IJCRR - 14(4), February, 2022
Date of Publication: 15-Feb-2022
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Comparative Study of the Antiurolithiatic Activity of Leea macrophylla, Alangium lamarkii, Saccharum officinarum, Marsdenia tenacissima, Chonemorpha macrophylla, Maerua oblongifolia
Author: Nangare N B, Deshpande M M, Arulmozhi S, Kurulkar M A
Abstract:Introduction: Incidence of urinary tract disorders and kidney disorder including nephropathies is on the rise. In spite of tremendous development of modern medicine, still there is no appropriate medical treatment for these problems.
Aim: In Ayurved, the plant Morat mentioned in Veeratarvadi gana' [type of classification], which has prime indication in Ashmari (Urolithiasis). Under the name of Morat, botanical identity such as Leea macrophylla, Saccharum officinarum, Alangium lamarkii, Marsdenia tenacissima, Maerua arenaria, Chonemorpha macrophylla are considered as they exhibit same medicinal properties
Methodology: The purpose of this work was to compare anti-urolithiatic activity of these plants by Ethylene glycol induced model method. Also, comparison of antiurolithiatic property of these plants and Cystone is studied.
Result: In this article effect on urine output, pH, and concentrations of urinary calcium, phosphate, oxalate & total protein, serum concentrations of Creatinine, Uric Acid, Calcium Phosphate and BUN is studied. Saccharum officinarum L, Chonemorpha macrophylla (Roxb) G. Don, Maerua arenaria Hook, Marsdenia tenacissima exhibited significant antiurolithiactic activity.
Conclusion: Comparison suggests that Chonemorpha macrophylla is the most suitable source of Morat for Antiurolithiatic Activity is.
Keywords: Morat, Veeratarvadi gana, Ashmari, Cystone, Antiurolithiactic activity, Ayurveda
The incidence of urinary tract disorders and kidney disorders including nephropathies is on the rise. The incidence is higher in developing countries including India. UTI, Urolithiasis, Diabetic Nephropathy & Nephrotoxic drugs are the most common conditions which cause injury to the kidney. Prevalent environment, lifestyle, and socioeconomic conditions have contributed to the rising incidence of the same. In spite of the tremendous development of modern medicine, still there is no appropriate medical treatment for these problems. There is, therefore continuous rising demand for safe & effective herbal drugs. Hence in recent years, plants documented in Ayurvedic literature like Charaka Samhita, Sushruta Samhita, and Ashtanga Hridaya has been received much attention. In Ayurved, many categories of drugs (Gana) are mentioned which could be useful in kidney disorders. ‘Veeratarvadi gana’ is one of them, which has prime indication in Ashmari (Urolithiasis).1 Morat is one of the controversial plants, which is mentioned in this group. Plants can be considered as Morat are of L. macrophylla, A. lamarkii, S. officinarum, M. tenacissima, C. macrophylla. M. oblongifolia.2 This study was conducted to evaluate & the anti-urolithiatic activity of species that are mentioned under the name of Morat in animal.
Material and Method:
Plant collection and authentication: All plants self-collected from their natural habitats & identified on the basis of their morphological characters with the help of by Dr. S. D. Jagtap (Senior Taxonomist & Head, Herbal Biotechnology, IRSHA, Bharati Vidyapeeth, Pune, India.) & flora. Voucher specimens of all six plants have been deposited in Regional Ayurveda Institute for Fundamental Research(RAIFR), Pune, India.
Processing of plant material: Collected plant samples were shade dried, powdered with a mechanical grinder, sieved through 80# mesh and stored in air tight glass vessel. These powders were utilized for various experimental studies. Kwatha (decoction) of test drugs were freshly prepared prior to administration to the animals. It was prepared according to the procedure mentioned in the “Sharngadhar Samhita” Madhyam khand 2/77 i.e. 16 parts water and one part drug which was boiled on low flame till 1/8th part remained. This was filtered and allowed to cool before administering to the animals.9
Pharmacological screening for antiurolithiatic activity
Animals: Wistar strain albino rats of either sex; weighing 180 to 250 g were obtained from the National Institute of Biosciences, 1091/ABC/07/CPCSEA & maintained in the animal house of Bharati Vidyapeeth University, Poona College of Pharmacy, Pune. Animals were housed on straw bedding 6 per cage & exposed to natural day and night cycles with ideal laboratory conditions in term of ambient temperature and humidity. The Institutional Animal Ethics Committee approved the protocol of this study (CPCSEA Number: CPCSEA/PCP/PCL05/2018)
Grouping and posology: Animals were divided into nine groups containing 6 animals in each group. The dose for experimental animals was calculated on the basis of body surface area ratio by referring to the standard table of Paget and Barnes (1969). 10
Ethylene glycol-induced urolithiasis model: The method of Ethylene glycol-induced model 11 was employed for the assessment of antilithiatic activity. Rats were divided in nine groups containing six in each. Ethylene glycol (0.75% v/v) in drinking water was feed to all groups except control for induction of renal calculi till the 28th day. In the study all the groups except the control received test drugs from 15th day till 28th day.11 Cystone was used as a standard drug. During the study animals were allowed free access to food.12, 13
From day 1 to day 14, ethylene glycol (0.75%) in drinking water was fed orally (q.s.) to rats from group II to IX for rendering them hyperoxaluric. The state of hyperoxaluria was checked and confirmed by collection and examination of urine after day 14. Then onwards till day 28, rats from group III to IX received curative treatments with individual drugs and group I and II received saline water (5 ml/kg). All doses were administered once daily by oral route.
