PROTECTIVE EFFECT OF EVENING PRIMROSE OIL AND EXTRA … · Evening primrose oil has a high concentration of polyunsaturated fatty acids, and has been used for more than 30 years as

JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 2.417, ISSN: 2320-5083, Volume 4, Issue 5, June 2016

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PROTECTIVE EFFECT OF EVENING PRIMROSE OIL AND EXTRA VIRGIN OLIVE OIL AGAINST AUTOIMMUNE HEPATIC INFLAMMATION

BASANT M. MORSY*

*Biochemistry Division, Dept. of Chemistry, Faculty of science, Beni-Suef University, Egypt

ABSTRACT

The evening primrose plant (OenotherabiennisL.) is commonly known as tree

primrose and sun drop.Evening primrose oil (EPO) has been used to treat a variety of

ailments.It is classified as a "dietary supplement" under the Dietary Supplement Health and

Education Act of 1994.These wide uses related to its high content of omega-6 (n-

6)polyunsaturated fatty acids specially γ-linolenic acid (GLA) also beneficial effects of Extra

Virgin Olive oil (EVOO) are not only related to its high content of oleic acid, but also to the

antioxidant potential of its polyphenols. Also Extra Virgin Olive Oil (EVOO) is the primary

source of fat in the Mediterranean diet.The current study performed to evaluate the effect of

EPO and EVOO on acute hepatic inflammation results from CFA administration. Also we

summarize recent findings emphasizing the role of main inflammatory markers and oxidative

stress in acute hepatic inflammation case. Hepatic inflammation was inducted by a single

dose of CFA (100 µl), injected subcutaneously. These rats received EPO and EVOO through

gastric intubation daily for 21 days after CFA injection. The current results revealed that EPO

and EVOO treatment ameliorated significantly the elevated levels of the hepatic cytokines

which elevated as a result to CFA injection. Moreover, EPO and EVOO treatment

ameliorated the non-enzymatic antioxidant, liver malondialdehyde and glutathione (GSH)

concentration and the enzymatic antioxidant, liver catalase, supper oxide dismutase and liver

GSH-peroxidase activities.

KEYWORDS: CFA; Primrose oil; Olive oil; Liver Injury; Inflammation; Oxidative Stress.

1- INTRODUCTION:

The incomplete Freund adjuvant (IFA) and complete Freund adjuvant (CFA), which is heat-

killed Mycobacterium tuberculosis bacilli (Mtb) dissolved in IFA. (Billiau and Matthys,

2001). This killed mycobacteria contain various pathogen-associated molecular patterns

including toll-like receptor 2,4 and 9 agonists (Akira and Takeuchi, 2006).These adjuvants

have been used extensively to establish experimental animal models of autoimmune diseases,

e.g. experimental autoimmune encephalitis (EAE), neuritis (EAN), uveitis (EAU), thyroiditis


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(EAT) and orchitis, and to produce antibodies (Billiau and Matthys, 2001).Although liver is

the primary target organ in host–microbe interaction and a major response organ in systemic

inflammation as it is highly responsive to microbial toxins. Studies on the effects of Freund

adjuvants on the liver have been largely neglected. The liver is regarded as a privileged site

of immune tolerance. The disruption of immune tolerance in the liver can lead to immune-

mediated liver diseases (Kumikoet al., 2015).Autoimmune hepatitis (AIH) is increasingly

recognized as a liver-specific autoimmune disease that results, at least in part, from the

perturbation of immune tolerance in the liver (Czaja and Manns, 2010). The disease is

characterized histologically by chronic infiltration of inflammatory cells in the liver and the

de- struction of hepatocytes. AIH is characterized clinically as a chronic liver disorder and

sometimes as an acute onset and severe liver disorder. AIH can lead to liver cirrhosis and

hepatocellular carcinoma, which are often indications for liver transplantation. However, the

pathogenesis of this disease remains poorly understood (Kumikoet al., 2015).Progressive

systemic TNF-α, IL-1β, IL-6 and NO increase the generation of reactive oxygen species

(ROS) and reactive nitrogen species (RNS), and decrease the activities of antioxidant

enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in tissues.

