Keywords
diabetes mellitus - Streptozotocin - Acacia catechu - nephroprotective
Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, glycosuria,
and negative nitrogen balance and it is mainly due to absolute deficiency or diminished
effectiveness of insulin. Survey reports had suggested that currently, 366 million
people are diabetic in the world and also been predicted that it will reach up to
552 million people by 2030.[1] With the current antidiabetes therapies, it cannot be completely curable. The mortality
in DM is accounted for its complications, such as nephropathy, neuropathy, and retinopathy.
It is estimated that approximately world’s 30% of diabetic patients progress to diabetic
nephropathy (DNP). The high concentrations of blood sugar damages the kidney tissues
thus leading to altered kidney function in the patients, causing DNP and develop an
end-stage renal disease.[2] DM is an important etiopathological factor in oxidative stress.[3] As a result of lipid and protein oxidation, the levels of superoxide dismutase (SOD),
glutathione peroxidase (GSH-Px) increases in kidneys.[4]
[5]
[6] Previous studies have demonstrated that DM exhibits enhanced oxidative stress and
highly reactive oxygen species (ROS) in pancreatic islets due to persistent and chronic
hyperglycemia, thereby depletes the activity of the antioxidative defense system,
and thus promotes the free radical generation.[7]
In DM patients, there is an increase in the level of type-IV collagen fibers with
the concomitant decrease in the level of laminin and heparan sulfate, thus affecting
the pore size and selectivity, causing kidney damage. The kidney damage in DNP is
manifested histologically by the thickening of the glomerular basement membrane, mesangial
matrix expansion, macrophage infiltration, podocyte loss, and tubular epithelial degeneration.[8] Existing therapy for DM is known to support glycemic control but it is believed
to do little in regard to the complications to the various organs. Besides, these
antidiabetic drugs are associated with mild-to-moderate sideeffects.[9]
Though different types of oral hypoglycemic agents are available along with the insulin
for the treatment of DM, there is an increased demand by patients to use natural products
which have antidiabetic activity. Therefore, herbal drugs are gradually gaining popularity
in the treatment of DM. The major qualities of herbal medicines are less costly, easily
available, efficacious, and have low incidence of serious adverse effects.
In India, since time immemorial, patients with noninsulin dependent diabetes are treated
orally with a variety of herbal drugs extracts. In Ayurveda literature, the numbers
of plants were mentioned which have antidiabetic properties.
In view of this, the present study has investigated the effect of an ethanolic extract
of leaves of Acacia catechu (A. catechu) in the management of DM in STZ-induced Wistar rats. STZ through its toxic effects
induces oxidative stress in the β cells of the pancreas, [10] so it is frequently used to induce DM in experimental animals. The diabetogenic
action of STZ is the direct result of irreversible damage to the pancreatic β cells
resulting in degranulation and loss of capacity to secrete insulin.[11] The STZ effect on different organs has been extensively studied. Various studies
have been done by using STZ to establish rat model of diabetic nephropathy.[12]
Acacia catechu wild belongs to Fabaceae family and mimosoideae subfamily. The generic name, “Acacia,” comes from the Greek word akis, meaning a point[13] which is distributed mainly in south India. Its bark root and heartwood has medicinal
uses. The main chemical constituents of A. catechu are flavonoids, alkaloids, sugars, glycosides, and tannins.[14]
A. catechu wild has been shown to possess multifarious medicinal properties such as antibacterial,[15] anticancer,[16] hypoglycemic, antidiarrhoeal[17]
[18] anti-inflammatory, antioxidant,[19] hepatoprotective,[20] sore throat, wound healing, etc.
However, systemic and scientific reports on the investigation of ethanolic extract
of leaves of A. catechu for its effect on renal function are scarce. This study was designed to know the
nephroprotective effects based on histopathological changes and antioxidant status
in A. catechu with STZ-induced nephrotoxic rats.
Materials and Methods
Plant Material
The leaves of A. catechu were identified, collected, and authenticated by a Botanist. The leaves were dried
in shade and powdered in our research laboratory with the help of pulverizer. The
powder was subjected to soxhlet extraction with 95% ethyl alcohol for 72 hours at
a temperature of 70 to 80°C. The extract was concentrated to a small volume and then
evaporated to dryness. This was then dissolved in sterile saline and administrated
orally to the rats. Plant extract dose for experimental rats were selected based on
in vivo acute toxicity study and its in vitro antioxidant potential compared with
vitamin C.
Experimental Animals
Male albino rats 9 to 11 weeks old, weighing between 200 and 250 g were used for the
experiment. All animals were maintained under standard laboratory conditions, with
a constant 12-hour light/dark cycle and controlled temperature (25 ± 2°C) with free
access to drinking water and pellet diet ad libitum.