Collection and assessment of urine: Rats were kept in individual metabolic cages for collection of 24 h urine. They had free access to drinking water during the urine collection period. During the entire study period, rats were subjected to 24 h urine collection twice. After day 14, urine was collected and analyzed to check and confirm the extent of Urolithiasis. After completion of study period (i.e. day 28), urine was collected and analyzed for biochemical parameters.
Routine urinalysis: Routine urinalysis was carried out using diagnostic reagents & kits (Coral Clinical Systems, Gitanjali, Tulip Block, Dr Antonio DO Rego Bagh, Bambolim Complex P.O. Goa) including quantitative determination of pH and specific gravity along with the presence of occult blood, bilirubin, urobilinogen, ketone bodies, proteins, nitrite, glucose and leucocytes in urine.
Urine biochemistry: A drop of concentrated hydrochloric acid was added to the urine before being stored at 4ºC. Using biochemical estimation kits, urine was analyzed for calcium and phosphate content; while, urinary oxalate content was estimated using a modified method of Hodgkinson and Williams (1972).
Assessment of serum: After the experimental period, blood was collected from the retro-orbital vein under anesthetic conditions. Serum was separated by centrifugation at 10,000 rpm for 10 min and analyzed for creatinine, uric acid and urea nitrogen.
Assessment of kidney: Finally all rats were sacrificed using an appropriate method of euthanasia. The abdomen was cut open to remove either kidney from each rat. Isolated kidneys were cleaned off extraneous tissue and preserved in 10% neutral formalin. One kidney (randomly selected) from each rat was processed for preparing kidney homogenate. From the remaining kidneys, one kidney as representative of the whole group was selected randomly and processed for a histopathology examination.
Kidney homogenate analysis: Each selected kidney was dried at 80ºC in a hot air oven. A sample of 100 mg of the dried kidney was boiled in 10 ml of 1 N hydrochloric acid for 30 min and homogenized. The homogenate was centrifuged at 2000 rpm for 10 min and the supernatant was separated. The calcium, phosphate and oxalate content in kidney homogenate were determined as mg/g of kidney weight using the methods as described earlier.
Histopathological examination of kidney: Each selected kidney was embedded in paraffin using conventional methods and cut into 5 µm thick sections and stained using hematoxylin-eosin dye and finally mounted in diphenyl xylene. Then each section was observed under a microscope for histopathological changes in kidney architecture and their photomicrographs were taken at x50 magnification. By visualizing different fields, a general method of scoring was adopted to observe the extent of nephritic damage and recovery. A minimum of 10 fields for each kidney slide were examined and assigned for the severity of renal damage and progression of recovery using scores on a scale of none (ND), mild (+), moderate (++) and severe (+++) damage.
Statistical analysis Results were expressed as mean ± SEM. Differences among data were determined using one-way ANOVA followed by Dunnett’s posttest using Graph pad Prism software (Graph pad Prism software Inc., Version 4.00.255). Differences between the data were considered significant at p<0.05.
Effect on urine output and pH in Ethylene glycol induced urolithiasis: Details of the effect of the Cystone and other test drugs in urolithiasis animals are summarized here in table 3. It shows that, in untreated animals, urine output was reduced. There was increased urine output observed in the groups treated with cystone, S. officinarum, M. tenacissima, C. macrophylla & M. arenaria. It was observed that there was increase in urinary pH of 7.117 in calculi-induced Group II, but it was brought down gradually toward normal value in the groups treated with L. macrophylla, A. lamarkii, S. officinarum, M. tenacissima, C. macrophylla. M. oblongifolia and Cystone
Values are expressed as mean ± SEM.*p<0.05,**p<0.01,***p<0.001 when compared to vehicle control. ns – Nonsignificant
Effect on Calcium, Phosphate, Oxalate & total protein: Details of the concentrations of urinary calcium, phosphate, oxalate & total protein are presented in Table 4. In the present study, group treated with S. officinarum M. tenacissima, C. macrophylla. & M. oblongifolia and Cystone shows a significant decrease in concentrations of urinary calcium, phosphate, oxalate & total protein.
