Ecological and health risk assessment of exposure to atmospheric heavy metals.

In the present study, we assessed the concentration of airborne HMs (Zn, Cu, Pb, and Cd) and their probable sources using the bark of Pinus eldarica as a bio-indicator. Hence, 47 tree bark samples were harvested according to the land uses and biomonitoring techniques in the city of Yazd, Iran. The potential health risks in 13 age groups, ecological risk, as well as the possible relationship between HM concentrations and traffic indicators, were evaluated. The order of average HM concentrations in the P. eldarica bark samples was as Zn > Pb > Cu > Cd. The mean values of non-carcinogenic risks of all HMs in entire age groups were within secure range (HQ < 1); however, the carcinogenic risk of Cd was higher than the allowed level (TCR > 1 × 10-6). About Pb, it was in the safe level. The main element causing potential ecological risks was Cd, indicating moderate to very high ecological risk in most of the study areas. There was an inverse significant association between distance from major roads and Pb concentration (ß = -0.011 95% confidence interval (CI): 0.022, -0.0001). All HMs in bark samples render the negative Moran's index, representing a random spatial distribution pattern. Besides, according to principal component analysis (PCA), the first component accounted for 36.55% of the total variance, dominated by Cd, Pb, Cu, and Zn, respectively, and characterized by vehicle and industrial emissions. Our results infer that industrial activities and traffic are the main sources of HMs pollution in urban environments that should be considered by decision-makers.

Chemical Composition and Antimicrobial Activity of Essential Oils from the Aerial Parts of Pinus eldarica Grown in Northwestern Iran.

Pinus eldarica (Pinaceae), an evergreen plant, is distributed across the warm and dry climates of western Asia, including Asia Minor, the Middle East, and land surrounding the Caspian Sea. Essential oils (EOs) from different aerial parts of this tree have been used in traditional medicine. We aimed to investigate the chemical profile and antimicrobial activity of the EO from P. eldarica grown in northwestern Iran. EO from the needles, bark, and pollen were extracted with boiling water using a Clevenger apparatus at yield of 0.7-1.2 cm3/100 g of dry plant material. The main chemical components of the EO from the needles were D-germacrene (18.17%), caryophyllene (15.42%), γ-terpinene (12.96%), and ß-pinene (10.62%); those from the bark were limonene (16.99%), caryophyllene oxide (13.22%), and drimenol (13.2%); and those from the pollen were α-pinene (25.64%) and limonene (19.94%). In total, 83 constituents were characterized in the EOs, using gas chromatography mass spectrometry analysis; mainly, sesquiterpene hydrocarbons in needle EO and monoterpene hydrocarbons in pollen and bark EOs. ß-Pinene, ß-myrcene, limonene, and caryophyllene were identified in the EOs from all three plant parts. The antibacterial and antifungal properties of the EOs were examined: pollen EO exhibited antibacterial activity against Escherichia coli; bark EO inhibited the growth of Candida albicans and Staphylococcus aureus; and the needle EO inhibited the growth of S. aureus. Thus, the EOs from aerial parts of P. eldarica can benefit the EO industry and antibiotic development.

Anti-nociceptive and anti-inflammatory effects of hydroalcoholic extract and essential oil of Pinus eldarica in animal models.

OBJECTIVE: Several species of Pinus have shown anti-nociceptive and anti-inflammatory properties. This study was aimed to evaluate anti-nociceptive and anti-inflammatory effects of hydroalcoholic extract of bark and essential oil of leaves of P. eldarica in animal models. MATERIALS AND METHODS: Hydroalcoholic extract of bark and essential oil of leaves of P. eldarica were prepared and phenolic content and essential oil composition were analyzed using Folin-Ciocalteu method and GC/MS, respectively. Anti-nociceptive effect was assessed using acetic acid, formalin and hot plate tests in male Swiss mice (25-30 g) and for evaluation of anti-inflammatory activity, carrageenan test in male Wistar rats (180-200 g) and croton oil-induced ear edema in male mice, were used. Involvement of opioid, α2-adrenergic, 5-HT3 receptors and adenosine triphosphate (ATP)-dependent K+ channels in pain relief was tested using naloxone, ondansetron, yohimbine and glibenclamide. RESULTS: The total phenolic content of the extract in terms of gallic acid equivalent was 404.9±7.7 mg/g of the extract powder. P. eldarica hydroalcoholic extract (200 and 400 mg/kg) and essential oil (100 and 200 µl/kg) significantly (all, p<0.001) decreased pain behavior in acetic acid and formalin tests but not in hot plate test. The extract and essential oil suppressed edema in carrageenan and croton tests. Glibenclamide partially reversed the anti-nociceptive effect of hydroalcoholic extract while the other antagonists were ineffective. CONCLUSION: Hydroalcoholic extract of bark and essential oil of leaves of P. eldarica significantly decreased acute and chronic pain as well as inflammation. ATP-dependent K+ channels mediate a part of the observed anti-nociceptive effect.

