INTRODUCTION:

Pre-eclampsia is actually a gestational syndrome, which is noted by gestational hypertension and proteinuria at the 20^th week of gestation. One possible mechanism is the imbalance between free radicals and antioxidants due to the failure of spiral arteries remodeling. Spiral arteries are exclusively designed to bear systemically lower peripheral resistance to allow maternal-fetal circulation regarding the exchange of nutrition, gas and metabolic waste products of the fetus¹.

The failure of this remodeling process causes an ischemia process within placental tissues. These ischemic tissues will promote the release of oxidants and free radicals, mainly the hydroxyl radicals which is highly toxic. This radical is responsible for the damage of endothelial lining which triggers the formation of lipid peroxide. Lipid peroxide will further damage the cell wall, as well as cell nuclei and proteins. To make matters worse, this highly toxic chemical may spread into the blood stream and lead to endothelial dysfunction². This condition then causes a decrease in nitric oxide formation and an increase in endothelial-1 formation which then leads to increased blood pressure³. Thus, this chemical, lipid peroxide/ Malondialdehyde (MDA) can be seen as the oxidative stress marker in pre-eclampsia⁴.

Currently, there is still no proven solution to solve pre-eclampsia, but many more studies showed the importance of antioxidants, such as vitamin C and E which may prevent the incidence of pre-eclampsia⁵.

In the other hand, this study examined one of emerging antioxidants, extra virgin olive oil(EVOO) which is known by its high content of tocopherol (vitamin E). The purification of olive oil also yields some other beneficial chemicals, such as hydroxytyrosol and tyrosol. EVOO is also shown to trigger more formation of MUFA (monounsaturated fatty acid) which in turn inhibits the formation of lipid peroxide and normalize the concentration of antioxidants in the blood, thus preventing damaging oxidative stress effect. Additionally, the hydrophilic region of olive oil shows an effective mechanism of lowering oxidative stress effect and directly works in hepatocytes⁶.

METHODS:

This study was carried in laboratory of molecular genetics, animal study laboratory of pharmacology department in the medical faculty of Padjajaran University, Bandung. MDA was measured following TBARs method (thiobarbituric acid reactive substances) spectophotometry which requires a blood sample, 200 µl SDS solutions (Sodium Deodecyl Sulphate) which function as protein denaturation agent, EDTA as an anticoagulant, 50µl of BHT (Butylated Hydroxytoulene) to stabilize MDA products, 1500 µl of acetate acid, 1500µl of TBA to detect MDA and 700ml of aquabidest. We used SmartSpec^TMPlus spectrophotometry from board which was recently recalibrated in November 2016, micropipette of 100- 1000 µl size, cuvet, water-bath and centrifuge. Meanwhile blood pressure was measured by using a specially designed noninvasive sphygmomanometer for mice.

This true experimental study is designed in the form of post test with control group design which was initiated by the acclimatization process of pregnant mice for the first seven days. Those mice were fed with AIN93-M and clean water, which was administered without any extents (ad libitum/ abundantly). Food was given as 10% of body weight, which is around 10-15g/day for each mouse. Feeding time was in each evening, at 04.00 PM. Drinking water was renewed each day. The cage was made from covered plastic box, size of 1 cm^2, which was conditioned to reach room temperature of 20⁰ – 24.5⁰ C and relative humidity of 50 ± 10% and was set to get exact periodicity of light (12 hour period of light and 12 hour period of darkness). The cage was equipped with feeding box and each cage was used only for one mouse. The floor of the cage was covered with husks and the husk was replaced each day to keep it clean. The cage was also equipped with a special glass pipe and a bottle to reduce the chance of contamination and feeding box was ensured to be always clean by covering it with gauze.

The mice were set up to mate by placing one male mouse for 3 female mice. The estrous phase of each female mouse was noted from their morphological and behavioural changes. When a female mouse reached her estates phase, her vaginal will show changes, it will become swollen and reddened. The female will become more irritated and active, in other words, attracting the opposite sex to copulate. Both sexes were then placed in the same cage at 05.00 PM, and mating process will usually be completed within the next three days. If copulation is successful, immediately in the next morning, copulatory plug will be seen covering the vagina, from the cervix to the vulva. Hence that day will be counted as the first day of gestation. After gestation is confirmed, the male mouse will then be displaced from the cage. Pregnant mice were then identified by placing some marker on their tail. Every 5 of them were then grouped into one cage. On the 6^th day of gestation 3ml/day of NaCl, 6% was injected until the 12^th day of gestation.

