INTRODUCTION:

One of the oldest and most common disorders is kidney stone production. People in various states of India use different herbs to treat urolithiasis. The urinary tract is thought to be the third most frequent ailment.

Urinary stone disease affects the biggest number of individual’s worldwide, accounting for about 4–15 percent of the global population. Urinary stones are predicted to impact 12% of the population in India, with 50% of those affected suffering from kidney failure or injury. Urolithiasis is a multifactorial illness that is very unexpected and has a complicated etiology. A stone is an accumulation of urine solute components such as calcium, phosphate, uric acid, and oxalate. Calcium oxalate is determined to be the most common element of urolithiasis in India¹. India's population suffers from urinary tract and kidney stones caused by calcium, phosphate, and oxalates deposits. These stones can last indefinitely, generating subsequent issues with catastrophic ramifications for the patient's life². It is excruciatingly uncomfortable, and a correct remedy is required to resolve the issue. Depending on where they are detected, kidney stones are referred to as urinary calculi, urinary tract stone disease, renal calculi, ureterolithiasis nephrolithiasis, and urolithiasis. Appropriate and timely treatment of kidney stones can prevent major complications such as partial or complete renal function loss³. Urine is a chemical solution containing a range of chemical components, urinary tract stones are a common issue. These chemicals crystallise readily and expand in size to create stones. Because there is no adequate medical treatment for such stone diseases, Except for few composite herbal medications and plants, the rationale behind their usage in ancient systems of medicine, including Ayurveda, has not been adequately established via rigors pharmacological and clinical investigations⁴. These plant components are said to be useful in lowering the reappearance rate of urinary stones. In indigenous medicine, the leaves of Tridax procumbens are used to cure a range of diseases, including urinary stones. There has been no scientific research published on the in vitro anti-urolithiatic efficacy of different extracts of Tridax procumbens flowers against calcium stones⁵.

METHODS AND MATERIAL:

Materials:

Drangendroff reagent, 1% Ferric chloride, 2% Lead acetate, Ethanol Rankem Pvt. Ltd. Distilled water purchased from Shree Ganesh scientific syndicate, Amravati, Maharashtra, India.

Collection of plant material and authentication:

In February 2022, the flowers of Tridax procumbens were taken from the nursery in the Amravati in the state of Maharashtra and the plant is authenticated at P.R.Pote Patil College of Agriculture, Amravati (281/2021) dried in the shade and the botanist made sure that the plant was real. The dried leaves were ground up into a rough powder (passed through sieve no. 40). After complete drying, the dried leaves were extracted using hot maceration with solvent ethanol, distilled water, and hydroalcoholic solution in various concentrations.

Figure 1- Flower of Tridax procumbens⁸

Plant Profile

Scientific name: Tridax procumbens

Family: Asteraceae

Kingdom: Plantae

Order: Asterales⁷.

METHOD:

Extraction:

The dried flowers were shade dried and powdered in a mixer grinder and stored in an airtight jar for study. The extraction was performed using the hot maceration technique according to standard procedure^9-11. The solvent used was ethanol and distilled water. First, the samples were divided into five groups for extraction, each containing a sample of 30g. The first sample was extracted with 200ml of pure ethanol, then in the second group, samples were extracted with pure distilled water and hydroalcoholic preparations were prepared for the remaining samples in different concentration ratios. The first was 70:30, which contained 140ml of ethanol and 60 ml of water; the second was 50:50, which contained 100ml of ethanol and 100ml of distilled water; and the last one was 70:30, which contained 140ml of distilled water and 60ml of ethanol. Each extract was subsequently filtered, and the filtrate was evaporated. All five samples were assembled for hot maceration for 24 hours and after that, the filtrate was collected and kept for evaporation. On evaporation, the solid form was obtained as a product.

