Plant Material and Preparation of Extract
The barks of P. eldarica were obtained from pine trees in Isfahan (Isfahan Province of Iran) on July 2018. The specimen voucher (No. 3318) was authenticated by a botanist and located at the Herbarium of our School.
The hydroalcoholic extract was prepared by maceration method using ethanol (70%) at room temperature for 72 hr in three times. After filtration and removing the solvent with a vacuum rotary evaporator, the yield extract was freeze-dried and the extract powder was kept at -20ºC.

Determination of Total Phenolic Content
Folin-Ciocalteu colorimetric assay was used for the measurement of total phenolic content in the bark extract of P. eldarica using a mixture solution of phosphomolybdate and phosphotungstate. In brief, 20 µL of the extract (2.5 mg/mL) was diluted with distilled water to 1.58 mL and mixed with Folin-Ciocalteu reagent (100 μL) and sodium carbonate (300 μL, 20%). After 2 hr incubation, the absorbance was determined using spectrophotometer at 765 nm. The results were calculated using a calibration curve of gallic acid and stated as milligram of gallic acid equivalent (GAE) per gram of the extract (16).

Animals
Male Wistar albino rats weighing 250 ± 20 gr were maintained under the standard environmental conditions including room temperature range of 20-25ºC and regular light/dark cycle of 12 hr/12 hr. They were allowed for free access to water and feed. The rats were acclimatized with the experimental environment for 1 week before the test. The experimental protocol was approved by the Biomedical Researches Ethics Committee of Isfahan University of Medical Sciences (ethical approval ID: IR.MUI.REC.1396.2.091).

In vivo Antioxidant Assay
For evaluation of the antioxidant effect of P. eldarica bark extract in rats, doses of 100, 200 and 400 mg/kg of the extract were administered daily intraperitoneally (i.p.) for 28 days (11). The control rats received i.p. injection of the vehicle daily. Each group consisted of 6 rats. At the end of the experiment, the blood was taken from orbital sinus plexus under anesthesia and the plasma was isolated for further testing.

Measurement of Hydroperoxides Concentration
The effect of P. eldarica bark extract on the plasma hydroperoxides of rats as the indicator for reactive molecules was measured using xylenol orange reagent-1 (FOX-1) method. In brief, the FOX1 reagent containing ammonium ferrous sulfate and xylenol orange in aqueous medium with sorbitol was added to 10 µL of the plasma samples. After incubation of mixture for 30 min at 37ºC, the absorbance of solution was determined at 540-560 nm. The level of hydroperoxides in plasma samples was calculated in term of µmole/L of hydrogen peroxide (H₂O₂) equivalents using a standard curve of H₂O₂ (17).

Ferric Reducing Antioxidant Power Assay
Ferric reducing antioxidant power (FRAP) assay was used for evaluation of the total antioxidant capacity in rats’ plasma samples after treatment with different doses of P. eldarica bark extract. This colorimetric assay evaluates the reduction of ferric-tripyridyl triazine (TPTZ) complex in FRAP reagent to ferrous form.  Briefly, plasma samples (10 µL) were mixed with FRAP reagent and incubated for 40 min at 37°C. Then, the absorbance was measured at 570 nm. The FRAP value in plasma samples was calculated in term of mmol/L of ferrous sulphate equivalents using a standard curve of FeSO₄ (18).

Evaluation of Antidyslipidemic Effects 
Dexamethasone-induced dyslipidemia was used for evaluation of the possible antidyslipidemic effects of P. eldarica bark extract. Dexamethason (10 mg/kg) was injected subcutaneously (s.c.) for 7 days in dyslipidemic control rats (19). Different doses of the extract (100, 200 and 400 mg/kg) were given orally plus dexamethasone in the treatment groups. Control group received s.c. injection of normal saline and orally administration of the vehicle extract. The reference group received dexamethasone and standard treatment of atorvastatin (40 mg/kg, orally) for 7 days. At the end of the experimental period, the blood samples of overnight fasted rats were taken and the serum was isolated for biochemical analysis. Then, animals were sacrificed and the livers were separated and kept immersed in 10% formalin and processed for histopathological examinations.

Biochemical Analysis for Dyslipidemia
Serum lipid profile including triglycerides (TG), total cholesterol (TC) and high-density lipoprotein (HDL) was enzymatically estimated using particular biochemical kits (Pars Azmoon Co., Iran). The following formula was used to calculate LDL: LDL = Cholesterol - (Triglyceride/5) – HDL. The blood glucose level was measured using glucose oxidase method.

Lipid Peroxidation Assay
The serum lipid peroxidation was assessed through measuring malondialdehyde (MDA) concentration. First, the serum samples were mixed with trichloroacetic acid (20%). Then, H₂SO₄ was added for the dispersion of precipitate. After adding a solution of thiobarbituric acid (0.67%) in Na₂SO₄ to the samples and heating the solution in a boiling water bath for 1 hr, the mixture was rapidly cooled and mixed with n-butanol. The absorbance of the colored solution was detected at 532 nm using a spectrophotometer. Finally, the results were estimated in term of nmol/mL of MDA equivalents using a calibration curve of MDA tetrabutyl ammonium (20).

