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Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 112 Copyright @ 2023 Authors
EVALUATION OF ANTIUROLITHIATIC ACTIVITY OF CANTHIUM DICOCCUM
ETHANOLIC EXTRACT IN RATS.
I Veena Rani Department of Pharmacology, SSJ College of Pharmacy, Gandipet, Hyderabad,
Telangana, India.
Veena Gadicherla Department of Pharmacology, Sri Indu Institute of Pharmacy, Sheriguda,
Ibrahimpatnam, R.R. District, Telangana, India-501510.
Email Id: vina913@gmail.com
Abstract:
Objective:
The aim of the present study was to evaluate the effect of ethanolic extract of Canthium dicoccum on
Gentamicin and Caliculi Producing Diet (CPD) induced urolithasis in rats.
Methods:
Thirty male albino wistarrats were divided randomly into six groups. Group I were treated with normal
saline, Group II were treated with Gentamicin for a period of 7 days, Group III treated with Cystone
(p.o), Group IV,V,VI was treated with plant extract 150, 300,600 mg/kg respectively for 14 days with
gentamicin treatment from day 7 to 14. All the animals were fed with caliculi Producing diet for entire
study period. Later, urine samples were collected at the end of the studyand analysed for renal function
parameters like Creatiinine, BUN, Serum Urea etc. The kidneys of the sacrificed animals were isolated
and sectioned for histopathological studies.
Results:
The rats treated with ethanolic extract of canthiumdicoccum at doses 150,300,600 mg/kg significantly
showed improved effect and prevented urolithasis in rats which is evident in the reduction of creatinine,
Urea and BUN levels when compared to gentamicin treated group. The histopathological studies also
showed less degenerative changes.It also decreased crystal deposition markedly into the renal tubules
in number as well as size and prevented damage to the renal tubules.
Conclusion:
The results demonstrated that the ethanolic extract had significantly reduced the urolithiasis that was
caused by an CPD diet combined with gentamicine injection in rats.
Keywords: Gentamicin, Anti-Urolithiatic activity, Canthiumdicoccum, ethanol.
Introduction:
Urolithiasis additionally known as kidney stones or nephrolithiasis or renal calculi occurs when
super saturation of the urine occurs with stone forming salts.Less commonly it occurs from persistent
urinary tract infection with urease producing bacteria (1).Formation of kidney stones is a complex
process which involves the physicochemical events like crystal nucleation, aggregation and with
retention in the urinary tract. The formation of kidney stones are multiple types, 80 percent of stones
are composed of calcium oxalates. Other types include 10 percent of stuvite, 9 percent of uric acid, 1
percent of cystone significantly. Different types of stones are formed due to risk factors like diet, prior
personal, environmental factors, medication history etc (2).
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 113 Copyright @ 2023 Authors
Renal calculi are common condition that affects about 1 out of 11 people in the United states
with its prevalence of 12 percent worldwide. In India it is relatively common in northern part of India
with 15% of increase in calculi cases (3). The rising of calculi cases is primarilyaffecting the working-
age population (4). Men present more common than women with 10.6% vs 7.5% obese and overweight
individuals compared to normal-weight individuals (5). A recurrence rate of up to 50% has been seen
in five years from the initial episodes of kidney stone formation. Many patients with kidney stones can
be managed with expectant management, analgesic and anti-emetic medication; however, stones that
are associated with obstruction, renal failure and infection require further increasingly critical
interventions (1).
Canthiumdicoccum is a flowering plant belonging to the family Rubiaceae. It is commonly
known as Nallabalusu in Telugu, NallaMandharam in Tamil Nadu. It is mostly cultivated in India (6).
Traditionally it was used for the treatment of diarrhoea, fever, fungal infections (7). Scientifically
reported activtities ofCanthiumdicoccuminclude antibacterial activity (8), antihelmentic,
antihypertensive activity (9), antimicrobial, anti-tuberculosis (10), anti-Inflammatory (11), anticancer
activity (12) and hepatoprotective (13, 14). Based upon the literature sources, the present study was
planned to evaluate the therapeutic potential of ethanolic extract of the plant Canthiumdicoccumon
Gentamicin and calculi producing diet induced urolithiasis since there was no study reported on this
activity.
Materials and Methods:
Collection of plant material and preparation of plant extract:
The plant of Canthium dicoccum was collected from the forest area of Tirupathi, Andhra
Pradesh and was authenticated by a botanist. The fresh whole plant material collected was shade dried
and made into coarse powder. The powder of Canthium dicoccum obtained was subjected to Soxhlet
extraction using 95% of ethanol for 72 hrs. The extract obtained was kept for drying and stored in
vacuum desiccators till use.
