Research Article | Open Access
Ebrahim Abbasi Oshaghi, Iraj Khodadadi, Fatemeh Mirzaei, Mozafar Khazaei, Heidar Tavilani, Mohammad Taghi Goodarzi, "Methanolic Extract of Dill Leaves Inhibits AGEs Formation and Shows Potential Hepatoprotective Effects in CCl4 Induced Liver Toxicity in Rat", Journal of Pharmaceutics, vol. 2017, Article ID 6081374, 9 pages, 2017. https://doi.org/10.1155/2017/6081374
Methanolic Extract of Dill Leaves Inhibits AGEs Formation and Shows Potential Hepatoprotective Effects in CCl4 Induced Liver Toxicity in Rat
The research was aimed at evaluating the antiglycation, antioxidant, and hepatoprotective properties of methanolic extract of Anethum graveolens (dill). The antioxidant properties, photochemical characteristics, and antiglycation effects of dill extract were measured. Carbon tetrachloride-induced hepatotoxic rats were used to show the hepatoprotective activity of dill leaves. Different concentration of dill extract (0.032, 0.065, 0.125, 0.25, 0.5, and 1 mg/mL) showed potential antioxidant ability. The extract of dill leaves significantly reduced AGEs formation and also fructosamine and protein carbonyl levels in rats’ liver. Thiol groups’ oxidation, amyloid cross-β, and protein fragmentation () significantly reduced in treated rats. Liver damage markers significantly reduced in dill-treated animals (). Dill with potential antioxidant, antiglycation, and hepatoprotective effects can be suggested for treatment of diabetes complications.
Free radicals are involved in many chronic and acute disorders such as diabetes, cancer, cardiovascular disease, immunosuppression, and neurological problems . The detrimental effects of the free radicals can be blocked by natural antioxidants . Numerous kinds of herbal medicine have studied for their antioxidant and antiradical properties . Anethum graveolens L. (dill) belongs to Apiaceae family and grows mostly in Europe, Mediterranean region, and Asia . Dill is used for various purposes in many countries and traditionally used for medicinal purpose such as digestive disorders, reduction of the bad breath, and stimulation of lactation and also known as a lipid lowering, anticancer, antimicrobial, antidiabetic, antigastric irritation, anti-inflammatory, and antioxidant agent [4, 5]. Administration of dill in human and animal models had antioxidant activity and normalized blood glucose and lipid profile [4–9]. Dill also showed potential antidiabetic activity . The exact antidiabetic mechanism of dill has not been recognized until now. The previous reports have not investigated all of the antioxidant indices of Anethum graveolens, neither its antiglycation effects. Furthermore, variance in cultivating area and the method of extraction cause different antioxidant ability . Consequently, this study was planned to assess the antiglycation and oxidant scavenging as well as hepatoprotective effects of dill cultivated in Hamadan (west of Iran).
2. Materials and Methods
2.1. Extraction of Plant Materials and Phytochemical Screening
Anethum graveolens was prepared from Hamadan (west of Iran) and identified by our colleague in the Buali-Sina University, Hamadan, Iran. For preparation of methanolic extract, dill leaves powder was dried and crushed. Dried dill powder (100 g) was mixed with 300 mL of methanol at room temperature for 48 hours. The prepared solution was filtered and subsequently concentrated and evaporated to dryness in vacuum. The extract was kept in dark vials at −20°C until analysis .
2.2. Phytochemical Screening
Phytochemical screening was performed according to Salmanian et al.  and Abbasi Oshaghi et al.  method. Total phenolic content of methanolic extract was determined using Folin-Ciocalteu reaction. Briefly, one milligram of methanolic extract was dissolved in the reaction solution (3.8 mL of deionized water + 2 mL of 2% Na2CO3 + 100 μL of 50% Folin-Ciocalteu). The prepared mixture was incubated at room temperature for 30 min and the absorbance of the sample was determined at 750 nm. Flavonoids content of dill was determined by using AlCl3 assay. Briefly 500 μL of the dill extract (1 mg/mL in methanol) was mixed with the reaction solution (1.5 mL of 95% alcohol + 100 μL of 10% AlCl3 + 100 μL of 1 M potassium acetate + 2.8 mL of deionized water). After 40 min of incubation at room temperature the absorbance of the samples was measured at 415 nm. Total flavonols content of dill was determined by adding of 1 mg/mL dill extract to the reaction solution (200 μL of 20 mg/mL AlCl3) + 6 mL sodium acetate solution (50 mg/mL). After 2.5 hours of incubation at room temperature the absorbance of the prepared solution was measured at 440 nm. The results were calculated per mg equivalents of gallic acid (for phenolic) and quercetin (for flavonoids and flavonols) per gram of each extract.
