Anti-urolithiatic effect of Ethanolic extract of Bryophyllum pinnatum (Lam) against chemicals induced Renal calculi in rats

 

S.K. Shinde, V.J. Chaware, V.K. Redasani

Department of Pharmacology, YSPM’s Yashoda Technical Campus, Satara, Maharashtra, India.

*Corresponding Author E-mail: shinde9922297739@gmail.com

 

Abstract:

Objective: The purpose of research was to establish whether an ethanolic extract of Bryophyllum pinnatum (Lam) helps to protect rats against chemically induced urolithiasis. Materials and methods: Ethylene glycol-induced urolithiasis: For 28 days, an oral dose of 0.75% v/v ethylene glycol was given to induce urolithiasis. The animals were split up into six groups, each with six animals. For 28 days, Group I animals were administered with distilled water. For a period of 28 days, drinking water containing 0.75% v/v ethylene glycol was given to all groups except group I. Group III animals (standard group) were given 500mg/kg of Cystone from the 15th to the 28th day, Group IV, V, and VI were given oral doses of 50, 100, and 200mg/kg of Bryophyllum pinnatum. Urolithiasis produced by sodium oxalate: In this model, the animals were split into six groups, each consisting of six animals. Group I animals (control group) were administered with distilled water. Group II was given sodium oxalate (70mg/kg i.p.) as a lithiatic control. Group III animals were administered with 500mg/kg of the standard drug Cystone. Groups IV, V, and VI animals were administered Bryophyyum pinnatum orally at a dosage of 50, 100, and 200mg/kg. Result: The outcome showed that, in comparison to lithiatic control rats, the ethanolic extract of Bryophyllum pinnatum significantly decreased the concentrations of calcium, uric acid, magnesium, urea and creatinine in blood serum as well as oxalate, calcium, uric acid, magnesium, urea and creatinine in urine. Conclusion: By inhibiting kidney crystal deposition, the ethanolic extract of Bryophyllum pinnatum (lam) exhibits favorable action against urolithiasis.

 

KEYWORDS: Ethylene glycol, Sodium oxalate, Urolithiasis, Bryophyllum pinnatum, kidney.

 

 

INTRODUCTION:

Urolithiasis is a common clinical disorder, sometimes known as urinary stones. Urolithiasis is the term for the development of stones anywhere in the urinary tract. Uric acid, calcium, and phosphate make up the majority of stones. Approximately 12% of the human population is affected, with 80% of cases involving calcium oxalate calculi. There was a 70–80% recurrence rate1. A number of physio-chemical processes, including nucleation, supersaturation, crystal development, aggregation, and retention, combine to generate the intricate process of renal stone formation2. It is believed that when the urine becomes saturated with insoluble materials, due to an elevated excretion rate, aggregates and crystals will develop, eventually forming a stone.

 

Due to the increased risk of kidney stone recurrence, urolithiasis requires treatment that is both preventative and curative3. While several stone inhibitors are used in cases of renal stones to prevent the formation of new stones, their long-term usage is limited by pharmacological factors. As a result, they overlooked the most efficient way to remove stone4. How kidney stones are treated varies on their size and location. Numerous treatments, such as probiotics, diuretics, chelating agents, and citrate, are prescribed; however, they have drawbacks and adverse consequences when used frequently. Thus, surgical procedures such as ureteroscopy, percutaneous nephrolithotomy, and ESWL are used to remove renal stones in the majority of cases; however, a rate of stone recurrence has been noted5. Numerous herbal plants have reportedly been utilized to treat urolithiasis in Ayurveda.

 

Common names for Bryophyllum pinnatum (lam) include "air plant" and "life plant." These plants are primarily found in Africa, America, Australia, China, and India. It has anti-inflammatory, anti-hypertensive, anti-cancer, anti-fungal, and anti-ulcer properties in conventional medicine6. The leaves of Bryophyllum pinnatum are widely utilized in ethanomedical and traditional medicine to alleviate stone formation and urinary insufficiency. This plant's leaves have crucial ingredients in poly-herbal remedies intended to treat lithiasis7. The entire plant (leaves, bark, stem, and roots) of Bryophyllum pinnatum (lam) is being used in this study to assess the plant's anti-lithiatic efficacy. The pharmacological and therapeutic qualities of the Bryophyllum pinnatum plant are attributed to the presence of alkanes, alkanols, triterpenes, sterols, triterpenoids, phenanthrenes, flavonoids, alkaloids, glycosides, and lipids8.