Pregnant mice were put into five separate groups (each group consists of five mice) and were classified into control and experiment groups. P0 as the control did not get any intervention. As for the experimental groups; P1 was not given any EVOO, as placebo, this group was given 2ml of water instead of EVOO, in the other hand, P2, P3, P4 were given EVOO in varieties of dose (mg) per body weight per day (0.30, 0.60, and 1.21 respectively). EVOO was administered via an oral tube on the 13^th to 19^th day of gestation.

Postest I was conducted on the 13^th day of gestation in which the blood pressure was measured after administration of NaCl, 6% on 6^th to 12^th days of gestation. Posts II was conducted on 2oth day of gestation after the given acute stress stimulant and EVOO. Blood pressure and MDA level in both control and experiment groups were measured.

The serum MDA level was measured by using TBARs method. A 2ml of blood sample from each individual mouse was taken and mixed with 2 drops of EDTA before being put into centrifuge machine. Centrifugation was set to 3000 RPM and it lasted for 30 minutes. After plasma is derived, we combined all reagents (200 µl SDS, 50 µl EDTA, BHT 50 µl, 1500 µl Asetat) with plasma; the mixture was then put into vortex for 10 minutes before being centrifuged with 300 RPM in 10 minutes. After centrifugation, the supernatant was taken and 1500 µl TBA was added. 6 other tubes consisting of standard solution (all reagents without plasma) and 1 tube consisting of 700 µl aquabidest were prepared; 700 µl TBA added to these and these tubes were put into vortex for 10 minutes. All reaction tubes were covered with gauze before being put into water bath for an hour. The water bath was set to reach temperature of 94⁰C. All tubes, except those with standard solution and aquabidest were then again centrifuged for 10 minutes before being examined. 1500 µl of the sample was piped out from the tube and put into cuvet to before getting examined on“SmartSpec^TMPlus” from BioRad spectrophotometry with 532nm wave length.

Blood pressure measurement was done with especially non invasives phygmomanomether which was applied on the mouse tail. The first mouse was put into the holder (replaced with PVC pipe) with its tile outside of the pipe, then wait for 5 minutes to get it to settle. Cuff and pulse censors were placed on the tail, the cuff was pumped into a maximum pressure of 300 mm Hg or until pulse is not palpable any longer. Then the pressure was released until the pulse is palpable again. The result will then appear on the screen (Arcana and Sugiritama, 2009). Accordingly, we used wrist sphygmomanometer from Omron which exactly worked with the same principle as explained earlier. The mice were derived from the Sprague Dawley line, age of 8-11 weeks with an approximate weight of 200 grams (difference may vary as much as 10% accordingly). All mice were healthy, active and did not show any physical deformation. The sample size was calculated by using Federer formula and we got a minimal sample size of 4 mice. As we decided to make 5 groups, then we actually need 20 mice, but we purposely added one mouse for each group to anticipate any chance of dropping out, therefore total sample being used in this study is 25 mice. Groups were randomly selected and ethical clearance was granted by the Ethical Committee of Animal Study at Faculty of Mathematics and Sciences of North Sumatera University. All data were then analyzed using the Paired t Test and Anova, continued with LSD and Pearson Product Moment (PPM) correlation. In case the data were shown to be abnormally distributed, non-parametric test will be applied.

RESULTS AND DISCUSSION:

Following the administration of EVOO, we found the approximate serum MDA level as follows:

Figure1. Error bar of serum MDA level in mice after administration of EVOO in both control and experiment groups. All same lowercase letters are showing insignificant difference (P value< α 0.05).

Serum MDA level in mice (Rattusnorvegicus) can also be seen on the following table:

Table1. Serum MDA level in pregnant mice after administration of EVOO

No	Group	Mean MDA plasma (mmol/µl)	SD	Kruskal Wallis (p value)
1	P0	7.51	3.04	0.036
2	P1	18.19	13.01	0.036
3	P2	6.22	1.89	0.036
4	P3	10.41	3.39	0.036
5	P4	10.04	3.21	0.036

According to figure 1 we found that the mean serum MDA level was decreased after administration of EVOO in the experimental groups. P2 shows higher decrease (6.22+1.89) compared to control group. Kruskal Wallis analysis showed p value 0.036 < α 0.05; CI 95%, which indicates that there is a significant difference in serum MDA level among groups. As for P1 which wasn’t given any dose of EVOO, it showed a higher serum MDA level compared to other experimental groups, while in the other hand, P2 showed nearly the same MDA level as in the control group. Mann Whitney test showed that there is a significant difference in MDA level among P2, P3 and P4 which indicates that even at the lowest dose of EVOO given to experimental animals is able to lower the level of MDA.

