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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 2  |  Page : 314-320

The effects of gilaburu (Viburnum opulus) juice on experimentally induced Ehrlich ascites tumor in mice


1 Genome and Stem Cell Center, University of Erciyes, Kayseri, Turkey
2 Department of Biology, School of Arts and Sciences, University of Akdeniz, Antalya, Turkey
3 Department of Anatomy, School of Medicine, University of Erciyes, Kayseri, Turkey
4 Department of Histology and Embryology, School of Medicine, University of Erciyes, Kayseri, Turkey
5 Department of Pharmacognosis, School of Pharmacy, University of Erciyes, Kayseri, Turkey

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Dilek Ceylan
Genome and Stem Cell Center, University of Erciyes, Kayseri 38039
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.181173

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 > Abstract 


Objective: The aim of study was to investigate anticancer effect of Viburnum opulus (VO) on Ehrlich ascites carcinoma (EAC) bearing mice that treated with different concentrations of VO.
Materials and Methods: For tumor transplantation; mice were inoculated with 1 × 106 EAC cells intraperitoneally and than divided into five groups (n = 9). Two hours after inoculation; experimental groups were treated daily with VO extract at doses of 1000 mg/kg, 2000 mg/kg, 4000 mg/kg.
Results: Extracts obtained from gilaburu juice can have hinder effect on tumor cell growth.
Conclusion: As far as we known, this is the first study about in vivo antitumoral activity of VOon Ehrlich ascites tumor model, and consequently VO extract exhibited anticancer activity against EAC-bearing mice.

Keywords: Antitumor activity, Ehrlich ascites tumor, experimental cancer, histopathology, Viburnum opulus


How to cite this article:
Ceylan D, Aksoy A, Ertekin T, Yay AH, Nisari M, Karatoprak G&, Ülger H. The effects of gilaburu (Viburnum opulus) juice on experimentally induced Ehrlich ascites tumor in mice. J Can Res Ther 2018;14:314-20

How to cite this URL:
Ceylan D, Aksoy A, Ertekin T, Yay AH, Nisari M, Karatoprak G&, Ülger H. The effects of gilaburu (Viburnum opulus) juice on experimentally induced Ehrlich ascites tumor in mice. J Can Res Ther [serial online] 2018 [cited 2019 Nov 13];14:314-20. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/314/181173




 > Introduction Top


Cancer is perhaps the most progressive and devastating disease posing a threat of mortality to the entire world despite significant advances in medical technology for its diagnosis and treatment.[1] The conventional therapies commonly used today for the treatment or prevention of cancer include the surgery, chemotherapy, and radiotherapy. Patients do not normally cope well with these therapies as they involve chemical and physical agents, separately or combined, that produce side effects or toxic reactions, and have a high cost.[2] Recently, considerable attention has been focused on identifying naturally occurring chemopreventive substances capable of inhibiting, retarding, or reversing the process of multistage carcinogenesis. The majority of these naturally occurring phenolics retain antioxidative and antiinflammatory properties which appear to contribute to their chemopreventive activity.[3] Antioxidants, such as vitamins and polyphenols, include a variety of compounds that can capture reactive oxygen species. Therefore, antioxidants have been proposed to have potential benefits for the prevention and treatment of diseases associated with reactive oxygen species. Antioxidants have been demonstrated to be effective in reducing the risk of carcinogenesis.[4]

Viburnum opulus (VO) belongs to the plant family Adoxaceae,[5] VO can be found in Eastern, North-Eastern, Western and Central Europe and Central Anatolia of Turkey.[6] It is called varying region to region as gilaburu, gileburu, girabolu, girebolu, gülebru and gili gili in Turkey and called as Cranberrybush in Europe.[7] The fruit of VO has a red color and astringed taste.[8] The juice of VO fruit and fruit can be used as drink and jellies by people.[9] The fruit of VO is highly acidic and contains a high amount of polyphenols,[10],[11] ascorbic acid[12] and L-malic acid.[13] As reported by several authors VO juice is a source of flavonoids which contain (+)-catechin and (−)-epicatechin, quercetin glycosides.[12] Therefore, VO juice includes a high proportion of chlorogenic acid and 54% of the total phenolic compound[8] and contain carotenoids.[13] It has been reported that ıts antioxidant effects in accordance with the compounds and amount of the compounds.[11],[14],[15],[16],[17],[18] Because of these properties, it can be used for the treatment of some diseases as the menstrual cramps, nervous system disorders, the liver and biliary disorders.[17] However, more clinical researches are necessary for whether it can be used or not as a drug. Also, recent study has reported that it has preventive effect on cancer by the time using as a natural drink of gilaburu fruit.[19] Simultaneously, recent investigations have reported that it has high antimicrobial potential and antioxidant activity that considered effective in reducing the risk of developing cancer.[20]

