Composition and Anti-Toxicity Effects of Cichorium intybus Distillate on Serum Antioxidant Status in Carbon Tetrachloride-Treated Rats

Introduction

Reactive oxygen species (ROS) can be produced by the normal metabolism of cells or environmental factors ( Mulla et al., 2018 ). Oxidative stress (OS) may result in multiple reproductive pathologies, such as polycystic ovarian syndrome, endometriosis, spontaneous abortion, infertility, and prenatal disorders ( Darché et al., 2017 ).

The natural antioxidants in the body consist of both non-enzymatic and enzymatic systems, including catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione reductase (GR), ( Gupta et al., 2006 , Showell et al., 2017 ). Nowadays, high costs of treatment and safety problems have stirred up growing interest in the administration of alternative medicine for reproductive disorders ( Ahangarpour et al., 2014 ).

Traditional medicine is considered a safe and natural therapeutic alternative among the public ( Rashidi et al., 2017 ). Plant antioxidants have gained much attention for their ability to protect the cells from oxidative stress and are widely used for reproductive purposes ( H Sekhon et al., 2010 ; Darché et al., 2017 ). There are various methods of traditional medicine preparation, including extraction, purification, fractionation, fermentation, concentration, and distillation.

Distillation products are among the most favorite drinks widely used in Iran, especially in Shiraz ( Gohari et al., 2017 ). Cichorium intybus L. (Chicory or Kasni) belongs to the genus Cichorium L. and the Asteraceae family. The polyphenols-rich fraction of the plant exhibits antioxidant activity and inhibits hydrogen peroxide ( Das et al., 2016 ). Chicory plants or extract are extensively used for the treatment of various diseases and improvement of reproductive organ status ( Saric-Kundalic et al., 2011 ). C. intybus distillate (CI) has also been used for reproductive aspects of health in different regions of Iran. Nonetheless, the possible effects of CI on antioxidant status, as well as distillate composition, have not yet been investigated scientifically.

Therefore, to evaluate the effect of CI on antioxidant status in females, the present study aimed to assess the activity level of antioxidant enzymes, including malondialdehyde (MDA) and glutathione (GSH), in the serum of carbon tetrachloride (CCl₄)-treated female rats and the chemical composition of C. intybus hydro-distillate.

Material and Methods

Preparation of Cichorium intybus Distillate.Cichorium intybus was collected from farms around Kashan, Isfahan province, Iran, and the genus and species were approved at the Herbarium of the Department of Botany, University of Isfahan, Iran. To obtain 1 liter of CI, 87.5 g of the dried plant was placed in a boiler with 1.75 liters of water as previously described ( Seghatoleslam et al., 2014 ). The outgoing steam was cooled, collected, and kept light-protected at 4ºC until use.

Extraction of Essential Oil. The essential oil was obtained from dried powder (100 g) of the aerial parts of C. intybus that was subjected to steam distillation using a Clevenger-type apparatus for 3h. Subsequently, the volatile fraction was isolated by hexane, dried by anhydrous sodium sulfate, and stored at 4°C in a closed vial until use for gas chromatography-mass spectrometry (GC/MS) analysis

Gas Chromatography-Mass Spectrometry Analysis. The oil was analyzed by GC/MS using Agilent technologies model 7890 B connected to a 5977A MSD. The separation was carried out by HP-5MS capillary column (5% phenyl methyl polysiloxane, 30 m×0.25 mm, and film thickness 0.25µm). The carrier gas was helium at a flow rate of 1ml/min (split; 10:1). The mass spectrometer was acquired in EI mode with ionization energy of 70 eV in a mass range of 50-550 m/z. The column temperature was maintained at 60°C for 4 min and then increased to 280°C at a rate of 5ºC/min and held at 280°C for 2 min. A sample volume of 50µl was diluted with 1000µl of hexane. Furthermore, 1µl was injected with a running time of 50 min. The components were identified based on a comparison with NIST (05a.L) and Willey (nl7) libraries spectra and the literature.

DPPH scavenging assay

The ability of the essential oil to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was assessed according to Moon et al. ( Moon and Shibamoto, 2009 ). The oil IC₅₀ value was calculated using an inhibition curve.

Animals. Adult female Sprague Dawley rats (180-200g) were obtained from Animal Breeding Center, Shiraz University of Medical Science, Shiraz, Iran. They were kept under standard conditions (12:12h light⁄dark, 25-35% humidity, and 20-22°C). All procedures were approved by the Institutional Animal Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC). To determine whether they had regular cycles, vaginal smears were obtained from all the rats before any drug administration.

Experimental Protocol. In this experimental study, 40 female rats were assigned to four groups: Control (receiving oral saline for four weeks), CI group (receiving 12.5 ml/kg/day CI orally for four weeks), CCl₄ group (receiving 1 ml/kg body weight CCl₄ via intraperitoneal (IP) injection twice a week for two weeks), and CC_l4+CI group (receiving both CI and 1 ml/kg of CCl₄ for two weeks). At the end of the experiment, all rats were anesthetized, blood samples were collected by cardiac puncture, and the sera were kept at -80°C for biochemical analysis.