A significant group responsible for MDA lowering effect of EVOO is phenol, a polar compound which is shown to have the capability of scavenging oxidants. Another simpler structure is hydroxytrosol which is found to be higher in EVOO compared to other antioxidants, such as vitamin E or C⁷. EVOO also contains high level of phenol of around 579.2 mg/kg and 90% of tocopherol⁶. Interestingly, EVOO contains of mostly α tocopherol, which is known for its ability to eliminate hydrogen phenolate from the oxidized proxy of PUFA which has undergone peroxidation process⁸. This α-tocopherol (vitamin E) is most biological active form of antioxidants in the body.

α-tocopherolpossess its role as an antioxidant by donating its hydrogen ion into lipid peroxidation reaction, hence transforming radical proxy which is less active. This in turn inhibits the activation of other peroxide that finally blocks the lipid peroxidation process^{7, 8, 9}. It is also postulated that EVOO modulates changes in the gene expression and enzyme activation, in which EVOO stimulates the formation of SOD, Gpx and catalase.In another study, EVOO showed its ability to reduce the oxidative stress level in the pancreas after consecutively administered for 3 days¹⁰.

Furthermore, tocopherol also reduces the process of fatty acid oxidation by sustaining cell membrane function and acts as scavenging antioxidants, along with other hydrophilic scavenging antioxidants, such as vitamin C, glutathione reductase, dismutase and peroxidase. Another type of lipophilic scavenger antioxidants is tocopherol (vitamin E) and β caroten⁹. Tocopherol is also shown to be able to inhibit cell proliferation and differentiation^{10, 11}.

Another study also showed that MUFA in EVOO protects again excessive activation of lipid peroxide by maintaining the level of hepatic antioxidant enzyme levels as close as possible to normal. The hydrophilic structure of EVOO is also shown to have direct and most potential ability as antioxidants to hepatocytes⁶. Another unique feature of EVOO is that its hydroxytyrosol structure is left unaffected even after digestion⁷.

As seen in our study, EVOO exerts a potential lowering effect of the MDA level, even within the lowest dose of 0.30mg/KgBW/day. Our study showed that even within this very low dose, EVOO is able to reduce serum MDA level by 36.02% to 53.12%.

Figure 2 shows a blood pressure measurement after administration of NaCl, 6% on day 13^th.

In the figureno 2. Error bar of blood pressure after administration of NaCl, 6% in both control and experiment groups. All same lowercase letters are showing insignificant difference (P value < α 0.05).

The above figure shows that systole was increased significantly in pregnant mice after the administration of 3ml NaCl 6% via intramuscular injection.

Table2. Systolic blood pressure of pregnant mice after administration of NaCl, 6%

No	Group	Mean TD systole (mmHg)	SD	Kruskal Wallis (p value)

1	P0	120.20	9.01	0.038
2	P1	142.40	15.76
3	P2	140.60	9.79
4	P3	143.60	8.96
5	P4	154.20	18.97

Kruskal Wallis test showed that there is a significant difference in blood pressure measurement between control and experiment groups (P value 0.038 < α 0.05). Furthermore, Mann Whitney test showed that there is no difference among experiment groups. Increased blood pressure on day 13^th of gestation is shown to be also proportionally related to increased level of MDA, as confirmed by Spearman correlation test(CI 95%: P value 0.026). Statistically the correlation showed only 44.4% strength which is considered a weak correlation, instead of that, we may still conclude that increased MSA also plays a role in increasing blood pressure.Figure3. Error bar of blood pressure after administration of NaCl, 6% in both control and experiment groups. All same lowercase letters are showing insignificant difference (P value < α 0.05).

Table 3. Mean blood pressure of all groups after administration of NaCl 6% on day 20^th of gestation

No	Group	Mean TD systole (mmHg)	SD	Kruskal Wallis (p value)
1	P0	102.20	22.06	0.010
2	P1	135.60	3.21
3	P2	101.40	17.52
4	P3	114.40	17.04
5	P4	110	9.92

Kruskal Wallis analysis showed P value 0.010 < α 0.05 that indicates significant difference of blood pressure among groups. Mann Whitneytest also showed that control group didn’t seem to have a significant difference from all other experiment groups, except P1(P value > α 0.05). It is worth noted that decreased of blood pressure was shown on the 20^th day of gestation, which accounts for 7 days (one week) from the last administration of NaCl 6% (on day 13^th). Accordingly, we conducted the Wilcoxon test to examine whether the administration of EVOO for one week may be related to decreased blood pressure. From that test we found that administration of EVOO for just one week in a variety of dose exerts a lowering effect on blood pressure (Figure 3).