These consequent events further aggravate protein and lipid oxidation, and injury tissues.

Thus, oxidative damage of the vital organs, particularly the liver, is considered as secondary

complications of arthritis (Sundaramet al., 2014). It is observed that the release of hepatic

transaminases such as glutamic oxaloacetic transaminase (GOT) and glutamate-pyruvate

transaminase (GPT) into the blood was found in AIA-mediated liver damage (Comaret al.,

2013 and Shusonget al., 2015).

Olive oil is an integral ingredient in the Mediterranean diet. There is growing

evidence that it may have great health benefits including the reduction in coronary heart

disease risk, the prevention of some cancers and the modification of immune and

inflammatory responses. Olive oil, a widely applied omega-9 enriched dietary lipid, has

attracted much interest in its effects against liver injuries (Tanaka et al., 2009 and Hualinet

al., 2014).

Evening primrose oil has a high concentration of polyunsaturated fatty acids, and has

been used for more than 30 years as a dietary supplement. Simultaneously there were many

reports published about the antitumor and immunotropic activity of evening primrose oil

(Guanget al., 2013).


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Therefore the aim of this study was to evaluate the protective effect of Evening primrose

oil and extra virgin olive oil against hepatic inflammation in rheumatic rats.

2- Materials and methods:

2.1. Experimental animals:

White male albino rats (Rattusnorvegicus) weighing between 100 g and 120 g were

used as experimental animals in the present investigation. They were obtained from the

animal house of National Research Institute, El-Giza, Egypt. They were kept under

observation for about 15 days before the onset of the experiment to exclude any inter current

infection. The chosen animals were housed in metal (stainless steel) separate bottom cages at

normal atmospheric temperature (25±5°C) as well as under good ventilation and received

water and standard balanced diet. All the procedures were performed in accordance with the

Institutional Animal Ethics Committee in Beni-Suef University recommendations.

2.2. Chemicals and oils:

Complete Freund adjuvant (CFA), evening primrose oil and extra virgin olive oil

were purchased from Sigma Chemical Company, St. Louis, MO, USA. They were stored at

2- 4ºC and protected from sun light. The dose selection for each compound was based on

previously published studies.

2.3. Doses and Treatment:

A single dose of CFA used in this study was (100 µl), injected subcutaneously

(Liviaet al., 2011). Evening primrose oil (EPO) and Extra virgin olive oil (EVOO) were

administered to male albino rats by gastric intubation at dose 5 mg / kg. b.w. / day (Silva et

al., 2014 and Nakbiet al., 2010 respectively) the treatment began on the day of CFA injection

and continued daily up to the 21th day after arthritis induction.

2.4. Experimental design:

The number of rats used in the present study is 24. They were allocated into 4 groups

designed as follow:

Group 1: This group was regarded as control group and given distilled water by gastric

intubation for 21 days

Group 2: It was given single dose of CFA (100 µl)injected subcutaneously in male rats.

Group 3: The rats of this group were administered single dose of CFA (100 µl) at beginning of

the experiment and were also treated with evening primrose oil (EPO) (5 mg /kg b. w. /day), by

gastric intubation for 21 days.


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Group 4:

The rats of this group were administered single dose of CFA (100 µl) at beginning of the

experiment and were also treated with extra virgin olive oil(EVOO) (5 mg /kg b. w. /day), by

gastric intubation for 21 days.

At the end of 21 days, six animals of normal, CFA-administered control rats and CFA-

administered rats treated with EPO and EVOO were sacrificed under mild diethyl ether

anesthesia. Liver from each animal was rapidly excised after dissection. 0.5g was

homogenized in 5ml 0.9% sterilized NaCl (10% w/v) using Teflon homogenizer (Glas-Col,

Terre Haute, USA).

