A Powerful ELISA Technique to Test The Potential of Extra Virgin Olive Oil in Reducing TNF- Level and Edema Volume in Male Rattus norvegicus Exposed to Carrageenan Andina Putri Aulia1.
Suprapto Maat2.
Aryati3 Department of Clinical Pathology.
Faculty of Medicine.
Universitas Islam Sultan Agung.
Semarang.
Indonesia Department of Medical Laboratory Technology.
Faculty of Health.
Universitas Nahdlatul Ulama Surabaya.
Surabaya.
Indonesia Department of Clinical Pathology.
Faculty of Medicine.
Universitas Airlangga.
Surabaya.
Indonesia Correspondence:
Andina Putri Aulia.
Jl.
Kaligawe Raya Km.
4 Semarang.
Central Java.
Indonesia Zip Code : 50112 Email: auliaputri.
dr@unissula.
Received: November 30, 2020 Revised: March 3, 2020 Accepted: March 31, 2020 Abstract Extra virgin Olive oil is extracted from fruit that can be used as anti-inflammatory agent.
This research aimed to test the potential of extra virgin olive oil in reducing edema volume and TNF- plasma in carrageenan-induced rats.
This research was purely experimental research with the post test control group design.
A total of twenty eight Wistar rats were divided randomly into four treatment groups.
Group I was a control negative group, while the group II, i, and IV were orally administered with extra virgin Olive oil at the dose of 0.
7 mL/day, respectively.
Paw edema was measured one hour before the rats was induced to carrageenan and every hour until four hours after it was induced to carrageenan.
TNF- plasma was measured at four Analysis of the data was done by calculating the presentation of edema inhibition in every group, then the data was statistically analyzed by Anova.
Repeated Anova.
LSD and Kruskal Wallis test with 95% confidence interval.
The result showed that extra virgin olive oil has an antiinflammatory effect.
The highest decrease in edema volume percentage was in group i .
21%).
There was a significant difference in the edema volume of all treatment groups at each time of the experiment with TNF- value .
< 0.
In conclusion, the administration of extra virgin olive oil can lower the volume of carrageenan-induced edema in rats depend on the dose.
Also, the administration of extra virgin olive oil can be dose-dependent in reducing the levels of TNF- in carrageenan-induced edema in rats.
Keywords Anti inflammation, extra virgin olive oil, edema volume.
TNF-, carrageenan INTRODUCTION and it involves many mediators .
Inflammation is a physiological response Inflammation is a beneficial response but it to various stimuli such as infection, irritation can be detrimental to the host because it or tissue injury.
Inflammation is also known contributes to numerous pathogenesis of as a type of non-specific immune response, diseases including allergic, autoimmune.
Andina Putri Aulia, et al.
stress, apoptosis or necrosis that occur in the osteoporosis, diabetes, myopathy and cancer This cytokine induces acute phase .
During the inflammatory process, pro- inflammatory and cytokines are secreted.
The fibroblasts, so the cascade of inflammatory process is initiated by vascular, cellular and elements, leukocytes, mediators and pro- humoral immune system .
The vascular inflammatory cytokines to accumulate at the changes due to pro inflammatory cytokines site of injury to remove harmful agents and to induction that will cause the movement of repair damaged tissue.
The cytokines are fluid to interstitial tissue called edema, one of the cardinal signs of inflammation .
interactions between cells and stimulating The prevalence of inflammation is immune reactivity, either in a specific or non- associated with place, race and disease.
Non- Inflammatory steroidal anti-Inflammatory drugs (NSAID.
cytokine is a small peptide secreted primarily is one of the most commonly prescribed for by macrophages and lymphocytes that will the treatment of inflammation .
Thirty activated the tissue in response to trauma inflammatory drugs are sold in the United complexes, physical and chemical trauma States annually.
This number reflects the .
The .
non-steroidal pro-inflammatory dependency on anti-inflammatory drugs .
Additionally, the incidence of inflammatory (LPS), disease such as osteoarthritis and gout disease cytokines, and UV radiation that will further has increased.
More than fifty percent of induces the activation of Nuclear factor- NSAIDs are administrated to patients over 60 kappa B (NF-kB) and activator protein-1 years old, so it leads to the increase in the side (AP-.