 

MATERIALS AND METHODS:

Drugs and chemicals:

Chemicals of analytical grade were utilized in the experiment. Standard drug Cystone (Himalaya Herbal Healthcare, Banglore, India) were used. Kits for creatinine, urea, and calcium were purchased from Bomax Enterprises Private Limited, Hyderabad, Telangana, India.

 

Plant material:

Fresh plant of Bryophyllum pinnatum (Lam) (Class- Mangnoliopsida/Family- Crassulaceae) were collected from Kas Plateau Reserved Forest area in Satara, Maharashtra, India. Authentication and botanical identification were carried out at Botany Department of YCIS Collage, Satara, Maharashtra, India.

 

Animals:

Adult Albino wistar rats weighing 180–200g were obtained from the Animal House at Yashoda Technical Campus, Satara. Rats were kept in polypropylene cages with a 12hour light/dark cycle, at a temperature of 23±2°C and a relative humidity of 45–55°C. They were given a regular food and free access to water.

 

Acute toxicity studies:

The effective dose for rats in the acute oral toxicity trial (420) was chosen based on the lethal dose (LD50), in accordance with the recommendations established by the Organization for Economic Corporation and Development (OECD).

 

EXPERIMENTAL MODELS FOR ANIMALS:

1.     Ethylene glycol- induced urolithiasis (Group-1):

Thirty six Albino wistar rats were divided into six groups, each group contained six animals. Group I served as control received regular food and water. Group II- Group VI received ethylene glycol 0.75% v/v with ammonium chloride 2% w/v for stone formation till 28 days. Group II served as lithiatic control group received 0.75% v/v ethylene glycol with 2% w/v ammonium chloride for 28th days. Group III served as standard control group received standard antiurolithiatic drug Cystone (500mg/kg p.o.) from 15th to 28th day. Group IV, V, VI received test drug ethanolic extract of Bryophyllum pinnatum (BPEE) plant at a dose of 50mg/kg, 100mg/kg, 200mg/kg respectively from 15th to 28th day9.

 

2.     Sodium oxalate-induced urolithiasis (Group-2):-

In this experimental model 36 Albino wistar rats were divided into six groups, each contained six animals, Group I served as control received regular food and water. Group II - Group VI received sodium oxalate (70mg/kg i.p.) treatment for 14 days. Group II served as lithiatic control received 70 mg/kg sodium oxalate through intraperitonial route (i.p.) for 14 days. Group III served as standard control group received Cystone (500mg/kg p.o.) from 8th to 14th day. Group IV, V and VI served as test control groups received ethanolic extract of Bryophyllum pinnatum (BPEE) plant at a dose of 50mg/kg, 100 mg/kg, 200mg/kg of body weight respectively10

 

Body weight and urine output analysis:

The change in body weight of animals was noted along with that urine output in 24 hrs was also determined.

 

Urine analysis:

A sample of urine was obtained 24 hours before the experiment's end. Glass beakers were used to collect the sample. Measuring cylinders were used to determine the urine volume. Several urine parameters including oxalate, calcium, uric acid, creatinine, urea, and magnesium were measured in addition to the urine's pH11.

Serum analysis:

Blood samples of each animal were withdrawn from retro-orbital plexus using capillary method under mild anesthesia. Blood sample was centrifuged at 10000 rpm for 10 min and serum was separated for the analysis of creatinine, urea, calcium, magnesium and uric acid11.