2.5. Biochemical analyses:

Hepatic Tumor necrosis factor-α (TNF-α) was measured according to the method of

Seckingeret al., (1998), Lantezet al., (1990) and Engelmann et al., (1990).Interleukins 6 and

1β were determined by the method of Dinarello, (1990) and Herzyket al., (1992) by using

specific ELISA kits Quantikine Rat Total Adeponectin Immunoassay (USA). Liver lipid

peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS)

according to the method of Preusset al., (1998). Catalase activity determined according to

Cohen et al., (1970) which follows the first-order kinetics as given by the equation: K = log

(S0 / S3) X 2.3 / t. The SOD activity was measured in liver homogenate according to the

method described by Marklund and Marklund,(1984).Liver glutathione (GSH) was

determined according to the method of Beulteret al., (1963) and finally Glutathione

peroxidase determined according Matkovicset al., (1998).

2.6. Statistical analysis of the results

The Statistical Package for the Social Sciences (IBM SPSS for WINDOWS7, version 20;

SPSS Inc, Chicago) was used for the statistical analysis. Comparative analysis was conducted

by using the general linear models procedure (IBM SPSS). p>0.05 were considered

statistically non significant, while p<0.05 were considered statistically significant.

3- Results:

Data summarized in Table 1 and figures 1, 2 and 3 show the effect of CFA administration

and treatment with EPO and EVOO on hepatic cytokines, TNF-α,IL-6 and IL-1β markers

.our results revealed that the administration of CFA produced marked impairment

demonstrated by significant increase in these hepatic inflammatory factors as compared to

normal rats, while Oral administration of primrose oil and olive oil significantly decreased

these elevated levels of Hepatic TNF-α,IL-6 and IL-1β when compared with the CFA-

administered rats recording noticeable amelioration as compared to normal ones .


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Table 2 and figures 5-8 shows the effect of the tested evening primrose oil (EPO) and extra

virgin olive oil (EVOO) on the liver oxidative stress markers and antioxidant defense system

of CFA-administered rats. Hepatic lipid peroxidation (LPO) product was significantly

increased as a result of CFA administration while the treatment of CFA-administered rats

with EPO and EVOO for 21 days significantly decreased these elevated values and becomes

near to those of normal values. On the other hand, rats injected with CFA exhibited a

noticeable decrease in values of catalase (CAT) activity, supper oxide dismutase (SOD)

activity, total glutathione content (GSH) and glutathione peroxidase (GPX) level as compared

to normal rats. while, the treatment with EPO and EVOO after CFA administration produced

a significant increase of these oxidative stress values as compared to the corresponding CFA

administered group pointing to a marked normalization as compared to normal group.

Table 1: Effect of evening primrose oil and extra virgin olive oil on hepatic cytokines, TNF-α, IL-6 and IL-1βactivities in CFA-administered rats.

*Values significantly different to control at (p≤0.05). *Data are expressed as mean ± SE.

*Values which share the same superscript symbol are not significantly different. *F-Probability: P < 0.05

Interleukin- 1β

IL-1β (pg/ml)

Interleukin6

IL-6 (pg/ml)

Tumor Necrosis Factor-α

TNF-α(pg/ml)

Parameter

Groups

28.74±1.29 a 33.47±1.48 a 31.29±1.01a Normal

114.29±1.35 d 124.45±1.24 c 122.12±1.43d CFA

75.78±0.90 c 58.55±1.44 b

52.92±1.93 c Evening Primrose Oil

( EPO)

63.23±1.57 b 49.64±2.06 b

60.06±1.22 b Extra virgin olive oil

(EVOO)

1.84 2.24 2.42 LSD value at 0.05


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Table 2: Effect of evening primrose oil and extra virgin olive oil on hepatic oxidative stress in CFA-administered rats.