Evaluation of Antiurolithiatic activity for Tridax procumbens:

Preparation of experimental kidney stone (Calcium phosphate, Calcium oxalate):

In 100ml distilled water, 1.47 gm calcium chloride dihydrate was dissolved, while 1.42gm disodium hydrogen phosphate was dissolved in 100ml of 2N H2SO4. To precipitate calcium phosphate, all were mixed evenly in a beaker with agitation. The calcium phosphate that resulted was washed with distilled water and dried for 2hours. The calcium phosphate stone was then punched into tablets and served as stones in the study¹². In 100mL of distilled water, 2.94g of calcium chloride dihydrate was dissolved, while 2.84g of disodium hydrogen phosphate was dissolved in 100mL of 2N H2SO4. To precipitate calcium phosphate, all were mixed evenly in a beaker with agitation. The calcium phosphate that resulted was washed with distilled water and dried for 2 hours. The synthesised calcium oxalate stone was then punched into tablets, which were used as stones in the study¹³.

Tablet Punching: By Direct Compression Method: Calcium oxalate and calcium phosphate are shaped into spherical kidney stones by direct compression, per usual method¹⁴.160mg of calcium oxalate and calcium phosphate are combined with the binder HPMC (Hydroxy Propyl Methyl Cellulose-E-3) and then compressed in a tablet compression machine.

Figure 2: Calcium oxalate and Calcium Phosphate tablets.

In vitro Antiurolithiatic study:

In vitro activity was assessed by changing the conventional procedure^15-17. Weight fluctuation and dissolving of calcium phosphate and calcium oxalate tablets were carried out in this study. A pH 7.4 phosphate buffer was made, and accurately weighed calcium oxalate and calcium phosphate tablets were inserted in each extract solution (concentrations of 100 mg/ml and 200mg/ml, respectively) and packed individually in the semi permeable membrane and sutured. A standard solution was also produced and utilised. The semi permeable membrane was allowed to float in the beaker independently. A negative control (perfectly weighted calcium phosphate and calcium oxalate tablet) was inserted in the semi-permeable membrane with 100ml of normal saline solution in the same way. For 12 days, all of the beakers were exposed to a percent dissolution investigation. The weight loss of calcium phosphate and calcium oxalate tablets was measured in each bag at a two-week interval. The dissolving rate was calculated by multiplying the beginning and end weights of the tablets using the following formula: -

% Dissolution = (W initial – W final) 100/W initial

W – Before and after weights of tablets^18-22.

Phytochemical constituent:

Standard techniques were used to test the extracts for the presence of active phytochemical components.

1. Phenols test: 1 ml of extract was mixed with 1% ferric chloride solution. The green colour showed that phenols were present.

2. Flavonoids test: 3-5 drops of a lead acetate solution with a concentration of 2 Percent were added to 1ml of alcoholic extract. The appearance of orange or yellow colour shows that flavonoid is there.

3. Alkaloid test: Alkaloids were detected by adding one drop of Drangendroff reagent to one millilitre of extract and looking for a yellow precipitate.

4. Triterpenoid Saponins Test: Test of Froth Formation In a test tube, when 1ml of extract was mixed with 1ml of water, foam formed¹⁹.

5. Bendict's Test: The extract mixed with Bendict's reagent and boiled until a red colour showed. This showed that the extract contained carbohydrate.

6. Biuret Test: A few drops of a 5 percent Sodium Hydroxide solution and a 1 percent Copper Sulphate solution were added to 2-3 ml of the extraction. When there is protein, the colour will be violet or pink^23-25.

RESULT AND DISCUSSION:

Extraction yield:

The extraction yield of all extracts in the solvent of different concentrations was different, as we carried out extraction in different solvents at different concentration ratios. All the extraction yield of all extracts of different solvents was in grams, and it was converted into percentages as shown in Table 1. From the sample of pure ethanol, we got a 20% yield, and from the sample of pure water and 70:30 HA, we got a high amount of yield, i.e., 90% and 88%.

Table 1 Extraction yield of different solvent extracts.

Extracts	Percentage Yield
Pure water	90%
Pure ethanol	20%
70:30 HA	88%
30:70 HA	58%
50:50 HA	62%

Phytochemical screening:

The result of phytochemical screening is given in Table 2. We performed phytochemical screening by standard procedure and we found the presence of bioactive compounds like alkaloid, phenol, flavonoids, carbohydrates, protein, triterpenoid saponin, and alkaloid.