 

Histopathological studies:

Rats were sacrificed under deep anaesthesia at the end of the experiments, and the kidneys were removed. The isolated kidneys were then cleansed of extraneous tissues and rinsed in ice-cold physiological saline. Following the infiltration of paraffin, the tissue slices were sectioned at a 5µm thickness and stained using eosin and hematoxylin for histological analysis12. The slides were inspected under a binocular microscope to check for calcium oxalate crystals, renal cellular alterations, tubular necrosis, and other histological changes in the kidney.

 

Statistical analysis:

The results are expressed as mean ± SEM. The statistical significance was assessed using One-way analysis of variance (ANOVA) followed by Dunnette’s multiple comparison test and value of p< 0.05 was considered statistically significant.

 

RESULT:

1.     Ethylene glycol induced urolithiasis model:

a.     Measurement of body weight and urine output:

Prior to the trial getting started, the rats' body weight and urine output capacity were established, and these values were nearly identical for each group. Table 1 displays the parameters that were recorded following treatment. The animals in the lithiactic control group had lost body weight as a result of the urolithiasis, while the animals in the other group had significantly gained body weight following the trial. With the exception of the lithiatic control group, which had a much lower urine output than the control group, there were no significant differences found between the groups.

 

Table 1: Effect of ethanolic extract of Bryophyllum pinnatum on body weight and urine output parameters of ethylene glycol induced urolithiasis in rats.

Parameters (unit)

Group I

(Normal Control)

Group II (Lithiatic Control)

Group III (Standard Control)

Group IV (Test 1)

Group V (Test 2)

Group VI (Test 3)

Body weight (%)

7.6 ± 0.5

-2.3 ± 0.07

5.12 ± 0.3

2.4 ± 0.1

3.94 ± 0.2

3.72 ± 0.25

Urine output (ml/24hr)

9.9 ± 0.65

7.2 ± 0.42

11.0 ± 0.5

12.6 ± 0.45

11.8 ± 0.72

10.3 ± 0.51

Values were expressed in mean±SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’ multiple comparison test p-values *p <0.05.

 

b.    Urine analysis:

When comparing ethylene glycol-induced rats to normal rats, there was a significant (p < 0.05) decrease in urine volume but no significant changes in pH. Rats treated with Cystone had significantly (p < 0.05) higher urine volumes than the lithiatic control group. When compared to rats receiving ethylene glycol, animals treated with BPEE at doses of 50mg/kg, 100mg/kg, or 200mg/kg body weight showed a considerable increase in urine volume without a significant change in pH. The current study found that giving ethylene glycol 0.75% v/v (p.o.) as a stone inducer results in hyperoxaluria, which is characterised by increased excretion of creatinine, oxalate, and uric acid in the urine and decreased magnesium excretion (Table 2).

 

Table 2: Effect of ethanolic extract of Bryophyllum pinnatum on urine parameters of ethylene glycol induced urolithiasis in rats.

Groups

Calcium mg/dl

Oxalate mg/dl

Uric acid mg/dl

Magnesium mg/dl

Urea mg/dl

Creatinine mg/dl

I

6.84 ±0.3

0.91 ±0.06

2.53 ±0.07

4.6 ± 0.04

39.61 ±1.2

1.76 ± 0.07

II

11.6 ±0.7

2.93 ±0.1

5.01 ±0.12

1.56 ± 0.02

48.02 ±2.4

3.32 ± 0.02

III

9.07 ±0.63

0.93 ±0.03

3.96 ±0.05

2.02 ± 0.1

32.9 ± 1.7

1.92 ± 0.05

IV

11.2 ±0.55

1.56 ±0.07

5.07 ±0.10

1.74 ± 0.08

43.64 ±2.9

2.84 ± 0.34

V

10.3 ±0.45

1.12 ±0.06

3.66 ±0.09

1.66 ± 0.05

36.12 ±2.3

2.20 ± 0.07

VI

9.10 ±0.42

0.97 ±0.02

4.02 ±0.2

1.93 ± 0.06

34.54 ±1.8

1.97 ± 0.1

Values were expressed in mean ± SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’ multiple comparison test p-values *p <0.05.