This study was performed in a Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA) approved laboratory under registration number 115/1999/CPCSEA
following all ethical practices as laid down in the guidelines for animal care. This
study has been approved by the Institutional Animal Ethics Committee (IAEC; reference
number KSHEMA/AEC/31/2011).
Chemicals
All the chemicals including STZ to induce DM and ether were purchased from Sri Durga
Laboratory Equipment Supplies at Chilimbi main Road in Mangalore, citrate buffer (pH:
4.5) was used as a solvent to dissolve STZ.
Induction of DM
The animals were fasted for 16 to 18 hours with free access to water prior to the
experiment. STZ of 45 mg/kbw was dissolved in 0.1 M citrate buffer (pH 4.5) and the
same was given a single dose of intraperitoneal administration (IP) to induce DM.[21] Then 5% sucrose was supplemented for 24 hours to prevent the animals from fatal
hypoglycemia. After 72 hours of STZ administration, fasting blood glucose (FBS) level
using the glucometer from the tail vein was determined. The rats with an FBS more
than 300 mg/dL were considered diabetic and included in the study.
Methodology
Male Wistar albino rats were selected based on their days of acclimatization. The
rats were divided into three groups, namely, control (group A), STZ-induced diabetes
mellitus (group B), and STZ-induced diabetes mellitus rats with A. catechu orally of 75 mg/kg body weight for 35 days, (group C) with each group having six
rats (n = 6) weighing between 200 to 250 g each. They were kept fasting overnight (but with
the free access to water). On the test day group A received only water orally, group
B received a single dose of STZ at 45 mg/kg body weight IP, and group C received a
single dose of STZ intraperitoneal and oral A. catechu for 35 days.
At the end of the 35th day of the observation period, the animals were deeply anesthetized
with ether. All the animals were observed for any gross/macroscopic pathological changes,
and the kidneys from the representative groups were removed and processed for the
histological studies and tissue homogenate for antioxidants, GOT (glutamic oxaloacetic
transaminase) and GPT (glutamic pyruvic transaminase).
Preparation of Kidney Homogenate
Kidneys were excised and cleaned with ice-cold saline and stored at–20°C in the freezer.
Tissues were thawed and homogenized in phosphate-buffered saline pH, 7.4, centrifuged
at 10,000 rpm for 15 minutes using refrigerated centrifuge and supernatant was stored
at–20°C. The supernatant was subjected to determination of GOT and GPT by Mohun and
Cook method,[22] SOD assay by Beauchamp and Fridovich method[23] and GSH-Px by Ellman’s method.[24]
Histopathological Examination
Kidneys were kept in 10% formalin for 48 hours (postfixation). By using standard histological
procedures, paraffin blocks were prepared; sections were taken at 5 μ thickness, stained
with hematoxylin and eosin, and observations were done under the light microscope
with ×40 magnification for cytoarchitecture.[25]
Statistical Analysis
Statistical analysis is performed using Student’s t-test and one-way analysis of variance (ANOVA) where ever it is applicable by SPSS,
Version 22.0 software.
Results
SOD activity and renal GSH in group C is closer to group A when compared with group
B where SOD and GSH activities were significantly elevated (p < 0.05; [Table 1]).
GOT and GPT in group C is closer to group A when compared with group B where GOT and
GPT activities were significantly elevated (p < 0.05; [Table 1]).
Histological studies in group C have revealed that the kidney were showing almost
normal cytoarchitecture with group A ([Fig. 1]). But in group B, rats’ kidneys have shown moderate-to-severe degenerative features
like dilated tubules, degenerated tubules, glomerular congestion, interstitial inflammatory
infiltration, and atrophy of glomerulus seen with dilated glomerular space. The degenerated
tubules cells with pyknotic nuclei and vacuolated cytoplasm. Sloughing of the epithelium
was seen in tubular lumens ([Fig. 2]). There was a significant change seen in the in tissue cytoarchitecture of group
C ([Fig. 3]) which clearly showed the antioxidant potency of the plant extract. This might be
the major reason behind oxidative stress management in diabetic rats in this study.
It shows that the plant extract has a nephroprotective effect which has favored the
normal level of these biochemical parameters followed by the treatment.
Table 1
The effect of Acacia catechu on functional enzymes in kidney tissues
|
|
SOD (U/g)
|
GSH (µmole/g)
|
GPT (U/g)
|
GOT (U/g)
|
|
Abbreviations: GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase;
GSH, glutathione; SOD, superoxide dismutase.