Table 2: Result of Phytochemical Analysis of extracts.

Sr. No	Phytochemicals	Test	Result (Leaves)
01	Alkaloids	Dragandroff test	+
02	Flavonoids	Lead. Acetate test	+
03	Phenols	Ferric Chloride test	+
05	Carbohydrate	Benditc’s test	+
06	Protien & Amino acid	Ninhydrin test	+
07	Triterpenoid Saponin	Froth formation test	+

[ (+) = Present (-) = Absence]

On the basis of this fraction, we compared the in vitro anti-urolithiatic activity of several Tridax procumbens leaf extracts with the standard Cystone against calcium phosphate and calcium oxalate stones. The second table compares the dissolving of calcium phosphate tablets by Tridax procumbens extract with Cystone.

Table 2: Weights of calcium phosphate tablets before and after treatment (100mg)

Sample	Concentration	Initial Weight of Calcium phosphate	Weight of Calcium phosphate after 4 days	Weight of Calcium phosphate after 8 days	Weight of Calcium phosphate after 12 Days
Cystone	100mg	0.163	0.1529	0.1496	0.1485
Control ^(NaCl)	100mg	0.163	0.095	0.085	0.08
Pure Ethanol	100mg	0.163	0.111	0.106	0.109
Pure Water	100mg	0.163	0.1443	0.1456	0.1449
50:50 HA	100mg	0.163	0.1409	0.1396	0.1397
70:30 HA	100mg	0.163	0.1325	0.1368	0.1305
30:70HA	100mg	0.163	0.1216	0.1263	0.1256

Figure 3- Percent Graphical representation of dissolution of calcium phosphate (100 mg)\

Table 3: Weights of calcium phosphate tablets before and after treatment (200mg)

Sample	Concentration	Initial Weight of Calcium Phosphate	Weight of Calcium phosphate after 4 days	Weight of Calcium Phosphate after 8 days	Weight of Calcium Phosphate after 12 Days
Cystone	200mg	0.163	0.1523	0.1482	0.1472
Control ^(NaCl)	200mg	0.163	0.095	0.085	0.08
Pure Water	200mg	0.163	0.144	0.13	0.125
Pure Ethanol	200mg	0.163	0.104	0.102	0.1
50:50 HA	200mg	0.163	0.1406	0.1355	0.132
70:30 HA	200mg	0.163	0.1322	0.1245	0.1215
30: HA	200mg	0.163	0.1255	0.1212	0.1133

Figure 4- Percent Graphical representation of dissolution of calcium phosphate (200 mg)

Table 4: Weights of calcium oxalate tablets before and after treatment (100mg)

Sample	Concentration	Initial Weight of Calcium oxalate	Weight of Calcium oxalate after 4 days	Weight of Calcium oxalate after 8 days	Weight of Calcium oxalate after 12 Days
Cystone	100mg	0.163	0.1552	0.1489	0.1469
Control	100mg	0.163	0.092	0.082	0.079
Pure Ethanol	100mg	0.163	0.115	0.111	0.106
Pure Water	100mg	0.163	0.148	0.145	0.135
50:50 HA	100mg	0.163	0.142	0.138	0.125
70:30 HA	100mg	0.163	0.135	0.128	0.122
30:70 HA	100mg	0.163	0.127	0.125	0.121

Figure 5- Percent Graphical representation of dissolution of calcium oxalate (100 mg

Table 5: Weights of calcium oxalate tablets before and after treatment (200mg)

Sample	Concentration	Initial Weight of Calcium oxalate	Weight of Calcium oxalate after 4 days	Weight of Calcium oxalate after 8 days	Weight of Calcium oxalate after 12 Days
Cystone	200mg	0.163	0.159	0.149	0.145
Control	200mg	0.163	0.098	0.088	0.083
Pure Ethanol	200mg	0.163	0.124	0.121	0.111
Pure Water	200mg	0.163	0.1443	0.1419	0.1409
50:50 HA	200mg	0.163	0.1325	0.1318	0.1311
70:30 HA	200mg	0.163	0.124	0.1226	0.119
30:70 HA	200mg	0.163	0.122	0.116	0.111

Figure 6- Percent Graphical representation of calcium oxalate (200 mg)

Figures 5 and 6 show graphical representations of percent calcium oxalate dissolution by Tridax procumbens extract in comparison to Cystone pure water and 50:50 hydroalcoholic extract, which produced higher calcium oxalate dissolution than other fractions and the standard. While standards show higher dissolution as compared to others, while ethanol extract shows the lowest dissolution of calcium oxalate as compared to other.

