INTRODUCTION:
The use of medicinal plants is an ancient
practice, forming the foundation of herbal medicine concept. In general, herbal
medicine has various advantages, including widespread availability with
abundant supply, relatively safe, minimal side effects, and lower costs
compared to conventional treatment1.
However, the utilization of plants as raw
materials is usually based only on empirical experience and requires scientific
investigations to determine the effective concentration of chemical contents to
convince the public2.
The increasing popularity of the back-to-nature lifestyle also
drives the resurgence of plant utilization as herbal medicine, which has a
positive effect on health. This trend leads to more frequent studies on
medicinal plants by scientists, covering cultivation aspects, chemical
contents, pharmacological effects, and several uses for health3. Herbal
medications plays a big role in the treatment of diseases of urinary system and
contribute to the improvement of the functional state of kidneys and urinary tract4. Herbal medicines have very little or no side effects as compared to the allopathic
drugs. Hence search for a new diuretic agent that retains therapeutic efficacy and yet having minimum side effects
is justified5.
A popular plant with the potential to be
developed as herbal medicine is bilimbi fruit (Averrhoa bilimbi L.),
efficacious in facilitating urine excretion (diuretic). Additionally, the
plant is believed to treat various diseases such as cough, diabetes,
mouth sores, toothache, bleeding gums, acne, pain relief (analgesic), and high
blood pressure (antihypertensive)6. Averrhoa bilimbi L.
contains chemical
compounds including oxalic acid and potassium, as well as flavonoids, oxalates,
volatile oils, phenols, saponins, and pectin. Fruit of Averrhoa bilimbi L. have 53 different
components were identified as the volatile constituens. Aliphatic
acids constitute 47.8% of the total volatiles. The main constituents were
hexadecanoic acid (palmitic acid) (20.4%), 2-furaldehyde (19.1%), and
(Z)-9-octadecenoic acid (10.2%). Twelve of the compounds identified were esters
among which butyl nicotinate (1.6%) and hexyl nicotinate (1.7%) were present in
higher quantities7. In the other research sixty-two compound were
identified, of which nonanal and (Z)-3-hexenol were dominant8. Flavonoid compounds, particularly
flavonols, are known for biological activity as diuretics. The mechanism of
action entails inhibiting the reabsorption of Na+, K+, and Cl-, leading to an
increase in electrolytes such as Na+ and water volume in the tubules, thereby
causing diuresis. The increased excretion enhances water excretion, leading to
an increase in urine volume9.
According to Jouad et al. (2001),
flavonoids can increase urine volume by raising the glomerular filtration rate
(GFR)10. In the other research Pongamia pinnata Linn
shows diuretic activity in
phyto chemical screening indicated the presence of Alkaloids, Phyto sterols,
Flavonoids and Saponins11. Averrhoa
bilimbi L. contains
flavonoids capable of acting as natural diuretics, namely substances that can
increase the rate of urine volume excretion.The other research of Shelvia conducted a study on the
effectiveness of Averrhoa bilimbi L11. infusion as a diuretic on male
white rats12. The results of testing infusion at
concentrations of 5%, 10%, and 20% showed significant activity in increasing
urine excretion. Furthermore, Andriyanto et al. (2013) used ethanol extract of Averrhoa
bilimbi L13. at three doses, namely 1.75 g/kgBW, 0.88g/kgBW, and
0.44g/kgBW. The results showed that doses of 0.88 and 1.75g/kgBW had strong
diuretic activity accompanied by an increase in sodium and potassium excretion
in the urine. This proves that ethanol extract of Averrhoa bilimbi L. has
the potential to be a natural diuretic.Similar results were reported by Poniman
(2011) who aimed to determine the potential of Averrhoa bilimbi L. ethanol
extract as a natural diuretic in male Sprague-Dawley white rats. The
results showed that doses of 0.44 and 0.88g/kg body weight had the potential as
a natural diuretic due to constituent flavonoid compounds, alkaloids, and
saponins14. This was demonstrated by the increased diuretic activity
and the excretion of sodium-potassium levels as well as reduced pH value in the
urine. In the other research Xanthium strumarium L shows diuretic activity15. Diuretic effect its potential diuresis is a
vital function for the living being by which the organism excretes all the
waste resulting from the cellular combustion, once it has kept the necessary
substances to feed itself and produce energy, it is considered among the
functions of elimination16
In this study,
fractionation methods were used to specifically extract flavonoid compounds,
particularly flavonols. In general, fractionation is a process of separating
compounds based on the level of polarity. The number and types of compounds
separated into fractions vary depending on the plant species. Flavonol
compounds, which are less polar, tend to dissolve more easily in non-polar
solvents, while ethyl acetate was used as the solvent. Fractionation with ethyl
acetate, a low-toxicity semi-polar solvent, is capable of extracting compounds
with a wide range of polarity, from polar to nonpolar. Ethyl acetate solvent
can also be used for extraction due to the affinity for compounds such as
flavonoids, alkaloids, saponins, tannins, polyphenols, and triterpenoids, with
significant diuretic properties17,18
Therefore, this study aimed
to test the diuretic activity of bilimbi (Averrhoa bilimbi L.) fruit
ethyl acetate fraction in male rats (Rattus norvegicus).
