The Review of Certain In Vivo Antioxidant Effects on Essential Oils of Origanum Minutiflorum O Schwarz-Ph Davis , Juniperus Excelsa Bieb . subsp . Excelsa and Histopathologic Changes

Essential oil of plants called Juniperus excelsa Bieb. (JE), Origanum minutiflorum O. Schwarz and P.H. Davis (OM) were used in this study. In order to determine experimental doses, LD50 values of essential oils were determined on mice. Taking into consideration the LD30 range, the experimental toxic doses were calculated for each rat (rat/kg). The toxic dosages thus determined were adapted to rats for active substances (rat/kg). Using commercially available pure virgin olive oil (VOO) as the solvent and diluting agent, OM oil (n=10), JE fruitoil (n=10), carvacrol (CRV) (n=10), VOO (n=10) and normal saline SF (n=8) were administered on the basis of 12 days into intraperitoneal (IP). Enzyme activities of Glucose-6-Phosphate dehydrogenase (G6PDH), malate dehydrogenase (MDH), Superoxide Dismutase (SOD), Glutathione-S-transferase (GST), Adenosine-deaminase (ADA) and Catalase were studied in isolates of kidney, brain and liver tissues. The data was statistically analyzed through Kruskal Wallis variance analysis. Elevated levels of GST and catalase have been found statistically important, as have both essential oil activities of OM and JE in the kidney tissue (p<0.005). All of the enzymes except the levels of ADA and SOD led to a statistically significant change in the brain and liver. There was sinusoidal hyperemia and capsular adhesion in the liver as histopathological were found to be statistically significant (p<0.005). It did not observe any important changes in the other organs. Findings were scored and analyzed by using x2(chi-square) test and Fisher’s definite variance analysis.


IntRoduCtIon
There are about 70 kinds of Juniperus L. (Cupressaceae) in the world.The type of Juniperus is represented by 10 taxons with seven sub types in Turkey.Juniperus excelsa Bieb (gray tall juniper), (Cupressaceae) which is an evergreen and coniferous tree, covers 82% of juniper forests in Turkey.It has disseminated to all countries within Iranian-Turan climate zone.The fruits of this tree are registered as juniper berries in Turkish codex (Demirhan, 2011); and the researches conveyed show that it involves alpha-pinene as active ingredient in its compound (Adams et al., 2013).
The fruits of Juniperus excelsa Bieb (J.E.) are being used as diuretic, stimulant, antiseptic and for treatment of injuries by Anatolians since the ancient ages.It is known that, its extracts bear the effects of being anti-inflammatory, anti-microbial, insecticide, anti-termite, hypotensive, diuretic; and also used as antiseptic for urinal system (Adams 1990a;Adams 1990b;Adams 2011;Demirhan 2001;Adams et al., 2013).Also Çetinus ŞA, Saygin H, Ercan N
Some kinds of Origanum are also used as spice in food, and in medicine among people against indigestion, inappetency and cough.It bears the features such as being antiseptic, sedative, expectorant and cramp healing.It is used in cosmetics, alcoholic or alcohol free beverages.Due to its anti-bacterial effect on bacteria which causes food spoil and food poisoning, it is a most wanted medicinal plant.It is generally known for its rich essential oils, antioxidant, antibacterial, antiviral and antifungal activities (Baser et al., 1993;Davis et al., 1998;Baytop 1999;Baser, 2002;Dadalıoğlu & Evrendilek, 2004;Baydar, 2005;Baser, 2008;Goze et al., 2010;Oke 2010;Kılıçgün & Korkmaz 2014).Owing to this fact, for the natural active ingredients to be defined in plants, which are used for medication or food, it is important to convey researches that examine the biological activity, toxicological and pharmacological dose-response relation.
This study aims to determine the in vivo antioxidant activity of O.M. and J.E. on kidneys which are used orally and pursuit possible tissue changes histopathologically.Although there are many studies about in vitro antioxidant and antibacterial activities of active ingredients of O.M. and J.E., there are not many studies of in vivo.Therefore, the research findings are the first one in this field.Plant samples which were dried in shade and pulverized, were held in Clevenger apparatus for approximately 3 hours being exposed to hydro distillation.The oil yield of 500gr dried plant was determined as 2.1 ml for JE and 10 ml for OM.