According to a comprehensive phytochemical investigation, it was shown that the crude extracts of Tridax procumbens flowers contain a significant number of bioactive components. These chemicals include alkaloids, phenols, flavonoids, carbohydrates, proteins, and saponins. An in vitro technique for measuring the dissolving percentage of kidney stones was used to test the anti-urolithiatic activity of Tridax procumbens flowers, and the results were compared to those of a reference medication. It has been shown that many extracts are the most efficient in urolithiatic activity.

CONCLUSION:

The study's goal is to learn more about the beneficial effects of Tridax procumbens plant, particularly leaves, in preventing and treating medical issues such kidney stones, which are becoming more common in the younger population as a result of sedentary lifestyles and poor eating habits. The aqueous extract of leaves was discovered to have strong anti-urolithiatic efficacy in the current investigation. Calcium phosphate and calcium oxalate stones dissolve well in aqueous extract of Bryophyllum pinnatum. The decrease in calcium phosphate and calcium oxalate stones is reduced in ethanolic extract and other hydroalcoholic solutions (70:30, 50:50, 30:70). As a result of the above research, we can infer that the anti-urolithiatic activity of Tridax procumbens flowers was satisfactorily assessed.

REFERENCES:

1. Malviya V, Ladhake V, Gajbiye K, Satao J, Tawar M. Design and Characterization of Phase Transition System of Zolmitriptan Hydrochloride for Nasal Drug Delivery System. International Journal of Pharmaceutical Sciences and Nanotechnology. 2020 May 31; 13(3):4942-51.

2. Malviya VR, Pande SD, Bobade NN. Preparation and Evaluation of Sustained Release Beads of Zolmitriptan Hydrochloride. Research Journal of Pharmacy and Technology. 2019 Dec 30; 12(12):5972-6.

3. Malviya VR, Pande SD. Road CKN. Preparation ad Evaluation of Zolmitriptan Hydrochloride Lozenge. J Pharma Res. 2019; 8(8):624-9.

4. Malviya VR, Tawar MG. Preparation and Evaluation of Oral Dispersible Strips of Teneligliptin Hydrobromide for Treatment of Diabetes Mellitus. International Journal of Pharmaceutical Sciences and Nanotechnology. 2020 Jan 31; 13(1):4745-52.

5. Fürer K, Simões-Wüst AP, von Mandach U, Hamburger M, Potterat O. Tridax procumbens and related species used in anthroposophic medicine: constituents, pharmacological activities, and clinical efficacy. Planta Medica. 2016 Jul; 82(11/12):930-41.

6. Malviya V, Manekar S. Design, Development and Evaluation of Aceclofenac and Curcumin Agglomerates by Crystallo Co-Agglomeration Technique. Research Journal of Pharmacy and Technology. 2021 Mar 1; 14(3):1535-41.

7. Burange PJ, Tawar MG, Bairagi RA, Malviya VR, Sahu VK, Shewatkar SN, Sawarkar RA, Mamurkar RR. Synthesis of silver nanoparticles by using Aloe vera and Thuja orientalis leaves extract and their biological activity: a comprehensive review. Bulletin of the National Research Centre. 2021 Dec; 45(1):1-3.

8. Malviya V. Preparation and Evaluation of Emulsomes as a Drug Delivery System for Bifonazole. Indian Journal of Pharmaceutical Education and Research. 2021 Jan 1; 55(1):86-94.

9. Malviya V, Pande S. Development and Evaluation of Fast dissolving Film of Fluoxetine hydrochloride. Research Journal of Pharmacy and Technology. 2021 Oct 31; 14(10):5345-0.