MATERIALS AND METHODS:
Materials:
The materials used in this study include
bilimbi fruits (Averrhoa bilimbi L.), distilled water, 96% ethanol, 40
mg furosemide tablets (Furosemide®), n-hexane, ethyl acetate, 1% Sodium carboxymethyl
cellulose (Na CMC), concentrated sulfuric acid (H2SO4), glacial
acetic acid (CH3COOH), Mayer's reagent, magnesium powder (Mg),
ferric chloride (FeCl3) 1%, urea, concentrated hydrochloric acid
(HCl), and 2N HCl.
Methods:
Sampling of Bilimbi Fruit:
Fresh bilimbi fruits (Averrhoa bilimbi L.)
were collected from Surau Tinggi Utara Village, Rambah Hlir Sub-district, Rokan
Hulu Regency, Riau Province.
Figure 1. Fresh bilimbi fruits (Averrhoa bilimbi L.)
Identification of Sample Plants:
Plant identification was carried out at the
Botany Laboratory, Biology Department, Faculty of Mathematics and Natural
Sciences (FMIPA), University of Riau (UNRI), Pekanbaru.
Preparation of Test Animals:
The animals used were 5 male white rats per
group, 25 of which were in good health with a body weight between 100-250grams.
The rats were first acclimatized to laboratory conditions for 7 days by being
given sufficient food and drink as well as adapting for 1 hour before treatment
in metabolic cages. Before the experiment, the rats were declared healthy when
significant deviations did not occur in body weight (≥10%).
Preparation of Bilimbi Fruit Extract:
The extraction process started with the
preparation of the simplicia, the fruits were sliced with a thickness range of
0.2-0.5cm, dried, and placed in an oven at 50˚C until completely dried.
This was followed by grinding into powder using a blender, which was then
sieved through a No. 40 mesh sieve to obtain fine simplicia powder. The
finished powder was stored in a tightly closed container. Extraction of ethanol extract from Averrhoa bilimbi L. was
carried out using the maceration method. This entailed soaking the simplicia
powder in a dark glass maceration vessel and immersing in 96% ethanol solution
until the simplicia was completely submerged. The maceration vessel was tightly
sealed and left to stand for 3 days. During the soaking period, stirring was
carried out once daily, repeated 3 times, subsequently, the soaked material was
filtered to obtain the filtrate, and the residue was soaked again using fresh
solvent. This was repeated until the extraction process was complete. The
obtained macerate was then precipitated, and the precipitate was discarded,
while the filtrate was collected. The filtrate was concentrated using a rotary
evaporator at 50ºC until a thick extract was obtained19.
Preparation of Ethyl Acetate Fraction:
The fractionation process started by weighing
30grams of the thick extract from bilimbi fruits (Averrhoa bilimbi L.)
and dissolving in 90ml of distilled water in a beaker glass until completely
dissolved, then transferring into a separating funnel. The ethyl acetate
fraction was the filtrate located above, while the water fraction was below.