Phytochemical screening of the extract:
The obtained Extract of Canthium dicoccum (ECD) was subjected for screening of
phytochemical constituents by using standard procedures.
Animals:
Thirty healthy male wistar rats weighing 200-300gms were housed at 22-250
C room
temperatures with 12 hours dark and light condition. They were given standard laboratory feed with
water ad libitium. All the procedures of the study were approved by the Institutional Animal Ethical
committee with approval number is SSJP/CPCSEA/2020/04.
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 114 Copyright @ 2023 Authors
Acute Toxicity Studies:
The rats were fasted overnight with free access to drinking water. The animals were divided
randomly in to six groups with a dosage range as per OECD guideline 423 and administered orally
through gastric lavage. The animals were observed for the first 2 hrs continuously to monitor any
neurological, behavioural changes. At the end of the study period the mortality was defined to calculate
the LD50 of the extract. The LD50 of the extract was found to be above 2000mg/kg.
Experimental Design:
Thirty male wistar rats were divided into 6 groups, each containing 5 rats and were housed
properly with calculi producing diet (CPD) and water ad libitum. The study period was 14 days with
Urolithiac induction done by administration of Gentamicin subcutaneously at a dose off 40 mg/kg from
day 7 of the study till day 14. The randomization of the experiment is as follows
Group I (Control Group) : Normal saline orally
Group II (Toxic Control group) :Gentamicin (40mg/kg) S.C.
Group III (Standard Group) :Cystone (750mg/kg) P.O + Gentamicin (40mg/kg) S.C
Group IV (Test Group I) : ECD (150/kg) P.O. Gentamicin (40mg/kg) S.C.
Group V (Test Group II) : ECD (300mg/kg)P.O.+ Gentamicin (40mg/kg) S.C.
Group VI (Test Group III) : ECD (600mg/kg)P.O.+ Gentamicin (40mg/kg) S.C.
Assessment of Antiurolithiatic Activity:
Collection and analysis of urine:
At the end of the study period urine samples of the rats were collected by placing them in
metabolic cages. During the urine collection period, animals were provided access to drinking water.
The collected urine was subjected to estimation of specific gravity, urinary pH, urine volume.
Collection of Blood and analysis of serum:
At the end of the study period, animals were anaesthetized under light ether anaesthesia and
blood samples were collected from retro-orbital plexus. The serum separated was subjected for
estimation of creatinine, uric acid, blood urea nitrogen (BUN).
Histopathological studies:
The rats were sacrificed by decapitation and the kidneys were isolated. The isolated kidneys
were stored in 10% buffered formalin solution until the histopathological examination. The tissue
sections were stained using heamotoxylin and eosin dyes and subjected to microscopical examination.
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 115 Copyright @ 2023 Authors
Statistical Analysis:
The results obtained in the study were anlaysed by applying One way ANOVA followed by
Dunnet’s t test for comparative analysis with p<0.05. All the data values were expressed as Mean ±
SEM.
Results:
Phytochemical Screening:
The results of qualitative phytochemical analysis of ethanol extract of Canthium dicocum
revealed the presence of taninns, cardiac glycosides, steroids, saponins and triterpenes. Mucilage and
proteins were absent.
Serum biochemical parameters:
The blood was collected from the retro orbital plexus under anaesthetic condition and serum
was estimated for creatinine, uric acid, urea and BUN. In present study, administration of gentamicin
in toxic group increased the levels of urea, uric acid and BUN when compared to control group. These
levels were significantly decreased in test groups (IV,V,VI) on treatment with plant extract of
Canthium dicoccum and prevented changes in stone formation in kidneys.
Table 1: Preliminary qualitative phytochemical analysis of Canthium dicoccum extract
Phytochemical Test Methanolic extract
Alkaloids
Dragandroffs test
Mayers test
Wagners test
+
+
+
Flavonoids
Shinoda test
Alkaline reagent test
+
+
Glycosides Conc.H2SO4 +
Cardiac glycosides Keller-kilianni test +
Saponins Frothing test +
Steroids Libbermann-Burchard test +
Tannins FeCl3 test +
Triterpentoids H2SO4 test +
Carbohydrates Molisch’stet +
Mucilage Ruthenium red test -
Proteins Biuret test -
(-): absent, (+): present.
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 116 Copyright @ 2023 Authors
Table 2: Effect of administration of gentamicin and ethanolic extract of canthium dicoccum for
7 days on serum creatinine level, urea, uric acid, BUN, urine volume.
