Preclinical Trial of Propolis Extract in Prevention of High Salt Diet- Induced Hypertension

Hypertension is a serious public health problem and the major cause of premature death worldwide.1 WHO (2019) reported around 1.13 billion people had hypertension and mostly lived in low-middle income countries. Hypertension is risk factor for several diseases, including heart attack, stroke, kidney damage, diabetes and even cancer.3-6 In addition, hypertension also may reduce cognitive performance and cause economic lost in either micro or macro scale.7-8


INTRODUCTION
Hypertension is a serious public health problem and the major cause of premature death worldwide. 1 WHO (2019) reported around 1.13 billion people had hypertension and mostly lived in low-middle income countries. Hypertension is risk factor for several diseases, including heart attack, stroke, kidney damage, diabetes and even cancer. [3][4][5][6] In addition, hypertension also may reduce cognitive performance and cause economic lost in either micro or macro scale. [7][8] Prevention of hypertension by controlling modifiable risk factors would have beneficial effect on the public health. 9 High intake of salty food is one of the important risk factor for developing hypertension. [10][11][12] The estimated salt intake is about 9-12 g per day in most countries which is higher than WHO recommendation of less than 5 g. 11 High salt intake causes the impairment of renin angiotensin-aldosterone (RAA) system and oxidative stress leading to the elevation of blood pressure. 13 The management of hypertension is currently by pharmaceutical therapies, however, it is costly, has adverse effect, and requires medical intervention. 9 The triple therapies, including diuretics, a calcium channel blocker and an angiotensinconverting enzyme inhibitor/angiotensin receptor blocker, cannot reduce the risk for resistant hypertension due to high salt intake. 13 In addition, the current management seems to be not effective in controlling hypertension, thus it needs another therapies, including dietary, lifestyle and nutritional supplement strategies. 14 Meanwhile, the study on bioactive natural components has been increased describing the mechanism of action in the prevention of hypertension. 15 Propolis is a natural product manufactured by bee from plant resins. 16 The biological activities of propolis have been widely studied, namely antioxidant, antibacterial, antiviral, antifungal, anti-inflammatory, anticancer, antidiabetes, immunomodulatory, antiemetic properties, and also to show antihypertensive property. [16][17][18][19][20] The previous research showed that the constituents of Brazilian propolis ameliorated the hypertension in rats. 21 However, there are some challenges on propolis research due to its diverse chemical constituents which depends on botanical origin, bee species and the preparation of extract. 15 Therefore, each region possibly has their unique properties.
Indonesia is a country with high biodiversity which has the potential to produce a great variety of propolis. Several studies on Indonesian propolis have showed that Indonesia propolis possessed antioxidant, immunomodulatory, anti-inflammatory, and anticancer activities. 20,[22][23][24] However, there has The rats were categorized to have hypertension when systolic blood pressure (SBP) ≥ 140 mmHg. [29][30] The hypertensive rats then administered with propolis for 1 week, while high-NaCl diet was continued. Body weight and blood pressure was weekly measured from week 0 until week 4. In addition, the rats were euthanized at week 4 to collect urine and blood samples for analysis. Heart, liver, kidney, spleen, lungs and urinary bladder were harvested to measure the weight. All experimental procedures have been approved by the Animal Care and Use Committee, IPB University (No. 176-2020 IPB).

Blood pressure measurement and urine collection
The procedures of blood pressure measurement referred to Malkoff et al. with slight modification. The blood pressure was weekly measured through a tail cuff in the morning using non-invasive blood pressure system (CODA, Kent Scientific, USA). The animals were placed in a holder for acclimatization for 10-15 minutes, whereas room temperature was kept at 26 o C. Blood pressure was read 10 times and the means were determined. 31

Statistical analysis
Data were expressed as mean ± standard deviation. The differences between groups were analysed with ANOVA followed by post-hoc Tukey's test. The significance was considered at p-value < 0.05.

