The diabetic condition has an impact on
renal function. Diabetes can adversely affect renal function by causing damage
to the kidneys through processes such as glomerular damage, hypertension,
inflammation, the formation of advanced glycation end-products, changes in
renal blood flow, and impaired tubular function (1, 2). Over time, these factors contribute to the development of diabetic
nephropathy, characterized by progressive kidney damage and a decline in
function. This condition can change biochemical parameters in the bloodstream,
including the concentrations of creatinine and urea (3). Creatinine, a byproduct of creatine metabolism primarily from
muscle tissue, serves as a reliable marker for renal function. Urea, the end
product of protein metabolism, is excreted through urine, and its accumulation
in the blood is indicative of kidney dysfunction. Herbal treatments may offer a
holistic approach, potentially aiding in maintaining renal health and managing
these crucial biochemical markers in individuals with diabetes. Indeed, herbal
remedies, often containing multiple compounds, have the potential to work
synergistically to improve renal conditions and regulate metabolites (4).
Sonchus arvensis L., known as Tempuyung in Indonesia, is widely used as a
traditional medicine against nephrolithiasis (5). Sonchus arvensis L. ethanolic extract (SALEE) contains
mineral ions such as potassium, magnesium, silica, and sodium. Organic
compounds found in SALEE include
saponins, flavonoids, taraxasterol, inositol, mannitol, coumarin, polyphenols,
α-lactocerol, β-lactocerol, and phenolic acids (6). SALEE is said to have an adaptive immune response effect (5), increase potassium oxalate solubility (7), and reduce pro-inflammatory cytokines (8). It has also been reported to lower creatinine levels and improve
glomerular cell impairment through histopathological observations (9). Although SALEE holds promise as a supplement, direct extract
consumption might deter patients due to taste or impracticality for daily use.
Hence, a more convenient and practical formulation of SALEE is desirable for
ease of regular administration.
Suspensions, a pharmaceutical formulation
where active ingredients like plant extracts are broken down into small
particles and dispersed in a liquid medium, offer several benefits for
enhancing the effectiveness of extract usage (). Firstly, they enhance solubility by breaking down the extract into
tiny particles, facilitating better contact with the dispersing medium (). This improves the overall stability, preventing settling or
separation of the extract over time. The even distribution of small particles
ensures consistency in dosage across the formulation, aiding in absorption
through cell membranes for improved bioavailability. Additionally, suspensions
provide dosing flexibility and ease of use for patients, especially when
dealing with extracts with strong tastes or odors, allowing for efficient
utilization of the active ingredients (, ).
Declarations
Conflict of Interest
The authors declare no conflicting interest.
Data Availability
The unpublished data is available upon request to the corresponding author.
Ethics Statement
The study received approval from the Research Ethics Committee at the Faculty of Medicine, Tadulako University, with the assigned approval number 2576/UN.28.1.30/K/2019.
Based on the information provided, this
research aimed to investigate the potential therapeutic effects of SALEE in
suspension form on renal function and biochemical markers associated with
kidney health, specifically focusing on creatinine and urea levels, in a
diabetic rat model induced by streptozotocin.
Experimental Section
Material
Sonchus arvensis L. leaves were collected from
Pasangkayu district, West Sulawesi, Indonesia. Male Wistar rats were obtained from test animal suppliers based at Tadulako University. Materials used were NaCMC (Sigma Aldrich, St.
Louis, MO, USA), hydrochloric acid (PT Lamurindo, Jakarta, Indonesia), aqua pro injection (Bernofarm
Pharmaceutical Company, Sidoarjo, Indonesia), iron
(III) chloride (SinarLab, Jakarta,
Indonesia), citrate-buffer solution (Sigma Aldrich, St. Louis, MO, USA), dragendrof LP (Merck, Kenilworth,
NJ, USA), and
96% absolute ethanol (Sigma Aldrich, St. Louis, MO, USA).
Sonchus arvensis L leaves extraction
The extraction of Sonchus arvensis L. leaves was carried out
utilizing the maceration method. Initially, 1,050 grams of sifted simplicia
powder of Sonchus arvensis L leaves (from no. 40 mesh sifter) was weighed. Subsequently,
this powder was subjected to extraction using 96% ethanol solvent in a quantity
of 7.5 liters over a period of three days. The extraction process was divided
into three maceration vessels, with occasional stirring to prevent saturation.
The resulting filtrate was concentrated using a rotary evaporator at a
temperature of 60°C. Following this, the concentrated extract underwent further
evaporation using a water bath until a thick extract was obtained. The extract was sampled for initial
phytoscreening, it includes alkaloid, flavonoid,
saponin, fenol, and tannin (14, 15).