Since three centuries, especially transplanted with chemical carcinogens and spontaneous tumor models have come fore in experimental cancer research. These models have been preferred frequently because they can be applied and also produced more easily in research centers.[21] In the last 2–3 decades, studies on cancer which can be transplanted have numerous increased. The Ehrlich tumor was initially described as a spontaneous murine mammary adenocarcinoma. It is a rapidly growing carcinoma with very aggressive behavior and can grow in almost all mice strains.[22],[23] It has been used as a transplantable tumor model to investigate the antineoplasic effects of several chemical compounds. After intraperitoneal inoculation of Ehrlich tumor cells, the ascitic volume and cells number increase drastically.[24] The aim of this study was to investigate the antitumor properties of different concentrations of gilaburu juice powder extract on both in vitro using Ehrlich tumor cells and in vivo using murine ascites Ehrlich tumors model.


 > Materials and Methods Top


Plant material and preparation of extract

The bunches with fruits of VOwere collected from Kayseri of Turkey in October 2011. The bunches with fruits were combined and packed in plastic bags. The stalks were removed and the fruits were crushed with a glass stick. The mash was centrifuged at 6000-gauge for 20 min. The supernatant of clear juice was poured into glass bottles. After centrifuged extracts from the fruits were lyophilized with Labconco freeze dryer (model 117-A65312906; FreeZone 2.5) for 24 h, and then stored in plastic vials at −80°C until analysis. On the analysis day, VO extract was sterilized with 0.45 and 0.20 μm filter in which sterile phosphate buffer saline (PBS).

Experimental animals

All animal procedures and experimental protocols were approved by the Experimental Animals Ethics Committee, Erciyes University, Turkey (12/89-12/08/2012). Totally 45 Balb/c mice of about 6–8-week-old with an average body weight of 25–30 g were procured from Laboratory Animal Unit of Experimental and Clinical Research Center, Erciyes University and housed under controlled conditioning (25 ± 1°C constant temperature, 55% relative humidity, 12 h dark/light cycles). Food and water were allowed ad libitum during the study period. The mice were acclimatized to laboratory conditions for 7 days before commencement of the experiment.

Tumor cells

Stock animal that has Ehrlich ascites carcinoma (EAC) obtained from Biology Department of Science Faculty, Gaziantep University. EAC is a murine spontaneous breast cancer that served as the original tumor from which an ascites variant was obtained. The tumor cells were maintained in our laboratory by serial intraperitoneal (i.p.) passage in male Balb/c mice at 7–10 days interval. EAC cells were tested for viability and contamination using trypan blue dye exclusion technique. Cell viability was always found to be 95% or more. Tumor cell suspensions were prepared in PBS.[25]

Tumor transplantation and treatment schedule

Mice were inoculated with 1 × 106 EAC cells (0.2 ml/mouse) i.p. Day of tumor implantation was assigned as day “0.” Two hours after inoculation the animals were randomized and divided into five groups (n = 9). The control and experimental groups were as follows:

  • Group 1: Negative control group (n = 9); PBS was injected i.p. daily for 10 days
  • Group 2: Positive control group (n = 9); 1 × 106 EAC cells were injected i.p. and PBS was injected i.p. daily for 10 days
  • Group 3: n = 9; 1 × 106 EAC cells were injected i.p., and then animals were treated daily with VO extract at dose of 25 mg (1000 mg/kg) i.p. to be within 200 μL PBS for 10 days
  • Group 4: n = 9; 1 × 106 EAC cells were injected i.p., and then animals were treated daily with VO extract at dose of 50 mg i.p. (2000 mg/kg) to be within 200 μL PBS for 10 days
  • Group 5: n = 9; 1 × 106 EAC cells were injected i.p., and then animals were treated daily with VO extract at dose of 100 mg i.p. (4000 mg/kg) to be within 200 μL PBS for 10 days.