Phytochemical Screening of Ethyl Acetate
Fraction from Bilimbi Fruits:
Alkaloid Test, Flavonoid Test, Saponin Test, Terpenoid and Steroid Tests,Tanin and Polifenol Tests
Preparation of Ethyl Acetate Fraction
Suspension of Bilimbi Fruits:
Test preparations were made with 3 dose
variations for rats with a body weight of 200grams. The administration process
was carried out orally with each dose composed of 20ml: Dose 0.44g/kgBW, Dose 0.88g/kg BW, Dose 1.76g/kgBW
Dosage Determination:
The dose of Averrhoa bilimbi L. fraction
given to experimental rats was divided into three treatment groups with a dose
of 0.44g/kgBW, 0.88g/kgBW, and 1.76g/kgBW. The
volume used for administration was 1% of the rat’s body weight.
Grouping Experimental Animals:
The experimental animals were randomly
divided into 5 groups, each consisting of 5 male white rats, with a weight
difference of no more than 20%. The rats were fasted for approximately 18hours
before the experiment, and 30 minutes before the treatment, 1 ml/100g BW of
warm water was administered. The treatment was conducted once a day per group,
with each group receiving the following oral treatment:
Testing the Effectiveness of Averrhoa
bilimbi L. Fraction using the Lipschitz Method.
The diuretic test method was carried out
according to Lipschitz et al (1943) with slight modifications20.
Before the test, the rats were acclimatized for 7 days and then fasted for 18
hours with only water (distilled) provided. Additionally, warm water was given
orally at a dose of 1 ml/100g body weight. For rats weighing 200grams, warm
water was given at a dose of 2ml/200g body weight to each 30 minutes before the
treatment as a loading dose. The rats were divided into 5 groups, each
consisting of 5 members, and given treatment once a day per group every
morning.
The procedure for testing the diuretic effect
according to the Lipschitz method is as follows:
1. Rats were grouped randomly into each group and
were fasted for 12-18 hours before the experiment.
2. Before the body weight experiment, the rats
were weighed and the volume given to each was calculated.
3. After giving the test preparation orally
according to the group, the rats were placed in individual metabolic
cages and the volume of urine excreted was recorded.
Urine Volume Measurement:
The measurement of urine volume was conducted
by placing individual rats in metabolism cages equipped with urine collection
containers. The urine volume of each rat was measured every hour of observation
at 1, 2, 3, 4, 5, and 6 hours. Subsequently, using a disposable syringe, the
urine collected was drawn and the volume was recorded throughout the
observation period21,22
Data analysis:
The parameters observed include the
measurement of urine volume, percentage of urine excretion, diuretic work, and
diuretic activity induced by each group. The diuretic activity data obtained
were compared with the diuretic scale of Gujral19. The
"Lipschitz Value" was also obtained, which refers to the comparison
between the excretion of the test animal and urea control (T/U). An index of
1.0 or more is considered to have a positive diuretic effect, and a diuretic is
considered strong when the index is 2.0 or more. Qualitative data are verbal
sentence-based information rather than numerical symbols or numbers.
The urine volume data obtained were used to
calculate the observed parameters according to the methods proposed by Mamun et
al. (2003) and Mahmood et al. (2004)23,24. The formulas used for
each variable are presented as follows Quantitative data are
information in the form of numerical symbols or numbers25. The
quantitative data in this study include the measured urine volumes. The data
obtained were analyzed using the statistical program SPSS (Statistical
Package for the Social Sciences) Version 26. Data analysis was conducted by
measuring urine volume at 1, 2, 3, 4, 5, and 6 hours for each group.
Significant differences between treatments were tested using a two-way analysis
of variance (Two-way ANOVA) and followed by the post hoc Tukey test. Normal
distribution of the data was tested using the Shapiro-Wilk test and homogeneity
of variances was assessed with Levene's test with a confidence level of 95%.