The NF-kB activates a number of effect of NSAIDs.
molecules involved in the inflammatory .
roinflammatory There are several ways to prevent or slow down the progress of inflammation, either by including iNOS.
COX-2.
TNF-.
IL-1, and using drugs or medicinal plants.
Up to now.
IL-6 .
Meanwhile, tumor Necrosis it is estimated that the Indonesian people still Factor- (TNF-) is a cytokine that has a use a variety of plants for an alternative different reaction in different cells.
The use of plants as drugs are involved in all process of inflammations and expected to have relatively low side effects it can be used as an indicator of oxidative compared to anti-inflammatory drugs.
The Ina J Med Lab Sci Tech 2020.
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Andina Putri Aulia, et al.
long term use of NSAIDs can cause erosion NO in carrageenan-induced pleura in rats and bleeding in the lower gastrointestinal .
Based on the afore mentioned reasons.
It was reported that NSAIDs cause the researcher wanted to analyzed the effects injury and affect the integrity of the mucous of extra virgin olive oil on carrageenan- membrane of the gastrointestinal tract .
induced paw edema volume and levels of One of the plants widely used as a drug is TNF- in rats.
oliveAos fruit.
Olive can be processed into olive oil, virgin olive oil and extra virgin olive oil.
Extra virgin olive oil has different characteristic from other types of olive oil because of its refining process and its composition .
Olive oil is used as a dietary component by the Mediterranean community to reduce the risk of illness and The Mediterranean people highly value the high oleic acid in olive oil as well as its minor components.
the phenolic compounds .
Phenolic compounds in extra virgin olive oil have been shown to have anti-inflammatory anti-oxidant properties, as well as anti-microbial activity.
Various phenolic compounds in extra virgin olive oil play an anti-inflammatory role in .
, hydroxytyrosol, flavonoid, and oleoropein .
The phenol compound of extra virgin olive oil has been shown to decrease the concentration of Interleukin-6 (IL-.
, a pro-inflammatory cytokine secreted by response to trauma.
Other studies have shown that phenolic compounds in olive oil can inhibit the cyclooxygenase-2 (COX-.
Impellizzer et al.
also reported that Ina J Med Lab Sci Tech 2020.
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This research method is an experimental with post-test only design only randomized control group design.
The population in this study were 28 male Wistar rats (Rattus norvegistu.
aged 2 - 3 months, weighing about 200 grams.
Animals were acclimatized for 7 days and were divided into 4 groups.
Group I was the control group.
Group II, i and IV were orally administrated with a single dose of extra virgin olive oil at 0.
mL/day, 1.
8 mL/day, and 2.
7 mL/day.
All groups were given 2% of carrageenan injection .
1 mL).
One hour before the injection of carrageenan, all labolatory animals were subjected to paw volume evaluation.
The Extra virgin olive oil was produced in Italy with the brand Bertolli.
It was administrated 30 minutes after carrageenan injection, and was followed by edema paw volume measurement at h1 .
fter 1 hou.
, h2 .
fter 2 hou.
, h3 .
fter 3 hou.
and h4 .
fter 4 hou.
after injection.
In h4, all groups were sacrificed under ether anesthesia.
Blood samples were taken through the heart after The blood samples were centrifuged oleuropein could reduce TNF-.
IL-1 and MATERIALS AND METHODS Andina Putri Aulia, et al.
at 1500 rpm for 30 minutes to obtain serum second and third hours after induction of for TNF- levels analysis.
This research has been approved by the Research Ethics decrease in edema volume at the h4.
In Group committee of the Faculty of Veterinary II and i, edema volume increased up to 1 Medicine.
Airlangga Animal Care and Use hour after injection of carrageenan, and it Committee began to decrease in the second hour after the (ACUC) Ethical Clearance Number 576-KE.
Afterwards, injection of carrageenan (Figure .
The All data was analyzed using Saphiro wilk groups were sacrificed by anesthetizing using normality test .
> 0.
and homogeneity of ether, and the blood samples were taken variance Levene's .
> 0.
The differences through the heart after surgery.
Blood between unpaired groups were analyzed samples were centrifuged at 1500 rpm for 30 using ANOVA.