The doses were selected based on a previous study done in our lab.[19] All the animals were sacrificed on 11 d, 24 h after the last dose and ascites fluid was collected from peritoneal cavity of Groups 2–5 for the evaluation of tumor growth. Anticancer effects of VO were assayed by observation of change with respect to daily body weight, viable and nonviable tumor cell count in the peritoneal cavity of treated groups and the control group. Following sacrification, liver and kidney tissues were removed and examined for biochemical parameters. In addition, we examined histologically liver, kidney, colon, and small bowel tissues for evaluation of the tumor inhibitior effect of VO extract.

In vitro study

For the in vitro cytotoxicity, the EAC cells were used to study the cytotoxicity of VO extract after collected from the peritoneal cavity of tumor-bearing mice. 5 × 105 EAC cells were seeded into 6-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated at 37°C in 5% CO2 incubator for 24 h. After 24 h incubation in the growth medium, the cells were exposed to different volumes of VO (125, 250, 500 μg/ml) which sterilized with 0.45 and 0.20 μm filter, for 48 h. After the 48 h cell suspension and a trypan blue solution was mixed 1:1 and the viability of the cells was estimated using a hemocytometer. The cytotoxicity was calculated as number of viable cells over number of total cells and was presented as percentage of cytotoxicity.

Evaluation of biochemical parameters

Antioxidant enzymes were evaluated in the liver and kidney tissues collected from the experimental animals after excision. After rinsing twice in buffer, the tissues were weighed and homogenized with a Teflon homogenizer in measured volumes of phosphate buffer (pH 7.4). The homogenate was centrifuged at 10.000 rpm for 10 min, and the supernatant was used for the analysis. The levels of thiobarbituric acid reactive substances (TBARS) in the liver and kidney tissues were measured as reported.[26] The levels of lipid peroxides (TBARS) were expressed as μmoles of malondialdehyde (MDA)/g of liver and kidney tissues. Superoxide dismutase (SOD) was analyzed according to the methods described by Sun[27] catalase (CAT) activity was performed according to the method proposed by Aebi.[28]

Evaluation of histopathological parameters

Tissues samples were fixed in neutral 10% buffered formalin (pH 7.2) at room temperature. After fixation, tissues were dehydrated through graded alcohol solutions, and embedded in paraffin. Sections (5 μm thickness) were stained with hematoxylin and eosin and examined under a light microscope (Zeiss Axiolab) for histopathological analysis.

Statistical analysis

Compliance with the normal distribution of data and variance homogeneity were assessed by the Shapiro–Wilk and Levene test, respectively. Comparisons between groups were evaluated using Kruskal–Wallis H-tests and one-way variance analysis. Multiple comparisons were evaluated by Tamhan T2 and Siegel–Castell tests. Data analyses were evaluated using IBM SPSS Statistics 20.0 commercial package programs (IBM Inc., Chicago, IL, USA) and significance level was assumed roughly P < 0.05. Values were expressed as n (%), mean ± standard deviation or median (25th and 75th percentiles).


 > Results Top


Anticancer activity of VO extract against EAC-bearing mice was evaluated with parameters as changes at body weight and viable and nonviable cell count. After the tumor control group, the maximum weight gain was observed in Group 3 where the least weight gain were observed in Group 5 among the experimental groups. The tumor weight was found to be significantly increased in EAC control group when compared with the other groups that treated with VO (P< 0.05). Among the experiment groups, the tumor weight was found to be significantly decreased in Group 5 (P< 0.05) [Table 1].
Table 1: The average weight changes in control and experiment groups

