F2-Isoprostane Levels in Deoxycorticosterone Acetate (DOCA)-Salt Induced Hypertensive Rats Administered with Coffee-Corn Mixture

 

Sugiyanta Sugiyanta1,2, Harianto Notopuro3*, Jusak Nugraha4, Retno Handajani3

1Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.

2Department of Biochemistry, Faculty of Medicine, Universitas Jember, Jember, Indonesia.

3Department of Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia.

4Department of Clinical Pathology, Faculty of Medicine, Universitas Airlangga,

Dr. Soetomo General Hospital, Surabaya, Indonesia.

*Corresponding Author E-mail: hariantonotopuroprof@gmail.com

 

ABSTRACT:

The purpose of this study is to analyze F2-Isoprostane levels in Deoxycorticosterone Acetate (DOCA)–Salt induced hypertensive rats that is given Coffee-Corn mixture. Measurement of blood pressure was carried out by non-invasive methods using CODA instruments. The coffee-corn mixture was made from Robusta (Coffea canephora) obtained from Kaliwining, Jember Regency, and Yellow Corn (Zea Mays) bought from the local market in Jember, East Java, Indonesia. F2-Isoprostane levels were obtained by ELISA examination. The mice’s blood pressure was measured using a non-invasive CODA® blood pressure gauge. Serum F2-isoprostane levels were measured using an F2-isoprostane-ELISA kit that was read with an Elisa reader at a wavelength of 450nm. The results showed that there was a decrease in mean systolic blood pressure in each treatment group, with the most decrease by 41.88mmHg occured in the group that was given a coffee-corn mixture with a ratio of 50% Robusta coffee (Coffea canephora) Kaliwining and 50% yellow corn (Zea Mays). The highest decrease in diastolic blood pressure by 35.25mmHg was also attained in the same group. From the results of serum F2 isoprostrane level examination, there was a decrease in the average level of serum F2 isoprostrane in the treatment group with the most reduction by 51.34pg/mL occured in the group given a coffee-corn mixture with a ratio of 50% Robusta (Coffea canephora) Kaliwining and 50% yellow maize (Zea Mays). Coffee-corn mixture can reduce blood pressure in hypertensive mice through anti-oxidant effects.

 

KEYWORDS: CODA Instrument, Coffe-Corn Mixture, Deoxycorticosterone Acetate (DOCA)-Salt Hypertensive Rats, F2-Isoprostane, ELISA.

 

 


INTRODUCTION:

Hypertension is considered as the most common risk factor for cardiovascular disease is considered as a major cause of mortality and morbidity worldwide. Increased blood pressure is a major risk factor for coronary and ischemic heart disease, as well as hemorrhagic stroke. In 2015, the prevalence of hypertension in women aged 18 years and over was around 20% and men around 24%1. This situation shows that hypertension management is paramount.

 

The mechanisms underlying the pathological process of hypertension are not fully explained. However, there is growing evidence that increased oxidative stress plays an important role in the pathophysiology of hypertension2.

 

The mechanism that underlies oxidative stress in hypertension happens through increased ROS formation by pro-oxidant enzymes, such as NADPH oxidase, mitochondrial oxidase, xanthine oxidase and nitric oxide synthase (NOS), and decreased degradation due to decreased activity of antioxidant systems, such as superoxide dismutase (SOD), Catalase, peroxidase and antioxidants regulated by Nrf-23.

 

Coffee is rich in compounds with high antioxidant activity such as polyphenols and its derivatives. One result of the degradation of phenolic acids from coffee and corn is ferulic acid4,5. Studies show that ferulic acid can be used as a therapeutic agent to prevent damage caused by hypertension due to interference with vascular smooth muscle cells6. Ferulic acid also has the potential to inhibit angiotensin converting enzyme (ACE), an enzyme that converts the Angiotensin I enzyme to Angiotensin II7. Roasted corn kernels are commonly used as a substitute for coffee8.

 

The tradition of drinking coffee-corn mixture has lasted a long time in Indonesia. Since coffee became one of the primary commodities of the Dutch East Indies Colonial Government, coffee beans produced by plantations were almost all exported. Low quality and non-exported coffees are given to garden workers to be used as drinks. These coffees were then consumed by indigenous people. However, because coffee is so expensive, corn is added to the roasting. It is this tradition that was later carried on and remains in Indonesia until today.