RESULT:
The results showed that 13 kg of fresh Averrhoa
bilimbi L. fruits were used to make the sample, resulting in 500 grams of
dried powder simplicia. Furthermore, extraction through maceration of ethanol
extract (104.93 g; 21% yield). Fractionation of the thick extract yielded 8.69
grams of ethyl acetate fraction with a yield of 9.66% as shown in Table 1.
The phytochemical screening results of fresh Averrhoa
bilimbi L. fruit samples, ethanol extract, and ethyl acetate fraction are
presented in Table 2.
Table 4. Calculation results of diuretic power
(potency) or percentage of rat urine excretion per hour of observation
|
Treatment
|
Urine Excretion (%) Every Hour To -
|
|
1
|
2
|
3
|
4
|
5
|
6
|
|
K (-)
|
44
|
33
|
18
|
10
|
10
|
0
|
|
K (+)
|
124
|
148
|
162
|
146
|
132
|
95
|
|
KP1
|
82
|
114
|
126
|
114
|
88
|
80
|
|
KP2
|
91
|
118
|
130
|
138
|
83
|
84
|
|
KP3
|
104
|
127
|
147
|
130
|
106
|
96
|
|
KPurea
|
34
|
42
|
57
|
66
|
68
|
131
|
The diuretic activity of each observation
group was calculated using the formula, and the results are presented in Table
5.
Table 5. Calculation results of diuretic action
|
Treatment
|
Diuretic Action (mL) Every Hour To-
|
|
1
|
2
|
3
|
4
|
5
|
6
|
|
K (-)
|
-
|
-
|
-
|
-
|
-
|
-
|
|
K (+)
|
2.81
|
4.48
|
9
|
14.6
|
13.2
|
|
|
KP1
|
1.89
|
3.45
|
7
|
11.4
|
8.8
|
∞
|
|
KP2
|
2.07
|
3.58
|
7.22
|
13.8
|
8.3
|
∞
|
|
KP3
|
2.36
|
3.85
|
8.17
|
13
|
10.6
|
∞
|
|
KPurea
|
0.77
|
1.27
|
3.17
|
6.6
|
6.8
|
∞
|
The diuretic activity test aims to determine
the potency of formulations used as diuretics. The results obtained are presented
in Table 6.
Table 6. Calculation results of rat diuretic
activity
|
Treatment
|
Diuretic Activity (mL) Every Hour To-
|
|
1
|
2
|
3
|
4
|
5
|
6
|
|
K (-)
|
-
|
-
|
-
|
-
|
-
|
-
|
|
K (+)
|
3,64
|
3,52
|
2,83
|
2,21
|
1,94
|
∞
|
|
KP1
|
2,45
|
2,71
|
2,2
|
1,72
|
1,29
|
∞
|
|
KP2
|
2,68
|
2,81
|
2,27
|
1,09
|
1,22
|
∞
|
|
KP3
|
3,07
|
3,04
|
2,58
|
1,97
|
1,56
|
∞
|
The diuretic potential was determined by
calculating the percentage of cumulative urine volume over 6 hours to the
initial volume of warm water given orally to the rats. The calculation of
diuretic potential is presented in, while the results or percentage of rat
urine excretion are shown in Table 4.
To examine the differences in means for each
treatment group, statistical analysis was conducted using ANOVA. The two-way
ANOVA test results showed a significant difference in rat urine volume
measurements, indicated by a significance value of P > 0.05. Homogeneity
test results showed a significance value greater than (0.05) (P>0.05),
indicating that the data were normally distributed and homogeneous. This
implies that the testing can proceed after meeting the criteria for two-way
ANOVA. The results showed a significant value for treatment groups on urine
volume (p <0.05), a significant difference in time (p <0.05), and a
significant influence of treatment groups on time (p <0.05). To determine
the significant mean values between each treatment group, the Tukey Post Hoc
test was used. The results showed that the formulation with an effective dose
was ethyl acetate fraction group with a dose of 1.76 g/kgBW and the best
diuretic effect occurred at 4 hours.