Glutathione

Peroxidase

(U/g)

Total

glutathione

content

(n mole/mg)

Super oxide

dismutase

(n mole/mg)

Catalase

(u/g.tissue)

Lipid

peroxidation

(n mole/mg)

Parameter

Groups

43.69±1.81 c 54.64±0.5 d 2.70±0.11 c 121.08±1.07 d 1.49±0.08 a Normal

8.82±0.64 a 22.81±0.97 a 0.28±0.01 a 64.25±1.18 a 17.44±0.93 d CFA

30.39±0.98 b 41.49±0.98 b 1.35±0.1 b 108.69±1.24 c 5.64±0.26 c Evening Primrose

Oil ( EPO)

28.75±0.48 b 44.39±1.09 c 1.62±0.13 b 97.71±1.1 b 3.56±0.2 b Extra virgin olive

oil (EVOO)

1.56 1.29 0.14 1.62 0.7 LSD value at 0.05

*Values significantly different to control at (p≤0.05). *Data are expressed as mean ± SE.

*Values which share the same superscript symbol are not significantly different. *F-Probability: P < 0.05

Fig. 1: The effect of primrose oil and olive oil administration on hepatic Tumor Necrosis Factor-α (TNF–α)

in liver inflammated rats

Fig. 2: The effect of primrose oil and olive oil administration on hepatic interleukine -6 in liver inflammated

rats


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Fig. 3: The effect of primrose oil and olive oil administration on on hepatic interleukine-1β in liver

inflammated rats

Fig. 4: The effect of primrose oil and olive oil administration on hepatic lipid peroxidation in liver

inflammated rats

Fig. 5: The effect of primrose oil and olive oil administration on on hepatic Catalase Activity in liver

inflammated rats


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Fig. 6: The effect of primrose oil and olive oil administration on on hepatic supper oxide dimutase Activity in

liver inflammated rats

Fig. 7: The effect of primrose oil and olive oil administration on on hepatic total glutathione content in

liver inflammated rats

Fig. 8: The effect of primrose oil and olive oil `administration on on hepatic glutathione peroxidase activity

in liver inflammated rats


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Discussion

The liver has a unique endothelium that consists of fenestrated endothelial cells lining

hepatic sinusoids, allowing hepatocytes to contact immune cells directly. The sinusoids

contain endothelial cells, Kupffer cells (resident macrophages), stellate cells (important in

remodeling and fibrosis) and intrahepatic lymphocytes. The liver plays a critical role in first-

line host defense against incoming foreign antigens absorbed through the gut into the portal

venous system. It must maintain a balance between tolerance to incoming antigens and

generation of an immune response. Tolerance is essential to avoid food allergy and explains

graft survival of liver transplants across major histocompatibility complex (MHC) antigen

differences. Loss of tolerance or a hyper immune response may lead to autoimmunity

(Marion, 2002).

Freund adjuvants are used extensively to establish experimental animal models of

autoimmune diseases and to produce antibodies. However, studies on their mechanisms of

action have been largely neglected, particularly their effects on liver, the primary target organ

for host–microbe interaction (Sai-Kiang Lim, 2003).

Our results obtained in the present work can be discussed in two main aspects: first,

the development of autoimmune hepatic inflammatory response and metabolic changes in the

liver of animals induced by complete Freund adjuvant (CFA); second, the effects of EPO and

EVOO against these inflammatory processes in the liver of induced rats. According to the

first aspect, it was possible to observe that the injection of CFA at the dose of 100 µg of

Mycobacterium tuberculosis induce autoimmune hepatic inflammation. These results run

parallel to those of Sai-Kiang Lim.,(2003) whose reported that Freund adjuvant induced a 5–

10-fold increase in toll-like receptor (TLR) 2 mRNA but not TLR4 mRNA in livers of mice.