Groups Creatinine Urea Uricacid BUN
Control Group
0.34±0.01**
3.26±0.06**
1.37±0.06*
38.70±0.16**
Toxic group
0.06±0.06 40.30±0.07 4.02±0.02 50.02±0.14
Standard Group
0.33±0.02**
23.74±0.04*
1.72±0.02**
36.72±0.25**
Test Group I
0.46±0.01*
22.56±0.05**
1.93±0.04*
40.43±0.12*
Test Group II
0.40±0.02**
20.52±0.04***
1.82±0.07**
36.25±0.23**
Test Group III
0.36±0.01**
17.03±0.04***
1.70±0.07**
33.02±0.02***
Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by
Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, Creatinine, BUN, Uric
acid, Urea levels were compared with toxic group
Kidney Histopathology:
No CaOx crystal deposits in the kidney of the control group (control group) as shown in (Figure
1-A) were seen. In Group II, many CaOx crystal deposits were seen in the lumen of PCT. Congestion
and dilation of the parenchymal blood vessels were also seen in the renal tissue of (toxic group) as
shown in (Figure 1-B). In the standard group, the kidney showed normal architecture with mild dilation
of tubules in the cortico-medullary junction with minimal interstitial inflammation, (Figure 1-C). In
test groups I, II and III, the kidney showed normal architecture and few renal tubules that revealed
vacuolar degeneration with mild CaOx crystal deposits (Figure 1-D, E, F) with minimal inflammation
in renal tubules with absence of CaOx crystal deposits.
Table 3: Urine analysis of the collected urine from rats treated with Canthium dicoccum extract
Groups
Specific gravity
of Urine
Urine pH
Urine volume
Weight of
kidney g/100 g
Control Group 1.009 ± 0.003**
7.6 ± 0.45
7.99±0.13** 1.03 ± 0.09**
Toxic Group 1.21 ± 0.004 6.3 ± 0.18
4.75±0.16
1.67 ± 0.08
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 117 Copyright @ 2023 Authors
Standard Group 1.007 ± 0.003**
7.3 ± 0.32
7.0±0.03** 1.13 ± 0.02**
Test Group I 1.016 ± 0.003**
6.9 ± 0.44
7.1±0.04* 1.51 ± 0.05
Test Group II 1.021 ± 0.004**
7.0 ± 0.25
7.12±0.01** 1.47 ± 0.09*
Test Group III 1.029 ± 0.004**
7.2 ± 0.43
7.01±0.06*** 1.31 ± 0.04*
Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by
Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, were compared with
toxic group.
Table 4: Evaluation of antioxidant enzymes from rats treated with Canthium dicoccum extract
Groups Glutathione peroxidase Lipid peroxidation
Control Group 3.59 ± 1.23***
22.25 ± 8.72***
Toxic Group 1.56 ± 0.31 73.43 ± 10.32
Standard Group 16.56 ± 2.43***
24.32 ± 5.67***
Test Group I 5.67 ± 1.46*
46.34 ± 2.34**
Test Group II 9.87 ± 2.01*
37.87 ± 3.43***
Test Group III 11.97 ± 1.89**
31.25 ± 3.78***
Figure 1: Histopathological studies of Isolated Kidney from rats treated with gentamicin and
ethanolic extract of Canthium dicoccum
A. Control Group B. Negative Control group
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 118 Copyright @ 2023 Authors
C. Positive Control Group D. Test Group I
E. Test Group II F. Test group III
DISCUSSION:
In essence, the pathophysiology of calcium oxalate (CaOx) stone formation is a multi-step
process that involves urine saturation, urinary super saturation, nucleation, crystal development, crystal
aggregation, and crystal retention. Different substances in the body have an impact on one or more of
the aforementioned stone-forming processes, which affects a person's body's capacity to encourage or
inhibit stone development. Stone formation is aided by stone formation promoters and prevented by
stone formation inhibitors. Stone formation is known to be aided by low urine volume, low urine pH,
calcium, salt, oxalate, and urate. Numerous chemical and inorganic compounds, including citrate,
magnesium, urinary prothrombin fragment 1, glycosaminoglycans, and osteopontin, have been shown
to prevent the formation of stones. Organic inhibitory substances adhere to the crystal's surface and
prevent crystal development (15).
We understand only a little about the occurrence and characteristics of spontaneous urolithiasis
in rats. Because early studies have demonstrated decreased incidence of calculi deposition with 3%
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 119 Copyright @ 2023 Authors
ammonium oxalate, 5% ammonium oxalate is employed in the current study instead of 3% ammonium
oxalate as reported by Sanjay Kumar et al. This regimen of gentamicin plus an ammonium oxalate-
rich diet causes renal tubular damage, increases calcium and oxalate super saturation, and creates
conditions that favour the development and proliferation of CaOx stones (16). Chronic hyperoxaluria
appears to be the main contributor to CaOx stone development. In the current investigation,
hyperoxaluria caused by gentamicin and an ammonium oxalate-rich diet enhanced CaOx deposition
in the kidney and, as previously reported, also caused papillary damage and incrustations. This
treatment schedule of gentamicin and ammonium oxalate increase calcium and oxalate super
saturation, renal tubular injury and produce favourable condition to the formation and growth of CaOx
stones (17).