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On the 11th day, ascites fluid was collected from peritoneal cavity of experimental and tumor control groups. The survival rate of Ehrlich ascites tumor (EAT) cells was reported to be 100% viability at Group 2. For the other groups (Groups 3–5) this index was found to be 88.72%, 69.02% and 51.87% respectively [Table 2]. The % viability in EAC-bearing group when compared with the experimental groups significantly decreased in Groups 4 and 5 (P< 0.05).
Table 2: Viable cell counts and percentage viability in control and experiment groups

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In assay for in vitro cytotoxicity, VO extract cytotoxicity was evaluated in EAC cells using the trypan blue exclusion test after 48 h of exposure in culture. The IC50 value was determined as 199.58 μg/ml. The lowest concentration of VO extract (199.58 μg/ml) had significant effect on cell death as the percentage of viable cells was reduced to 50% compared to control group. The percentage of viable cells reduced with increase of VO extract concentration. More than 79% cells were found to be dead at 500 μg/ml concentration [Table 3]. In cancer control group when compared with experimental groups, the cytotoxicty were found to be significantly increased among all of three treated groups with VO (P< 0.05).
Table 3: Cytotoxicity effect of Viburnum opulus on control and experiment groups

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Biochemical findings

The antioxidant activity of VO was evaluated on liver and kidney tissues. In this study, MDA levels of liver and kidney increased at EAC control group when compared with experimental groups, but this increase was not statistically significant (P > 0.05). CAT activity decreased in EAC-bearing control mice when compared with normal control group in both tissues (P< 0.05). When these mice were treated with three different concentrations of VO, the levels of this enzyme were observed to be elevated depending on the dose of VO in liver tissue. In kidney tissue, CAT activity increased at experimental Groups 4 and 5 (P< 0.05). On the other hand, SOD activity decreased at EAC control group when compared with normal control group and this activity increased at experimental groups in both tissues, but this difference was not found to be statistically significant (P > 0.05) [Table 4] and [Table 5].
Table 4: Antioxidant potential of Viburnum opulus on kidney tissue

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Table 5: Antioxidant potential of Viburnum opulus on liver tissue

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Histological findings

Tumor tissue

When we opened the peritoneal cavity of EAC control group mice, we determined whitish masses in only some mice of EAC control group. We removed these masses and stained by H and E. We determined that EAC cells were solidified in the peritoneal cavity [Figure 1]. Ahead in connective tissue capsule had been seen abnormal cell collapse and tissue-specific damage [Figure 1].
Figure 1: The histopathological estimation of whitish masses in Group 1 (Ehrlich ascites carcinoma positive control) Ehrlich ascites carcinoma cells which hyperchromatic large nuclei and eosinophilic cytoplasm with different shapes and sizes. Numerous blood vessels and blood cells (H and E, ×40)

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Liver tissues

According to histopathological findings, liver tissue of normal control group exhibited normal parenchymal structure features and normal architecture of hepatocytes radiating chord from central vein. Liver of EAC-bearing mouse and treated with 50 and 100 mg dose of VO showing mostly normal hepatocytes with normal nucleus and narrow blood sinusoids [Figure 2].
Figure 2: (a) Section of liver obtained from control mice. (b) Section of liver obtained from mice bearing Ehrlich ascites carcinoma cells. (c and d) Sections of liver collected from experiment administrated respectively 50 and 100 mg Viburnum opulus (H and E, ×20)

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Kidney tissues

Examples of the kidney histopathology are shown in [Figure 3]. According to histopathological findings, mice of negative control group exhibited normal histological structure on their kidney sections. Glomerular structures in the cortex were regular, and bowman's cavity was normal. In EAC control group, the EAC cells which at varying density with hyperchromatic large nuclei and eosinophilic cytoplasm, different shapes and sizes were established at the kidney capsule. It has been observed that agregation of tumor cells. Group 3 treated with 25 mg VO had been shown almost the same features with EAC control groups. Apparently, the other experimental groups treated with VO, diminished most of the pathological changes in EAC control group.
Figure 3: (a) Section of kidney obtained from control mice. Normal histological structure was seen in the renal cortex in the control group. Ehrlich ascites carcinoma cells were seen at the kidney capsule in other groups. (b) Section of kidney obtained from mice bearing Ehrlich ascites carcinoma cells. (c and d) Sections of kidney collected from experiment administrated respectively 25 and 100 mg Viburnum opulus (H and E, ×10). Ehrlich ascites carcinoma cells with hyperchromatic large nuclei and eosinophilic cytoplasm, different shapes and sizes