Values are expressed as mean ± SEM.*p<0.05,**p<0.01,***p<0.001 when compared to vehicle control. ns - Nonsignificant
Effect on Serum Levels of Creatinine, Uric Acid, Calcium Phosphate and BUN: Details of the serum concentrations of Creatinine, Uric Acid, Calcium Phosphate and BUN are presented in table 5. In the present study, in calculi-induced rats (Group II), marked renal damage was seen by the elevated serum levels of creatinine, uric acid and Blood Urea Nitrogen. However group treated with S. officinarum, M. tenacissima, C. macrophylla. & M. oblongifolia and Cystone restored the elevated serum levels of creatinine, uric acid and BUN
The Calcium, Phosphate, Total Protein and Oxalate content in the kidney homogenate: Details of the Calcium, Phosphate and Oxalate content in the kidney homogenate is presented in table 6. Kidney homogenate analysis revealed increased amount (P< 0.001) of stone-forming components Calcium, Phosphate and Oxalate content in diseased animals(Group II vs. Group I).
Kidney homogenate analysis revealed increased amount of stone-forming components calcium, phosphate, and oxalate in diseased animals (Group II vs. Group I). The animals treated with S. officinarum, M. tenacissima, C. macrophylla & M. oblongifolia & cystone showed decreased levels of stone-forming components. Whereas animals treated with L macrophylla & A. lamarckii didn’t show reduced content of calcium, phosphate, and oxalate.
Many medicines have been employed during the ages to treat urinary stones. In Ayurveda, most of the medicines were plant origin and they were shown to be beneficial, though the rationale behind their practice is not well proven through systematic pharmacological studies. Morat is one of the effective drug, which was mentioned in conditions such as Mutrakruchra, Mutraghata & Ashmari.
Urolithiasis is one of the common kidney disorders. The majority of stones found very often in humans are composed mainly of calcium oxalate or calcium oxalate stones mixed with calcium phosphate. So in the present study, Ethylene glycol-induced model was employed for the assessment of the activity.
Antiurolithiatic plants show multidimensional action & effectiveness at different stages of urolithiasis. In the present study, there was increased urine output observed in the groups treated with Cystone, S. officinarum, M. tenacissima, C. macrophylla and M. arenaria. So it can be stated that the above plants shows effectiveness in urolithiasis as their diuretic action increases the quantity of fluid going pass through the kidney & flush out the deposits.
Oxalate, calcium and phosphate elimination were increased significantly in the Ethylene glycol induced animals (Group II) which caused hyperoxaluria. It indicates stone formation in ethylene glycol-fed animals.
Group treated with S. officinarum M. tenacissima, C. macrophylla, M. arenaria and Cystone shows significant decrease in concentrations of urinary calcium, phosphate, oxalate & total protein.
Accumulation of the CaOx crystals in the kidney increased the urinary pH, which is one of the indications of urolithiasis. It was observed that there was increase in urinary pH of 7.117 in calculi-induced Group II, but it was brought down gradually toward normal value in the groups treated with L. macrophylla, A. lamarkii, S. officinarum, M. tenacissima, C. macrophylla. M. oblongifolia and Cystone.
The glomerular filtration rate (GFR) is significant parameter for confirming renal function and it gets reduced in urolithiasis due to the obstruction to the urine output by stones in urinary system, which leads to increase in urea, creatinine, and uric acid in blood. In Ethylene glycol induced animals (Group II), noticeable renal impairment was seen by the raised serum levels of Creatinine, Uric acid and Blood Urea Nitrogen (BUN). However, the groups treated with L. macrophylla, A. lamarkii, S. officinarum, M. tenacissima, C. macrophylla. M. arenaria and Cystone restored the elevated serum levels of creatinine, uric acid and BUN.
Kidney homogenate analysis revealed increased amount (P< 0.001) of stone forming components Calcium, Phosphate and Oxalate content in diseased animals (Group II vs. Group I), which indicating Ethylene glycol induced urolithiasis.
The animals treated with S. officinarum, M. tenacissima, C. macrophylla. & M. arenaria & cystone showed decreased levels of stone-forming components. Whereas animals treated with L macrophylla & A. lamarckii didn’t show reduced content of calcium, phosphate, and oxalate.
Saccharum officinarum L, Chonemorpha macrophylla (Roxb) G. Don, Maerua arenaria Hook, and Marsdenia tenacissima exhibited significant antiurolithiactic activity in animal model. Plants mentioned in name of Morat, except Marsdenia tenacissima exhibited significant diuretic activity in animal model. The inter-group comparison suggests that the most suitable source of Morat is Chonemorpha macrophylla
The authors would like to express their sincere thanks to Dr. S. D. Jagtap (Senior Taxonomist & Head of Department, Herbal Biotechnology, IRSHA, Bharati Vidyapeeth, Pune, India.) for helping in the authentications of plant samples.
Conflicts of Interests: NIL
Source of funding: NIL
Nangare N performed the experiments, drafted the manuscript, analysed and interpreted the data.
Deshpande M, Arulmozhi S., designed the experiments, supervised, analysed, critically revised and finally approved the manuscript.
Kurulkar M interpreted the data & technically supported the study.
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