 

Figure 1: Effect of ethanolic extract of Bryophyllum pinnatum on urine parameters in ethylene glycol induced urolithiasis

 

c.     Serum analysis:

When compared to the normal group, the current study's ethylene glycol treatment considerably altered the levels of creatinine, urea, calcium, and uric acid in the serum. When compared to the lithiatic control group, treatment with BPEE (50, 100, and 200mg/kg) and cystone (500mg/kg) substantially reduces the levels of creatinine, urea, calcium, and uric acid while increasing the levels of magnesium (Table 3).

 

Table 3: Effect of ethanolic extract of Bryophyllum pinnatum on serum parameters of ethylene glycol induced urolithiasis in rats.

Groups

Calcium mg/dl

Uric acid mg/dl

Magnesium mg/dl

Urea mg/dl

Creatinine mg/dl

I

6.54±0.2

3.02±0.1

3.38±0.03

32.7±1.2

0.38±0.01

II

10.38±0.6

6.94±0.05

1.30±0.02

51.3±3.5

1.56±0.03

III

7.10±0.3

2.67±0.04

2.51±0.1

35.4±2.4

0.72±0.01

IV

9.45±0.09

5.08±0.3

1.02±0.02

44.61±2.5

1.31±0.02

V

8.20±0.2

4.32±0.03

2.98±0.03

27.67±1.7

0.67±0.009

VI

7.66±0.06

3.18±0.02

2.72±0.05

38.52±1.5

0.93±0.05

Values were expressed in mean ± SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’ multiple comparison test p-values *p <0.05.

 

Figure 2: Effect of ethanolic extract of Bryophyllum pinnatum on serum parameters of ethylene glycol induced urolithiasis in rats

 

d.    Histopathology of kidneys:

The kidney's microscope section demonstrated normal architecture in the normal group, but the kidneys of the lithiatic control group showed significant damage to the glomeruli, medulla, tubules, interstial space, and mononuclear cell infiltration. While renal damage was almost restored and crystal deposition was not observed in the cystone-treated group, substantial renal damage was recovered and intratubular space crystal deposition was prevented in the groups that received BPEE (50, 100, and 200mg/kg).

 

 

2.     Sodium oxalate induced urlithiasis:

a.     Measurement of body weight and urine output:

The body weight and urine output capacity of rats were determined before start of experiment which was almost same for all the groups. The parameters recorded after experiment are shown in Table 4. The urolithiatic treatment had shown loss in body weight of the animals in the lithiactic control group, whereas the other group animal as showed significant gain in their body weights after the experiment. Urine output was not significantly different among the groups except the lithiatic control group which was significantly low as compared to the control group.

 

Table 4: Effect of ethanolic extract of Bryophyllum pinnatum on body weight and urine output parameters of sodium oxalate induced urolithiasis in rats.

Parameters

(Unit)

Group I

(Normal Control)

Group II

(Lithiatic Control)

Group III (Standard Control)

Group IV (test 1)

Group V (test 2)

Group VI (test 3)

Body weight (%)

5.72±0.3

-2.70±0.06

6.84±0.3

1.9±0.04

3.66±0.065

4.2±0.08

Urine output (ml/24hr)

8.6±0.5

6.8±0.08

9.7±0.04

12.4±0.52

7.3±0.05

10.4±0.4

Values were expressed in mean ± SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’ multiple comparison test p-values *p <0.05.

 

b.    Urine analysis:

In the current investigation, the administration of sodium oxalate at a dose of 70mg/kg (i.p.) as a stone inducer results in hyperoxaluria, which is characterised by a decrease in magnesium and an increase in the excretion of calcium, creatinine, uric acid, and oxalate in the urine. When rats were given 70mg/kg (i.p.) of sodium oxalate, their urine volume decreased significantly (p<0.05) compared to rats given normal treatment. Rats treated with cystone had significantly (p<0.05) higher urine volumes than the lithiatic control group. When compared to rats given sodium oxalate, animals treated with BPEE at doses of 50mg/kg, 100mg/kg, and 200mg/kg body weight exhibited a substantial increase in urine volume (Table 5).