Liver Function Tests. Liver injury induced by CCl₄ was evaluated by the measurement of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) enzymes activities using Biorex kit (Shiraz, Iran).

Measurement of Malondialdehyde Concentration. The MDA concentration was evaluated by a colorimetric method as previously described ( Mashhoody et al., 2014 ). It was calculated in µmol/mg protein using 1,1,3,3-Tetraethoxypropane as a standard.

Measurement of Glutathione Concentration. GSH assay with DTNB [5, 5′-dithiobis-(2-nitrobenzoate)] dye was performed, followed by a standard Ellman's method with some modifications to evaluate GSH in µmol/mg protein ( Mashhoody et al., 2014 ). The absorbance of the products was observed at 412 nm after 5 min.

Determination of Glutathione Peroxidase Activity. The GPx activity was measured using the method of Fecondo and Augusteyn with minor changes ( 29 )(Zal et al., 2014). The enzyme activity was expressed as mU/mg of the protein using the molar extinction coefficient of 6.22 x 10⁶ M^-1 cm^-1 for NADPH.

Determination of Catalase Activity. CAT activity was estimated by monitoring H₂O₂ decomposition using the procedure of Aebi with minor modifications ( 26 )(Yarahmadi et al., 2017). It was expressed as mmol of H₂O₂ consumed per min/mg of protein using the molar extinction coefficient of 43.6/ M/cm for H₂O₂.

Determination of Glutathione Reductase Activity. GR activity was examined using the method of Carlberg and Mannervik with some modifications ( 28 )(Zal et al., 2018). GR catalyzes the reduction of GSSG to GSH using NADPH for the reduction of the GSSG molecule. The results were based on the molar extinction coefficient of 6.22×10⁶ M^-1cm^-1 for NADPH.

Statistical Analysis. Statistical analysis was performed in SPSS software (version 19), and the graphs were obtained using GraphPadPrism5 software (San Diego, CA, USA). Curve expert 1.3 was used for IC₅₀ values. The data were depicted as mean±SEM. One-way ANOVA and Tukey’s post-hoc tests were used for between-groups comparisons (n=10). A p-value less than 0.05 was considered statistically significant.

Results

Chemical Composition of C. intybus. The chemical composition of CI is presented in Table 1. A total of 68 compounds were identified using the GC-MS analytical method and literature comparison. They were mainly antioxidants, such as terpene and terpenoid, as well as flavonoid and phenolic compounds.

Antioxidant Activity. The IC₅₀ value of chicory hydrodistillate was found to be 75µg /l using the DPPH method.

Liver Function Tests. As suggested by the results, the activities of ALT and AST were significantly increased in the serum of CCl₄-treated rats, compared to those in the control group. The administration of CI decreased the levels of these liver enzymes (P<0.05; Figure1 A and B).

Figure 1.Liver function enzyme activities in female rats (n=10): ALT (A) and AST (B). The control group received saline, the CI group received Cichorium intybus distillate orally for four weeks, the CCl₄ group received 1 ml/kg BW CCl₄ via IP injection twice a week for two weeks, and the CCl₄+CI group received CI and 1 ml/kg BW of CCl₄ for two weeks. * stands for P<0.05 compared to the control group and Φ for P<0.05, in comparison with the CCl₄ group.

Antioxidant Enzyme Activity. As displayed in Figure 2A, the GPx activity significantly decreased in the CCl₄ group, compared to that in the control group (P<0.05). Nevertheless, CI significantly increased the activity of GPx (approximately 33%), in comparison with that in the CCl₄ group (p<0.05).

Figure 2.Serum enzyme activity in female rats (n=10): Glutathione peroxidase (GPx) (A), glutathione reductase (B), and catalase (C). The control group received saline, the CI group received Cichorium intybus distillate orally for four weeks, the CCl₄ group received 1 ml/kg BW CCl₄ via IP injection twice a week for two weeks, and the CCl₄+CI group received CI and 1 ml/kg BW of CCl₄ for two weeks. * stands for P<0.05, compared to the control group and Φ for P<0.05, in comparison with the CCl₄ group.

A significant reduction was observed in GR activity (P<0.05) by CCl₄ administration, and CI administration ameliorated (P<0.05) its activity (Figure 2B). Moreover, the CAT level significantly reduced in the CCl₄ group, and it was restored (P<0.05) by CI treatment (Figure 2C). Nonetheless, the CI did not change the activities of enzymes in normal rats.

Glutathione and Malondialdehyde Levels. As illustrated in Figure 3, the administration of CCl₄ decreased GSH levels, while no significant change was observed in the CI-treated group. The rats treated with CCl₄ showed significantly increased levels of MDA (48%), compared to the control group (Figure 4). Moreover, the administration of CI in the CCl₄-treated group ameliorated the adverse effects of CCl₄ (P<0.05).

