Effectiveness of avocado leaf extract (Persea americana Mill.) as antihypertensive

Introduction

Hypertension is an increase in blood pressure measured twice in resting conditions, with systolic blood pressure (SBP) and diastolic blood pressure (DBP) of >140 mmHg and >90 mmHg in adults, respectively.¹ Various risk factors or their interactions lead to hypertension, such as obesity, unhealthy diet (excessive sodium salt, low potassium diet, and excessive intake of saturated fatty acids), heavy alcohol consumption, tobacco use, genetics, physical inactivity, persistent stress, and unhealthy environments.^2,3

Hypertension is a cardiovascular risk factor that causes many deaths worldwide. Chronic hypertensive conditions can lead to kidney failure, stroke, and ischemic heart disease.^1,4,5

Recent basic health research (Riskesdas) reported that the prevalence of hypertension in Indonesia increased from 25.8% in 2013 to 34.11% in 2018.⁶ A chronic hypertensive state may lead to complications; thus, hypertension should be managed and treated properly. Management of hypertension is essential because high blood pressure is also a risk factor for several diseases.¹ Treatment includes pharmacological and non-pharmacological methods.⁷ However, pharmacologically, prolonged use of drugs such as diuretics, calcium channel blockers, angiotensin receptor blockers, angiotensin-converting enzyme (ACE) inhibitors, and beta-blockers can cause side effects. The body may not be able to tolerate the appropriate drug levels. The drugs cannot completely cure the disease, so the drugs must be maintained for long periods. Moreover, hypertension drugs have high economic value because they must be purchased regularly over the course of the patient’s life.⁸

In Indonesia, Malays utilize diverse plant species that have shown effectiveness in treating various diseases. For example, residents of the Jambi Province, Indonesia, treat hypertension using natural ingredients that have been processed into traditional medicines, such as avocado leaves (Persea americana Mill.). Malays switched from using chemical drugs to herbal medicines because of the belief that traditional medicines that come from nature are easily tolerated by the body, have low economic value, and have relatively high safety despite long-term use. P. americana leaves are considered an effective antihypertensive agent because they are rich in flavonoids and quercetin compound, which are considered effective in reducing high blood pressure.^5,7,9–11

Various studies in hypertensive rats have shown that quercetin can exert a diuretic effect by increasing urine volume, thereby decreasing blood pressure.^12,13 Antihypertensive therapy using quercetin compound administered continuously can inhibit conversion of ACE from angiotensin I to angiotensin II, which causes vasoconstriction in blood vessels and consequently hypertension.^5,7,14,15 Inhibition of ACE, along with the increase in nitric oxide and nitrate oxide levels, inhibits oxidative stress due to decreased levels of antioxidants.^15–17

The Wistar rat, which has been bred at the Wistar Institute since 1906, is one of the most widely used animal model in biomedical research.¹³ Rats are mammals; therefore, the treatment response may be similar to that of other mammals. The use of rats as experimental animals is also based on economic considerations, and that the rat’s life span is only 2-3 years with a reproduction time of 1 year. The advantages of white rats over wild rats are that they mature quickly, do not show seasonal mating, and reproduce faster. Other advantages of using a laboratory animal include that it is effortless to handle, it can be left alone in a cage as long as it can hear the sounds of other mice, and it is large enough to facilitate observation.¹⁸

A previous study by Auwal et al. (2017) demonstrated that increasing the in vivo efficacy of antihypertensive biopeptides of chitosan nanoparticles using the ionic gelation method in spontaneously hypertensive rats proved that the angiotensin converting enzyme (ACE) inhibitory biopeptide stabilized by chitosan nanoparticles effectively reduced blood pressure for a long period.¹⁹ Mariangela’s (2011) research related to a new formulation in the treatment of hypertension proved that the nanoparticle method was effective as an antihypertensive in the kidney, heart, or smooth muscle organs.²⁰ The results of Yuan’s (2012) study revealed that nanoparticles have better properties for pharmacokinetic drugs in vivo because the nanoscale size can help penetrate tissue through capillary blood vessels and epithelial layers.²¹Another study by Sun’s (2014) showed that nanoparticles were durable and had a significant antihypertensive effect on spontaneously hypertensive rats.²²