 

Table 5: Effect of ethanolic extract of Bryophyllum pinnatum on urine parameters of sodium oxalate induced urolithiasis in rats.

Groups

Calcium mg/dl

Oxalate mg/dl

Uric acid mg/dl

Magnesium mg/dl

Urea mg/dl

Creatinine mg/dl

I

7.02±0.3

1.02±0.02

2.21±0.03

3.90±0.06

35.8±2.5

1.43±0.01

II

13.8±0.7

2.31±0.05

4.93±0.02

1.34±0.025

47.36±1.8

4.06±0.02

III

10.5±0.6

0.97±0.01

3.54±0.09

1.79±0.03

29.82±1.4

1.66±0.01

VI

11.4±0.2

1.81±0.02

5.38±0.5

1.41±0.05

41.24±3.4

2.91±0.07

V

9.8±0.6

0.87±0.01

2.98±0.1

2.28±0.04

34.07±2.25

1.98±0.04

VI

10.9±0.2

1.06±0.02

3.62±0.04

1.82±0.01

32.80±1.3

2.02±0.03

Values were expressed in mean ± SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’multiple comparison test p-values *p <0.05.

 

Figure 3: Effect of ethanolic extract of Bryophyllum pinnatum on urine parameters of sodium oxalate induced urolithiasis in rats.

 

c.     Serum analysis:

When compared to the normal group, the current study's sodium oxalate 70mg/kg (i.p.) treatment resulted in significant evaluation in the serum levels of creatinine, urea, calcium, and uric acid. When compared to the lithiatic control group, treatment with BPEE (50, 100, and 200mg/kg) and cystone (500mg/kg) significantly lowers the levels of creatinine, urea, calcium, and uric acid while increasing the levels of magnesium (Table 6).

Table 6: Effect of ethanolic extract of Bryophyllum pinnatum on serum parameters of sodium oxalate induced urolithiasis in rats.

Groups

Calcium mg/dl

Uric acid mg/dl

Magnesium mg/dl

Urea mg/dl

Creatinine mg/dl

I

6.32±0.2

2.98±0.45

3.14±0.03

36.02±1.14

0.42±0.01

II

9.87±0.3

5.17±0.052

1.26±0.02

45.62±3.12

3.32±0.2

III

7.72±0.05

3.32±0.012

2.84±0.09

31.87±1.15

0.8±0.02

IV

9.30±0.1

5.56±0.5

1.12±0.08

42.13±2.25

1.76±0.04

V

7.81±0.07

4.01±0.02

2.97±0.06

29.70±1.67

1.02±0.01

VI

8.12±0.4

3.94±0.1

2.82±0.03

34.57±2.4

0.97±0.002

Values were expressed in mean ± SEM (n=6); Statistical significant test for comparisons was done by ANOVA, followed by Dunnet’ multiple comparison test p-values *p <0.05.

 

Figure 4: Effect of ethanolic extract of Bryophyllum pinnatum on serum parameters of sodium oxalate induced urolithiasis in rats.

 

d.    Histopathological studies:

The normal control group's histopathology showed no signs of damage or crystal deposition. The lithiatic control group had the highest degree of crystal deposition and damage. When compared to other groups, the group treated with cystone exhibits the least amount of crystal deposition and cell damage, while the group treated with BPEE (50, 100, and 200mg/kg) exhibits a medium amount of crystal deposition.

 

DISCUSSION:

An imbalance between the kidney's promoters and inhibitors typically results in urolithiasis. Increased tubular damage in the kidneys causes the intracellular calcium to be excreted through the urine, which raises calcium levels. Higher magnesium loss is caused by hypercalciuria. The production of calcium phosphate crystals, which directly produce CaOx deposition, appears to be facilitated by increased urine phosphorus excretion associated with oxalate stress13. Urinary tract stones cause obstructions to urine outflow in urolithiasis, which lowers the glomerular filtration rate. As a result, waste products, primarily nitrogenous ones like urea and creatinine, uric acid accumulate in the blood14.