Histopathological Analysis
The formalin fixed liver samples were dehydrated and cleared in xylene. Then paraffin blocks were prepared and cut at 5 μm thickness, stained with hematoxylin & eosin and evaluated microscopically for histopathological changes.

Statistical Analysis
The results were stated as the mean ± standard error of mean (SEM). The experimental groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey post-hoc test using the SPSS 18 (SPSS Inc., Chicago, IL., USA). The P-value<0.05 was considered as the level of significance.

Discussion

Findings of the present investigation demonstrated that the hydroalcoholic extract of P. eldarica bark has antioxidant, hypoglycemic and antihy-perlipidemic effects in dexamethasone-induced dyslipidemia. It is well known that oxidative stress, hyperglycemia, dyslipidemia and metabolic syndrome are considered as common problems during long-term treatment with high doses of glucocorticoids which are mainly due to the high production of free radicals, insulin resistance and abnormalities in lipid metabolism (21,22).
In this study, high amounts of phenolic compounds were detected in P. eldarica bark extract suggesting its potential therapeutic efficacy in various oxidative-induced disorders especially cardiovascular diseases. Our results indicated in vivo antioxidant activities for P. eldarica bark extract through reducing the plasma hydroperoxides concentration and improving the total antioxidant capacity in normal rats and also declining the lipid peroxidation in dyslipidemic rats. Similarly, Bargi et al., discussed the protective effects of P. eldarica extract against the oxidative damage of hippocampal tissues in pentylenetetrazole-induced seizure model among rats (23).

These antioxidant properties may be related to the great quantities of phenolic compounds containing hydroxyl groups with high reactivity to metal ions and free radicals (15).

In the study of Sadeghi and co-workers which evaluated the bark, seed and needle of P. eldarica for the antioxidant components, the highest amount of total polyphenols was identified in the bark extract suggesting the valuable potential of this part of the plant for clinical use (24).
In the present study, the data related to assessing the antidyslipidemic effect revealed the ability of P. eldarica bark extract in decreasing serum TG, TC and blood glucose level and also improving the liver histopathological changes in dexamethasone-induced dyslipidemic rats.
Some investigations have described antidiabetic, anti-atherosclerotic and anti-hyperlipidemic activities for different parts of Pinus species including leaves, nuts, branches and barks. In this regard, antidiabetic effect has been reported for the essential oil of P. koraiensis leaves and the bark extract of P. pinaster and P. roxburghii (25-27). The branches extract of P. roxburghii has also shown the strong α-glucosidase inhibitory activity in vitro (28).
The bark extract of P. pinaster has displayed antidislipidemic effect by reducing LDL and increasing HDL in animal and clinical studies (29,30). Further, Kim et al, reported that the essential oil obtained from the extract of P. koraiensis leaves inhibits acyl-coenzyme A: cholesterol acyltransferase and up-regulates LDL receptor representing hypolipidemic activity (31).
Concerning P. eldarica, Fallahhuseini et al., have defined the antihyperglycemic effect for the nut extract of this plant in alloxan-induced diabetic rats without any effect on triglyceride and cholesterol levels (11). However, in their recent study, P. eldarica nut extract resulted in a significant decrease in blood cholesterol level in hypercholesterolemic rabbits (12).
The presence of bioactive constituents in the bark of P. eldarica including polyphenolics such as catechin, taxifolin, caffeic acid and ferulic acid and some monoterpenes and sesquiterpenes such as α-pinene and β-caryophyllene may be responsible for the hypoglycemic and antihyperlipidemic activities of this plant (15). The beneficial effects on serum glucose and lipid profile have been recognized for catechins by controlling hemoglobin A(1c) and lowering cholesterol and LDL levels (32). Taxifolin modulates the production and secretion of hepatic lipoproteins (33). Further, it protects diabetic animals by decreasing blood glucose and serum insulin level and improving the pathological alterations of kidneys via NF-κB pathway (34). Caffeic acid reduces the blood glucose level by elevating the glucose uptake and stimulating the insulin receptor signaling cascade, and inhibits hyperlipidemia by controlling the lipogenic enzymes in the liver (35,36). Ferulic acid recovers glucose and lipid dysregulation through regulating the expression of lipogenic and gluconeogenic genes in the liver (37).
The major limitation of the present study was the lack of evaluating the mechanisms of antihyperglycemic and antihyperlipidemic effects of the P. eldarica bark extract because of financial budget restriction.

Conclusion

The present study indicated the in vivo antioxidant, hypoglycemic and hypolipidemic properties of hydroalcoholic extract of P. eldarica bark in dexamethasone-induced dyslipidemia. The observed effects are probably due to bioactive constituents especially the high amounts of phenolic compounds in the bark extract of this plant.

 

Acknowledgements

The authors are thankful to the Isfahan University of Medical Sciences for all supports. The project was supported by a grant No. 296091 from Vice-Chancellery for Research and Technology (Isfahan University of Medical Sciences).

 

Conflicts of Interest

Authors declared no conflict of interest.