الفهرس 
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Abstract
Lead (pb) is a toxic heavy, bluish-gray metal that is used in several industries. Over the past century lead has contaminated the environment as a result of human activity. In the last several decades it became apparent that both acute and chronic exposure is harmful, with adverse effects on neurodevelopment and cognition if exposure exceeds tolerable levels. Lead can affect individuals of any age, but it has a disproportionate effect on children because their behavioral patterns place them at higher risk for exposure to lead. Their bodies absorb a larger percentage of the lead that they ingest, and they exhibit lead toxicity at lower level of exposure than adults do. Traces of lead occur in many rocks, in addition to those that qualify as over lead, and thus, lead finds its way into soil and water and hence into food, animals, and human tissues even in remote places where there is no use of the metal or its compounds. In spite of its widespread distribution in tissues, there is no indication that has no beneficial effect, but it causes many problems to the plant, food industry, and animal health. Although various countries have established legislation regulating their concentration, they are still sometimes a danger for consumer health.
Lead is translocated through the food chain to man and animals, and its toxicity depends on its chemical form administrated to the animal, the route of administration, and the frequency and duration of administration to animals. Lead induce overproduction of reactive oxygen species (ROS) and consequently enhance lipid peroxidation, decrease the saturated fatty acids, and increase the unsaturated fatty acid contents of membranes. Also, it has been shown to enhance the production of ROS in a variety of cells resulting to oxidative stress. ROS are the by-products of many degenerative reactions in many tissues, which will affect the regular metabolism by damaging the cellular components. In addition, ROS are highly reactive to membrane lipids, protein, and DNA. They are believed to be the major contributing factors to stress injuries and to cause rapid cellular damage causing brain toxicity.
Currently, natural products play an important role in the prevention, limitation and treatment of brain diseases due to their ability to scavenge the free radicals. Black chia oil and peppermint oil are among the natural products that have neuroprotective effect against lead acetate toxicity due to their antioxidant and anti-inflammatory properties.
Black chia is an herbaceous plant (Salvia hispanica L.) of the family Lamiaceae. Black chia is one of the highly nutritive value plants. It contains several chemical compounds that are known to have antioxidant, disease preventing and health promoting properties. Black chia is effective in curing memory impairment and learning disabilities due to its medical properties.
Peppermint (Mentha piperita L.) is actually a crossed-hybrid mint of water mint (M. aquatica L.) and spearmint (M. spicata L.) in the Lamiaceae family. Peppermint volatile oil extract is a good source of natural antioxidants, as indicated by their high contents of polyphenols, flavonoids and terpenoids (monoterpenes and sesquiterpenes), which one effective in prevention and treatment of neurodegenerative diseases.
The aim of the present work was to investigate the active chemical constituents and antioxidant capacities of black chia oil and peppermint volatile oil extracts (COE and POE), respectively. Lead acetate deleterious brain effects as well as the role of COE, POE and their mixture supplementation against lead acetate intoxication were evaluated on growing male rat’s brain.
Black chia oil and peppermint volatile oil extracts were chemically analyzed for their main active constituents of black chia oil and peppermint volatile oil extracts were determined using GC-MS technique. Hexanolic black chia oil extract (COE), volatile peppermint oil extract (POE) and their mixtures (COE+POE) were administrated to each rat according to its body weight.
Brain deleterious effects were induced by oral intubation with a daily dose of lead acetate (75 mg / kg B.W) which was dissolved in saline immediately before administration to rats for 7 days.
Eighty healthy growing wistar male rats (Rattus norvegicus) strains (from 2 or 3 weeks) weighing (90±5) g received the standred diet and water for 7 days (acclimatization period) and continued to have the same diet throughout the 4 weeks of the experiment (experimental period). All rats were divided into eight groups each of ten rats and divided as the following; group (1) CG: Control group, rats were given distilled water (1 ml) by oral intubation daily (28 days). group (2) COECG: Chia oil extract control group, rats were given chia oil extract for 28 days by oral intubation (0.9 g/kg BW). group (3) POECG: Peppermint oil extract control group, rats were given peppermint volatile oil extract for 28 days by oral intubation (0.04 g/kg BW). group (4) MOsECG: Mixture oils extract control group, rats were given chia oil extract (0.45 g/kg BW) and peppermint volatile oil extract (0.02 g/kg BW) for 28 days by oral intubation. group (5) LACG: Lead acetate control group, rats were given lead acetate (75 mg/kg BW) for 7 days followed by distilled water for 21 days by oral intubation. group (6) LA+COE, group (7) LA+POE, group (8) LA+MOsE: Lead acetate intoxicated rats (75 mg/kg BW) for 7 days followed by chia oil extract (0.9 g /kg BW) for 21 days by oral intubation, peppermint volatile oil extract (0.04 g /kg BW/21 days orally) and mixture of chia oil extract (0.45g /kg BW/ 21days orally) and peppermint volatile oil extract (0.02 mg/kg BW/ 21days orally) respectively.