2.3. Antioxidant Activity
To measure the antioxidant activity of prepared dill extract different tests were carried out including ferric reducing antioxidant power (FRAP), DPPH radical scavenging, superoxide anion and hydrogen peroxide scavenging, metal chelating, reducing power, and nitric oxide scavenging activity, according to the previously published methods .
2.4. Glycation of BSA and Fructosamine
Glycated BSA was prepared using treatment of BSA with different concentration of fructose (200 and 500 mM) at different time periods (1, 2, 3, and 4 weeks) . Aminoguanidine (AG) a known antiglycation agent was used as a positive control. After dialysis in PBS, glycated BSA formation was determined using a fluorometry method at an excitation wavelength of 440 nm and emission wavelength of 460 nm (spectrofluorometer, Jasco FP-6200) . Nitroblue tetrazolium (NBT) reaction was used to measure the fructosamine level .
2.5. Thiol Group and Protein Carbonyl Content
The free thiol and carbonyl contents in glycated BSA were determined according to Adisakwattana et al.’s report .
2.6. Protein Aggregation and Fragmentation
Amyloid cross-β structure, which is recognized as an indicator of protein aggregation, was determined using Congo red dye . The fragmentation of protein was estimated and shown using SDS-PAGE .
2.7. In Vivo Studies
2.7.1. Hepatoprotective Activity
Male Wistar rats weighing 210–220 g were divided randomly into four groups (): (1) normal rats that received 30% CCl4 in olive oil (1 mL/kg body wt i.p) every 72 hours for a period of 10 days (hepatotoxic group); (2) CCl4 hepatotoxic induced rats that received 100 mg/kg dill extract for 10 days; (3) CCl4 induced hepatotoxic rats that received 300 mg/kg dill extract for 10 days; (4) normal rats that received distilled water (1 mL/kg body wt) orally for 10 days . After that, the animals were anesthetized and blood was collected from their heart. All of biochemical assays were performed using commercial kits (Pars Azmun, Iran) . All procedures were approved by ethics committee of Hamadan University of Medical Science, Hamadan, Iran.
2.7.2. Histopathological Examination
The pieces of rats’ liver were excised and then fixed in 10% formalin solution and processed by standard way. Liver sections with thickness of 5 μm were stained with haematoxylin and eosin (H&E). The stained slides were evaluated under a light microscope.
2.8. Statistical Analysis
Data are expressed as means ± SEM of three duplicate measurements and then analyzed by SPSS package (version 16, SPSS, Inc). One way analysis of variance (ANOVA) followed by Tukey test was used to analyze the results. The values less than 0.05 were regarded as statically significant.
3.1. In Vitro Antioxidant Study
Dill extract showed strong DPPH radical scavenging activity in a dose dependent manner with IC50 of 0.064 mg/mL. Dill extract also had potential FRAP value and reducing power ability (Figure 1). Dill showed potential super oxide anion-, hydrogen peroxide- and NO-scavenging activity and metal chelating with IC50 of 0.110, 0.125 mg/mL, 0.064, and 0.056 mg/mL, respectively (Figure 1). The total phenols, flavonoids, flavonols, alkaloid, anthocyanin, tannins, and saponin contents were , , , , , , and mg/g of extract, respectively.
In vitro antiglycation study of dill extract at different concentration significantly declined AGEs formation at 1, 2, 3, and 4 weeks of incubation. Dill also significantly declined fructosamine levels (Table 1) and carbonyl content (Table 2) and also inhibited thiol groups oxidation (Table 2), amyloid cross-β structure, and protein fragmentation rate (Figure 2).
| when compared to BSA/fructose at the same incubation time.|
| when compared to BSA/fructose at the same incubation time.|
In the in vivo study the serum levels of LDH, ALP, AST, ALT, γ-GT, total bilirubin, direct bilirubin, triglycerides, total cholesterol, and liver weight were significantly increased, whereas total protein, albumin, and body weight significantly reduced in CCl4 group. These values normalized in the animals which were pretreated with dill extract ( for all factors) (Table 3).