10. Malviya V. Design and Characterization of Thermosensitive Mucoadhesive Nasal Gel for Meclizine Hydrochloride. International Journal of Pharmaceutical Sciences and Nanotechnology. 2022 Feb 28; 15(1):5782-93.

11. Malviya V, Burange P, Thakur Y, Tawar M. Enhancement of Solubility and Dissolution Rate of Atazanavir Sulfate by Nanocrystallization. Indian Journal of Pharmaceutical Education and Research. 2021 Jul 1; 55(3):S672-80.

12. Malviya, Vedanshu, Mukund Tawar, Prashant Burange, and Rahul Jodh. A Brief Review on Resveratrol. 2022: 157-162.

13. Lin K, Wu C, Chang J. Advances in synthesis of calcium phosphate crystals with controlled size and shape. Acta Biomaterialia. 2014 Oct 1; 10(10):4071-102.

14. Shangraw RF. Compressed tablets by direct compression. Pharmaceutical Dosage forms: Tablets. 1989 Jun 5; 1:195-246.

15. Bansode P, Pawar P, Babar M. Invitro Urolithiatic Activity Of Tridax procumbens Against Experimentally Designed Calcium Oxalate And Calcium Phosphate Stones.

16. Yadav M, Gulkari VD, Wanjari MM. Tridax procumbens leaf extracts prevent formation of renal calculi in lithiatic rats. Ancient Science of Life. 2016 Oct; 36(2):90.

17. Nagarajan Y, Boopathi R, Yahoob SA, Venkatraman A. In Vitro Evaluation of Anti Urolithiatic Activity of Tridax procumbens Lam. In Vitro. 2019 Aug; 5(8):97-102.

18. Malviya V, Tawar M, Burange P, Bairagi R. Preparation and Characterization of Gastroreten-tive Sustained Release In-situ Gel of Lafutidine. International Journal of Pharmaceutical Sciences and Nanotechnology (IJPSN). 2022 Dec 12; 15(6):6216-28.

19. Jagtap PN, Vyapari BR, Nimbalkar YH, Kale SV, Nigade GB. Evaluation of antiurolithiatic activity of polyherbal formulation (Lithout Tablets) by in-vitro inhibition of calcium oxalate crystallization. Research Journal of Pharmacy and Technology. 2019 Nov 1; 12(11):5477-8.

20. Malviya V, Arya A, Burange P, Gajbhiye K, Rathod G, Tawar M. To Evaluate the Cardioprotective effect of Hydroalcoholic Extract of Matricaria chamomilla Linn in Isoproterenol Induced Myocardial Infarction in Wistar Rats. Research Journal of Pharmacy and Technology. 2022 Sep 28; 15(9):3887-92.

21. Malviya V, Thakur Y, Gudadhe SS, Tawar M. Formulation and evaluation of natural gum based fast dissolving tablet of Meclizine hydrochloride by using 3 factorial design 2. Asian Journal of Pharmacy and Pharmacology. 2020; 6(2):94-100.

22. Kamakshi UN, Rao BG, Rao BV. Comparison of in vitro antiurolithiatic activity of Aerva lanata, Sphaeranthus indicus, Merremia emarginata. Research Journal of Pharmacy and Technology. 2017; 10(6):1653-6.

23. Kalita S, Kumar G, Karthik L, Rao KV. A Review on Medicinal Properties of Lantana camara Linn. Research Journal of Pharmacy and Technology. 2012; 5(6):711-5.

24. Kayalvizhi D, Sivakumar V, Jayanthi M. Phytochemical screening and antinephrolithiasis activity of ethanol extract of Aerva lanata on ethylene glycol induced renal stone in rats. Research Journal of Pharmacy and Technology. 2015 Nov 1; 8(11):1481.

25. Dhimmar N, Patel NM, Gajera V, Lambole V. Pharmacological activities of Moringa oleifera: an overview. Res J Pharm Tech. 2015 Apr 28; 10:476-80.

Received on 08.11.2022 Modified on 13.12.2022

Res. J. Pharma. Dosage Forms and Tech.2023; 15(3):157-162.

DOI: 10.52711/0975-4377.2023.00026