DISCUSSION:
This study was conducted to investigate the
diuretic activity of the ethyl acetate fraction of Averrhoa bilimbi L.
fruits on male white rats. Secondary metabolites suspected to be abundant in
the plant are flavonoids, particularly flavonol compounds, known to possess
biological activity as diuretics. The mechanism of action entails inhibiting
the reabsorption of Na+, K+, and Cl-. This leads to an increase in electrolytes
such as Na+ and water volume in the tubules, resulting in increased urine
volume (diuresis). Flavonoids can raise urine volume by increasing the
glomerular filtration rate (GFR)21.
The Averrhoa bilimbi L. fruits used
were obtained from Surau Tinggi Utara Village, Rokan Hulu Regency, Riau
Province. In the initial stage of the study, plant identification was conducted
at the Botany Laboratory of the Biology Department, Faculty of Mathematics and
Natural Sciences (FMIPA), Universitas Riau (UNRI) in Pekanbaru.
The maceration process was carried out for 3
days with 3 repetitions to ensure maximum compound extraction. Proper
maceration leads to a high yield of extracted compounds and a short maceration
time may result in incomplete solubility of all compounds in the solvent. On
the other hand, prolonged maceration time may lead to compound degradation3.
The maceration produced 104.93g of extract with a percentage yield of 21%.
After obtaining the crude extract, fractionation was performed to specifically
extract flavonoid compounds, particularly flavonols, using liquid-liquid
fractionation with a separating funnel. The purpose of fractionation is to
obtain specific parts (fractions) of the extract26. The
fractionation produced 8.69g of ethyl acetate fraction with a percentage yield
of 9.66%. The calculation of yield aims to determine the ratio of extract
obtained to the initial weight of the simplicia as well as to determine the
amount of bioactive compound content in the extract after extraction with the
sample weight used. The yield is considered good when the value is greater than
10%. The higher the yield, the higher the content of the attracted substances
in a raw material27.
The phytochemical screening results showed
that fresh Averrhoa bilimbi L. fruits contain
alkaloids, flavonoids, saponins, and terpenoids. The screening of ethanol
extract indicated the presence of alkaloids, flavonoids, saponins, steroids,
and terpenoids, while the ethyl acetate fraction was found to contain
flavonoids and saponins. The difference in the number of secondary metabolites
in each phytochemical screening may be attributed to the extraction process,
where the solvent used is adjusted to the polarity level of the compound groups
to be extracted. The selection of the solvent should be appropriate depending
on the type of plant material compounds to be tested because each solvent has
the ability to attract different molecule compounds depending on the similarity
of the polarity properties28.
This study used male white rats (Rattus
norvegicus) as experimental animals due to the ability to maintain a more
stable biological condition and not being influenced by estrus (menstrual)
cycles and pregnancy compared to females. Another reason for selecting male
white rats is the potential for faster drug metabolism rates and larger body
weights, allowing for larger urine output. A total of 25 male white rats
weighing approximately ± 200grams, and divided into 5groups were used. This is
in accordance with the World Health Organization (WHO) guidelines in the
Research Guidelines for Evaluating the Safety and Efficacy of Herbal Medicine,
stating that studies using rodents should consist of at least 5 animals in each
treatment group. Each rat was placed in a separate cage. The cage floor was
covered with sawdust evenly, while food and water were provided daily. The food
provided was standard rat feed, and the drinking water was placed in plastic
bottles with standard aluminum pipes commonly used for rats.
Before testing, the rats were acclimatized for
±7 days, adjusting to the acclimatization period required by the USDA
Guidelines Stabilization/Acclimation Times for Research Animals29,30.
The purpose was to adapt the rats to the test environment conditions, without
causing stress.
The positive control used in this study was
furosemide which has a rapid onset of action and is a potent diuretic commonly
applied as a standard comparator in diuretic testing. The mechanism of action
is to increase urine production by inhibiting the absorption of Na+, K+, and Cl
ions in the ascending limb of the loop of Henle, which has a high capacity for
NaCl reabsorption, leading to a significant diuretic effect. Furosemide was
suspended with 1% Na CMC because it is practically insoluble in water at a ratio
of 1: >10,000 according to the Indonesian Pharmacopoeia third edition.