Sonchus arvensis L leaves extract suspension
The SALEE was weighed to prepare suspension dosage, each containing
0.4 g, 0.6 g, and 0.8 g.
Subsequently, 0.5% NaCMC was added to each extract, and the volume was adjusted with
distilled water to reach 25 ml. The suspension was then thoroughly shaken until
a homogeneous mixture was achieved.
Animal preparation
All experiments were conducted in adherence
to the animal welfare standards outlined by the World Organisation for Animal
Health (OIE) and were approved by the Research Ethics Committee, Faculty of Medicine, Tadulako
University,
under approval number 2576/UN.28.1.30/K/2019. Male Wistar rats weighing between
200–250 g were acclimated in specific local animal cages. These rats were given
a 14-day adaptation period. The selection criteria for the rats included being
approximately three months old male rats with body weight within the range of
200-250 g, having white fur, and displaying active behavior.
Animals treatment
Streptozotocin powder was weighed and
dissolved in pH 4.5 citrate-buffered saline with a final concentration of 10
mg/mL. All rats (in group II-V) were induced intraperitoneally (IP) at a dose
of 40 mg/kg BW (Tuldjannah et al., 2018). After the induction, blood samples were collected
and the creatinine and urea levels were measured spectrophotometrically. For the treatment, healthy control group (Group
I) and control negative group (Group II) received only 0.5% NaCMC. Group III-V
received 200, 300, and 400 mg/kg BW SALEE. All treatments were done daily for
14 days. Blood sample was taken from the tail on day 0, 7, 14, and 21.
Statistical
analysis
Statistical analysis was conducted to
determine a significance of difference between groups of treatments. ANOVA test was
used for multivariate comparison analysis, followed by Post Hoc tests (Tukey). All statistical analyses
were performed using R Studio (Version 4.2.1, RStudio Inc, Boston, USA).
Result and Discussion
Extract yield and
pythochemicals
The extract derived from 1,050 grams of
dried Sonchus arvensis L. leaves yielded 71 grams, which is a 6.76%
yield. A qualitative analysis of this extract confirmed the presence of
flavonoids, phenols, alkaloids, tannins, and saponins, aligning with existing
literature (16). Refer to Table 1 for a detailed analysis of the SALEE.
Table 1. Phytochemical test results for Sonchus arvensis L. leave ethanol extract suspension.
No
Secondary metabolite compounds
Result
SALEE
characteristics after reaction
Present
1
Alkaloid
An orange precipitate
formed
+
2
Flavoniod
Formed brick red color
+
3
Saponin
Formed froth that
persists for at least 1 minute
+
4
Fenol
Blackish green color
formed
+
5
Tannin
Blackish blue color
formed
+
Creatinine level
Elevated levels of creatinine serve as a
parameter for kidney failure. Creatinine is a byproduct of phosphocreatine
breakdown, a compound utilized by muscles for energy production. The kidneys
function to filter the blood and excrete creatinine through urine. In individuals
with diabetes, particularly poorly controlled type 2 diabetes, high blood
glucose levels can damage small blood vessels in the kidneys. This damage
impairs the kidneys' ability to efficiently filter the blood, causing
creatinine and other substances that should be excreted in the urine to persist
in the bloodstream. Accumulation of creatinine in the blood indicates a decline
in kidney function. The normal serum creatinine levels in rats range
from 0.4 to 0.8 mg/dL (17).
In Figure 1, it can be observed that all streptozotocin-induced groups
exhibit significantly higher levels of creatinine compared to the normal
control group (Group I, non-diabetic and no treatment) on day 0. This suggests
that streptozotocin can effectively induce an elevation in creatinine levels.
This aligns with the initial expectation that diabetes conditions can impact
kidney function, ultimately resulting in the accumulation of creatinine in the
blood. On day 0, it is also evident that the negative control group (Group II)
and Group III show a significant increase in creatinine levels compared to
Group I, whereas the other groups exhibit a non-significant increase in
comparison to Group I. This indicates that the immediate effects of the
high-dose SALEE (300 and 400 mg/kg BW) can be observed right after
administration.
Figure 1. Blood creatinine levels in male white rats. Note: (*, p<0.05) shows significant difference to Group I. (#, p<0.05) shows significant difference to Group V. (&, p<0.05) shows significant difference to group III.