 

One of the biomarkers/markers of oxidative stress is F2-isoprostane. F2-isoprostane is a stable end product of lipid peroxidation and can be measured in all tissues and biological fluids  including urine, plasma and cerebrospinal fluid. Because of its stability and sensitivity to changes in redox conditions, F2-isoprostane is considered the most reliable marker for tracking oxidative stress. Plasma and urine F2-isoprostane levels correlate with circulating ROS levels in hypertension9. The purpose of this study was to analyze the level of F2-Isoprostane in Deoxycorticosterone Acetate (DOCA) – Salt induced hypertensive rats that is given Coffee-Corn mixture. The coffee-corn mixture was made from Robusta (Coffea canephora) obtained from Kaliwining, Jember Regency, and yellow corn (Zea Mays) bought from the local market in Jember, East Java, Indonesia. F2-Isoprostane levels were obtained by ELISA examination.

 

MATERIAL AND METHODS:

Experimental animals:

Male Wistar strain mice (250±20g) were obtained from the animal house of the Laboratory of Biochemistry, Faculty of Medicine, Airlangga University, Surabaya. They were maintained under standard laboratory conditions including room temperatures of 20-25ºC. Experimental animals were given water and pellets ad libitum. The mice were given 1 week before the experiment to adapt to laboratory conditions. All experimental protocols were approved by the Animal Research Ethics Committee of the Faculty of Veterinary Medicine, Airlangga University, Surabaya (Ethical Clearance Information No: 2.KE.116.07.2019).

 

Coffee-corn mixture extraction:

Robusta coffee solution (Coffea canephora) Kaliwining is made from 5grams of coffee powder added with 60ml of distilled water at 100°C and stirred until homogeneous11. After homogeneous, the extracted water was filtered and the filter supernatant was freeze dried. The supernatant was put in a freeze dryer at −72°C for 17 hours and produced 1.12grams. The conversion dose to mice is 200gr to 20.16mg. The average body weight of rats in this study was 250g, so the dose used was 250/200 x 20.16mg = 25.2mg/250g or 100.8mg/kgbw. To simplify, the calculation of the dose was rounded to 100mg/kgbw. Coffee-corn mixture consists of 2 ratios, namely: a mixture with a ratio of 75% Robusta coffee, 25% yellow corn and 50% Robusta coffee, 50% yellow corn.

 

Experimental protocol:

Animals were randomly divided into 4 groups (eight mice in each group) as follows: Group 1: Normotence group, group 2: hypertension control. Hypertensive rats were prepared by induction of deoxycorticosterone acetate (DOCA), twice a week for five weeks (10 injections). In its administration, DOCA was dissolved in 0.5ml 12 corn oil, then injected subcutaneously into the rat cervical spine with the first five doses of 20 mg/kg, and the last five doses of 10mg/kg. Rats were given 1% NaCl through their drinking water. Group 3: hypertension group was given a mixture of coffee-corn extract with a composition of 75% coffee and 25% corn for 2 weeks, and Group 4: hypertension group was given a mixture of coffee-corn extract with a composition of 50% coffee and 50% corn for 2 weeks. At the end of the second week, animal blood samples were taken intracardially under mild ether anesthesia and the serum was separated for further experiments.

 

Blood pressure measurement:

Measurements were made twice using a non-invasive CODA® blood pressure gauge. The non-invasive blood pressure measurement method is done with tail cuffs, namely Volume Pressure Recorder (VPR) cuff and an Occlusion cuff. Mice restrainment was done in a particular place using animal holders. Prior to measurement, the mice were warmed with a temperature of 37 on the heating pad until the temperature of the mice reached the optimum temperature. Each measurement was carried out ten repetitions for each experimental animal which was then taken on average13.

 

Biochemical analysis:

Serum F2-isoprostane levels were measured using the F2-isoprostane-ELISA kit (Elabscience) with Catalog No: E-EL-0041. This ELISA Kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit had been pre-coated with 8-epi-PGF2α. During the reaction, 8-epi-PGF2α in the sample or standard competes with a fixed amount of 8-epi PGF2α in the solid phase in Biotinylated Detection Ab specifically for 8-epi-PGF2α. The excess sample or standard unbound conjugate was washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate and incubated. Then TMB substrate solution is added to each well. Enzyme-substrate reaction was ended with the addition of a stop solution and the color change is measured spectrophotometrically with an Elisa reader at a wavelength of 450nm. The 8-epi-PGF2α concentration in the sample is then determined by comparing the sample OD with a standard curve. Isoprostane F2 concentration is in pg/mL14.

 

Statistical analysis:

Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey post-hoc test using SPSS software version 23.0. Data are expressed as mean±SD. P values <0.05 are considered statistically significant.