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Colon and small bowel tissues

[Figure 4] and [Figure 5] demonstrate the histological results of each group. Colon tissues of mice at negative control group were seen normal histological features. The mucosa contains numerous straight tubular intestinal glands (crypts of Lieberkühn) that extend through the full thickness of the mucosa in the control group. The general architecture of small bowel was also structurally normal in the control groups. After the inoculation, tumor cells were spreaded to serosa and mucosa layers in colon and small intestine tissues respectively in the EAC-bearing control group. After the treatment with 25 mg VO, in Group 3, it has been observed that aggregation of tumor cells and had been shown almost the same features with EAC control groups at small bowel tissue. However, Group 4 that treated with 50 mg VO extract showed that there was no tumor inhibition at both tissues. Group 5 diminished most of the pathological changes in EAC control group at colon tissue. These observations suggest that 50 mg VO alleviated histopathologic damage in both small bowel and colon.
Figure 4: (a) This photomicrograph of an H and E preparation shows the mucosa, submucosa and part of the muscularis layer. The surface epithelium is continuous with tubular intestinal glands (crypts of Lieberkühn) in control group (H and E, ×20). (b) Tumor cells had been invased to serosa layer at the Ehrlich ascites carcinoma bearing control mice.(H and E, ×10). (c) Treatment with 50 mg Viburnum opulus attenuated cell invasion (H and E, ×20). (d) Treatment of mice with 100 mg Viburnum opulus showing normal colonic mucosa and submucosa appearance (H and E, ×20)

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Figure 5: Representative photomicrographs of H and E-stained small intestine tissue sections from experimental groups. (a) Small intestine microscopic image of normal mice with intact epithelial and mucosal layer (H and E, ×20). (b) The Ehrlich ascites carcinoma bearing control mice with extensive damage, tumor cells were invasing to mucosa layers (H and E, ×40). (c) Histological small intestine sections of 25 mg Viburnum opulus group showing attenuated severity of the histological damage (H and E, ×20). (d) Histological small intestine mucosal sections of mice administrated with only 50 mg VO showing normal small intestine mucosa and submucosa appearance (H and E, ×10)

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 > Discussion Top


Recently, it is used to natural compound dietary with chemotherapy agents in cancer therapy. Many publications have reported that these diets contain flavonoids and phenolic compounds that related to inhibition of cancer. Phenolic compounds have anticancer effects which are associated with their antioxidants, antiinflammation properties. Polyphenols when given before or during the carcinogen treatment are effective in the preventing of cancer. Transplanted tumor models may be relevant to therapy with polyphenolics.[29] EAC model that was transplanted is very aggressive and specific for mice. Thus, EAC is convenient tumor model to evaluation the anticancer effects of VO extract. Earlier studies related to VOreported that it has antioxidant,[10] strong radical scavenging due to high phenolic concentration all of the extracts.[15]

In recent study, the effects of gilaburu (VO) juice on colon tumorogenesis were investigated. Experimental colonic tumors had been induced by 1,2-dimethylhydrazine (DMH), subcutaneous once a week for 12 weeks. Treatment groups received gilaburu juice for 30 weeks (started with first DMH injection) and for 18 weeks (started after last DMH injection). The sites and incidences of tumoral lesions (low-grade dysplasia, high-grade dysplasia, intramucosal carcinoma, and invasive carcinoma) were analyzed and compared with control. There was a reduction in the mean total number of tumor lesion in treatment groups when compared to cancer control group. This study has been reported gilaburu juice could prevent the progress of established tumors but could not prevent the chemical induction of colonic tumors in mice.[19]