REPEATED ANOVA and min to obtain serum for TNF- levels LSD test for rat paw edema volume, and one- way ANOVA .
< 0.
and LSD .
< 0.
In Group IV, there was an increase in test for the TNF-.
All statistical tests were edema volume occurred shortly after the performed with SPSS program.
injection of carrageenan .
and there was a decrease in edema volume at 1 hour after RESULTS injection of carrageenan .
Table 1 shows The increased volume of edema (Table .
the percentage of a decline in edema volume showed that the control group experienced an in each group.
The percentage of reduction in increase in edema volume during the first, edema volume in the group IV was 14.
Table 1.
Mean and Standard Deviation of RatAos Paw Edema Volume .
L) Group 63 A 0.
34 A 0.
P1*
53 A 0.
14 A 0.
P2*
47 A 0.
13 A 0.
P3*
59 A 0.
23 A 0.
*Anova test : p < 0.
42 A 0.
17 A 0.
22 A 0.
16 A 0.
48 A 0.
99 A 0.
06 A 0.
16 A 0.
6 A 0.
88 A 0.
94 A 0.
08 A 0.
29 A 0.
87 A 0.
8 A 0.
85 A 0.
iVolume (%) 10,25 A 5,6 11,8 A 11,6 13,28 A 6,0 14,21 A 4,5 Mean edema volume .
L) 4,00 3,50 CONTROL
3,00
KP1
2,50
KP2
2,00
KP3
t before Interval time of measurement .
Fig 1.
Mean Increase in Edem Volume .
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Andina Putri Aulia, et al.
The ANOVA test showed a significant between h-1 and h0 .
= 0.
Therefore, it difference in all groups.
Thus, there was a can be concluded that all groups has edema in significant difference among the time of the rat paw after injection of carrageenan.
administration in each group.
LSD test of The LSD results also showed that there edema volume variables showed that there were significant differences in edema volume was much difference in edema volume in the based on the time of repeated measurement time between h-1 and h3 .
= 0.
In the which was found between Group I and II .
Group I, the most significant difference in = 0.
, and Group I and i .
= 0.
edema volume was found between h-1 and h3 The percentage of reduction in edema .
= 0.
Meanwhile, in the treatment volume has a normal distribution yet the group i, the most different in edema volume variance was not homogeneous, so that the was the time between h-1 and h1 .
= 0.
different test used was Kruskal Wallis test.
and time between h-1 and h1 .
= 0.
The different test results of the percentage of Furthermore.
In the treatment group IV, the most different in edema volume was the time administration of extra virgin olive oil between h-1 and h0 .
= 0.
showed that there was no significant A repeated ANOVA test was used to difference .
= 0.
among the groups (Table .
Thus, there was no difference in differences in the repeated measurements.
the percentage of reduction in edema volume The results showed an overall significant in all of groups.
difference between the time of measurement In the variable of TNF- level (Figure .
, .
= 0.
and a significant difference in the the highest mean was found in the control mean of edema volume between the groups group .
,736.
6 A 1,535.
2 pg/mL) while the based on the time of measurement .
= lowest one was found in the group P3 .
A 90.
0 pg/mL) ANOVA test result P = 0.
The LSD test based on the time of It indicated that there were significant measurement that showed a significant differences between groups.
The result of difference were: the time group between h-1 LSD test showed that there was a significant and h0 .
= 0.
, h-1 and h1 .
= 0.
, h- difference between Group I and Group i .
1 and h2 .
= 0.
, h-1 and h3 = 0.
, h- = 0.
Group I and Group IV .
= 0.
1 and h4 .
= 0.
, h0 and h4 .
= 0.
Group II and Group i .
= 0.
Group II h1 and h3 .
= 0.
, h1 and h4 .
= 0.
and Group IV .
= 0.
, as well as Group h2 and h4 .
= 0.
and h2 and h4 .
= i and Group IV .
= 0.
The most Ina J Med Lab Sci Tech 2020.
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There was a significant difference Andina Putri Aulia, et al.
significant difference was between Group I and Group IV, along with Group II and IV.
TNF-
TNF- .
g/mL) Group Fig 2.
Difference in The Mean of TNF-
DISCUSSION