Phytochemical standardization, formulation and evaluation of oral hard gelatin capsules from Pinus eldarica bark extract.

OBJECTIVE: The extract of Pinus eldarica bark contains many polyphenolic compounds that were studied due to their high antioxidant, anti-inflammatory and anti-mutagenic effects. Therefore, the purpose of the present study was to conduct phytochemical standardization and develop hard gelatin capsules from the extract of P. eldarica bark. MATERIALS AND METHODS: Extraction was carried out by maceration method at room temperature for 72 hr using ethanol 70% followed by freeze drying. Quantification and standardization tests were performed using Folin-Ciocalteu method. Then, nine formulations were prepared containing different amounts of stearic acid (1-3%) and corn starch (3%, 10%, and 25%). Each formulation was characterized by FTIR and pharmacopoeial tests such as drug content, disintegration time, flowability parameters and drug release percent. The optimized formulation underwent stability studies at 75±5% humidity and 40±2°C. RESULTS: The total phenolic content of the extract in terms of gallic acid equivalent was 362.8±5.4 mg/g and the total procyanidin content in the extract was 174.386±2.5 mg/g. FTIR revealed no interaction between the components. The results presented that the best formulation of the capsules was achieved they contained 3% of stearic acid and 25% of corn starch. This formulation showed 91.69±0.33% of drug content, 9.36±0.02 min disintegration time and 83.02±0.81% release percent. Moreover, it showed good flowability. Stability studies on the optimized formulation displayed that the formulation was stable within 6 months in the accelerated condition. CONCLUSION: In conclusion, results of the present phytopharmaceutical evaluations confirmed this product as a promising herbal capsule formulation.

Neuroprotective effects of Pinus eldarica in a mouse model of pentylenetetrazole-induced seizures.

OBJECTIVE: Oxidative stress has pernicious effects on the brain. Pinus eldarica has antioxidant properties. We explored neuroprotective effect of P. eldarica against pentylenetetrazole (PTZ)-induced seizures. MATERIALS AND METHODS: Male mice (BALB/c) were grouped as control, PTZ, Soxhlet (Sox) 100, Sox 200, Macerated (Mac) 100 and Mac 200 groups. Sox and Mac extracts (100 and 200 mg/kg) were injected during 7 days. Delay in onset of minimal clonic seizure (MCS) and generalized tonic- clonic seizure (GTCS) was measured. Number of dark neurons (DN) and levels of oxidative stress indicators in the hippocampus were evaluated. RESULTS: Onset of MCS and GTCS was later in groups treated with the extracts than the PTZ group (p<0.01 and p<0.001). Number of DN in the hippocampus in the PTZ group was higher than the control group (p<0.001) while in the extract groups, was lower than the PTZ group (p<0.05, p<0.01 and p<0.001). MDA level was higher whereas total thiol level and activity of SOD and CAT were lower (p<0.001) in the PTZ group than the control group. MDA level in the Sox 100 (p<0.01), Sox 200 (p<0.001) and Mac 200 (p<0.01) groups was less than the PTZ group. Total thiol level in the Sox 200 (p<0.001), SOD in the Sox 100 (p<0.05), Sox 200, and Mac 200 and CAT in the Sox 200 (p<0.001) groups were higher than the PTZ group. CONCLUSION: P. eldarica prevented neuronal death and reduced seizures caused by PTZ via improving brain oxidative stress.

Investigating the effects of bark extract and volatile oil of Pinus eldarica against cisplatin-induced genotoxicity on HUVECs cell line.