Since CFA is essentially made of killed Mycobacterium tuberculosis bacilli (Mtb) dissolved

in Incomplete Freund adjuvant (IFA), it is the solvent in CFA that induced an increase in

TLR2 expression. As TLR2 is the receptor activated by killed Mtb, this solvent-mediated

increase in TLR2 expression will result in enhanced recognition of killed Mtb by hepatocytes

during CFA administration. We propose that the potency of Freund adjuvant in eliciting an

immune response lies in their ability to induce expression of the appropriate TLR, TLR2, for

the active ingredient, killed Mtb, in CFA. Hence, adjuvant administration has been known to

stimulate innate immunity, induce expression of cytokines, enhance phagocytosis and trigger

autoimmune-like diseases (Billiau and Matthys, 2001). Consequently, the inflammatory

indicators as TNF-α, IL-1β, IL-6 were increased significantly in the liver of CFA-induced


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rats, and then oral administration with EPO and EVOO reduced their accumulation in a dose-

dependent manner, these results are in accordance with Shusong et al,.(2015).

The liver resembles a central organ of cytokine activity due to the fact that it hosts

hepatocytes, which are highly susceptible to the activity of cytokines in a variety of

physiological and pathophysiological processes. Moreover, the non-parenchymal cells of the

liver, in particular Kupffer cells (KCs), the resident tissue macrophages of the liver, are able

to synthesize a variety of cytokines that may act systemically on any other organ of the body,

or in a paracrine manner on hepatocytes and other non-parenchymal liver cells. (Giuliano and

Thomas, 2001).In the last few years, it has been suggested that in most cases hepatocellular

injury is due not to the damaging agent itself but to the inflammatory cells that have been

attracted by the stressed hepatocytes. In contrast to sustained hepatocellular damage, acute

hepatitis is a temporary event that finishes with normal liver histology and function (restitutio

ad integrum). In fact, hepatotoxins (drugs, infectious agents) may induce a stress situation in

hepatocytes with subsequent release of chemokines followed by accumulation of

inflammatory cells and subsequent hepatocellular damage (Fig. 9)(Diehl, 2000). Hepatotropic

infectious agents such as viruses or activated T-cells may act in a similar manner (Tiegs,

1997) and induce accumulation of inflammatory cells that kill hepatocytes (Arii and

Imamura, 2000).

Fig 9.Acute liver injury.(Diehl, 2000)


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Hepatocellular stress may activate resident liver macrophages. Moreover, KCs can be

activated directly, e.g. by binding of endotoxins via the CD14 receptor. Proinflammatory

cytokines such as IL-1α, IL-1β and TNF-α are released from KCs, which induces cell

adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on sinusoidal

endothelial cells involved in the recruitment of inflammatory cells, such as blood monocytes

(Neubaueret al., 1998). Moreover, pro-inflammatory cytokines may be released from

mesenchymal liver cells. In fact, it is debated whether mononuclear phagocytes of the liver

are the main source of pro-inflammatory cytokines. Further induction of chemokines

amplifies the inflammatory cascade (Marra et al., 1998) Deteriorated hepatocytes may then

be removed by mononuclear phagocytes. The classical hypothesis that toxic liver injury is

permitted by hepatocellular death and subsequent attraction of inflammatory cells, the latter

removing dead hepatocytes, has come into debate, since newer data have shown that ICAM-

1, which is crucial for immigration of inflammatory cells into the liver tissue, may be

expressed from sinusoidal cells before the appearance of necrotic hepatocytes in the rat

model of carbon tetrachloride-induced acute liver injury (Neubaueret al., 1998). TNF- α

released early after carbon tetrachloride intoxication participates in the down-regulation of

platelet endothelial cell adhesion molecule 1, which may represent an important event in the

sinusoidal transmigration of inflammatory cells (Neubaueret al., 2000). According to all these

findings we can say Cytokines plays a critical role in many aspects of immune system

development, immune response regulation, and T cell-mediated tissue injury specially TNFα

that has both proinflammatory and immunoregulatory properties. Also it considered as a

critical growth factor for thymocytes and plays an important role in the peripheral immune

system in antigen-presenting cell function and in regulating apoptosis of potentially

autoreactive T cells. It may also foster tissue regeneration in the liver (Diehl , 2000).