The present study indicates that there is and increase in weight of kidneys in toxic group than
compared to control group of animals which is in accordance with the histopathological studies which
showed degenerative changes and deposition of stones were observed (Figure 1). Further treatment of
ECD reduced the changes and reduced the weight of kidneys which suggested the reduction of stone
formation and is in accordance with the H & E sections showing intact nephrotic cells.
The Glomerular Filtration Rate (GFR) decreases as a result of the obstruction caused by stones
that prevent urine from exiting the body. The accumulation of waste products, especially nitrogenous
compounds like urea, creatinine, and uric acid in blood, is caused by a decrease in GFR. The level of
blood urea nitrogen is regarded as a reliable sign of nitrogen metabolism equilibrium. It usually gets
better as tissue catabolism rises. In the current investigation, the treated group reduced considerably in
blood urea level than that of toxic group. Increased serum creatinine is a sign of hyperoxaluria-related
renal impairment. Treatment with ECD reduced the serum creatinine levels significantly than
compared to toxic control group. Similar results were shown in levels of Urea and uric acid (18).
According to studies, oxalate, a key ingredient in the formation of stones, interacts with
polyunsaturated fatty acids in cell membranes to cause tissue damage and induce lipid peroxidation.
The kidney is vulnerable to damage by reactive oxygen species (ROS) because of its high amount of
polyunsaturated fatty acids. Oxidative Stress (OS) is caused by an imbalance between the levels of
oxidants and anti-oxidants. Low levels of renal cellular glutathione are thought to promote lipid
peroxidation and cause the kidney to retain calcium and oxalate (19). In the current investigation, it
was found that feeding rats an ammonium oxalate-rich diet along with gentamicin injections caused a
considerable rise in the level of lipid peroxidation in the kidney tissue of the negative control group
compared to the normal control group. Administration of ECD significantly reduced the lipid
Dogo Rangsang Research Journal UGC Care Group I Journal
ISSN : 2347-7180 Vol-13, Issue-6, June 2023
Page | 120 Copyright @ 2023 Authors
peroxidation levels and improved the glutathione levels respectively, attributing towards its actions in
reducing the formation of stones (20).
Thus, the present study suggests that the antiurolithiac actions of the ECD may be by improving
the antioxidant status and integrity of cell membrane with increased urine volume and inhibition of
crystal formation, growth and aggregation.
Conclusion:
The alcoholic extract if Canthium dicoccium showed a marked antiurolithiac activity in a dose
dependent manner and further studies is required to know the in depth molecular mechanisms and
exploration of active principles of the plant.
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3. Singh S, Gupta S, Mishra T, et al. (February 27, 2023) Risk Factors of Incident Kidney Stones in
Indian Adults: A Hospital-Based Cross-Sectional Study. Cureus 15(2): e35558).
4. Charles D. Scales, Alexandria C. Smith, Janet M. Hanley, Christopher S. Saigal, Prevalence of
Kidney Stones in the United States. Eur Urol. 2012 Jul; 62(1): 160–165.
5. Taylor EN, Stampfer MJ, Curhan GC. Obesity, Weight gain, and the risk of kidney stones. JAMA.
2005 Jan 26:293(4):455-62.
6. BhaargaviVuyyuri, ReshmaTripurana, JyotsnaGangeyula, Firasat Ali, Anti- inflammatory activity
of ethanolic extract of Canthiumdicoccum. VuyyuriBhaargaviint.
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extract of roots of Canthiumdicoccum in streptomycin induced nephrotoxicity in animal model;
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8. K.S. Meghashree, K.P. Latha. Potential antibacterial activity of canthiumdicoccum (gaertn.)