Based on the results, the lowest urine
volume was observed in the negative control group given 1% Na CMC suspension
compared to other treatment groups. This occurred because the negative control
did not contain active ingredients that could increase urine volume, leading to
minimal urine excretion (diuresis)31. Meanwhile, the positive
control group treated with furosemide had the highest mean cumulative urine
volume compared to other treatment groups, indicating that furosemide had a
strong diuretic effect. The ethyl acetate fraction group at a dose of 1.76g/kgBW
had the second-highest average urine volume output after furosemide, suggesting
the treatment demonstrated the strongest diuretic effect among the three doses
given. Urine volume measurements were used to calculate diuretic activity and
the results showed that the larger the dose of the administered formulation,
the greater the impact on urine volume excretion (diuresis).
In line with the method used by Mahmood et al.
(2004), the diuretic potency or percentage of urine excretion was determined by
calculating the percentage of total cumulative urine volume collected over 6
hours to the initial volume of warm water given25. The highest
percentage was obtained by the group treated with Averrhoa bilimbi L. fruits
fraction at a dose of 1.76g/kg BW, with a maximum urine excretion percentage
occurring at the third hour of administration, reaching 147%.
Further calculations were performed to obtain
the diuretic activity, where the comparison results obtained were used as a
measure of the level of diuresis induced by the test substances. Following the
method by Mamun et al. (2003), diuretic activity was obtained by dividing the
percentage of urine excretion in the treatment groups by the percentage in the
normal or negative control group (1% Na CMC). The diuretic activity, with the
average closest to the furosemide group, was obtained in the ethyl acetate fraction
group at a dose of 1.76 g/kg BW. At the 6th hour, the diuretic activity of all
treatments had an undefined value. This was because the divisor factor in the
diuretic activity of the negative control group (1% Na CMC) had a percentage of
urine excretion at zero or no longer producing urine.
The diuretic activity calculation was
performed to determine the strength of the preparation used as a diuretic,
based on the scale by Gujral23. This parameter was evaluated using
the result of the diuretic work value of the treatment groups compared to the
urea group as a reference. The diuretic activity in the urea group has a value
of 1 (Lipschitz, 1943) and is easily soluble, which can increase osmotic
pressure, leading to greater excretion of water and electrolytes. Based on the
the scale by Gujral et al. (1955), a value < 0.72, 0.73-1.0, 1.1-1.5, and
>1.5 implies no diuretic activity, weak, moderate, and strong diuretic
activity respectively. In a study conducted by Gujral et al. (1955), the
results showed that the ethyl acetate fraction of Averrhoa bilimbi L. fruit
was effective as a diuretic at a dose of 1.76g/kg BW. The second-highest
diuretic activity was closer to the value of the positive control group
(furosemide). Despite the high dose, the rats remained stable, supporting the
empirical experience of the community that Averrhoa bilimbi L. fruit has
diuretic properties.
Consuming a large amount of medicinal plants
without considering the optimal dosage may not provide the desired benefits,
and cause harmful consequences. In herbal studies, dosage increments should be
ensured because an increase may not necessarily raise the urine volume output.
This indicates that some plants have a threshold dosage capable of providing
efficacy. It is necessary to consume herbal plants in appropriate amounts while
also considering the effective dosage. The diuretic activity of ethyl acetate
fraction from Averrhoa bilimbi L. fruit is due to the active
ingredients. When used properly, traditional medicine is considered safer than
synthetic drugs. The abundance of substances found in Averrhoa bilimbi L.
fruit, specifically the flavonoid content, alters the body's fluid balance and
triggers the kidneys to mobilize fluids. The increase in osmotic pressure
within the tubule lumen due to the administration of the ethyl acetate fraction
caused water diffusion from inside the cells toward the lumen. This event leads
to an increase in the volume of water in the urine32,33. Therefore,
it is suspected that the ethyl acetate fraction belongs to the category of
osmotic diuretics.
The data analysis used a two-way ANOVA to
examine the influence of treatment groups and time on urine volume output with
a significance level of α = 0.05. The Tukey test was only continued when
the p-value was <0.05 to determine the significantly different groups. The
results indicate a significant difference in the average urine volume output
among the test groups given the ethyl acetate fraction of Averrhoa bilimbi L. fruits.