Figure3. Error bar of systolic blood pressure on day 13^th and 20^th. Wilcoxon tests showed the P value:P0= 0.104, P1= 0.498, P2= 0.043, P3= 0.043, P4= 0.043.

A decrease of systolic blood pressure was actually seen in experiment group and the control group which were not EVOO dosed. After the administration of NaCl, 6% on day 13^th, analysis with Kruskal Wallis showed a significant increase of blood pressure, in which experimental groups showed a more prominent increase compared to control group. The experiment group showed mean blood pressure of 140/90 mmHg to 154/85 mmHg (this is higher than mean blood pressure in mice; 130/90 mmHg). On the other hand, the control group showed mean blood pressure of 120.20 mmHg±9.01. A studied showed that high intake of salt dosage increases the blood pressure among the experimental mice^{12, 13}.

Thus the administration of NaCl, 6% is a logical way of inducing pre-eclampsia in experimental animals which is confirmed throughWilcoxon test which showed that increased blood pressure really exists due to administration of NaCl, 6% (P value< α 0.05).

Meanwhile, systolic blood pressure was decreased significantly in the experimental groups. It was confirmed that among the experimented micegroups which were given EVOO, the blood pressure was decreased significantly and their mean blood pressure was nearly the same with that in the control group. In contrast, systolic blood pressure in P1 which wasn’t given any dose of EVOO showed an insignificant decrease, but is still higher than the overall mean value of blood pressure.This phenomenon proves that EVOO, which is read in tocopherol and many kinds of unsaturated fatty acid, such as oleic acid, phenolicacid play a critical role in improving systemic circulation by exerting a cardio protective effect and enhancing endothelial vasomotor response that is against the effect of increased MDA level¹³. Polyphenol, hydro cortisol and tyrosol in EVOO are also known to stimulate the production of NO which plays an important role in inhibiting the formation of peroxynitrite (an accident causing oxidative stress), as well as reducing the formation of ROS and stimulates vasodilation¹³, thus improving systemic blood pressure of the mice. The administration of EVOO as little as 0.30 mg/kgBW/dayis able to improve blood pressure on the 18^th day of gestation.

EVOO also exerts another positive and beneficial effect in lowering blood pressure. In another study, it is shown that daily consumption of 2 tablespoon of EVOO (around 30-40 gram) in 6 months may help a chronic hypertensive patient dependency upon anti hypertension¹⁵. In addition, unsaturated fatty acids in EVOO also exert inhibitory action of ACE (Angiotensin-Converting Enzim) and an 8-isoprostanes(a prostaglandin like free radicals in the urine) which in turn helps to lower blood pressure¹⁵.

ACE is an enzyme in renin-angiotensin system which regulates extracellular volumes (plasma, blood, lymph and interstitial fluid) and promotes vasoconstriction of arteries. ACE works by catalyzing the transformation of angiotensin I (AT I) into AT II, and facilitates the degradation of bradykinin (a peptide which is responsible to dilate blood vessels). Angiotensin I receptor is found in various organs, such as kidneys, adrenal glands, heart, blood vessels and brain¹⁴. By inhibiting ACE, EVOO is able to inhibit the activation of angiotensin and degradation of bradykinin which in turn stimulating the formation of prostaglandin E2 that facilitates vasodilatation, thus lowering blood pressure¹⁵.

CONCLUSION:

This research confirms the administration of 6% of NaCl is able to stimulate the incidence of pre-eclampsia in pregnant experimental mice. Meanwhile, the administration of EVOO, has showed hypotensive impact at a lower dose,Particularly in P2 group, serum MDA level and blood pressure were seen to be close to those in the control group. However, further in depth studies are suggested to understand the role of anti-oxidants, such as GPx, SOD, and cocktails, which would allow a better understanding of the role of EVOO as an antioxidant.

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Received on 19.09.2017 Modified on 10.01.2018

Research J. Pharm. and Tech. 2018; 11(3):1053-1057.

DOI: 10.5958/0974-360X.2018.00197.X

Currently, there is still no proven solution to solve pre-eclampsia, but many more studies showed the importance of antioxidants, such as vitamin C and E which may prevent the incidence of pre-eclampsia5.

Currently, there is still no proven solution to solve pre-eclampsia, but many more studies showed the importance of antioxidants, such as vitamin C and E which may prevent the incidence of pre-eclampsia⁵.