against certain bacterial species. Plant Archives. Volume 20 (2), 2020. 8818-8820
9. Meghashree KS, Latha KP, Vagdevi HM, Ajish A, jayanna N, arunkumar N. screening of
phytochemical content and in vitro biological investigation of canthiumdicoccum (gaertn.) and
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10. C.Mahendraa, Mohana N, ChandraM, MuralicM.R, AbhilashS. BrijeshSinghcS, SatishM,
Sudarshan. Phyto-fabricated ZnO nanoparticles from Canthiumdicoccum (L.) for antimicrobial,
anti-tuberculosis and antioxidant activity.Process Biochemistry. 89, February 2020, 220-226
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13. Ravi Kumar Vakkalagadda, ParameshwarRavula, KondapuramParameshwar, KaspaSaraswathi, P
Sindhuri, RajalaSrikala, KosikaSandeep Protective Potential of CanthiumdicoccumMethanolic
Extract Against Hepatic Injury in Rats.Pharmacognosy Journal. 2021, 13(6),1648-1655
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Evaluation of Antiurolithiatic Activity of Canthium dicoccum Ethanolic Extract In Rats.pdf

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  • 1. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 112 Copyright @ 2023 Authors EVALUATION OF ANTIUROLITHIATIC ACTIVITY OF CANTHIUM DICOCCUM ETHANOLIC EXTRACT IN RATS. I Veena Rani Department of Pharmacology, SSJ College of Pharmacy, Gandipet, Hyderabad, Telangana, India. Veena Gadicherla Department of Pharmacology, Sri Indu Institute of Pharmacy, Sheriguda, Ibrahimpatnam, R.R. District, Telangana, India-501510. Email Id: vina913@gmail.com Abstract: Objective: The aim of the present study was to evaluate the effect of ethanolic extract of Canthium dicoccum on Gentamicin and Caliculi Producing Diet (CPD) induced urolithasis in rats. Methods: Thirty male albino wistarrats were divided randomly into six groups. Group I were treated with normal saline, Group II were treated with Gentamicin for a period of 7 days, Group III treated with Cystone (p.o), Group IV,V,VI was treated with plant extract 150, 300,600 mg/kg respectively for 14 days with gentamicin treatment from day 7 to 14. All the animals were fed with caliculi Producing diet for entire study period. Later, urine samples were collected at the end of the studyand analysed for renal function parameters like Creatiinine, BUN, Serum Urea etc. The kidneys of the sacrificed animals were isolated and sectioned for histopathological studies. Results: The rats treated with ethanolic extract of canthiumdicoccum at doses 150,300,600 mg/kg significantly showed improved effect and prevented urolithasis in rats which is evident in the reduction of creatinine, Urea and BUN levels when compared to gentamicin treated group. The histopathological studies also showed less degenerative changes.It also decreased crystal deposition markedly into the renal tubules in number as well as size and prevented damage to the renal tubules. Conclusion: The results demonstrated that the ethanolic extract had significantly reduced the urolithiasis that was caused by an CPD diet combined with gentamicine injection in rats. Keywords: Gentamicin, Anti-Urolithiatic activity, Canthiumdicoccum, ethanol. Introduction: Urolithiasis additionally known as kidney stones or nephrolithiasis or renal calculi occurs when super saturation of the urine occurs with stone forming salts.Less commonly it occurs from persistent urinary tract infection with urease producing bacteria (1).Formation of kidney stones is a complex process which involves the physicochemical events like crystal nucleation, aggregation and with retention in the urinary tract. The formation of kidney stones are multiple types, 80 percent of stones are composed of calcium oxalates. Other types include 10 percent of stuvite, 9 percent of uric acid, 1 percent of cystone significantly. Different types of stones are formed due to risk factors like diet, prior personal, environmental factors, medication history etc (2).
  • 2. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 113 Copyright @ 2023 Authors Renal calculi are common condition that affects about 1 out of 11 people in the United states with its prevalence of 12 percent worldwide. In India it is relatively common in northern part of India with 15% of increase in calculi cases (3). The rising of calculi cases is primarilyaffecting the working- age population (4). Men present more common than women with 10.6% vs 7.5% obese and overweight individuals compared to normal-weight individuals (5). A recurrence rate of up to 50% has been seen in five years from the initial episodes of kidney stone formation. Many patients with kidney stones can be managed with expectant management, analgesic and anti-emetic medication; however, stones that are associated with obstruction, renal failure and infection require further increasingly critical interventions (1). Canthiumdicoccum is a flowering plant belonging to the family Rubiaceae. It is commonly known as Nallabalusu in Telugu, NallaMandharam in Tamil Nadu. It is mostly cultivated in India (6). Traditionally it was used for the treatment of diarrhoea, fever, fungal infections (7). Scientifically reported activtities ofCanthiumdicoccuminclude antibacterial activity (8), antihelmentic, antihypertensive activity (9), antimicrobial, anti-tuberculosis (10), anti-Inflammatory (11), anticancer activity (12) and hepatoprotective (13, 14). Based upon the literature sources, the present study was planned to evaluate the therapeutic potential of ethanolic extract of the plant Canthiumdicoccumon Gentamicin and calculi producing diet induced urolithiasis since there was no study reported on this activity. Materials and Methods: Collection of plant material and preparation of plant extract: The plant of Canthium dicoccum was collected from the forest area of Tirupathi, Andhra Pradesh and was authenticated by a botanist. The fresh whole plant material collected was shade dried and made into coarse powder. The powder of Canthium dicoccum obtained was subjected to Soxhlet extraction using 95% of ethanol for 72 hrs. The extract obtained was kept for drying and stored in vacuum desiccators till use. Phytochemical screening of the extract: The obtained Extract of Canthium dicoccum (ECD) was subjected for screening of phytochemical constituents by using standard procedures. Animals: Thirty healthy male wistar rats weighing 200-300gms were housed at 22-250 C room temperatures with 12 hours dark and light condition. They were given standard laboratory feed with water ad libitium. All the procedures of the study were approved by the Institutional Animal Ethical committee with approval number is SSJP/CPCSEA/2020/04.