Note: Values are expressed as mean ± standard deviation, n = 6.
aValues are significantly different from the normal control group at (p < 0.05).
|
|
Normal
|
312.32 ± 10.13
|
1.906 ± 0.17
|
0.523 ± 0.0144
|
1.613 ± 0.025
|
|
Diabetic control
|
437.5 ± 29.46a
|
240.124 ± 38.90a
|
0.988 ± 0.245a
|
1.789 ± 0.035
|
|
Diabetic + Acacia catechu extract (75 mg/kg b.w.)
|
337.41 ± 70.18a
|
1.994 ± 0.55a
|
0. 686 ± 0.063a
|
1.643 ± 0.169
|
Fig. 1 A sectional representation of normal rat kidney (group A) at 40x magnification (hematoxylin
and eosin stain) showing normal Glomeruli (G) with an intact Bowman’s capsule, proximal
convoluted tubules, and Distal convoluted tubules.
Fig. 2 A representative section of STZ diabetic control rat (Group B) kidney at 40x magnification
(hematoxylin and eosin stain) showing increased Bowman’s space, glomerular congestion,
atrophy of glomerulus seen with dilated glomerular space and dilated tubules, degenerated
tubules, interstitial inflammatory infiltration.
Fig. 3 A representative section of AC (75 mg/kg) extract treated group (Group C) kidney
at 40x magnification (hematoxylin and eosin stain) showing sections with reduction
in tubule dilation and degeneration with reduced Bowman’s space, compared with STZ
group. Glomerular congestion seen. There was no interstitial inflammatory infiltrate
in any of the sections.
Discussion
The present work was aimed to study the antidiabetic activity of ethanolic extract
of A. catechu leaves in STZ-induced diabetic rats. In our study, 72 hours after the injection of
STZ to animals, significant hyperglycemia was observed in all rats. Hyperglycemia
exerts its adverse effects by generation of ROS which then, in turn, can oxidize many
other important biomolecules inducing “oxidative stress” (OS), [26] and the increment of free radicals may lead to kidney cells damage,[27] and the condition further deteriorates when the levels of antioxidants like GSH,
GPx, SOD, vitamin C, etc., decrease which is usually observed in DM individuals,[28] but some of the studies done had different observation, they had seen an increased
levels,[29]
[30] thus the role of antioxidants in DM is controversial.
On the contrary, the present investigation revealed an increase in the activity of
antioxidant enzyme SOD and the concentration of GSH in the kidney tissue homogenates
of STZ-induced DM. This may be because the diabetes was induced in healthy rats and
the study period was very short; so, group B rats effectively increased the expression
of antioxidant GSH and the enzyme SOD as a defense to reduce the oxidative stress
developed by STZ in the kidney. The overexpression of these antioxidants might be
an adaption response. SOD, for instance, is an important antidote to the toxic effect
of superoxide anion is also increased due to the increasing dismutation of superoxide
to hydrogen peroxide which is generally taken care of by the glutathione system. But
in DM patients due to insulin deficiency, there is more of β oxidation of fatty acids
due to which hydrogen peroxide formation will increase.[31] The higher free radical production and increased activity of antioxidant defense
system during the early phase of the disease may be a phase of compensation which
fails to sustain and hence leads to harmful effects of the unchecked oxidants causing
complications of DM later on. In our study, after administration of A. catechu for 35 days to the STZ-induced DM rats, the antioxidant enzymes level reaches near
to the normal level.
Evaluation of important housekeeping enzymes GOT and GPT activities in kidney tissues
showed a significant increase in the activity in untreated STZ-induced DM rats compared
with healthy group A ([Table 1]). While the activity of these enzymes reduced significantly in group C in comparison
to group A. These alterations in the activity of mentioned enzymes in untreated DM
rats may be due to the metabolic abnormalities or cellular injuries.[32] It has been reported that the increase in GOT and GPT activities in the kidney tissues
of STZ treated rats is due to the subtle membrane changes that allow to the passage
of intracellular enzymes to the extracellular space.[33]
Our data have shown that activity of the above-mentioned enzymes in the STZ-induced
DM was nearly normalized by the A. catechu.
In accordance with the results obtained in biochemical analysis, the histological
analysis in DM rats supplemented with extract of A. catechu showed reduction in tubule dilation and degeneration with normal glomerular space
compared with STZ group. Glomerular congestion was also reduced. There was no interstitial
inflammatory infiltrate in any of the sections compared with STZ treated group. By
virtue of its antioxidant property, A. catechu extract was able to render nephroprotection in these models by attenuating oxidative
stress. So, it was speculated that the nephroprotective effect of A. catechu might be due to its antioxidant property. The plant extract might contain bioactive
components that have the potential to reverse the undesirable changes in the kidney
associated with hyperglycemia-induced oxidative stress.[34] Thus, a corrective measure even on the histology of the kidney was noticed.
Conclusion
A. catechu was evaluated for its anti-diabetic property in STZ-induced DM rats for 35 days at
the dose of 75 mg/kg body weight. The antioxidants levels GOT and GPT and histological
studies have suggested that the A. catechu have some active principals which are antidiabetic and nephroprotective. Further
studies are to isolate the active components of A. catechu which is needed for the treatment of DM.