In this study, a speedy increase was observed in viable cells counts, % viability value of cells and body weight of EAC tumor-bearing mice due to cell multiplication and growth of EAC while these parameters were reduced at the experimental groups which treated with VO extracts. In experimental groups; decrease in the number of viable cells and body weights and increase in nonviable cell count was dependent on dose of VO extracts. These results indicated the anticancer effects of VO against EAC cells and also there was statistically significant difference between experimental and control groups. The in vitro cytotoxicity effect of VO was demonstrated by the trypan blue method. The IC50 value was found as 199.58 (μg/ml). This assay also confirmed the reduction on cell viability and cell number was due to the cytotoxic effect of VO against the EAC cells. Anticancer agents when used to treat cancer cell lines (in vitro-in vivo), they showed very different effects on cell cycle as apoptosis and necrosis. It could be suggested that the therapeutic role of flavonoids ultimately was the induction of apoptosis in cancer cells[30] inhibition of cell proliferation[31] and modulation of a number of cellular signaling pathways activity.[32] According to these results, VOshowed antiproliferative effect against EAC cells in vitro and was a promising extensive screen in experimental animal models. The antitumor effect of VO may be possibly derived from alkaloids, phenolic compounds, flavonoids or their synergistic effects that contained in VO extract.

It is reported to play an important role of oxygen-derived radicals in the etiology of cancer development.[33] These free radicals which have the mutagenic capacity occur as a result of the interaction between the chemical molecules that highly reactive and DNA.[34]

Cell protection from oxidative damage is achieved by enzymatic or nonenzymatic antioxidant systems. Liver and kidney tissues have antioxidants which prevent damage caused by the excess oxygen metabolites. These antioxidants neutralize both peroxides and free radicals.[35] MDA the final product of lipid peroxidation occurred during degenerative decomposition of the cancerous tissue, is a biomarker of oxidative stress that exhibited higher levels in cancer tissues compared to normal tissues.[36] The activity of this enzyme is known to increase when composed the oxidant stress including cancer with adaptive mechanism.[37] The results of the present study, MDA levels in the EAC-bearing mice had found higher than treated groups, but this increase was not statistically significant.

CAT is a peroxidase which converted to water and oxygen using H2O2 as substrate. Superoxide is converted to hydrogen peroxide by the enzyme SOD.[36] It is well-known that cells exhibit enzymatic antioxidant mechanisms, such as the generation of SOD and CAT, which are involved in the elimination of free radicals. SOD and CAT are involved in the scavenging of superoxide and hydrogen peroxide. Researchers reported that decreased levels of SOD activity were detected in EAC-bearing mice in response to the loss of Mn2+-SOD activity and mitochondria in EAC cells, resulting in a decrease in the amount of total SOD activity in the liver.[38],[39],[40] The inhibition of SOD and CAT activity as a consequence of tumor growth has also been reported.[41]

Similar results were found in our study on EAC control group. CAT and SOD activity decreased on liver and kidney tissues of EAC-bearing control mice when compared with normal control group. The levels of CAT and SOD activity increased in a dose-dependent manner toward normal levels at treated with VO extract. The decrease of SOD and CAT activities and the increase of MDA levels could be considered as aggressively of free radicals resulted from the development of cancer. However, it is considered to be converted free radicals to more stable products or stimulate the antioxidant enzymes directly because of including amount of antioxidant compounds of VO extract particulary phenolic compounds.

These results were supported by the histopathological findings in liver, kidney, colon, and small bowel tissues of EAC-bearing mice and administrated VO extract mice. Histopathological findings in EAC control and treated groups were observed normal liver histology. Hence, metastasis was not observed in the liver. This effect is probably related to content of VOextract. However, it has been observed that aggregation of tumor cells in the other tissues of EAC control group. On the other hand, experimental groups treated with VO showed that diminished pathological changes owing to the antioxidant features of VO that were cytotoxic toward EAC cells. According to these pathological findings, there was tumor inhibition in all VO extract given groups, compared to the EAC-bearing mice.

As far as we known, we reported the in vitro and in vivo antitumoral activity of VOon EAT model for the first time.


 > Conclusion Top


VO extract exhibited possibly antitumor activity against EAC by modulating lipid peroxidation and augmenting endogenous antioxidant defense systems. Future studies are required to investigate which compounds were effective and responsible for antitumor activity of VO extract.

Acknowledgments

The authors would like to thank Professor Mehmet Özaslan, Department of Biology, Gaziantep University, Turkey for providing the EAC cells.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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