RESULTS
To confirm whether high-NaCl consumption affects body weight, we weekly measured the weight of animals. The weight changes during the administration of high-NaCl diet and propolis are shown in Table 1. However, the weight of animals during administration of a high-NaCl diet for 3 weeks did not change statistically (p>0.05). After propolis administration for 1 week, the weight of animals was also not affected (p>0.05).
A high-NaCl diet successfully caused hypertension in rats after 3 weeks of intervention. Either systolic or diastolic blood pressure was statistically raised at week 3 in all high-NaCl groups compared to standard diet group (P<0.05). All propolis samples remarkably reduced SBP and DBP compared to high-NaCl only group after 1 week of administration (p<0.05). The biggest changes were found in groups administered with propolis from Riau Archipelago and South Sulawesi, where the effect seemed to be similar with positive control group. The blood pressure of animal during the treatments can be seen in Table 2 and Table 3.
Result of urine test showed that propolis seems to increase the 24-h urine volume. Propolis from Riau Archipelago and Lampung increased significantly the 24-h urine volume compared to standard diet group (p<0.05). It was confirmed by lighter urine colour in both groups. However, the other parameters (density, pH, consistency, leukocytes, nitrite, glucose, ketone, urobilinogen, bilirubin, erythrocytes, and haemoglobin) showed not statistically significant difference. Nevertheless, protein were relatively higher in all propolis groups. The result of urine test can be seen in Table 4. Table 5 shows lipid profiles of rats at the end of intervention. The present study found no differences in triglyceride and cholesterol concentrations between the groups. However, among the groups with high-NaCl diet, LDL concentrations were significantly decreased in group administered with propolis from Lampung compared to high-NaCl only group (p<0.05). In addition, HDL concentrations significantly increased in group administered with propolis from South Sulawesi compared to standard diet (p<0.05).
Result of haematological analysis is presented in Table 6. Almost all haematological parameters did not differ compared to standard diet group, except lymphocytes levels. The concentration of lymphocytes increased significantly in several groups, including positive control group, group administered with propolis from Riau Archipelago and Lampung compared to standard diet group. Nevertheless, we found relatively low levels of WBC, monocytes, granulocytes, RBC, haemoglobin, HCT, MCHC, PLT and PCT in high-NaCl only group compared to propolis group(s).
The weight of organs was measured to see the possibility of pathological    Data are mean ± standard deviation (n=6) 1 Weight difference = weight at week 3 -weight at week 0 2 Weight difference = weight at week 4 -weigh at week 3 changes. Relative weight of organs is depicted in Table 7. Heart seemed to be altered during the treatments. Decreased relative weight of heart was found in all groups administered with high-NaCl. Meanwhile, the relative weight of liver, spleen, lungs and urinary bladder did not differ significantly between the groups.