  • 3. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 114 Copyright @ 2023 Authors Acute Toxicity Studies: The rats were fasted overnight with free access to drinking water. The animals were divided randomly in to six groups with a dosage range as per OECD guideline 423 and administered orally through gastric lavage. The animals were observed for the first 2 hrs continuously to monitor any neurological, behavioural changes. At the end of the study period the mortality was defined to calculate the LD50 of the extract. The LD50 of the extract was found to be above 2000mg/kg. Experimental Design: Thirty male wistar rats were divided into 6 groups, each containing 5 rats and were housed properly with calculi producing diet (CPD) and water ad libitum. The study period was 14 days with Urolithiac induction done by administration of Gentamicin subcutaneously at a dose off 40 mg/kg from day 7 of the study till day 14. The randomization of the experiment is as follows Group I (Control Group) : Normal saline orally Group II (Toxic Control group) :Gentamicin (40mg/kg) S.C. Group III (Standard Group) :Cystone (750mg/kg) P.O + Gentamicin (40mg/kg) S.C Group IV (Test Group I) : ECD (150/kg) P.O. Gentamicin (40mg/kg) S.C. Group V (Test Group II) : ECD (300mg/kg)P.O.+ Gentamicin (40mg/kg) S.C. Group VI (Test Group III) : ECD (600mg/kg)P.O.+ Gentamicin (40mg/kg) S.C. Assessment of Antiurolithiatic Activity: Collection and analysis of urine: At the end of the study period urine samples of the rats were collected by placing them in metabolic cages. During the urine collection period, animals were provided access to drinking water. The collected urine was subjected to estimation of specific gravity, urinary pH, urine volume. Collection of Blood and analysis of serum: At the end of the study period, animals were anaesthetized under light ether anaesthesia and blood samples were collected from retro-orbital plexus. The serum separated was subjected for estimation of creatinine, uric acid, blood urea nitrogen (BUN). Histopathological studies: The rats were sacrificed by decapitation and the kidneys were isolated. The isolated kidneys were stored in 10% buffered formalin solution until the histopathological examination. The tissue sections were stained using heamotoxylin and eosin dyes and subjected to microscopical examination.
  • 4. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 115 Copyright @ 2023 Authors Statistical Analysis: The results obtained in the study were anlaysed by applying One way ANOVA followed by Dunnet’s t test for comparative analysis with p<0.05. All the data values were expressed as Mean ± SEM. Results: Phytochemical Screening: The results of qualitative phytochemical analysis of ethanol extract of Canthium dicocum revealed the presence of taninns, cardiac glycosides, steroids, saponins and triterpenes. Mucilage and proteins were absent. Serum biochemical parameters: The blood was collected from the retro orbital plexus under anaesthetic condition and serum was estimated for creatinine, uric acid, urea and BUN. In present study, administration of gentamicin in toxic group increased the levels of urea, uric acid and BUN when compared to control group. These levels were significantly decreased in test groups (IV,V,VI) on treatment with plant extract of Canthium dicoccum and prevented changes in stone formation in kidneys. Table 1: Preliminary qualitative phytochemical analysis of Canthium dicoccum extract Phytochemical Test Methanolic extract Alkaloids Dragandroffs test Mayers test Wagners test + + + Flavonoids Shinoda test Alkaline reagent test + + Glycosides Conc.H2SO4 + Cardiac glycosides Keller-kilianni test + Saponins Frothing test + Steroids Libbermann-Burchard test + Tannins FeCl3 test + Triterpentoids H2SO4 test + Carbohydrates Molisch’stet + Mucilage Ruthenium red test - Proteins Biuret test - (-): absent, (+): present.