 

RESULTS AND DISCUSSION:

Effect of DOCA-salt induction on mice’s blood pressure profiles:

The results showed that there was an increase in the mean value of systolic and diastolic blood pressure profiles in Wistar strain white mice that were induced by DOCA-salt compared to normal mice. The measurement results are presented in Table 1 and Figure 1.

 

Table 1: Average blood pressure profile values before and after the induction of deoxycorticosterone acetate (DOCA)-salt.

No

Groups

Mice

Blood Pressure

Systolic (mmHg)

Diastolic (mmHg)

1

A

8

125.50±12.82

85.00±14.52

2

B

8

160.25±14.65

118.62±5.24

3

C

8

145.13±15.26

106.63±8.89

4

D

8

166.38±16.19

115.12±10.68

Note: Group A. Without DOCA-salt induction. Groups B, C, D. DOCA-salt induced.

 

Figure 1: Mean blood pressure profile values before and after deoxycorticosterone acetate (DOCA)-salt induction. White bars: Systolic blood pressure. Black bars: Diastolic blood pressure. Group A. Without DOCA-salt induction. Groups B, C, D DOCA-salt induced. *: p <0.05 Systolic blood pressure compare Group A. #: p <0.05 Diastolic blood pressure compare Group A.

 

Normality and homogeneity tests revealed p >0.05, so it was continued with the One-Way Anova parametric test and Post-Hoc tests. The test results showed p <0.05 so that it was concluded there were differences in the mean systolic and diastolic blood pressure between treatment groups. The biggest increase in systolic blood pressure in group D was 40.88mmHg compared to group A. While the largest increase in diastolic blood pressure in group B was 33.62mmHg compared to group A.

 

Effect coffee-corn mixture administration on mice’s blood pressure profiles:

After the induction of DOCA-salt was completed, coffee-corn mixture was given to groups C, and D for 2 weeks. Groups A and B were only given Aquades. The results can be seen from Table 2, Figure 2, and Figure 3.

 

Table 2: Mean blood pressure profile values before and after administration of coffee-corn mixture.

No

Groups

Mice

Blood pressure

Pre-test

Post-test

Systolic (mmHg)

Diastolic (mmHg)

Systolic (mmHg)

Diastolic (mmHg)

1

A

8

125.50±

12.82

85.00±

14.52

114.63±

7.42

91.75±

12.77

2

B

8

160.25±

14.65

118.62±

5.24

160.62±

9.53

112.87±

8.83

3

C

8

145.13±

15.26

106.63±

8.89

116.75±

10.93

85.25±

10.56

4

D

8

166.38±

16.19

115.12±

10.68

114.50±

4.04

79.87±

4.64

Note: Group A dan B were only given aquades. A. Normotensive group; B. Hypertensive group; C. hypertensive group+(75% coffee and 25% corn) for 2 weeks; D. Hypertensive group+(50% coffee and 50% corn) for 2 weeks.

 

 

Figure 2: Average systolic blood pressure profile before and after coffee-corn mixture administration. Group A dan B were only given aquades. White bars: prior to coffee-corn mixture administration, Black bars: post coffee-corn mixture administration (groups C, dan D); A. Normotensive group; B. Hypertensive group; C. hypertensive group+(75% coffee and 25% corn) for 2 weeks; D. Hypertensive group+(50% coffee and 50% corn) for 2 weeks. *: p <0,05 Systolic blood pressure compare prior to coffee-corn mixture administration.

 

Figure 3: Average diastolic blood pressure profile before and after coffee-corn mixture administration. Group A dan B were only given aquades. White bars: prior to coffee-corn mixture administration, Black bars: post coffee-corn mixture administration (groups C, dan D); A. Normotensive group; B. Hypertensive group; C. hypertensive group+(75% coffee and 25% corn) for 2 weeks; D. Hypertensive group+(50% coffee and 50% corn) for 2 weeks.*: p <0.05 Systolic blood pressure compare prior to coffee-corn mixture administration.

 

From Figure 2, it was found that there was a decrease in mean systolic blood pressure in groups C and D, with the most reduction in group D by 41.88mmHg. Figure 3 shows a decrease in mean diastolic blood pressure in groups C and D, with the most reduction in group D by 35.25mmHg. Statistical analysis T-test in paired samples resulted in a p value of p <0.05, so it could be inferred that there were differences in mean systolic and diastolic blood pressure before and after coffee-corn mixture administration.

 

Quantitative analysis of serum F2-isoprostan levels:

The results of the F2-Isoprostan content analysis can be seen in the table and figure below:

 

Table 3: Mean serum F2 isoprostane levels (pg/mL).