DISCUSSION
Obesity has been confirmed as one of the main risk factors for developing hypertension. [33][34] In the other hand, high consumption of salty food is strongly associated with obesity and probably due to increase in leptin production, leptin resistance and endogenous fructose production. [35][36] However, the present study found no significantly change in weight of animals after high-NaCl diet intervention and propolis administration. Consumption of salty diet is associated with osmotic stress and will be compensated with increase in water consumption which may limit food intake. 37 The present result is also in line with the prior study  5.9 ± 0.5 a 6.0 ± 0. Data ara mean ± standard deviation (n=6) The different superscript in the same column are statistically significant (p<0.05) SD : standard diet group NaD : high-NaCl diet group PD : high-NaCl diet + captopril (25 mg/ kg) NaDP1 : high-NaCl diet + propolis from Riau Archipelago (200 mg/ kg) NaDP2 : high-NaCl diet + propolis from Lampung (200 mg/ kg) NaDP3 : high-NaCl diet + propolis from South Sulawesi (200 mg/ kg)  Data ara mean ± standard deviation (n=6) The different superscript in the same column are statistically significant (p<0.05) which reported that salt inhibited obesogenic effect of high fat diet by decreasing adipocytes size and leptin concentrations. 38 In addition, we speculated that difference in NaCl concentration might be responsible for this result. The studies which showed obesogenic effect of high-NaCl diet were not more than 4%. [35][36] In contrast, our present study and the study of Pitynski-Mille et al. administered the diet with aroud 8% NaCl. This concentration might cause too salty diet and could decrease food intake. 39 The present study found that Indonesian propolis successfully decreased either SBP or DBP of hypertensive rats. Indeed, SBP was normalized after 1 week of propolis administration. Propolis is rich in phytochemical compounds, such as phenolics, flavonoids, and terpenes. 19 The prior studies also reported the antihypertensive activity of propolis from several regions, including Brazil, Australia, and Tunisia. 21,[40][41][42][43] Several compounds with antihypertensive activity have also been isolated from propolis, including dihydrokaempferide, kaempferide, butelelol, isosakuranetin, and caffeoylquinic acid. 21,41 Indonesian propolis is a tropical-type propolis containing phenolic and flavonoid compounds and possesses strong antioxidant activity. 20,[44][45] . The antioxidant activity of Indonesian propolis was proposed to underlie its antihypertensive activity through enhancing bioavailability of NO which responsible for vessel effect vasodilation. 46 Furthermore, low antioxidant levels are associated with hypertension. 47 We further analyzed the urine of rats after 1 week of intervention. We found some propolis samples including propolis from Riau Archipelago and Lampung had diuretic effect which might also be responsible for its antihypertensive effect. The evidences have showed the diuretic agents as promising antihypertension by removing any extra fluid and widening blood vessel. 48 Unfortunately, we also found relatively higher concentrations of urine protein in all propolis groups, which in contrast to the current knowledge. 49 However, one toxicological study reported Brazilian propolis induced acute renal failure. 50 Our present results were similar to antihypertensive effect of amlodipine. 51-53 Therefore, our data suggest the possible mechanism of Indonesian propolis as antihypertension may be similar to the antihypertensive effect of calcium channel blockers (CCBs).
The other possible mechanism of propolis in lowering blood pressure is probably through lipid metabolism. Propolis from Lampung decreased significantly LDL levels compared to high-NaCl only group. We also found propolis from South Sulawesi increased significantly HDL levels when compared to standard diet group. The previous studies showed that propolis from Nigeria and Brazil could improve LDL and HDL levels. [54][55] LDL levels have been reported to correlate with blood pressure in Japanese women that probably due to its association with leptin resistance. 56 In addition, low HDL levels are also associated with reduced kidney function and may contribute to hypertension. 57 The haematological parameters data mainly showed no significant changes between interventions and control group. Nevertheless, increased lymphocyte concentrations in propolis groups possibly were related to its immunomodulatory effect. 58 In addition, our present study found relatively low concentrations of several haematological parameters in high-NaCl only group compared to propolis group(s). Increase in blood volume due to high-NaCl diet might cause relatively low concentration of several haematological parameters. 59 In fact, the prior study reported protective effect of propolis on haematological alterations. [60][61] Chronic high-salt diet has been found to alter weight of several organs which may indicate pathological changes. 62 From the relative weight of organs, however, we did not find the possibility of alterations in liver, kidney, spleen, lungs, and urinary bladder. We found only decrease in relative weight of heart which might be attributed to hypertonic effect of NaCl in plasma result in decreased water contents in organs. 63 However, changes in relative weight of organs are not always correlated with pathological condition. 64

CONCLUSION
The present study found that high-NaCl diet did not cause weight gain. All propolis samples ameliorated high-NaCl diet-induced hypertension, where the biggest changes found in both group administered with propolis from Riau Archipelago and those with propolis from South Sulawesi. According to 24-h urine volume, propolis from Riau Archipelago and Lampung possessed diuretic effect. Increased urine protein was found in all propolis groups. Nevertheless, the other parameters (density, pH, consistency, leukocytes, nitrite, glucose, ketone, urobilinogen, bilirubin, erythrocytes, and haemoglobin) were not affected. Propolis from Lampung and South Sulawesi are seemed to improve LDL and HDL concentrations, respectively. Moreover, haematological parameters mainly did not change after the treatments.
In addition, we only found decrease in relative weight of liver in all groups administered with high-NaCl diet.
Our study suggests that the antihypertensive effect of Indonesian propolis samples through different pathways. Care should be taken in use of propolis among patients with proteinuria. Histopathological changes need further investigation.