Figure 3. Level of serum reduced glutathione in female rats (n=10). The control group received saline, and the CI group received Cichorium intybus distillate orally for four weeks. The CCl₄ group received 1 ml/kg BW CCl₄ via IP injection twice a week for two weeks. The CCl₄+CI group received both CI and 1 ml/kg BW of CCl₄ for two weeks. * stands for P<0.05, compared to the control group and Φ for P<0.05, in comparison with the CCl₄ group.

Figure 4. Level of serum malondialdehyde (MDA) in female rats (n=10). The control group received oral saline for four weeks. The CCl₄ group received 1 ml/kg BW CCl₄ via IP injection twice a week for two weeks. The CCl₄+CI group received both CI and 1 ml/kg BW of CCl₄ for two weeks. * stands for P<0.05, compared to the control group and Φ for P<0.05, in comparison with the CCl₄ group.

Discussion

As evidenced by the results of the present study, the administration of CCl₄ increased ALT and AST levels, while CI administration improved the enzyme levels.   Carbon tetrachloride is extensively used to induce liver toxicity via lipid peroxidation ( Sharma and Agrawal, 2017 ). In line with the current study, several experiments reported that CCl₄ administration increased ALT and AST levels. They also indicated that treatment with antioxidant herbal plants, such as Echium Amoenum, Terminalia bellirica fruits, Ficus religiosa, and Syzygium samarangense, improved liver function enzymes ( Kuriakose et al., 2017 ; Sobeh et al., 2018 ).

The results of CI GC-MS identified various classes, such as terpenes, polyphenols, and flavonoids. In the present study, the pattern and amount of certain substances in GC-MS analysis of hydrodistillate of C. intybus aerial parts differed from those obtained in other studies ( Gol, 2014 ). It was reported that ( Būdienė, 2008 ) the predominant compositions of C. intybus in Lithuania were aliphatic hydrocarbons and their derivatives, while the quantities of terpenoids were minor.

Another study ( Gol, 2014 ) revealed that the major and minor compositions were γ -terpenes and aliphatic hydrocarbons, respectively. In a similar vein, the present study detected the same pattern, except for the slight differences in values. Some data suggested that ( Būdienė, 2008 ; Zahid Khorshid Abbas a and Nahla Zidan d, 2015 ) the hydro-alcoholic extract of chicory leaves possess higher values of flavonoids and phenolic acids. In agreement with previous reports ( Haghi et al., 2012 ; Muhammad et al., 2014 ), the findings of the current study confirmed the presence of some terpenes, flavonoids, and polyphenols in hydrodistillates.

Nevertheless, the results of the present demonstrated comparatively lower amounts of flavonoid and phenolic contents. This discrepancy can be ascribed to differences in seasonal, climatic, and geographical conditions, species, and the use of distillation as the method of plant preparation. The results of the current study indicated that chicory distillate was rich in terpene and terpenoid, as well as flavonoid and phenolic compounds, which might be responsible for the observed antioxidant activity of the distillate.

Therefore, the present study for the first time demonstrated that the essential oil of chicory hydrodistillate as the widely used herbal remedy in traditional medicine exhibited antioxidant activity and introduced it as a new potential source of natural antioxidants. Anti-oxidant and anti-inflammatory effects had been reported for sesquiterpenes in previously conducted studies ( Chadwick et al., 2013 ). The anti-oxidant and radical scavenging effects of CI flavonoids and sesquiterpene might be responsible for the ameliorative effects of C. intybus distillate on the CCl₄-induced oxidative stress.

In the present study, the observed decreases in the levels of the antioxidant enzymes in the CCl₄ groups might be due to the induced oxidative stress. Furthermore, the level of GSH was reduced by CCl₄ administration. In agreement with the results of the present study on female rats, it was reported that CCl₄ administration also decreased CAT, GPx, and GR activity in male rats ( Al-Rasheed et al., 2016 ). In 2013, consistent with the present study regarding distillate, it was reported that C. intybus leaf powder also increased CAT activity in rats ( Street et al., 2013 ).

The elevated MDA level after hepatic injury and its decrease after CI administration were in line with previous studies conducted on other plants ( Gupta et al., 2011 ; Asirvatham and Usha, 2017 ; and Sobeh et al., 2018 ).

Conclusion

Based on the obtained results, CI could be suggested as a safe medicine supplement to improve antioxidant status in females via the attenuation of oxidative stress due to its beneficial effects and lack of hepatotoxicity. Nonetheless, the safety, dosage, stability, and efficacy of distillates, as well as their exact constituents need further investigations.

Authors' Contribution

Study concept and design: A. S. and F. Z.

Acquisition of data: Z. Kh., R. Gh. and M. M.

Analysis and interpretation of data: A. S., F. Z. and M. M.

Drafting of the manuscript: All the authors

Critical revision of the manuscript for important intellectual content: : A. S., F. Z., M. N., Sh. F. and R. Gh.

Statistical analysis: A. S., F. Z. and Z. Kh.

Administrative, technical, and material support: A. S., F. Z.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Grant Support

The present study was financially supported by a research grant (93-7244) from Shiraz University of Medical Sciences, Shiraz, Iran. This article was extracted from an MSc thesis.

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