Our results exhibited a marked amelioration in hepatic cytokines (TNF-α, IL-6 and

IL-1β) as a result of EPO and EVOO administration; referring to their highly potent immune-

modulatory and anti-inflammatory effects. These results are in agreement with those of (Yan

et al., 2013; Sekhon-Looduet al., 2014 and Claudio et al., 2015). Beneficial effects for EPO

have been reported in several diseases as eczema, asthma, rheumatoid arthritis, breast

problem, fibromyalgia syndrome, premenstrual, menopausal syndrome, diabetic neuropathy,

mastalgia, atopic dermatitis, several autoimmune conditions and gastrointestinal

symptoms(Rodgers et al., 2009; Ola and Omran, 2012). EPO is a rich source of the ω-6


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essential fatty acid, linoleic acid, and γ-linolenic acid (GLA), precursors of the series-1

prostaglandins (FIG. 10).

N-6 FATTY ACIDS Linolenic Acid (LA)

Vegetable oils

γ -Linolenic Acid (GLA) human milk

evening primrose oil

DIHOMOGAMMA-LINOLENICACID (DHLA)

ARACHIDONIC ACID PG 2 Series , LT 4 Series

LONG CHAIN FATTY ACIDS

FIG. 10 long chain fatty acids in evening primrose oil. (Jennifer, 2003).

Essential fatty acids are incorporated into cell membranes where they play a vital role in the

structure of cell membranes, influencing membrane flexibility, fluidity, and the behavior of

membrane-bound proteins. Essential fatty acids serve as a source of eicosanoids.

Consumption of GLA (18:3 ω-6) favors an increase in the dihomogammalinolenic acid

(DGLA) content of cell membranes without a corresponding increase the arachidonic acid

concentration (Fan andChapkin, 1998). Ingestion of EPO elevates concentrations of DGLA

(20:3 ω-6), enhancing production of eicosanoids of the prostaglandin 1 series (PG1). In

addition, DGLA, which itself cannot be converted to leukotrienes, can form a 15-hydroxyl

derivative that blocks the transformation of arachidonic acid to leukotrienes. Increased

DGLA may act as a competitive inhibitor of the proinflammatory eicosanoids, prostaglandin

2 and leukotriene 4 series, which promotes TNF-α, IL-6 and IL-1β production (Cuiyinget

al.,2016) are produced from arachidonic acid (20:4 ω-6) (see Figure10). Membranes rich in

DGLA favor formation of the prostaglandin 1 series of eicosanoids and reduce leukotriene

synthesis. Membranes rich in DGLA favor a less inflammatory state. On stimulation, DGLA

can be converted by inflammatory cells into compounds that possess both anti-inflammatory

and anti-proliferative properties. Chronic inflammation is reduced by suppression of T

lymphocytes. EPO is also used to correct an ω-6 fatty acid deficiency (Jennifer, 2003).


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Linoleic acid GLA DGLA AA

PG1 PG2 LT4

Fig. 11 Omega-6 eicosanoids.(Jennifer, 2003)

The olive fruit and virgin olive oil are essential components of the Mediterranean diet, a

nutritional regimen gaining ever-increasing recognition for its beneficial effects on human

health (Urpi-Sardaet al., 2012 and Escrichet al., 2011). There is some evidence that the

effects of olive oil on immune function in animal studies are due to high content of oleic acid,

but there is also growing evidence that The high content of phenolic compounds in virgin

olive oil have demonstrated anti-inflammatory, antioxidant and antineoplastic activities

(Procopioet al., 2009 and Yaqoob, 2013). Among these, hydroxytyrosol (HT), which is

abundant in the aqueous fraction of olive pulp, is a simple phenolic compound with marked

antioxidant activity (Jemai et al., 2009 and Tutino et al., 2012).