On the 7th day, the
creatinine levels in Group II significantly increased, indicating a sustained
accumulation in the blood. All diabetes-induced groups still maintained
elevated levels on that day, but only Group III (receiving 200 mg/kg BW of
SALEE) did not show a statistically significant difference in creatinine levels
compared to healthy group. This suggests that a smaller dose of SALEE
demonstrates better and consistent long-term effects, even though these effects
are not immediately visible after administration as observed on day 0. By the
14th day, a decrease in creatinine levels began to be noticeable across all
groups, though it remained significant compared to both Group I and III. A
significant decrease in creatinine levels became evident on the 21st day, with
Group III displaying the lowest creatinine levels. Based on these observations,
it can be concluded that SALEE at a dose of 200 mg/kg BW is more efficient in
reducing the accumulation of creatinine in the blood.
Urea level
Urea is the main metabolite resulting from
the breakdown of dietary proteins and tissue protein turnover. It is relatively small
molecules (around 60 Dalton) and it disperses throughout the body's total water content. Serum urea nitrogen (BUN) can be quantified from
blood urea levels and constitutes roughly half (28/60 or 0.446) of the blood
urea. Normal BUN levels range from 5-20 mg/dL, and the BUN-to-serum creatinine
ratio is approximately 15:1 (18). Based on this literature, the healthy control
group (non-diabetic and untreated) demonstrates consistent results where their
blood urea levels range from 15-20 mg/dL, resulting in BUN levels of
approximately 6-9 mg/dL and serum creatinine levels of around 0.4 mg/dL. The
obtained BUN-to-serum creatinine ratio ranges from 15:1 to 22.5:1. However, the
observed blood urea levels appear to fluctuate from day to day. This
fluctuation aligns with existing literature, which confirms that BUN is more
influenced by dietary and physiological conditions than kidney function,
leading to fluctuations over time (18).
Figure 2. Blood urea levels in male white rats. Note: (*, p<0.05) shows significant difference to group IV, (#, p<0.05) shows significant difference to group I, ($, p<0.05) shows significant difference to group II, and (@, p<0.05) shows significant difference to group III.
In Figure 2, it can be observed that in Group IV (receiving 300 mg/kg BW of
SALEE), the blood urea levels are the lowest and significantly different
compared to the healthy control group (Group I). However, it is not possible to
draw any conclusions at this point because the negative control group also
experiences a reduction in blood urea. Nonetheless, the pattern of increase in
blood urea is more evident in Group V, followed by Group IV on the final day of
observation. As discussed earlier, although BUN accumulation can depict kidney
function, BUN levels can also be influenced by dietary and physiological
conditions (19). The increase in the BUN-to-serum creatinine
ratio observed on days 14 and 21 in Group V may be influenced by a higher
protein intake (18). This is supported by a study conducted by Yelvia
N. et al. (2014), where the body weight of mice significantly increased on day
90, and an increase in appetite was indeed observed (20).
Conclusion
The ethanol extract of Sonchus
arvensis L leaves contains various secondary metabolite compounds, such as
alkaloids, flavonoids, phenols, tannins, and steroids. SALEE has an impact on creatinine
and urea levels in white male rats induced by streptozotocin. A dose of 200
mg/kg BW of the SALEE shows a gradual and consistent decrease in serum
creatinine levels up to day 21, although immediate effects after administration
were not observed as seen with doses of 300 and 400 mg/kg BW. Interestingly,
BUN levels in the test subjects actually increased with higher doses of SALEE
(400 mg/kg BW). However, BUN levels also showed fluctuations in the
other groups, suggesting that the increase in BUN is influenced more by the
test subjects' appetite and higher protein intake. Based on the findings, it
can be concluded that suspension of SALEE (200 mg/kg BW) is a potential
treatment or supplement for diabetics-induced renal failure.
The study focused on analyzing the secondary metabolites present in the ethanol extract of Sonchus arvensis L. leaves (SALEE) and their impact on creatinine and urea levels when administered in suspension form at different doses. Phytochemical screening of SALEE identified alkaloids, flavonoids, phenols, saponins, and tannins. In the experiment, twenty-five white male rats were divided into five groups: a healthy control group (Group I), a negative control group (Group II), and three test groups (Groups III, IV, and V) receiving SALEE at doses of 200, 300, and 400 mg/kg BW, respectively. After inducing diabetes with streptozotocin and a 21-day follow-up period, creatinine and urea levels were assessed. The results demonstrated that SALEE administration significantly reduced creatinine levels and increased urea levels. Notably, the 200 mg/kg BW dose of SALEE exhibited a consistent and long-term effect. It was observed that blood urea levels fluctuated even in healthy and negative control groups during the 21-day observation period. In the group receiving the highest dose of SALEE (400 mg/kg BW), blood urea levels increased significantly on days 14 and 21, indicating a potential increase in the rats' appetite rather than renal function failure. Overall, the findings suggest that SALEE has promising potential to regulate creatinine and urea levels in the blood of individuals with diabetes, highlighting its potential for therapeutic and supplemental use.
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