Cisplatin is used for treating multiple types of cancers. Alongside its therapeutic effects, there are side effects, including cytotoxicity and genotoxicity for healthy cells, which are mainly related to radical oxygen species (ROS) production by the drug. These side effects could troublesome the treatment process. Previous studies have suggested that members of Pinaceae family are rich sources of antioxidant components. This article investigates the antioxidant activity (AA) of Pinus eldarica (Pinaceae) along with its cyto/genoprotective effects following cisplatin exposure on human umbilical vein endothelial cells (HUVECs) cell line. Pinus eldarica's hydroalcoholic bark extract (PEHABE) and P. eldarica's needle volatile oil (PENVO) were prepared using maceration and hydrodistillation methods, respectively. PENVO was analysed via gas chromatograph-mass spectrometry, and the total phenolic content of PEHBAE was measured by folin-ciocalteu reagent. AA of both PEHABE and PENVO were determined using DPPH assay. Moreover, MTT test was used to determine the cytoprotective effects of both agents. Comet and micronucleus (MN) tests were also performed to investigate the genoprotective effect of P. eldarica. Germacrene D (35.72%) was the main component of PENVO. PEHABE showed higher AA compared with PENVO, with the highest AA observed at 25 and 250 µg/ml, respectively. Both PENVO and PEHABE were cytoprotective, with the latter having mitogenic effects on cells at 75, 100, and 200 µg/ml concentrations (P < 0.01 and P < 0.001). Also, both PEHABE and PENVO showed genoprotective effects against cisplatin in comet assay (P < 0.001). As PEHABE's concentrations were increased, a reduced number of MN formation was observed after cisplatin's exposure (P < 0.001). In conclusion, PEHABE had higher AA compared with PENVO, and both agents had cyto/genoprotective effects on HUVECs.

Evaluation of acute and sub-acute toxicity of Pinus eldarica bark extract in Wistar rats.

OBJECTIVE: Pinus eldarica (P. eldarica) is one of the most common pines in Iran which has various bioactive constituents and different uses in traditional medicine. Since there is no documented evidence for P. eldarica safety, the acute and sub-acute oral toxicities of hydroalcoholic extract of P. eldarica bark were investigated in male and female Wistar rats in this study. MATERIALS AND METHODS: In the acute study, a single dose of extract (2000 mg/kg) was orally administered and animals were monitored for 7 days. In the sub-acute study, repeated doses (125, 250 and 500 mg/kg/day) of the extract were administered for 28 days and biochemical, hematological and histopathological parameters were evaluated. RESULTS: Our results showed no sign of toxicity and no mortality after single or repeated administration of P. eldarica. The median lethal dose (LD50) of P. eldarica was determined to be higher than 2000 mg/kg. The mean body weight and most of the biochemical and hematological parameters showed normal levels. There were only significant decreases in serum triglyceride levels at the doses of 250 and 500 mg/kg of the extract in male rats (p<0.05 and p<0.01, respectively) and in monocyte counts at the highest dose of the extract in both male and female rats (p<0.05). Mild inflammation was also found in histological examination of kidney and liver tissues at the highest dose of extract. CONCLUSION: Oral administration of the hydroalcoholic extract of P. eldarica bark may be considered as relatively non-toxic particularly at the doses of 125 and 250 mg/kg.

Cytotoxic effects of Pinus eldarica essential oil and extracts on HeLa and MCF-7 cell lines.

Several attempts have so far been made in the search of new anticancer agents of plant origin. Some studies have reported that different species of Pine genus possess cytotoxic activities against various cancer cell lines. In the present study, we evaluated the cytotoxic effects of Pinus eldarica bark and leaf extracts or leaf essential oil on HeLa and MCF-7 tumor cell lines. Hydroalcoholic and phenolic extracts and the essential oil of plant were prepared. Total phenolic contents of the extracts were measured using Folin-Ciocalteu reagent. Essential oil components were determined by gas chromatography-mass spectroscopy (GC-MS). Cytotoxic activity of the extracts and essential oil against HeLa and MCF-7 tumor cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The polyphenolic content of hydroalcoholic and phenolic extracts of the bark and hydroalcoholic extract of the leaf were 48.31%, 47.2%, and 8.47%, respectively. According to the GC-MS analysis, the major components of the leaf oil of P. eldarica were: ß -caryophyllene (14.8%), germacrene D (12.9%), α-terpinenyl acetate (8.15%), α -pinene (5.7%), and -α humulene (5.9%). Bark extracts and leaf essential oil of P. eldarica significantly reduced the viability of both HeLa and MCF-7 cells in a concentration dependent manner. However, leaf extract showed less inhibitory effects against both cell lines. The essential oil of P. eldarica was more cytotoxic than its hydroalcoholic and phenolic extracts. The terpenes and phenolic compounds were probably responsible for cytotoxicity of P. eldarica. Therefore, P. eldarica might have a good potential for active anticancer agents.