During the experimental period, feed intake was recorded and animals were weighed weekly to calculate body weight change. At the end of the experimental period (4weeks) all rats fasted over night. the experimental animals were sacrificed under sodium barbiturate anesthesia. The brain and liver were removed, washed with saline and brain was weighed. Random brain and liver samples were fixed in 10% neutral formalin solution for histological study, the remaining brain and liver samples were prepared to form tissue homogenate for other analysis.
The Results of the Current Study are Summarized as Following:
1- The Main Active Constituents of black Chia Oil and Peppermint Volatile Oil Extracts
These results concluded that main active constituents of black chia oil extract by using GC-MS technique were Z-citral, α-citral, oleic acid, n- Hexadecanoic acid, estragole, erucic acid, neric acid, octadenoic acid, linoleic acid ethyl ester and other constituents. Z-citral represent the highest concentration (19.59%) of black chia oil extract when compared to all other components while 9, 12-octadecadienoic acid (Z,Z)-,Z-hydroxy-1-1(hydroxmethyl) ethyl ester represent the lowest concentration (0.84%), also this result concluded that main constituents of peppermint volatile oil extract by GC-MS technique were menthol, menthone, menthofuran, menthyl acetate, camphene, D-limonene, eucalyptol, 1-Hexade canol,2-methyl, geranyl acetate, myrcene and other constituents. Menthol represent the highest concentration (35.47%) of peppermint volatile oil extract when compared to all other components while germacerene represent the lowest concentration (0.02%).
2- Body Weight Change, Food Intake, Feed Efficiency Ratio, Absolute and Relative Brain Weight
Administration of lead acetate to rats caused a significant decrease in body weight, food intake, slightly decreased in absolute and relative brain weight, and it was noticed that there were slightly significant change between groups in feed efficiency ratio. COE and POE served as good sources of phenols, flavonoids and also high free radical scavenging activity which could have great effects on body weight, feed intake, absolute and relative brain weight, also reducing the oxidative stress and the inflammation resulted from lead acetate administration, so decreasing the deleterious brain effects induced by lead acetate as showed by marked elevation in body weight with a significant increase in relative brain weight.
3- Neural Cell Markers
Administration of lead acetate to rats caused a state of oxidative stress resulting in increased loss of brain neurotransmitters leading to decreased brain functions as shown by a significant decrease in the brain butyryl cholinesterase (BuChE), sertonine (5-HT) and dopamine (DA) levels. Administration of COE, POE and (COE+POE) to intoxicated rats caused improvement in the levels of neural cell markers due to presence of active components in the oils extracts that restored and attenuated the loss of brain neurotransmitters. Treatment with mixture of (COE and POE) caused the most significant improvement in brain BuChE activity, 5-HT and DA levels compared to other treatments as a result of the synergistic effects between black chia oil and peppermint volatile oil extracts active constituents.
4- Oxidative Stress and Antioxidant Status in Brain
Administration of lead acetate remarkably elevated brain oxidative biomarker MDA levels and significantly declined brain antioxidant markers as SOD activity, brain CAT activity, TAC level, GPx and GST levels as a result of oxidative stress caused by lead acetate. Supplementation with COE, POE and COE+POE decreased the oxidative stress induced by lead acetate and caused a significant enhancement in the brain SOD activity, brain CAT activity, TAC level, GPx and GST levels, where the supplementation with both of (COE and POE) caused the most significant improvement in brain MDA content, SOD activity, CAT activity, TAC level GPx and GSH levels followed by (COE) and finally (POE) supplemented groups.