S. No

Group

Mice

F2 Isoprostan Level

1

A

8

442.887±22.156

2

B

8

465.669±2.051

3

C

8

445.961±17.300

4

D

8

414.329±31.034

 

Figure 4: Mean serum F2 isoprostane levels (pg/mL).*: p <0.05 compare Group B. #: p <0.05 compare Group C.

From the above results, there was a decrease in mean serum F2 levels of isoprostran in groups C and D with the most reduction in group D compared to group B by 51.34pg/mL. The normality test revealed a p value of p >0.05, followed by the One-Way Anova test with a p value of p<0.05. There are differences between treatment groups.

 

Measurement of blood pressure was done without anesthesia using the tail-cuff auto-pickup method. Measurements were made using a non-invasive CODA® akinrinde blood pressure gauge. Normal blood pressure (normotension) in white mice is 129mmHg for systolic pressure, and 90mmHg for diastolic pressure11. Systolic blood pressure in hypertensive condition is above 145 to 200mmHg15.

 

Deoxycorticosterone Acetate (DOCA)-salt induction can increase systolic and diastolic blood pressure. This model is ideal for defining the role of two main pathways that are very important in the pathogenesis of essential hypertension, namely the active sympathetic nervous system and imbalances in the renin angiotensin system16. This induction causes kidney sodium imbalance where the amount of sodium and water that is reabsorbed by the kidneys is greater, causing hypervolemia. Interestingly, renin, an enzyme that converts angiotensinogen to angiotensin (Ang)-I, was observed to be low in this salt-sensitive population. This is important because the same phenomenon is observed in animals given DOCA-salt, which shows that this model not only incorporates a high-salt diet but also produces low-renin hypertension, similar to what is observed in human population17.

 

The mice model induced by DOCA salt is also a model of endocrine hypertension which can cause rapid severe hypertension and oxidative stress18. Oxidative stress contributes to the pathogenesis of hypertension through vascular dysfunction, vascular smooth muscle remodeling, and atherosclerotic plaque formation. In addition, ROS mediates the effects of angiotensin II vasoconstrictors19. Cellular damage to ROS is mainly through lipid peroxidation which causes structural changes in biological membranes. Isoprostane F2 is the result of lipid peroxidation which can reflect the oxidative stress status of hypertension. Because of its stability and sensitivity to changes in redox conditions, F2-isoprostane is considered the most reliable marker for tracking oxidative stress. Plasma and urine F2-isoprostane levels correlate with circulating ROS levels in hypertension.

 

One result of the degradation of phenolic acids from coffee and corn is ferulic acid4,5. Ferulic acid shows strong antioxidant properties. Phenolic rings provide strong resonant stability and can accept electrons more easily from free radicals such as hydrogen peroxide, superoxide, and hydroxyl radicals. The anti-free radical activity of ferulic acid is much better compared to caffeic acid20.

 

Ferulic acid also increases the activity of antioxidant enzymes and is responsible for capturing free radicals. Ferulic acid also inhibits free radicals that produce enzymes in the tissue. Previous studies have shown that the activity and expression of cardiac superoxide dismutase, glutathione peroxidase (GPx) and catalase (CAT) are increased by ferulic acid and p-coumaric acid21.

 

How ferulic acid shows beneficial effects on the cardiovascular system and hypertension is not fully understood. Ferulic acid might inhibit the aortic NADPH oxidase enzyme in spontaneous hypertensive mice22. Ferulic acid also acts as an antagonist of nonselective NADPH oxidase by reacting with the sulfhydryl group23. In addition, ferulic acid has the potential to inhibit Angiotensin converting enzyme (ACE), an enzyme that converts the enzyme Angiotensin I to Angiotensin II7.

 

CONCLUSION:

Coffee-corn mixture can reduce blood pressure in hypertensive model mice through anti-oxidant effects. The antihypertensive and antioxidant effects of the coffee-corn mixture are thought to be caused by the content of ferulic acid in the Robusta (Coffea canephora) Kaliwining coffee mix in Jember Regency and Yellow Corn (Zea Mays).

 

ACKNOWLEDGEMENT:

This study was funded by the Indonesian Ministry of Finance's Educational Fund Management Institute (LPDP) through the Indonesian Domestic Lecturer Scholarship (BUDI-DN). We thank EJA – Professional Translation Services for editing the manuscript.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 25.05.2020           Modified on 30.06.2020

Accepted on 25.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(6):3353-3357.

DOI: 10.52711/0974-360X.2021.00583