 

Urolithiasis was produced in albino wistar rats for the current investigation. Wistar rats' urinary systems are similar to human urinary systems13. The first model we choose to cause urolithisis is the administration of ethylene glycol. Ethylene glycol metabolizes into glycoaldehyde by the action of alcohol dehydrogenase, further it converted into glycolic acid and glycoxalic acid which then metabolized to oxalic acid due to action of glycolic acid dehydrogenase on which action of intracellular Ca2+ takes place to form calcium oxalate15. Rats receiving ethylene glycol 0.75% v/v in drinking water to develop hyperoxaluria and hypercalciuria15. Additionally, intratubular crystal deposits are found in the cortex and medulla from day 1 onward along with tubular damage, dilatation, regeneration, and interstitial inflammation16. In this model, every group with the exception of the normal control group has kidney crystal presence, which results in urolithiasis.

 

Previous research shown that injection of sodium oxalate (NaOx) causes hyperoxaluria17. Since hyperoxaluria had been accepted as a significant risk factor in a etiology of renal stone18. In the current investigation, calcium crystal formation within tubules was caused by sodium oxalate (NaOx) at a dose of 70mg/kg (i.p.). Crystals are first intraluminated and then found in the interstitial space and tubular cells19. If endolysis of crystals was performed, then crystal uptake and transfer was increased which forms interstitial crystal growth; if cell damage or death occurs, then crystal agglomeration was increased which forms luminal crystal growth which leads to stone formation20. Sodium oxalate alters either membrane properties or cell surface proteins which leads to cell injury further it leads to crystal nucleation or crystal adhesion. With the exception of the normal control group, all animal groups exhibit crystal deposition as a result.

 

In some parts of India, Bryopyllam pinnatum is referred to as panphuti or Pattharcatta, which suggests that it has the ability to dissolve stones. The plant's leaves have historically been used to cure stones21. The test medication in this study is an extract of the entire plant, including the leaves, stem, bark, and roots. As a solvent, ethanol was utilized to extract the medication. According to the current investigation, the ethanolic extract of the Bryophyllum pinnatum plant reduced the alterations in urine and serum parameters as well as the kidney's histology that were caused by stones.

 

The ethanolic extract of the Bryophyllum pinnatum plant possesses alkaloids, flavonoids, phenols, carbohydrates, proteins, and amino acids, according to phytochemical analysis studies. These phytochemicals are responsible to dissolve or heal stones22.The current investigation demonstrates that Bryophyllum pinnatum (BPEE) ethanolic extract administration increased urination and prevented the supersaturation of stone forming salts in the urinary tract.

 

Urine magnesium levels and creatinine clearance are gradually decreasing showing that these are accumulating in the blood and raising the risk of urolithiasis. While decreasing crystallisation, BPEE treatment raised urine magnesium levels and creatinine clearance. Chemicals that produce glycolate and oxalate, which raise free radicals, and nitrogenous compounds that cause acute tubular necrosis in rats enhance the activity of glycolic acid oxidase23. Serum creatinine, urea, and uric acid levels were all elevated in lithiatic control rats, indicating severe damage to the kidneys. After receiving Cystone and Bryophyllum pinnatum extract, lower levels of these nitrogenous substances show that renal damage can be prevented.

 

CONCLUSION:

The ethanolic extract of the Bryophyllum pinnatum plant has anti-urolithiatic activity in chemically induced urolithiasis by suppressing calcium, oxalate, uric acid and urea in kidney tissues and promoting various inhibitors like citrate and magnesium in serum and urine. This conclusion can be drawn from the discussion above. Additionally, BPEE has a strong diuretic effect that is helpful in promoting calcium oxalate stone dissolution and inhibiting the formation of new stones. According to the current study, BPEE has the potential to be an effective treatment and may assist with preventing and treating kidney stones. However, more research is need to fully understand the mechanism of the Bryophyllum pinnatum plant.

 

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Received on 15.06.2024      Revised on 27.08.2024

Accepted on 08.10.2024      Published on 14.12.2024

Available online on December 05, 2024

Research J. Science and Tech. 2024; 16(4):283-290.

DOI: 10.52711/2349-2988.2024.00040

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