Various studies used the extract of P. americana leaves in to investigate its effectiveness in treating hypertension. However, the previous studies did not assess the effect of nanoparticles extract.^5,9,10,23,24 The number of hypertension cases is increasing worldwide,²⁵ and there is a need for information regarding herbal medicine to treat hypertension. Therefore, this study is relevant nowadays and, in the future, to provide information regarding hypertension treatment. Moreover, there is a need to examine differences in the effectiveness of modern and herbal antihypertensive medicines in vivo through measuring the reduction in SBP and DBP and increase in urine volume. Thus, this study aimed to investigate the antihypertensive effects (such as inhibition of ACE, decreasing SBP, decreasing DBP, as well as increasing urine volume and increase in nitric oxide and nitrate oxide levels) of P. americana leaf extracts and nanoparticles in vivo involving male Wistar rats to develop potential natural materials of P. americana leaves in an attempt to control the prevalence of hypertension, especially in Sarolangun Regency, Jambi.

Methods

Results

Results of P. americana leaf extraction

Processing of P. americana leaves was conducted at the texture analysis laboratory of the UNDIP Integrated Laboratory. Figure 1 shows solid preparation of P. americana leaf extracts. As shown in Figure 1, extracts were obtained from solid preparations of P. americana leaves. This preparation was also used as a sample for the phytochemical test to determine levels of quercetin, mineral compound, and antioxidant activity.

Figure 1. Solid preparation of P. americana Mill leaves extracts.

Phytochemical test results

Quantification of Quercetin

High-performance liquid chromatography (HPLC) was performed for the quercetin test of samples of P. americana leaf extract. Based on the result, samples contained quercetin of 1129.597 ppm.

Antioxidant activity

The analysis of antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl method showed that the antioxidant activity at IC₅₀ was 44.734 ppm.

Mineral compound analysis

Figure 2 presents detailed test results of the analysis of mineral compounds using the 2,2′-azinobis-3-ethyl benzothiazolin–sulfonic acid method. Levels of mineral compounds in leaf extracts (P. americana) tested using the 2,2′-azinobis-3-ethyl benzothiazolin–sulfonic acid method are shown in Table 1. Table 1 shows that the extract contains 10 mineral compounds including potassium as the highest content, chlorine, sulfur, silicone, calcium, phosphorus, magnesium, iron, rubidium, and zinc.

Figure 2. Graph of mineral compound.

Table 1. Mineral compounds in P. americana leaves extract.

No	Component	Symbol	Result (%)
1.	Potassium	K	1.0100
2.	Chlorine	Cl	0.1210
3.	Sulfur	S	0.1150
4.	Silicone	Si	0.1030
5.	Calcium	Ca	0.1020
6.	Phosphorus	P	0.0470
7.	Magnesium	Mg	0.0171
8.	Iron	Fe	0.0068
9.	Rubidium	Rb	0.0042
10.	Zinc	Zn	0.0016

Hypertension subjects

Four white male Wistar rats were utilised in each group, not including the six Wistar rats excluded in the analysis due to death. Rats’ blood pressure was measured before and after treatment with a 16% NaCl solution continuously administered for 14 days, followed by 7 days of receiving the preparations. Blood pressure was measured non-invasively using a CODA™ mouse rat tail-cuff system. To induce above-normal blood pressure, rats were administered with a 16% NaCl solution for 14 days. Furthermore, furosemide treatment (K3), P. americana leaf extract (K4), P. americana leaf extract nanoparticles (K5), and chitosan nanoparticles (K6) were administered orally in each group. In all groups except K1, the 16% NaCl solution was administered to maintain hypertensive conditions. Blood pressure was measured again after 7 days of receiving the preparations (day 22). When the rats’ blood pressure decreased, 2 mL blood samples were taken to test the activity of ACE inhibitors and measure serum levels of nitric oxide. Subsequently, rats were left to stand for 3 h after collection of blood samples and put into individual metabolic cages for 24 h to check for the diuretic effect between treatment groups.

Changes in SBP, DBP, urine volume, and DAI

In this study, two stages of SBP and DBP changes were observed. In the first stage, SBP and DBP were measured on day 0 (before the experiment). Then, K2–K6 rats were administered 16% NaCl solution to induce hypertension. At the second stage (day 15 to 21), K2–K6 rats still received 16% NaCl solution, while K3–K6 were given treatment according to their respective groups for 7 days. The SBP and DBP was re-measured on day 22.

