prepared from Razi Vaccine and Serum Research Institute and divided into three groups of 10 (control, treatment group with thyme aqueous extract and treatment group with hydroalcoholic extract). On the second day of incubation in completely sterile condition eggs in the control group received 10 microliters of sterile normal saline, in the first group 10 microliters of Zataria multiflora extract with a dose of 250 µg/ml and for the second group 10 microliters of hydroalcoholic extract with a dose of 0.001 µl/ml. On the twelfth day of incubation, images of blood vessels on the surface of the chorioallantoic membrane of all specimens were prepared using a photo-stereomicroscope and the number and length of vascular bifurcations were measured for all specimens using image J software. At the end of the study, the morphological status and weight of embryos in each group were assessed and recorded. For statistical analysis of the results with the help of SPSS software, version 22 and using one-way analysis of variance ANOVA and a significant level of P <0.05 was considered.
Results: After culturing vero cells with different dilutions, 250 µg/ml and 0.001 µl/ml were the best dose of  aqueous extract and hydroalcoholic extract respectively. Because these doses did not show a significant difference compared to the control group ( P> 0.05). After 12 days of incubation the number and length of vessels from the chorioallantoic membrane were calculated. In a comparative study of the mean total length of vascular branches, a significant difference was observed between the control group and the treatment group with aqueous extract and also with the group treated with hydroalcoholic extract. P = 0.00 and P = 0.00, respectively. Regarding the number of vascular branches, a significant difference was observed between the group treated with thyme aqueous extract and the control group P = 0.00 and this difference between the group treated with hydroalcoholic extract and the control group was also seen P = 0.00. Although the use of the extract reduced the length of the embryos, but this difference was not statistically significant (P = 0.10). In the case of hydroalcoholic extract, no significant difference was seen with the control group (P = 0.67). Regarding the mean embryos weight, there was no significant difference between the control group and the groups treated with aqueous extract and hydroalcoholic extract, P = 0.19 and P = 0.097, respectively. No morphological abnormalities were observed in the embryos of all three groups.
Conclusion: The results of this study showed that Zataria multiflora extract can significantly affect the rate of angiogenesis on the chorioallantoic membrane of embryos and reduce this process. In angiogenesis, blood vessel endothelial cells are stimulated by angiogenic factors such as vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), which secrete plasminogen activators and proteases that eventually lead to the release of endothelial cells become new blood vessels by destroying the basement membrane of blood vessels. Due to the importance and relationship of these factors with vascular angiogenesis in tumors, one of the therapeutic goals of tumors is the use of compounds that can reduce the expression of these angiogenic factors. In the present study, the use of Zataria multiflora extract significantly reduced the length and number of blood vessel branches in the chorioallantoic membrane and this significant reduction has been observed when using its active ingredient, thymol and carvacrol, which is probably due to the role of thymol and carvacrol in reducing the expression of genes involved in angiogenesis stimulants. In the present study, despite the reduction of angiogenesis, morphological abnormalities were not observed in treated embryos, which was probably due to the presence of some soluble antioxidants in the extract, including carvacrol and various polyphenols. However, there is still a need for further research into the effects of Zataria multiflora in animal models with different doses, gene expression, intracellular signals and histological studies.
Ethical Considerations
Compliance with ethical guidelines
This study was supported by Razi Vaccine and Serum Research Institute, Shiraz Branch, Agricultural Research, Education and Extension Organization (AREEO) and the experiment was accomplished under the approval of the state committee on animal ethics.
Conflicts of interest
The authors declare that they have no conflict of interest.
Type of Study: Research | Subject: Pharmacology

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