  • 5. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 116 Copyright @ 2023 Authors Table 2: Effect of administration of gentamicin and ethanolic extract of canthium dicoccum for 7 days on serum creatinine level, urea, uric acid, BUN, urine volume. Groups Creatinine Urea Uricacid BUN Control Group 0.34±0.01** 3.26±0.06** 1.37±0.06* 38.70±0.16** Toxic group 0.06±0.06 40.30±0.07 4.02±0.02 50.02±0.14 Standard Group 0.33±0.02** 23.74±0.04* 1.72±0.02** 36.72±0.25** Test Group I 0.46±0.01* 22.56±0.05** 1.93±0.04* 40.43±0.12* Test Group II 0.40±0.02** 20.52±0.04*** 1.82±0.07** 36.25±0.23** Test Group III 0.36±0.01** 17.03±0.04*** 1.70±0.07** 33.02±0.02*** Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, Creatinine, BUN, Uric acid, Urea levels were compared with toxic group Kidney Histopathology: No CaOx crystal deposits in the kidney of the control group (control group) as shown in (Figure 1-A) were seen. In Group II, many CaOx crystal deposits were seen in the lumen of PCT. Congestion and dilation of the parenchymal blood vessels were also seen in the renal tissue of (toxic group) as shown in (Figure 1-B). In the standard group, the kidney showed normal architecture with mild dilation of tubules in the cortico-medullary junction with minimal interstitial inflammation, (Figure 1-C). In test groups I, II and III, the kidney showed normal architecture and few renal tubules that revealed vacuolar degeneration with mild CaOx crystal deposits (Figure 1-D, E, F) with minimal inflammation in renal tubules with absence of CaOx crystal deposits. Table 3: Urine analysis of the collected urine from rats treated with Canthium dicoccum extract Groups Specific gravity of Urine Urine pH Urine volume Weight of kidney g/100 g Control Group 1.009 ± 0.003** 7.6 ± 0.45 7.99±0.13** 1.03 ± 0.09** Toxic Group 1.21 ± 0.004 6.3 ± 0.18 4.75±0.16 1.67 ± 0.08
  • 6. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 117 Copyright @ 2023 Authors Standard Group 1.007 ± 0.003** 7.3 ± 0.32 7.0±0.03** 1.13 ± 0.02** Test Group I 1.016 ± 0.003** 6.9 ± 0.44 7.1±0.04* 1.51 ± 0.05 Test Group II 1.021 ± 0.004** 7.0 ± 0.25 7.12±0.01** 1.47 ± 0.09* Test Group III 1.029 ± 0.004** 7.2 ± 0.43 7.01±0.06*** 1.31 ± 0.04* Values expressed as Mean ± SD (n = 5), statistical analysis is done by ANOVA followed by Dunnet’s test to find out significance ***p < 0.001, **p< 0.01 and *p <0.5, were compared with toxic group. Table 4: Evaluation of antioxidant enzymes from rats treated with Canthium dicoccum extract Groups Glutathione peroxidase Lipid peroxidation Control Group 3.59 ± 1.23*** 22.25 ± 8.72*** Toxic Group 1.56 ± 0.31 73.43 ± 10.32 Standard Group 16.56 ± 2.43*** 24.32 ± 5.67*** Test Group I 5.67 ± 1.46* 46.34 ± 2.34** Test Group II 9.87 ± 2.01* 37.87 ± 3.43*** Test Group III 11.97 ± 1.89** 31.25 ± 3.78*** Figure 1: Histopathological studies of Isolated Kidney from rats treated with gentamicin and ethanolic extract of Canthium dicoccum A. Control Group B. Negative Control group
  • 7. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 118 Copyright @ 2023 Authors C. Positive Control Group D. Test Group I E. Test Group II F. Test group III DISCUSSION: In essence, the pathophysiology of calcium oxalate (CaOx) stone formation is a multi-step process that involves urine saturation, urinary super saturation, nucleation, crystal development, crystal aggregation, and crystal retention. Different substances in the body have an impact on one or more of the aforementioned stone-forming processes, which affects a person's body's capacity to encourage or inhibit stone development. Stone formation is aided by stone formation promoters and prevented by stone formation inhibitors. Stone formation is known to be aided by low urine volume, low urine pH, calcium, salt, oxalate, and urate. Numerous chemical and inorganic compounds, including citrate, magnesium, urinary prothrombin fragment 1, glycosaminoglycans, and osteopontin, have been shown to prevent the formation of stones. Organic inhibitory substances adhere to the crystal's surface and prevent crystal development (15). We understand only a little about the occurrence and characteristics of spontaneous urolithiasis in rats. Because early studies have demonstrated decreased incidence of calculi deposition with 3%