5- Inflammatory Biomarkers
Lead acetate toxicity induced a massive increase in the levels of brain TNF-α and IL-6. Administration of COE, POE, COE+POE attenuated the inflammatory effects of lead acetate. Supplementation with (COE+POE) caused the most significant improvement in brain TNF-α and IL-6 levels followed by (COE) and finally (POE) supplemented groups. These improvements were due to the anti-inflammatory effect of black chia oil and peppermint volatile oil extracts.
6- Liver Enzymes Activities
Administration of lead acetate remarkably elevated liver enzymes activities resulted in significant increase in serum AST, ALT and ALP activities. Administration of COE and POE lowered the activities of liver enzymes when compared with LA group. The most significant ameliorating effect on serum AST, ALT and ALP were seen in (COE+POE) followed by (POE) and finally (COE) due to their protective activity against hepatotoxicity.
7- Kidney Functions
Lead acetate toxicity induced a massive increase in the levels of serum creatinine and urea. Administration of COE, POE, COE+POE attenuated the toxicity effects of lead acetate. Supplementation with (COE+POE) caused the most significant improvement in serum creatinine and urea levels followed by (POE) and finally (COE) supplemented groups. These improvements were due to the effect of black chia oil and peppermint volatile oil extracts on kidney function.
8- Microscopic Examination
a) Brain Tissues
Microscopic examination for brain tissues from the lead acetate group showed numerous neuropathological alterations which described as diffuse neuronal damage, pyknotic perikarea accompanied with moderate perineuronal edema, reactive astroglial cells and microglial cells infiltrates. Meanwhile, administration of black chia oil and peppermint oil extracts showed improvement in the histopathological picture and minimal neuroprotective efficacy. The most significant improvements in the microscopic examination of brain tissue were recovered in LA+MOsE (COE+POE) group followed by LA+COE group and finally LA+POE group respectively.
b) Liver Tissues
Microscopic examination for liver tissues from the lead acetate group showed hepatocellular damage vacuolar degenerative changes, necrotic hepatocytes, dilatation of hepatic vasculatures including hepatic sinusoids and perivascular inflammatory cells infiltrates. Meanwhile, administration of black chia oil and peppermint oil extracts showed improvement in the histopathological features of hepatic parenchyma. The most significant improvements in the microscopic examination of liver tissue were recovered in LA+MOsE (COE+POE) group followed by LA+POE group and finally LA+COE group respectively.
7. Conclusion
from the Results of this Study, it can be concluded that:
Lead is a pervasive environmental contaminant. Lead accumulates in the body, impairing a molecular level various cellular processes. Lead exposure during childhood causes adverse and permanent neurodevelopmental consequences, sometimes even with “low” blood lead levels. Symptoms are frequently silent, making lead exposure an often unrecognized and underestimated threat for pervasive neurocognitive disorders. It also confirmed a neurotoxin, but questions remain about lead associated intellectual deficits at blood lead levels<10 lg=dL and whether lower exposures are, for a given change in exposure, associated the greater deficits.
Administration of black chia oil extract (COE) and peppermint oil extract (POE) and their mixture (COE+POE) caused significant improvements in biological, biochemical, microscopic change in all affected rat groups and alleviate brain toxicity induced by lead acetate, hence protecting the functional bioavailability of brain neurons.
This improvement is due to the synergistic effect between black chia and peppermint oils extracts active constituents that of high antioxidant capacity and anti-inflammatory effects protecting brain tissue against lipid peroxidation and free radicals generation caused by lead acetate administration.
8. Recommendations
Recently, a close attention has been paid on health –promoting antioxidants from natural origin, with economic, medical implications and low side effects. Hence, the following recommendations are prescribed according to the results of this study:
• Lead widely used in industry and causes health problems, it can pass through the blood brain barrier accelerating brain tissue degeneration and attenuating brain growth so using of it should be avoided. Exposure to less doses of lead may affect the child´s mental awareness so the findings of the current research reinforce the international efforts to prevent the use of lead in all products that children deal with.
• Preparation of black chia and peppermint oils extracts for human consumption in markets and advising people for regular daily or day after day to prevent oxidative stress and inflammation, retard brain aging, improving memory and learning ability in patients with brain diseases.
• Developing a food product at a lower economic cost and recommended to be applied as nutritional product for children inin pre-school and primart stages future that contains equal quantities of both chia and mint oils to increased their effectiveness, enhanced health benefits and reduced exposure to lead which is considered one of the most important causes of academic delay and learning difficulties.
• Producing awareness brochures that include an introduction to the effects of exposure to lead and how to prevent these harms.