MAteRIAl And Method
In the study, 40 healthy 180gr and approximately 4 weeks old adult albino Wistar rats (Rattus Norvegicusvar.Albino) were used; and in order to determine the LD 50 dose, again, 40 healthily 25-30gr, 4 weeks old rats (MusMusculusvar.Albino) were used.The experimental animals were provided from Experimentary Animals Laboratory at Cumhuriyet University.Experimental Animals Ethical Committee's approval was gained (approval date and no: 1/9/2005-116).The animals were preserved under standard laboratory circumstances, with free feed and water at room temperature (21 ± 3 ºC).
During literature searches no studies about OM and JE, LD 50 doses were found.Therefore, LD 50 dose of oils was determined by using rats.The toxic dose was calculated as rat/kg for each and found as LD 30 (for JE -LD 30 :160 ul).The animals which were tried on with Pure OM and pure carvacrol died at all the levels of doses given.Thus, OM and carvacrol (CRV) were mixed into olive oil and a depot/store solution was prepared (1 OM+3 Olive oil); every rat was given 100u (LD 30 ) i.p. from this solution and this dose was determined as LD 30 for OM.In the same way, by applying 100ul to each rat from the store solution which was prepared by using carvacrol (1 carvacrol +6 olive oil), LD 30 value was found for the carvacrol.
This experiment was made on rats.The rats were divided into 5 groups.To the 1 st group of rats JE (160ul), to the 2 nd group only olive oil (40 ul), to the 3 rd group OM (100ul), to the 4 th group carvacrol (100ul) were injected for 12 days with i.p. and 10 rats were used as control group (SF).At the end of 12 days the rats were killed with dislocation and their organs were taken into cooled boxes.
The tissues samples which were sent to a pathology laboratory for routine follow up in automatic tracking device via handon Path Centre (Thermo Electron Corporation, UK).It observed adhesions among the intestine kidney, brain and liver of rats which were given JE in the macroscopic examination.After scored of findings analyzed with X 2 test and precise analysis of variance Fisher (Akgül, 2005).

Results of kidney homogenates
The activities of GST and catalase were found important as a statistics for OM and JE (p<0.005).
The difference between groups was not significant as G 6 PDH, MDH, ADA and SOD enzymes values compared with in pairs (p>0.05)Apart from these, there were significant difference in values (p<0.05).Between groups of OM and CRV, JE and CRV, JE and olive found significant differences to pairwise comparisons are made in terms of value in GST (p<0.005).
When catalase activity compared in pairs of groups OM and CRV, OM and VOO, JE and CRV, JE and olive oil values difference was found statistically significant as shown in Table1 and Table 2.

Results of brain homogenates
The level of G6PDH between groups of VOO /JE and JE/SF (p<0.005); the level of GST between groups of JE-CRV (p<0.005); the level of MDH between groups of OM-JE and CRV-JE (p<0.005); the level of catalase between groups of OM-VOO and JE-VOO-SF, CRV-JE and SF were found important if statistically compared in pairs (Table 2).

Results of liver homogenates
The activity of G6PDH in between groups of OM-SF and JE-VOO and CRV/VOO/SF; the activity of GST between groups of JE-CRV and JE-VOO and JE-SF; the activity of MDH between groups of OM and VOO, the activity of catalase between groups of OM/VOO and SF were found statistically important.The enzymes of G6PDH, MDH, ADA and SOD did not show significant differences between the groups compared in pairs (p>0.005)(Table3).

Table1 -Analyzes of enzymes activities in kidney homogenates
In histopathological examination, a finding was not seen in kidney tubule epithelia cell except cloudy swelling (p>0.05).After scored of findings analyzed Çetinus ŞA, Saygin H, Ercan N