  • 8. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 119 Copyright @ 2023 Authors ammonium oxalate, 5% ammonium oxalate is employed in the current study instead of 3% ammonium oxalate as reported by Sanjay Kumar et al. This regimen of gentamicin plus an ammonium oxalate- rich diet causes renal tubular damage, increases calcium and oxalate super saturation, and creates conditions that favour the development and proliferation of CaOx stones (16). Chronic hyperoxaluria appears to be the main contributor to CaOx stone development. In the current investigation, hyperoxaluria caused by gentamicin and an ammonium oxalate-rich diet enhanced CaOx deposition in the kidney and, as previously reported, also caused papillary damage and incrustations. This treatment schedule of gentamicin and ammonium oxalate increase calcium and oxalate super saturation, renal tubular injury and produce favourable condition to the formation and growth of CaOx stones (17). The present study indicates that there is and increase in weight of kidneys in toxic group than compared to control group of animals which is in accordance with the histopathological studies which showed degenerative changes and deposition of stones were observed (Figure 1). Further treatment of ECD reduced the changes and reduced the weight of kidneys which suggested the reduction of stone formation and is in accordance with the H & E sections showing intact nephrotic cells. The Glomerular Filtration Rate (GFR) decreases as a result of the obstruction caused by stones that prevent urine from exiting the body. The accumulation of waste products, especially nitrogenous compounds like urea, creatinine, and uric acid in blood, is caused by a decrease in GFR. The level of blood urea nitrogen is regarded as a reliable sign of nitrogen metabolism equilibrium. It usually gets better as tissue catabolism rises. In the current investigation, the treated group reduced considerably in blood urea level than that of toxic group. Increased serum creatinine is a sign of hyperoxaluria-related renal impairment. Treatment with ECD reduced the serum creatinine levels significantly than compared to toxic control group. Similar results were shown in levels of Urea and uric acid (18). According to studies, oxalate, a key ingredient in the formation of stones, interacts with polyunsaturated fatty acids in cell membranes to cause tissue damage and induce lipid peroxidation. The kidney is vulnerable to damage by reactive oxygen species (ROS) because of its high amount of polyunsaturated fatty acids. Oxidative Stress (OS) is caused by an imbalance between the levels of oxidants and anti-oxidants. Low levels of renal cellular glutathione are thought to promote lipid peroxidation and cause the kidney to retain calcium and oxalate (19). In the current investigation, it was found that feeding rats an ammonium oxalate-rich diet along with gentamicin injections caused a considerable rise in the level of lipid peroxidation in the kidney tissue of the negative control group compared to the normal control group. Administration of ECD significantly reduced the lipid
  • 9. Dogo Rangsang Research Journal UGC Care Group I Journal ISSN : 2347-7180 Vol-13, Issue-6, June 2023 Page | 120 Copyright @ 2023 Authors peroxidation levels and improved the glutathione levels respectively, attributing towards its actions in reducing the formation of stones (20). Thus, the present study suggests that the antiurolithiac actions of the ECD may be by improving the antioxidant status and integrity of cell membrane with increased urine volume and inhibition of crystal formation, growth and aggregation. Conclusion: The alcoholic extract if Canthium dicoccium showed a marked antiurolithiac activity in a dose dependent manner and further studies is required to know the in depth molecular mechanisms and exploration of active principles of the plant. REFERENCES: 1. Scales CD, Smith AC, Hanley JM, Saigal CS., Urologic Diseases in America Project. Prevalence of kidney stones in the United States. Eur Urol.2012 Jul;62(1): 160-5. 2. Chung MJ. Urolithiasis and nephrolithiasis. JAAPA. 2017 Sep;30(9):49-50. 3. Singh S, Gupta S, Mishra T, et al. (February 27, 2023) Risk Factors of Incident Kidney Stones in Indian Adults: A Hospital-Based Cross-Sectional Study. Cureus 15(2): e35558). 4. Charles D. Scales, Alexandria C. Smith, Janet M. Hanley, Christopher S. Saigal, Prevalence of Kidney Stones in the United States. Eur Urol. 2012 Jul; 62(1): 160–165. 5. Taylor EN, Stampfer MJ, Curhan GC. Obesity, Weight gain, and the risk of kidney stones. JAMA. 2005 Jan 26:293(4):455-62. 6. BhaargaviVuyyuri, ReshmaTripurana, JyotsnaGangeyula, Firasat Ali, Anti- inflammatory activity of ethanolic extract of Canthiumdicoccum. VuyyuriBhaargaviint. J.Pharm.Phytopharmacol.Res.2013;3(3):226-230. 7. MalothuNagulu; Mangilal, T. ; Kishore, R.N.; Abinayani, G, Sravya, N, Investigation of aqueous extract of roots of Canthiumdicoccum in streptomycin induced nephrotoxicity in animal model; WJPPS:2014:3(9):314-320. 8. K.S. Meghashree, K.P. Latha. Potential antibacterial activity of canthiumdicoccum (gaertn.) against certain bacterial species. Plant Archives. Volume 20 (2), 2020. 8818-8820 9. Meghashree KS, Latha KP, Vagdevi HM, Ajish A, jayanna N, arunkumar N. screening of phytochemical content and in vitro biological investigation of canthiumdicoccum (gaertn.) and amischophacelusaxillaris (l.). Asian J Pharm Clin Res, 13(1), 2020, 109-114.
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