Based on Figure 3 and Figure 4, the mean of SBP and DBP in K3–K6 groups declined after the treatment (days 22) compared with day 15. The greatest decrease of SBP and DBP was experienced by the K5 group with the P. americana leaf extract nanoparticles intervention by 68.75 mmHg (175.00 mmHg to 106.25 mmHg) and 55.25 mmHg (128.42 mmHg to 73.17 mmHg), respectively, followed by K3 in which the changes of SBP and DBP were 57.58 mmHg (165.42 mmHg to 107.83 mmHg), and 50.83 mmHg (122.67 mmHg to 71.83 mmHg). On the other hand, the SBP and DBP in K4 groups declined by 48.08 mmHg (164.92 mmHg to 116.83 mmHg), and 35.58 mmHg (118.42 mmHg to 82.83 mmHg), while the SBP and DBP changes in K6 group were 48.58 mmHg (168.33 mmHg to 119.75 mmHg), and 36.25 mmHg (118.50 mmHg to 82.25 mmHg), respectively (Figure 3 and Figure 4).

Figure 3. Change trend of systolic blood pressure (Mean) before the experiment and after treatment (A), Blood pressure (Standard Deviation) before the experiment and after treatment (B).

Figure 4. Change trends in diastolic blood pressure (Mean) before the experiment and after treatment (A), Blood pressure (Standard Deviation) before the experiment and after treatment (B).

Increased urine volume

Diuretic effects can be seen through the increase in urine volume between the groups treated by measuring the urine volume after all rats were placed in individual metabolic cages for 24 h. Figure 5 shows that K2 and K6 have lower urine volumes than K3–K5. This is because K2 and K6 do not contain active substances that can increase urine volume, causing little urine excretion.

Figure 5. Urine volume after 24 h of treatment.

Figure 5 reveals that the groups with the highest to lowest diuretic activities were K5, K3, K4, K6, and K2, respectively. K5 had the highest DAI value of 2.25, which indicates that K5 had high diuretic activity (Figure 6).

Figure 6. Graph of diuretic activity index.

Test results of ACE inhibitions, nitrite oxide, and nitrate oxide levels

The results of the ACE inhibition test using the ELISA method in rat serum are shown in Figure 7. The ACE inhibition test was conducted according to the instructions on the ACE ELISA kit. Validation of the ACE inhibition test shows the performance required in the ACE ELISA kit. The calibration graph shows a linear equation (line y=0.1889x+0.2189) and linearity value (R²=0.9944) (Figure 7). The mean ACE inhibitions in the ELISA test are presented in Table 2. As depicted in Table 2, on mean, ACE was inhibited by the P. americana leaf extract and the nanoparticle extract were 60.0±12.1%, and 59.5±3%, respectively.

Figure 7. Results of the ACE inhibition test.

Table 2. Mean ACE inhibition, mean nitrate serum levels, and mean nitrite serum levels.

Sample	ACE inhibition		Serum nitrate		Serum nitrite
	% ACE inhibition	Mean (μmol/L) ± SE	μmol/L	Mean (μmol/L) ± SE	μmol/L	Mean (μmol/L) ± SE
P. americana leaf extract (K4)	73.3	60.0±12.1	70.5	44.0±9.0	155.5	83.7±24.0
	66.6		39.8		62.3
	76.4		30.9		52.8
	24.1		34.6		64.2
Nanoparticle extract (K5)	51.8	59.5±3.3	43.8	41.1±11.5	81.6	81.0±23.2
	56.1		72.2		146.3
	64.7		19.6		42.5
	65.6		28.8		53.7
Chitosan nanoparticles (K6)	53.9	63.8±5.7	39.5	60.4±26.0	60.2	120.1±66.3
	80.4		34.0		58.0
	59.4		29.6		43.2
	61.6		138.4		318.8

The results of measuring nitric oxide serum level using the ELISA method were divided into two, namely, nitrate level test and nitrite level. Concentrations of serum nitrate are shown in Figure 8. The nitrate level test graph was conducted according to the NO Assay kit instructions. Validation of the nitrate test shows that the test followed the requirements in the NO Assay kit. The calibration graph shows a linear equation (line y=0.0021x+0.065) and a linearity value of R² of 0.967 (Figure 8). The mean levels of nitrate oxide are shown in Table 2. As shown in Table 2, chitosan nanoparticles (K6) caused the highest mean serum nitrate with 60.4±26.0 μmol/L, while the mean serum nitrate as a result of P. americana leaf extract (K4), and nanoparticles of P. americana leaf extract (K5) were 44.0±9.0 μmol/L, and 41.1±11.5 μmol/L, respectively.