The Shapiro-Wilk normality test was conducted
to determine whether the data was normally distributed or not. Based on the results,
the significance value (p> 0.05) indicates that the data was normally
distributed. The Shapiro-Wilk method is a normality test used for samples with
a small sample size (<50). Data is considered normally distributed when the
significance value (p) is >0.05, but when (p) is <0.05, data is
considered not normally distributed.
The homogeneity test, which is a
requirement to proceed with the ANOVA aims to determine data homogeneity. Data
is considered homogeneous when the significance value (p) is >0.05. Based on
the results, the obtained data was homogeneous with a significance value (p
> 0.05). Subsequently, the two-way ANOVA test was conducted to examine
significant differences with a significance value (p) <0.05. The results
showed a significant effect of the treatment on urine volume, as indicated by
the significance value (p) of < 0.05. Furthermore, there was a significant
difference in time, indicated by (p < 0.05). This implies that the timing of
drug administration has a significant effect on urine volume.
The Post Hoc Tukey analysis was conducted
to determine whether the obtained data has significant differences and to test
all pairs of treatments. Data was considered to have a significant difference
when the harmonic mean value was in different subsets. In the treatment groups,
the dose of 1.76g/kg body weight was in the same subset as the positive control
group of furosemide. This implied that the dose was effective because it did
not differ significantly from the positive control group. The doses of 0.44g/kg body weight, 0.88 g/kg body
weight, 1.76g/kg body weight, and the positive control differed significantly
from the negative control, proving that the ethyl acetate fraction at a dose of
0.44g/kg body weight has diuretic activity. However, the most effective dose
was 1.76g/kg body weight which was in the same subset as the positive control.
The results for the time groups showed that the highest amount of urine was
produced in the 4th hour, indicated by the subset differences. Averrhoa bilimbi L. fruits
can be diuretic plants help us
get rid of excess water as well as cleanse our body of harmful toxins and
metabolitic waste through increased urination34.
CONCLUSION:
In conclusion, the ethanol extract of Averrhoa
bilimbi L. yielded 104.93 grams with a yield
percentage of 21%, while the ethyl acetate fraction yielded 8.69 grams with a
percentage of 9.66%. Phytochemical screening showed the presence of alkaloids,
flavonoids, saponins, and terpenoids. The ethanol extract was found to contain
alkaloids, flavonoids, saponins, steroids, and terpenoids, while the ethyl
acetate fraction was composed of flavonoids and saponins. Based on the results
of secondary metabolite testing, flavonoids, specifically flavonol compounds
may contribute to the diuretic effect.
The urine volume measurements were used to
calculate the diuretic potency, with the highest percentage found in the ethyl
acetate fraction group at a dose of 1.76 g/kg body weight. The ethyl acetate
fraction group at a dose of 1.76g/kg body weight showed effective diuretic
activity. Furthermore, the fraction was effective as a diuretic, showing the
highest diuretic activity after the positive control group. The effective dose
in increasing rat urine volume was found to be the ethyl acetate fraction of
1.76g/kg body weight, which showed urine volume comparable to the positive
control. The ANOVA analysis results showed that the ethyl
acetate fraction had diuretic activity at doses of 0.44g/kg body weight, 0.88g/kg
body weight, and 1.76g/kg body weight, as evidenced by significant differences
(p< 0.05) compared to the negative control. Furthermore, Post hoc Tukey test
results showed that the effective dose was in the group given ethyl acetate
fraction at a dose of 1.76g/kg body weight, and the best diuretic effect was
produced at the 4th hour.
CONFLICT OF INTEREST:
The
authors have no conflicts of interest regarding this investigation.
ETHICAL
CONSIDERATION:
The study was approved by the Ethics Committee of The
Abdurrab University No.079/KEP-UNIVRAB/VII/2023.
ACKNOWLEDGMENTS:
The authors would like to thank to Abdurrab University
for the funding of this research and their kind support during research and
all other lab studies.
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