The Review of Certain In Vivo Antioxidant Effects on Essential Oils of Origanum Minutiflorum O Schwarz-Ph Davis, Juniperus Excelsa Bieb.subsp. Excelsa and Histopathologic Changes
with X 2 test and precise analysis of variance Fisher.Fuzzy swelling (FS) in surface of tissue sample according to where tissue is turned off as semi quantitative (%), focal necrosis (FN) and the group of cell necrosis as a number (present or absent), Kupffer cell hyperplasia (present or absent), periportal inflammation (present or absent), sinusoidal hyperemia (SHR) (present or absent) were evaluated (Table 4).The sinusoidal hyperemia (SH) and capsular adhesion (CA) in liver were found statistically important (p<0.05)(Figure1).There are not significant changes which in other organs were observed.dISCuSSIon Serum enzyme activities are widely used in determining of organ functions in mammals.Also these are very important for environmental biology, searching for the effects of environmental pollutants on living organisms, the toxic effects of orally consumed plants and for bioavailability in recent years.These tests have been of great importance in recent years, particularly in the context of bioavailability, to explore the aquatic effects of environmental biology and environmental pollutants and the toxic effects of orally consumed plants.
The changes that may occur in the activities of these important enzymes working in the energy metabolic pathways may affect the formation of energy by the   Superoxide dismutase (SOD), catalase (CAT), Glutathione reductase (GST) and malate dehydrogenase (MDH) are enzymatic compounds.Superoxide dismutase (EC 1.15.1.1,EC-SOD), as a superoxide anion is a form that part of the cellular antioxidant defense system by catalyzing the conversion in hydrogen peroxide to molecular oxygen.It plays an important role in controlling cellular superoxide levels in the compartment.It consists in high concentrations in the brain, liver, heart, kidney and erythrocytes (Fridovich, 1989).
In this study, SOD was found stable in all of the tissues.The mature erythrocytes don't include ribosome, mitochondria and nucleus.Enzymes of SOD, catalase, GSH-Px and pentose phosphate pathway protected against O 2 radicals.It can consider that OM and JE don't cause inhibition of the metabolic pathway.
Glucose-6-phosphate dehydrogenase (G-6-PD) (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, E.C. 1.1.1.49)is the first and control enzyme of the pentose phosphate pathway.G 6 PHD; it is an enzyme that plays a key role in the pentose, phosphate and glycolysis (Scott 1975).Also it has an anti-oxidant activity.G 6 PDH enzyme activity was found to decrease in homogenates of all tissues.According to these results, it can be observed that essential oils may generate inhibiting change.
Adenosine deaminase is purine metabolism enzyme which is responsible for immune mechanism (Wilson et al., 1991).Significant changes weren't observed in ADA activity in all organs except for the brain.In this case, the tested substances aren't effective in purine metabolism and suggest no creating damage.
MDH; is taken office in the maintenance of cell membrane integrity and the regulation of enzyme activity (Musrati et al., 1998).In this research, the levels of MDH was high in the liver and in the brain but was stabile in the kidney.
GST, glutathione plays an important role against sourced interior and exterior toxic chemicals in the cellular defense system (Armstrong, 1990).Reversible damage may have been created during the period of JE and OM application by various mechanisms in all tissues.In this case, it can be considered to change their enzyme activations to maintain the redox state of cells and especially repairing damage membranes by keeping to a minimum damage.It is very important for all of tissues.Catalase (H2O2: H2O2 oxidoreductase, EC 1.11.1.6)has 4 units with a group hemoprotein and localized in intracellular organelles as peroxisomes.Its task is to destroy hydrogen peroxide to oxygen and water (Kono & Fridovich, 1982).This is quite low compared to the catalase activity in the kidneys of the control group that can cause essential oils erythrocyte catalase activity by inhibiting the accumulation of H 2 O 2 and consequently hemolysis which is very important for all of tissues.
As a result, the activity of catalase was very meaningful in all tissues while the level of SOD was stable.The levels of GST and catalase were in kidney, ADA and SOD were important in liver.Statistical differences were found in all enzymes except for SOD.It can be observed that these types of plants consumed as a food of toxic effects should have more comprehensive studies.Additionally, the LD value for each study which uses the plant´s essential oils, need to be determinate.Because the composition of the plant essential oils vary depending on many reasons such as seasonal, droughts and temperature difference.

ACknowledge
This study was supported by The Scientific Research Project Fund of Cumhuriyet University under project number SHMYO-5.
JE fruit and OM were harvested from Isparta, Sütçüler flora.The samples of the plant were registered under JE (CUFH Voucher No. ED11003) and OM (CUFH Voucher No. ED 1101) numbers at the Herbarium of Cumhuriyet University Faculty of Science and Literature Department of Biology.

Figure1-
Figure1 -A general view of adhesions in the liver capsule (HEx40).

The Review of Certain In Vivo Antioxidant Effects on Essential Oils of Origanum Minutiflorum O Schwarz- Ph Davis, Juniperus Excelsa Bieb.subsp. Excelsa and Histopathologic Changes per
1 gram.The homogenates were centrifuged (Beckman Model J2) for 15 min at 4800 rpm.Glucose 6 phosphate dehydrogenase (G 6 PDH),

Table 3 -
Analyzes of enzymes activities in liver homogenates GST U/mg pro; MDH U/mg pro; ADA U/mg pro; SOD U/g pro; CATALASE U/mg pro