Figure 8. Serum level of nitrates.

Nitrite levels using the ELISA method are shown in Figure 9. As presented in Figure 9, the graph of the nitrite level test was created correctly according to the NO Assay kit instructions. Validation of the nitrite test shows that the test followed the requirements of the NO Assay kit. The calibration graph shows a linear equation (line y=0.0024x+0.0542) and a linearity value (R²=0.9984) (Figure 9). The mean levels of nitrite oxide are shown in Table 2. Table 2 reveals that chitosan nanoparticles (K6) resulted in the highest mean of serum nitrite with 120.1±66.3 μmol/L, while the mean of serum nitrite was led by P. americana leaf extract (K4), and nanoparticle of P. americana leaf extract (K5) were 83.7±24.0 μmol/L, and 81.0±23.2 μmol/L, sequentially.

Figure 9. Result of the nitrite level test.

Bivariate analysis

Results of the data normality test showed that only the SBP group had a normal data distribution. Table 3 presents results of the data normality test of group data for SBP, DBP, and urine volume.

Table 3. Group data normality test for SBP, DBP, and urine volume.

Treatment group	SBP	DBP	Volume urine
K1 (Normal)	0.682	0.875	0.683
K2 (Control−)	0.230	0.596	0.001
K3 (Control +)	0.805	0.875	0.001
K4 (Extract)	0.377	0.141	0.001
K5 (Nano extract)	0.945	0.260	0.272
K6 (Nano chitosan)	0.771	0.046	0.001

Based on Table 3, the data normality test used the Shapiro–Wilk test since the number of samples was less than 50. The data distribution was considered normal if p>0.05 so the SBP group had normal data distribution. Since the data distribution was normal, the data variant test was further tested using the variant homogeneity test. Results of the variant homogeneity test in the SBP group aim to determine which variants come from the same variant and do not show significant differences from one another. Significance was set at p<0.05 for different data variants. The homogeneity test results of SBP data variance had a p-value of 0.001 (p<0.05), which indicated that the data variance was different, so a one-way ANOVA Tamhane’s test was performed.

SBP differences between groups

The one-way ANOVA test in the SBP group gained p-value of 0.000 (p<0.05). The test results showed significant difference in SBP between the treatment groups ( Table 4). Results of post hoc Tamhane analysis for comparison of SBP between groups obtained significance value of p<0.05 between K1 and K2, K2 and K4, K2 and K5, and K2 and K6, which showed significant differences in SBP between two treatment groups (Table 5).

Table 4. SBP between groups tested using one–way ANOVA.

Treatment group	n	Mean±SE	Minimum	Maximum	p-value
K1 (Normal)	4	112.3±3.1	105.7	119.3	0.000*
K2 (Control −)	4	165.7±1.5	161.3	168.0
K3 (Control +)	4	116.2±12.6	87.7	144.0
K4 (Extract)	4	124.8±2.0	120.3	128.7
K5 (Nano extract)	4	105.8±4.3	95.7	116.3
K6 (Nano chitosan)	4	112.0±5.0	109.3	133.7

* Significant.

The measurement was conducted post treatment on the Day 22.

Table 5. SBP comparison between groups analyzed using post hoc Tamhane’s.

Treatment group	Mean±SE	Minimum	Maximum	p-value
K1 and K2	−53.3±3.4	−73.6	−33.0	0.001*
K2 and K3	49.5±12.7	−54.9	153.9	0.353
K2 and K4	40.9±2.5	28.5	53.2	0.000*
K2 and K5	59.8±4.5	29.9	89.7	0.004*
K2 and K6	43.6±5.2	7.2	80.0	0.027*
K3 and K4	−8.5±12.7	−110.7	93.5	1.000
K3 and K5	10.3±13.3	−79.5	100.2	1.000
K3 and K6	−5.8±13.5	−92.0	80.3	1.000
K4 and K5	18.9±4.7	−8.6	46.4	0.189
K4 and K6	2.7±5.3	−30.9	36.4	1.000
K5 and K6	−16.1±6.5	−47.1	14.8	0.533

* Significant.

The measurement was conducted post treatment on the Day 22.