Silvaet al., reported thathydroxytyrosol has the most bioavailability in olive oil

components (Silvaet al., 2014). Olive oil phenolic compounds are mainly absorbed in the

small intestine (Visserset al., 2002) and therefore the increase of hydroxytyrosol

bioavailability, in olive oil, might be related to the rate of gastric emptying and slow release

of hydroxytyrosol from the oil matrix (Gonzalezet al., 2010) Moreover hydroxytyrosol is

protected from degradation in the gastrointestinal tract, before absorption, due to the presence

of other antioxidants, in olive oil, thus improving bioavailability (Tucket al., 2001).

Claudio et al., observed that HT not only increases PPAR-α levels, but also restores the

expression of its downstream regulated gene FGF2, a cytokine/hormone, in the liver (Claudio

et al., 2015). It was found that PPAR-α is an important regulator of inflammatory process

(Gervois and Mansouri, 2012). The anti-inflammatory effects of all classes of PPARs rely

primarily on genomic mechanisms of transrepression of transcriptional factors ,i.e. nuclear

transcription factor-kB( NF-kB), which induce the reduction of pro-inflammatory cytokines

and enzymes (Wahli and Michalik, 2012). Therefore, similarly to PPAR-γ, it is conceivable

that PPAR-α activity could modulate metabolic disorders associated with inflammation either

through its metabolic activity or its anti-inflammatory effects. Recently, the current

knowledge concerning the main biological properties of HT was summarized, including its

antinflammatory effects (Granados et al., 2010) Here, we demonstrated that HT significantly

reduces the expression of inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the liver,


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confirming its anti-inflammatory activity, likely associated to its ability in controlling

metabolic alterations through PPAR- α recover.

Antioxidants are substances that either directly or indirectly protects cells against adverse

effects of xenobiotics, drugs, carcinogens and toxic radical reactions (Kamesh and Sumathi,

2012). Oxidative stress results from an imbalance between the cellular production of reactive

oxygen species (ROS) and the antioxidant mechanisms that remove them(Posadas et al.,

2009). Reactive oxygen species are different forms of activated oxygen inevitably produced

in living organisms as by-products of regular metabolism or from external sources. These

oxygen forms can easily react with most biological molecules including proteins, lipids,

lipoproteins and nucleic acids, and cause progressive decline in cell function resulting in a

variety of pathophysiological disorders (Botsoglouet al., 2010).

Liver is the primary site of drug metabolism and has one of the highest antioxidant

enzyme capacities in the body. These antioxidant enzymes limit the effects of oxidant

molecules on tissues and are active in the defense against oxidative cell injury thanks to the

fact that they are free radical scavengers (Nakbiet al., 2010).Hence, our study can suggest

that the significant decrease of the antioxidant enzyme activities and the marked increment of

MDA contents in the liver proved the failure of antioxidant defense system to overcome the

influx of ROS generated by CFA administration which result in increased oxidative stress,

protein damage and lipid damage. Theseresults run parallel with Amandaet al.,

(2016).Moreover, the oral administration of EPO and EVOO recovered the activities of

hepatic antioxidant enzymes (CAT, SOD and GPx), and reduced the product of lipid

peroxidation (e.g.TBARS) and total glutathione content, which may have resulted from the

stabilization of plasma membrane as well as the repair of the hepatic tissue damage induced

by CFA which result in increased oxidative stress, protein damage and lipid damage. These

results are in agreement with those of Shahidi and Ambigaipalan, (2015) and Kasdallah et

al.,(2008).Evening primrose contain three major low molecular-weight phenolic compounds,

namely (+) catechin, (−) epicatechin, and gallic acid (Wettasinghe et al., 2002). Shahidi

(2000) reported that the stronger antioxidant activity of evening primrose may be attributed to

its tannin components. More recent studies have suggested that the antioxidant properties of

evening primrose may arise from phenolic acids such as gallic, caffeic,p-hydroxybenzoic,

vanillic, ferulic and salicylic acids, as well as proanthocyanidins and flavanols(Puri, 2004),

catechin and epicatechin derivatives (Wettasinghe and Shahidi, 2002) or protocatechuic and

gallic acids and esters (Peschel et al.,2007). Salicylic, p-hydroxybenzoic, 2-hydroxy-4-


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methoxybenzoic, vanillic, m- and p-coumaric, gallic, ferulic and caffeic acids are found in

smaller quantities in evening primrose seeds (Ribas-Agustiet al.,2011). Muratet al., 2011

reported that theses phenolic constetuents result in a tendency to bind to free radicals.