Anti-pseudomonas activity of essential oil, total extract, and proanthocyanidins of Pinus eldarica Medw. bark.

Pinus eldarica Medw. (Iranian pine) is native to Transcaucasian region and has been vastly planted in Iran, Afghanistan, and Pakistan. Various parts of this plant have been widely used in traditional medicine for the treatment of various diseases including infectious conditions (e.g. infectious wounds). In this study we aimed to investigate the antibacterial activity of P. eldarica bark extract, essential oil and proanthocyanidins on three important bacteria, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Antibacterial analysis was performed using standard disk diffusion method with different concentrations of essential oil, bark total hydroalcoholic extract, and bark proanthocyanidins (0.5, 1, 2 and 3 mg/ml). After incubation at 37°C for 24 h, the antibacterial activity was assessed by measuring the zone of growth inhibition surrounding the disks. The results indicated that the essential oil, total hydroalcoholic extract, and proanthocyanidins of the bark of the P. eldarica were effective against the gram negative bacteria, P. aeruginosa, and significantly inhibited its growth in disk diffusion method (P<0.001) of which the essential oil had the most potent inhibitory effect. However, none of the bark preparations could significantly inhibit the growth of S. aureus or E. coli. Our findings showed that P. eldarica bark components have significant anti-pseudomonas activity having potentials for new sources of antibacterial agents or antibacterial herbal preparations.

Cytoprotective Effect of Hydroalcoholic Extract of Pinus eldarica Bark against H2O2-Induced Oxidative Stress in Human Endothelial Cells.

BACKGROUND: Pinus eldarica is a widely growing pine in Iran consisting of biologically active constituents with antioxidant properties. This study investigates the effect of hydroalcoholic extract of P. eldarica bark against oxidative damage induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs). METHODS: The total phenolic content of P. eldarica extract was determined using Folin-Ciocalteu method. The cytotoxicity of P. eldarica extract (25-1000 µg/ml) on HUVECs was assessed using 3-(4,5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. Cytoprotective effect of P. eldarica extract (25-500 µg/ml) on H2O2-induced oxidative stress was also evaluated by MTT assay. The intra- and extra-cellular hydroperoxides concentration and ferric reducing antioxidant power (FRAP) were measured in pretreated cells. RESULTS: The total phenolic content of P. eldarica extract was estimated as 37.04±1.8% gallic acid equivalent. P. eldarica extract (25-1000 µg/ml) had no cytotoxic effect on HUVECs viability. The pretreatment of HUVECs with P. eldarica extract at the concentrations of 50-500 µg/ml significantly reduced the cytotoxicity of H2O2. P. eldarica extract decreased hydroperoxides concentration and increased FRAP value in intra-cellular fluid at the concentration range of 100-500 µg/ml and in extra-cellular fluid at the concentration range of 25-500 µg/ml. CONCLUSIONS: This study revealed the antioxidant and cytoprotective effects of P. eldarica extract against H2O2-induced oxidative stress in HUVECs. Concerning the high content of phenolic compounds in P. eldarica, more research is needed to evaluate its clinical value in endothelial dysfunction and in other oxidative conditions.

Effects of ethanolic extract of pine needles (Pinus eldarica Medw.) on reserpine-induced depression-like behavior in male Wistar rats.

BACKGROUND: In this study, the antidepressant activity of ethanolic extract of Pinus eldarica Medw needles was assessed using forced swimming test (FST) in rats. MATERIALS AND METHODS: MALE WISTAR RATS WERE RANDOMLY DIVIDED INTO SIX GROUPS AND TREATED AS FOLLOWS: first group was received only reserpine (6 mg/kg, i.p.), second group was received reserpine (6 mg/kg, i.p.) and imipramine (10 mg/kg, i.p.), three experimental groups received reserpine (6 mg/kg, i.p.) and three doses of pine needle extract (100, 300, and 500 mg/kg, p.o.) respectively and the final group (control group) received only vehicle (5% DMSO, i.p.). RESULTS: Acute oral administration of ethanolic extract of P. eldarica Medw needles at a dosage of 300 mg/kg reduced reserpine-induced increase in immobility time in the FST, demonstrating an antidepressant effect in the FST. Additionally, extract treatment did not modify the ambulation and rearing evaluated in open field test, indicating that antidepressant effect found in the forced swimming test was not based on the stimulation of locomotor activity. CONCLUSION: These results indicate that ethanolic extract of Pinus eldarica needles possesses an antidepressant activity.