Differences in DBP between groups

Owing to the non-normal data distribution, the Kruskal–Wallis test was used to determine differences in DBP between groups. The analysis gained value of p=0.03, which means there was a significant difference in DBP between the treatment groups ( Table 6). Thereafter, a post hoc Mann–Whitney test was performed to determine differences between treatment groups. In the post hoc Mann–Whitney test comparing DBP between groups, the p-value was <0.05, which indicated differences in DBP between K1 and K2, K2 and K3, K2 and K4, K2 and K5, and K2 and K6 (Table 7).

Table 6. DBP differences between groups.

Treatment group	Mean±SE	Median	Minimum	Maximum	p-value
K1 (Normal)	69.4±2.3	70.0	63.3	74.3	0.03*
K2 (Control −)	126.5±6.0	129.3	110.0	137.6
K3 (Control +)	77.6±11.7	78.3	50.3	103.6
K4 (Extract)	82.5±3.1	84.8	73.3	87.3
K5 (Nano extract)	73.5±8.0	78.6	50.3	86.3
K6 (Nano chitosan)	83.3±9.0	90.5	56.3	96.0

* Significant.

The measurement was conducted post treatment on the Day 22.

Table 7. DBP comparison analyzed using post hoc Mann–Whitney.

Treatment group	p-value
K1 and K2	0.02*
K2 and K3	0.02*
K2 and K4	0.02*
K2 and K5	0.02*
K2 and K6	0.02*
K3 and K4	1.00
K3 and K5	0.66
K3 and K6	0.56
K4 and K5	0.38
K4 and K6	0.24
K5 and K6	0.14

* Significant.

The measurement was conducted post treatment on the Day 22.

Urine volume test

From the Kruskal–Wallis test results on urine volume, we obtained a p-value of 0.002 (p<0.05). The value indicated a significant difference in the increase in urine volume between the treatment groups ( Table 8). Furthermore, the post hoc Mann–Whitney test was utilized to examine differences between the treatment groups. Results of the post hoc Mann–Whitney test (p<0.05) showed a significant difference in the increase in urine volume between K2 and K3, K2 and K4, K2 and K5, K3 and K5, K3 and K6, K4 and K5, K4 and K6, and K5 and K6 (Table 9).

Table 8. Kruskal–Wallis test on urine volume.

Treatment group	n	Mean±SE	Median	Minimum	Maximum	p-value
K1 (Normal)	4	1.5±0.20	1.50	1.00	2.00	0.002*
K2 (Control −)	4	1.3±0.12	1.50	1.00	1.50
K3 (Control +)	4	2.2±0.25	2.00	2.00	3.00
K4 (Extract)	4	2.1±0.12	2.00	2.00	2.50
K5 (Nano extract)	4	3.3±0.23	3.25	3.00	4.00
K6 (Nano chitosan)	4	1.6±0.12	1.50	1.50	2.00

* Significant.

The urine volume increase measurements were performed 24 hours after the decrease in blood pressure on Day 22.

Table 9. Post hoc Mann Whitney on urine volume increase.

Treatment group	p-value
K2 and K3	0.01*
K2 and K4	0.01*
K2 and K5	0.01*
K2 and K6	0.18
K3 and K4	0.85
K3 and K5	0.03*
K3 and K6	0.04*
K4 and K5	0.01*
K4 and K6	0.04*
K5 and K6	0.01*

* Significant.

The urine volume increase measurements were performed 24 hours after the decrease in blood pressure on Day 22.

Discussion

One of the factors that cause primary hypertension is excessive salt intake and increased circulation of natriuretic hormone, which inhibits intracellular sodium transport and results in an increase in extracellular fluid volume due to salt accumulation in the body.^27,28 The decrease in SBP and DBP can be influenced by the contents of P. americana leaf extract, namely, flavonoids and quercetin compound.^7,14 Flavonoids and quercetin can reduce SBP and DBP because these compounds can inhibit ACE, which converts angiotensin I to angiotensin II causing vasoconstrictions and thus increasing blood pressure. Quercetin compound can inhibit ACE activity by 60.0%, increasing endothelial relaxation and widening blood vessels, so blood is smoothly supplied to the heart. Inhibition of ACE activity by P. americana leaf extract proves that the bioactivity of quercetin compound is functionally excellent for antihypertensives.^7,11,29