Furthermore, essential fatty acids inhibitory effect on certain enzymes (i.e. cyclooxygenase

and elastase), which trigger the generation of free radicals in the organism, by means of

competitive binding to the enzymes, and its strengthening the glutathione-dependent

antioxidant defense system, could be mentioned to explain the mode of action of evening

primrose oil(Hamburger et al., 2002 and Puri, 2004).Kasdallahet al., showed that the oral

supplementation of olive oil to rats administered ethanol chronically restored damage caused

to the liver by inhibiting lipid peroxidation and improving enzymatic activities (Kasdallah et

al., 2008). The mechanism proposed to explain the positive effects of olive oil may be

attributed to its richness in MUFA, mainly oleic acid which has different effects on lipid

profiles and peroxidation in hepatic mitochondria (Ochoaet al., 2001). Indeed, EVOO

contains a considerable amount of phenolic compounds as oleuropein, hydroxytyrosol,

tyrosol and caffeic acid (Fig. 12), which all have potent inhibition effects against ROS

(Owenet al., 2000 and Fenget al., 2008).Hydroxytyrosol is highly effective against DNA

damage by peroxynitrite in vitro. Caffeic acid phenethyl ester and its related compounds limit

the functional alterations of the isolated mouse brain and liver mitochondria submitted to in

vitro anoxia-reoxygenation(Fenget al., 2008).

Fig12. Chemical structures of some phenolics found in olive oil.(Shahidi and Ambigaipalan, (2015)

Mitochondrial impairment causes enhanced ROS production, which in turn self-

sustains organelle damage. In particular, products of cellular lipid peroxidation (i.e. MDA)

associated to inflammatory cytokines (i.e. TNF- α), contribute to mitochondrial dysfunction

by interfering with mitochondrial respiratory chain and by forming superoxide anion


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(Sanchezet al., 2000). Indirectly, TNF-α promotes mitochondrial dysfunction, increasing

RNS as consequence of iNOS induction (Wuet al., 2009). For this reason, the antioxidant

activity of hydroxytyrosol (HT) has been well examined. HT significantly reduces ROS

production, MDA levels and protein nitrosylation. Our data are in agreement with (Claudio et

al., 2015and Zhenget al., 2015), where, HT improves mitochondrial function and reduce

oxidative stress potentially through activation of the activated protein kinase (AMPK)

pathway in the brain. In particular, AMPK induces the phosphorylation of acetyl CoA

carboxylase (ACC), a modification that inactivates the enzyme, reducing the formation of

malonyl-CoA. The decrease in malonyl-CoA synthesis, in turn, reduces liver carnitine

palmitoyltransferase1 (CPT1) inhibition, thus prompting adequate shutting of fatty acids into

the mitochondria and hence their oxidation (Aguilera et al., 2008). Here, we show that HT

increases the phosphorylation of ACC and the transcription of liver CPT1, both remarkably

reduced by EVOO. The restoration of CPT1 was consistent with the normalization of its

transcriptional regulatory factor PPAR-α. Our results are in agreement with a previous study

(Alberdiet al., 2013).

Conclusion:

EPO and EVOO administration exhibited a beneficial therapeutic effect on rats with

hepatic inflammation due to the presence of omega-3 Fatty acids and polyphenols, which

showed anti-inflammatory and antioxidant action.

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PROTECTIVE EFFECT OF EVENING PRIMROSE OIL AND EXTRA … · Evening primrose oil has a high concentration of polyunsaturated fatty acids, and has been used for more than 30 years as