This study showed that the extract inhibited ACE by >50%. ACE needs to be inhibited because it converts angiotensin I to angiotensin II as a vasoconstrictor of precapillary arterioles and postcapillary venules. Inhibited production of angiotensin II can lead to lowering the blood pressure.^7,10,24,30 In addition, the study reveals that the extract contains potassium, and magnesium. Potassium afffect the blood pressure lowering through improvement of endothelial function, NO release, and vasodilatation.³¹ Magnesium plays role to blood pressure regulation partly via its sympatholytic property and vasodilator action. Beside, the antihypertensive effect is related to calcium channel blockage, increases in NO, and better endothelial function. Increasing NO level in serum lead to relaxing vascular smooth muscle cells and reducing blood pressure.^31,32

The current study reveals that the extract resulted in serum nitrite and nitrite means of >40 μmol/L and >80 μmol/L. Regarding the antihypertensive mechanism of action, an increase in nitrate and nitrite levels is important because they are related to blood pressure; a lower blood pressure indicates higher NO levels in the blood or vice versa.^17,33 Nitrates and nitrites increase level lead to the capacity dilating of the arteries.³⁴ The antihypertensive effects are related to reduced NADPH oxidase-derived oxidative stress. NADPH oxidase has an important role in the nitroso-redox imbalance.³⁵ Nitrite also works as a precursor for the systemic generation of vasodilatory nitric oxide, and exogenous nitrate administration leads to lowering blood pressure.³⁶

Moreover, SBP and DBP decreased because P. americana leaf extract has high antioxidant activity.²³ Antioxidant activity is related to Nitric Oxide Synthase (NOS) levels where quercetin can increase NOS activity in endothelial cells acting on arteries by stimulating or activating endothelium-derived relaxing factor, causing vasodilation of endothelial cells.^4,17 The results of this study showed that P. americana leaf extract was effective in reducing SBP and DBP until normal blood pressure is attained. Compared with furosemide, P. americana leaf extract showed lower SBP and DBP-reducing effect, but it was only slightly different. The nanoparticle method significantly reduces the frequency of doses related to pharmacodynamics but optimizes the efficacy on target organs related to pharmacokinetics.^37–41

The results of this study also show that the use of P. americana leaf extract decreased SBP and DBP owing to the diuretic activity of quercetin compound, as shown in the increased urine volume measured after treatment. Diuretics are compounds or drugs that can increase urine volume.^42,43 Flavonoid compounds and quercetin increase urine volume by inhibiting sodium reabsorption, triggering its discharge. As a result, the kidneys quickly remove waste products from the body.^4,44

There were some limitations to this study. Firstly, the potential zeta value in the P. americana Mill leaf extract nanoparticles is not yet stable, and there was no measurement of the potential zeta value for nano chitosan. Secondly, rats weighing less than 200 grams are difficult to adjust to the holder and rubber on CODA™ mouse rat tail-cuff system, causing blood pressure measurements to be more difficult than rats weighing over 200 grams. Thirdly, measuring the blood pressure of rats on day 0 before treatment was difficult because the rats had not adapted to the CODA™ mouse rat tail-cuff system causing the measurement time to be longer. Besides, the 16% NaCl induction group was placed in one cage where rats under stress conditions due to hypertension should be separated into individual cages to avoid death in rats.

This research was only limited to the potential of extracts and nanoparticles of P. americana Mill leaf chitosan extract. Further research is needed to test the toxicity of the kidneys and liver of test animals and on LD50 to determine the optimum dose of P. americana Mill leaf extract nanoparticles as a curative antihypertensive. Furthermore, additional research is needed to manufacture more modern nanoparticles such as capsules or tablets to produce products that can be applied to the public.

Conclusion

The use of P. americana leaf extract is effective in reducing SBP and DBP and thus helps achieve normal blood pressure. Though its blood pressure-reducing effect is lower than that of modern medicine (furosemide), the nanoparticles extract had the most significant result in lowering SBP and DBP. Administration of P. americana leaf extract significantly increased urine volume in tested rats.

Author contributions

Sutiningsih D: Conceptualization, Investigation, Resources, Project Administration, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Sari DP: Investigation, Data Curation, Formal Analysis; Adi MS: Methodology, Supervision, Formal Analysis; Hadi M: Methodology, Formal Analysis, Validation; Azzahra NA: Writing – Original Draft Preparation.

Data availability